Gastrointestinal Microbiota of Seabirds
By
Meagan Dewar
Bachelor of Science, (Marine Biology) (BSc)
Bachelor of Applied Biology (Hons)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Deakin University
28th September, 2012
i
DEAKIN UNIVERSITY
ACCESS TO THESIS - A
I am the author of the thesis entitled
Gastrointestinal Microbiota of Seabirds
submitted for the degree of Doctorate of Philosophy
This thesis may be made available for consultation, loan and limited copying
in accordance with the Copyright Act 1968.
'I certify that I am the student named below and that the information provided in the form is
correct'
Full Name:
Meagan Dewar
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Date:
28th September 2012
ii
DEAKIN UNIVERSITY
CANDIDATE DECLARATION
I certify that the thesis entitled
Gastrointestinal Microbiota of Seabirds
submitted for the degree of Doctorate of Philosophy
is the result of my own work and that where reference is made to the work of
others, due acknowledgment is given.
I also certify that any material in the thesis which has been accepted for a
degree or diploma by any university or institution is identified in the text.
'I certify that I am the student named below and that the information provided in the form is
correct'
Full Name:
Meagan Dewar
Signed
Date
28th September 2012
i
Earthworms are more important than humans. The fact is that earthworms are more
“
important than people from an ecological perspective for the simple reason that
earthworms can survive without people but people will not be able to survive without
earthworms. People are dependent on earthworms; earthworms are not dependent
upon humans.
Worms
can live on the planet without people; people can’t live on the planet
without worms. Honeybees are more important than people, bacteria are more
important than people – any species that is actually a foundation species which
allows for the other species to survive is more important than the species up top.
The fact is that the ecological law of diversity dictates that diversity of species is
essential for the survival of each species within that diversity. Humans are equal to
other species for the simple reason that if the other species were removed from the
eco-systems that people inhabit, humans could not survive."
Captain Paul Watson, Sea Shepherd
ii
ACKNOWLEDGEMENTS
Over the past 4.5 years a lot of blood, sweat and tears have gone into achieving one
thing, this thesis, and now that journey must come to an end. Throughout this time
there have been numerous people who have helped me achieve my goal. First and
foremost, I would like to thank my supervisors Dr Stuart Smith and Associate
Professor John Arnould for this opportunity. I wish to thank Stuart for his
encouragement, guidance, expertise and support over the past 4.5 years. Your
assistance has been instrumental in achieving my goal. I wish to thank John for his
advice, expertise and guidance. Your support has also been instrumental in helping
me achieve my goal. I would also like to thank John Reynolds for your statistical
assistance and advice.
I would also like to thank my collaborators Dr Peter Dann (Phillip Island Nature
Parks), Dr Phil Trathan (British Antarctic Survey) and Dr Rene Groscolas (Centre
National de la Recherche Scientifique) for your logistical support, sample collection
and editorial assistance. I would also like to thank the support crew from BAS;
Stacey Adlard and Fabrice Le Bouard and CNRS; Vincent Viblanc, Nelly Mallosse,
Goetz Eichhorn and René Groscolas for assisting with sample collection from
Possession and Bird Island.
I would also like to acknowledge my funding partners Holsworth Wildlife Research
Endowment, Molecular Nutrition Group Strategic Research Centre (Deakin
University), Phillip Island Nature Parks, Snowtown Valley, the French Polar Institute
(IPEV) (project N° 119) and the British Antarctic Survey. Without your financial
assistance this project would not have been possible.
A special thank you to Dr Tiana Preston, Dr Louise Kelly, Joel Maloney, David,
Kaye & Belinda Dewar for their assistance in the field at Phillip Island and to Nicole
Schuman, Marcus Salton and Julie Baxter for their assistance in the field on Notch
Island. Also to Tiana and Louise thank you for all the great penguin nights on the St
Kilda breakwater and thank you for your support and advice throughout this time.
To the Hutchesson family, Damien, Vanessa, Max and Oliver, thank you for your
support, endless trips to the zoo, photography outings and the endless laughter. To
my dearest friends Kathryn Trevaskis, Sarah Troia, Dimi Paraskevas, Suree Morrison
and Marty Durkan I want to thank you for your love and support over the years and
iii
thank you for the many nights of gossip and laughter, but most of all thank you for
your friendship. To my beautiful angels Bevan, Remy, Luca and Dylan, you have
brought so much joy and happiness to my life. You always know how to make your
Aunty Megs laugh, so thank you for making the hard times so much easier. To my
family (Aunties, Uncles and Cousins), thank you for your support and believing in
me over the years it has meant the world to me.
To my sister, thank you believing in me, for your support, our wonderful holidays
together and for putting up with me over the past 4.5 years. I know at times it hasn’t
been easy. But thanks.
To the Deakin crew Amy, Aimee, Sheena, Marissa, Paul and Lisa, thank you for
sharing this journey with me. Thank you for your support, endless gossip sessions
and daily coffee breaks.
To my animals, Kitty, Claw, Pepper, Jessie, Sultan and Sebastian, you have been my
source of comfort and joy over the past 23 years. I will never forget your endless
love. Rest in Peace. To my new kitty, Nala, thank you for your love and cuddles
these past few months.
Finally, to the two most important people, my parents David and Kaye Dewar,
without you this dream would never have become a reality. Thank you so very much
for all the sacrifices you have made throughout my life to help me achieve my
dreams. Thank you for your enduring love, support and belief in me.
iv
I dedicated this thesis to my parents: David and Kaye Dewar.
Thank you for everything, without you this would not have been possible.
v
PUBLICATIONS FROM Ph.D.
Dewar, M.L. Arnould, J.P.Y. Dann, P. Trathan, P. Groscolas, R and Smith, S.C.
(2012). Inter-specific variations in the gastrointestinal microbiota of penguins.
MicrobiologyOpen (Early View)
http://onlinelibrary.wiley.com/doi/10.1002/mbo3.66/full
FUTURE PAPERS FROM THESIS
Dewar, M.L. Arnould, J.P.Y. Dann, P. Reynolds, J. Smith, S.C (2012) Variations in
the Gastrointestinal Microbiota of Little Penguins (Eudyptula minor) During
Development (In Preparation)
Dewar, M.L. Arnould, J.P.Y. Trathan, P. Dann, P. Smith, S.C (2012) Influence of
Fasting During Moult on the Gastrointestinal Microbiota of Penguins. (In
Preparation)
Dewar, M.L. Arnould, J.P.Y. Dann, P. Smith, S.C (2012) Inter-Specific Variations
in the Gastrointestinal Microbiota of Procellariiformes (In Preparation)
Dewar, M.L. Arnould, J.P.Y. Dann, P. Reynolds, J. Smith, S.C (2012) Variations in
the Gastrointestinal Microbiota of Short-Tailed Shearwaters (Puffinus tenuirostris)
During Development. (In Preparation)
OTHER PUBLICATIONS
Dewar, M.L., and Scarpaci, C. (2011). Microbial implications associated with
stomach flushing of Little Penguins. The Victorian Naturalist 128 (4), 2011, 128–131
Saunders, S. Smith, S.C. Oppedisano, F. Dewar, M.L. Tang, M. (2012) Alteration of
the intestinal microbiota by Lactobacillus rhamnosus GG and Peanut Oral
Immunotherapy (P-POIT) in children with peanut allergy. Gut Pathogens (In
preparation)
Smith, S.C. Chalker, A. Dewar, M.L. Arnould, J.P.Y (2012). Age-related
differences revealed in Australian fur seal Arctocephalus pusillus doriferus gut
microbiota. FEMS Microbiology Ecology (In Review)
vi
COMMUNICATIONS
Oral Communications of PhD results
Dewar, M.L., Arnould, J.P.Y., Dann, P., Trathan, P., Smith, S.C. (2012). Variations
in the Gastrointestinal Microbiota of Moulting and Non-Moulting Penguins.
International Polar Year: Knowledge to Action. Montreal Canada 22-27 April 2012
Dewar, M.L., Arnould, J.P.Y., Trathan, P., Smith, S. (2011). Microbial Ecology of
Penguins in Extreme Polar Regions. CAREX Life in Extreme Environments. 18-20th
October 2011
Dewar, M.L, Arnould, J.P.Y., Trathan, P., Groscolas, R., Smith, S.C. (2010).
Gastrointestinal microbiota of King Penguins (Aptenodytes patagonicus) and the
Influence of geographical separation. 7th International Penguin Conference
Abstracts. Boston, Massachusetts. 30 August – 3rd September 2010
Dewar, M.L., Arnould, J., Dann, P., Trathan, P., Groscolas, R., Smith, S.C.
Exploring the gastrointestinal microbiota of king and little penguins using high
throughput sequencing. (2010) 7th Annual Exercise and Nutrition Sciences Higher
Degrees by Research Student Symposium, Deakin University Melbourne Australia.
(Awarded best presentation)
Poster Presentation of PhD results
Dewar, M.L., Arnould, J.P.Y., Dann, P., Trathan, P., Groscolas, R., Smith, S.
(2010). Changes in the gastrointestinal microbiota during fasting in penguins. 1st
World Seabird Conference. Victoria, British Columbia, 7th -11th of September 2010
(Poster)
Dewar, M.L., Arnould, J.P.Y., and Smith, S. (2009). Gastrointestinal Tract of Little
penguins. Seabird Group 10th International Seabird Conference. Brugge, Belgium
27th -30th March 2009 (Poster).
vii
ABSTRACT
The gastrointestinal (GI) tract contains a highly diverse and complex microbial
ecosystem that consists of residential and transient microbes. At birth, the GI tract in
avian species is devoid of microorganisms. Immediately after birth however, the GI
microbiota is rapidly colonised by microbes. The GI microbiota plays a significant
role in gut development and maturation, host biochemistry, physiology, metabolism,
synthesis and secretion of essential vitamins, minerals and nutrients, energy
extraction, fat metabolism and storage. To date, studies on the avian GI microbiota
have primarily been focused on poultry, with a limited number of studies conducted
on avian species living under natural conditions. This thesis aimed to address this
knowledge gap through a study on the gastrointestinal microbiota of seabirds using
faecal samples collected via cloacal swabs. The study was designed to characterise
the gastrointestinal microbiota of marine seabirds under natural conditions and to
examine the variation in microbial composition during development and to examine
the influence of fasting during moult on the GI microbiota. The study species in this
thesis included; king (Aptenodytes patagonicus), gentoo (Pygoscelis papua),
macaroni (Eudyptes chrysolophus) and little penguin (Eudyptula minor), short-tailed
shearwater (Ardenna tenuirostris), fairy prion (Pachyptila turtur) and common
diving petrel (Pelecanoides urinatrix). DNA extracted from faecal samples was
analysed using quantitative Real Time PCR (qPCR) and 16S rRNA pyrosequencing.
Quantitative data obtained from the qPCR analysis highlighted significant
differences in the abundance of Firmicutes, Bacteroidetes, Actinobacteria and
Proteobacteria between all penguins (Chapter 2), Procellariiformes (Chapter 5),
during development of little penguins (4) and in fasting penguins (study 4). No
significant differences
were observed during development in short-tailed
shearwaters. The pyrosequencing analysis identified that the penguin microbiota was
dominated by Firmicutes, Bacteroidetes, Fusobacteria and Proteobacteria. However
there were significant differences in the relative abundance of the major phyla. There
was a relatively high level of similarity between gentoo and macaroni penguins. In
common diving petrels (CDP) the microbiota was dominated by Firmicutes,
Bacteroidetes and Proteobacteria, whereas the microbiota of short-tailed shearwaters
(STS) and fairy prions (FP) was dominated by Firmicutes. There was a high level of
similarity between the oil producing seabirds (STS & FP) and a low level of
similarity between the oil producing (STS & FP) and non-oil producing (CDP)
viii
seabirds. Potential factors influencing the microbiota of penguins are most likely to
be associated with diet, prey associated microbiota and host phylogeny, whereas the
microbiota of Procellariiformes are most likely to be associated with digestive
physiology (production of stomach oil, digestive anatomy) and host phylogeny. The
development of the seabird microbiota, differs greatly between species (little penguin
& short-tailed shearwater). In the little penguin, there were significant differences in
the microbial composition throughout development and a significant upward trend in
the abundance of Firmicutes and Bacteroidetes. In the short-tailed shearwater,
however, there were no significant differences throughout development and appears
relatively stable throughout development with a high level of similarity between each
age class. In addition, fasting during moult significantly influenced the microbial
composition of both king and little penguins, with the degree to which moult impacts
the microbial composition differing between the two species. In little penguins, there
is still a high level of similarity between early and late moult (60%), with little
variation in the abundance of the major phyla observed with both the qPCR and
pyrosequencing analysis. In contrast, there were significant differences between early
and late moult with a 75% increase in the level of Fusobacteriaceae. In addition,
there was a low level of similarity (20%) between early and late moult in king
penguins. This research provides a baseline for future work regarding the microbial
composition of seabirds. This thesis has identified that a complex community
inhabits the GI tract of seabirds and provides insight into the successional changes
that occur during development and how fasting influences the microbial composition.
ix
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
iv
ABSTRACT
ix
LIST OF FIGURES
xiv
LIST OF TABLES
xvi
CHAPTER 1
1
Introduction
1
Gastrointestinal microbiota
1
Role of Gastrointestinal Microbiota
1
Gastrointestinal Physiology
1
Immune System
2
Metabolism & Adiposity
2
Establishment of Gastrointestinal Microbiota
3
Factors Influencing Gastrointestinal Microbiota
5
Gastrointestinal Microbiota & Fasting
6
Avian Gastrointestinal Microbiota
7
Spheniscidae Taxonomy and Distribution
8
King Penguin (Aptenodytes patagonicus)
9
Macaroni Penguin (Eudyptes chrysolophus)
10
Gentoo Penguin (Pygoscelis papua)
11
Little Penguin (Eudyptula minor)
12
Procellariiform Taxonomy and Distribution
14
Family Procellariidae
15
Short-tailed Shearwater (Ardenna tenuirostris)
15
Fairy Prion (Pachyptila turtur)
16
Family Pelecanoididae
17
Common Diving Petrel (Pelecanoides urinatrix)
17
Penguins as a model of Avian Gastrointestinal Microbiota
17
Procellariiformes as a model of Avian Gastrointestinal Microbiota
18
Research Objectives and Thesis Structure
26
Preface
27
CHAPTER 2
28
Inter-specific variations in the gastrointestinal microbiota of Penguins 28
x
Introduction
29
Methods
32
Results
35
Discussion
45
CHAPTER 3
49
Variations in the Gastrointestinal Microbiota of Little Penguins 49
(Eudyptula minor) During Development
Introduction
50
Methods
51
Results
55
Discussion
64
CHAPTER 4
67
Influence of Fasting During Moult on the Gastrointestinal Microbiota 67
of Penguins
Introduction
68
Methods
69
Results
71
Discussion
82
CHAPTER 5
85
Inter-Specific Variations in the Gastrointestinal Microbiota of 85
Procellariiformes
Introduction
86
Methods
87
Results
89
Discussion
97
CHAPTER 6
101
Variations in the Gastrointestinal Microbiota of Short-Tailed 101
Shearwaters (Puffinus tenuirostris) During Development
Introduction
102
Methods
103
Results
105
Discussion
116
CHAPTER 7
119
General Discussion
119
xi
CHAPTER 8
126
Future Studies
128
Bibliography
134
Appendix
153
Appendix 1, Taxonomic breakdown of bacteria present in the GI 153
microbiota of penguins
Appendix 2, Taxonomic Breakdown of bacteria present in little penguins 165
during development
Appendix 3, Taxonomic Breakdown of bacteria present in moulting 173
penguins
Appendix 4, Taxonomic Breakdown of bacteria present in Procellariiform 185
Seabirds.
Appendix 5, Taxonomic Breakdown of bacteria present in short-tailed 196
shearwaters during development.
xii
LIST OF FIGURES
Figure 1.1, King Penguin
Figure 1.2, Macaroni Penguin
Figure 1.3, Gentoo Penguin
Figure 1.4, Little Penguin
Figure 1.5, Short-tailed Shearwater chick
Figure 2.1 Variation in the abundance of the major phyla; Firmicutes,
Bacteroidetes, Proteobacteria and Actinobacteria in king, gentoo, little
and macaroni penguins
Figure 2.2, Similarity of the major bacterial phyla of penguin species
using qPCR.
Figure 2.3 Relative abundance of major phyla from the GI microbiota of
gentoo, king, little and macaroni penguins
Figure 2.4, MDS Analysis of the gut microbiota of penguin species
Figure 2.5, Analysis of Similarity of the GI microbiota of gentoo, king,
little and macaroni penguins
Figure 2.6, Relative abundance of major families from the GI microbiota
of gentoo, king, little and macaroni penguins.
Figure 2.7, Variation in species diversity in king, gentoo, little and
macaroni penguins.
Figure 3.1, Artificial nest box used by little penguin in this study
Figure 3.2, Adult little penguins and chicks in nesting box
Figure 3.3, Changes in the abundance of the major phyla; Actinobacteria
(a), Bacteroidetes (b), Firmicutes (c) and Proteobacteria (d) during
development in little penguin chicks.
Figure 3.4, Similarity of the major bacterial phyla in little penguins
during development using qPCR.
Figure 3.5, Relative abundance of major phyla during development in
little penguin chicks.
Figure 3.6, Similarity of the gut microbiota of little penguins during
development.
Figure 3.7, Relative abundance of major Families during development in
little penguin chicks.
Figure 3.8, Variation in species diversity during development and in
adult little penguins
Figure 4.1, Variation in the abundance of the major phyla; Firmicutes,
Bacteroidetes, Proteobacteria and Actinobacteria during moult in little
penguins.
Figure 4.2, Variation in the abundance of the major phyla; Firmicutes,
Bacteroidetes, Proteobacteria and Actinobacteria during moult in king
penguins.
Figure 4.3, Similarity of the major bacterial phyla in little penguins
during moult using qPCR.
Figure 4.4, Similarity of the major bacterial phyla in king penguins
during moult using qPCR.
Figure 4.5, Relative abundance of major phyla in moulting little penguins
using 16S rRNA pyrosequencing
Figure 4.6, Relative abundance of major families from the GI microbiota
of moulting little penguins
9
11
12
13
16
37
38
40
41
42
43
44
52
53
57
58
60
61
62
63
72
73
74
75
77
78
xiii
Figure 4.7, Relative abundance of major phyla in moulting king penguins
using 16S rRNA pyrosequencing
Figure 4.8, Relative abundance of major families from the GI microbiota
of moulting king penguins.
Figure 5.1, Variation in the abundance of the major phyla; Firmicutes (a),
Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) in Common
Diving Petrels, Fairy Prions and Short-tailed Shearwaters
Figure 5.2, Similarity of the major bacterial phyla in Procellariiform
seabirds using qPCR.
Figure 5.3, Relative abundance of major phyla in three Procellariiform
Seabirds; a) Common Diving Petrels, b) Fairy Prions, and c) Short-tailed
Shearwaters
Figure 5.4, Cluster Analysis of the GI microbiota Common Diving
Petrels (CDP), Fairy Prions (FP) and Short-tailed Shearwaters (STS).
Figure 5.5, Relative abundance of major Families in common diving
petrels, fairy prions and short-tailed shearwaters.
Figure 5.6, Species diversity of Procellariiformes Microbiota
Figure 6.1, Short-tailed shearwater breeding colony. Sticks represent nest
sites used in this study
Figure 6.2, Natural burrow of short-tailed shearwater
Figure 6.3, Short-tailed shearwater chick
Figure 6.4, Changes in the abundance of the major phyla; Firmicutes (a),
Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) during
development in short-tailed shearwater chicks.
Figure 6.5, Similarity of the major bacterial phyla in short-tailed
shearwater chicks during development using qPCR
Figure 6.6, Relative abundance of major the phyla in short-tailed
shearwater chicks during development
Figure 6.7, Similarity of the gut microbiota of short-tailed shearwater
chicks during development
Figure 6.8, Relative abundance of major Families during development in
short-tailed shearwater chicks.
Figure 6.9, Species diversity at different age classes
Figure 7.1, MDS/Cluster analysis of seabirds
Figure 7.2, Relative abundance of the major phyla for all seabird species
using 16S rRNA pyrosequencing
80
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90
91
93
94
95
96
106
107
108
109
110
112
113
114
115
121
122
xiv
LIST OF TABLES
Table 1.1, Metabolites contributed by gut bacteria
Table 1.2 Microbial compositions of avian species from previous 16S
rRNA studies
Table 1.3, Bacteria isolated from penguins from previous studies
Table 1.4, Microorganisms isolated from Procellariiform seabirds from
previous studies
Table 2.1 Quantitative real time PCR primer sequences used in this
study to detect and quantify major phyla present in penguin faeces.
Table A1, Taxonomic breakdown of bacteria present in the GI
microbiota of penguins
Table A2, Taxonomic Breakdown of bacteria present in little penguins
during development
Table A3.1, Taxonomic Breakdown of bacteria present in moulting
little penguins
Table A3.2, Taxonomic Breakdown of bacteria present in moulting
king penguins
Table A4, Taxonomic Breakdown of bacteria present in Procellariiform
Seabirds.
Table A5 Taxonomic Breakdown of bacteria present in short-tailed
shearwaters during development.
4
8
19
23
34
153
165
173
178
185
196
xv
CHAPTER 1
INTRODUCTION
Gastrointestinal Microbiota
The gastrointestinal (GI) tract contains a highly diverse and complex microbial
ecosystem that consists of residential and transient microbes [67, 143, 163, 257]. The
GI microbiota was once considered to be a “collection of freeloading commensals”
[162] that did not provide benefit to their host. However, recent studies have
identified that a complex and mutually beneficial relationship exists between a host
and its microbiota. This complex relationship is said to be the result of a long and
complex coevolution [154]. The GI microbiota plays a considerable role in gut
development and maturation, host biochemistry, physiology, metabolism, synthesis
and secretion of essential vitamins, minerals and nutrients [168, 180, 299].
Role of Gastrointestinal Bacteria
The use of germ free animals has highlighted the importance of the GI microbiota to
its vertebrate host. These studies have shown that the GI microbiota has a profound
influence on the nutritional, physiological, immunological and metabolic processes
of the host [168, 299], playing a significant role in energy harvest, fat metabolism,
secretion and synthesis of nutrients, vitamins, amino acids and the production of
short chain fatty acids from the diet consumed [67, 94, 248, 257, 267]. In the absence
of bacteria the GI tract does not fully mature and an animal’s ability to secrete and
absorb essential vitamins and nutrients from their diet is significantly reduced [25,
199, 214, 215]. Germ free animals are not only more susceptible to disease, but also
require a greater caloric intake to achieve and maintain a normal body weight [9].
This indicates that members of the GI microbiota modulate fat deposition [185].
Gastrointestinal Physiology
The use of germ free models such as mice; chickens and zebrafish have revealed a
range of functions that are influenced by the presence of a commensal microbiota.
Rawls et al [215] used zebrafish, to examine the influence of the GI microbiota on
the development of the GI tract. Zebrafish are a useful germ free model to examine
the influence of the GI microbiota on gut development. Firstly, the structure of the
GI tract is similar to that of human hosts and secondly because they are completely
transparent during the fry and juvenile stages. When examining the GI tract of germ
1
free Zebrafish, Rawls et al [215] noted that in the absence of a commensal GI
microbiota, the epithelium of the GI tract is arrested in aspects of differentiation. In
addition, the absence of a GI microbiota reduces a host’s ability to secrete and absorb
essential nutrients and extract energy from dietary sources [38].
Immune System
The GI microbiota has also been shown to be responsible for the establishment and
maintenance of the host’s immune system through its effect on gut morphology,
nutrition, and pathogenesis of intestinal diseases [257]. The indigenous microbiota
also suppresses the colonisation of invading bacteria which provides protection to the
host against pathogenic species [143, 159, 240] and can prevent enteric infection by
secreting defensins, while phagocytes and M cells continually survey the microbiota
in the lumen for potential pathogens. In recent studies, microbial components of the
indigenous microbiota have been shown to stimulate the immune system [215].
Metabolism & Adiposity
Animal studies have demonstrated that the gastrointestinal microbiota has a profound
influence on the modulation of host metabolism. The GI microbiota appears to be
able to modify a number of lipids in serum, adipose tissue and in the liver, with
drastic effects on triglycerides and phosphatidylcholine. The resident microbiota is
also responsible for the production of metabolites that contribute to host fitness and
survival [132]. Some of these metabolites are listed in table 1.1 below.
There have been multiple studies linking the presence of gut bacteria to adiposity,
which have noted that changes to the microbial composition can significantly
increase the level of adiposity experienced by the host causing obesity [9, 89, 126,
152, 156, 177, 180, 213, 260, 263]. When germ free animals are inoculated with the
GI microbiota of conventionally raised hosts, the body fat level of the germ free host
rapidly increases, despite decreased food intake [9]. Conventionalisation of germ free
mice increases the production of SCFA’s, which are absorbed and metabolised by the
body and supresses the expression of fasting induced adipose factor (Fiaf). The
suppression of Fiaf, leads to increased enzymatic activity of lipoprotein lipase (LPL),
increasing the uptake of lipids into adipocytes and other tissues [38]. In addition,
both the GI microbiota and host genetics have been identified as risk factors in the
development of obesity. Research has indicated that obesity is associated with a
2
higher proportion of Firmicutes and a decreased proportion of Bacteroidetes
increasing energy production [9, 153, 154, 263]. Turnbaugh et al [264] documented
that members from the Phylum Firmicutes were associated with the breakdown of
complex carbohydrates, polysaccharides, and sugars into simple sugars and fatty
acids which are then absorbed and utilised as an energy source by the host. In
chickens Panda et al [196] documented that the supplementation of diet with butyrate
increased their overall body mass. As butyrate is a SCFA derived from microbial
fermentation, this observation indicates that butyrate producing microbes could
contribute to host adiposity.
Establishment of Gastrointestinal Microbiota
In nature, bacteria are found everywhere and with the availability of nutrients are
able to colonise new habitats, including the GI tract [6]. At birth, the GI tract is
considered to be devoid of microorganisms. In mammals, the microbial community
is said to be inherited from the mother through intimate contact with faecal and
vaginal microbes during birth and from breast milk [194]. In birds, however, new
born chicks, acquire their microbiota from the surface of the egg, surrounding
environment (i.e. nest) and their first meal [6, 99, 101, 159, 162, 217, 256, 291]. In
addition, vertical transmission of microbes via regurgitation of an undigested or
partially digested meal is also said to influence the microbiota of the chick [110, 142,
205]. Through ecological succession, the sterile GI tract, is rapidly colonised forming
a dense and complex microbial community within 2 years in humans
[164, 166,
168, 199, 240, 268] and 40 days in chickens [16, 17, 68, 90, 217].
3
Table 1.1, Metabolites contributed by Gut Bacteria (table modified from Nicholson
et al [182]).
Metabolites
Related Bacteria
Short-chain fatty
acids: acetate,
propionate, butyrate,
isobutyrate, 2methylpropionate,
valerate, isovalerate,
hexonate
Clostridial clusters IV
and XIVa of
Firmicutes, including
species Eubacterium,
Roseburia,
Faecalibacterium
Bile acids
Lactobacillus,
Bifidobacteria,
Enterobacteria,
Bacteroides,
Clostrium
Indole derivatives
Clostridium
sporogens, E.coli
Lipids: conjugated
fatty acids, LPS,
peptidoglycan,
acylglycerols,
cholesterol,
triglycerides
Campylobacter
jejuni, Clostridium
saccharolyticum,
Bifidobacterium,
Roseburia, Klebsiella,
Enterobacter,
Citrobacter,
Clostridium
Potential Biological
Function
Decreased colonic pH,
inhibit growth of
pathogens; stimulate
water and sodium
absorption; cholesterol
synthesis; provide energy
to the colonic epithelium
cells; implicated in obesity
Absorb dietary fats and
lipid soluble vitamins,
facilitate lipid absorption,
maintain intestinal barrier
function, regulate
triglycerides, cholesterol,
glucose and energy
homeostasis
Protect against stressinduced lesion in the GI
tract, modulate
expression of
proinflammatory genes,
increase expression of
anti-inflammatory genes,
and strengthen epithelial
cell barrier properties.
Implicated in GI
pathologies brain-gut axis,
and a few neurological
conditions
Impact intestinal
permeability, active
intestine-brain-liver
neural axis to regulate
glucose homeostasis, LPS
induces chronic systemic
inflammation, enhance
the immune system, alter
lipoprotein profiles
Reference
[39, 196, 231,
288]
[117, 223,
251]
[20, 139, 292]
[43, 235]
4
At birth, the neonate gut is an aerobic environment that is rapidly colonised by
aerobic and facultative anaerobes such as Escherichia coli and Streptococci [5, 68,
168, 217, 268]. Within the first few days both E. coli and Streptococci are present in
extremely high numbers of around 108 – 1010 CFU of faeces [168]. Research suggests
that these pioneering bacteria reduced the amount of oxygen present in the GI tract
through oxidation/reduction, creating an environment that is highly favourable to
facultative and obligate anaerobes [168, 199] such as Bacteroides, Clostridia, and
Bifidobacterium [168, 245].
Although the microbial succession in the developing vertebrate has been studied in
many animals, including humans, chickens, and pigs [168] to date, little is known
about the microbial succession in the developing seabird.
Factors Influencing Gastrointestinal Microbiota
Throughout life, the GI tract is continuously exposed to microbes from the
surrounding environment, diet and through colonial living [87, 262, 299]. In
addition, other factors such as host phylogeny, physiology, gut morphology, nesting
material, diet, prey associated microbiota, antibiotic use and weight influence the
microbial composition [73, 87, 88, 131, 153, 154, 181].
In a revolutionary study on the microbial composition of 60 species of terrestrial
mammals, Ley et al [154] discovered that host phylogeny, diet and gut physiology
influenced the composition [154, 262]. In addition, Ochman et al [186] and Yildrim
et al [290] also showed that evolutionary lineages determine the composition of the
gut microbiota of a vertebrate species. However, more recent evidence points to diet
being the main driving force behind differences in the gut microbiota of
phylogenetically similar hosts [178, 181]. In accordance with Ley et al [154], Nelson
[181] identified that both host phylogeny and diet influence the gut microbiota of
Antarctic seals. In addition, Banks et al [12] documented that host phylogeny was a
major driving force behind differences in the GI microbiota of adéliepenguins. These
differrences however, need to be examined in seabirds in more detail.
During development, nesting environment has been identified as a major source of
microbial colonisation avian species [162, 256]. Lucas and Heeb [162] discovered
that when blue tit chicks were fostered in to the nest of great tits the blue tit chick
5
acquired the bacterial microbiota of their foster siblings. Whilst Torok [256],
identified that the microbial composition of the litter (nest) material influenced the
microbial composition of chickens during the first week of development.
Whilst host environment and phylogeny have been shown to influence the microbial
composition of the infant, diet factor influencing the microbial composition
throughout life [140]. Dietary changes modify the composition of the GI microbiota
by altering the intestinal environment and metabolic activity of the GI tract. When
examining the microbiota of 60 mammals Ley et al [153, 154] identified that
individuals clustered together according to their dietary preference (i.e. herbivore,
carnivores, omnivore). In addition, Blanco et al [24] identified significant changes in
the microbial composition of red kites in response to changes in diet, within an
increase
in pathogenic species of bacteria Thus diet influences GI microbiota
profoundly.
Gastrointestinal Microbiota and Fasting
Previous studies examining the effect of fasting on the GI microbiota of vertebrate
(hamsters, python, mice, termites), have shown that fasting not only alters the
composition and diversity of the GI microbiota, and influence the host’s immune
defences [73, 74, 241]. Research has also shown that complex interrelationships exist
between a host, its GI microbiota and the hosts nutritional status, diet and
physiological state [73, 74]. These studies however, have concentrated on animals
that are relatively inactive during times of nutrient deprivation. Unlike other
vertebrates, penguins do not hibernate during times of fasting. In penguins fasting
occurs during the breeding season (incubation of eggs and chick brooding) and
moult, where penguins replace their entire plumage whist fasting on land. The
moulting fasts can last anywhere from 2 to 5 weeks. Therefore, penguins must
survive long periods of starvation while also coping with increased metabolic
demands for feather synthesis and thermoregulation [102, 281]. Therefore, penguins
provide an attractive model for investigating the influence of fasting that is
associated with increased metabolic demands. To date the influence of fasting on the
microbiota of a seabird has not been examined.
6
Avian Gastrointestinal Microbiota
In comparison to mammals, there is limited information available on the avian
microbiota with the exception of poultry. Most avian based studies on microbial
composition have used selective culture based techniques to investigate specific
bacterial populations and/or to identify the presence of known human and veterinary
pathogens [142, 158]. From the limited number of studies conducted on avian
species using 16S rRNA sequencing to profile the GI microbiota, has identified
similarities between mammals and birds especially at the higher taxonomic level
(phylum). However, at lower taxonomic levels (genus and species) there are
significant differences, with avian specific genera and species being identified [101,
142, 158, 160, 161]. From these 16S rRNA studies it has been identified that the
avian GI microbiota is dominated by Firmicutes, Bacteroidetes, Proteobacteria,
Actinobacteria and Tenericutes; however the relative abundances of these phyla
differ from species to species as shown in table 1.2.
7
Table 1.2 Microbial compositions of avian species from previous 16S rRNA studies
(table modified from Kohl [142])
Phyla
Adélie
Gull
Penguins
Canada
Turkey
Chicken
Parrot
Geese
Firmicutes
39.2
54.6
71.6
32.3
70
72.9
Bacteroidetes
14.7
1.1
10.1
54.2
1.9
0.2
Actinobacteria 32.3
6.4
7.0
<0.1
4.9
12.0
Proteobacteria
9.8
23
10.4
3.4
21.5
14.9
Tenericutes
3.9
8.9
0.2
<0.1
1.6
14.9
Reference
[12]
[161]
[160]
[234]
[297]
[289]
Spheniscidae Taxonomy and Distribution
The family Spheniscidae are a group of flightless seabirds, containing 16-19 species
[76, 247]. Penguins occupy an extensive range including Antarctica, the Galapagos
Islands, South Africa, Australia, New Zealand and South America [27]. Rough
estimates of penguin abundance suggest that there are 23.6 million breeding pairs of
penguins worldwide [218, 286] with 10 of the 19 species, considered vulnerable to
extinction (www.iucnredlist.org) [27, 218].
Penguins are long lived seabirds, spending most of their lives at sea, only returning
to land to breed and moult. Penguins undergo prolonged periods of fasting during
breeding and moult [31, 55, 119, 283]. Prior to undergoing their annual fasts,
penguins build up large reserves of white adipose tissue and protein for long periods
of fasting during incubation and moult [124]. During the breeding season, most of
the fasting occurs during incubation and brooding of the chick. Throughout this time,
penguins must rely on their endogenous fat stores for nourishment [49]. In addition,
penguins are able to store copious amount of undigested food in their stomach for
long periods of time, which is used to provision their chicks [124]. During moult
penguins replace their entire plumage whilst fasting on land and cannot return to sea
because of the consequences of reduced waterproofing and thermal insulation [105,
119]. Similarly to the breeding season, penguins must rely on endogenous fat and
protein reserves for feather synthesis and nourishment for the duration of moult
which can take between 2-5 weeks [50, 218].
8
This thesis will examine the GI microbiota of four members of the family
Spheniscidae,
the
king
(Aptenodytes
patagonicus),
macaroni
(Eudyptes
chrysolophus), gentoo (Pygoscelis papua) and little (Eudyptula minor).
King Penguin (Aptenodytes patagonicus)
Figure 1.1, King penguin (Photography by Meagan Dewar)
Of the 19 species of penguins, the king penguin (Aptenodytes patagonicus) is the
second largest species, standing between 85-90cm tall and is one of two species
comprising the genus Aptenodytes. King penguins have an extensive geographical
range; breeding throughout the sub-Antarctic and Antarctic Islands between 45° and
65° south, including South Georgia Islands, Crozet Archipelago, Macquarie Island,
Prince Edward Islands, and Kerguelen Islands [70, 84]. Current estimates on the
global population size of king penguin’s ranges from 1.5 million breeding pairs [14].
At sea king penguins forage north of Antarctic pack ice near where the
Antarctic/sub-Antarctic waters meet the Indian, Australasian and Atlantic Ocean
regions. The diet of king penguins consists of Myctophid fish (90%), with
crustaceans and cephalopods [52, 53, 192, 211, 224].
King penguins have a long and complex breeding cycle and are the only penguin
species, whose breeding cycle lasts more the a year. After a period of courtship,
female king penguins lay one large egg (November), with both parents taking turns
to incubate the egg (54 days) [247]. During guard stage, one parent must remain with
the chick, whilst the other resumes foraging. Once chicks are able to thermoregulate,
9
both parents return to sea to resume foraging, whilst chicks join crèches within the
colony. From January to the end of the Austral summer (April), king penguin chicks
will have built up large fat reserves [105, 118]. Due to sharp declines in food
availability during the winter, adults must forage further away from the colony,
leaving their chicks to fasting for extensive periods of time, which can last for up to 5
months [86, 218, 278]. In September, parents return to the colony and feeding
resumes with both parents provisioning their chicks until fledging in Nov/Dec. In its
entirety, the breeding season for a king penguin takes between 12-14 months, with
king penguins breeding once every 2 years. After breeding king penguins return to
sea to replenish their energy reserves to prepare for moult which can last up to 5
weeks [218].
Macaroni Penguin (Eudyptes chrysolophus)
Eudptid or Crested penguins are characterised by their crests of yellow or orange
plumes and red or red-brown eyes. Of the eudyptids, macaroni penguins are the
second largest [218]. Macaroni penguins are the most abundant penguin species [71,
82] with 9-11.8 million breeding pairs globally [286].
Macaroni penguins are the most southerly breeding of all the eudyptids. There are
approximately 216 colonies of macaroni penguins, breeding at 50 sites throughout
the Antarctic and Sub-Antarctic islands, including; South Georgia, Chile, South
Sandwich Islands, Crozet, Kerugelen Islands, Heard and McDonalds Islands and
locally on the Antarctic peninsula [15, 75, 285, 287]. During the breeding season, the
diet of macaroni penguin consists mainly of the Euphausiid krill Euphausia superb
(>90%), with small fish and cephalopods becoming more predominant at the end of
the breeding season [32, 76, 82].
10
Figure 1.2 Macaroni Penguin (photography by M. Dewar 2012)
In September/October every year macaroni penguins, return to the colony for the
beginning of the breeding season. Macaroni penguins breed on steep terrain such as
lava flows, rock falls, and side of cliffs, caves and hills. Their nests are made out of
rocks, sticks and pebbles. Macaroni breeding sites are extremely crowded, which can
contain anywhere from 1-2 million breeding pairs [285]. In contrast to other penguin
species, the eggs of eudyptid penguins are not uniform in size. The first egg is
significantly smaller than the second egg, and is abandoned before hatching, with up
to 50% being abandoned prior to the second egg being laid [270, 281, 282, 284].
Following an incubation period of 5 weeks, one parent remains at the nest to brood
the chick whilst the other returns to sea to forage. During the guard stage, male cares
for the chick whilst the female does most of the foraging, returning to the colony
every 2-3 days to provision the chick Post guard stage (25 days), and both parents
return to sea. In macaroni penguins, chick rearing takes approximately 60-70 days
[282]. Once the chicks fledge, adult macaroni penguins return to sea to build-up large
reserves of protein and lipid in preparation for moult. In macaroni penguins the
moulting can take up to 40 days [282, 284].
Gentoo Penguin (Pygoscelis papua)
Gentoo penguins (Pygoscelis papua) are the largest of all Pygoscelis penguins [218,
283]. Gentoo penguins have a circumpolar distribution mainly located on Sub11
Antarctic Islands and along the Antarctic Peninsula [218], with 80% of the global
population of gentoo penguins breed at the Falkland Islands [95], South Georgia
[258], and the Antarctic peninsula [218]. Globally there is an estimated abundance
of between 300,000 to 387,000 breeding pairs [165, 218]. Their diet consists mainly
of rock cod, crustaceans and cephalopods, with females consuming more crustaceans
than males and males consuming more fish than females [208].
Figure1.3, Gentoo Penguin (Photography by Meagan Dewar)
In mid to late October, Gentoo penguins return to their colonies to breed. Like other
Pygoscelis penguins, gentoo penguins construct a pebble nest and lay two eggs, with
both the male and female taking turns to incubate their eggs. Similar to other penguin
species, chicks remain with their parents until approximately 3-4 weeks of life.
Chicks then enter a crèche until fledging at around 14 weeks of age [218]. In
January, adult gentoo penguins begin their annual moult which lasts for a period of
15-21 days [218].
Little Penguin (Eudyptula minor)
Little penguins (Eudyptula minor) are the smallest of all living species at only 3545cm tall with an average body mass of 0.8-1.3kg and are the only species within
the genera Eudyptula [7, 78, 218, 244].
Little penguins are distributed along the southern coast of Australia, New Zealand
and the Chatham Islands [77, 218]. In Victoria, little penguins can be found along
the entire coast from Portland to Gabo Island. To date, no global population size has
been estimated for the little penguins. However, in Australia the current population is
12
estimated to be just under 1 million breeding pairs, with the largest colonies located
on Gabo Island (35,000 birds) and at the Summerland Peninsula on Phillip Island
(25,000) [81, 84]. At the Summerland Peninsula, Phillip Island little penguins are
present in the colony throughout the year including the winter and non-breeding
season [77]. On average the lifespan for little penguins is 6.5 years. However, these
penguins have been recorded to live over 20 years of age at St Kilda (Zoe Hogg,
Earth Care, Personal Communication) and Summerland Peninsula [79, 80, 219] The
diet of little penguins predominantly consists of
Clupeiformes with Engraulis
australis and Sardinops neopilchardus dominating [56, 58, 170, 218], with the
remainder of the diet including; barracouta, warehou, cod and cephalopods [56, 58].
Figure 1.4, Adult little penguin with chicks (Photography by Meagan Dewar)
The breeding season for little penguins occurs from August to February [172] Unlike
most other penguins, egg laying is not synchronised within a little penguin colony,
with egg laying occurring in some individuals as early and June and July. However,
in most penguins the peak egg laying time usually occurs during September-October.
Little penguins produce a clutch of two eggs with both parents alternating between
incubation and foraging [218]. The incubation period lasts on average between 31-40
days [59, 220]. Similar to all penguin species, little penguin chicks are unable to self
thermoregulate, and therefore parents must take turns guarding their chick whilst the
other forages. The guard stage in little penguins occurs during the first 2-3 weeks
[218, 244]. During the guard stage, chicks are fed on average every 1-2 days [60].
13
whereas during the post-guard stage adults alternate between short and long foraging
trips [230]. Fledging occurs between 9-10 weeks of age [220].
Procellariiformes Taxonomy and Distribution
The order Procellariiformes is made up of four families; Diomedeidae (Albatrosses),
Procellariidae (Petrels and Shearwaters), Hydrobatidae (Storm petrels) and
Pelecanoididae (Diving petrels), 23 genera and 128 species. Procellariiformes are
long lived seabirds, that are characterised by their tubular nostrils, grooved, hooked
beaks and their feet are well set back on their bodies and their ability to produce
stomach oils (all except the Pelecanoididae) [76]. Procellariiformes are the most
abundant seabirds in the Southern Ocean [172, 279] and are the most endangered
bird taxa, with 36 species threatened with extinction (www.iucnredlist.org).
The ability to produce stomach oils is a unique trait restricted to all members of the
Order Procellariiformes, except for diving petrels. Stomach oils are produced in the
proventriculus through partial digestion of prey. The oil fractions of the prey are then
concentrated and retained for long periods of time via delayed gastric emptying [225,
271]. This adaptation reduces the cost of travelling long distance to provide their
chick with a highly concentrated food source that is high in energy and fats [76, 271,
273]. Stomach oils have also been shown to greatly influence the health and
development of the Procellariiform chick [226, 271]. Stomach oils are neutral dietary
lipids. The chemical composition of stomach oils differs from species to species, but
almost always contains wax esters and triglycerides [271].
Procellariiformes lay a single egg with both parents sharing incubation duties (6-12
weeks). Similarly to penguins, Procellariiformes brood their chicks during the early
stages of development due to their inability to thermoregulate, with both parents
taking turns to guard the chick whilst the other forages for food [11, 255, 272]. After
this initial guard period, Procellariiformes use a twofold breeding strategy which
incorporates both short and long foraging trips [48, 275, 276]. Short foraging trips
are used to provision the chick, whilst the long foraging trips are used by adults to
replenish their dwindling fat reserves [275]. Procellariiform chicks accumulate large
fat reserves as a result of over feeding by their parents during the early stages of
development. As a result Procellariiform chicks are often obese and can accumulate a
peak mass of 136% of their parents body mass [272]. Fat accumulation in chicks is
14
essential to enable them to survive prolonged periods of fasting in the later stages of
development [125, 222]. Procellariiform chicks fledge at around 3 months for the
smaller species and 9 months for the larger species [11, 255, 272].
Family Procellariidae
The procellariids are the most diverse and numerous of the Order Procellariiformes
[272]. The family Procellariidae consists of four distinct groups; the fulmarine
petrels, the gadfly petrels, the prions and the shearwaters [272]. This thesis will
examine the GI microbiota of two members of the family Procellariidae; the Shorttailed Shearwater (Ardenna tenuirostris) and the Fairy Prion (Pachyptila turtur).
Short-tailed Shearwater (Ardenna tenuirostris, formally Puffinus tenuirostris)
The Short-tailed shearwater (Ardenna tenuirostris) is a medium sized (40-45cm),
long lived, pelagic seabird that spend the bulk of its life at sea [23]. Short-tailed
shearwaters are the most abundant of all Procellariiformes with an estimated global
population size of 23 million individuals, with the majority breeding in Tasmania and
Victoria [36].
All members of the family Procellariidae, including the Short-tailed shearwater nest
in simple scrapes or burrows, lined with vegetation. Short-tailed shearwaters spend
the winter months in the northern hemisphere foraging in the northern Pacific and
Arctic Ocean during the winter months [123, 237]. In October, short-tailed
shearwaters return to the southern hemisphere to begin the breeding season. Prior to
egg laying, adult shearwaters embark on long foraging journeys to replenish their
energy and fat reserves [236]. Egg laying is synchronised over a two to three week
period, with all eggs laid by the last week in November. Males take the first
incubation shift which lasts for up to 10-14 days [237], with the entire incubation
period lasting 50-55 days Short-tailed shearwaters only lay one egg per season,
therefore if breeding fails it is not replaced [44, 272]. In contrast to other seabirds,
the guard stage in Procellariiformes is extremely short, lasting for between 2-3 days.
This is because Procellariiformes are able to regulate body temperature and reduce
brooding time. This ability is in part due to the subcutaneous fat deposits laid down
during the first few days [272].
15
Figure 1.5, Short-tailed shearwater chick (Photography by M. Dewar 2010)
Like all Procellariiformes, short-tailed shearwaters use a twofold foraging strategy
with birds alternating between two short foraging trips of 1-4 days, with one long
foraging trip of approximately 8-19 days [141, 277]. Weimerskirch & Cherel [277]
documented the dominance of Australian krill Nyctiphanes australis in the stomach
contents of short-tailed shearwaters from Tasmania when returning from short
foraging trips and Antarctic krill and myctophid fish dominating the stomach
contents after long foraging trips. [277]. However, in Victorian shearwaters, there is
little evidence to suggest that shearwaters forage on Antarctic species (Schumann et
al, unpublished; [173]). Although chicks do not fledge until around 97days of age,
adults begin their migration north when their chicks are around 85 days old at the
beginning of April. For the next 2-3 weeks chicks must survive off their endogenous
fat stores until fledging at 97 days of age [190].
Fairy Prion (Pachyptila turtur)
Fairy prions are the smallest of six species within the genera Pachyptila [37]. Fairy
prions found throughout the southern hemisphere, breeding on coastal islands
including; South Georgia, the Chatham, Snares Island, Antipodes and Bass Strait
Islands (Australia), Crozet Islands [84] with a current global population estimate of 5
million individuals [35]. Recent studies have identified the diet of Fairy Prions at
Lady Julia Percy Island, in Victoria consists exclusively of the Australian krill
16
Nyctiphanes australis (Schumann et al, unpublished). Eggs are laid between October
and November with adults sharing incubation duties with eggs hatching around 25
days later. Similarly to other Procellariidae’s, Fairy prions have a short guard stage
of 1-5 days, with chicks fledging after 31-45 days
[128]. Unlike many other petrel species, there is limited information on the biology
of Fairy Prions.
Family Pelecanoididae
In contrast to other members of the Order Procellariiformes, diving petrels
(Pelecanoididae), do not produce stomach oils [272]. Pelecanoides is the only genus
within the family Pelecanoididae, and contains four species including; P. urinatrix,
P. garnotii, P. magellani and P. georgicus. In this thesis we will be examining the GI
microbiota of the Common Diving Petrel (P. urinatrix).
Common Diving Petrel (Pelecanoides urinatrix)
Common diving petrels are the only diving petrel known to breed in southern
Australia, ranging from the from Seal Island group and nearby islands off Wilsons
promontory, to west of Lady Julia Percy Island and Lawrence rocks [202]. Common
diving petrels have a global population size of 16 million individuals [35]. The diet
of Common Diving Petrels used in this study on Notch Island, consists entirely of
Nyctiphanes australis which is a Euphausiid Krill [233]. In contrast to many other
Procellariiformes, common diving petrels commence their breeding in mid-winter, as
opposed to spring, with chicks fledging between November and December [172].
Similarly to other Procellariiformes, common diving petrels lay only one egg, with
both parents sharing incubation. Guard stage on average lasts 6.7 days, with chicks
fledging at 45-60 days old [172]. Similarly to the fairy prion, to date there is limited
data available on the biology of this species.
Penguins as a Model of Avian Gastrointestinal Microbiota
Although penguins are one of the most extensively studied species in the southern
hemisphere, little is known about the resident microbiota. To date the composition
and role of this ecosystem remains uncharacterised for many seabird species,
including penguins, with earlier studies based upon the use of culture dependent
techniques to provide description of the composition and abundance of members of
the microbial community [28, 205, 254, 298, 299] or specific microbial pathogens
17
such as Salmonella [191, 195] Campylobacter [28, 34, 116, 133, 151, 210] and
Pasturella multocida [85, 149, 150, 274]. However, cultivation techniques do not
accurately reflect the actual microbial composition but only those microbes that can
be cultured using selective media and as a result, provide an inaccurate account on
the composition and abundance of microbes from complex ecological systems such
as the gastrointestinal tract. Therefore, earlier studies on microbial composition of
penguins are considered incomplete. For this reason many microbiologists have
turned to molecular methods to characterise and explore the microbial composition
of complex ecosystems such as the gastrointestinal tract, and marine ecosystems. In
penguins, the use of molecular based methods to examine the microbial composition
has been limited to just two studies on adélie penguins [12, 294]. Zdanowski et al
[294] examined the microbial composition of freshly deposited adélie penguin guano
while Banks et al [12] examined the influence of geographical separation and host
phylogeny on the microbial composition of adélie penguins. Both studies,
documented that adélie penguins are highly dominated by Firmicutes (41%),
Actinobacteria (35%), Proteobacteria (12.5%). However, Banks et al [12] also noted
the presence of Bacteroidetes (5%). Table 1.2 below lists microbes isolated from
penguins.
Procellariiformes as a Model of an Avian Gastrointestinal Microbiota
The order Procellariiformes is made up of four families; Diomedeidae (Albatrosses),
Procellariidae (Petrels and Shearwaters), Hydrobatidae (Storm petrels) and
Pelecanoididae (Diving petrels). These birds are characterised by the following traits;
they are long lived, have external tubular nostrils, have the ability to produce
stomach oils (all except the Pelecanoididae), grooved, hooked beaks and their feet
are well set back on their bodies [76].
The production of stomach oils occurs during the digestion of food [271]. This
adaptation reduces the cost of travelling long distances to provide their chick with a
highly concentrated food source that is high in energy and fats [76]. Unlike penguins,
there are no previous publications examining the microbial composition of
Procellariiform seabirds, with all previous investigations isolating pathogenic
microorganisms and identifying the cause of mortality. In table 1.3, below lists the
microorganisms previously isolated from Procellariiform seabirds.
18
Table 1.3, Bacteria isolated from Penguin from previous studies
Penguin
Common Name
Bacteria
Reference
Aptenodytes
Emperor
Chlamydia spp
[41]
forsteri
Penguin
Salmonella spp
[191, 195]
Escherichia
[135]
intermedia
Bacillus spp
[135]
Alcalgenes
Pygoscelis papua Gentoo Penguin
aquamarines
Achromobacter
[28]
spp
Actinomyces
Campylobacter
lari
Salmonella spp
[28]
[29]
Yersinia spp
Campylobacter
[28]
jejuni
[29]
Edwardsiella
[29]
species
Alcaligenes
[242]
faecalis
Pseudomonas
[242]
Escherichia coli
[242]
19
Table 1.3, Continued
Pygoscelis
Adélie Penguin
adeliae
Campylobacter lari
[28]
Campylobacter jejuni
Pasteurella
[149]
multocida
Alcaligenes faecalis
[242]
Citrobacter freundii
[242]
Enterobacter spp
[242]
Escherichia coli
[184, 242]
Staphylococcus spp
[184]
Clostridium spp
Salmonella spp
[28, 176, 187]
Yersinia spp
[28]
Mycoplasma spp
[148]
Chlamydia spp
[174]
Bacillus spp
[212]
Brachybacterium spp
Streptomyces spp
Arthrobacter spp
Psychrobacter spp
[217]
Pseudomonas spp
Moraxellaceae/
[294]
Pseudomonadaceae
Flavobacteriaceae
[294]
Micrococcaceae
Actinobacteria
[12]
Firmicutes
Bacteroidetes
Proteobacteria
Pygoscelis
Chinstrap Penguin
Bacillus spp
[135]
antarctica
20
Table 1.3 continued.
Pygoscelis antarctica
Alcalgenes
cont.
aquamarines
[135]
Micrococcus spp
Staphylococcus spp
Eubacterium spp
Sarcina ureae
Streptococcus spp
Escherichia coli
Corynebacterium spp
Salmonella spp
[28]
Yersinia spp
Campylobacter lari
Campylobacter jejuni
Pasteurella multocida [72]
Eudyptes chrysocome
Southern
Rockhopper
Escherichia coli
[242]
Chlamydia spp
[174]
Escherichia coli
[242]
Enterobacter spp
[242]
Pasteurella multocida [85, 175]
Eudyptes
Macaroni Penguin
Chrysolophus
Eudyptes Schlegeli
Bacillus spp
[40]
Campylobacter jejuni
[33]
Pasteurella multocida [72]
Royal Penguin
Chlamydia spp
[174]
Bacillus spp
[40]
21
Table 1.3, Continued
Eudyptes Schlegeli
Royal Penguin
Escherichia coli
[242]
Megadyptes antipodes
Yellow-eyed
Corynebacterium
[2-4]
Penguin
amycolatum
Spheniscus
Magellanic
Corynebacterium
magellanicus
Penguin
spheniscorum
Eudyptula minor
Little Penguin
[113]
Corynebacterium spp
[205]
Erysipelothrix
[26]
rhusiopathiae
Spheniscus demersus
African Penguin
Escherichia coli
[242]
Mycoplasma
[96]
sphenisci
Salmonella
[65]
typhimurium
Aptenodytes
patagonicus
King Penguin
Escherichia coli
[242]
Citrobacter freundii
22
Table 1.4, Microorganisms isolated from Procellariiform seabirds from previous
studies
Procellariiform
Common Name
Bacteria
Reference
Thalassarche
Black-Browed
Salmonella
[195]
melanophrys
Albatross
Thalassarche
Grey-Headed
Salmonella
[195]
chrysostoma
Albatross
Pasteurella
[274]
Thalassarche
Atlantic Yellow-
chlororhynchos
nosed Albatross
multocida
Erysipelothrix
rhusiopathiae
Macronectes
Southern
giganteus
Petrel
Giant Pasteurella
[150]
multocida
Escherichia coli
Enterococcus
[135]
faecalis
Bacillus subtilis
Brevibacterium
brunneum
Alcaligenes faecalis
Mycoplasma
[148]
gallisepticum
Mycoplasma
synoviae
Salmonella
23
Table 1.4, continued.
Campylobacter lari
[28]
Campylobacter
jejuni
Yersinia
Catharacta
lonnbergi
Brown Skua
Chlamydophila
[130]
abortus
Campylobacter lari
[28]
Alcaligenes faecalis
[135]
Micrococcus
Eubacterium
alactolyticum
Staphylococcus
epidermidis
Enterococcus
faecalis
Proteus
Escherichia coli
[13, 135]
Streptococcus
24
Table 1.4 Continued
Pseudomonas
[13]
Acinetobacter
Micrococcus
Pasteurella
[13, 149, 197]
multocida
Salmonella
[28]
Campylobacter lari
[151]
Campylobacter
[28]
jejuni
Yersinia
Catharacta
maccormicki
South Polar Skua
Salmonella
[28, 187]
Campylobacter lari
[28]
Yersinia
Campylobacter
jejuni
25
Research Objectives and Thesis Structure
The overall objective of this thesis was to characterise the microbial composition of
the gastrointestinal microbiota of Spheniscidae and Procellariiformes. More
specifically, the aims of this thesis were to:
Characterise the microbial composition of sub-Antarctic and temperate penguins;
Investigate the influence of fasting during moult on the faecal microbiota of king and
little penguins;
Investigate the microbial colonisation of the faecal microbiota of little penguin
chicks from birth to fledging (as a model for development);
Characterise the microbial composition of Procellariiform seabirds; and
Investigate the microbial colonisation of the faecal microbiota of short-tailed
shearwater chicks from birth to fledging.
The following Chapters in this thesis are written as a series of five standalone
manuscripts (Chapters 2-6) addressing particular aspects of the objectives and aims
stated above. The collection of manuscripts means that some repetition and overlap
occurs in the introduction and methods sections of some Chapters.
In Chapter 2, the microbial composition of king, gentoo, macaroni and little penguins
was investigated using Quantitative Real Time PCR (qPCR) and 16S rRNA
amplicon sequencing. Using qPCR and 16S rRNA amplicon sequencing the
influence of fasting during moult was investigated in little and king penguins in
Chapter 3. Chapter 4 investigated the variations in the gastrointestinal microbiota of
little penguin chicks from hatching to fledging. In Chapter 5 the microbial
composition of short-tailed shearwaters, fairy prions and common diving petrels was
investigated using Quantitative Real Time PCR (qPCR) and 16S rRNA amplicon
sequencing. Chapter 6 investigated the variations in the gastrointestinal microbiota of
Short-tailed Shearwater chicks during development. The major findings from these
Chapters are compiled and discussed in Chapter 7.
26
Preface
All opinions and concepts presented in this thesis are my own. Dr Stuart Smith and
Associate Professor John Arnould provided structural and editorial suggestions in a
supervisory capacity and have therefore been included as co-authors of all
manuscripts. In Chapter 2, Dr Rene Groscolas (CNRS) coordinated and collected
samples from king penguins, from Possession Island, Crozet Archipelago. In
Chapters 2 and 3 Dr Phil Trathan (BAS) coordinated and collected samples from
king, gentoo and macaroni penguins from Bird Island, South Georgia and has made
some editorial suggestions. In Chapters 2-5 Dr Peter Dann (PINP) supervised field
work, provided logistical support and organised ethics and permit for field work
conducted at Phillip Island Nature Parks. As such, Dr Groscolas, Dr Trathan and Dr
Dann have been included as co-authors of manuscripts resulting from the above
Chapters.
All work was carried out under the approved Department of Sustainability and
Environment Wildlife Research Permits (#10004713 and #10005156) and approval
by the Phillip Island Nature Parks Animal Ethics Committee (#1.2008, #2.2009,
#3.2009) and Deakin University Animal Welfare Committee (#A31/2008). SubAntarctic samples collected from Crozet Archipelago were obtained under Permit
N°2008-69. All sub-Antarctic samples were imported under AQIS Permit
(#IP090088774).
27
CHAPTER 2
Inter-specific variations in the gastrointestinal microbiota of
penguins
A version of this chapter has been accepted for publication in MicrobiologyOpen Dewar,
M.L. Arnould, J.P.Y, Dann, P, Trathan, P, Groscolas, R and Smith, S.C. (2013).
MicrobiologyOpen, Early View (http://onlinelibrary.wiley.com/doi/10.1002/mbo3.66/full)
Summary
Despite the enormous amount of data available on the importance of the
gastrointestinal microbiota in vertebrate (especially mammals), information on the
GI microbiota of seabirds remains incomplete. As with many seabirds, penguins have
a unique digestive physiology that enables them to store large reserves of adipose
tissue, protein and lipids. This study used quantitative real time polymerase chain
and 16S rRNA gene pyrosequencing to characterise the inter-specific variations of
the gastrointestinal microbiota of four penguin species: the king, gentoo, macaroni
and little penguin.
The qPCR results indicated that there were significant differences in the abundance
for all of the major phyla. A total of 132,340, 18,336, 6,324 and 4,826 partial length
16S rRNA gene sequences were amplified from faecal samples collected from king,
gentoo, macaroni and little penguins respectively. A total of 13 phyla were identified
with Firmicutes, Bacteroidetes, Proteobacteria and Fusobacteria dominating the
composition, however, there were major differences in the relative abundance of the
phyla. In addition, this study documented the presence of known human pathogens
such as Campylobacter, Helicobacter, Prevotella, Veillonella, Erysipelotrichaceae,
Neisseria and Mycoplasma. However, their role in disease in penguins is unknown.
To our knowledge, this is the first study to provide an in depth investigation of the
gastrointestinal microbiota of penguins.
28
Introduction
Penguins are a distinctive group of flightless seabirds found exclusively in the
southern hemisphere, occupying an extensive geographic range extending from the
Galapagos Islands to the Antarctic continent [247]. Penguins like all seabirds, spend
most of their lives at sea, only coming to land to breed and moult [218, 227, 247].
Penguins have a unique digestive physiology that enables them to store large
amounts of undigested food, build up large reserves of adipose tissue (fat) and store
large amounts of protein and lipids for long periods of fasting during breeding and
moult [124].
The diet of all penguin species is derived from the sea and can include; crustaceans,
cephalopods, and fish, with composition varying from species to species and season.
The diet of the king penguin predominantly consists of myctophid fish (family
Myctophidae), For king penguins at Possession Island, (Crozet), two species of
myctophid (Krefftichthy andersoni and Electrona carlsbergi) make up over 90% of
the overall diet, whilst the remainder is composed of squid and other myctophid
species [30, 49, 53, 54, 105, 211, 224]. At the beginning of the breeding season, the
diet of macaroni penguin predominantly consists of the krill Euphausia superba
(95%), but is then switched to a diet myctophid fish (Krefftichthy andersoni) [51,
104, 115, 224]. The diet of gentoo penguins is comprised of fish (nototheniids),
cephalopods (Longio gahi) and Krill (Euphausia superba), with both temporal and
spatial variation observed within the diet [208]. Whilst little penguins eat a variety of
fish and small cephalopods, including anchovy (Engraulis spp), pilchard (Sardinops
sagax), sandy sprat (Hyperlophus vittatus), juvenile barracouta (Thyrsites atun) and
Krill (Nyctiphanes autralis) [56, 57, 103].
The fish and krill based diet of all four penguin species are a rich source of lipids,
proteins, in n-3 and n-6 polyunsaturated fatty acids (PUFA) such as EPA,
arachidonic acid and DHA. Krill are also a rich source of
provitamin E,
phospholipids, flavonoids, vitamin A, alpha-linolenic acid, astacin and other essential
nutrients [137, 295] which are all essential for penguins during the pre-breeding and
pre-moulting [53, 112, 192, 252] with penguins storing high levels of n-3 PUFA in
their sub-cutaneous adipose tissue [211, 243]. In addition, diets high in n-3 and n-6
PUFA’s are essential for normal metabolism, growth, heart health and neurological
development [243]. In avian species, arachidonic is a polyunsaturated omega-6 fatty
29
acid that is important for the development of tissues in the chick embryo, with roles
in signal transduction and eicosanoid synthesis [243].
The gastrointestinal (GI) tract contains a diverse and complex microbial ecosystem
made up of hundreds of different species of micro-organisms, that has coevolved
with its host [67, 143, 163, 257]. The GI microbiota has a profound influence on the
nutritional, physiological, immunological and metabolic processes of the host [168,
180, 298, 299], playing a significant role in energy harvest, fat metabolism, secretion
and synthesis of nutrients, vitamins, amino acids and the production of short chain
fatty acids from the diet consumed by the host [67, 94, 248, 257]. Rawls et al [214,
215]. identified that in the absence of microbial colonisation the GI tract results in an
immature and arrested differentiation. Furthermore, research has shown that the
absence of a functional GI microbiota reduces an animal’s ability to secrete and
absorb essential vitamins and nutrients from their diet [25, 199].
To date, the vertebrate GI microbiota has been extensively studied and shown to be
dominated by two main phyla; Firmicutes (usually 60-80% of total composition) and
Bacteroidetes (usually 20-40% of total composition) in multiple vertebrate species
including mammals (such as humans, rodents and pinnipeds), poultry, and livestock
(i.e. cattle, pigs). [101, 108, 109, 152, 154-156]. The avian microbiota however, is
comprised of approximately 640 species from 140 genera, with only 10% of the
avian microbiota being cultured in the laboratory [257]. However, much of what we
know about the avian microbiota is based from research on poultry [6, 83, 297].
In a study on the microbial composition of 60 species of terrestrial mammals, Ley et
al [154] identified that host phylogeny, diet and gut morphology influenced the
composition [154, 262] In addition, Ochman et al [186] and Yildrim et al [290] also
showed that evolutionary lineages determine the composition of the gut microbiota
of a vertebrate species. However, more recent evidence points to diet being the main
driving force behind differences in the gut microbiota of phylogenetically similar
hosts [178, 181]. In accordance with Ley et al [154], Banks et al [12] documented
that host phylogeny is the main driving force behind differences in the GI microbiota
of adélie penguins in the Ross Sea. In addition, to host phylogeny and diet, Nelson
[181] identified that prey associated microbiota significantly dominated the predator
microbiota.
30
Despite the enormous amount of data available on the importance of microbes in
mammals, it is surprising that so little research has been carried out on the avian
microbiota, with most studies focusing on the poultry microbiota (chicken, turkey)
[6, 297] To date the composition and role of this ecosystem remains incomplete for
seabirds, including penguin, with the exception of culture dependant techniques
which have been used to describe the composition and abundance of specific bacteria
within the microbial community [28, 205, 254, 298, 299], or specific microbial
pathogens such as Salmonella [191, 195] Campylobacter [28, 34, 116, 133, 151, 210]
and Pasturella multocida [85, 149, 150, 274]. However, traditional cultivation
techniques do not accurately reflect the microbial composition of the GI ecosystem,
and therefore, earlier studies on microbial composition of penguins are considered
incomplete. For this reason many microbiologists have turned to molecular methods
to characterise and explore the microbial composition of complex ecosystems such as
the gastrointestinal tract, and marine ecosystems. In penguins, the use of molecular
based methods to examine the microbial composition has been limited to just two
studies on adélie penguins [12, 294]. Zdanowski et al [294] examined the microbial
composition of freshly deposited adélie penguin guano while Banks et al [12]
examined the influence of geographical separation and host phylogeny on the
microbial composition of adélie penguins. Both studies, documented that adélie
penguins are highly dominated by Firmicutes (41%), Actinobacteria (35%),
Proteobacteria (12.5%). However,
Banks et al [12] also noted the presence of
Bacteroidetes (5%).
Given the ease in obtaining faecal samples from penguins, compared to the
alternative which would require the euthanisation of penguins to obtain to observe
the microbial composition of the entire GI tract, we chose to analyse the microbial
compositon of the penguin GI tract via faeces collected via rectal swab as performed
in previous studies [156, 194, 199, 262]. It has been shown that bacterial cells within
a faecal material are representative of the microbiota in the colonic lumen, colonic
mucosa and bacteria originating from other regions of the GI tract [91, 268]. Thus,
although faeces do not fully represent the entire microbial community from the GI
tract, a substantial proportion of bacterial species from this environment may be
present [91, 268] and thus the analysis of faecal material should be considered
adequate for investigating the microbial community of different species.
31
In order to understand the complex relationship between a host and its microbiota,
one must first characterise the GI microbiota of healthy individuals at baseline before
elucidating the role of microbiota, and the influence alterations of the GI microbiota
will have on health and disease of an individual or population [157, 262]. Therefore,
the goal of this study was to exanine variations in the microbial compostion of four
penguin species; king, gentoo macaroni and little (Eudyptula minor). The aims of
this study were; 1) Examine the variation in the abundance of four dominant phyla
within the vertebrate GI microbiota using quantitative real time PCR and 2) to
examine the community composition of the GI microbiota of four penguin species.
Materials and Methods
Sample Collection
Faecal samples were collected from king (Aptenodytes patagonicus) (n = 12), gentoo
(Pygoscelis papua) (n = 12), macaroni (Eudyptes chrysolophus) (n = 12) and little
penguin (Eudyptula minor) (n = 12) upon returning to the colony from a recent
foraging trip during the breeding season. Samples from gentoo and macaroni
penguins were collected from Bird Island, South Georgia (54 00′S, 38 03′W).
Samples from king penguins were collected from Baie du Marine, Possession Island
Crozet Archipelago (46°25'S, 51°52 E). Samples from little penguins were collected
from Phillip Island, Australia (38.4833° S, 145.2333° E). Upon return to the colony,
birds were captured by hand, and a cloacal swab was collected (Copan Eswabs,
Copan™ Italy) and stored in liquid nitrogen for transport.
Sample Analysis
DNA Extraction and Real Time PCR
DNA was extracted using the Qiagen™ QIAamp DNA Stool Mini Kit (Hilden,
Germany) following the manufacturer’s instructions. The major phyla selected for
analysis in this study were chosen from previous studies that had examined the
predominant gastrointestinal microbiota of vertebrates (mammals, chickens, and
adélie penguins) [91], which included Firmicutes, Bacteroidetes, Actinobacteria and
Proteobacteria. Due to the absence of seabird specific bacterial primers, the primer
sequences used in this study were obtained from previously published papers (see
Table 2.1). The primer sequences and annealing temperature for the chosen bacterial
groups are listed in table 2.1. The quantitative real time PCR was performed on the
32
Stratagene MX3000P. Each PCR reaction mixture comprised of 5µl of Brilliant II
SYBR green (Stratagene™), 20 pmol/µL of forward and reverse primer, 2ng of
template DNA and made up to a final volume of 20µl with nuclease free water. The
cycling conditions were 95⁰C for 2 mins, followed by 40 cycles of 95⁰C for 5 secs,
followed by annealing temperature (listed in table 2.1) for 30 secs with all samples
were run in triplicate. Bacterial concentration was determined by comparing the
threshold value (Ct. Values) with a standard curve. The standard curve was created
by using a serial 10 fold dilution from DNA extracted from a pure culture of E.coli
ranging from 102 – 1010 CFU.
PCR amplification of 16S ribosomal RNA gene sequences
Gentoo, Little & Macaroni Penguin Samples
From the original samples, 4 individuals per species were chosen for 16S rRNA
pyrosequencing. All samples/species were pooled together with the attachment of
MID tag barcodes (i.e. Barcode 338R_BC0495 “TCACTGGCAGTA” was attached
to all little penguin samples). Samples were then amplified using universal primers
Roche adapter A (5’GCC TCC CTC GCG CCA TCA GT-3’) and reverse 338R (5’CAT GCT GCC TCC CGT AGG AGT-3’) to amplify the V2-V3 region. Following
amplification, samples were sequenced on the Roche/454 GS FLX Titanium Genome
Sequencer by Engencore (USA) according to Fierer et al (2008). All sample
preparation and sequencing was performed by Engencore (USA) according to the
Roche 454 and Fierer et al (2008) protocol. Following sequencing, barcodes were
removed using Roche SFF software (Roche Applied Science, Indianapolis, IN). King
Penguin Samples
Results from a previously unpublished study on the microbial composition of king
penguins were included in this study. In the previous study, the V2-V3 region was
amplified using primers 8F (5’ AGAGTTTGATCCTGG 3’) [12, 22, 91, 152-154,
221] and 519R (5’ TTACCGCGGCTGCT 3’) [146]. Following PCR amplification,
samples were pooled and run on the Roche 454 FLX genome sequencer at CSIRO.
33
Phylogenetic Target
Forward
Reverse
Reference
Firmicutes
GGC AGC AGT RGG GAA TCT
ACA CYT AGY ACT CAT CGT TT
[92]
TC
Bacteroidetes
Actinobacteria
GGA RCA TGT GGT TTA ATT
AGC TGA ACG ACA ACC ATG
CGA TGA T
CAG
ACG GGC GGT GTG TAC A
TCC GAG TTR ACC CCG GC
[121]
AGT GTA GAG GTG AAA TT
ACG GGC GGT GTG TAC A
[22]
CTC GTG TAG CAG TGA
ACG GGC GGT GTG TAC A
[22]
CMA TGC CGC GTG TGT
ACT CCC CAG GCG GTC DAC
GAA
TTA
AGC GTT AYT CGG AAT CAC
CCC CGT CTA TTC CTT TGA GTT
TGG
TT
[179]
Alphaproteobacteria,
Deltaproteobacteria
Fusobacterium
Betaproteobacteria
Gammaproteobacteria
Epsilonproteobacteria
[22]
[179]
Table 2.1 Quantitative real time PCR primer sequences used in this study to detect
and quantify major phyla present in penguin faeces.
34
Bioinformatics
Quality control, removal of chimera’s, sequence alignment, identification and
Operational Taxonomic Units (OTU) classification were performed by Ribocon
GmbH (Germany) as per the protocol of Prusse et al [206, 207]. The 16S rRNA
sequences reported in this study have been submitted to EMBL under accession
number ERP001504.
Statistical Analysis
To determine if there were significant differences between all penguin species for the
major phyla for qPCR analysis, a one-way ANOVA with Tukeys HSD for pairwise
comparison was performed in SPSS, and was considered to be significantly different
if P = < 0.05. A multidimensional scaling plot (MDS), species diversity and cluster
analysis was performed on both the qPCR and pyrosequencing data using Primer E
v6 as per Clarke [64]. The samples that share the highest similarity will be
represented on the plot by points plotted closest together, while samples that share
limited similarity will be represented on the plot by points that are plotted farthest
apart [221].
Diversity Indices
Microbial diversity in the faecal samples of the four penguin species was estimated
with the Shannon-Weiner diversity index (H’). This index was calculated by the
following equation:
Shannon-Weiner index H’ = -∑i pi log(pi)
Where pi is the proportion of the total count arising from the ith species
Results
Quantitative Real Time PCR (qPCR)
The abundance of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were
detected from DNA extracted from faecal samples of king, gentoo, macaroni and
little penguins and using phyla specific primers. Proteobacteria dominates the GI
microbiota of gentoo, macaroni and little penguins, whereas Bacteroidetes,
dominated the GI microbiota of king penguins. Bacteroidetes was the second most
abundant phyla in gentoo, macaroni and little penguins, followed by Firmicutes and
Actinobacteria. In king penguins, Proteobacteria was the second most abundant
phyla, followed by Firmicutes and Actinobacteria. The abundance of Firmicutes,
Bacteroidetes, Proteobacteria and Actinobacteria in little penguins were significantly
35
lower in comparison to other penguin species (P = 0.0001). Firmicutes was
significantly higher in macaroni penguins in comparison to king (P = 0.002) and
gentoo (P = 0.008) penguins. While, significant differences in the abundance
Proteobacteria were observed between king and gentoo penguins (P = 0.041). For
Actinobacteria there were no significant differences observed between macaroni,
king and gentoo penguins (Figure 2.1).
The results from the MDS analysis, shows evidence that individual little penguins are
clustering together, indicating limited variation within this species. Some individual
gentoo, king and macaroni penguins appear to be clustered together, whilst other
individuals appear to be separated, indicating similarities between the three penguin
species and also the potential for individual variation, within each species (Figure
2.2).
16S rRNA gene Pyrosequencing
Microbial Composition of penguins
A total of 132,340, 18,336, 6,324 and 4,826 partial 16S rRNA gene sequences were
amplified from faecal samples collected from king, gentoo, macaroni and little
penguins respectively. With 97% sequence similarity a total of 1331, 2195, 1362 and
561 Operational Taxonomic Units (OTU) were identified respectively.
From the phylogenetic analyses, there were 13 phyla represented in penguin gut
microbiota; Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria,
Chloroflexi, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Fibrobacteres,
Planctomycetes, Spirochaetes and Synergistetes and four recently classified
candidates (BD1-5; OP10, SR1 and TM7) were also represented. The most abundant
phyla present in all penguin populations included the Bacteroidetes, Firmicutes,
Proteobacteria and Fusobacteria. Although all four species of penguin are dominated
by similar phyla, there are differences in the abundance of each phylum between all
four penguin species. Gentoo penguins were dominated by Fusobacteria (55%),
Firmicutes (18%), Proteobacteria (18%) and Bacteroidetes (7%). In king penguins,
Firmicutes (48%), Fusobacteria (24%) and Bacteroidetes (18%) dominate. The
dominant phyla in little penguins were Proteobacteria (30%), Firmicutes (24%),
Bacteroidetes (22%), Planctomycetes (11%) and Actinobacteria (7%). Whilst in
36
macaroni penguins, Firmicutes (45%), Proteobacteria (29%) and Bacteroidetes
(19%) dominate (Figure 2.3).
*P = 0.0001, **P < 0.01, ***P < 0.05
Figure 2.1 Variation in the abundance of the major phyla; Firmicutes (a),
Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) in king, gentoo,
little and macaroni penguins. Proteobacteria dominates the GI microbiota of
gentoo, macaroni and little penguins, followed by Firmicutes, Bacteroidetes and
Actinobacteria. Whereas Bacteroidetes dominates the king penguin microbiota,
followed by Proteobacteria, Firmicutes and Actinobacteria.
37
Figure 2.2, Similarity of the major bacterial phyla of penguin species using
qPCR. Similarity of faecal microbiota of penguin species displayed in MDS plot
performed in Primer E. There is a high level of similarity between individual little
penguins and gentoo penguins (with some outliers), indicating limited individual
variation. Although king and macaroni penguins do cluster together, it is not a tight
as little and gentoo, indicating potential individual variation within the population.
38
MDS analysis is used to examine the similarity between samples, with highly similar
samples clustering closely together on the plot, whereas samples of limited similarity
are further apart on the plot. The MDS analysis of the penguin microbiota
demonstrates a high level of similarity between gentoo and macaroni penguins. For
king and little penguins, there is little similarity with other penguin species (Figure
2.4). The analysis of similarity results also indicates a high level of similarity
between gentoo and macaroni penguins, sharing approximately 50% of the microbial
species. Little penguins share approximately 40% of their microbiota with gentoo
and macaroni penguins. Whereas king penguins have the lowest level of similarity,
sharing less than 10% of their microbiota with the other penguin species (Figure 2.5).
At family level, gentoo penguins were highly dominated by Fusobacteriaceae (55%)
with Fusobacterium the dominant genus. In king penguins, Leuconostocaceae,
Campylobacteriaceae and Porphyromonadaceae were the most dominate families
with Leuconostoc, Weissella, Helicobacter, Proteiniphilum and Petrimonas the
predominant
genera.
In
little
penguins
the
dominant
families
were
Enterobacteriaceae, Porphyromonadaceae and Phycisphaeraceae with Enterobacter,
Escherichia
and
Klebsiella
the
dominant
genera
within
the
family
Enterobacteriaceae. Barnesiella was the dominant genera of Porphyromonadaceae,
while Phycisphaeraceae was dominated by an unclassified genus. In macaroni
penguins Leuconostocaceae and Porphyromonadaceae were the dominant families
with Leuconostoc and Petrimonas the predominant genera (Figure 2.6).
Diversity
Diversity index was calculated for total microbial composition and for each major
phylum for each penguin species using the Shannon-Weiner index in Primer E. For
total microbial composition, little penguins had the highest microbial diversity with
H = 3.543, followed by macaroni, king and gentoo penguins with a diversity index
of H = 3.379, H = 2.889 and H = 2.363 respectively (Figure 2.7).
39
Figure 2.3, Relative abundance of major phyla from the GI microbiota of
gentoo, king, little and macaroni penguins using 16S rRNA pyrosequencing.
Bacteroidetes, Firmicutes and Proteobacteria dominate the microbial composition of
king, little and macaroni penguins. Fusobacteria, Firmicutes and Proteobacteria
dominate the GI microbiota of gentoo penguins.
40
Figure 2.4, Similarity of the gut microbiota of penguin species.
There is a high level of similarity between gentoo and macaroni penguins, as
indicated by the close clustering. For king and little penguins, there is a low level of
similarity to other penguin species.
41
Figure 2.5, Analysis of Similarity of the GI microbiota of gentoo, king, little and
macaroni penguins. The results from the analysis of similarity of bacterial profile
indicate that the similarity between gentoo and macaroni penguins is approximately
50%. In little penguins there is a similarity of approximately 40% between little,
gentoo and macaroni penguins. Whereas king penguins have a very low level of
similarity to all other penguin species with less than 10% similarity.
42
Figure 2.6, Relative abundance of major families of gentoo, king, little and
macaroni penguins using 16S rRNA pyrosequencing. Fusobacteriaceae dominates
the GI microbiota of gentoo penguins. In king penguins, Leuconostocaceae,
Campylobacteriaceae
and
Porphyromonadaceae
dominate.
Little
penguins
Enterobacteriaceae, Porphyromonadaceae and Phycisphaeraceae dominate the GI
microbiota. Macaroni penguins Leuconostocaceae, Porphyromonadaceae and
Moraxellaceae were the most dominant families.
43
Figure 2.7, Variation in species diversity in king, gentoo, little and macaroni
penguins. Species diversity was calculated by Shannon-Weiner index using Primer
E. Little penguins have the highest level of species diversity with H = 3.543,
followed by macaroni (H = 3.379), king (H = 2.889) and gentoo (H =2.363).
44
Discussion
Prior to this study, limited information was available on the microbial composition
and diversity of the penguin microbiota and the variation that exists between
different penguin species. In the current study, the microbial composition of king,
gentoo, macaroni and little penguins were characterised using Quantitative Real
Time PCR (qPCR) and 16S rRNA amplicon pyrosequencing. Quantitative Real Time
PCR was used to quantify the abundance of specific phylogenetic groups and to
examine the influence of individual variation, whilst the 16S rRNA amplicon
sequencing was used to provide a more in-depth characterisation of the microbial
diversity of four different penguin species.
Quantitative Real Time PCR
The results from the qPCR demonstrated that there was significant variation between
all penguin species, especially between little penguins and the three sub-Antarctic
species for all major phyla. In gentoo, macaroni and little penguins, Proteobacteria
appears to be the most dominant phyla with an abundance of 1.5 x 108, 8.0 x 107 and
1.82 x 106 CFU (colony forming units) respectively. Whilst in king, Bacteroidetes is
the most abundant phyla present with an abundance of 6.6 x 107 CFU. Actinobacteria
had the lowest abundance level for all penguin species ranging from 8.8 x 103 to 7.7
x 105 in king penguins (Figure 2.1).
The MDS results indicate that there is some clustering within the different species,
with the majority of individuals for each species clustering together, indicating that
there is some variation in the microbial composition of different penguin species
(Figure 2.2). However, the clustering of individuals within each species does not
appear to be tight and therefore could indicate individual variation within each
species (Figure 2.2).
16S rRNA gene pyrosequencing
Influence of “Primer Sequences” on Pyrosequencing results
In this study, we included data from a previously unpublished study on the microbial
composition of king penguins (Dewar et al, unpublished) and although the primer
sequence used to amplify the V2-V3 region analysis of the sequence data produced
has shown that the primer sets used in this study have a similar coverage of the
bacterial taxa present in all penguin species as shown in table A1 (Appendix). Thus
45
we feel that the sequences used in the king penguin study do not influence the
variation in microbial composition between king penguins and the other penguin
species.
Microbial Composition of penguins
Pyrosequencing provided a more detailed characterisation with 161,826 sequence
reads a total of 5,449 OTUs were identified from all penguin species, from13 phyla;
Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria, Chloroflexi,
Cyanobacteria,
Deferribacteres,
Deinococcus-Thermus,
Fibrobacteres,
Planctomycetes, Spirochaetes and Synergistetes and four recently classified
candidates (BD1-5; OP10, SR1 and TM7). Firmicutes, Bacteroidetes, Proteobacteria
and Fusobacteria were found to dominate the penguin microbiota. There were
significant differences in the results from the qPCR and 16S rRNA pyrosequencing,
with the qPCR results indicating that Bacteroidetes and Proteobacteria were the most
dominant phyla in all four species of penguins, whilst the pyrosequencing has
identified that the proportion of Firmicutes and Bacteroidetes are more dominant
than Proteobacteria. These differences could be due to the qPCR primer sets being
designed based upon data from terrestrial vertebrate microbiota, (such as humans,
rodents and poultry) and that they do not cover the entire microbial composition of
the penguin microbiota. As the 16S rRNA pyrosequencing amplifies all bacterial
DNA within the V2-V3 bacterial region, it is likely to provide a more accurate and
thorough coverage of the bacteria present.
The dominance of Bacteroidetes and Firmicutes at phylum level in penguins is in
accordance with previous studies on other vertebrate species; including many
mammalian species [91, 121, 145, 153, 249] . Members of the phylum Firmicutes are
associated with the breakdown of complex carbohydrates, polysaccharides, sugars
and fatty acids, which are then utilised by the host as an energy source with high
abundances of Firmicutes being linked to increased adiposity [93, 253]. Members of
the phylum Bacteroidetes in humans have been associated with vitamin synthesis,
polysaccharide metabolism, and membrane transport [120]. Although the penguin
microbiota in this study is similar to that of other vertebrate species (i.e. dominance
of Firmicutes and Bacteroidetes), they do differ from other vertebrates in regards to
the relatively high abundance of Fusobacteria (2-55%) and Proteobacteria (5-30%)
and their functional roles in penguins is not known.
46
The composition of the faecal microbiota varies amongst all four penguin species,
with differences observed at both the phylum and family level. These results indicate
that there are significant variations.
However, the cause of these variations is
unknown. Possible factors such as diet, prey associated microbiota, phylogenetic
differences, and external environment, have all been found to influence a hosts
microbial composition [12, 120, 154, 181]. In adélie penguins, Banks et al [12]
identified that host phylogeny had a greater influence over host microbial
composition in comparison to geographical location. Similarly, Ley et al [154] noted
that host phylogeny and diet appear to majorly influence the composition at a species
and population level, observing distinct differences between herbivores, omnivores
and carnivores. In Antarctic pinnipeds, Nelson [181] identified that not only did diet
and host phylogeny influence the GI microbiota, but that prey associated microbiota
dominated the GI microbiota of the higher predator.
The pyrosequencing data from this study have identified a significant proportion of
“unclassified” bacteria, indicating penguins harbour a large novel group of resident
bacteria in their digestive systems. Earlier studies in terrestrial vertebrates have
discovered that previously ‘unclassified’ bacteria to be responsible for important
metabolic, immunological or physiological functions [203, 296]. These bacteria
could have the potential to be involved in significant functions relating to digestive
physiology (synthesis and storage of proteins, lipids, PUFA’s), health and disease in
penguins and need to be analysed further.
The 16S rRNA pyrosequencing analysis also highlighted the presence of potential
pathogens
such
as
Campylobacter,
Helicobacter,
Prevotella,
Veillonella,
Streptococcus, Erysipelotrichaceae, Neisseria, Ureaplasma and Mycoplasma. The
identification of these pathogens is of significant concern due to their potential
impact on wildlife health and survival. Although these are known pathogens in
humans and other vertebrates there is limited data available linking these pathogens
to disease except for a member of the family Erysipelotrichaceae, which was
associated with the death of a little penguin in captivity [26]. Also, Campylobacter
spp have previously been identified in many penguin species, however, there have
been no studies linking the presence of this bacterium to disease in sub-Antarctic
penguins [28, 34, 116, 151] In addition, Helicobacter spp are widely distributed
amongst many vertebrate species including marine mammals (i.e. pinnipeds,
47
cetaceans) [111, 193] and although some species have been associated with disease
in humans and some marine mammals [127, 193] there is no data linking the
presence of Helicobacter to illness or disease in marine seabirds. The presence of
these pathogens may also indicate transmission from humans, which needs further
investigation. Because of their dense colonial living, penguins are more susceptible
to rapid pathogen transfer, which could potentially lead to major disease outbreaks
[116]. Therefore, the presence of any known pathogen warrants further investigation
to ascertain if these organisms are in fact pathogenic to penguins and if so, what
would the potential impact be on the population.
Conclusion
This study has identified, that there is large variation within the faecal microbiota of
sub-Antarctic and temperate penguin species and that these microbiota appear to be
dominated by five major phyla; Firmicutes, Bacteroidetes, Proteobacteria,
Fusobacteria and Actinobacteria. Although, the cause of these differences is yet to be
determined, host phylogeny, and diet could potentially play a major role in
determining the final microbial composition of an individual [12, 154]. This study
also identified the presence of known mammalian pathogens that could potentially
cause illness or disease within a penguin population and these findings warrant
further investigation as to the roles of these pathogenic bacteria in penguins.
48
CHAPTER 3
Variation in the Gastrointestinal Microbiota of Little Penguins
(Eudyptula minor) During Development
Abstract
At birth (emergence from egg), the gastrointestinal (GI) tract of all avian species is
considered to be a completely sterile environment that is rapidly colonised. Factors
such as microbial load on the eggshell, nesting environment and the first meal are all
known to influence the microbial colonisation of the GI tract of the chick.
Immediately after birth, the microbiota changes from a completely sterile aerobic
environment to a complex anaerobic environment dominated by anaerobic bacteria.
To date the development of the microbiota in a long lived seabird has not been
examined. Therefore this study aimed to characterise the GI microbiota of little
penguin chicks from birth to fledging (56-70 days), using Quantitative Real Time
PCR (qPCR) and 16S rRNA pyrosequencing sequencing. Results from the qPCR
analysis showed an upward trend for Firmicutes and Bacteroidetes during developing
in the little penguin chick. No trend was observed for Proteobacteria or
Actinobacteria. 16S rRNA pyrosequencing data identified that there are significant
fluctuations in the microbial composition of little penguins from hatching to
fledging. The similarity between all age classes is around 20%, with similarity
slightly increasing to 40% between weeks 5, 7 and 9. The newly hatched chick is
quickly
colonised
by
aerobic
and
facultative
anaerobic
bacteria,
with
Enterobacteriaceae dominating the microbiota throughout the first three weeks of
development. By week 5, the composition is dominated by Fusobacteriaceae and
Clostridiaceae, while Fusobacteriaceae and Clostridiaceae continue to dominate
throughout development until fledging. Similarly to other vertebrates, there is an
initial increase in species diversity, followed by a steady decline by fledging. This
study describes the first molecular examination of the microbial community of the
digestive tract of little penguin chicks throughout development. Little penguin chicks
display similar ecological successional changes as other vertebrates with aerobic and
facultative anaerobes being the first microbes to colonise the GI tract and slowly
transitioning to a microbiota dominated by strict anaerobes. However, unlike other
vertebrate species, the little penguin microbiota at fledging is still relatively unstable
and does not resemble that of the adult microbiota.
49
Introduction
In nature, bacteria are found everywhere and with the availability of nutrients are
able to colonise new habitats, including the GI tract [6]. At birth (emergence from
egg), the GI tract is considered to be devoid of microorganisms. In mammals, the
microbial community is said to be inherited from the mother through intimate contact
with faecal and vaginal microbes during birth and from breast milk [194]. In birds,
however, newborn chicks, acquire their microbiota from the surface of the egg,
surrounding environment (i.e. nest) and their first meal [6, 99, 101, 159, 217, 256,
291]. In addition, vertical transmission of microbes via regurgitation of an undigested
or partially digested meal is also said to influence the microbiota of the chick [110,
142, 205]. Through ecological succession, the sterile GI tract is rapidly colonised
forming a dense and complex microbial community within 2 years in humans [164,
166, 168, 199, 240, 268] and 40 days in chickens [16, 17, 68, 90, 217].
At birth, the neonate gut is an aerobic environment that is rapidly colonised by
aerobic and facultative anaerobic bacteria such as Escherichia coli and Streptococci
immediately after birth [5, 68, 168, 217, 268]. After initial colonisation, aerobic
bacteria modify the GI tract environment, by reducing the level oxygen through
oxidation/reduction. This process creates an environment that is highly favourable to
facultative and obligate anaerobes [168, 200], such as Bacteroides, Clostridia, and
Bifidobacterium [168, 245] before stabilising as an anaerobic environment.
To examine the development of the GI microbiota in a long lived seabird, the little
penguin was used as a model. Little penguins (Eudyptula minor) breed between
August to February [172] with peak egg production occurring between September
and October [220]. Little penguins nest in burrows, rocks, crevices and artificial nest
boxes lined with vegetation. Little penguins produce two eggs which are incubated
by both parents for 31-40 days [59, 220]. During the early stages post hatch (2-3
weeks), little penguin chicks are unable to self thermoregulate, and therefore one
parent must remain with the chick during this period (guard stage) [218, 244], During
the guard stage, chicks are fed on average every 1-2 days [60], whereas during the
post-guard stage adults alternate between short and long foraging trips [230].
Fledging occurs between 9-10 weeks of age [218].
50
To date, there have been no previous publications exploring the microbial
colonisation of the GI microbiota in long lived birds, especially birds that derives
their entire nutrition from the marine environment. Therefore, the goal of this study
was to characterise the colonisation of the GI microbiota of the little penguin
hatching to fledging (7-10 weeks) using DNA derived from cloacal material.
Quantitative Real Time PCR and 16S rRNA gene amplicon sequencing was used to
identify bacteria at each stage during development and the samples were compared to
examine changes in community structure over time.
Materials and Methods
Study site and Sample Collection
To characterise the microbial composition of the little penguin chick during
development, faecal samples were collected from little penguin chicks from the
Phillip Island Nature Park. The study was conducted within the Summerland
Peninsula, a previous housing estate, which had been reclaimed for penguin habitat.
Due to severe compaction of the ground, burrows in the area consist of artificial
wooden burrows. Burrows are lined with vegetation and excrement from previous
breeding seasons (Figures 3.1 and 3.2). At the beginning of the breeding season, 10
nest boxes were randomly selected with the restriction that the nest contained 2 eggs
at the beginning of the breeding season. Of the 20 eggs, 16 successfully hatched,
with only 9 successfully fledging at the end of the breeding season. Following
hatching, cloacal swabs were collected from each chick directly from the cloacae
using Eswabs (Copan, Italy). On the same day every week, penguin chicks were
removed from their nest, weighed, their stomach was inked (red or green food dye)
for identification purposes and swabbed and then placed back into the nest within a
5-10 minutes. Samples were placed into an amine solution for preservation of the
DNA and stored in liquid nitrogen (-196°C) for shipment and then stored at -20 until
DNA extraction.
51
Figure 3.1, artificial nest box used by little penguins in this study (Photography by
M. Dewar 2008).
52
Figure 3.2, inside of an artificial nesting box, with adult little penguin and chicks
53
Quantitative Real Time PCR
DNA extractions and quantitative Real Time PCR analysis were conducted as per the
protocol described in chapter 2. The qPCR primers used to analyse the abundance of
major bacterial phyla and classes are outlined in Table 2.1 in chapter 2.
16S rRNA Pyrosequencing,
Samples from 4 little penguin chicks at weeks 1, 3, 5, 7 and 9 were chosen for further
analysis by 16S rRNA pyrosequencing. All samples/species were pooled together
with the attachment of MID tag barcodes (i.e. Barcode 338R_BC0500
“CAGCTCAACTA” was attached to all week 1 samples). Samples were amplified
and sequenced as per the protocol described in chapter 2. Results from Adult little
penguins from chapter 2 were included for comparison.
Bioinformatics, Statistical Analysis and Diversity
Quality control, removal of chimera’s, sequence alignment, identification and
Operational Taxonomic Units (OTU) classification was performed by Ribocon
GmbH (Germany) as per the protocol of Prusse et al [206, 207]. The 16S rRNA
sequences reported in this study have been submitted to EMBL under accession
number ERP001967.
In order to explore the variation in bacterial counts, linear mixed models with
individual birds as a random effect and common assessment times (weeks) as a fixed
effect were fitted to the log-transformed data. Bacterial counts were log transformed
(base 10), to uncouple apparent variance-mean relationships, after inspection of
diagnostic plots of Studentized residuals versus predicted values [238].
The linear mixed models were restricted to six types corresponding to the six
combinations of the following:
Random effects (penguin chicks):
No autocorrelation in the repeated counts from the penguin chicks and homogeneous
variances at each assessment (scaled identity model). No autocorrelation in the
repeated counts from the penguin chicks and heterogeneous variances at each
assessment (diagonal model). First order autoregressive process for the co-variation
in the repeated counts (AR (1) model) and homogeneous variances.
54
Fixed effects (weeks):
Week as a categorical factor.
Week as a linear trend.
For each abundance measure, the fitted model with the smallest value of the Akaike
Information Criterion (AIC) [144] was selected and the significance of the
association of the common assessment times with variation in abundance was
assessed using the F-test. If the selected model included week as a categorical factor,
weekly means and their standard errors were reported. Otherwise, if the selected
model included week as a linear trend, the estimated intercept and slope coefficients,
and their standard errors, were reported.
All analyses used the mixed model
procedures in the IBM SPSS Statistics package (Version 20).
Principal Coordinates Analysis, diversity and cluster analysis was performed on
qPCR, whilst a Multidimensional scaling plot (MDS), was performed on the
pyrosequencing data. The samples that share the highest similarity will be
represented on the plot by points plotted closest together, while samples that share
limited similarity will be represented on the plot by points that are plotted farthest
apart
Microbial diversity in the faecal samples of the four penguin species was estimated
with the Shannon-Weiner diversity index (H’). This index was calculated by the
following equation:
Shannon-Wiener index H’ = -∑i pi log (pi)
Where pi is the proportion of the total count arising from the ith
Results
Quantitative Real Time PCR
The abundance of Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria were
detected from DNA extracted from faecal samples collected from little penguin
chicks during development using phyla specific primers. Bacteroidetes and
Firmicutes dominate the microbial composition throughout development, followed
by Proteobacteria. Actinobacteria appeared to have the lowest abundance with less
than 103 CFU.
During weeks 1-3, 5, 7-9 Firmicutes dominated the microbial
composition. Whilst during weeks 4, 6 and 10 Bacteroidetes dominated with
55
statistical differences observed throughout development in both Firmicutes and
Bacteroidetes. When analysing the data using a linear mixed model with time as a
covariate, a significant increasing linear trend was observed for both Firmicutes and
Bacteroidetes (Figure 3.3), indicating that there was an underlying linear increase
common to the observed abundance profiles for the 9 chicks/birds from hatching to
fledging. The MDS results show a strong clustering of all individuals throughout
development indicates that there is little variation according to qPCR results (Figure
3.4).
16S rRNA gene pyrosequencing
A total of
17,106, 19,386, 9,705, 11,792 and 31,418 partial 16S rRNA gene
sequences were amplified from faecal samples collected from little penguin chicks
on weeks 1, 3, 5, 7 and 9 during development respectively. With 97% sequence
similarity a total of 1,127, 1,099, 1,190, 1,998, and 1,694 Operational Taxonomic
Units (OTU) were identified from little penguin chicks respectively. A total of 4,826
partial 16S rRNA gene sequences and 561 OTU’s were amplified from adult little
penguins during the breeding season.
From the phylogenetic analyses, there were 10 phyla represented in developing little
penguin gut microbiota Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria,
Proteobacteria,
Planctomycetes,
Chlorobi,
Cyanobacteria,
Tenericutes,
Verrucomicrobia and five recently classified candidates (Candidate division BRC1,
OP10, TM7, SM2F11 and TM6) were also represented (Figure 3.5). Fusobacteria,
Firmicutes and Proteobacteria dominate the developing microbiota. Proteobacteria
(87%) dominated the microbiota of little penguin chicks during early development
(week 1-3), with Fusobacteria increased in abundance by week 3. After week three,
Fusobacteria, Firmicutes and Proteobacteria dominate the microbiota until fledging.
In the adult penguin, the predominant phyla were Proteobacteria (30%), Firmicutes
(24%), Bacteroidetes (22%), Planctomycetes (11%) and Actinobacteria (7%).
Although Bacteroidetes is a dominant member of the adult microbiota, its presence in
chicks is negligible.
56
Figure 3.3, Changes in the abundance of the major phyla; Actinobacteria (a),
Bacteroidetes (b), Firmicutes (c) and Proteobacteria (d) during development in little
penguin chicks. A significant upward trend was detected in both Firmicutes and
Bacteroidetes.
57
Figure 3.4, Similarity of the major bacterial phyla in little penguins during
development using qPCR. The PCoA cluster analysis was performed using Calypso
and shows no clear clustering, indicating no significant differences between chicks
during development.
58
At Family level, the microbiota of the week old chick was dominated by Gram
negative aerobic and facultative anaerobic bacteria such as Enterobacteriaceae (69%)
and Moraxellaceae (11%) with Enterobacter, and Psychrobacter the predominant
genera. Similarly at week three, the microbiota was dominated by Enterobacteriaceae
(44%), with the level of Fusobacteriaceae (33%) and Unclassified (14%) bacteria
increasing. Escherichia again was the dominant genera. In weeks 5 and 9, the
microbiota was dominated by Fusobacteriaceae (56%) and Clostridiaceae (17%),
with Fusobacteria the predominant genera. In week 7, there is a sharp increase in the
abundance of Vibrionaceae (34%), with Vibrio the dominant genera. In the adult
penguin
the
predominant
families
were
Enterobacteriaceae
(16%),
Porphyromonadaceae (13%), and Phycisphaeraceae (11%) (Figure 3.7).
The MDS/Cluster analysis indicates that level of similarity between the different age
classes increases with age. During the early stages of development, the level of
similarity between age classes is approximately 20%. In the later stages of
development, there is an increase in similarity, with weeks 5, 7 and 9 sharing 40% of
their microbiota. However, by fledging, the there is still a low level of similarity
between the adult and chick microbiota (Figure 3.6).
Diversity
The bacterial diversity in the little penguin chick gradually increases from week one
(H = 1.797) to week 7 (H = 2.556), but then declines by week 9 (H = 1.21) and
significantly increases in the adult penguin (H = 3.543) (Figure 3.8).
59
Figure 3.5, Relative abundance of major phyla during development in little penguin
chicks. In weeks 1 and 3, Proteobacteria highly dominates the microbial
composition. In weeks 5 and 8 Fusobacteria dominates the microbial composition. In
week 7 Firmicutes and Proteobacteria dominate the microbial composition.
60
Figure 3.6, Similarity of the gut microbiota of little penguins during development
using 16S rRNA Pyrosequencing. The results indicates that there is limited similarity
between the different stages of development in little penguin chicks and also between
little penguin chicks and adult penguins.
61
Figure 3.7, Relative abundance of major Families during development in little
penguin chicks. Microbial composition of the developing GI tract changes from a
dominance of Enterobacteriaceae and Fusobacteriaceae to a domination of
Fusobacteriaceae
over
time.
The
adult
microbiota
is
dominated
by
Enterobacteriaceae, Porphyromonadaceae and Phycisphaeraceae.
62
Figure 3.8 Variation in species diversity during development and in adult little
penguins. Species diversity was calculated by Shannon-Wiener Index using Primer
E. Species diversity increases with age until week 7. After week 7 we see a steep
decline in species diversity. Adult little penguins have the highest level of species
diversity in comparison to little penguin chicks.
63
Discussion
Establishment and early colonisation of the GI tract has been recognised as a crucial
stage in neonatal development with pioneering species responsible for influencing
the GI pH, oxygen levels, mucosal structure and immunity and therefore, these
pioneering species influence a hosts overall health and disease status throughout life
[136, 138, 198]. In mammals the establishment and colonisation of the neonate GI
tract has been extensively studied [168, 199, 268]. In avian species, however, data is
restricted to the microbiota of poultry [17, 68, 134, 171, 256, 266]. To date there is
no data available on the GI microbiota of long lived seabirds, nor the microbial
succession that occurs during development. Therefore this study aimed to examine
the establishment and microbial succession that occurs in little penguin chicks during
development.
Quantitative data obtained by qPCR for Firmicutes, Bacteroidetes, Proteobacteria
and Actinobacteria respectively demonstrated that the microbiota establishment
occurs rapidly. However, unlike humans and chickens, the microbiota did not remain
stable over time, indicating that the microbiota of little penguins stabilises after
fledging. From overall abundance of the four major phyla examined in this study,
that the total microbial population in a week old chick is over 1.3 x 107 CFU. By
fledging the total microbial population is estimated at over 3.0 x 106. These estimates
are consistent with other neonate vertebrates, a total population estimate of 108- 1010
CFU 7 days post birth [136, 168], with a final population size of around 1014 by the
end of development. Throughout development an upward trend was observed for
Firmicutes and Bacteroidetes, but no trend was observed for Proteobacteria and
Actinobacteria. The qPCR analysis indicates that an abundant population of microbes
have colonised the GI microbiota of little penguin chicks by the first week of life.
Results obtained from pyrosequencing identified that the microbiota of little
penguins is dominated by aerobic and facultative anaerobic bacteria from the phylum
Proteobacteria, with Escherichia and Psychrobacter the major pioneering
populations, followed by members of the phylum Firmicutes, with members of the
phyla Bacteroidetes and Planctomycetes present in lower numbers. Although the
presence of anaerobic bacterial populations begins to flourish in week 3,
Enterobacteriaceae still dominates the microbiota. From week 3 to 5, there is a
significant transition from an environment dominated by aerobic and facultative
64
anaerobic bacteria to an environment dominated by strict anaerobes with
Fusobacteria and Clostridium dominating the microbiota for the remainder of
development, except in week 7, when Vibrionaceae dominates.
The microbial composition of the little penguin during early development is
dominated by microbes that are known to colonise the infant microbiota of many
other vertebrates. Members of the family Enterobacteriaceae such as Escherichia,
Enterobacter, Klebsiella, and Citrobacter are responsible for the absorption of
dietary fats, lipids, and soluble vitamins, maintain intestinal barrier function, regulate
triglycerides, impact intestinal permeability, activate intestine-brain-liver neural axis,
enhance the immune system, alteration of lipoprotein profiles, protection against
stress-induced lesions, modulation of proinflammatory and anti-inflammatory gene
expression, and strengthen epithelial cell barrier properties. However, these microbes
have also been implicated in GI pathologies, brain-gut axis, and a few neurological
conditions [43, 117, 139, 223, 235, 251, 292]. While Psychrobacter, which has
previously not been isolated from the infant microbiota, has been shown to have
probiotic properties boosting the immune system and weight gain of Groupers [250].
By week 3, butyrate producing bacteria begin to flourish and by week 5 dominate the
microbiota [204].
Butyrate is an essential short-chain fatty acid produced in the colon. The main effects
butyrate has on the intestinal tract in humans include, influencing ion absorption, cell
proliferation and differentiation, immune regulation, and is an important antiinflammatory agent and is an essential energy source for colonocytes and has been
associated with colonic health [42]. In chickens, butyrate supplementation can lead to
a significant increase in host defence peptide gene expression, enhance antibacterial
properties of monocytes against pathogenic bacteria, boost host immunity and
increase host adiposity [196].
Conclusion
Similar to other infant vertebrates, the little penguin microbiota is dominated by
aerobic and facultative anaerobic bacteria, with the total population, estimated at
over 107 CFU by 7 days, with a significant upward trend observed throughout
development in Firmicutes and Bacteroidetes populations [134, 166]. During the
early stages of development the microbiota is dominated by members of the family
65
Enterobacteriaceae which have been identified as pioneering populations associated
with gut maturation and development, absorption of dietary fats and lipids, and
soluble vitamins, enhance the immune system, alteration of lipoprotein profiles,
modulation of proinflammatory and anti-inflammatory gene expression, and shave
also been implicated in GI pathologies [20, 43, 117, 139, 223, 235, 251, 292]. These
pioneering microbes are said to alter the GI environment towards an environment
that favours the growth of anaerobic bacteria. Similarly to other studies, the
microbiota of the little penguin began to transition from an aerobic/facultative
anaerobic microbial population, to a strict anaerobic population by week 3, and
continued throughout development, with the anaerobic population being dominated
by butyrate producing microbes. In poultry, butyrate has been shown to not only
boost the immune system and protect against the colonisation of pathogenic bacteria,
but has also been shown to influence host adiposity. In little penguins, there is a
strong link between host body mass and fledging survival. Therefore, the high
abundance of butyrate producing microbes in little penguin chicks could influence
chick body mass and therefore fledging survival. However, this would require further
analysis.
66
CHAPTER 4
Influence of Fasting During Moult on the Gastrointestinal
Microbiota of Penguins
Summary
Many seabirds including penguins are adapted to long periods of fasting, due to
reproduction, prey availability and moult, which are all a normal part of their
lifecycle. In the past many studies have focused their attention on understanding the
physiology of fasting; with limited research being done to understand the impact
fasting has on the composition of the gastrointestinal (GI) microbiota. Previous
studies examining the influence of fasting in other vertebrates (Syrian hamsters, mice
and pythons) identified that fasting not only influences the microbial composition
and diversity, but can also influence the immune response. However, the influence of
fasting on the GI microbiota has not been investigated in seabirds. Therefore, the
present study aimed to examine the microbial composition and diversity of the GI
microbiota of fasting penguins during early and late moult. Faecal samples were
collected from little (Eudyptula minor) and king penguins (Aptenodytes patagonicus)
during moult and analysed using Quantitative Real Time PCR (qPCR) and 16S
rRNA gene pyrosequencing.
The results from this study indicated that there was little change in the abundance of
the major phyla during moult, except for a significant increase in the level of
Proteobacteria in king penguins when using qPCR. In accordance with these results,
the 16S rRNA gene pyrosequencing did not identify any significant changes in the
relative abundance of the major phyla in moulting little penguins. However, there
were significant differences between early and late moulting little penguins at lower
taxonomic levels (i.e. Family). In king penguins, however, significant variation in the
microbial composition was observed at all taxonomic levels (i.e. phylum, family)
with a significant increase of Fusobacteria from 1 - 75% of the total composition.
Similarly to mammals and reptiles, our result indicated that fasting during moult,
alters the microbial composition of both little and king penguins and that the
microbial composition during early and late moult differs for each penguin species,
indicating that responses to moulting could be species specific.
67
Introduction
An intricate and complex relationship exists between a host and its microbiota. The
GI microbiota plays a significant role in energy extraction, fat metabolism and
storage, production of short chain fatty acids and host adiposity [9, 259, 293, 298]
and has a profound influence on the modulation of host metabolism. The GI
microbiota has the ability to modify a number of lipids in serum, adipose tissue and
in the liver, with drastic effects on triglycerides and phosphatidylcholine. The
resident microbiota is also responsible for the production of metabolites that
contribute to host fitness and survival [132]. The use of germ free animals has
highlighted the importance of the GI microbiota on its vertebrate host. Germ free
animals are not only more susceptible to disease, but also require a greater caloric
intake to achieve and maintain a normal body weight [9]. However, when germ free
animals are inoculated with the microbiota of conventionally raised hosts, the body
fat level of the germ free host rapidly increases, despite decreased food intake [9],
indicating that members of the GI microbiota may modulate fat deposition [185].
Previous studies examining the effect of fasting on the GI microbiota of vertebrate
(hamsters, python, mice), have shown that fasting not only alters the composition and
diversity of the GI microbiota, but it also influences the host’s immune defences [73,
74, 241] and that interrelationships exist between a host, its microbiota and the host’s
nutritional status, diet and physiology [73, 74] These studies however, have
concentrated on animals that are relatively inactive during times of nutrient
deprivation. Unlike other vertebrates, penguins have to survive long periods of
starvation while also coping with increased metabolic demands for feather synthesis
and thermoregulation [102, 281]. Therefore, penguins provide an attractive model for
investigating the influence of fasting that is associated with the increased metabolic
demands of moult.
Many seabirds, including penguins, are adapted to long periods of fasting due to
periodic fluctuations in nutrient availability, breeding and moult [31, 55, 119]. In
general, moult occurs post breeding in most penguin species and can last anywhere
from 2-5 weeks depending on the species. During moult penguins replace their entire
plumage whilst fasting on land and cannot return to sea because of the consequences
of reduced waterproofing and thermal insulation [105, 119]. Throughout moult
penguins must rely on endogenous fat and protein reserves for feather synthesis and
68
nourishment and is considered to be the most stressful and energetically costly period
within the penguin life cycle due to increased metabolic demands for feather
synthesis and thermoregulation [50]. Many penguin species experience high rates of
mortality during moult as a consequence of inadequate storage of fat and protein
reserves [50, 77, 114]. Because of its role in host adiposity, immune function and
regulation and metabolism, the GI microbiota could potentially influence host health
and survival during this stressful period and therefore needs to be investigated. The
king and little penguin experience different periods of moult, with moult lasting 2-3
weeks in little penguins, and 5 weeks in king [218, 247]. Therefore, these two
species provide an opportunity to examine the influence of moult and the length of
moult in the microbiota. The goal of this study was to gain an accurate understanding
of how fasting during moult influences the microbial composition of king
(Aptenodytes patagonicus) and little (Eudyptula minor) penguins.
Materials and Methods
Sample Collection
Faecal samples were collected from king (Aptenodytes patagonicus) (n = 12) and
little penguins (Eudyptula minor) (n = 9) during early and late moult. King penguins
were located at Bird Island, South Georgia (54 00′S, 38 03′W), while little penguins
were located at the Summerland Peninsula, Phillip Island, Australia (38.4833° S,
145.2333° E). A cloacal swab was collected from all birds during the early and late
moult using Copan E-swabs (Copan™, Italy), and placed into an amine solution for
preservation of the DNA and frozen for storage.
DNA Extraction and Real Time PCR
DNA was extracted using the Qiagen™ QIAamp DNA Stool Mini Kit (Hilden,
Germany) following the manufacturer’s instructions.
DNA was extracted using the Qiagen™ QIAamp DNA Stool Mini Kit (Hilden,
Germany) following the manufacturer’s instructions. The major phyla selected for
analysis in this study were chosen from previous studies that had examined the
predominant gastrointestinal microbiota of vertebrates (mammals, chickens, and
adélie penguins) [91], which included Firmicutes, Bacteroidetes, Actinobacteria and
Proteobacteria. Due to the absence of seabird specific bacterial primers, the primer
sequences used in this study were obtained from previously published papers (see
69
Table 2.1). The primer sequences and annealing temperature for the chosen bacterial
groups are listed in table 2.1. The quantitative real time PCR was performed on the
Stratagene MX3000P. Each PCR reaction mixture comprised of 5µl of Brilliant II
SYBR green (Stratagene™), 20 pmol/µL of forward and reverse primer, 2ng of
template DNA and made up to a final volume of 20µl with nuclease free water. The
cycling conditions were 95⁰C for 2 mins, followed by 40 cycles of 95⁰C for 5 secs,
followed by annealing temperature (listed in table 2.1) for 30 secs with all samples
were run in triplicate. Bacterial concentration was determined by comparing the
threshold value (Ct. Values) with a standard curve. The standard curve was created
by using a serial 10 fold dilution from DNA extracted from a pure culture of E.coli
ranging from 102 – 1010 CFU.
16S ribosomal RNA gene pyrosequencing
Individual samples were amplified using universal primers Roche adapter A (5’GCC
TCC CTC GCG CCA TCA GT-3’) and reverse 338R (5’-CAT GCT GCC TCC CGT
AGG AGT-3’) to amplify the V2-V3 region. Samples were pooled following PCR
amplification with the attachment of MID tag barcodes to ensure that bacterial DNA
from all members of each species was equally represented. Samples from the same
species were pooled together for sequencing on the Roche/454 FLX Genome
Sequencer by Engencore (USA). All sample preparation and sequencing was
performed by Engencore (USA) according to the Roche 454 protocol.
Bioinformatics, Statistical Analysis & Diversity
Quality control, removal of chimera’s, sequence alignment, identification and
Operational Taxonomic Units (OTU) classification was performed by Ribocon
GmbH (Germany) as per the protocol of Prusse et al [206, 207]. The 16S rRNA
sequences reported in this study have been submitted to EMBL under accession
number ERP0001595
To determine if there was significant differences between all penguin species for the
major phyla for qPCR analysis, paired samples T-test were performed in SPSS, and
were considered to be significantly different if P<0.05. A Principal Coordinates
Analysis (PCoA), diversity and cluster analysis was performed on the qPCR, whilst a
Multidimensional scaling plot was performed on the pyrosequencing data. The
samples that share the highest similarity will be represented on the plot by points
70
plotted closest together, while samples that share limited similarity will be
represented on the plot by points that are plotted farthest apart [12, 22, 91, 152-154].
Microbial diversity in the faecal samples of the two penguin species was estimated
with the Shannon-Weiner diversity index (H’). This index was calculated by the
following equation:
Shannon-Wiener index H’ = -∑i pi log (pi)
Where pi is the proportion of the total count arising from the ith species
Results
Quantitative Real Time PCR
The abundance of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria from
DNA obtained from faecal samples collected from king and little penguins during
early and late moult were examined using qPCR. In little penguins a significant
decrease in the abundance of Bacteroidetes was observed during moult (Figure 4.1).
In king penguins a significant increase in the abundance of Proteobacteria was
observed during moult (Figure 4.2). In both king and little penguins, Bacteroidetes is
the most abundant phyla followed by Firmicutes, Proteobacteria and Actinobacteria
in little penguins and Proteobacteria, Firmicutes and Actinobacteria in king penguins.
The PCoA results indicate that during early moult, all individuals cluster together
indicating little variation in the microbial composition in little penguins, with 1
outlier. However, by late moult, individuals appear to cluster into two main groups
and two outliers, indicating the presence of individual variation in response to moult
(Figure 4.3). The cluster analysis also indicates a low level of similarity between
early and late moulting little penguins. For king penguins, the PCoA results shows
that most individuals cluster together during both of moult, but especially during
early moult, with most individuals tightly clustered together indicating that there is
little variation between individuals during the early moult. This clustering indicates
that the microbial compositions between early and late moult in general are different
(Figure 4.4).
71
*P = 0.02
Figure 4.1, Variation in the abundance of the major phyla; Firmicutes, Bacteroidetes,
Proteobacteria and Actinobacteria during moult in little penguins. The abundance of
the major bacterial phyla was determined by comparing the threshold value (Ct
values) with a standard curve.
72
*P = 0.005
Figure 4.2, Variation in the abundance of the major phyla; Firmicutes, Bacteroidetes,
Proteobacteria and Actinobacteria during moult in king penguins. Proteobacteria
significantly increases during moult.
73
Figure 4.3, Similarity of the major bacterial phyla in little penguins during moult
using qPCR. The PCoA results indicate that there is a low level of similarity between
early and late moulting little penguins. There is also a high level of individual
variability.
74
Figure 4.4, Similarity of the major bacterial phyla in king penguins during moult
using qPCR. There is distinct clustering of early and late moult. This clustering
indicates that there is a high level of variation between individuals during the late
moult state, including two outliers
75
16S rRNA gene, pyrosequencing
Variations in the microbial composition of little penguins during moult
A total of 4,986 and 5,856 partial 16S rRNA gene sequences were amplified from
faecal samples collected from little penguins during the early and late moult
respectively. With 97% sequence similarity a total of 954 and 1,003 Operational
Taxonomic Units (OTU) were identified in little penguins during the early and late
moult respectively. A total of 10 phyla were represented in the GI microbiota of both
early and late moulting little penguins. These included Actinobacteria, Bacteroidetes,
Chloroflexi, Firmicutes, Fusobacteria, Planctomycetes, Proteobacteria, Spirochaetes,
Tenericutes and 1 recently classified candidate (division TM7). In addition, members
from the phyla Cyanobacteria and Deferribacteres were also present during early
moult, while Synergistetes and two additional classified candidates (Candidate
division BRC1 and Candidate division SR1) were present during the late moult.
The microbiota of little penguins during early moult was dominated by Firmicutes
(33%), Proteobacteria (19%), Fusobacteria (17%), Bacteroidetes (10%) and
Actinobacteria (8%). Similarly, little penguins during late moult have a similar
composition to individual little penguins in early moult, with Firmicutes (32%),
Proteobacteria (21%), Bacteroidetes (20%) and Actinobacteria (10%), except, for the
relative abundance of Fusobacteria which had significantly declined from 17% to 1%
(Figure 4.5). The similarity in the microbial composition between early and late
moulting little penguins was approximately 55%, indicating that microbial
composition varies during moult.
The predominant families dominating the microbial composition of little penguins
during the early moult were Fusobacteriaceae, Bacilliaceae and Porphyromonadaceae
with Fusobacterium, Oceanobacillus and Petrimonas the dominant genera. During
late moult, Porphyromonadaceae, Neisseriaceae and Lachnospiraceae dominate the
population (Figure 4.6). The dominant genera included Proteiniphilum, Petrimonas,
uncultured Neisseriaceae and Lachnospiraceae.
76
Figure 4.5, Relative abundance of major phyla in moulting little penguins using 16S
rRNA pyrosequencing. Firmicutes, Proteobacteria Bacteroidetes and Fusobacteria
dominate the microbial composition during early moult in little penguins. By late
moult, Fusobacteria has completely disappeared.
77
Figure 4.6, Relative abundance of major families from the GI microbiota of
moulting little penguins using 16S rRNA pyrosequencing. Fusobacteriaceae,
Bacilliaceae and Porphyromonadaceae dominate the GI microbiota during early
moult, while, Porphyromonadaceae, Neisseriaceae and Lachnospiraceae dominate
during late moult.
78
Variations in the microbial composition of king penguins during moult
A total of 15,151 and 16,749 partial 16S rRNA gene sequences were amplified from
faecal samples collected from king penguins during the early and late moult
respectively. With 97% sequence similarity a total of 2196 and 1551 Operational
Taxonomic Units (OTU) were identified in king penguins during the early and late
moult respectively. A total of 6 phyla were represented in gut microbiota of both
early and late moulting king penguins; Bacteroidetes, Firmicutes, Fusobacteria,
Proteobacteria, Spirochaetes, Tenericutes, and four recently classified candidates
(BD1-5, Candidate division OD1, Candidate division SR1, Candidate division TM7).
In addition, members from the phyla Verrucomicrobia, Acidobacteria, Chloroflexi,
Cyanobacteria, Planctomycetes, Synergistetes were represented in early moulting
king penguins, while members from the phyla Chlorobi and Lentisphaerae were
represented in late moulting king penguins.
There is a significant difference between early and late moult in king penguins. In
early moult the microbiota is dominated by Firmicutes (37%), Proteobacteria (34%)
and Bacteroidetes (20%) dominated the microbiota of king penguins during early
moult, with Leuconostocaceae, Campylobacteriaceae and Porphyromonadaceae
(11%), the dominant families. By late moult, however, Fusobacteria (75%)
dominates (Figure 4.7), with Fusobacteriaceae the dominant family (Figure 4.9). The
level of similarity between early and late was less than 30%. A significant decline in
the level of species diversity was also observed (3.41 to 1.1).
Diversity
The overall diversity in the microbial composition of little penguins declines slightly
from 3.635 to 3.231 in early to late moult. However, in king penguins, the level of
diversity significantly decline from 3.409 to 1.157 in early and late moult
respectively.
79
Figure 4.7, Relative abundance of major phyla in moulting king penguins using 16S
rRNA pyrosequencing. During early moult, Proteobacteria, Firmicutes and
Bacteroidetes dominate the microbial composition. By late moult, Fusobacteria
dominates the microbial composition, comprising over 70% of the total composition.
80
Figure 4.8, Relative abundance of major families from the GI microbiota of
moulting
king
penguins.
During
early
moult
Leuconostocaceae,
Campylobacteriaceae and Porphyromonadaceae are the dominant families, whilst
Fusobacteriaceae dominates the GI microbiota during late moult.
81
Discussion
Previous studies examining the effect of fasting on the GI microbiota of vertebrate
(hamsters, python, mice, termites), have shown that fasting not only alters the
composition and diversity of the GI microbiota, and influence the host’s immune
defences [73, 74, 241]. Research has also shown that complex interrelationships exist
between a host, its GI microbiota and the hosts nutritional status, diet and
physiological state [73, 74]. These studies, however, have concentrated on animals
that are relatively inactive during times of nutrient deprivation. Unlike other
vertebrates, penguins do not hibernate during times of fasting. In penguins fasting
occurs during the breeding season (incubation of eggs and chick brooding) and
moult, where penguins replace their entire plumage whist fasting on land. The
moulting fasts can last anywhere from 2 to 5 weeks. Therefore, penguins must
survive long periods of starvation while also coping with increased metabolic
demands for feather synthesis and thermoregulation [102, 281]. Penguins provide an
attractive model for investigating the influence of fasting that is associated with
increased metabolic demands. To date the influence of fasting on the microbiota of a
seabird has not been examined. Therefore, this study aimed to examine the influence
of fasting during moult in king and little penguins using qPCR and 16S rRNA
pyrosequencing.
Quantitative Real Time PCR
The quantitative results from the qPCR analysis showed that moult significantly
decreased that abundance of Bacteroidetes in little penguins and significantly
increased the abundance of Proteobacteria in king penguins. In accordance with other
fasting vertebrates, there is a low level of similarity between early and late moult in
both penguin species, indicating that moult does alter the microbial composition of
king and little penguins [73, 74, 241].
16S rRNA gene Pyrosequencing
Results obtained from pyrosequencing from the 16S rRNA gene sequencing show
significant differences to that of the qPCR, with the 16S rRNA sequencing
identifying significant differences in the microbial composition and diversity
between early and late moulting penguins. Similar to other vertebrate species, the
most predominant phyla in early moulting penguins were Firmicutes (33-37%),
Bacteroidetes (10-20%) and Proteobacteria (19-34%) [73, 91, 147, 154]. However,
82
unlike
other
vertebrates,
Leuconostocaceae,
king
penguins
were
Campylobacteriaceae,
dominated
by
families
Porphyromonadaceae,
and
Helicobacteriaceae, while, little penguins were dominated by Fusobacteriaceae,
Bacilliaceae, Porphyromonadaceae and Neisseriaceae.
Increased levels of members from the phyla Firmicutes are associated with increased
adiposity, by enhancing energy extraction and through modulation of the genes that
regulate fat storage [9, 152, 156, 259, 260, 263] . Although Firmicutes dominates the
microbial composition during early moult in both king and little penguins, it does not
constitute a large proportion of the total composition, as in other vertebrates that
have large fat stores (i.e. Australian sea lions, polar bears) [108, 147]. This is quite
surprising, considering that penguins build up large reserves of fat prior to moult.
Therefore, one would expect the microbiota of penguins during early moult to have a
microbial profile similar to that of other vertebrates that are able to store large fat
deposits (i.e. pinnipeds). In accordance with previous studies, a significant shift in
the microbial composition was observed in king and little penguins. In Syrian
hamsters Sonoyama et al [241] documented that the microbial composition during
fed, fasted and hibernating hamsters were all highly dominated by the Phylum
Firmicutes. However, the abundance of the class Clostridia was lower in fasted
hamsters, while Akkermansia mucinphila a mucin degrader were significantly
increased in the fasted state. In Burmese pythons, Costello et al [73] documented that
the microbial composition during feeding was dominated by Clostridium,
Lactobacillus, and Peptostreptococcus, whilst during fasting, the microbial
community
was
dominated
by
Bacteroidetes,
Rikenella,
Synergistes
and
Akkermansia. Whereas Gupta et al [122] documented a 35-fold and 12-fold increases
in the levels of Campylobacteriaceae and Helicobacteriaceae in malnourished
children when comparing them to healthy children. In this study we saw the
complete disappearance of Campylobacteriaceae and Neisseriaceae by late moult and
a 52% decline in the abundance of Helicobacteriaceae in king penguins. Whilst in
little penguins we see a 60% increase in the level of Neisseriaceae and a 58% decline
in the level of Enterobacteriaceae from early to late moult.
Due to the absence of data on the functional role of microbes in penguins we can
only infer what impact these changes to the microbiota will have on penguins.
Throughout moult the little penguin microbiota was associated with potentially
83
Butyrate producing microbes (Fusobacteria and Clostridia) [8, 204], known
gastrointestinal commensals and known human and veterinary pathogens [169, 216]
such as Campylobacteriaceae which has also been associated with disease in
penguins [113]. Whilst the king penguin microbiota is dominated by microbes that
are associated with butyrate production, chitin degradation, a novel probiotic
(Psychrobacter) and known gut commensals [8, 204, 250]. Known pathogens such as
Campylobacter, Escherichia coli, and Helicobacter also dominate the microbiota
during early moult [204]. By late moult, the microbiota is dominated by
Fusobacteria, which is a known butyrate producer. In mammals Butyrate is an
essential short-chain fatty acid produced in the colon. The main effects butyrate has
on the intestinal tract in humans include, influencing ion absorption, cell proliferation
and differentiation, immune regulation, and is an important anti-inflammatory agent
[42]. In chickens, butyrate supplementation leads to a significant increase in host
defence peptide gene expression, enhance antibacterial properties of monocytes
against pathogenic bacteria, boost host immunity and increase host adiposity [196].
Therefore the presence of butyrate producing microbes could influence host
adiposity levels prior to moult.
Conclusion
The results from the qPCR and pyrosequencing both indicate that fasting does alter
the microbial composition of both king and little penguins during moult. However, it
appears that the microbial composition of king penguins is more affected by fasting
than little penguins with the length of fast the most probable cause for this difference.
The reduction in the level of ‘potential’ pathogenic bacteria could be in part due to
the anti-microbial properties of butyrate. However, further investigation of more
species is required to examine the influence of moult on GI microbiota.
84
CHAPTER 5
Inter-Specific Variations in the Gastrointestinal Microbiota of Procellariiform
Seabirds
Summary
Despite the enormous amount of data available on the importance of the
gastrointestinal microbiota in vertebrate (especially mammals), to date there is
currently no information available on the microbiota of seabirds, with the unique
ability to produce and store stomach oils. All Procellariiformes, except diving petrels
have the unique ability to produce and store ‘stomach oils’ in their proventriculus.
Stomach oils are produced in the proventriculus through partial digestion of prey.
The oil fractions of the prey are then concentrated and retained for long periods of
time via delayed gastric emptying. Therefore, short-tailed shearwaters, fairy prions
and common diving petrels offer the unique opportunity to examine the microbiota
of seabirds that consume the same diet, but differ in digestive physiology. Therefore
this study compared the microbiota short-tailed shearwaters, fairy prions and
common diving petrels using quantitative real time PCR and 16S rRNA gene
pyrosequencing. The qPCR results indicated that overall there were significant
differences between all Procellariiform seabirds for Firmicutes and Bacteroidetes,
but no significant difference in Proteobacteria and Actinobacteria. From the
phylogenetic analyses, a total of 16 classified phyla represented in the
Procellariiform microbiota, with Firmicutes and Proteobacteria dominating the
microbial composition of all three Procellariiform seabirds, and Bacteroidetes also
dominating the microbial composition of common diving petrel. The microbial
composition of short-tailed shearwater and fairy prion are highly similar sharing 5060% of the same operational taxonomic units OTUs, whereas the similarity between
short-tailed shearwaters, fairy prions and common diving petrels is approximately
20%. As all three species share a similar diet, it can be assumed that diet is not the
major influence for the difference in microbial composition. Therefore, other
possible causes for these differences are likely to be host phylogeny, digestive
physiology or retention time.
85
Introduction
An intricate and complex relationship exists between a host and its microbiota. The
GI microbiota plays a significant role in energy extraction, fat metabolism and
storage, production of short chain fatty acids and host adiposity [9, 259, 293, 298]
and has a profound influence on the modulation of host metabolism. The GI
microbiota has the ability to modify a number of lipids in serum, adipose tissue and
in the liver, with drastic effects on triglycerides and phosphatidylcholine. The
resident microbiota is also responsible for the production of metabolites that
contribute to host fitness and survival [132]. The use of germ free animals has
highlighted the importance of the GI microbiota on its vertebrate host. Germ free
animals are not only more susceptible to disease, but also require a greater caloric
intake to achieve and maintain a normal body weight [9]. However, when germ free
animals are inoculated with the microbiota of conventionally raised hosts, the body
fat level of the germ free host rapidly increases, despite decreased food intake [9],
indicating that members of the GI microbiota may modulate fat deposition [185].
Many metabolites that are essential host nutrition are the product of microbial
fermentation. These metabolites are responsible for the absorption of dietary fats
lipids, and soluble vitamins, maintain intestinal barrier function, regulate
triglycerides, impact intestinal permeability, activate intestine-brain-liver neural axis,
enhance the immune system, alteration of lipoprotein profiles, protection against
stress-induced lesions, modulation of pro-inflammatory and anti-inflammatory gene
expression, and strengthen epithelial cell barrier properties and have been associated
with influencing host adiposity [43, 117, 139, 223, 235, 251, 292]. However, to date
there is currently no information available on the microbiota of seabirds, with the
unique ability to produce and store stomach oils. Therefore, this study offers the
unique opportunity to examine the influence of stomach oils on the microbiota.
Procellariiformes are long lived seabirds, that consume a diet high in dietary lipids
and are characterised by their ability to produce stomach oils (all except the
Pelecanoididae), [42]. Stomach oils are produced in the proventriculus through
partial digestion of prey. The oil fractions of the prey are then concentrated and
retained for long periods of time via delayed gastric emptying. [76]. This adaptation
reduces the cost of travelling long distances to provide their chick with a highly
concentrated food source that is high in energy and fats [225, 271]. The absence of
stomach oils in diving petrels is explained by differences in their digestive
86
morphology in comparison to other Procellariiformes. Procellariiformes, have a
highly extensible proventriculus, small gizzard and a unique arrangement of their
duodenal loop. The proventriculus acts as a separating funnel, separating the lighter
lipid layer from the denser aqueous layer. The dense aqueous layer is the first to pass
through the digestive tract, while the lipid layer is retained longer. In diving petrels,
however, the dorsal positioning of the pylorus and gizzard reduces the retention time
of the digesta, with the lighter lipid layers emerging first [243]. In diving petrels the
retention time of digesta is said to preclude the formation of stomach oils [225]. The
diet of short-tailed shearwaters, fairy prions and common diving petrels is dominated
by Australian krill Nyctohpanes australis. Their krill based diet is a rich source of n3 polyunsaturated fatty acids (PUFA), proteins and minerals [271]. Krill are also a
rich source of provitamin E, phospholipids, flavonoids, vitamin A, Alpha-linolenic
acid, astacin and other essential nutrients [69, 183, 269]. In avian species diets high
in n-3 PUFA’s are essential for normal metabolism, growth, heart health and
neurological development [137, 295].
In order to understand the complex relationship between a host and its microbiota,
one must first characterise the GI microbiota of healthy individuals at baseline before
elucidating the role of microbiota, and the influence alterations of the GI microbiota
will have on health and disease of an individual or population [157, 262]. The goal
of this study was to examine variations in the microbial compostion of three
members of the Order Procellariiformes; short-tailed shearwater, fairy prion and
common diving petrel using quantitative real time PCR and 16S rRNA
pyrosequencing.
Methods
Sample Collection
Faecal samples were collected from Short-tailed Shearwaters (Puffinus tenuirostris)
from Phillip Island, (Australia), Common Diving Petrels (Pelecanoides urinatrix)
from Notch Island, (Bass Strait, Australia) and Fairy Prions (Pachyptila turtur) from
Lady Julia Percy, (Australia) when returning to their colonies at night. Short-tailed
shearwaters were captured by hand, whereas Common Diving Petrels and Fairy
Prions were captured by mist netting.
87
To obtain faecal samples a sterile Copan E-swab (Copan™, Italy) was inserted in to
the cloaca. The samples was then placed into an amine solution for preservation of
the DNA and frozen for storage and stored at -80 degrees until analysis.
Sample Analysis
Quantitative Real Time PCR
DNA extractions and quantitative Real Time PCR analysis were conducted as per the
protocol described in chapter 2. The qPCR primers used to analyse the abundance of
major bacterial phyla and classes are outlined in Table 2.1 in chapter 2.
16S rRNA Pyrosequencing
Faecal samples were analysed using the 16S rRNA pyrosequencing protocols
described in chapter 2.
Bioinformatics, Statistical Analysis and Diversity Index
Quality control, removal of chimera’s, sequence alignment, identification and
Operational Taxonomic Units (OTU) classification was performed by Ribocon
GmbH (Germany) as per the protocol of Prusse et al [206, 207], as per Chapter 2.
As the data was considered to be non-parametric all data was log transformed in
SPSS. To determine if there was significant differences between Procellariiform
seabirds for the major phyla for qPCR analysis, the data was log transformed and a
one-way ANOVA, was performed in SPSS, and was considered to be significantly
different if P<0.05. Multidimensional scaling plot (MDS), diversity and cluster
analysis was performed on both the qPCR and pyrosequencing data.
Microbial diversity in the faecal samples of the three Procellariiformes was estimated
with the Shannon-Weiner diversity index (H’). This index was calculated by the
following equation:
Shannon-Wiener index H’ = -∑i pi log (pi)
Where pi is the proportion of the total count arising from the ith species
88
Results
Quantitative Real Time PCR (qPCR)
The abundance of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were
detected from DNA extracted from faecal samples of common diving petrels, fairy
prions and short-tailed shearwaters using phyla specific primers (Table 2.1). Overall
there were significant differences between all Procellariiform seabirds for Firmicutes
and Bacteroidetes, but no significant difference in Proteobacteria and Actinobacteria.
Short-tailed shearwaters had a significantly higher abundance of Firmicutes in
comparison to fairy prions (P = 0.0001) and a significantly high abundance of
Bacteroidetes in comparison to fairy prions (P = 0.021) and common diving petrels
(P = 0.023). In addition, fairy prions had a significantly higher abundance of
Firmicutes in comparison to common diving petrels (P = 0.016) but no significant
difference between Bacteroidetes, Proteobacteria and Actinobacteria (Figure 5.1).
The results from the MDS analysis indicates a strong clustering between all fairy
prion samples indicating little variation amongst individual fairy prions. For shorttailed shearwaters and common diving petrels there is loose clustering of all
individuals indicating that there is more individual variation within these two species
(Figure 5.2).
16S rRNA gene pyrosequencing
A total of 10,993, 9,877 and 23,967 partial 16S rRNA gene sequences were
amplified from faecal samples collected from common diving petrel, fairy prion and
short-tailed shearwater respectively. With 97% sequences similarity a total of 2,684,
809 and 1,800 Operational Taxonomic units (OTU) were identified for common
diving petrel, fairy prion and short-tailed shearwater respectively.
89
Figure 5.1, Variation in the abundance of the major phyla; Firmicutes (a),
Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) in Common Diving
Petrels, Fairy Prions and Short-tailed Shearwaters. There were significant differences
between all seabirds for Firmicutes and Bacteroidetes but no significant difference
between Actinobacteria and Proteobacteria.
90
Figure 5.2, Similarity of the major bacterial phyla in Procellariiform seabirds using
qPCR. MDS analysis shows tight clustering individual fairy prions indicting little
individual variation of the main phyla. While most individuals are clustering together
for short-tailed shearwaters and common diving petrels, at least one third do not
cluster, indicating potential individual variation.
91
From the phylogenetic analyses, a total of 16 phyla represented in the Procellariiform
microbiota; Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria,
Planctomycetes,
Chlorobi,
Deinococcus-Thermus,
Chloroflexi,
Spirochaetes,
Cyanobacteria,
Synergistetes,
Deferribacteres,
Tenericutes,
and
Verrucomicrobia and four recently classified candidates (BD1-5, OP10, SR1, and
TM7). Firmicutes and Proteobacteria dominated the microbial composition of all
three Procellariiform seabirds, with Bacteroidetes also dominating the microbial
composition of common diving petrel (Figure 5.3). The level of similarity between
short-tailed shearwater and fairy prion is relatively high (50-60%). In comparison,
the level of similarity between common diving petrels and short-tailed shearwaters
and fairy prions is relatively low (20%) (Figure 5.4).
At family level Ruminococcaceae, Lachnospiraceae and Porphyromonadaceae
dominate
the
microbial
composition
of
common
diving
petrels
with
Faecalibacterium, Uncultured Lachnospiraceae, Petrimonas and Barnesiella the
dominant genera. In fairy prions and short-tailed shearwaters Leuconostocaceae and
Streptococcaceae are the dominant families, with Leuconostoc, Weissella, and
Lactococcus the dominant genera in fairy prions and Leuconostoc and Lactococcus
the dominant genera in short-tailed shearwaters (Figure 5.5).
Diversity
Common diving petrels have a high level of diversity in comparison to the shorttailed shearwater and fairy prion (Figure 5.6).
92
Figure 5.3, Relative abundance of major phyla in three Procellariiform Seabirds; a)
Common Diving Petrels, b) Fairy Prions, and c) Short-tailed Shearwaters.
Firmicutes, Bacteroidetes and Proteobacteria dominate the common diving petrel
microbiota, whereas, Firmicutes dominates the GI microbiota of short-tailed
shearwaters and fairy prions.
93
Figure 5.4, Cluster Analysis of the GI microbiota Common Diving Petrels (CDP),
Fairy Prions (FP) and Short-tailed Shearwaters (STS). The results from the cluster
analysis indicate that the similarity of the GI microbiota between fairy prions and
short-tailed shearwaters is approximately 50%, whilst the similarity between
common diving petrels and fairy prions and common diving petrels and short-tailed
shearwaters is relatively low with only 20% similarity.
94
Figure 5.5, Relative abundance of major Families in common diving petrels, fairy
prions and short-tailed shearwaters. Common diving petrels are dominated by
Ruminococcaceae, Lachnospiraceae and Porphyromonadaceae. Fairy prions and
short-tailed shearwaters are dominated by Leuconostocaceae and Streptococcaceae.
95
Figure 5.6, Species diversity of Procellariiformes microbiota. Common diving
petrels have significantly higher species diversity compared to short-tailed
shearwaters and fairy prions.
96
Discussion
An intricate and complex relationship exists between a host and its microbiota. The
GI microbiota plays a significant role in energy extraction, fat metabolism and
storage, production of short chain fatty acids and host adiposity [9, 259, 293, 298]
and has a profound influence on the modulation of host metabolism. The GI
microbiota has the ability to modify a number of lipids in serum, adipose tissue and
in the liver, with drastic effects on triglycerides and phosphatidylcholine. The
resident microbiota is also responsible for the production of metabolites that
contribute to host fitness and survival [132]. However, to date there is currently no
information available on the microbiota of seabirds, with the unique ability to
produce and store stomach oils. Therefore, this study offers the unique opportunity to
examine the influence of stomach oils on the microbiota. In the current study, the
microbial composition of short-tailed shearwaters, fairy prions and common diving
petrels were characterised using Quantitative Real Time PCR (qPCR) and 16S rRNA
pyrosequencing.
The quantitative results from the quantitative qPCR analysis demonstrated that the
microbial composition of all Procellariiformes was significantly different.
Specifically, there was a significant difference in the abundance of Firmicutes and
Bacteroidetes. In all three Procellariiformes, Proteobacteria was the most abundant
phyla with an abundance of 2.66 x 108, 2.54 x 106 and 1.20 x 108 CFU (colony
forming units) for short-tailed shearwater, fairy prion and common diving petrel.
Actinobacteria had the lowest abundance level for all Procellariiformes ranging from
1.74 x 101 to 4.93 x 103 (Figure 5.1). The MDS results show strong clustering of
individual fairy prions. Most individuals of common diving petrels and short-tailed
shearwaters loosely cluster together with almost one third of individuals not
clustering together (Figure 5.2).
Results obtained from pyrosequencing identified that the microbiota of that the
microbiota of all three Procellariiformes were dominated by Firmicutes and
Proteobacteria, with Bacteroidetes also dominating the microbiota composition of
common diving petrels, which is in contrast to the qPCR results. These differences
could be due to the qPCR primer sets being designed based upon data from terrestrial
vertebrate microbiota, (such as humans, rodents and poultry) and that they do not
cover the entire microbial composition of the microbiota of Procellariiformes.
97
In accordance with previous studies on other marine vertebrates such as polar bears
and Australian sea lions the GI microbiota of short-tailed shearwaters and fairy
prions is highly dominated by the phylum Firmicutes [108, 147]. [91]. Whereas the
domination of Firmicutes and Bacteroidetes in common diving petrels is similar to
that of other vertebrate species; including penguins (Chapter 2), terrestrial mammals,
Arctic and Antarctic pinnipeds and avian species [73, 109, 142, 145, 154, 181]
Members of the phylum Firmicutes are associated with the breakdown of complex
carbohydrates, polysaccharides, sugars and fatty acids, which are then utilised by the
host as an energy source [93, 253] with high abundances of Firmicutes being linked
to adiposity. Members of the phylum Bacteroidetes in humans have been associated
with vitamin synthesis, polysaccharide metabolism, and membrane transport [120].
Although the common diving petrel microbiota in this study is similar to that of other
vertebrate species they do differ from other vertebrates in regards to the relatively
high abundance of Proteobacteria (5-30%).
In humans and mice the ratio of Firmicutes to Bacteroidetes in obese and lean
individuals differs, with obese individuals having a higher abundance of Firmicutes
and relatively lower abundance of Bacteroidetes in comparison to their lean
counterparts [152, 156, 263]. In oil producing Procellariiformes the relative
proportion of Firmicutes (77%) to Bacteroidetes (1-2%) is similar to the relative
proportions found in obese humans and mice. [152]. This ratio of Firmicutes to
Bacteroidetes has also been found in another marine vertebrate that also has a
predisposition to obesity the Australian Sea Lion (80% Firmicutes, 2%
Bacteroidetes). The high level of body fat in Procellariiformes therefore may be a
combination of a rich lipid based diet and a microbiome that is high in Firmicutes. In
the common diving petrels however, the proportion of Firmicutes to Bacteroidetes is
almost 1:1, as opposed to 77:1in oil producing Procellariiformes. Unlike most
Procellariiformes, common diving petrels do not migrate over large distances and are
thought to be fairly sedentary remaining close to coastal waters all year round and
therefore may not require large fat reserves [84].
At family level short-tailed shearwaters and fairy prions are dominated by
Leuconostocaceae and Streptococcaceae, with Leuconostoc, Weissella, and
Lactococcus the dominant genera of short-tailed shearwaters and Leuconostoc and
Lactococcus are the dominant genera of fairy prions. There is little similarity
98
between common diving petrels and the oil producers (20%). Due to the absence of
data on the functional role of microbes in seabirds we can only infer what impact
these changes to the microbiota will have on Procellariiformes. The microbiota of
short-tailed shearwaters and fairy prions are dominated by species that are not
commonly found in the vertebrate gastrointestinal tract (Leuconostoc &
Lactococcus), are commonly used as starter cultures for dairy products and are
associated with the skin microbiota of animals [21, 46, 188]. Weissella is associated
with the vertebrate GI tract and is also used as a probiotic [21] The microbiota of
common diving petrels is associated with butyrate producing microbes (Fusobacteria
and Ruminococcus) [8, 204] and microbes associated with anti-inflammatory
properties. Butyrate is an essential short-chain fatty acid produced in the colon. The
main effects butyrate has on the intestinal tract in humans included, influence of ion
absorption, cell proliferation and differentiation, immune regulation, and is an
important anti-inflammatory agent [42]. In chickens, butyrate supplementation leads
to a significant increase in host defence peptide gene expression, enhance
antibacterial properties of monocytes against pathogenic bacteria, boost host
immunity and increase host adiposity [196].
As all three species share a similar diet, it can be assumed that diet is not the reason
for the difference in microbial composition. Therefore, the cause of these differences
is likely to be due to differences in host phylogeny, digestive physiology or retention
time.
Ley et al
[154] identified that host phylogeny and gut physiology are
significant predictors of host microbial composition. In addition, however retention
time of digesta can also influence the microbial composition of the GI tract [246]. It
is well known that the digestive physiology in diving petrels differs to that of other
Procellariiformes, but retention time of digesta is also significantly shorter than other
Procellariiformes [225]. For example in the diving petrel Pelecanoides georgicus the
retention time for lipids was 2.3hrs, whereas in Shy Albatross (Macronectes
giganteus) and other large Procellariiformes the retention time is 12.5h and in
smaller oil producing Procellariiform Antarctic Prion (Pachyptila desolata) the
average retention time was 15hrs [225].
Penguins Vs. Procellariiformes
The penguin microbiota is dominated by Firmicutes, Bacteroidetes, Fusobacteria and
Proteobacteria (Chapter 2), while the GI microbiota of Procellariiformes is
99
dominated by Firmicutes, Proteobacteria and in common diving Bacteroidetes is also
dominant (Chapter 5) (Figure 7.2). In penguins, diet, and diet associated microbiota
could potentially be a major factors influencing the microbiota of seabirds, with the
krill eating penguins clustering closely together on the MDS plot, in comparison to
the king and little penguins (Figure 2.4) [12, 153]. Whereas, in the Procellariiformes,
digestive physiology and retention time of digesta appear to be the major factors
influencing the microbial composition (Figure 5.4), with a high level of similarity
between the two oil producing seabirds and a low level of similarity between the oil
and non-oil producing seabirds.
In contrast to the sequencing results of penguins (Chapter 2), the Procellariiformes
did not have a large proportion of “unclassified” bacteria present and most sequences
were able to be identified down to genus level. In addition, although opportunistic
pathogens
such
as
members
from
the
family
Enterobacteriaceae,
Campylobacteriaceae, Helicobacteriaceae and Neisseriaceae are present in the
microbiota of all Procellariiformes, they are not present in significant numbers <1%.
Conclusion
This study has identified that there is large variation within the faecal microbiota of
Procellariiformes which can be divvided into oil producing and non-oil producing
such as common diving petrels. Firmicutes and Proteobacteria dominate the GI
microbiota of oil producing Procellariiformes, whilst Firmicutes, Proteobacteria and
Bacteroidetes dominate the GI microbiota of common diving petrels. Although, the
cause of these differences is yet to be determined, host phylogeny, digestive
physiology and retention time could potentially play major roles in determining the
final microbial composition of individual seabird species [12, 153, 246].
100
CHAPTER 6
Variations in the Gastrointestinal Microbiota of Short-Tailed Shearwaters
(Puffinus tenuirostris) During Development
Summary
At birth (emergence from egg) the gastrointestinal (GI) tract of all avian species is
considered to be completely sterile and is colonised immediately after emerging from
the egg. Factors such as microbial load on the eggshell, nesting environment and
their first meal are all said to influence the microbial colonisation of the GI
microbiota of the chick. Immediately after birth, successional changes occur within
the microbiota until a climax community is achieved. To date, the development of
the microbiota in a long lived seabird has not been examined. Therefore, this study
aimed to characterise the microbiota of short-tailed shearwater chicks from hatching
until fledging (98 days), using Quantitative Real Time PCR (qPCR) and 16S rRNA
pyrosequencing sequencing. Results from the qPCR and 16S rRNA pyrosequencing
analysis identified no significant differences or trends in the microbiota of shorttailed shearwaters from hatching to fledging. The pyrosequencing analysis identified
that the GI microbiota of short-tailed shearwater chicks was dominated by gram
positive facultative anaerobes and obligate anaerobes from the phylum Firmicutes
(73-85%) throughout development. The ratio of Firmicutes to Bacteroidetes in shorttailed shearwater chicks (77:1) were similar to that of obese humans and mice
(100:1), indicating that the GI microbiota may play a role in fat deposition during
development. This study describes the first molecular examination of the microbial
community of the digestive tract of short-tailed shearwater chicks throughout
development. Short-tailed shearwater chicks display similar ecological successional
changes as other vertebrates with aerobic and facultative anaerobes being the first
microbes to colonise the GI tract and slowly transitioning to a microbiota dominated
by strict anaerobes.
101
Introduction
In nature, bacteria are found everywhere and are able to colonise new habitats with
the availability of nutrients. As bacteria are dispersed by water, soil and wind there
are very few places that are unable to be colonised by bacteria, including the GI tract
[6]. At birth the GI tract is considered to be devoid of microorganisms. In mammals,
the microbial community is said to be inherited from the mother through intimate
contact with their mothers faecal and vaginal microbes and from breast milk [194].
In birds however, the new born chick, acquires its microbiota from the surface of the
egg, surrounding environment (i.e. nest) and their first meal [6, 99, 101, 159, 162,
217, 256, 291]. In addition, vertical transmission of microbes via regurgitation of an
undigested or partially digested meal is also said to influence microbial colonisation
[110, 205]. Through ecological succession, the sterile GI tract, is rapidly colonised
overtime forming a dense and complex microbial community. In humans this process
usually takes around 2-3 years [164, 166, 168, 240], whilst in chickens, stabilisation
of the composition of the GI microbiota occurs within 40 day [16, 17, 68, 90, 217].
At birth, the infant gut is an aerobic environment that is rapidly colonised by aerobic
and facultative anaerobic bacteria, such as Escherichia coli and Streptococci [5, 68,
168, 217, 268]. Within the first few days both E.coli and Streptococci are present in
extremely high numbers of around 108 – 1010 CFU of faeces [168]. Research suggests
that these pioneering bacteria reduce the amount of oxygen present in the GI tract
through oxidation/reduction, creating an environment that is highly favourable to
facultative and obligate anaerobes [168, 199], such as Bacteroides, Clostridia, and
Bifidobacterium [168, 245], which flourish in a low oxygen environment.
To date, the development of the GI microbiota in a long lived seabird has not been
examined. Therefore, the short-tailed shearwater was used as a model to examine the
development of the GI microbiota in a long lived seabird. Short-tailed shearwaters
breed between September and April with egg laying occurring in the last week of
November [237]. Prior to egg laying, adult shearwaters embark on long foraging
journeys to replenish their energy and fat reserves [236]. Short-tailed shearwater nest
in simple scrapes or burrows, lined with vegetation. Short-tailed shearwaters lay one
egg, which is incubated for a period of 50-55 days [44, 272]. In contrast to other
seabirds, Procellariiformes are able to regulate body temperature much sooner than
other seabirds, reducing the guard stage to 2 - 3 days. This ability to regulate body
temperature is in part due to the subcutaneous fat deposits laid down during the first
102
few days [272]. Like all Procellariiformes, short-tailed shearwaters (Ardenna
tenuirostris)use a twofold foraging strategy during the breeding season, with adults
alternating between two short foraging trips of 1 - 4 days, with one long foraging
trips of approximately 8 - 19 days [141, 277]. Previous studies have documented that
the diet of short-tailed shearwaters is dominated by Australian krill Nyctiphanes
australis [44, 141, 173]. However, in contrast to previous studies, Weimerskirch &
Cherel [277], discovered that during long foraging trips, Antarctic krill and
Myctophid fish dominated the diet of short-tailed shearwaters. In Victorian
shearwaters, however, there is little evidence to suggest that shearwaters forage on
Antarctic species with only Nyctiphanes australis present in dietary samples
(Schumann et al, unpublished [173]). Although chicks do not fledge until around
97days of age, adults begin their migration north when their chicks are around 85
days old at the beginning of April. For the next 2-3 weeks chicks must survive off
their endogenous fat stores until fledging at 97 days of age [190].
The goal of this study was to characterise the microbial community of the shorttailed shearwaters digestive tract during development from newly hatched chicks to
fledging juveniles (7-10 weeks) using DNA derived from faecal material.
Quantitative Real Time PCR and 16S rRNA gene amplicon sequencing was used to
identify bacteria at each stage during development and the samples were compared to
examine changes in community structure over time. To our knowledge, this is the
first study to investigate the microbial composition of short-tailed shearwater chicks.
This
Methods
Sample Collection
To characterise the microbial composition of the short-tailed shearwater chick during
development faecal samples were collected from short-tailed shearwater chicks from
the Phillip Island Nature Park. On Phillip Island, short-tailed shearwaters burrow
among the sand dunes and line their nests with surrounding vegetation (Figure 6.1).
At the beginning of the breeding season, 20 burrows were randomly selected with the
restriction that the nest contained an egg at the beginning of the breeding season. Of
the 20 eggs, 17 successfully hatched, with only 10 chicks successfully fledging at the
end of the breeding season. Following hatching, cloacal swabs were collected from
each chick directly from the cloacae using Eswabs (Copan, Italy). On the same day
103
every week, short-tailed shearwater chicks would be removed from their nest,
weighed, inked (red or green food dye) for identification purposes and swabbed and
then placed back into the nest within 5-10 minutes to avoid stress. Samples were
placed into an amine solution for preservation of the DNA and stored in liquid
nitrogen (-196°C) for shipment and then stored at -20°C until DNA extraction.
Sample Analysis
Quantitative Real Time PCR
DNA extractions and quantitative Real Time PCR analysis were conducted as per the
protocol described in chapter 2. The qPCR primers used to analyse the abundance of
major bacterial phyla and classes are outlined in Table 2.1 in chapter 2.
16S rRNA Pyrosequencing
Samples from 4 short-tailed shearwater chicks for week 2, 5, 8, 11 and 14 were
analysed 16S rRNA pyrosequencing as per protocols described in chapter 2. Results
from adult short-tailed shearwaters in Chapter 5 were included for comparison.
Bioinformatics, Statistical and Diversity analysis
Quality control, removal of chimera’s, sequence alignment, identification and
Operational Taxonomic Units (OTU) classification was performed by Ribocon
GmbH (Germany) as per the protocol of Prusse et al [206, 207], as per Chapter 2
methodology.
In order to explore the variation in bacterial counts, linear mixed models with
individual birds as a random effect and common assessment times (weeks) as a fixed
effect were fitted to the log-transformed data. Bacterial counts were log transformed
(base 10), to uncouple apparent variance-mean relationships, after inspection of
diagnostic plots of Studentized residuals versus predicted values [238].
The linear mixed models were restricted to six types corresponding to the six
combinations of the following:
Random effects (short-tailed shearwater chicks):
No autocorrelation in the repeated counts from the short-tailed shearwater chicks and
homogeneous variances at each assessment (scaled identity model).
104
No autocorrelation in the repeated counts from the short-tailed shearwater chicks and
heterogeneous variances at each assessment (diagonal model).
First order autoregressive process for the co-variation in the repeated counts (AR (1)
model) and homogeneous variances.
Fixed effects (weeks):
Week as a categorical factor.
Week as a linear trend.
For each abundance measure, the fitted model with the smallest value of the Akaike
Information Criterion (AIC) [239] was selected and the significance of the
association of the common assessment times with variation in abundance was
assessed using the F-test. If the selected model included week as a categorical factor,
weekly means and their standard errors were reported otherwise, if the selected
model included week as a linear trend, the estimated intercept and slope coefficients,
and their standard errors, were reported.
All analyses used the mixed model
procedures in the IBM SPSS Statistics package (Version 20). A Principal
Coordinates Analysis (PCoA) diversity and cluster analysis was performed on the
qPCR whilst a Multidimensional scaling plot (MDS), was performed on the
pyrosequencing data.
Microbial diversity in the faecal samples of the four penguin species was estimated
with the Shannon-Weiner diversity index (H’). This index was calculated by the
following equation:
Shannon-Wiener index H’ = -∑i pi log (pi)
Where pi is the proportion of the total count arising from the ith species
Results
Quantitative Real Time PCR
The abundance of Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria were
detected from DNA extracted from faecal samples collected from short-tailed
shearwater chicks during development and using phyla specific primers. Throughout
development Firmicutes dominated the microbial composition of the short-tailed
shearwater chick. In contrast to little penguins (Chapter 3) no significant trends of
differences in the abundance of the major phyla were detected (Figure 6.4). The
105
PCoA results, also did not detect any significant differences between age classes with
no distinct clustering of age classes or individuals (Figure 6.5).
Figure 6.1, Short-tailed shearwater breeding colony. Sticks represent nest sites used
in this study (Photography by Meagan Dewar).
106
Figure 6.2, Natural burrow of short-tailed shearwater (Photography by Meagan
Dewar)
107
Figure 6.3, Short-tailed shearwater chick (Photography by Meagan Dewar).
108
Figure 6.4, Changes in the abundance of the major phyla; Firmicutes (a),
Bacteroidetes (b), Proteobacteria (c) and Actinobacteria (d) during development in
short-tailed shearwater chicks.
There were no significant differences or trend
detected during development.
109
Figure 6.5, Similarity of the major bacterial phyla in short-tailed shearwater chicks
during development using qPCR. No clustering of age classes or individuals was
observed.
110
16S rRNA gene pyrosequencing
A total of 12,522, 5,089, 24,222, 20312, and 16,131 partial 16S rRNA gene
sequences were amplified from faecal samples collected from short-tailed shearwater
chicks on weeks 2, 5, 8, 11 and 14 respectively. With 97% sequence similarity a total
of 1,040, 655, 1,786, 1,469 and 1,124 Operational Taxonomic Units (OTU) were
identified from short-tailed shearwater chicks respectively. A total of 23,967 partial
16S rRNA gene sequences and 1,800 OTU’s were amplified from adult short-tailed
shearwaters at the beginning of the breeding season
From the phylogenetic analyses, there were 13 phyla represented in developing
short-tailed shearwater gut microbiota; Firmicutes, Fusobacteria, Proteobacteria,
Planctomycetes,
Bacteroidetes,
Acidobacteria,
Actinobacteria,
Chloroflexi,
Cyanobacteria, Gemmatimonadetes, Synergistetes, Tenericutes and seven recently
classified candidates (Candidate division BRC1, OD1, OP10, OP11, TM7, BD1-5
and SM2F11). Throughout development Firmicutes dominated the microbiota,
comprising over 80% of the total composition except for weeks 8 and 11 where
Firmicutes comprises 62 and 73% (Figure 6.6). There was a high level of similarity
in the microbial composition of short-tailed shearwater chicks throughout
development with 60% similarity between all weeks except for week 5, where the
level of similarity declined to 40%. (Figure 6.7). In the adult short-tailed shearwater,
Firmicutes and Proteobacteria were the dominant phyla (Figure 6.6). The level of
similarity between the adult short-tailed shearwaters and its offspring was 40%
(Figure 6.6).
At family level, Leuconostocaceae and Planococcaceae dominated throughout
development, with Leuconostoc and Lysinibacillus the dominant genera. During
weeks 2 and 5 Leuconostocaceae is the most dominant family, followed by
Planococcaceae. However by week 8, Planococcaceae dominated the microbiota
until fledging in week 14 (Figure 6.8). In adult shearwaters, Leuconostocaceae and
Streptococcaceae (Figure 6.6) are the dominant families, with Leuconostoc,
Weissella, Lactococcus and Streptococcus the dominant genera (Figure 6.8).
Species Diversity
The species diversity in the short-tailed shearwater chick fluctuates throughout
development. The adult short-tailed shearwater has a high level of diversity (Figure
6.9).
111
Figure 6.6, Relative abundance of the major phyla in short-tailed shearwater chicks
during development. Firmicutes dominates the microbiota of short-tailed shearwater
chicks throughout development and in the adult microbiota.
112
Figure 6.7, Similarity of the gut microbiota of short-tailed shearwater chicks during
development. The result indicates that there is a high level of similarity between
weeks 2, 8, 11 and 14 with 60%, with limited similarity between week 5 and the
other weeks with 40%. In addition, there is around 40% similarity between adult
short-tailed shearwaters and their offspring.
113
Figure 6.8, Relative abundance of major Families during development in short-tailed
shearwater chicks. Planococcaceae and Leuconostocaceae dominate the microbial
composition of short-tailed shearwater chicks throughout development, whereas,
Leuconostocaceae and Streptococcaceae dominate the adult microbiota.
114
Figure 6.9, Species diversity at different age classes. Species diversity fluctuates in
the short-tailed shearwater chick, throughout development.
115
Discussion
Establishment and early colonisation of the GI tract has been recognised as a crucial
stage in neonatal development with pioneering species responsible for influencing
the GI pH, oxygen levels, mucosal structure and immunity and therefore, these
pioneering species influence a hosts overall health and disease status throughout life
[136, 138, 198]. In mammals the establishment and colonisation of the neonate GI
tract has been extensively studied [168, 199, 268]. In avian species, however data is
restricted to the microbiota of poultry [17, 68, 134, 171, 256, 266]. To date there is
no data available on the GI microbiota of long lived seabirds, nor the microbial
succession that occurs during development. Therefore this study aimed to examine
the establishment and microbial succession that occurs in little penguin chicks during
development. This study aimed to analyse the microbial composition of the
developing short-tailed shearwater from hatching to fledging using Quantitative Real
Time PCR and 16S rRNA pyrosequencing.
Quantitative data obtained by qPCR analysis for Firmicutes, Bacteroidetes,
Actinobacteria and Proteobacteria demonstrated that the microbiota in short-tailed
shearwater chicks occurs rapidly after birth, with over 4.1 x 106CFU present at 2
weeks of age (estimation calculated from abundance of Firmicutes, Bacteroidetes,
Actinobacteria and Proteobacteria), which is similar to little penguin chicks (Chapter
3) and other vertebrates with the microbial population estimated at around 2 x 106 in
little penguins and 108 -1010 in humans [136, 168]. In contrast to other vertebrates
however, the short-tailed shearwater microbiota appears to stabilise quite rapidly
with the absence of any significant changes in bacterial abundance.
In accordance with the qPCR results, the pyrosequencing analysis, demonstrated that
there was little change in the microbiota of short-tailed shearwater chicks during
development. In previously examined neonates including penguins (Chapter 3), the
GI microbiota is initially colonised by gram negative aerobic and facultative
anaerobes, followed by facultative and strict anaerobes [5, 68, 168, 217, 268]. In the
short-tailed shearwater however, the microbiota at 2 weeks of age is colonised by
gram positive facultative and obligate anaerobes. However, as samples were not
analysed during the first week post hatch, there is a possibility that these early
pioneering species could have been present. In comparison to other marine
vertebrates, the microbial composition of short tailed shearwater chicks appears to be
116
more similar to that of Australian fur seal pups than other seabirds including the little
penguin (Chapter 3). In Australian fur seal pups the microbiota at 2, 6, and 9 months
is heavily dominated by gram positive bacteria from the phylum Firmicutes (>80%)
including; members from the Order Clostridia and Bacilli (Smith et al, unpublished).
In addition, little variation was observed in the microbial composition of both the
short-tail shearwater and Australian fur seal (>60%), with high similarity throughout
development (60%) except for week 5 (Smith et al unpublished). Even during the
final stages of development when adults abandon their chicks to return to the
northern hemisphere, leaving chicks to survive off their endogenous fat reserves
[190] a high level of similarity is maintained. There is low level of similarity
between the adult and chick indicating that the adult has negligible influence of the
GI microbiota of the chick.
Throughout development the microbiota of short-tailed shearwater chicks is
dominated by Firmicutes. In humans and mice, the ratio of Firmicutes to
Bacteroidetes has been shown to influence a host’s adiposity level by enhancing
energy extraction and modulating genes that regulate fat storage [152, 156, 259, 260,
263] and is currently considered to play a crucial role in obesity. In obese individuals
the ratio of Firmicutes to Bacteroidetes is 100:1, whereas in their lean counterparts
the ratio is 10:1 [152, 156, 263]. Similarly to adult short-tailed shearwater (Chapter
5), the ratio of Firmicutes to Bacteroidetes in short-tailed shearwater chicks ranges
from 73:1 to 85:1 and is similar to that of obese humans, mice and Australian sea
lions, except for week 5, where the ratio is 62:18 [152, 156, 263]. Following birth,
the body mass of short-tailed shearwater chicks rapidly increases and by week 7, the
body mass of chicks exceeds that of the adult shearwater by 15-50% [189]. While
many factors, such as a high lipid diet and gut physiology, may influence body mass,
the high abundance of Firmicutes of the short-tailed shearwater chick may confer a
predisposition towards developing excess body fat stores. A chick’s ability to rapidly
accumulate large fat reserves enables adults to feed them less regularly and enables
chicks to survive the long periods between feeds and is essential for survival post
fledging [201].
Due to the absence of data available on the role of microbes in seabirds, the potential
role of the microbiota in short-tailed shearwaters can only be elucidated from
previous research. At genus level the microbiota of short-tailed shearwater chicks is
117
dominated by Lysinibacillus, a known soil bacterium. Other genera known to
dominate the microbiota of short-tailed shearwater chicks are Leuconostoc and
Lactococcus, which are lactic acid bacteria commonly used in the food industry as
starter cultures for dairy products and have been associated with fermentation [1, 21,
188]. However, to date their role in the GI microbiota is still unknown and requires
further investigation.
Conclusion
The microbial composition of short-tailed shearwater chicks is highly dominated by
the phylum Firmicutes. In contrast to other infants, the short-tailed shearwater
microbiota is dominated by gram positive facultative and obligate anaerobic bacteria
and is stable throughout development. The ratio of Firmicutes to Bacteroidetes in the
short-tailed shearwater chick strongly resembles that of obese humans, mice and
Australian sea lions and fur seals. However, due to the absence of information on the
functional role of the dominant microbiota in short-tailed shearwaters and other
vertebrates, it is extremely difficult to elucidate the role of the microbiota short-tailed
shearwater development.
118
CHAPTER 7
General Discussion
Introduction
In order to understand the complex relationship between a host and its microbiota,
one must first characterise the GI microbiota of healthy individuals at baseline before
elucidating the role of microbiota, and the influence alterations of the GI microbiota
will have on health and disease of an individual or population [157, 262]. Therefore,
the main goal of this project was to characterise the assemblage and community
composition of the seabird microbiota in its natural environment. Seabirds are a
group of birds that obtain their entire nutrition from the marine environment [100,
105, 232]. The diet of seabirds is high in lipids, proteins and polyunsaturated fatty
acids [211]. This research to my knowledge is the first to use both Quantitative Real
Time PCR and 16S rRNA pyrosequencing to characterise the GI microbiota from
members of the Orders Sphenisciformes and Procellariiformes.
In recent years, research has been carried out on the mammalian [108, 109, 147,
154, 181, 290], avian [12, 162, 289, 297], human [155, 156, 168], reptilian [73, 167]
and fish [18, 214, 215, 228] gut microbiota. These studies have increased our
knowledge and understanding on the complex relationship that exists between a host
and its microbiota and that the GI microbiota has a profound influence on the
nutritional, physiological, immunological and metabolic processes of the host. The
GI microbiota is essential for the development maturation of the immune system
[214, 215], developmental regulation of the intestinal physiology, energy harvest, fat
metabolism and production of short-chain fatty acids [66, 167, 168, 180, 215, 298,
299]. The colonisation process during infancy has also been shown to affect a hosts
predisposition to disease later in life [66].
Over the past decade, a large number of internal and external factors have been found
to influence the colonisation and stabilisation of the GI microbiota. These factors
include, diet, host phylogeny, intestinal physiology, age, external environment (i.e.
nesting material), antibiotics and stress [73, 87, 88, 131, 153, 154, 181]. However,
the major limitation in being able to link the composition of the GI microbiota to host
function is an absence of knowledge concerning the indigenous community that is
present under natural conditions.
119
Therefore, characterisation of the vastly diverse ecosystem of the GI tract is the first
step in exploring its role in digestive physiology, gut development and maturation,
immune function and disease [91]. Seabirds live in an unpredictable environment and
have had to adapt to fluctuations in availability of prey, oceanic currents and periods
of fasting during breeding and moult. Therefore, knowledge on the composition and
role of the GI microbitoa is essential to further understand the ecology of seabirds.
This thesis provides greater understanding of the composition of the GI microbiota of
seabirds under natural conditions during different life stages (i.e. chicks, adults,
moult). This discussion outlines the general conclusions of the research that has been
covered in this thesis.
Gastrointestinal Microbiota of Seabirds
The results from this thesis identified that the gastrointestinal microbiota of seabrids
differ not only between avian orders (Spheniscidae & Procellariiformes) but also
between species, with a relatively low level of similarity between all seabirds
examined in this study (Figure 7.1). The penguin microbiota is dominated by
Firmicutes, Bacteroidetes, Fusobacteria and Proteobacteria (Chapter 2), while the GI
microbiota of Procellariiformes is dominated by Firmicutes, Proteobacteria and in
common diving Bacterodetes is also dominant (Chapter 5) (Figure 7.2). In penguins,
diet, and diet associated microbiota could potentially be a major factors influencing
the microbiota of seabirds, with the krill eating penguins clustering closely together
on the MDS plot, in comparison to the king and little penguins (Figure 2.4) [12, 154].
In contrast, the Procellariifomres, digestive physiology and rention time digesta
appear to be the major factors influencing the microbial composition (Figure 5.4),
with a high level of similarity between the two oil producing seabirds and a low level
of similarity between the oil and non-oil producing seabirds. Although diet is not a
major factor influencing the differences in Procellariiform seabird microbiota, it
appears to be a major influential factor in seabirds with the krill eating seabirds
(short-tailed shearwaters, fairy prions, macaroni & gentoo penguins and common
diving petrels) clustering closesly together (Figure 7.1) [12, 153, 228, 246].
120
Figure 7.1, Level of similarity between seabirds. Short-tailed shearwaters and fairy
prions are highly similar in their microbial composition. In addition, gentoo and
macaroni penguins are highly similar to common diving petrels. Little and king
penguins are highly disimilar to other seabird species.
121
Figure 7.2, Relative abundance of the major phyla for all seabird species using 16S
rRNA pyrosequncing. Little, king and macaroni penguins and common diving petrels
are dominated by Fimicutes, Bacteroidetes and Proteobacteria. Gentoo penguins are
dominated by Fusobacteria. Short-tailed shearwaters and fairy prions are dominated
by Firmicutes.
122
The dominance of Bacteroidetes and Firmicutes in penguins and common diving
petrels, is in accordance with previous studies on other vertebrate species; including
Antarctic seals [91, 121, 145, 153, 249]. However, the relatively high abundance of
Fusobacteria (2-55%) and Proteobacteria (5-30%) differ from other vertebrates. The
domination of Firmicutes in oil producing Procellariiformes is very similar to that of
other marine vertebrates including; polar bears and Australian sea lion [108, 147]. To
date, there is no knowledge available in the literature on the functional role of the GI
microbiota in seabirds and therefore, information on the role of these microbes must
be elucidated from previous research in other vertebrates. The penguin and common
diving petrel microbiota appears to be associated with microbes that are responsible
for responsible for the absorption of
dietary fats, lipids, and soluble vitamins,
maintain intestinal barrier function, regulate triglycerides, impact intestinal
permeability, activate intestine-brain-liver neural axis, enhance the immune system,
alteration of lipoprotein profiles, protection against stress-induced lesions,
modulation of proinflammatory and anti-inflammatory gene expression, and
strengthen epithelial cell barrier properties. However, these microbes have also been
implicated in GI pathologies, brain-gut axis, and a few neurological conditions [43,
117, 139, 223, 235, 251, 292], butyrate production [204] and microbes with probiotic
properties [250]. In contrast, the Procellariidae microbiota appears to be dominated
by lactic acid bacteria that are commonly used in the food industry and the skin
microbiota of some vertebrates [22, 47, 188].
Development of the Gastrointestinal Microbiota in Seabirds
Establishment and early colonisation of the GI tract has been recognised as a crucial
stage in infant development with pioneering species responsible for influencing the
GI tract pH, oxygen levels, mucosal structure and immunity [136, 138, 198].
Immediately after birth, the GI microbiota is rapidly colonised and undergoes
successional changes until a dense and stable community is achieved. In most
vertebrates this process can take anywhere from 40 days [16, 17, 68, 90, 217] to 2
years
[164, 166, 168, 240]. The results from this thesis identified that the
development of the seabird microbiota, differs greatly between species. In the little
penguin, there were significant differences throughout development and significant
upward trends in the abundance of Firmicutes and Bacteroidetes, whereas, in the
short-tailed shearwater there were no significant differences throughout development
and appears relatively stable throughout development with a high level of similarity
123
between each age class. There is low similarity between adults and chicks for both
the short-tailed shearwaters and little penguin, indicating that the adult microbiota
has a negligible influence over the chick’s microbiota.
The microbial composition of the little penguin is dominated by microbes that are
responsible for the absorption of dietary fats, lipids, and soluble vitamins, maintain
intestinal barrier function, regulate triglycerides, impact intestinal permeability,
activate intestine-brain-liver neural axis, enhance the immune system, alteration of
lipoprotein profiles, protection against stress-induced lesions, modulation of
proinflammatory and anti-inflammatory gene expression, and strengthen epithelial
cell barrier properties. However, these microbes have also been implicated in GI
pathologies, brain-gut axis, and a few neurological conditions [43, 117, 139, 223,
235, 251, 292], butyrate production [204] and microbes with probiotic properties
[250]. In the short-tailed shearwater chick, however the GI microbiota appears to be
associated with soil and lactic acid bacteria that are not commonly associated with
the GI tract.
In seabirds, pre-fledging body mass is associated with juvenile survival and
therefore, the accumulation of fat reserves is important for post fledging survival. In
little penguin chicks the relatively high abundance of butyrate producing microbes
could influence body mass, as previously observed in poultry [196]. Although the
ratio of Firmicutes to Bacteroidetes strongly resembles that of obese humans, mice
and Australian pinnipeds, the microbes within the phylum Firmicutes (Lysinibacillus,
Leuconostoc and Lactococcus) have previously not been associated with energy
extraction, lipid accumulation or obesity.
The differences between little penguins and short-tailed shearwaters could be in part
due to differences diet composition, digestive physiology, nesting environment and
host phylogeny, all of which have been previously shown to strongly influence the
microbial composition of vertebrates [12, 154, 246, 256]. Due to the absence of
information on the functional role of the dominant microbiota, we are unable to
elucidate the role of the GI microbiota in fledging survival of little penguins and
short-tailed shearwaters.
124
Influence of Fasting on the Gastrointestinal Microbiota of Penguins
Once a year all penguins must undergo moult to replace their old warn plumage with
new feathers. Prior to moult penguins spend large amounts of time at sea building up
their protein and lipid reserves necessary for the upcoming moult [218]. During
moult penguins replace their entire plumage whilst fasting on land and cannot return
to sea because of the consequences of reduced waterproofing and thermal insulation
[105, 119]. Similarly to the breeding season, penguins must rely on endogenous fat
and protein reserves for feather synthesis and nourishment for the duration of moult
which can take between 2-5 weeks [50, 218].
This study aimed to examine the influence of fasting during moult on the GI
microbiota of two penguins; the king (Aptenodytes patagonicus) and little penguin
(Eudyptula minor). The results from this thesis identified significant changes in the
microbial composition of both king and little penguins. This study also documented
that the degrees to which moult impacts the microbial composition differs between
the two penguin species. In little penguins, there is still a high level of similarity
between early and late moult (60%), with little variation in the abundance of the
major phyla observed with both the qPCR and pyrosequencing analysis. In contrast,
there were significant differences between early and late moult with a 75% increase
in the level of Fusobacteriaceae. In addition, there was a low level of similarity
(20%) between early and late moult in king penguins. Due to the absence of
knowledge in the literature regarding the role of microbes in a seabird host, we are
unable to elucidate the consequences of fasting as a result of microbial changes to the
microbial composition.
Quantitative Real Time PCR Vs. 16S rRNA Pyrosequencing.
The primer sets used in this study appear to be limited in their ability to provide an
accurate estimate of the abundance of major phyla within the faecal samples of
seabirds. The limitation of the primers could be in part due to the fact that they have
been designed from bacterial sequences obtained from human and chickens and
therefore, do not cover all members of the bacterial phyla found in the penguin
microbiota. The use of the pyrosequencing data from this project to design more
accurate primer sets could be useful in providing more accurate abundance estimates
of the major phyla present in seabirds.
125
CHAPTER 8
Conclusions and Future Directions
Conclusions
This research provides a baseline for future work regarding the microbial
composition of seabirds. This thesis has identified that a complex community
inhabits the GI tract of seabirds and provides insight in to the succession changes that
occur during development and how fasting influences the microbial composition.
The results obtained in this thesis allow us to conclude;
The results from this thesis identified that the gastrointestinal microbiota of seabrids
differs not only between avian orders (Spheniscidae & Procellariiformes) but also
between species, and at different life stages
The microbiota of penguins and common diving petrels, is dominated by Firmicutes,
Bacteroidetes and Proteobacteria, with Fusobacteria
The GI microbiota of oil producing Procellariiformes (short-tailed shearwaters and
Fairy Prions) is highly dominated by Firmicutes and Proteobacteria to a less extent.
Diet and prey associateed microbiota appear to be major contributing factors
influencing the GI microbiota of seabird species with strong clustering of krill eating
seabirds. However, within the Order Procellariiformes, digestive physiology appears
to be the major factor influencing microbial composition, with high similarity
between the two oil-producing Procellariiformes and a low level of similarity
between oil and non-oil producing Procellariiformes. As diet between these three
seabirds, is highly similar, diet and prey associated microbiota do not appear to
explain these differences.
Signficant differences in the colonisation of the GI microbiota during development in
different seabird species, with significant changes observed in the little penguin
microbiota throughout development, whereas, no signifcant changes were observed
in the short-tailed shearwater microbiota throughout development. Therefore, is no
‘core’ microbiota colonising the GI microbiota of seabirds during development.
Fasting during moult influences the microbial composition of both king and little
penguins, but to differing degrees. In little penguins these changes appear to be
subtle, with only a 40% difference in the microbial composition. In king penguins
126
however, not only is there a significant increase in the level of Fusobacterium but
there is little similarity between the early and late moult (20%). These differences,
could in part be due to the differences in moult length (i.e. 2 weeks in little penguins
vs. 5 weeks in king penguins).
Limitations
Quantitative Real Time PCR Primer Sets
Due to the absence of ‘seabird’ specific oligonucleotide primers, the primers used in
this study were from previous research on the dominant microbiota of other
vertebrate hosts including humans and chickens. Although, Zdanowski et al [294] did
use ‘penguin specific’ bacterial primers in their study, upon further testing of these
primers in our laboratory, these primers were not non-selective towards the particular
primer as stated in the paper (i.e. Firmicutes primer was able to detect
Bifidobacteria). Therefore, for this thesis we used bacterial primers that were specific
to a given phylum. However, the results from the 16S rRNA pyrosequencing of this
study, which was conducted after the qPCR analysis, identified that the primer sets
used in this thesis, are unable to detect all members of the major phyla in the study
animals of this thesis and therefore, are limited in their ability to provide an accurate
estimate of the abundance of major phyla within the faecal samples of seabirds. If
more time and funds were available, the data from the 16S pyrosequencing could
have been used to design primer sets that were more specific to seabirds.
16S rRNA gene pyrosequencing
Due to the availability of limited funds and the high costs associated with
pyrosequencing, samples were pooled together following amplification and prior to
sequencing on the Roche GS FLX 454 sequencer. By pooling samples, after
amplification, ensured that bacterial DNA from all members of each species was
equally represented and enable us to look at changes in the microbiota at a
community level. However, by pooling samples we were unable to examine
individual differences and also prevented us from removing potential outliers that
may influence the overall result.
127
Future Studies
This thesis has provided knowledge of the resident GI microbiota of two seabird
Orders, the Sphenisciformes and Procellariiformes. To further our knowledge of the
GI microbiota of seabirds and its role in health, digestive physiology, survival and
disease the following studies are recommended.
Microbiome of penguins
Microbiome of fasting penguins
Introduction of alien species by humans in Antarctic penguins
Microbiome of Procellariiformes
The developing microbiota and immune system in Seabirds
Study 1: Microbiome of Penguins
Summary: In the past, only two studies have focused on examining the “residential”
microbiota of penguins using molecular based technologies [12, 294], by examining
the microbial ecology of adélie penguins. Whereas other research has focused on
identifying the presence of known human and veterinary pathogens, or the microbial
composition using traditional culture techniques. Whereas for temperate penguins,
there have been no investigations into the microbial ecology utilising molecular
based techniques. To date there is no data available on; 1) the stability of the penguin
microbiome over time and 2) role of microbes in penguins; including the role of
potential pathogens, except when death has been caused by an infectious agent (i.e.
Avian Cholera) [26, 45, 62, 63, 85, 106, 113, 149, 151].
The recent advances of metagenomic techniques allows for a more comprehensive
and unbiased assessments of the microbial diversity by allowing the examination of
microbes that are not easily cultured in a laboratory [264]. Metagenomics allows for
the characterisation of the microbial community within the gut [107] and can
elucidate important processes for the gut microbes and the host. In addition,
metagenomics can provide insight into the insight into the relationships between the
host and its gut microbiota [10, 209, 264, 280]. By coupling metagenomics with
metabolomics (study of metabolites) will shed light on how microbial communities
function in the gastrointestinal tract of penguins [261].
128
Methods: Samples from penguins will be collected from Antarctic (i.e. emperor,
royal, gentoo, and adélie), Sub-Antarctic (i.e. macaroni, king), Temperate (i.e. little,
rockhopper, snares, yellow-eyed) and Tropical (i.e. Humboldt, Galapagos) penguin
species.
For
statistical
significance,
a
population
size
between
10-20
individuals/species will be required. Metagenomic analysis of samples will be
completed on the Roche 454 GS FLX Titanium. Metagenomic analysis will elucidate
information on the microbial population and potential functionality of the microbial
population. Metabolites present in samples are identified by GC mass spectroscopy.
Results are then run through genomic and metabolite databases to identify microbes
and metabolites present in the sample.
Significance: This research is significant because it starts to elucidate the role of
microbes in penguin health, metabolism and digestive physiology which has not
previously been done before. In addition, this study would also provide valuable
information on forces influencing the GI microbiota of penguins (i.e. host phylogeny,
diet, location).
Study2: Microbiome of fasting penguins
Summary: Like many seabirds, penguins have had to adapt to periodic fluctuations
in nutrient availability and to prolonged periods of fasting [31, 55, 119] especially
during moult and breeding. Previous studies examining the effect of fasting on the GI
microbiota of vertebrate (hamsters, python, mice), have shown that fasting not only
alters the composition and diversity of the GI microbiota, but it also influences the
host’s immune defences [73, 74, 241] and that interrelationships exist between a
host, its microbiota and the hosts nutritional status, diet and physiology [73, 74].
Unlike many other vertebrates, penguins undergo two distinct of fasting. During the
breeding season, adult penguins undergo long periods of fasting during egg
incubation and during guard and post-guard stage of chick rearing, alternating
foraging trips between short and long foraging trips. In little penguins, long foraging
trips are often associated with adult penguins replenishing their energy reserves
[230]. In contrast, during moult penguins replace their entire plumage whilst fasting
on land and cannot return to sea because of the consequences of reduced
waterproofing and thermal insulation [105, 119]. During both the breeding and
moulting fast penguins must rely on endogenous fat and protein reserves built up
129
prior to fast [50]. Inadequate storage of fat and protein reserves during moult can
result in high rates of mortality [50, 77, 114] whereas inadequate reserves during the
breeding season, can result in abandonment of chicks. To date no studies have
examined the changes in microbial functionality during fasting vertebrates. By
coupling metagenomics with metabolomics, this study will shed light on how fasting
during moult affects the composition of the microbiota and identify changes in the
function and presence of metabolites in the gastrointestinal tract of penguins [261].
Methods: Because of their availability throughout the year, the fact that little
penguins are present in the colony all year round, and the ease of following the same
individual over time, make the little penguin the perfect penguin species to examine
the influence of fasting during breeding and moult. Metagenomic analysis of samples
will be completed on the Roche 454 GS FLX Titanium. Metagenomic analysis will
elucidate information on the microbial population and potential functionality of the
microbial population. Metabolites present in samples are identified by GC mass
spectroscopy. Results are then run through genomic and metabolite databases to
identify microbes and metabolites present in the sample.
Significance: This study will enable scientists to examine if the GI microbiota
influences the penguins ability to store large reserves of proteins and lipids prior to
the fast and how the microbial composition changes during the fasting process.
Study 3: Impact of Humans on the microbial composition of Antarctic penguins
Summary: A recent article highlighted the introduction of alien species to the so
called ‘pristine’ environment of the Antarctic, since the arrival of man. Chown et al
[61] discovered that both scientists and tourists inadvertently brought alien species
with them on their clothing to the Antarctic. In addition, the study identified that
scientists were the main vector introducing alien species such as seeds, plant
structures, and propagules. But what about microbes? Frenot et al [98] identified that
alien microbes, fungi, plants and animals have managed to invade sub-Antarctic
islands and some parts of the Antarctic continent, with the main routes of
introduction being associated with human activity in the region. However, when
Bonnedahl et al [28] examined the faeces of penguins at six tourist locations on the
Antarctic peninsula, for the presence of human associated pathogenic bacteria, failed
to isolate any human-associated pathogenic bacteria from penguins. However, this
study only concentrated on Campylobacter, Salmonella and Yersinia, therefore,
130
excluding other possible human-associated pathogens. Greikspoor et al [116] not
only identified the presence of Campylobacter jejuni in macaroni penguins, but that
the C. jejuni isolates were of a genotype common among humans, indicating the
bacterium was possibly of human origin. The use of molecular based methods such
as 16S rRNA in this thesis has helped identify the presence of known human and
veterinarian pathogens in seabirds. However, this thesis was unable to identify
whether or not these microbes posed any threat to penguin health. Therefore, by
incorporating methods such as metagenomics and metatranscriptomics scientists will
be able to examine the potential influence of human presence on the GI microbiota of
penguins.
Methods: To examine the impact of humans on the GI microbiota of penguins, we
will examine the GI microbiota of penguin species frequently visited by tourists
(king, gentoo, adélie). Samples will be collected from three populations. These
populations will be selected based on 1) frequency of tourisms, 2) presence of
scientists and 3) absence of human contact. To examine the microbial composition of
penguins, genomic DNA will be amplified using V2-3 region bacterial primers and
then run on the Roche 454 GS FLX Titanium.
Significance: This study will provide the first in depth investigation to see if
proximity to humans and civilisation negatively impacts penguin microbiota and if
scientists and tourists are capable of introducing alien microbes to penguins and
other Antarctic wildlife and the impacts these alien species can have on penguin
health and survival.
Study 4: Microbiome of Procellariiformes
Summary: Unlike penguins, there are no previous publications examining the
microbial composition of Procellariiform seabirds, with all previous investigations
isolating pathogenic microorganisms and identifying the cause of mortality.
The recent advancement of metagenomic techniques allows for a more
comprehensive and unbiased assessment of the microbial diversity by allowing the
examination of microbes that is not easily cultured in a laboratory [264].
Metagenomics allows for the characterisation of the microbial community within the
gut [107] and can elucidate important processes for the gut microbes and the host. In
addition metagenomics can provide insight into the insight into the relationships
131
between the host and its gut microbiota [10, 209, 264, 280]. By coupling
metagenomics with metabolomics’ (study of metabolites) will shed light on how
microbial communities function in the gastrointestinal tract of penguins [261].
Methods: Samples are run on the Roche 454 GS FLX Titanium to sequence the
microbial population, whilst metabolites present in samples are identified by GC
mass spectroscopy. Results are then run through genomic and metabolite databases
to identify microbes and metabolites present in the sample.
Significance: This research is significant because it starts to elucidate the role of
microbes in seabird health, metabolism and digestive physiology which has not
previously been done before. In addition, this research could elucidate the influence
of stomach oils on microbial community composition.
Study 5: The developing microbiota and immune system in Seabirds
Summary: Studies investigating the developing microbiota and immune system has
been limited to poultry. To date there are no publications examining the development
of the microbiota and its influence on the developing immune system in seabirds.
The composition of the microbial community within the gastrointestinal tract of
vertebrates is fundamental for the development and function of an effective and
competent immune system [19, 47, 229]. Research utilising gnotobiotic mice have
shown that in the absence of a diverse gastrointestinal microbiota, the mice had a
compromised immune system and a higher susceptibility to disease [97, 214, 215,
229, 265]. In addition, research has shown that any alteration of the host’s microbiota
during post natal development can have profound effects on the establishment of the
immune system [129, 229]. This thesis identified that the development of the GI
microbiota varies from one seabird species to another, with little penguins
experiencing relatively high variation throughout development, whereas short-tailed
shearwaters experience very little variation. Further research into the factors
influencing the colonisation process of the GI microbiota and the role the developing
GI microbiota has on the immune system will provide valuable information on the
influence of early microbial colonisation on health and disease state of adults.
Methods: Samples will be collected from little penguins and short-tailed shearwaters
from hatch and until fledging. To investigate the development of the immune system
in seabirds, we must first identify the lymphocyte and inflammatory markers such as
132
Tcell, Bcell and MHC II CD4, CD8, and cytokines IFNg, IL4, IL10, IL12, IL2, and
IL6. This will be achieved by taking blood and tissue samples from captive Adult
penguin species, using flow cytometric methods. Once the markers have been
identified, blood samples will be collected at weekly time points and analysed by
quantification of gene expression using qRT-PCR with primers that would be
developed in this study.
To investigate the developing microbiota, faecal samples would initially collected on
a daily basis from birth for the first week (to examine the microbial composition
immediately after birth), then weekly thereafter. Samples are run on the Roche 454
GS FLX Titanium to sequence the microbial population, whilst metabolites present
in samples are identified by GC mass spectroscopy. Results are then run through
genomic and metabolite databases to identify microbes and metabolites present in the
sample.
Significance: This study will help identify the role the developing GI microbiota has
on the development of the innate and adaptive immune system of seabirds. This
knowledge will be invaluable in providing information on factors that influence
survival and health of penguin chicks.
133
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Appendix 1
Table A1, Taxonomic Breakdown of bacteria present in the GI microbiota of
penguins. KP = King, GP = Gentoo, LP = Little, MP = Macaroni
Taxonomic group (SILVA tax, release 106)
KP
GP
LP
MP
Bacteria;Acidobacteria;Acidobacteria;11-24;
0
0
0
2
Bacteria;Acidobacteria;Holophagae;iii1-8;
0
6
0
0
Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales;
Acidimicrobineae;Acidimicrobiaceae
Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales;
Acidimicrobineae;Iamiaceae
Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales;
Acidimicrobineae;Iamiaceae;Iamia;
Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales;
Acidimicrobineae;Sva0996 marine group
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales
;Actinomycineae;Actinomycetaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Actinomycineae;Actinomycetaceae;Actinomyces;
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Actinomycineae;Actinomycetaceae;uncultured;
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales
;Actinomycineae;Actinomycetaceae;Varibaculum;
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Corynebacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Corynebacteriaceae;Corynebacterium;
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Dietziaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Family Incertae Sedis
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Mycobacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Frankineae;Frankiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Frankineae;Nakamurellaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Frankineae;Sporichthyaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;AKAU3644
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Dermacoccaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Dermatophilaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Dermatophilaceae;Dermatophilus
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Intrasporangiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Microbacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Micrococcaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micromonosporineae;Micromonosporaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
PeM15;
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Propionibacterineae;Nocardioidaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Propionibacterineae;Propionibacteriaceae;Tessaracoccus;
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Propionibacterineae;Propionibacteriaceae;uncultured;
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Streptosporangineae;Thermomonosporaceae
0
1
0
0
0
9
0
0
1
0
0
0
0
18
113
81
0
32
46
27
74
0
0
0
2
0
0
0
2
0
0
0
0
0
31
0
13
0
0
0
0
10
0
3
0
0
0
1
0
7
41
5
0
0
17
0
0
3
26
0
0
0
0
1
0
0
0
4
0
16
0
1
0
11
0
4
3
0
0
0
0
12
0
9
0
1
0
13
0
2
0
0
0
0
0
2
0
18
0
1
0
38
61
21
1
0
0
0
1
0
0
0
0
0
4
0
153
Bacteria;Actinobacteria;Actinobacteria;OPB41;
0
0
0
2
Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae;AKIW543;
0
2
0
0
Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae;Solirubrobacterales;
480-2;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;
Bacteroides;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;gir-aah93h0;
0
0
0
1
15
0
0
2
87
0
0
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Marinilabiaceae;
2
0
0
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Marinilabiaceae
;uncultured;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;p-2534-18B5 gut group;
0
0
480
2
99
0
0
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;
Dysgonomonas;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;
Odoribacter;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;
Paludibacter;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;
Parabacteroides;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;
Petrimonas;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;
Porphyromonas;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;
Proteiniphilum;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;
uncultured;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;
5
0
0
0
0
4
0
4
712
0
0
18
0
237
101
723
18262
87
18
13
1550
243
22
147
1387
2
0
2
0
13
88
5
6
2
0
6
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;RF16;
0
0
0
42
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae;RC9 gut
group;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7;
0
0
97
127
0
0
0
14
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;vadinHA21;
182
0
0
1
Bacteria;Bacteroidetes;BD2-2;
0
4
202
10
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;
Adhaeribacter;
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Fibrella;
0
0
4
0
0
0
0
8
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;
Hymenobacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;
Brumimicrobium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;
Brumimimicrobium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;
Crocinitomix;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;
Cryomorpha;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;
Fluviicola;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;
Owenweeksia;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
0
0
0
2
0
3
0
0
1
0
0
0
1
0
0
0
3
0
0
0
12
1
0
0
0
1
0
0
29
29
0
2
8
0
0
0
2
0
0
0
26
0
0
0
0
0
0
1
0
204
0
25
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Aequorivita;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Arenibacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Bergeyella;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Bizionia;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Capnocytophaga;
154
681
0
0
15
0
90
0
4
26
169
35
2
528
79
0
5
9
0
0
0
2
0
0
0
0
31
0
0
20
0
0
0
0
0
0
1
0
1
0
0
2
0
0
0
0
2
0
1
62
0
0
0
0
1
0
0
Bacteria;Bacteroidetes;SB-1;
0
1
0
0
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;hitinophagaceae;
Sediminibacterium;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;hitinophagaceae;
uncultured;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Cyclobacteriaceae;Aquiflexum;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Cytophagaceae;Leadbetterella;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;E6aC02;
0
3
3
19
32
12
0
0
1
0
0
0
2
0
0
0
5
0
0
0
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Saprospiraceae;Lewinella;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Saprospiraceae;uncultured;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;Parapedobacter;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;Pedobacter;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;Sphingobacterium;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;uncultured;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;WCHB1-69;
0
5
0
0
2
0
0
0
0
1
0
0
37
0
0
0
6
0
0
0
0
41
0
0
1
0
0
0
Bacteria;Bacteroidetes;VC2.1 Bac22;
362
0
0
0
Bacteria;BD1-5;
53
0
0
0
Bacteria;Candidate division BRC1;
0
3
18
0
Bacteria;Candidate division OP10;
1
0
0
0
Bacteria;Candidate division OP11;
0
10
2
0
Bacteria;Candidate division SR1;
937
8
3
4
Bacteria;Candidate division TG-1;Lineage IIb;
1
0
0
0
Bacteria;Candidate division TM7;
1278
7
0
0
Bacteria;Candidate division WS6;
0
0
0
1
Bacteria;Chlorobi;Chlorobia;Chlorobiales;SJA-28;
0
1
0
0
Bacteria;Chlorobi;Ignavibacteria;Ignavibacteriales;uncultured;
0
19
0
3
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Chryseobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Cloacibacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Coenonia;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Flavobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Gelidibacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Lacinutrix;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Myroides;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Ornithobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Psychroserpens;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Salinimicrobium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
uncultured;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Wautersiella;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Weeksella;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;NS9 marine group;
155
Bacteria;Chloroflexi;Anaerolineae;Anaerolineales;Anaerolineaceae;
uncultured
Bacteria;Chloroflexi;Caldilineae;Caldilineales;Caldilineaceae;Caldilinea;
37
0
0
5
0
0
0
5
Bacteria;Chloroflexi;Caldilineae;Caldilineales;Caldilineaceae;uncultured;
0
0
0
1
Bacteria;Chloroflexi;Thermomicrobia;JG30-KF-CM45;
0
4
12
13
Bacteria;Cyanobacteria;Chloroplast;
5
14
0
0
Bacteria;Cyanobacteria;SubsectionIV;Anabaena;
0
1
0
0
Bacteria;Deinococcus-Thermus;Deinococci;Deinococcales;Deinococcaceae;
Deinococcus;
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Amphibacillus;
0
3
0
0
0
0
0
3
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Anoxybacillus;
0
8
0
7
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Filobacillus;
0
0
0
2
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Geobacillus;
1
0
0
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Natronobacillus;
0
0
0
2
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Sediminibacillus;
0
0
16
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Virgibacillus;
0
5
0
0
Bacteria;Firmicutes;Bacilli;Bacillales;Family XI Incertae Sedis;Gemella;
0
3
0
6
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Incertae Sedis;
18
0
0
0
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Planomicrobium;
0
0
0
12
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Sporosarcina;
12
0
0
0
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;uncultured;
2
0
0
0
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Nosocomiicoccus;
0
11
0
0
Bacteria;Firmicutes;Bacilli;Bacillales;Thermoactinomycetaceae;
Thermoactinomyces;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Allofustis;
0
1
0
0
3
0
0
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Atopostipes;
0
3
0
3
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Carnobacterium
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Trichococcus;
1
0
0
0
2
4
0
1
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;uncultured;
17
2
0
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Catellicoccus;
424
22
0
29
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Enterococcus;
2
0
0
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Vagococcus;
3
8
316
82
Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus;
17
0
0
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Fructobacillus
0
1067
68
1100
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Leuconostoc;
380
91
57
785
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Weissella;
753
0
0
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;MOB164;
1
0
0
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;P5D1-392;
0
0
0
1
Bacteria;Firmicutes;Bacilli;Lactobacillales;PeH08;
0
3
0
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Rs-D42;
0
524
63
254
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Lactococcus;
121
131
95
32
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;
0
2
0
4
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;uncultured;
0
6
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Alkaliphilus;
3217
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Caloranaerobacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Caminicella;
0
0
41
0
7
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Candidatus Arthromitus;
0
8
27
5
156
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Clostridiisalibacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridium;
36
123
49
42
2256
12
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridium;
Candidatus Arthromitus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Proteiniclasticum
4
0
0
0
0
0
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Sarcina;
0
0
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;uncultured;
0
1
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Dethiosulfatibacter;
3
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Eubacteriaceae;Acetobacterium;
0
0
16
25
Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae
Sedis;Proteiniborus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;
0
5
0
9
617
33
29
42
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Finegoldia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Gallicola;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Helcococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Parvimonas;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Peptoniphilus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Soehngenia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Sporanaerobacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Tepidimicrobium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Tissierella;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae
Sedis;Fusibacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae
Sedis;Guggenheimella;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae Sedis;
15759
1
0
0
5758
0
0
5
63
2
0
3
188
0
0
0
66
17
0
22
2062
1
0
1
384
2
18
2
16
3
5
3
7
55
0
14
287
109
0
44
78
1
0
0
12
3
0
37
1291
3
0
0
8
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae
Sedis;Anaerovorax;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae
Sedis;Eubacterium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae
Sedis;Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae
Sedis;Mogibacterium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae
Sedis;uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
18
0
0
0
8
0
0
0
0
1
0
2
52
0
0
1
11
0
0
3
1
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Anaerostipes;
0
6
38
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Blautia;
5
9
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Catabacter;
0
2
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Coprococcus;
0
1
41
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Epulopiscium;
662
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Howardella;
0
709
14
10
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Incertae Sedis;
1604
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Moryella;
0
0
1
2
157
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Oribacterium;
0
7
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Pseudobutyrivibrio;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Roseburia;
0
0
0
2
1
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Shuttleworthia;
0
18
32
188
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;uncultured;
553
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;livecontrolB21;
5
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;Peptococcus;
8
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;uncultured;
0
0
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Filifactor
500
191
109
3
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;
Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;
Proteocatella;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Proteiniborus;
3876
0
0
0
0
0
0
1
1781
0
0
0
995
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
28
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Anaerofilum;
0
0
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Anaerotruncus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Anaerotruncus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Faecalibacterium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Fastidiosipila
815
0
0
0
0
0
0
1
0
16
19
20
222
2
49
9
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Incertae
Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Papillibacter;
2
0
0
0
0
1
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Saccharofermentans;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Subdoligranulum;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;uncultured;
0
0
0
5
4
6
12
20
1923
1
0
0
1
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Syntrophomonadaceae;
Pelospora;
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;
Phascolarctobacterium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Selenomonas;
0
0
0
3
0
21
41
13
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;uncultured;
407
0
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella;
8
0
0
1
Bacteria;Firmicutes;Clostridia;D8A-2;
0
0
1
2
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;
Coprobacillus;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;
Incertae Sedis;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;
Solobacterium;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;
Turicibacter;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae
;uncultured;
Bacteria;Firmicutes;Mollicutes;Acholeplasmatales;Acholeplasmataceae;
Acholeplasma;
Bacteria;Firmicutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;
Mycoplasma;
Bacteria;Firmicutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;
Ureaplasma;
Bacteria;Firmicutes;Mollicutes;RF9;
0
2
0
3
1
0
0
0
0
1
0
2
0
1
0
0
0
1
0
0
13
0
0
0
656
0
0
0
15212
0
0
0
605
0
0
0
158
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;ASCC02;
0
2
0
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;boneC3G7;
0
8
0
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7;
2825
45
0
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;
Cetobacterium;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;
Fusobacterium;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;
Psychrilyobacter;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;
Streptobacillus;
Bacteria;Gemmatimonadetes;Gemmatimonadetes;AT425-EubC11 terrestrial
group;
Bacteria;Gemmatimonadetes;Gemmatimonadetes;Gemmatimonadales;
Gemmatimonadaceae;Gemmatimonas;
Bacteria;Lentisphaerae;Lentisphaeria;BS5;
1
9956
82
1
28570
0
0
0
0
4
0
0
45
3
0
0
0
0
0
1
0
1
0
0
0
0
0
6
Bacteria;Nitrospirae;Nitrospira;Nitrospirales;OPB95;
0
0
0
3
Bacteria;Planctomycetes;OM190;
0
63
541
11
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;
Caulobacteraceae;Phenylobacterium;
Bacteria;Proteobacteria;Alphaproteobacteria;DB1-14;
0
8
0
0
3
0
0
0
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;alphaI cluster;
0
0
0
2
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Beijerinckiaceae;
uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;DUNssu371;
0
5
0
0
0
0
0
2
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Nordella;
1
0
0
0
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Phyllobacteriaceae
;Mesorhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae;
Ensifer;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae;
Rhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Xanthobacteraceae
;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Maritimibacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Paracoccus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales
;Rhodobacteraceae;Phaeobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Pontibaca;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Pseudorhodobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Roseibacterium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Roseobacter clade OCT lineage;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Roseobacter clade;Roseovarius;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Roseovarius;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Seohaeicola;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Sulfitobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Tateyamaria;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Thalassococcus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
0
0
0
2
0
13
0
1
0
2
0
0
0
0
0
5
0
1
0
0
0
0
0
7
57
0
0
0
0
5
0
0
0
65
0
3
19
0
0
2
0
0
0
2
0
9
0
0
3
0
0
0
0
6
0
0
0
4
0
0
0
2
0
0
0
15
0
0
0
9
0
0
1
0
0
3
159
Rhodobacteraceae;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;
Acetobacteraceae;Roseomonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Erythrobacteraceae;Altererythrobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Erythrobacteraceae;Erythrobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Sphingomonadaceae;Blastomonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Sphingomonadaceae;Sandarakinorhabdus;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Sphingomonadaceae;Sphingobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Sphingomonadaceae;Sphingopyxis;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;
Alcaligenes;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;
Castellaniella;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;
Derxia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;
Parasutterella;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;
Pusillimonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;
Sutterella;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;
uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Cupriavidus;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Limnobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Polynucleobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Ralstonia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Acidovorax;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Albidiferax;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Aquabacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales
;Comamonadaceae;Chlorochromatium;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Delftia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Diaphorobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Hydrogenophaga;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Paucibacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Piscinibacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Polaromonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Rhodoferax;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales
;Comamonadaceae;Simplicispira;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Variovorax;
0
0
2
0
0
13
0
0
1
0
0
0
2
0
0
1
1
0
0
0
0
29
0
0
1
0
0
0
0
0
0
1
0
23
0
0
1
14
0
0
0
5
0
1
0
3
0
0
7
0
0
0
1
208
0
0
0
6
0
0
0
1
0
0
0
1
5
1
1
24
0
2
7
39
0
51
1
36
0
4
0
0
0
1
0
0
10
0
0
0
112
2
0
3
0
4
0
21
0
1
1
2
0
0
0
0
5
1
0
0
0
2
3
0
0
2
0
10
0
3
0
8
0
2
1
0
0
0
0
4
0
4
160
0
1
0
0
0
7
0
7
0
0
0
1
0
7
0
4
0
1
0
0
0
0
0
2
0
55
0
5
0
0
0
2
3
0
0
0
0
7
0
0
0
2
1
0
0
19
0
20
171
0
0
0
4
29
65
139
33
0
0
0
111
0
0
0
0
6
0
5
0
0
0
3
0
22
62
199
0
3
0
0
0
0
0
2
0
2
0
7
0
2
0
2
3
0
0
0
0
1
0
0
0
2
0
4
0
37
0
35
0
1
0
1
0
1
0
0
0
0
0
1
0
2
0
0
Bacteria;Proteobacteria;Betaproteobacteria;UCT N117;
0
2
0
1
Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales;
Bdellovibrionaceae;Bdellovibrio;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;
Desulfobacteraceae;Desulfobacterium;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;
Desulfobulbaceae;Desulfobulbus;
0
7
0
0
0
14
55
28
91
2
0
0
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Herminiimonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales
;Oxalobacteraceae;Massilia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Naxibacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Undibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;H2SRC138;
Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenophilales;
Hydrogenophilaceae;Petrobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales;
Methylophilaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Aquitalea;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Conchiformibius;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Deefgea;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Eikenella;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Laribacter;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Microvirgula;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Neisseria;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Uruburuella;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Vitreoscilla;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;
Vogesella;
Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomonadales;
Gallionellaceae;Sideroxydans;
Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomonadales;
Nitrosomonadaceae;Nitrosomonas;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Azoarcus;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Azospira;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Dechloromonas;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Methyloversatilis;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Sterolibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Uliginosibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Zoogloea;
Bacteria;Proteobacteria;Betaproteobacteria;SC-I-84;
161
0
8
0
11
0
1
0
11
1
0
0
0
0
1
0
0
3
0
0
0
0
3
0
5
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;Cystobacterineae
;uncultured;
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;JG37-AG-15;
0
1
0
0
0
0
0
1
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;Nannocystineae;
Haliangiaceae;Haliangium
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;Nannocystineae;
Nannocystaceae;Nannocystis
Bacteria;Proteobacteria;Deltaproteobacteria;Order IncertaeSedis;
Syntrophorhabdaceae;Syntrophorhabdus;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;
Campylobacteraceae;Arcobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;
Campylobacteraceae;Campylobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;
Campylobacteraceae;Sulfurospirillum;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;
Helicobacteraceae;Helicobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;
Helicobacteraceae;Sulfurimonas;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;
Helicobacteraceae;Sulfurovum;
Bacteria;Proteobacteria;Gammaproteobacteria;Acidithiobacillales;
KCM-B-112;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;
Alteromonadaceae;Glaciecola;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;
Alteromonadaceae;Haliea;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;
Alteromonadaceae;Marinobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;NB-1d;
0
0
0
3
0
0
0
2
0
1
0
5
42
17
16
31
4651
3
0
0
2
66
0
0
638
2
0
0
0
49
23
21
1
0
0
0
0
4
0
2
0
0
1
0
0
88
0
0
0
0
0
3
0
0
0
1
0
93
0
0
0
4
22
14
0
4
1
5
9
0
0
0
4
0
0
1
0
1
0
0
2
2
0
1
0
96
0
106
0
0
4
0
0
104
516
57
0
183
104
31
0
0
143
2
0
5
12
0
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;
Desulfobulbaceae;Desulfotalea;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;
Desulfobulbaceae;uncultured;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;
Desulfohalobiaceae;uncultured;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;
Desulfovibrionaceae;Desulfovibrio;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfuromonadales;
GR-WP33-58;
Bacteria;Proteobacteria;Deltaproteobacteria;GR-WP33-30;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales
;Psychromonadaceae;Psychromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;B38;
Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;
Cardiobacteriaceae;Cardiobacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;
Cardiobacteriaceae;Suttonella;
Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;
Cardiobacteriaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;
Chromatiaceae;Thiocystis;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;
Granulosicoccaceae;Granulosicoccus;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Cedecea;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Citrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Edwardsiella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Erwinia;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Hafnia-Obesumbacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Klebsiella;
162
0
0
8
0
0
0
0
1
7
0
7
0
0
3
4
2
0
5
18
56
Bacteria;Proteobacteria;Gammaproteobacteria;NKB5;
34
1
0
0
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;
Alcanivoracaceae;Alcanivorax;
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;SAR86
clade;
Bacteria;Proteobacteria;Gammaproteobacteria;Order Incertae Sedis;Family
Incertae Sedis;Marinicella;
Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae
;Aggregatibacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae
;Haemophilus;
Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae
;Pasteurella;
Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae
;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;
Moraxellaceae;Acinetobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;
Moraxellaceae;Alkanindiges;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;
Moraxellaceae;Enhydrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales
;Moraxellaceae;Psychrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;
Pseudomonadaceae;Cellvibrio;
Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;Francisellaceae;
Francisella;
Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;Piscirickettsiace
ae;Methylophaga;
Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;Thiotrichaceae;
Leucothrix;
Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae;
Photobacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae;
Vibrio;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Sinobacteraceae;Steroidobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Arenimonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Ignatzschineria;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Luteimonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Lysobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Rhodanobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Stenotrophomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales
Xanthomonadaceae;Thermomonas;
Bacteria;Spirochaetes;Spirochaetes;
0
2
0
0
0
6
0
4
0
1
0
0
0
0
36
0
0
33
0
2
0
9
3
0
0
79
153
133
0
0
0
4
0
17
2
11
0
1364
0
635
8
0
0
0
0
234
63
29
27
0
0
0
20
0
0
0
0
1
0
0
0
0
0
3
0
0
0
1
0
3
0
0
0
3
0
0
0
13
0
4
0
3
0
0
0
17
0
1
3
4
0
23
0
1
0
0
1
6
0
2
0
0
0
1
0
3
0
96
45
0
0
0
10
2
0
0
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Morganella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Rahnella;
Bacteria;Proteobacteria;Gammaproteobacteria;Legionellales;Coxiellaceae;
Aquicella;
Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;
Crenotrichaceae;Crenothrix;
Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;Hyd24-01;
Bacteria;Spirochaetes;Spirochaetes;Spirochaetales;Spirochaetaceae;
Treponema;
Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Jonquetella;
163
Bacteria;Tenericutes;Mollicutes;Anaeroplasmatales;Anaeroplasmataceae;
Anaeroplasma;
Bacteria;Tenericutes;Mollicutes;Entomoplasmatales;Candidatus
Hepatoplasma;
Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;
Mycoplasma;
Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;
Ureaplasma;
Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;
Verrucomicrobiaceae;Akkermansia;
Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;
Verrucomicrobiaceae;uncultured;
Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;
Verrucomicrobiaceae;Verrucomicrobium;
Unclassified;
0
1
0
0
0
36
109
4
0
2
0
0
0
4
0
2
0
0
0
7
0
51
57
102
0
0
0
2
4434
0
0
0
164
Appendix 2
Table A2, Taxonomic Breakdown of bacteria present in little penguins during
development (Chapter 3). A = Adult, 1 = week 1, 3 = Week 3, 5 = week 5, 7 = Week 7,
9 = Week 9.
Taxonomic group (SILVA tax, release 106)
A
1
3
5
7
9
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Actinomycineae;Actinomycetaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Corynebacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Dietziaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Family Incertae Sedis
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Mycobacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Nocardiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;Fodinicola
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;Geodermatophilaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;Nakamurellaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;Sporichthyaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;uncultured
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Bogoriellaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Brevibacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Cellulomonadaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Dermabacteraceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Microbacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Micrococcaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Propionibacterineae;Nocardioidaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Propionibacterineae;Propionibacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Streptomycineae;Streptomycetaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Bifidobacteriales;Bifidobacteriaceae;Bifidobacterium
Bacteria;Actinobacteria;Actinobacteria;Coriobacteridae;
Coriobacteriales;Coriobacterineae;Coriobacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae;
Rubrobacterales;Rubrobacterineae;Rubrobacteriaceae
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;
Bacteroides;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Barnesiella;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Petrimonas;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Porphyromonas;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Proteiniphilum;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;uncultured;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;
Prevotella;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;
uncultured;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae;
Alistipes;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7;
113
0
1
11
12
1
46
0
15
8
5
5
31
0
0
1
8
3
0
0
0
2
1
0
0
0
0
0
9
0
41
0
0
0
1
0
0
2
0
0
0
0
17
0
0
0
0
0
0
0
0
0
1
0
26
0
0
0
0
0
0
0
0
0
1
0
0
0
0
3
1
0
0
0
8
21
23
2
0
0
0
0
1
1
0
2
4
6
12
1
0
2
1
12
4
8
0
0
3
8
19
6
0
0
0
0
0
1
61
4
0
1
2
0
0
0
0
1
0
0
4
0
0
0
0
1
0
0
0
0
4
28
0
5
1
0
2
0
202
5
1
5
2
13
480
0
0
0
0
0
101
0
6
498
109
12
18
0
0
0
6
0
22
0
0
50
31
5
0
0
0
2
0
0
88
0
0
1
0
0
0
1
0
0
3
2
0
5
0
0
5
0
97
10
1
6
1
2
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cyclobacteriaceae;Echini
cola;
0
0
0
0
0
1
165
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cyclobacteriaceae;uncult
ured;
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;
Arcicella;
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;
Hymenobacter;
Bacteria;Bacteroidetes;Cytophagia;Order III Incertae Sedis;Family
Incertae Sedis;Balneola;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Cryomorphaceae;Brumimicrobium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales
;Flavobacteriaceae;Aequorivita;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Chryseobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Cloacibacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales
;Flavobacteriaceae;Flavobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Gelidibacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales
;Flavobacteriaceae;Myroides;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Ulvibacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;uncultured;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Chitinophagaceae;uncultured;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Saprospiraceae;uncultured;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;Olivibacter;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;Pedobacter;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;Sphingobacterium;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;uncultured;
Bacteria;Candidate division BRC1;
0
0
0
0
1
0
4
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
1
0
0
0
0
1
2
0
0
0
0
0
2
0
0
9
30
5
8
3
0
3
0
0
1
0
35
0
0
0
3
0
0
0
0
0
4
0
0
246
0
0
0
0
0
0
0
1
36
0
0
0
1
0
0
0
3
0
0
0
17
1
0
0
0
0
4
1
0
1
0
0
0
0
0
3
0
1
4
0
0
86
51
0
0
0
0
0
0
2
5
0
0
0
0
1
0
0
Bacteria;Candidate division OD1;
18
0
0
0
0
0
Bacteria;Candidate division OP10;
0
1
0
0
0
0
Bacteria;Candidate division SR1;
2
0
0
0
0
0
Bacteria;Candidate division TM7;
3
2
0
3
2
0
Bacteria;Chlorobi;Chlorobia;Chlorobiales;OPB56;
0
0
0
0
1
1
Bacteria;Cyanobacteria;Chloroplast;
12
0
3
0
1
2
Bacteria;Cyanobacteria;SubsectionI;Synechococcus;
0
0
0
2
3
0
Bacteria;Firmicutes;Bacilli;Bacillales;Alicyclobacillaceae;
Alicyclobacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;
0
0
0
1
0
0
0
0
0
0
2
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Alkalibacillus;
0
0
0
0
3
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Amphibacillus;
0
0
0
0
2
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus;
0
0
1
0
32
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Filobacillus;
0
0
0
0
1
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Geobacillus;
0
0
0
0
28
8
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Gracilibacillus;
0
0
0
0
1
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Halobacillus;
0
0
0
0
8
1
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Lentibacillus;
0
0
0
5
14
1
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Natronobacillus;
0
0
0
0
1
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Oceanobacillus;
0
0
0
0
7
4
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Paucisalibacillus;
0
0
0
1
44
2
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Sediminibacillus;
0
0
1
0
1
1
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;uncultured;
0
0
4
5
220
43
166
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Virgibacillus;
16
0
0
3
50
10
Bacteria;Firmicutes;Bacilli;Bacillales;Family XI Incertae Sedis;Gemella;
0
0
2
7
20
21
Bacteria;Firmicutes;Bacilli;Bacillales;Family XII Incertae
Sedis;Exiguobacterium;
Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;
Ammoniphilus;
Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Brevibacillus
0
0
0
0
16
0
0
0
0
0
0
4
0
0
0
1
0
0
Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Paenibacillu;
0
8
0
2
1
0
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;IncertaeSedis;
0
0
0
0
8
0
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
Jeotgalibacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
Lysinibacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
Planomicrobium;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Sporosarcina;
0
0
0
0
20
0
0
0
0
4
14
0
0
0
0
2
0
0
0
0
7
3
407
4
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;uncultured;
0
0
0
0
4
0
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;
Jeotgalicoccus;
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;
Macrococcus;
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;
Staphylococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae;
Dolosicoccus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Atopococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Atopostipes;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Carnobacterium;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Dolosigranulum;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
uncultured;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;
Enterococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;
Vagococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;
Lactobacillus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;
Leuconostoc;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;
Weissella;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Rs-D42;
0
0
0
0
1
0
0
4
0
0
0
0
0
126
173
9
9
0
0
0
0
1
13
8
0
0
1
0
121
8
0
0
0
0
134
8
0
5
0
0
2
0
0
2
0
3
1
0
0
0
1
13
361
64
0
310
25
8
6
7
0
0
14
1
0
0
316
44
13
5
26
48
68
332
63
279
229
42
57
270
57
76
73
20
0
0
0
0
58
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;
Lactococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;
Streptococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Alkaliphilus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Candidatus Arthromitus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Clostridiisalibacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Clostridium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Sarcina;
63
64
31
35
61
14
95
17
2
10
8
51
0
0
0
0
1
0
1
0
0
0
14
0
41
0
0
0
8
166
27
0
0
0
80
0
49
262
587
139
8
284
5
4795
1
16
0
0
2
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae
Sedis;Proteiniborus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Finegoldia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Gallicola;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
16
0
0
4
7
1
29
0
0
0
18
0
0
0
0
0
1
0
0
0
0
0
93
0
0
0
0
0
0
1
167
Sedis;Parvimonas;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Peptoniphilus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Soehngenia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Sporanaerobacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Tepidimicrobium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Tissierella;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Blautia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae
;Caldicoprobacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Coprococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Dorea;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Oribacterium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Pseudobutyrivibrio;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;
Desulfonispora;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;
Peptococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;
Filifactor;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;
Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;
Peptostreptococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
0
0
0
1
0
0
0
0
0
1
0
0
18
0
0
0
0
0
5
0
0
0
8
1
0
0
0
1
155
7
0
0
0
0
9
0
38
0
0
0
1
4
0
0
0
0
4
0
0
0
0
0
0
1
41
0
0
0
0
0
14
6
59
463
145
2319
1
0
0
0
0
0
0
0
0
0
0
2
32
14
2
20
57
3
0
0
0
0
2
0
0
0
0
1
1
0
1
0
0
0
0
0
109
37
28
398
136
322
0
0
5
0
0
2
0
0
0
0
0
2
0
0
0
15
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Anaerotruncus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Faecalibacterium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Fastidiosipila;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Ruminococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Subdoligranulum;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;
Veillonella;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;Allobaculum;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;Erysipelothrix;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;Turicibacter;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;uncultured;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7;
0
0
0
1
0
0
0
0
1
2
0
5
19
0
0
3
0
0
49
0
0
0
0
1
0
0
0
0
0
3
0
0
0
0
0
9
12
1
1
11
9
87
41
23
2
7
2
3
1
0
1
1
0
0
0
0
0
0
15
2
0
0
0
0
0
1
0
0
0
0
0
2
0
0
4
67
2
37
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;
Cetobacterium;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;
Fusobacteriaceae;Fusobacterium;
Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales;
Phycisphaeraceae;CL500-3;
0
0
15
25
1
183
82
55
6441
548
0
992
2095
4
541
105
34
470
83
23
168
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;
Caulobacteraceae;Asticcacaulis;
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;
Caulobacteraceae;Brevundimonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Beijerinckiaceae;Chelatococcus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Bradyrhizobiaceae;Bradyrhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Brucellaceae;Pseudochrobactrum;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Hyphomicrobiaceae;Devosia;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Methylobacteriaceae;Methylobacterium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Phyllobacteriaceae;Mesorhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Rhizobiaceae;Rhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Paracoccus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Pontibaca;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Rhodobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Roseovarius;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Rubellimicrobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Thalassococcus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;
KCM-B-15;
Bacteria;Proteobacteria;Alphaproteobacteria;SAR11 clade;
Chesapeake-Delaware Bay;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
GOBB3-C201;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Sphingomonadaceae;Blastomonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Sphingomonadaceae;Novosphingobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Sphingomonadaceae;Sphingobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;
Sphingomonadaceae;Sphingomonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Achromobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Alcaligenes;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Castellaniella;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Parasutterella;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Pusillimonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Cupriavidus;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Ralstonia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Acidovorax;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Aquabacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Brachymonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Comamonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Ottowia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Pelomonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;uncultured;
0
0
0
4
0
0
0
4
1
1
0
0
0
0
0
0
1
0
0
0
2
0
0
0
0
3
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
2
0
0
0
0
4
0
0
0
0
0
4
5
3
0
0
0
0
7
1
0
0
0
0
1
0
0
0
0
0
6
0
0
0
0
0
5
0
0
0
0
0
12
5
0
0
0
1
5
0
2
0
0
0
0
0
0
0
2
0
0
0
0
3
0
0
0
0
0
0
0
0
6
0
0
3
0
0
0
0
0
0
0
5
0
0
0
0
0
0
3
1
0
0
0
0
1
0
0
0
0
0
34
0
0
0
0
3
8
0
0
0
0
2
0
0
0
0
0
3
3
0
0
0
0
0
25
2
0
0
2
0
0
0
5
5
2
1
3
0
0
7
1
1
2
1
0
3
0
0
0
0
10
0
0
0
0
0
112
3
0
2
0
1
0
0
0
0
5
0
5
1
0
0
0
0
0
0
1
0
0
0
169
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Herbaspirillum;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Janthinobacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Massilia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Undibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;H2SRC138;
0
0
0
5
1
7
0
0
0
3
0
0
0
0
0
5
1
0
0
250
0
0
3
0
0
6
0
0
0
0
0
0
0
4
0
0
Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales;
Methylophilaceae;LD28 freshwater group;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Eikenella;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Microvirgula;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Neisseria;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Vitreoscilla;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;Azoarcus;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;Propionivibrio;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;uncultured;
Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales;
Bdellovibrionaceae;Bdellovibrio;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;
Desulfobulbaceae;Desulfobulbus;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfuromonadales;
GR-WP33-58;
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;0319-6G20;
0
2
0
0
0
0
1
0
0
0
0
0
0
9
2
0
4
0
65
0
0
0
0
0
62
0
0
0
0
0
0
0
1
0
1
0
0
1
0
0
0
0
0
2
0
0
0
0
0
0
0
0
2
0
0
0
3
0
0
0
55
0
0
0
0
0
0
0
0
0
0
1
0
5
2
0
0
0
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;
Campylobacteraceae;Arcobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;
Campylobacteraceae;Campylobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;
Helicobacteraceae;Sulfurovum;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;
Aeromonadaceae;Aeromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;
Aeromonadaceae;Oceanimonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;
Aeromonadaceae;Oceanisphaera;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;
Aeromonadaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;
Aeromonadaceae;Zobellella;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;
Alteromonadaceae;Gilvimarinus;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;
Alteromonadaceae;Haliea;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;
Alteromonadaceae;Marinobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;
NB-1d;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;
Psychromonadaceae;Psychromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;B38;
0
4
3
0
2
1
16
0
0
11
5
0
23
0
0
0
0
0
0
7
0
1
1
0
0
0
0
0
1
0
0
0
0
0
2
1
0
0
1
1
142
4
0
0
0
0
1
0
0
0
0
0
2
0
1
0
0
0
0
0
0
2
0
0
15
1
0
0
0
0
1
0
0
8
0
0
0
0
0
71
2584
0
0
0
Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;
Cardiobacteriaceae;Cardiobacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;
Cardiobacteriaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;
Chromatiaceae;Nitrosococcus;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;
Chromatiaceae;Rheinheimera;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;
Ectothiorhodospiraceae;Thioalkalispira;
22
0
0
2
1
0
1
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
1
0
170
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Biostraticola;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales
;Enterobacteriaceae;Cedecea;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Citrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Cronobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Dickeya;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Edwardsiella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Enterobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Erwinia;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Escherichia-Shigella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Klebsiella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales
;Enterobacteriaceae;Kluyvera;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Pantoea;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Raoultella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Salmonella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Serratia;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Tatumella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;
Enterobacteriaceae;Yersinia;
Bacteria;Proteobacteria;Gammaproteobacteria;Legionellales;
Legionellaceae;Legionella;
Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;
Hyd24-01;
Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;
Methylococcaceae;Methylomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;
Alcanivoracaceae;Alcanivorax;
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;
Halomonadaceae;Cobetia;
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;
Halomonadaceae;Halomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;
Oceanospirillaceae;Pseudospirillum;
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;
Oceanospirillaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;
Pasteurellaceae;Haemophilus;
Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;
Pasteurellaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;
Moraxellaceae;Acinetobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales
;Moraxellaceae;Alkanindiges;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;
Moraxellaceae;Enhydrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;
Moraxellaceae;Psychrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;
Pseudomonadaceae;Pseudomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Salinisphaerales;
Salinisphaeraceae;Salinisphaera;
Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;
Vibrionaceae;Photobacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;
Vibrionaceae;Vibrio;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Sinobacteraceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Ignatzschineria;
0
0
0
0
0
1
0
16
0
1
0
8
0
245
59
9
19
2
4
0
21
0
0
0
0
0
2
0
0
1
0
0
15
2
0
1858
516
10510
25
6
8
6
0
0
14
0
1
0
104
640
8506
1
2
1
143
65
0
0
0
0
12
26
0
0
0
0
8
0
0
0
0
0
0
354
18
0
0
0
0
0
0
0
0
1
0
0
2
0
0
0
0
0
48
0
0
0
0
0
0
0
0
1
7
0
0
0
0
0
4
0
0
1
0
0
18
0
0
0
1
0
0
0
0
0
3
0
0
0
0
0
1
0
0
0
0
0
5
0
0
0
0
0
1
0
0
0
0
0
4
0
36
0
0
0
0
0
3
12
0
0
0
0
153
602
8
19
16
4
0
3
0
0
0
0
2
13
2
4
0
0
0
1309
101
32
47
9
63
656
0
2
138
1
0
0
0
0
1
0
0
5
0
0
0
0
0
0
2
23
400
2
68
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
32
0
171
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Luteibacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Lysobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Rhodanobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Stenotrophomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;Thermomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;
Xanthomonadaceae;uncultured;
Bacteria;SM2F11;
0
0
15
0
0
0
0
0
0
0
3
0
0
0
0
0
2
0
0
91
1
0
2
0
0
0
0
0
1
0
0
0
0
0
6
0
0
1
0
0
0
0
Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;
Mycoplasmataceae;Mycoplasma;
Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;
Mycoplasmataceae;Ureaplasma;
Bacteria;TM6;
109
0
0
0
0
0
0
0
1
23
0
0
0
1
0
0
0
0
Bacteria;Verrucomicrobia;OPB35 soil group;
0
0
0
2
0
0
Unclassified; (this class will be removed with SILVA 108)
57
69
238
14
120
22
172
Appendix 3
Table A3.1, Taxonomic Breakdown of bacteria present in moulting little penguins
Taxonomic group (SILVA tax, release 106)
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Actinomycineae;Actinomycetaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Corynebacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Dietziaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Family Incertae Sedis
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Mycobacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Nocardiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Frankineae;Sporichthyaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Brevibacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Dermabacteraceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Microbacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Micrococcineae;Micrococcaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Propionibacterineae;Propionibacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Bifidobacteriales;
Bifidobacteriaceae;Bifidobacterium
Bacteria;Actinobacteria;Actinobacteria;Coriobacteridae;Coriobacteriales;
Coriobacterineae;Coriobacteriaceae
Bacteria;Actinobacteria;Actinobacteria;MB-A2-108;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;Bacteroides;
Early
moult
34
Late
Moult
189
244
358
7
0
16
0
2
0
5
0
0
1
21
0
23
0
9
1
17
9
9
29
3
1
2
0
2
0
54
25
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Paludibacter;
0
8
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Petrimonas;
230
414
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;
45
18
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Proteiniphilum;
122
608
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;uncultured;
1
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;
9
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae;Blvii28
wastewater-sludge group;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7;
1
0
2
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;vadinHA21;
8
58
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cyclobacteriaceae;uncultured;
1
0
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Persicitalea;
1
0
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Chryseobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Flavobacterium
;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Ornithobacterium;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Chitinophagaceae;
Ferruginibacter;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Chitinophagaceae;uncultured
;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Sphingobacteriaceae;
Sphingobacterium;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;ST-12K33;
6
9
0
1
0
1
2
0
7
0
3
0
0
2
173
Bacteria;Bacteroidetes;VC2.1 Bac22;
0
2
Bacteria;Candidate division BRC1;
0
1
Bacteria;Candidate division SR1;
0
2
Bacteria;Candidate division TM7;
19
27
Bacteria;Chloroflexi;Anaerolineae;Anaerolineales;Anaerolineaceae;uncultured;
2
39
Bacteria;Chloroflexi;Caldilineae;Caldilineales;Caldilineaceae;Caldilinea;
1
0
Bacteria;Cyanobacteria;Chloroplast;
5
0
Bacteria;Deferribacteres;Deferribacteres;Deferribacterales;SAR406 clade(Marine
group A);
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;
1
0
2
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus;
10
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Halolactibacillus;
2
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Lentibacillus;
58
32
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Oceanobacillus;
92
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Paucisalibacillus;
17
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;uncultured;
62
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Virgibacillus;
162
3
Bacteria;Firmicutes;Bacilli;Bacillales;Family XI Incertae Sedis;Gemella;
83
2
Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Ammoniphilus;
0
1
Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Paenibacillus;
3
0
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Incertae Sedis;
0
1
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Sporosarcina;
11
0
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Jeotgalicoccus;
0
3
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Nosocomiicoccus;
0
12
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Staphylococcus;
2
55
Bacteria;Firmicutes;Bacilli;Bacillales;Thermoactinomycetaceae;Thermoactinomyces;
5
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae;Dolosicoccus;
26
1
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Atopococcus;
39
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Atopostipes;
7
3
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;uncultured;
105
2
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Enterococcus;
20
9
Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus;
3
48
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Leuconostoc;
162
221
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Weissella;
117
89
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Lactococcus;
40
62
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;
52
27
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;uncultured;
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Alkaliphilus;
0
6
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridiisalibacter;
0
28
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridium;
177
24
Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae Sedis;Proteiniborus;
36
44
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Finegoldia;
96
172
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Gallicola;
0
27
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Helcococcus;
1
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Parvimonas;
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Peptoniphilus;
9
27
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Soehngenia;
1
8
174
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Sporanaerobacter;
4
41
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Tepidimicrobium;
0
17
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Tissierella;
0
6
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;uncultured;
7
15
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae Sedis;Guggenheimella;
5
24
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae Sedis;Mogibacterium;
1
3
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Anaerostipes;
3
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Blautia;
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Incertae Sedis;
9
3
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;uncultured;
99
722
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;Peptococcus;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Filifactor;
11
16
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Incertae Sedis;
83
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Faecalibacterium;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Fastidiosipila;
16
79
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcus;
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Saccharofermentans;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;uncultured;
2
40
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella;
6
6
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Allobaculum;
0
1
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Turicibacter;
1
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7;
7
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Cetobacterium;
2
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium;
817
1
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;Streptobacillus;
1
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;uncultured;
0
4
Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales;Phycisphaeraceae;CL500-3;
43
29
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Bradyrhizobiaceae;
Bradyrhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Family Incertae
Sedis;Nordella;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Hyphomicrobiaceae;Devosia;
2
0
0
1
7
3
7
0
0
1
1
0
2
0
12
0
3
0
12
0
0
2
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae;
Sphingomonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;uncultured;
0
3
1
0
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;
Burkholderia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;
Polynucleobacter;
1
8
0
3
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
Paracoccus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
Pontibaca;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
Roseibacterium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
Rubellimicrobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
Thalassococcus
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;Rhodospirillaceae;
Thalassospira;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;GOBB3-C201;
175
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;Ralstonia;
0
2
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
2
0
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
Acidovorax;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
Comamonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
uncultured
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;
Herbaspirillum;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;
Janthinobacterium
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;Massilia;
1
6
13
0
4
0
2
0
11
0
0
1
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;
Undibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Eikenella;
0
6
2
3
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Microvirgula;
0
3
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;
42
142
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Stenoxybacter;
1
0
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;uncultured;
298
805
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Vogesella;
2
0
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Azoarcus;
0
1
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Azospira;
9
0
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Denitratisoma
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;uncultured;
0
1
0
2
Bacteria;Proteobacteria;Betaproteobacteria;SC-I-84;
0
9
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfobulbaceae;
Desulfobulbus;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulfohalobiaceae;
uncultured;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;
Campylobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae;
Sulfurovum
Bacteria;Proteobacteria;Gammaproteobacteria;Acidithiobacillales;Acidithiobacillaceae;
Acidithiobacillus;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;Aeromonadaceae;
Aeromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;Aeromonadaceae;
uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Alteromonadaceae;
Marinobacter
Bacteria;Proteobacteria;Gammaproteobacteria;B38;
26
7
5
2
8
21
12
3
1
0
0
1
4
1
1
0
2
0
6
12
0
2
1
0
1
0
22
22
1
0
100
34
135
54
25
9
Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;Cardiobacteriaceae;
Cardiobacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;Cardiobacteriaceae
;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;Chromatiaceae;
Rheinheimera;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Citrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Cronobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Enterobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Escherichia-Shigella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Klebsiella;
176
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Kluyvera;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Pantoea;
Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;Hyd24-01;
3
0
2
0
0
1
Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;Methylococcaceae;
Methylomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;Halomonadaceae;
Cobetia;
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;Halomonadaceae;
Halomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Pasteurella;
4
17
3
0
13
0
6
0
Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;uncultured;
1
0
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae;
Acinetobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae;
Psychrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae;
uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae;
Pseudomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;Thiotrichaceae;Thiothrix;
43
39
9
0
0
3
4
14
0
1
Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae;Vibrio;
49
7
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae;
uncultured;
Bacteria;Spirochaetes;Spirochaetes;Spirochaetales;Spirochaetaceae;Treponema;
7
0
27
83
Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Jonquetella;
0
15
Bacteria;Tenericutes;Mollicutes;Acholeplasmatales;Acholeplasmataceae;Acholeplasma;
2
0
Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;Mycoplasma;
273
287
Bacteria;Tenericutes;Mollicutes;RF9;
6
30
Unclassified;
315
467
177
Appendix 3
Table A3.2, Taxonomic Breakdown of bacteria presents in moulting king penguins
(Chapter 4).
Bacteria;Acidobacteria;Acidobacteria;SJA-149;
Early
Moult
6
Late
Moult
0
Bacteria;Acidobacteria;Holophagae;iii1-8;
2
0
Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;Acidimicrobiales;
Acidimicrobineae;Acidimicrobiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Actinomycineae;Actinomycetaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Corynebacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Dietziaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Corynebacterineae;Nocardiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Frankineae;
Frankiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Frankineae;
uncultured
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae
;Dermatophilaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae
;Intrasporangiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae
;Microbacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae
;Micrococcaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae
;Promicromonosporaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;
Propionibacterineae;Propionibacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Coriobacteridae;Coriobacteriales;Coriobacterineae;
Coriobacteriaceae
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;Bacteroides;
3
2
115
0
225
0
4
0
6
0
5
0
1
0
5
3
3
0
11
1
6
0
3
0
29
0
19
0
1
2
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Marinilabiaceae;Marinifilum;
4
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Marinilabiaceae;uncultured;
0
1
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Paludibacter;
0
2
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Petrimonas;
1367
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;
72
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Proteiniphilum;
296
230
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;uncultured;
4
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;
0
3
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;uncultured;
2
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7;
1
0
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;vadinHA21;
3
0
Bacteria;Bacteroidetes;BD2-2;
8
0
Bacteria;Bacteroidetes;Class Incertae Sedis;Order Incertae Sedis;Family Incertae
Sedis;Prolixibacter;
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cyclobacteriaceae;Algoriphagus;
0
1
6
2
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Flexibacter;
2
0
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Leadbetterella;
5
0
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;Brumimicrobium;
0
1
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;Cryomorpha;
0
2
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Cryomorphaceae;Owenweeksia;
2
0
Taxonomic group (SILVA tax, release 106)
178
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
3
0
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Aequorivita;
0
6
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;
Chryseobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Coenonia;
123
79
985
0
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Flavobacterium;
90
40
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Gelidibacter;
1
23
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Mariniflexile;
4
0
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Ornithobacterium
6
1
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Psychroserpens;
1
0
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;Flavobacteriaceae;Sufflavibacter;
2
0
Bacteria;Bacteroidetes;SB-1;
4
3
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Chitinophagaceae;
Ferruginibacter;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Chitinophagaceae;uncultured;
3
1
15
4
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;env.OPS 17;
7
0
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;KD1-131;
11
15
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;Sphingobacteriaceae;
Pedobacter
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;WCHB1-69;
9
0
16
25
Bacteria;Bacteroidetes;vadinHA17;
0
1
Bacteria;Bacteroidetes;VC2.1 Bac22;
2
0
Bacteria;Bacteroidetes;WCHB1-32;
0
1
Bacteria;BD1-5;
18
1
Bacteria;Candidate division OD1;
9
2
Bacteria;Candidate division SR1;
29
5
Bacteria;Candidate division TM7;
38
1
Bacteria;Chlorobi;Chlorobia;Chlorobiales;SJA-28;
0
2
Bacteria;Chloroflexi;Anaerolineae;Anaerolineales;Anaerolineaceae;uncultured;
1
0
Bacteria;Cyanobacteria;Chloroplast;
16
0
Bacteria;Cyanobacteria;SubsectionIII;
13
0
Bacteria;Cyanobacteria;SubsectionIII;Geitlerinema;
9
0
Bacteria;Cyanobacteria;SubsectionIII;Leptolyngbya;
26
0
Bacteria;Cyanobacteria;SubsectionIII;Phormidium;
9
0
Bacteria;Cyanobacteria;SubsectionIV;Anabaena;
11
0
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus;
10
0
Bacteria;Firmicutes;Bacilli;Bacillales;Family XII Incertae Sedis;Exiguobacterium;
0
1
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Planomicrobium;
1
0
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Sporosarcina;
16
1
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Trichococcus;
5
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Enterococcus;
15
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus;
310
2
Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Pediococcus;
4
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Leuconostoc;
1966
1
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;Weissella;
932
1
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Lactococcus;
842
2
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;
187
7
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;uncultured;
6
0
179
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Alkaliphilus;
5
3
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Caloranaerobacter;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridiisalibacter;
120
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Clostridium;
117
1718
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;Proteiniclasticum;
6
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae Sedis;Proteiniborus;
16
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;
0
16
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Finegoldia;
358
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Gallicola;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Parvimonas;
5
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Peptoniphilus;
32
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Soehngenia;
35
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Sporanaerobacter;
15
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Tepidimicrobium;
5
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;Tissierella;
83
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae Sedis;uncultured;
14
47
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae Sedis;Fusibacter;
2
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae Sedis;Guggenheimella;
6
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae Sedis;Anaerovorax;
0
1
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae Sedis;Incertae Sedis;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIV Incertae Sedis;Anaerobranca;
19
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Blautia;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Epulopiscium;
0
15
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;IncertaeSedis;
152
275
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;uncultured;
147
40
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;Peptococcus;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;IncertaeSedis;
9
122
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Proteocatella;
4
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
0
2
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Anaerotruncus;
0
4
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Fastidiosipila;
31
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Incertae Sedis;
8
63
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcus;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Saccharofermentans;
1
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;uncultured;
46
77
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella;
38
0
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Erysipelothrix;
0
1
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Incertae Sedis;
0
1
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;Erysipelotrichaceae;Turicibacter;
0
1
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;boneC3G7;
0
1
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7;
0
19
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Cetobacterium;
1
32
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Fusobacteriaceae;Fusobacterium;
137
12546
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;Leptotrichia;
1
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;Streptobacillus;
1
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;Leptotrichiaceae;uncultured;
6
0
180
Bacteria;Lentisphaerae;Lentisphaeria;BS5;
0
1
Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales;Phycisphaeraceae;CL500-3;
101
0
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;Caulobacteraceae;
Brevundimonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;Caulobacteraceae;uncultured
6
0
3
0
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Methylobacteriaceae;
Methylobacterium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Methylocystaceae;
Pleomorphomonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;Rhizobiaceae;Rhizobium;
1
0
3
0
17
0
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
5
0
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
Paracoccus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
Pseudorhodobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
Rhodobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;
uncultured
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;Acetobacteraceae;
Acetobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;AKIW852;
9
0
0
2
7
0
10
1
2
0
8
0
16
0
7
1
2
0
4
0
78
0
2
0
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae;
Sandarakinorhabdus;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae;
Sphingomonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphingomonadaceae;
Sphingopyxis;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Alcaligenaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;
Burkholderia
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;Ralstonia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
1
4
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Acidovorax
30
38
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Albidiferax
14
58
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
Chlorochromatium;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
Comamonas
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
Diaphorobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
Hydrogenophaga;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Malikia;
13
0
14
0
18
0
34
11
0
4
18
1
1
0
13
7
0
1
1
0
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
Simplicispira;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;uncultured;
2
2
83
3
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Variovorax
3
6
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;
Herminiimonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;
Janthinobacterium;
1
0
5
0
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Paucibacter
;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Pelomonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
Polaromonas
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;
Rhizobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Rhodoferax
181
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;Massilia;
1
0
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;Naxibacter;
1
0
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;uncultured;
4
0
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Oxalobacteraceae;
Undibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenophilales;Hydrogenophilaceae;
Thiobacillus;
Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenophilales;Hydrogenophilaceae;
uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales;Methylophilaceae;uncultured;
4
0
0
2
0
2
0
2
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Microvirgula;
27
0
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;
30
0
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;uncultured;
64
0
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Vitreoscilla;
13
0
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Vogesella;
3
0
Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomonadales;Gallionellaceae;
Sideroxydans
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
1
0
1
0
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Azospira;
5
1
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Dechloromonas
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Propionivibrio
0
3
0
1
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Sterolibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;Thauera;
Thauera
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;
Uliginosibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;Rhodocyclaceae;uncultured;
5
0
0
2
1
0
1
1
Bacteria;Proteobacteria;Betaproteobacteria;UCT N117;
0
1
Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales;Bacteriovoraceae;
Bacteriovorax;
Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales;Bdellovibrionaceae;
Bdellovibrio;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfobacteraceae;
Desulfobacterium;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfobulbaceae;
Desulfobulbus;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfobulbaceae;
uncultured
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulfohalobiaceae;
uncultured;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulfovibrionaceae;
Desulfovibrio;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfuromonadales;GR-WP33-58;
0
1
6
0
0
4
84
1
0
1
7
0
9
0
2
0
11
0
0
1
7
0
1725
0
9
0
1226
600
0
1
47
0
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;Nannocystineae;Haliangiaceae;
Haliangium
Bacteria;Proteobacteria;Deltaproteobacteria;Order IncertaeSedis;Syntrophorhabdaceae;
Syntrophorhabdus;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;
Arcobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;
Campylobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;
Sulfurospirillum;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae;
Helicobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae;
Sulfurimonas;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae;
Sulfurovum;
182
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Helicobacteraceae;
Wolinella;
Bacteria;Proteobacteria;Gammaproteobacteria;Acidithiobacillales;Acidithiobacillaceae;
Acidithiobacillus;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;Aeromonadaceae;
Aeromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;Aeromonadaceae;
Oceanimonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Pseudoalteromonadaceae;
Pseudoalteromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Psychromonadaceae;
Psychromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;Chromatiaceae;Thiocystis;
0
1
5
0
4
0
0
1
0
7
10
0
0
1
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
1
0
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Citrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Enterobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Escherichia-Shigella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Klebsiella;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Kluyvera;
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;
Serratia;
Bacteria;Proteobacteria;Gammaproteobacteria;Methylococcales;Methylococcaceae;
Methylomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Oceanospirillales;SAR86 clade;
103
0
96
0
15
51
20
0
62
0
9
0
26
0
5
0
Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Pasteurella;
10
0
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae;
Acinetobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae;
Alkanindiges;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae;
Enhydrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae;
Psychrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Moraxellaceae;
uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae;
Pseudomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae;Vibrio;
263
0
1
0
34
0
778
140
2
0
19
202
0
4
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Sinobacteraceae;JTB255
marine benthic group;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae;
1
0
8
0
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae;
Arenimonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae;
Dokdonella;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae;
Lysobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae;
Rhodanobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae;
Silanimonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae;
Thermomonas;
Bacteria;Spirochaetes;Spirochaetes;
8
0
4
3
0
1
5
4
0
2
0
3
0
1
Bacteria;Spirochaetes;Spirochaetes;Spirochaetales;Spirochaetaceae;Treponema;
41
0
Bacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Jonquetella;
3
0
Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;Mycoplasma;
24
6
Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;Ureaplasma;
18
10
183
Bacteria;Tenericutes;Mollicutes;RF9;
7
1
Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;Verrucomicrobiaceae;
uncultured;
Unclassified;
8
0
338
20
184
Appendix 4
Table A4, Taxonomic Breakdown of bacteria present in Procellariiform Seabirds.
CDP = Common diving Petrel, FP = Fairy Prion, STS= Short-tailed Shearwater
Taxonomic group (SILVA tax, release 106)
CDP
FP
STS
Bacteria;Acidobacteria;Acidobacteria;11-24;
4
0
0
Bacteria;Acidobacteria;Acidobacteria;Candidatus Chloracidobacterium;
3
0
0
Bacteria;Acidobacteria;Acidobacteria;Candidatus Solibacter;
1
0
0
Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;
Acidimicrobiales;Acidimicrobineae;OCS155 marine group
Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;
Acidimicrobiales;Acidimicrobineae;Sva0996 marine group
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Actinomycineae;Actinomycetaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Corynebacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Mycobacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Nocardiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;uncultured
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;Geodermatophilaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;Sporichthyaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;uncultured
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Kineosporiineae;Kineosporiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;AKAU3644
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Brevibacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Cellulomonadaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Dermabacteraceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Dermacoccaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Intrasporangiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Microbacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Micrococcaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Sanguibacteraceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micromonosporineae;Micromonosporaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Propionibacterineae;Nocardioidaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Propionibacterineae;Propionibacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Pseudonocardineae;Pseudonocardiaceae
0
0
7
1
0
0
208
13
55
342
24
44
13
0
0
5
0
0
7
0
1
0
19
7
5
0
0
0
8
0
1
0
13
4
0
0
6
0
0
1
0
0
3
0
0
2
0
0
5
0
0
7
0
26
1
34
21
1
0
0
2
0
0
3
0
0
39
36
60
0
1
0
185
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Streptomycineae;Streptomycetaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Streptosporangineae;Thermomonosporaceae
Bacteria;Actinobacteria;Actinobacteria;Coriobacteridae;
Coriobacteriales;Coriobacterineae;Coriobacteriaceae
Bacteria;Actinobacteria;Actinobacteria;OPB41;
Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae;
Rubrobacterales;Rubrobacterineae;Rubrobacteriaceae
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;Bacte
roides;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Barnesiella;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Odoribacter;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Petrimonas;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Porphyromonas;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Proteiniphilum;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;uncultured;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Prevotellaceae;Prevotella;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Prevotellaceae;uncultured;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;RF16;
2
0
0
2
0
0
8
24
0
1
0
0
1
10
0
265
10
19
488
0
0
4
0
0
570
0
0
4
0
18
315
0
0
1
0
0
409
0
13
1
0
0
96
0
0
368
4
12
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae;Alistipe
s;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Rikenellaceae;RC9 gut
group;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7;
2
0
0
17
74
74
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;vadinHA21;
30
0
0
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Adhae
ribacter;
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Arcice
lla;
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Flexib
acter;
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;Cytophagaceae;Hyme
nobacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Cryomorphaceae;Brumimicrobium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Cryomorphaceae;Fluviicola;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Cryomorphaceae;Owenweeksia;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Chryseobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Cloacibacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Coenonia;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales
;Flavobacteriaceae;Flavobacterium;
0
17
0
1
0
0
1
0
0
1
0
0
0
1
0
2
0
0
0
20
0
16
51
139
0
0
3
589
0
0
7
6
13
186
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Gelidibacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Krokinobacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Ornithobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Persicivirga;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;uncultured;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Wautersiella;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;NS9 marine
group;
Bacteria;Bacteroidetes;SB-1;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Chitinophagaceae;uncultured;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
env.OPS 17;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Saprospiraceae;Lewinella;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
ST-12K33;
Bacteria;Bacteroidetes;VC2.1 Bac22;
2
0
0
3
0
0
3
0
1
1
0
0
1
0
2
3
0
0
2
0
0
0
0
1
11
0
0
2
0
0
1
0
0
2
0
0
17
0
12
Bacteria;BD1-5;
2
0
0
Bacteria;Candidate division OP10;
1
0
0
Bacteria;Candidate division SR1;
3
0
0
Bacteria;Candidate division TM7;
37
0
3
Bacteria;Chlorobi;Chlorobia;Chlorobiales;OPB56;
4
0
0
Bacteria;Chloroflexi;Anaerolineae;Anaerolineales;
Anaerolineaceae;uncultured;
Bacteria;Chloroflexi;Caldilineae;Caldilineales;Caldilineaceae;
Caldilinea;
Bacteria;Chloroflexi;Thermomicrobia;JG30-KF-CM45;
6
0
0
0
7
0
2
0
0
Bacteria;Cyanobacteria;Chloroplast;
8
0
17
Bacteria;Cyanobacteria;SubsectionI;
1
0
0
Bacteria;Cyanobacteria;SubsectionI;Prochlorococcus;
0
8
0
Bacteria;Cyanobacteria;SubsectionI;Synechococcus;
0
7
10
Bacteria;Cyanobacteria;SubsectionII;SubgroupII;Pleurocapsa;
2
0
0
Bacteria;Deferribacteres;Deferribacteres;Deferribacterales;
Deferribacteraceae;Mucispirillum;
Bacteria;DeinococcusThermus;Deinococci;Deinococcales;Deinococcaceae;
Deinococcus;
Bacteria;Deinococcus-Thermus;Deinococci;Deinococcales;
Trueperaceae;Truepera;
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus;
0
0
1
4
0
0
1
0
0
0
0
40
0
0
1
0
0
20
1
0
0
6
0
0
Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae
;Paenibacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;
Saccharibacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Bhargavaea;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
Lysinibacillus;
187
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
Planomicrobium;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
Sporosarcina;
Bacteria;Firmicutes;Bacilli;Bacillales;Sporolactobacillaceae;
Sporolactobacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;
Macrococcus;
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;
Staphylococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae;
Aerococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Atopococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Atopostipes;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;
Catellicoccus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;
Enterococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;
Lactobacillus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;
Leuconostoc;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;
Weissella;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;
Lactococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;
Streptococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Alkaliphilus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Clostridiisalibacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Clostridium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Sarcina;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae
Sedis;Proteiniborus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Finegoldia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Peptoniphilus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Sporanaerobacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Tepidimicrobium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XII Incertae
Sedis;Guggenheimella;
0
0
19
4
0
0
0
26
0
0
19
0
2
0
36
0
0
11
0
0
2
2
0
0
0
0
5
0
72
0
4
13
320
17
51
117
92
5337
7278
88
47
6277
23
1069
3249
6
227
644
0
0
37
1
0
0
18
0
0
70
478
31
1
0
0
32
0
0
64
0
0
1
0
0
5
0
0
8
0
0
1
0
0
1
8
0
11
0
0
188
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae
Sedis;Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae
Sedis;Mogibacterium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII Incertae
Sedis;uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIV Incertae
Sedis;Anaerobranca;
Bacteria;Firmicutes;Clostridia;Clostridiales;Gracilibacteraceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Anaerostipes;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Blautia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Butyrivibrio;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Catabacter;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Coprococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Dorea;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Lachnospira;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Marvinbryantia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Oribacterium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Pseudobutyrivibrio;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Roseburia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;
Peptococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Peptococcaceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;
Peptostreptococcaceae;Filifactor;
Bacteria;Firmicutes;Clostridia;Clostridiales;
Peptostreptococcaceae;Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;
Peptostreptococcaceae;Peptostreptococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;
Peptostreptococcaceae;uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Anaerotruncus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Faecalibacterium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Fastidiosipila;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
4
0
0
1
0
0
10
0
0
1
0
0
3
0
0
22
0
0
183
0
7
0
0
6
1
0
0
21
0
0
40
0
0
425
24
6
17
0
0
15
0
6
1
0
0
176
0
1
63
0
0
547
4
40
2
0
0
4
0
0
9
0
0
48
171
25
13
0
0
1
0
7
58
0
0
962
0
0
7
0
4
72
0
2
57
0
9
189
Ruminococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Subdoligranulum;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;
Anaerospora;
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;
Dialister;
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;
Veillonella;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;Holdemania;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;Incertae Sedis;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;Turicibacter;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;uncultured;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;
Fusobacteriaceae;Cetobacterium;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;
Fusobacteriaceae;Fusobacterium;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;
Fusobacteriaceae;Propionigenium;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;
Fusobacteriaceae;Psychrilyobacter;
Bacteria;Fusobacteria;Fusobacteria;SHA-35;
Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales;
Phycisphaeraceae;CL500-3;
Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales;
Phycisphaeraceae;SM1A02;
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;
Caulobacteraceae;Brevundimonas;
Bacteria;Proteobacteria;Alphaproteobacteria;DB1-14;
Bacteria;Proteobacteria;Alphaproteobacteria;
Rhizobiales;alphaI cluster;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Bradyrhizobiaceae;Nitrobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Bradyrhizobiaceae;Rhodopseudomonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
DUNssu371;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Family Incertae Sedis;Nordella;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Hyphomicrobiaceae;Devosia;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Methylobacteriaceae;Methylobacterium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Phyllobacteriaceae;Aquamicrobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Rhizobiaceae;Rhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Loktanella;
243
0
0
497
2
27
0
0
1
43
0
0
4
44
115
2
0
0
5
0
0
0
13
0
46
0
3
2
0
0
0
8
3
518
97
60
1
0
0
3
0
0
0
6
0
38
268
525
1
0
0
6
10
17
1
0
0
1
0
0
0
8
0
0
2
0
47
0
0
1
0
0
1
0
0
9
13
120
0
0
18
2
0
14
1
0
0
190
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Maritimibacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Paracoccus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Pseudorhodobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Roseovarius;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Seohaeicola;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Tateyamaria;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Thalassococcus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;
Acetobacteraceae;Acidiphilium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;
Acetobacteraceae;Roseomonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;
Rhodospirillaceae;Thalassospira;
Bacteria;Proteobacteria;Alphaproteobacteria;Rickettsiales;
SM2D12;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Erythr
obacteraceae;Altererythrobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Erythr
obacteraceae;Porphyrobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;GOBB
3-C201;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphin
gomonadaceae;Novosphingobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphin
gomonadaceae;Sphingobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphin
gomonadaceae;Sphingomonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Sphin
gomonadaceae;Sphingopyxis;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Achromobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Parasutterella;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Sutterella;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Burkholderia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales
;Burkholderiaceae;Cupriavidus;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Polynucleobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Acidovorax;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Comamonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
1
0
0
15
0
0
0
14
0
2
0
0
1
0
0
1
0
0
3
0
0
9
0
5
1
0
0
1
0
0
0
0
8
1
0
0
7
0
0
2
0
0
2
0
0
4
0
0
2
0
0
2
0
43
0
0
42
3
0
0
1
0
0
177
0
0
2
0
0
0
0
161
0
0
5
2
0
0
1
0
147
29
0
1
0
1
0
191
Comamonadaceae;Delftia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Diaphorobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Hydrogenophaga;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Paucibacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Pelomonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Piscinibacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Rhizobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Variovorax;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Herminiimonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Janthinobacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Massilia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Undibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenophilales;Hydrog
enophilaceae;Thiobacillus;
Bacteria;Proteobacteria;Betaproteobacteria;Hydrogenophilales;Hydrog
enophilaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales;
Methylophilaceae;Methylophilus;
Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales;
Methylophilaceae;Methylotenera;
Bacteria;Proteobacteria;Betaproteobacteria;Methylophilales;
Methylophilaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Conchiformibius;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Microvirgula;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Neisseria;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Vitreoscilla;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Vogesella;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;Azoarcus;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;Azospira;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;Denitratisoma;
1
8
21
1
9
0
0
0
1
1
0
0
3
0
0
2
0
0
13
6
1
2
18
0
0
17
0
0
0
6
12
12
15
0
36
62
1
18
0
1
61
16
3
0
0
2
0
0
12
0
0
4
0
0
28
0
0
1
0
0
1
22
75
345
0
0
160
0
0
0
20
17
3
9
7
2
0
0
1
0
0
1
0
0
192
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;Methyloversatilis;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;UCT N117;
Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales;
Bacteriovoraceae;Bacteriovorax;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfo
bulbaceae;Desulfobulbus;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfobacterales;Desulfo
bulbaceae;Desulfotalea;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulf
ohalobiaceae;uncultured;
Bacteria;Proteobacteria;Deltaproteobacteria;Desulfovibrionales;Desulf
ovibrionaceae;Bilophila;
Bacteria;Proteobacteria;Deltaproteobacteria;
Desulfuromonadales;M113;
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;
0319-6G20;
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;
Cystobacterineae;uncultured;
Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Cam
pylobacteraceae;Arcobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;
Campylobacterales;Campylobacteraceae;Campylobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;
Campylobacterales;Campylobacteraceae;Sulfurospirillum;
Bacteria;Proteobacteria;Epsilonproteobacteria;
Campylobacterales;Helicobacteraceae;Helicobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;
Campylobacterales;Helicobacteraceae;Sulfurovum;
Bacteria;Proteobacteria;Gammaproteobacteria;
Acidithiobacillales;Acidithiobacillaceae;Acidithiobacillus;
Bacteria;Proteobacteria;Gammaproteobacteria;
Acidithiobacillales;KCM-B-112;
Bacteria;Proteobacteria;Gammaproteobacteria;
Aeromonadales;Aeromonadaceae;Aeromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Alteromonadales;Alteromonadaceae;Alteromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Alteromonadales;Alteromonadaceae;BD1-7 clade;
Bacteria;Proteobacteria;Gammaproteobacteria;
Alteromonadales;Alteromonadaceae;Glaciecola;
Bacteria;Proteobacteria;Gammaproteobacteria;
Alteromonadales;Alteromonadaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Pseu
doalteromonadaceae;Pseudoalteromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Alteromonadales;Psych
romonadaceae;Psychromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;B38;
Bacteria;Proteobacteria;Gammaproteobacteria;
Cardiobacteriales;Cardiobacteriaceae;Cardiobacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;
Chromatiaceae;Rheinheimera;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;
Bacteria;Proteobacteria;Gammaproteobacteria;
1
0
0
1
0
0
0
2
0
0
0
4
22
0
0
2
0
0
9
0
0
12
0
0
0
0
5
2
0
0
3
0
0
1
0
38
23
616
2
2
0
22
0
0
79
17
0
0
12
0
0
1
0
0
2
9
16
6
0
0
2
0
0
1
0
0
1
0
0
1
0
0
2
0
0
0
0
1
107
0
0
1
0
0
0
0
1
0
0
1
193
Enterobacteriales;Enterobacteriaceae;Cedecea;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Citrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Edwardsiella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Enterobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Escherichia-Shigella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Klebsiella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Kluyvera;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Morganella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Pantoea;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Pectobacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Plesiomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Rahnella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Raoultella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Methylococcales;Hyd24-01;
Bacteria;Proteobacteria;Gammaproteobacteria;
Methylococcales;Methylococcaceae;Methylomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;NKB5;
Bacteria;Proteobacteria;Gammaproteobacteria;
Oceanospirillales;Halomonadaceae;Cobetia;
Bacteria;Proteobacteria;Gammaproteobacteria;
Oceanospirillales;Halomonadaceae;Halomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Oceanospirillales;Halomonadaceae;Kushneria;
Bacteria;Proteobacteria;Gammaproteobacteria;
Oceanospirillales;Oceanospirillaceae;Marinomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pasteurellales;Pasteurellaceae;Haemophilus;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pasteurellales;Pasteurellaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pseudomonadales;Moraxellaceae;Acinetobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pseudomonadales;Moraxellaceae;Enhydrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pseudomonadales;Moraxellaceae;Psychrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria
Pseudomonadales;Moraxellaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria
;Pseudomonadales;Pseudomonadaceae;Cellvibrio;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pseudomonadales;Pseudomonadaceae;Pseudomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Salinisphaerales;Salinisphaeraceae;Salinisphaera;
Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;
25
139
749
18
0
0
8
81
388
71
0
79
0
4
29
0
25
80
0
0
1
0
0
49
0
0
66
4
0
0
0
14
0
0
8
21
11
0
0
6
0
12
1
0
0
4
0
0
1
0
0
8
0
0
7
0
0
1
0
0
381
0
0
56
218
1367
5
11
175
173
0
23
2
0
0
1
0
0
20
56
130
1
0
0
6
0
0
194
Piscirickettsiaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Thiotrichales;
Thiotrichaceae;Leucothrix;
Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;
Vibrionaceae;Photobacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;
Vibrionaceae;Vibrio;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Sinobacteraceae;JTB255 marine benthic group;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Dokdonella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Dyella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Frateuria;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Rhodanobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Stenotrophomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Thermomonas;
Bacteria;Spirochaetes;Spirochaetes;Spirochaetales;
Spirochaetaceae;Treponema;
Bacteria;Synergistetes;Synergistia;Synergistales;
Synergistaceae;uncultured;
Bacteria;Tenericutes;Mollicutes;Acholeplasmatales;
Acholeplasmataceae;Acholeplasma;
Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;
Mycoplasmataceae;Mycoplasma;
Bacteria;Tenericutes;Mollicutes;RF9;
2
0
0
0
15
0
8
0
0
6
0
0
2
0
0
2
0
2
3
0
0
21
0
10
158
0
24
1
0
0
36
0
0
1
0
0
1
0
0
22
0
0
6
0
0
Bacteria;Verrucomicrobia;OPB35 soil group;
1
0
0
Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;Verruc
omicrobiaceae;Prosthecobacter;
Bacteria;Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;Verruc
omicrobiaceae;Verrucomicrobium;
Unclassified
0
0
25
1
0
0
135
37
260
195
Appendix 5
Table A5, Taxonomic Breakdown of bacteria present in short-tailed shearwaters during
development.
A = Adult, 2 = Week 2, 5 = Week 5, 8 = Week 8, 11 = Week 11 and 14 = Week 4.
Taxonomic group (SILVA tax, release 106)
A
2
5
8
11
14
Bacteria;Acidobacteria;Acidobacteria;Acidobacteriales;
Acidobacteriaceae;Edaphobacter;
Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;
Acidimicrobiales;Acidimicrobineae;Iamiaceae
Bacteria;Actinobacteria;Actinobacteria;Acidimicrobidae;
Acidimicrobiales;Acidimicrobineae;OCS155 marine group
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Actinomycineae;Actinomycetaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Corynebacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Dietziaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;Mycobacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae
;Actinomycetales;Corynebacterineae;Nocardiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Corynebacterineae;uncultured
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;Frankiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;Geodermatophilaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;Sporichthyaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Frankineae;uncultured
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Kineosporiineae;Kineosporiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Bogoriellaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Brevibacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Dermabacteraceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Dermacoccaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Intrasporangiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Microbacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Micrococcaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Promicromonosporaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Micrococcineae;Ruaniaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae
;Actinomycetales;Propionibacterineae;Nocardioidaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Propionibacterineae;Propionibacteriaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Pseudonocardineae;Pseudonocardiaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae;
Actinomycetales;Streptomycineae;Streptomycetaceae
Bacteria;Actinobacteria;Actinobacteria;Actinobacteridae
;Bifidobacteriales;Bifidobacteriaceae;Bifidobacterium
Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae;
Solirubrobacterales;480-2;
Bacteria;Actinobacteria;Actinobacteria;Rubrobacteridae;
Solirubrobacterales;Solirubrobacteriaceae;Solirubrobacter
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Bacteroidaceae;Bacteroides;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Marinilabiaceae;Marinifilum;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Paludibacter;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales
Porphyromonadaceae;Petrimonas;
0
0
2
0
0
0
0
0
0
19
0
0
7
0
0
0
0
0
55
8
21
5
85
44
44
43
107
141
661
87
0
0
0
7
0
8
0
0
0
0
16
0
0
3
6
15
12
6
1
0
1
11
13
0
0
0
0
0
0
1
7
0
0
0
0
0
0
0
3
0
0
7
0
0
0
1
0
1
13
0
0
0
0
0
0
0
0
0
1
0
0
24
10
74
95
16
0
14
7
50
0
6
0
0
0
13
0
0
0
13
4
17
21
0
26
17
6
11
17
0
21
0
2
33
0
2
0
0
0
0
7
0
0
0
0
0
1
0
0
0
9
39
0
3
60
18
6
5
5
15
0
7
2
0
0
0
0
1
4
0
0
0
0
0
0
7
0
0
0
0
0
0
38
0
0
0
0
3
0
0
19
0
2
0
0
7
0
0
0
0
1
0
0
0
0
0
0
5
0
13
4
0
17
25
196
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Porphyromonas;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Porphyromonadaceae;Proteiniphilum;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Prevotellaceae;Prevotella;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Prevotellaceae;uncultured;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;
Rikenellaceae;Alistipes;
Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;S24-7;
18
6
0
0
2
0
0
1
1
11
6
7
13
0
4
0
25
12
0
15
0
0
3
6
12
1
0
4
0
4
74
28
0
5
41
0
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;
Cytophagaceae;Dyadobacter;
Bacteria;Bacteroidetes;Cytophagia;Cytophagales;
Cytophagaceae;Fibrella;
Bacteria;Bacteroidetes;Cytophagia;Cytophagales
;Cytophagaceae;Pontibacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Cryomorphaceae;Fluviicola;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Cryomorphaceae;Owenweeksia;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Aequorivita;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Bergeyella;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Bizionia;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Chryseobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Cloacibacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Coenonia;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Flavobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Gelidibacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Kriegella;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Ornithobacterium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Salinimicrobium;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;uncultured;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Vitellibacter;
Bacteria;Bacteroidetes;Flavobacteria;Flavobacteriales;
Flavobacteriaceae;Wautersiella;
Bacteria;Bacteroidetes;SB-1;
0
0
0
0
0
3
0
0
0
1
0
0
0
0
0
0
0
1
0
0
2
0
0
0
0
0
3
8
0
0
0
0
0
40
19
8
0
0
0
0
1
0
0
0
0
14
0
0
139
67
12
68
68
46
3
0
0
0
0
0
0
16
841
237
1218
17
13
0
16
9
10
2
0
0
11
70
0
0
0
0
0
2
0
0
1
7
4
7
16
0
0
0
0
36
0
0
2
0
3
3
0
0
0
0
6
19
0
0
0
0
3
0
0
0
1
0
0
0
0
0
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Chitinophagaceae;Sediminibacterium;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
KD1-131;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;Parapedobacter;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;Pedobacter;
Bacteria;Bacteroidetes;Sphingobacteria;Sphingobacteriales;
Sphingobacteriaceae;Sphingobacterium;
Bacteria;Bacteroidetes;VC2.1 Bac22;
0
22
2
12
27
0
0
0
0
0
35
0
0
0
0
7
0
0
0
0
9
19
0
13
0
0
0
4
0
0
12
0
0
0
0
0
Bacteria;BD1-5;
0
0
5
0
0
0
Bacteria;Candidate division BRC1;
0
0
0
2
0
0
Bacteria;Candidate division OD1;
0
0
3
4
3
0
Bacteria;Candidate division OP10;
0
0
0
3
0
0
Bacteria;Candidate division OP11;
0
0
0
0
0
2
Bacteria;Candidate division TM7;
3
31
0
23
0
22
Bacteria;Chloroflexi;KD4-96;
0
1
0
0
0
0
Bacteria;Cyanobacteria;Chloroplast;
17
42
16
0
20
0
197
Bacteria;Cyanobacteria;SM2F09;
0
0
0
2
0
0
Bacteria;Cyanobacteria;SubsectionI;Prochlorococcus;
0
3
0
0
0
0
Bacteria;Cyanobacteria;SubsectionI;Synechococcus;
10
0
0
0
0
0
Bacteria;Deferribacteres;Deferribacteres;Deferribacterales;
Deferribacteraceae;Mucispirillum;
Bacteria;Firmicutes;Bacilli;Bacillales;Alicyclobacillaceae;
Alicyclobacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus;
1
0
0
0
0
0
0
0
0
1
0
0
40
21
5
9
1
3
Bacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;uncultured;
0
0
2
11
6
0
Bacteria;Firmicutes;Bacilli;Bacillales;Family XII Incertae
Sedis;Exiguobacterium;
Bacteria;Firmicutes;Bacilli;Bacillales;Family XII Incertae Sedis;Incertae
Sedis;
Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;
Paenibacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;
Saccharibacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
Lysinibacillus;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
Planomicrobium;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
Sporosarcina;
Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;
uncultured;
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;
Macrococcus;
Bacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;
Staphylococcus
Bacteria;Firmicutes;Bacilli;Lactobacillales;
0
0
1
0
0
0
0
0
0
0
11
1
1
0
0
0
0
0
20
0
0
0
0
0
0
377
4
1271
1370
3
6457
726
6
19
0
0
1
1
0
0
0
15
64
0
14
0
0
0
6
0
0
0
9
2
0
0
0
36
105
12
78
26
36
11
0
0
0
0
0
Bacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae;
Aerococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Atopostipes;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Carnobacterium;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;
Trichococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae
uncultured;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;
Enterococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;
Lactobacillus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;
Fructobacillus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;
Leuconostoc;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Leuconostocaceae;
Weissella;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;
Lactococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;
Streptococcus;
Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Clostridiaceae;
Clostridium;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family Incertae
Sedis;Proteiniborus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Finegoldia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;Tissierella;
Bacteria;Firmicutes;Clostridia;Clostridiales;Family XI Incertae
Sedis;uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Blautia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Butyrivibrio;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Epulopiscium;
2
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
4
0
0
1
0
0
0
320
34
43
85
162
34
117
57
0
90
53
25
0
2
0
0
0
0
7278
371
2
1328
4936
5145
480
9
6277
116
34
110
186
145
3249
898
287
909
1974
998
644
259
57
173
375
300
37
0
2
0
0
0
31
46
13
65
67
41
0
0
0
0
3
0
0
15
0
3
33
1
0
10
2
11
3
0
0
3
9
0
0
18
7
3
0
0
0
0
6
0
0
2
0
0
0
0
5
0
0
0
198
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Marvinbryantia;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
Pseudobutyrivibrio;
Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;
uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;
Peptostreptococcaceae;Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;
Peptostreptococcaceae;uncultured;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
6
110
9
23
14
27
6
0
0
0
0
0
1
0
0
0
0
0
40
7
0
21
27
0
25
110
1
13
16
40
11
7
13
0
16
0
7
0
0
0
2
0
0
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Anaerotruncus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Fastidiosipila;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Incertae Sedis;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
Ruminococcus;
Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;
uncultured
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;
Anaerospora;
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;
Megamonas;
Bacteria;Firmicutes;Clostridia;Clostridiales;Veillonellaceae;
Veillonella;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;Allobaculum;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;Turicibacter;
Bacteria;Firmicutes;Erysipelotrichi;Erysipelotrichales;
Erysipelotrichaceae;uncultured;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;CFT112H7
0
0
1
0
0
11
4
2
0
0
0
0
2
0
0
8
18
0
9
0
0
0
0
0
27
1
0
0
33
0
1
0
0
0
0
0
0
0
0
24
0
0
115
42
36
98
257
39
0
0
1
0
0
0
0
0
0
1
0
0
3
0
0
0
0
0
0
0
0
109
0
0
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;
Fusobacteriaceae;Cetobacterium;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;
Fusobacteriaceae;Fusobacterium;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;
Leptotrichiaceae;Sneathia;
Bacteria;Fusobacteria;Fusobacteria;Fusobacteriales;
Leptotrichiaceae;uncultured;
Bacteria;Gemmatimonadetes;Gemmatimonadetes;
AT425-EubC11 terrestrial group;
Bacteria;Gemmatimonadetes;Gemmatimonadetes;S0134
terrestrial group;
Bacteria;Planctomycetes;Phycisphaerae;Phycisphaerales;
Phycisphaeraceae;CL500-3;
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;
Caulobacteraceae;Brevundimonas;
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;
Caulobacteraceae;Caulobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;
Caulobacteraceae;Phenylobacterium;
Bacteria;Proteobacteria;Alphaproteobacteria;Caulobacterales;
Caulobacteraceae;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Bartonellaceae;Bartonella;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Beijerinckiaceae;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Bradyrhizobiaceae;Bradyrhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Bradyrhizobiaceae;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Brucellaceae;Pseudochrobactrum;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Hyphomicrobiaceae;Devosia;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales
;Hyphomicrobiaceae;Pedomicrobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Methylobacteriaceae;Methylobacterium;
3
2
0
5
0
6
60
41
87
133
79
399
0
3
0
0
0
0
0
0
1
0
0
0
0
0
0
6
0
0
0
0
0
5
0
0
525
236
40
345
152
136
17
23
3
15
10
8
0
0
4
0
0
0
0
13
0
0
0
0
0
8
0
19
0
7
0
0
0
0
0
6
0
0
0
0
0
21
0
0
0
21
0
1
0
2
0
0
0
0
0
0
3
0
0
0
0
0
0
25
0
0
0
0
0
3
0
0
120
6
0
0
0
0
199
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
P-102;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Phyllobacteriaceae;Aquamicrobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Phyllobacteriaceae;Mesorhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Rhizobiaceae;Rhizobium;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales;
Xanthobacteraceae;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Paracoccus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Phaeobacter;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Pontibaca;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Roseovarius;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;Thalassococcus;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodobacterales;
Rhodobacteraceae;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;
MSB-1E8;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;
Rhodospirillaceae;Thalassospira;
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales;
Rhodospirillaceae;uncultured;
Bacteria;Proteobacteria;Alphaproteobacteria;
Sphingomonadales;Sphingomonadaceae;Sphingobium;
Bacteria;Proteobacteria;Alphaproteobacteria;
Sphingomonadales;Sphingomonadaceae;Sphingomonas;
Bacteria;Proteobacteria;Alphaproteobacteria;
Sphingomonadales;Sphingomonadaceae;Sphingopyxis;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Achromobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Alcaligenaceae;Pusillimonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Burkholderia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Cupriavidus;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Limnobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Burkholderiaceae;Ralstonia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Acidovorax;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Aquabacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Comamonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Delftia;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Diaphorobacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Paucibacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Pelomonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales
;Comamonadaceae;Piscinibacter;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Polaromonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Comamonadaceae;Variovorax;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Herminiimonas;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Janthinobacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Massilia;
0
0
0
0
0
6
18
0
0
0
0
0
0
13
0
0
0
0
14
0
0
8
0
7
0
0
0
0
0
6
0
0
6
17
0
0
0
0
0
11
0
0
0
0
0
27
0
0
0
0
0
1
0
0
0
0
0
21
0
0
5
0
0
22
0
0
0
0
0
0
0
1
8
0
0
0
0
0
0
0
5
0
0
0
0
19
0
0
0
0
43
0
0
1
2
0
42
0
0
0
0
0
0
0
0
9
0
0
0
0
0
20
0
0
161
554
151
527
1168
388
5
0
0
0
1
3
0
0
0
1
0
0
0
23
4
23
80
33
0
0
0
0
9
0
147
0
22
0
0
10
0
11
0
24
15
7
1
9
0
0
3
7
0
0
0
3
0
0
21
1
3
0
20
2
1
0
0
0
0
0
0
14
1
0
0
0
0
8
0
0
0
0
0
0
0
14
0
0
1
0
0
0
0
7
0
0
0
0
1
0
6
0
0
0
0
0
15
87
33
47
30
42
62
0
0
8
0
0
200
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;
Oxalobacteraceae;Undibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;DR-16;
0
0
2
0
0
0
16
0
0
0
0
20
0
1
0
0
0
0
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Aquitalea;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Microvirgula;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Neisseria;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Uruburuella;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Vitreoscilla;
Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;
Neisseriaceae;Vogesella;
Bacteria;Proteobacteria;Betaproteobacteria;Nitrosomonadales;
Nitrosomonadaceae;Nitrosospira;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;Thauera;Thauera
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;Uliginosibacterium;
Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales;
Rhodocyclaceae;uncultured;
Bacteria;Proteobacteria;Betaproteobacteria;TRA3-20;
0
2
2
0
0
10
75
7
10
53
15
15
0
0
0
0
1
0
0
0
1
0
10
0
0
0
0
0
0
10
17
6
2
9
10
10
7
0
0
0
0
0
0
0
5
0
0
0
0
18
0
0
0
0
0
0
0
5
9
0
0
0
0
3
0
0
0
0
1
0
0
0
Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales;
Bacteriovoraceae;Bacteriovorax;
Bacteria;Proteobacteria;Deltaproteobacteria;Bdellovibrionales;
Bdellovibrionaceae;Bdellovibrio;
Bacteria;Proteobacteria;Deltaproteobacteria;
Desulfuromonadales;GR-WP33-58;
Bacteria;Proteobacteria;Deltaproteobacteria;
Desulfuromonadales;M113;
Bacteria;Proteobacteria;Deltaproteobacteria;GR-WP33-30;
4
0
0
0
0
0
0
0
0
1
0
0
0
0
3
2
0
0
5
0
0
0
0
0
0
0
0
16
0
0
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;
0319-6G20;
Bacteria;Proteobacteria;Deltaproteobacteria;Myxococcales;
Sorangiineae;uncultured;
Bacteria;Proteobacteria;Epsilonproteobacteria;
Campylobacterales;Campylobacteraceae;Arcobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;
Campylobacterales;Campylobacteraceae;Campylobacter;
Bacteria;Proteobacteria;Epsilonproteobacteria;
Campylobacterales;Campylobacteraceae;Sulfurospirillum;
Bacteria;Proteobacteria;Epsilonproteobactea;
Campylobacterales;Helicobacteraceae;Helicobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Acidithiobacillales;Acidithiobacillaceae;Acidithiobacillus;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;
Aeromonadaceae;Aeromonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;
Aeromonadaceae;Oceanimonas;
Bacteria;Proteobacteria;Gammaproteobacteria;Aeromonadales;
Aeromonadaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;
Alteromonadales;Alteromonadaceae;Marinobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;B38;
0
0
2
14
0
0
0
0
0
0
0
1
38
0
0
15
16
3
2
4
4
0
18
34
22
9
0
0
0
0
79
8
0
64
0
0
0
22
28
20
35
1
16
11
2
27
0
0
0
7
8
110
6
0
0
0
0
12
2
0
0
0
3
34
9
0
1
0
0
0
0
0
Bacteria;Proteobacteria;Gammaproteobacteria;
Cardiobacteriales;Cardiobacteriaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;
Chromatiaceae;Nitrosococcus;
Bacteria;Proteobacteria;Gammaproteobacteria;Chromatiales;
Granulosicoccaceae;Granulosicoccus;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Cedecea;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Citrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
0
0
0
1
0
0
0
0
0
9
0
0
0
0
0
0
0
10
1
0
0
0
0
0
1
0
0
0
0
1
749
152
38
197
250
70
388
105
25
62
198
150
201
Enterobacteriales;Enterobacteriaceae;Enterobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Escherichia-Shigella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Klebsiella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Kluyvera;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Morganella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Pantoea;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Pectobacterium;
Bacteria;Proteobacteria;Gammaproteobacteria;
Enterobacteriales;Enterobacteriaceae;Raoultella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Legionellales;Coxiellaceae;Aquicella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Methylococcales;Crenotrichaceae;Crenothrix;
Bacteria;Proteobacteria;Gammaproteobacteria;
Methylococcales;Methylococcaceae;Methylomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Methylococcales;Methylococcaceae;Methylosoma;
Bacteria;Proteobacteria;Gammaproteobacteria;
Oceanospirillales;Halomonadaceae;Chromohalobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Oceanospirillales;Oceanospirillaceae;Pseudospirillum;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pasteurellales;Pasteurellaceae;Pasteurella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pseudomonadales;Moraxellaceae;Acinetobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pseudomonadales;Moraxellaceae;Enhydrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pseudomonadales;Moraxellaceae;Psychrobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Pseudomonadales;Pseudomonadaceae;Pseudomonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Thiotrichales;Piscirickettsiaceae;Cycloclasticus;
Bacteria;Proteobacteria;Gammaproteobacteria;
Thiotrichales;Piscirickettsiaceae;endosymbionts;
Bacteria;Proteobacteria;Gammaproteobacteria;
Thiotrichales;Thiotrichaceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;
Vibrionales;Vibrionaceae;Vibrio;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Sinobacteraceae;JTB255 marine benthic group;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Sinobacteraceae;Steroidobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Sinobacteraceae;uncultured;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Arenimonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Dyella;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Luteimonas;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Lysobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Rhodanobacter;
Bacteria;Proteobacteria;Gammaproteobacteria;
Xanthomonadales;Xanthomonadaceae;Stenotrophomonas;
Bacteria;SM2F11;
79
2
9
5
48
86
29
7
0
0
0
0
80
2
3
4
28
119
1
0
2
11
0
0
49
0
0
0
0
0
66
0
0
0
0
0
21
0
0
0
0
13
0
0
0
8
0
0
0
3
0
0
0
0
12
4
0
0
2
1
0
0
1
0
0
0
0
0
4
0
0
0
0
0
0
0
1
0
0
26
50
0
0
0
1367
113
61
229
422
174
175
19
9
16
8
15
23
6
90
17
19
0
130
28
7
86
43
26
0
0
0
0
12
0
0
0
0
0
1
0
0
2
0
0
0
0
0
9
0
65
46
0
0
0
1
0
6
8
0
0
0
0
0
13
0
0
0
6
0
0
0
6
0
0
0
0
2
0
0
0
0
0
0
0
2
9
0
0
0
0
8
9
0
0
10
0
4
11
0
2
24
7
1
11
0
3
0
0
0
0
3
0
Bacteria;Synergistetes;Synergistia;Synergistales;
Synergistaceae;Jonquetella;
Bacteria;Tenericutes;Mollicutes;Anaeroplasmatales;
Anaeroplasmataceae;Anaeroplasma;
Bacteria;Tenericutes;Mollicutes;Mycoplasmatales;
Mycoplasmataceae;Mycoplasma;
Bacteria;Verrucomicrobia;Verrucomicrobiae;
Verrucomicrobiales;Verrucomicrobiaceae;Prosthecobacter;
Unclassified;
0
0
1
0
0
0
0
0
0
8
0
0
0
0
0
0
0
1
25
0
0
0
0
0
260
122
51
76
147
66
202
203