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LSU Doctoral Dissertations
Graduate School
2015
The Role of Rhizobial Mutualisms in the
Establishment and Colonization of Exotic Acacia
Species in Novel Ranges
Metha Martine Klock
Louisiana State University and Agricultural and Mechanical College
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Klock, Metha Martine, "The Role of Rhizobial Mutualisms in the Establishment and Colonization of Exotic Acacia Species in Novel
Ranges" (2015). LSU Doctoral Dissertations. 2728.
http://digitalcommons.lsu.edu/gradschool_dissertations/2728
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THE ROLE OF RHIZOBIAL MUTUALISMS IN THE ESTABLISHMENT AND
COLONIZATION OF EXOTIC ACACIA SPECIES IN NOVEL RANGES
A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Biological Sciences
by
Metha Martine Klock
B.A., Sarah Lawrence College, 2001
M.S., Louisiana State University, 2009
December 2015
To my family and friends, for their support, laughter, and encouragement.
ii
ACKNOWLEDGEMENTS
I would first and foremost like to thank my major advisor, Dr. Kyle E. Harms for his
help, kindness, and supportive guidance throughout the course of my doctoral degree. He is one
of the most genuine people I know, and his authenticity and truthful care for his students, family,
as well as the health of the world have restored my hope for the future of our planet. His
openness to scientific inquiry, dedication to craft, and devotion to work-life balance have
provided a model for life that I can only hope to emulate as I further my own career. I would also
like to thank Drs. Peter H. Thrall and Luke G. Barrett at the Commonwealth Scientific and
Industrial Research Organisation in Canberra, Australia. I have been extremely lucky to have
their help, enthusiasm, knowledge, and friendship over the course of my dissertation. Their
support has been invaluable, teaching me independence and strength, how to do good research,
how to ask significant questions, and how to laugh, drink a beer, and move on when things get
tough.
My committee members (past and present), Dr. Meredith Blackwell, Dr. Sun Joseph
Chang, Dr. James T. Cronin, Dr. Bret Elderd, and Dr. Richard Stevens have helped me
throughout the years to develop as a scientist, challenged me to delve deeply into academic
query, and encouraged me to produce a dissertation of which I am proud. I am extremely
thankful for their help and guidance.
I would like to thank my lab mate Sandra P. Galeano. Her constant optimism, faith in
human kind, ability to step outside tense moments and see the good in all situations has been a
constant inspiration to me. She is a model scientist and person. She has supported me through
some of the most trying times in my life, and been always available when I needed a friend. I
hope that she has the chance to spread her knowledge, strength and kindness with students for
iii
generations to come. They would be lucky to have her as a teacher, as I have had her as a teacher
and a friend.
To my lab mates past and present, especially Katherine Hovanes, whose intelligence and
perseverance are inspirational, and to my student workers Elizabeth Weltman and Zach
Goodnow, whose help doing the not always fun stuff in the lab (even while I was far away in
Australia) were indispensable.
This dissertation would not have been possible without the timeless hours, patience, and
friendship of Dr. Hector Urbina. Hector has taught me not only the technical side of science, but
how to keep things fun, how to be patient and have faith in myself. He has been always available
to me as a friend and colleague, has been tirelessly patient and kind, and follows all that up with
humor and a genuine positive outlook on life. Hector has taught me so much about science,
friendship, and the true meaning of collaboration. I am eternally lucky to have him as friend and
colleague.
To the whole crew at CSIRO past and present: Alexandre de Menenzes, Shamshul
Hoque, Kristy Lam, Kelly Hamonts, Holly Vuong. The support and kindness these intelligent
and driven people have shown me over the years has shown me the true meaning of friendship
and collaboration. They are my family “down undah” and I will be eternally grateful for having
met them.
To my cohort and friends at LSU who I thank for making me laugh and for being bright,
generous, smart, and kind human beings.
To my LSU friends who have graduated and moved on to different pastures: Amy
Scaroni, you’re like, #1, Ringtones forever! Tracy Hmielowski, friends from the first day we met
in Plant Pop! Becky Carmichael, the best co-TA, boss, and wonder woman I know. Barbi
iv
Landano, amazing friend, field assistant, and confidant. Steven Wright, my favorite guy ever,
even though you kicked my butt in the field. Carlos Prada, scientist extraordinaire!
To Lindsay Rogers and Marisa Falcigno, who have been two of my biggest cheerleaders,
and who are two of the kindest and most generous people I know. I am fortunate to have them as
lifelong friends.
For financial assistance I would like to thank the LSU Biology Graduate Student
Organization, the Louisiana Environmental Education Coalition, Sigma Xi (the LSU and
National Chapters), the Louisiana Board of Regents, and the National Science Foundation.
I would like to thank my family, my parents Bob and Peggy Klock, who joined me in the
field, keeping me company and helping me on my travels through California searching for
acacias, my sister Gretchen Castets and her husband Nicolas Castets, my new niece Cora
Valentina Castets, and my Grandmother, Jean Madigan, who turned 90 years old this year.
Without their continued love, support, and belief in me this would never have been possible.
Finally, I would like to thank Kaitlin Cannava, my partner. She has shown me that this
world is an open book, which we can explore and write together. Her support through this last
year of my dissertation, reading drafts, watching my practice talks until we both never want to
hear them again, listening to my fears and successes and always treating them with value and
care has meant more to me than she can ever know. I am lucky to have found her (you were right
across the street all that time!). Now we get to move from across the street to around the globe. I
love you. Thank you for your patience and insight, your trust and inspiration.
v
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ........................................................................................................... iii
LIST OF TABLES ....................................................................................................................... viii
LIST OF FIGURES ....................................................................................................................... ix
ABSTRACT .................................................................................................................................... x
CHAPTER 1. INTRODUCTION ................................................................................................... 1
1.1. OVERVIEW AND RESEARCH OBJECTIVES ................................................................ 1
1.2. AUSTRALIAN ACACIAS INTRODUCED ABROAD..................................................... 2
1.3. ACACIA-RHIZOBIA SYMBIOSIS ................................................................................... 4
1.4. OVERVIEW OF CHAPTERS ............................................................................................. 5
CHAPTER 2. DIFFERENTIAL INVASIVENESS OF ACACIA SPECIES IN CALIFORNIA
IS NOT INFLUENCED BY HOST PROMISCUITY WITH RHIZOBIAL SYMBIONTS ......... 8
2.1. INTRODUCTION ............................................................................................................... 8
2.2. METHODS ........................................................................................................................ 13
2.3. RESULTS .......................................................................................................................... 18
2.4. DISCUSSION .................................................................................................................... 23
CHAPTER 3. HOST PROMISCUITY IN SYMBIONT ASSOCIATIONS CAN INFLUENCE
LEGUME ESTABLISHMENT AND COLONIZATION OF NOVEL RANGES ...................... 29
3.1. INTRODUCTION ............................................................................................................. 29
3.2. METHODS ........................................................................................................................ 33
3.3. RESULTS .......................................................................................................................... 39
3.4. DISCUSSION .................................................................................................................... 43
CHAPTER 4. PROVENANCE OF RHIZOBIAL SYMBIONTS IS SIMILAR FOR
INVASIVE AND NON-INVASIVE ACACIAS IN CALIFORNIA ........................................... 49
4.1. INTRODUCTION ............................................................................................................. 49
4.2. METHODS ........................................................................................................................ 53
4.3. RESULTS .......................................................................................................................... 61
4.4. DISCUSSION .................................................................................................................... 68
CHAPTER 5. CONCULSIONS ................................................................................................... 74
5.1. SUMMARY OF KEY RESULTS ..................................................................................... 74
5.2. IMPLICATIONS FOR CONSERVATION AND HABITAT RESTORATION ............. 76
LITERATURE CITED ................................................................................................................. 78
vi
APPENDIX A. SUPPORTING INFORMATION FOR CHAPTER 2 ........................................ 87
APPENDIX B. SUPPORTING INFORMATION FOR CHAPTER 3 ........................................ 95
APPENDIX C. SUPPORTING INFORMATION FOR CHAPTER 4 ...................................... 108
APPENDIX D. LETTER OF PERMISSION ............................................................................. 118
VITA ........................................................................................................................................... 120
vii
LIST OF TABLES
Table 1.1. Definition of the terms “invasive,” “naturalized,” and “non-invasive” as they relate
to the invasiveness categories of Acacia species introduced to novel ranges......................3
Table 2.1. Acacia species occurring in California ........................................................................11
Table 2.2. Summary of Analysis of Variance results testing the effects of host species and
soil treatment on aboveground biomass response ..............................................................19
Table 3.1. Average symbiotic response (i.e. average biomass of each invasive group per
rhizobial strain divided by the average biomass of the non-inoculated control for that
group) for each invasiveness category among rhizobial strains ........................................39
Table 4.1. Collection sites for rhizobial strains isolated from Acacia species in Australia and
California ...........................................................................................................................55
Table 4.2. Rhizobial genera associated with different Acacia species from different locations
based on the 16S rRNA gene .............................................................................................63
viii
LIST OF FIGURES
Figure 2.1. Aboveground biomass (g) response of invasiveness categories to different soil
inoculants ...........................................................................................................................18
Figure 2.2. Percent survival for invasiveness categories among soil treatments ..........................20
Figure 2.3. Percent nodulation for invasiveness categories among soil treatments......................21
Figure 2.4 Average nodulation index for different invasiveness categories among soil
treatments ...........................................................................................................................22
Figure 2.5. Ordination of the rhizobial ribotype richness in different invasiveness categories
(Jaccard similarity) based on the a) 16S and b) nifD genes ...............................................22
Figure 3.1. Distribution maps of Acacia species used in this experiment within Australia .........34
Figure 3.2. Symbiotic response of invasiveness categories to rhizobial strains ...........................40
Figure 3.3. Symbiotic response for species in limited versus widespread native range
distributions among rhizobial strains .................................................................................41
Figure 3.4. Percent nodulation for each invasiveness category among rhizobial
strains .................................................................................................................................42
Figure 4.1. Distribution maps of Acacia species used in this experiment in their native
continent of Australia .........................................................................................................54
Figure 4.2. Phylogenetic tree based on the 16S rRNA gene .........................................................62
Figure 4.3. Phylogenetic tree based on the nifD gene...................................................................65
Figure 4.4. Phylogenetic tree based on the nodC gene .................................................................66
Figure 4.5. Diversity of rhizobial strains associating with Acacia species in Australia
(native continent) and California (introduced region) .......................................................67
Figure 4.6. Ordination of rhizobial strain identity and abundance for the a) the nifD and
b) nodC genes for rhizobial strains collected from Acacia species in Australia and
California ...........................................................................................................................67
ix
ABSTRACT
Comparing species establishment and expansion across regional and global scales and
comparing congeners that differ in invasive capacity can provide novel insights into
mechanisms driving species invasions. Acacia species introduced from Australia around the
globe constitute an ideal group for comparing drivers of establishment across different regions
and species. Acacias form mutualistic associations with nitrogen-fixing root-nodule bacteria
(i.e. rhizobia), which may be a key factor in invasion success. In this dissertation, I examine the
acacia-rhizobia mutualism across a suite of host species to explore a key mechanism
influencing patterns and processes of acacia invasion.
I used three complementary approaches to examine the role of rhizobial mutualisms in
the invasiveness of Australian acacias. First, I examined acacia host promiscuity with rhizobia on
a regional scale, comparing plant performance and rhizobial symbiont diversity among hosts
grown in novel soils. This approach allowed me to examine whether acacias that are invasive in
California are more promiscuous rhizobial hosts. Second, I examined acacias that range in
invasiveness globally, pairing them with multiple rhizobial strains to examine whether global
invasiveness is influenced by promiscuity with rhizobia. Third, I examined the provenance of
rhizobia nodulating with acacias in their native and introduced ranges, which allowed me to
assess whether acacias were introduced to California concurrent with their native rhizobia. These
approaches comprise a comprehensive examination of a key mutualism that may influence acacia
invasion in a multi-species, large-scale framework.
Results from this dissertation suggest that host promiscuity with rhizobia delineates
acacia invasiveness on a global, but not regional scale. Acacia species categorized as globally
invasive were more promiscuous hosts than naturalized or non-invasive species. On a regional
x
scale, acacias differing in invasiveness within California did not differ in host promiscuity.
Regardless of invasive status in California, acacias appear to have been co-introduced with their
native Australian rhizobia.
This project contributes to clarifying the driving forces of exotic species invasion success
abroad. Mechanistic approaches to understanding the causes of species invasions are important
for informing management, control, and abatement of further introduction of species that have
proven records of, or the capacity to become, invasive when introduced abroad.
xi
CHAPTER 1. INTRODUCTION
1.1. OVERVIEW AND RESEARCH OBJECTIVES
Elucidating the mechanisms that allow certain species to outcompete others is a
fundamental goal in ecology and invasive species management. Examining invasive species in a
comparative framework across native and invasive ranges, as well as among suites of native and
invasive congeners has proven useful for understanding how some exotic species outcompete
native species (Hierro et al. 2005; Williams et al., 2010). By taking large-scale, multiple species
approaches to delineating the drivers of species invasions, we may achieve a more general
understanding of the mechanisms that allow species to invade, and be better equipped to translate
this information to useful and broadly applicable management strategies.
A unique opportunity for this approach arises when a suite of closely related species is
introduced to a novel region, yet the species vary in their success as invaders. Many Acacia
species native to Australia have been introduced abroad but vary in their establishment and
colonization of novel ranges. Those that have become invasive have been shown to outcompete
native plant species, increase soil erosion, alter soil chemistry, and facilitate invasion of nonnative grasses (Richardson et al., 2000; Richardson & Kluge 2008). The factors that influence
their ability to establish and expand in natural areas are still largely unknown (Gibson et al.,
2011; Richardson et al., 2011).
A key mechanism that may influence the invasiveness of Acacia species is their
symbiotic relationship with nitrogen-fixing soil bacteria (i.e. rhizobia). Rhizobia fix
atmospheric nitrogen and convert it to a form usable by the plant; in turn the plant provides
carbon and protection from desiccation to the bacteria (Begon et al., 2006; Sprent, 2001). Plant
growth, along with reproduction, is often limited by nitrogen availability (Kaye & Hart, 1997).
1
Therefore, species that can acquire symbiotic nitrogen-fixing bacteria in novel ranges may have
a competitive benefit over non-symbiotic native species. This suggests that the ability of Acacia
species to find suitable rhizobial symbionts, and the selectivity with which they can obtain
rhizobial partners, will influence their ability to establish and expand in novel ranges.
The goal of this dissertation is to examine a key mechanism driving species invasions,
focusing on the unique opportunity afforded by exotic acacias. This project takes a
biogeographical approach, using data collected in native and novel regions to look specifically at
the role that rhizobial mutualisms play in the establishment and colonization of Australian
Acacia species that have been introduced abroad and that vary in their establishment success in
novel regions. I pay particular attention to acacia mutualisms with rhizobia, and evaluate how
this key mutualism contributes to the differential establishment success of these species abroad.
1.2. AUSTRALIAN ACACIAS INTRODUCED ABROAD
Over 1,000 species of acacia are native to Australia. Of these, 386 have been introduced
outside their native range; 23 species have become invasive, 48 naturalized, and 315 have no
record of invasiveness (Richardson et al., 2011). Definitions of invasiveness categories can be
found in Table 1.1. Acacias have been distributed widely throughout the world; those that have
become invasive abroad vary in the number of regions they have invaded (Richardson et al.,
2011; Richardson & Rejmánek, 2011). Records of introduction, as well as details on life-history
traits are readily available in many areas where acacias have been introduced, making this an
ideal suite of species for comparative studies in invasion biology. The widespread introduction of
Australian Acacia species provides a large-scale framework to examine mechanisms of invasion.
Most Acacia species that have been introduced abroad have become invasive in areas
classified as Mediterranean ecosystems. This includes California, South Africa, Portugal,
2
Table 1.1. Definition of the terms “invasive,” “naturalized,” and “non-invasive” as they relate to
the invasiveness categories of Acacia species introduced to novel ranges.
Term
Invasive
Naturalized
Non-Invasive
Definition
Non-native species that: 1) have self-sustaining
populations which, for a minimum of 10 years
have reproduced by seed or ramets without (or
despite) human intervention, and 2) have spread
and established reproductive populations at large
distances from parent plants
Non-native species that have escaped cultivation
and established self-sustaining populations but
have not spread to the extent of invasive species
Non-native species that do not establish
populations without the aid of humans
Reference
Richardson et al.
2011
Richardson et al.
2011
Jepson eFlora 2015
and Spain, to name a few (Richardson et al., 1989; Holmes & Cowling, 1997; Marchante et al.,
2003). These ecosystems are characterized as having mild winters in which the concentration of
rainfall occurs and warm, dry summers (Aschmann, 1973). Some of the Acacia species that have
become invasive abroad are native to western and southern Australia, which are classified as
Mediterranean ecosystems (Commonwealth of Australia, 2015), however many acacias that have
become invasive abroad are native to eastern Australia, which is characterized as temperature
broadleaf and mixed forests (Commonwealth of Australia, 2015). While not all acacias that have
become invasive are from areas in Australia with Mediterranean ecosystems, introduction into
similar climactic conditions as Acacia species experience at home may influence their ability to
invade novel regions.
Multiple Acacia species have been introduced to California, where they vary in status
from invasive to non-invasive (Cal-IPC, 2006; Herbarium, 2013; CalFlora, 2015). Australian
acacias introduced to California provide a regional opportunity to explore drivers of invasive
species establishment and expansion. Sixteen Australian Acacia species currently occur in
California (Jepson eFlora, 2015). Acacias were first introduced to California and sold in the
3
nursery trade here in the mid-1800s (Butterfield, 1938). Acacias at this time were primarily
introduced from Australia as seed, although seedlings were also transported from Australia to
California (Butterfield, 1938). Acacias were desired ornamental plants that were widely sold in
plant nurseries in multiple regions in California during the late 1800s (Butterfield, 1938).
Multiple Acacia species introduced as horticultural plants escaped cultivation and became
naturalized or invasive in California. According to the most recent UC Berkeley Jepson
Herbarium treatment of Acacia, the California Invasive Plant Council Weed Assessment, CalFlora.org and personal observations, Acacia species currently span from non-invasive to
invasive, indicating that they differ in their capacity to establish and colonize natural areas in
California (Cal-IPC, 2006; Herbarium, 2013; CalFlora, 2015). The spectrum of invasiveness of
these species within California provides a novel framework for examining mechanisms of
invasiveness within a group of closely related exotic species on a regional scale.
In this dissertation, I examine acacia invasion on a global and regional scale, asking
whether the mechanisms driving or limiting invasion of Acacia species are consistent when
evaluated at different spatial scales.
1.3. ACACIA-RHIZOBIA SYMBIOSIS
Acacias develop a mutualistic relationship with nitrogen-fixing soil bacteria (i.e.
rhizobia), and this interaction may confer a competitive advantage to both organisms. The
interaction occurs when rhizobia infect the root hairs of plants they can associate with,
eventually triggering cell division in the root cortex, which leads to the development of nodules
on the roots in which the rhizobia are housed (this process is referred to as “nodulation”) (Sprent,
2001). Rhizobia enable acacia hosts to access nitrogen unavailable to non-symbiotic plants, and
host plants provide the products of photosynthesis to their rhizobial symbionts (Begon et al.,
4
2006). Acacias often vary in their host specificity with rhizobia (Thrall et al., 2000). Thus, lessspecific hosts may be able to nodulate with a wider suite of rhizobia, and may more easily find
suitable symbionts in their introduced ranges. More promiscuous hosts may acquire a
competitive advantage. Variation in effectiveness of symbiotic associations between rhizobia and
different Acacia species has been documented, with more promiscuous hosts showing at least
some evidence of being more widespread in their native continent (Thrall et al., 2000). Host
specificity may limit the ability of certain Acacia species to expand when introduced abroad.
Rhizobial strains that nodulate with introduced acacias may be of either native or novel
origin, and this too may be an important determinant of invasiveness. In California, acacias that
exhibit more invasive characteristics may have been introduced with their native Australian
rhizobia, as has been observed for A. longifolia and A. saligna in Portugal, (RodríguezEcheverría, 2010; Crisóstomo et al., 2013) and A. pycnantha in South Africa (RodríguezEcheverría et al., 2011). Alternatively, introduced acacias may form successful symbioses with
native California rhizobia. It is also possible, though less likely, that more invasive acacias are
capable of successful establishment even in the absence of appropriate rhizobial symbionts.
1.4. OVERVIEW OF CHAPTERS
The overarching question for this dissertation project is: Why are Australian acacias
differentially successful in colonizing natural areas? I examine this by exploring a key
mutualism that promotes acacia establishment in their native and introduced regions: the
acacia-rhizobia symbiosis.
In Chapter 2, I ask the question: What is the role of host promiscuity in the differential
establishment and colonization of Acacia species in California? I explored the following
hypothesis: Host promiscuity of Acacia species will influence invasiveness of different acacias
5
in California. I predicted that invasive acacias in California would be more promiscuous hosts
than less or non-invasive species, capable of nodulating with a greater diversity of rhizobial
strains. To examine this hypothesis, I conducted a glasshouse experiment in Australia in which
I paired seven Acacia species ranging in invasiveness within California with 10 soils collected
from areas in which these species did not occur. I grew each Acacia species with each soil and
measured plant performance, as well as the richness of rhizobial strains associating with
acacias, in each plant x soil combination. I did not find a difference in host promiscuity among
Acacia species differing in invasiveness in California, suggesting that the ability to associate
with a wider range of rhizobial symbionts does not differentiate invasive status in this region.
In Chapter 3, I investigate the role of host promiscuity with rhizobia in acacia
invasiveness on a global scale. All acacias introduced to California except for one have become
invasive in at least one region of the globe. I proposed the following hypothesis: Host
promiscuity of Acacia species will influence their ability to invade on a global scale. I predicted
that acacias invasive globally would be more promiscuous hosts than less or non-invasive
species. To test this hypothesis, I grew 12 Acacia species ranging in global invasiveness with 12
isolated rhizobial strains and compared plant performance among acacias in different
invasiveness categories. I found that acacias that are invasive globally are generally more
promiscuous hosts than naturalized and non-invasive species. These results suggest that, on a
larger-scale, host promiscuity with rhizobia influences the ability of acacias to invade novel
regions.
In Chapter 4, I examine the role of rhizobial provenance in the invasiveness of acacias in
California. I posed the following hypothesis: The symbiosis of Australian acacias with
Californian or Australian rhizobia will influence the invasiveness of different Acacia species.
6
This hypothesis predicts that invasive acacias in California would be found nodulating with
rhizobia of Australian origin more frequently than less or non-invasive species. To test this, I
collected root nodules from four Acacia species (two invasive and two non-invasive in
California) in their native range of Australia and introduced range of California. I isolated and
sequenced rhizobia collected in each region, and compared rhizobial identity among the Acacia
species examined. I found acacias in California, regardless of invasive status, associating with
rhizobia closely related to those of Australian origin. This suggests that acacias have been
introduced to California along with their native Australian rhizobial mutualists, and provenance
of acacia-associated rhizobial symbionts does not delineate invasiveness in this range.
In Chapter 5, I synthesize the results obtained among these chapters to provide a
conceptual framework for the role of the legume-rhizobia symbiosis in the invasiveness of
Australia Acacia species regionally and globally. I conclude that the capacity of Acacia species
to form a symbiotic relationship with rhizobia plays an important role in establishment of these
species abroad. Whereas their ability to differentially invade on a local scale is not driven by
acacia host promiscuity or rhizobial provenance, the ability to invade on a global scale does
appear to be at least partially influenced by acacia promiscuity with rhizobial symbionts. I also
propose avenues for future research, and suggest additional mechanisms that may be influential
in the differential establishment of Acacia species abroad.
7
CHAPTER 2. DIFFERENTIAL INVASIVENESS OF ACACIA SPECIES IN
CALIFORNIA IS NOT INFLUENCED BY HOST PROMISCUITY WITH RHIZOBIAL
SYMBIONTS
2.1. INTRODUCTION
Non-native species are a threat to native ecosystems, particularly when they colonize new
areas and rapidly expand in abundance. Collectively, these invasive species have local and
globally destructive impacts, threatening biodiversity, accelerating global change, and causing
economic hardship (D'Antonio & Vitousek, 1992; Vitousek et al., 1996; Mack et al., 2000;
Pimentel et al., 2000). Even though not all introduced species become invasive, those that do
have been shown to variously alter food sources for native wildlife, change fire regimes,
outcompete native species, and impact soil communities by altering microbial structure and soil
nitrogen levels, etc. (Mack & D'Antonio, 1998; Mack et al., 2000; Brooks et al., 2004). To better
understand how species become invasive in new environments, in-depth investigations of
mechanisms driving species invasions are needed.
Diverse mechanisms and theories have been proposed for why introduced species may
become invasive. Many of the better-investigated drivers of invasiveness are based on
antagonistic or competitive interactions (Blossey & Notzold, 1995; Callaway & Aschehoug,
2000; Keane & Crawley, 2002; Levine et al., 2003). Much work to date has investigated the role
of enemy-release in facilitating species invasions (i.e. invaders that prosper in new environments
because they leave their parasites, pests, and predators behind [Keane & Crawley, 2002]). For
example, DeWalt et al. (2004) found support for the Enemy-Release Hypothesis with the
invasive shrub Clidemia hirta, which suffered lower herbivore damage in novel habitats outside
its native range, suggesting release from pests and pathogens when introduced abroad. The
Evolution of Increased Competitive Ability Hypothesis predicts that adaptive evolution of
8
invaders provides a competitive advantage in the novel range (Blossey & Notzold, 1995).
Studies of the invasive tree Chinese tallow (Sapium sebiferum L. Roxb, synonym Triadica
sebiferum) indicate that invasive genotypes have reduced herbivore resistance and allocate more
resources to growth (Siemann & Rogers, 2003; Zou et al., 2008). Although overcoming
adversity imposed by antagonists and competitors may be the driver of invasiveness for some
species, mutualistic interactions may also play a key role for other species (Richardson et al.,
2000a).
A growing body of work has examined the role of mutualisms in the invasion of nonnative species (Richardson et al., 2000a; Wandrag, 2012; Birnbaum et al., 2012). The Enhanced
Mutualism Hypothesis suggests that species encounter novel beneficial symbionts in their native
range, which enhance their ability to survive and spread abroad (Richardson et al., 2000a). The
Accompanying Mutualist Hypothesis suggests that invasive species are introduced concurrent
with their native mutualistic partners, thereby enhancing their ability to survive in novel habitats
(Rodríguez-Echeverría, 2010). Mutualisms such as those between legumes and their symbiotic
nitrogen-fixing soil bacteria (i.e. rhizobia) may be particularly important in explaining the ability
of this group of species to establish and expand abroad. Elucidating the potential role that
mutualistic interactions play a role in species establishment and colonization may point towards
mechanisms driving differential levels of species invasion.
Australian Acacia species (Family: Fabaceae) are a diverse group of legumes that form
symbiotic relationship with rhizobia. They have been introduced throughout the world for a
variety of purposes, including ornamental use, fuel wood, erosion control, and forestry (Kull &
Rangan, 2008; Carruthers et al., 2011). Many species that have been introduced have become
invasive abroad (Richardson et al., 2011). Over 1,000 Acacia species occur in Australia (Miller
9
et al., 2011). Of these, ~400 species have been introduced outside their native range, with ~6%
becoming invasive, ~12% becoming naturalized, and ~82% non-invasive (Richardson et al.,
2011; Rejmánek & Richardson, 2013). Acacias vary in the number of regions they have invaded
globally [as defined by Richardson & Rejmánek (2011) and Rejmánek & Richardson (2013)]
and include: North America, Europe, Middle East, Asia, Indonesia, Pacific Islands, New
Zealand, Australia, Indian Ocean Islands, Africa (southern), Africa (rest), Atlantic Islands, South
America, Caribbean Islands, and Central America. Certain Acacia species have invaded almost
all regions (e.g., A. mearnsii: 12 regions), whereas other species (e.g., A. elata) have invaded
only one region (Richardson & Rejmánek, 2011; Rejmánek & Richardson, 2013). Acacias have
not consistently invaded all areas where they were introduced. The differential invasiveness of
introduced acacias provides an opportunity to examine mechanisms that may be limiting or
promoting their colonization and establishment in novel ranges (Klock et al., 2015).
Sixteen Australian Acacia species have been introduced to California (Jepson eFlora,
2015) (Table 2.1). While all these species except for one (A. cultriformis) are invasive in at least
one part of the world, they vary markedly in their ability to invade and expand population sizes
in California. Records indicate that Acacia species were first introduced to California for
ornamental purposes and sold through the nursery trade beginning in the mid-1800s (Butterfield,
1938). Two species, A. dealbata and A. melanoxylon, are currently designated as invasive in
California (Cal-IPC, 2006; CalFlora, 2015), signifying that they have established independently
reproducing populations that spread and outcompete native species in areas where they occur.
Five Acacia species are naturalized in California, and nine species are non-invasive (Jepson
eFlora, 2015). Understanding the mechanisms that enable multiple closely related species to
10
differentially establish and colonize natural areas in one particular region is important for
understanding what controls and promotes species establishment in general.
Table 2.1. Acacia species occurring in California. California invasiveness status compiled from
CalFlora (CalFlora, 2015), Cal-IPC (Cal-IPC, 2006), and Jepson Herbarium (Jepson eFlora,
2015). Regions invaded globally compiled from Richardson et al. (2011) and Rejmanek et al.
(2013). Acacia species included in this study are noted with an *.
Species
A. baileyana*
A. cultriformis*
A. cyclops
A. dealbata*
A. decurrens
A. elata
A. longifolia*
A. mearnsii
A. melanoxylon*
A. paradoxa
A. podalyriifolia
A. pycnantha*
A. redolens
A. retinodes
A. saligna
A. verticillata*
California status
Naturalized
Non-Invasive
Naturalized
Invasive
Non-Invasive
Non-Invasive
Naturalized
Non-Invasive
Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Naturalized
Non-Invasive
Naturalized
Non-Invasive
Regions invaded globally
2
0
4
6
3
1
7
12
10
4
2
2
0
2
4
2
One mechanism that may be important in the invasion of non-native acacias abroad is
their symbiotic relationship with rhizobia. The legume-rhizobia interaction has been long
recognized as important for the growth and establishment of many legumes (Sprent, 2001).
Rhizobia are gram-negative bacteria that convert atmospheric nitrogen to a form usable by the
plant (Bauer, 1981; Sprent & Sprent, 1990). Within nodules, the plant provides rhizobia access to
carbon substrates, micronutrients, and also protects rhizobia from desiccation (Sprent, 2001).
When legumes form an association with compatible symbiotic bacteria they obtain a source of
nitrogen unavailable to other plants. Soil nitrogen availability is often low (Masclaux-Daubresse
11
et al., 2010), so species that are able to form this association may have a competitive advantage
over other plant species, particularly in low-resource systems (Funk & Vitouskek, 2007).
The selectivity of different plant hosts for particular rhizobial symbionts (hereafter
referred to as “host promiscuity”) may contribute to the ability of certain Acacia species to
establish and expand abroad. Hosts that are more promiscuous (i.e. are able to associate with a
wider range of rhizobial strains) may have a competitive advantage when introduced to novel
areas, where they encounter unfamiliar nitrogen-fixing bacteria (Richardson et al., 2000b). The
ability of certain Acacia species to form symbiotic relationships with a wide range of rhizobial
symbionts may circumvent limitations imposed by encountering novel rhizobial strains
(Rodríguez-Echeverría, 2010; Birnbaum et al., 2012). Variation in host promiscuity among
Acacia species introduced to California may help explain why certain species have differentially
invaded this region.
The goal of this study was to examine host promiscuity of a suite of Acacia species that
have become differentially invasive within California. To examine this, I used multiple Acacia
species representing different invasiveness categories, and performed whole soil inoculation
experiments with a range of soils from different environments. I evaluated aboveground plant
growth (biomass), survival, and nodulation responses. I examined whether the interaction
between acacias and their rhizobial symbionts influenced plant performance. I also used
molecular techniques, specifically analysis of terminal restriction length polymorphisms (TRFLPs), to identify the diversity of rhizobial strains associating with acacias in different
invasiveness categories. I hypothesized that invasiveness of non-native acacias in California
would be influenced by host promiscuity with rhizobial strains, with the following predictions: 1)
Invasive acacias would have higher biomass, survival, and nodulation responses (i.e. plant
12
performance) across a greater number of soils than naturalized and non-invasive acacias; and 2)
invasive acacias in California would associate with a greater number of rhizobial strains (as
measured by number of ribotypes, or unique terminal restriction fragment lengths) than
naturalized or non-invasive species.
2.2. METHODS
2.2.1. Study species
The genus Acacia (Fabaceae: Mimosoideae) is native to Australia, with over 1,000
species occurring variously across the continent (Miller et al., 2011). I examined seven species
that have been introduced to California and have become invasive (A. dealbata and A.
melanoxylon), naturalized (A. baileyana and A. longifolia), and non-invasive (A. cultriformis, A.
pycnantha, and A. verticillata) (Cal-IPC, 2006; Jepson eFlora, 2015). All species used in this
study are native to southeastern Australia, and were introduced to California for ornamental
purposes beginning in the mid-1800s (Butterfield, 1938). They range from broadly distributed to
restricted within Australia (AVH, 2014).
2.2.2. Soil inoculant collection and preparation
Soil samples were taken from multiple sites to obtain a diverse range of rhizobial
communities for use in the glasshouse inoculation studies. I collected soils from ten sites within a
150 km radius of Canberra, ACT, during July 2011. Sites varied in disturbance regimes, from a
highly disturbed agricultural field, to an abandoned paddock, to an undisturbed diverse native
legume site (Table S2.1 in Supporting Information). I chose sites that did not contain any of the
Acacia species used in this study so as to challenge all of the study species with unfamiliar
rhizobial communities, in an attempt to mimic conditions these species might encounter when
introduced to a novel range. Soils were collected over the course of seven days. Soil was
13
excavated using a clean shovel and stored in paper bags until processing. I collected multiple
samples from within each site and then bulked the soil samples to make a single composite for
each of the 10 sites. Following collection, soils were dried for up to six days. Once dry, they
were sieved to 3 mm and stored in paper bags until use.
2.2.3. Glasshouse experiment
I conducted a glasshouse experiment to examine host promiscuity of Acacia species in
different invasiveness categories. Glasshouse facilities were located at the Commonwealth
Scientific and Industrial Research Organisation’s Black Mountain site in Canberra, Australia.
The experiment was conducted from July to November of 2011. Seeds of all Acacia species were
obtained from the Australian Seed Company in July 2011. Seeds received a boiling water
treatment to induce germination (boiling water was poured over seeds and they were left to
imbibe water for 24 hours). I transferred seeds to trays of steam sterilized vermiculite and
watered them daily with sterile water for 14 – 20 days, or until germination occurred. Seedlings
were grown in the glasshouse under natural light conditions.
Once germinated, seedlings were transferred to individual pots inoculated with soils
collected from each of the 10 sites. For each of the bulked soils, 10 replicates of each Acacia
species were planted in 8 x 15 cm pots filled ¾ with sterilized sand and vermiculite (1:1
volume), 50 g of an individual soil treatment, and topped with additional sterilized sand and
vermiculite (1:1 volume) to avoid cross contamination. A nitrogen free (N–) control was also
included in the study in which plants were not inoculated. Acacia species x soil combinations
were spatially randomized by glasshouse bench (i.e. each bench contained one replicate of each
species x soil combination, and placement on bench was randomized). All plants were watered
twice weekly with sterile N-free McKnight’s solution (McKnight, 1949) and sterile water as
14
needed. Plants were spaced well apart on glasshouse benches to avoid cross-contamination
during watering.
Plants were grown for 16 weeks in a temperature-controlled glasshouse (~20° C) and
harvested in November 2011. At harvest, seedlings were clipped at the soil surface and
aboveground material was stored in paper bags. Aboveground material was oven dried at 70° C
for 48 h and weighed. Belowground material (roots and attached nodules) of each plant was
stored individually in plastic bags and frozen at –20° C until processing for molecular analysis.
Roots were scored at harvest for nodulation quantity (0, <10, 10 – 50, >50) and quality (none,
ineffective [black or very small white nodules], intermediate [mixture of small to medium
white/pink nodules], and good [pink nodules]) (Thrall et al., 2011).
2.2.4. Isolation of DNA and T-RFLP
I used terminal restriction length polymorphism (T-RFLP) to identify community
composition and genotypic richness of rhizobia nodulating with Acacia species in the
glasshouse experiment. This technique is frequently used for examining taxon richness of
bacterial communities (Birnbaum et al., 2012). To extract DNA from root nodules collected
during harvest, two to ten intact nodules per plant were first snipped from roots stored at –20°
C. Nodules were surface sterilized by immersion in 90% ethanol for five to ten seconds,
transferred to 3% sodium hypochlorite and soaked for two to four minutes, and rinsed in five
changes of sterile water. Nodules were crushed using liquid nitrogen, and DNA was extracted
using Mo Bio PowerPlant® DNA Isolation kits following the manufacturer’s protocol (MO Bio
Laboratories, Inc., Carlsbad, CA, USA). I amplified the 16S rRNA gene using the primers GM3
(5'-AGA GTT TGA TCM TGG C-3') and GM4 (5'-TAC CTT GTT ACG ACT T-3') and the
following PCR program: Initial denaturation at 95 °C for 2 min, followed by 35 cycles of 95 °C
15
for 30s, 50 ° C for 30 s, and 72 °C for 90s, followed by a final extension step at 72 °C for 10
min, and a final holding temperature of 4 °C. I also amplified the nifD gene using the primers
nifD1R (5'-CCC AIG ART GCA TYT GIC GGA A-3') and nifD2F (5'-CAT CGG IGA CTA
CAA YAT YGG YGG-3'), which have reliably worked for rhizobia (Birnbaum et al., 2012),
and the following PCR program: Initial denaturation at 96 °C for 2 min, followed by 7 cycles of
96 °C for 30s, 55 °C for 45s, and 45 °C for 1 min, followed by 25 cycles of 96 °C for 30s, 48
°C for 45s, and 72 °C for 90s, followed by a final extension step at 72 °C for 5 min, and a final
holding temperature of 4 °C. I digested the PCR product using the restriction enzymes MspI
(16S), RsaI (16S), Hinf1 (nifD) and AluI (nifD) (New England BioLabs) in 30 l reaction
mixtures, and analysed the fragment sizes using a 3130xl genetic analyzer (Applied
Biosystems, Warrington, United Kingdom). I used GeneMapper v 5 (Life Technologies, Grand
Island, NY, USA) to examine T-RFLP profiles and included peaks over 50 bp for further
analysis. I quantified resulting peaks using the local southern method (Southern, 1979). Peaks
were binned using Ramette’s interactive binner script (Ramette, 2009) with R statistical
programming language version 3.2.0 (R Core Team, 2015).
2.2.5. Plant growth, survival, and nodulation response
I examined the responses of acacias representing three invasiveness categories to
inoculation with 10 different soils collected from habitats in which the acacias used in this
experiment do not occur. I measured differences among the invasiveness categories by assessing
aboveground biomass, survival, nodulation presence/absence, and nodulation index of
effectiveness (based on scores of nodulation quantity).
I examined these four variables for the entire data set using generalized linear mixed
models (GLMM) and used AIC to select the best models (Burnham & Anderson, 2002). Acacia
16
species was included in the model as a random effect to include individual variation of species in
each invasiveness category. Aboveground biomass and nodulation index were modeled as having
a Gaussian distribution, and nodule presence/absence and survival were modeled as having a
binomial distribution with a logit link function. I also examined biomass, nodulation
presence/absence, and survival for individual Acacia species to assess species-specific responses
to individual soil inoculants. I used ANOVA to compare biomass among species x soil
combinations and logistic regression to analyze survival and nodulation presence. I used a posthoc pairwise t-test with a Bonferroni adjustment to compare biomass of different species to each
soil inoculant. Analyses were conducted using R statistical programming language version 3.2.0
(R Core Team, 2015).
I used the R statistical package ‘lme4’ (Bates et al., 2012) to determine whether main
effects (biomass, nodulation presence and nodule index, survival, and invasiveness category)
contributed significantly to the models of interest, and whether there were interactions among
main effects. Acacia species was maintained in all models as a random effect. Models with the
lowest AIC score were selected for further analysis; models with a difference in AIC values of
<2 were considered equally likely (Burnham & Anderson, 2002; Bolker et al., 2009). I used the
R statistical package ‘multcomp’ (Hothorn et al., 2008) to determine whether there were
significant differences among invasiveness categories in the variables of interest for individual
soils.
2.2.6. Rhizobial community composition and richness
I analysed binary data obtained from T-RFLP analysis using nonmetric multidimensional
scaling (NMDS) based on a Jaccard similarity matrix. I used the R statistical package ‘vegan’
(Oksanen et al., 2015) to conduct ordination and permutational anova (PerManova; function
17
“ADONIS”) to test for differences in rhizobial community composition among invasiveness
categories and soil types. I used ANOVA to examine whether there were differences in ribotypes
richness among invasiveness categories. Analyses were conducted using R statistical
programming language version 3.2.0 (R Core Team, 2015).
2.3. RESULTS
2.3.1. Plant biomass
In general, plants in different invasiveness categories responded similarly to different
soils. I did detect a significant interaction between soil and invasiveness category for
aboveground biomass (ΔAIC = 19.1, wi = 1.00), indicating that the growth response of species
from different invasiveness categories depended on the soil in which they were grown (Table
S2.2 in Supporting Information). However, biomass was significantly greater for the naturalized
group for only one soil (Namadgi grassland). For all other soils, biomass did not differ
significantly among the invasiveness categories (Figure 2.1).
Biomass (g)
Invasive
Naturalized
Non-Invasive
5
5
5
4
4
4
3
3
3
2
2
2
1
1
1
0
0
CS
GE
HV
ND
NG
NL
PB
SG
SN
YC
0
CS
GE
HV
ND
NG
NL
PB
SG
SN
YC
CS
GE
HV
ND
NG
NL
PB
SG
SN
YC
Soils
Figure 2.1. Aboveground biomass (g) response of invasiveness categories to different soil
inoculants. The horizontal solid line indicates the point at which host species within a given
invasiveness category have the same biomass response as their least effective soil. The dashed
line is the average biomass response for all host species within a given invasiveness category
combined across all soils. Error bars represent standard errors (SE) of the means.
18
Biomass varied for individual Acacia species across soil treatments (Table 2.2). From
here on, individual Acacia species are indicated in the text as: I (invasive), N (naturalized), and
NI (non-invasive). Using as a comparison the soil where biomass was lowest for each species, A.
longifolia (N) and A. melanoxylon (I) had significantly greater biomass for three soils, A.
baileyana (N), A. cultriformis (NI), A. dealbata (I), and A. verticillata (NI) for two soils, and A.
pycnantha (NI) for one soil (Figure S2.1 in Supporting Information). Plant biomass also varied
among soil inoculations. Though not the explicit focus of this study, two soils (Sawyer’s Gully
and Yarrangobilly Caves) consistently had the highest biomass response among all individual
Acacia species tested.
Table 2.2. Summary of Analysis of Variance results testing the effects of host species and soil
treatment on the aboveground biomass response.
Source
Host species
Soil
Host x Soil
Residual
d.f.
6
9
9
54
545
SS
305.8
470.2
F
50.97
52.25
P
<0.001
<0.001
135.0
508.6
2.50
0.93
<0.001
2.3.2. Survival
Models including an interaction among soil and invasiveness category for survival did
not converge. This is because multiple soils had invasiveness categories in which survival was
100%. In these instances, with complete lack of variability in the response variable, models
continue permutations infinitely and cannot converge upon a solution (Agresti, 2007). The best
supported model included only soil inoculation as a main effect with species as a random
variable (ΔAIC = 3.87, wi = 0.87) (Table S2.3 in Supporting Information). I thus examined each
soil individually for differences in survival among invasiveness categories. Similar to the full
model, for four individual soils models did not converge due to certain invasiveness categories
19
having 100% survival. In the six remaining soils I did not detect a significant difference among
invasiveness categories for survival. Raw survival percentages for soils in which models did not
converge indicate little difference in survival among invasiveness categories (Figure 2.2).
Survival for individual species was generally high across soils, with over 50% survival
for each species in a minimum of seven soils (A. pycnantha [NI]) and a maximum of all ten soils
(A. longifolia [N] and A. melanoxylon [I]) (Figure S2.2 in Supporting Information).
YC
SN
SG
NL
PB
NG
HV
ND
YC
SN
SG
NL
PB
NG
0.00
HV
0.00
ND
0.00
CS
0.25
GE
0.25
YC
0.25
SN
0.50
SG
0.50
NL
0.50
PB
0.75
NG
0.75
HV
0.75
ND
1.00
CS
1.00
CS
Non-Invasive
GE
Naturalized
1.00
GE
% survival
Invasive
Soils
Figure 2.2. Percent survival for each invasiveness category among soil treatments.
2.3.3. Nodulation presence and index of effectiveness
Models including an interaction among soil and invasiveness category for nodulation
presence did not converge. The best supported model included only soil inoculation as a main
effect with species as a random variable (ΔAIC = 3.92, wi = 0.88) (Table S2.4 in Supporting
Information). I thus examined each soil individually for differences in nodulation presence
among invasiveness categories. As with survival, non-convergence occurred for four soils and in
the six remaining soils I did not detect a significant difference among invasiveness categories for
nodulation presence. Raw survival percentages for soils in which models did not converge
indicate little difference in nodulation presence among invasiveness categories (Figure 2.3).
20
Naturalized
SN
YC
SG
NL
PB
NG
HV
ND
CS
SN
YC
SG
NL
PB
NG
0.00
HV
0.00
ND
0.00
CS
0.25
GE
0.25
SN
0.25
YC
0.50
SG
0.50
NL
0.50
PB
0.75
NG
0.75
HV
0.75
ND
1.00
CS
1.00
GE
Non-Invasive
1.00
GE
% nodulation presence
Invasive
Soils
Figure 2.3. Percent nodulation for each invasiveness category among soil treatments.
Nodulation presence for individual species was generally high across soils, with over
50% nodulation presence for each species in a minimum of seven soils (A. baileyana [N]) and a
maximum of all ten soils (A. cultriformis [NI], A. longifolia [N], A. melanoxylon [I], and A.
verticillata [NI]) (Figure S2.3 in Supporting Information).
I found a significant interaction between soil and invasiveness category for nodulation
index of effectiveness (ΔAIC = 9.7, wi = 0.97) (Table S2.5 in Supporting Information). This
indicates that there was an effect of individual soil type on nodulation index, such that the
number of nodules of plants belonging to different invasiveness categories depended on the soil
with which they were grown. I therefore could not generalize nodulation index response for
invasiveness categories across all soils, and examined nodulation index for each soil
individually. When soils were examined individually, I found no significant difference in
nodulation index among invasiveness categories (Figure 2.4).
2.3.4. Rhizobial community composition and richness
Ordination diagrams indicated a difference in rhizobial community composition among
Acacia species invasiveness categories for the 16S gene (Figure 2.5, a) but not the nifD gene
21
Invasive I
Naturalized
N
Non-Invasive
W
Average nodule index
2.0
1.5
1.0
YC
SN
SG
PB
NL
NG
ND
HV
GE
CS
0.5
Soils
Figure 2.4 Average nodulation index for different invasiveness categories among soil
treatments. The different shapes depict different invasiveness categories (square = invasive,
circle = naturalized, triangle = non-invasive).
(Figure 2.5, b). The 16S rRNA gene is a highly conserved gene of prokaryotic organisms and is
capable of detecting genotypes other than rhizobia specifically. NifD, however, is more likely to
represent rhizobia alone (or at least only bacteria capable of fixing nitrogen) (Fedorov et al.,
2008).
1.5
(b)
0.5
−0.5
0.0
NMDS Axis 2
0.5
0.0
−1.5
−1.5
−1.0
−1.0
−0.5
NMDS Axis 2
IInvasive
N
Naturalized
W
Non−Invasive
1.0
IInvasive
N
Naturalized
W
Non−Invasive
1.0
1.5
(a)
−1.5
−1.0
−0.5
0.0
0.5
1.0
1.5
−1.5
NMDS Axis 1
−1.0
−0.5
0.0
0.5
1.0
1.5
NMDS Axis 1
Figure 2.5. Ordination of the rhizobial community composition in different invasiveness
categories (Jaccard similarity) based on the a) 16S and b) nifD genes from different soil
treatments derived from nonmetric multidimensional scaling (NMDS; stress = a) 0.13, b) 0.03).
Invasiveness categories more similar in rhizobial community composition are closer together in
ordination space.
22
PerManova results from T-RFLP analyses on the 16S rRNA gene indicate that
differences in rhizobial community composition occur between the invasive and non-invasive
(ADONIS, R = 0.08, P = 0.008) and naturalized and non-invasive categories (ADONIS, R =
0.05, P = 0.039). While the invasive category had the highest number of average ribotypes
(14.00), followed by the naturalized category (12.78), and the non-invasive category (10.74),
these differences were not statistically significant (F = 1.287, P = 0.284).
Biomass results indicated a difference among host species in response to specific soil
inoculants. PerManova results from T-RFLP analyses of the 16S rRNA gene did not indicate a
significant difference in rhizobial community composition among soils (ADONIS, R = 0.11, P =
0.836), however the nifD gene analysis did indicate a difference in rhizobial community
composition among soils (ADONIS, R = 0.36, P = 0.001). Further analysis indicated that this
difference was driven by three soils: Sawyer’s Gully, which had significantly different rhizobial
community composition from all other soils, Namadgi Legume (five soils), and Namadgi
Diverse (one soil).
2.4. DISCUSSION
The goal of this project was to examine whether host promiscuity with rhizobial
symbionts plays a role in the differential invasion of Acacia species in California. I found that for
hosts characterized as invasive or otherwise within this particular region, host promiscuity as
measured by plant growth, survival, and nodulation response did not differ among invasiveness
categories. While the rhizobial community composition for acacias in different categories of
invasiveness differed when examining the 16S rRNA gene, this was not consistent with results
from the nifD gene, which is more likely to specifically isolate bacteria capable of nitrogen
fixation, such as rhizobia (Fedorov et al., 2008). Plant growth response, paired with belowground
23
rhizobial diversity results indicate that, within California, host promiscuity with rhizobial
symbionts is not a driving factor delineating invasiveness of Australia Acacia species.
Scale-dependent categorizations of invasiveness may influence resulting mechanisms that
are found important for driving establishment and colonization of species. All Acacia species in
this analysis, while varying in invasiveness within California, are invasive in at least one region
globally (Richardson & Rejmánek, 2011; Rejmánek & Richardson, 2013). In other words, all
species tested here have been found capable of establishing self-sustaining populations that
spread and establish populations in new areas without human assistance (Richardson et al.,
2011). It is possible that the species used in this study are all promiscuous hosts with rhizobial
symbionts. This would explain why, when tested with multiple randomly collected soils from
their native continent, I did not detect a difference in plant performance and associated rhizobial
diversity among invasiveness categories.
Contrary to results found here, previous research has shown that more invasive Acacia
species may be more promiscuous rhizobial hosts (Klock et al., 2015). However, the previous
research categorized Acacia species invasiveness on a global, rather than regional scale. Klock et
al. (2015) paired 12 rhizobial strains ranging in effectiveness with 12 Acacia species differing in
global invasiveness (four species invasive, four naturalized, and four non-invasive). They found
that, in regard to plant growth, invasive acacias were generally more promiscuous hosts, able to
associate and have a positive growth response with more rhizobial strains than naturalized and
non-invasive acacias. As mentioned before, all Acacia species tested here except for one are
invasive in at least one region of the world (Richardson & Rejmánek, 2011; Rejmánek &
Richardson, 2013). Our results, combined with previous results suggest that all acacias examined
in this study may be promiscuous hosts, which may influence the lack of difference seen in plant
24
performance response. However, host promiscuity does not appear to be the key mechanism
delineating invasiveness of acacias in the more restricted region of California.
Differences in aboveground biomass, while not seen among acacias in different
invasiveness categories in California, were seen among different soil types, suggesting that soil
type (i.e. rhizobial community), rather than invasiveness category influenced plant growth
response. Inoculation with two soils in particular collected from two different Eucalyptus spp.
understory sites resulted in the highest biomass for all Acacia species in this study. For the
species examined here, soil treatment (or rather, the rhizobial community occurring in that soil)
appears to drive differences in acacia performance. The particular soils in which plant growth
was highest may host more diverse rhizobial communities, providing Acacia species with a
wider suite of rhizobial symbionts with which to form associations. Conversely, these soils may
contain generalist rhizobial strains, capable of effectively associating with a larger suite of
legume species (Burdon et al., 1999). T-RFLP analysis of rhizobial community composition of
individual soils suggests that both situations may be occurring. I observed higher biomass across
all Acacia species in one soil (SG) that had a significantly different rhizobial community
composition (based on the nifD gene) than all other soils. However, I also observed higher
biomass across all Acacia species in one soil that did not differ significantly in rhizobial
community composition from almost all other soil treatments. It may therefore be either rhizobial
community composition or availability of generalist strains driving patterns of plant
performance.
Although our results indicate that acacias ranging in invasiveness in California do not
differ in host promiscuity, this may be driven in part by the relatively geographically restricted
selection of soils with which they were paired. All soils used in this experiment were collected
25
from Acacia species’ native continent. Rhizobial strains found in these soils may be more closely
related to those that naturally occur with Acacia species used in this experiment. Should these
Acacia species be paired with soils from more distant regions, such as their invaded region of
California, differences in plant performance, and thus host promiscuity among invasiveness
categories may be observed.
I paired Acacia species with whole-soil inoculation treatments, which likely host a
variety of rhizobial symbionts, unlike isolated rhizobial strains. Barrett et al. (2014) found
evidence that acacias paired with multiple isolated rhizobial strains (greater than two and four
strains per plant) suffered diminished plant growth response, likely due to competition among
rhizobial strains. Interactions with multiple rhizobial strains in randomly collected soils may
result in reduced plant performance if those rhizobial strains are in competition for plant
resources. However, since each Acacia species tested here was paired with each soil, the
influence of multiple strain interactions would likely be the same across species.
Rhizobia-related mechanisms other than host promiscuity may influence invasiveness of
acacias introduced to novel regions. Increasing evidence has shown that legumes have been
introduced abroad with their native rhizobial symbionts (Rodríguez-Echeverría, 2010; Ndlovu et
al., 2013; Crisóstomo et al., 2013; Chapter 4 of this dissertation). Co-introduction of particular
species with their co-evolved beneficial symbionts may circumvent the need for introduced
species to develop novel symbiotic associations. Acacia pycnantha, a native Australian species,
was introduced to and has become invasive in South Africa (Ndlovu et al., 2013). In this region,
A. pycnantha has been found associating with rhizobial strains more closely related to those of
Australian origin (Ndlovu et al., 2013). Both A. longifolia and A. saligna have been found
associating with rhizobia of Australian origin in Portugal (Rodríguez-Echeverría, 2010;
26
Crisóstomo et al., 2013). Legumes native to Portugal were also found associating with rhizobial
strains of Australian origin in areas where A. longifolia occurred (Rodríguez-Echeverría, 2010).
Dual invasion of symbiotic plant and microbial species may thus be occurring in regions where
acacias have been introduced, which may result in above and belowground structural changes in
native habitat composition. These dual symbiotic invasions may promote establishment of both
novel species and influence the population structure of native species. In California, cointroduction of rhizobial symbionts may influence which Acacia species become invasive.
Acacias that become invasive in California may benefit from mutualistic interactions
other than the legume-rhizobia symbiosis that aid in their establishment and colonization. As
indicated here, host promiscuity with rhizobia does not appear to delineate invasiveness of
acacias in California. However, as a general trait promoting invasiveness, host promiscuity with
other taxa may be important to the establishment, colonization, and survival these species. Ant
mutualists may aid in seed dispersal and seed bank accumulation as well as protection from
herbivores for Acacia species that become invasive in their novel range (Holmes, 1990;
Montesinos & Castro, 2012). Acacia species that become invasive in California may also
develop successful mutualisms with avian seed dispersers (Glyphis et al., 1981; Underhill &
Hofmeyr, 2007; Aslan & Rejmánek, 2010). Gallager et al. (2011) found that invasive Acacia
species tend to be larger trees and shrubs, which may make them more visited by avian seed
dispersers. Being promiscuous hosts for a variety of mutualistic organisms may increase the
opportunity for Acacia species to develop self-sustaining, spreading populations that invade
novel ranges.
Species that have become invasive in multiple areas of the world may be constrained
from establishing and colonizing all regions where they are introduced. Identifying or ruling out
27
potential mechanisms limiting expansion of certain species that have become invasive globally
but are constrained regionally can inform management of species introduced abroad. I found that
acacias differing in invasiveness in California do not vary in host promiscuity, as evidenced by a
lack of difference in plant performance and rhizobial richness in different soil inoculants.
Invasive status of introduced acacias in California does not appear to be determined by their
ability to associate with larger numbers of rhizobial symbionts.
Species that are introduced around the world vary in their ability to colonize novel
ranges. Some species are so successful at establishing new populations and spreading in areas
where they are introduced that they alter and negatively impact native communities (D'Antonio
& Vitousek, 1992; Vitousek et al., 1996; Mack et al., 2000). These species are often categorized
as invasive. Just as species differentially establish in their native ranges, the levels of
invasiveness that species accomplish when introduced abroad may also vary. Understanding the
mechanisms that limit and/or promote species establishment abroad may help us understand what
leads to the differential invasion of certain species, as well as what precludes certain introduced
species from becoming invasive at all.
28
CHAPTER 3. HOST PROMISCUITY IN SYMBIONT ASSOCIATIONS CAN
INFLUENCE LEGUME ESTABLISHMENT AND COLONIZATION OF NOVEL
RANGES*
3.1. INTRODUCTION
Interactions between hosts and their mutualist partners, whether those partners are native
or introduced, can play an important role in successful establishment and expansion outside of
the hosts’ native ranges. Plants are often dependent on specific mutualisms with insects and other
animals for a diversity of functions, including pollination, seed dispersal, herbivore protection,
etc. (Bascompte & Jordano, 2007), and with fungi and bacteria (often symbionts) to acquire
nutrients such as phosphorous and nitrogen, which might be otherwise unavailable (Richardson
et al., 2000a). Positive species interactions take many forms and require empirical investigation
to fully understand their roles in invasion of species introduced to novel ranges.
One feature likely to be critical to establishment of mutualistic interactions in novel
environments is partner specificity. Several alternative hypotheses have been framed that make
specific predictions in this regard. The Generalist Host Hypothesis suggests that invasive hosts
are likely to be relative generalists in terms of associations with mutualists and are therefore less
constrained than more specialized species by absence of specific partners (Parker, 2001;
Rodríguez-Echeverría et al., 2011; Stanton-Geddes & Anderson, 2011; Birnbaum et al., 2012).
Species that are generalist hosts may be capable of associating with a wider range of symbionts
in their novel range. The Enhanced Mutualism Hypothesis predicts that introduced species that
become invasive form novel effective mutualisms with native species (Richardson et al., 2000a).
Species that become invasive when introduced abroad may be more capable of forming novel
______________
*Reprinted from Diversity and Distributions with permission from John Wiley & Sons Ltd.
Citation: Klock, M. M., Barrett, L. G., Thrall, P. H. and K. E. Harms. 2015. Host promiscuity in
symbiont associations can influence exotic legume establishment and colonization of novel
ranges. Diversity and Distributions 21:1193-1203.
29
mutualisms if they are less selective hosts. The Accompanying Mutualist Hypothesis posits that
species that become invasive are introduced concurrent with mutualistic symbionts from their
native ranges (Rodríguez-Echeverría, 2010). In all cases, the mutualistic association formed by
the invader is presumed to contribute to a competitive advantage over native species.
An important mutualistic interaction that may play a role in invasion of certain species is
between legumes (Family: Fabaceae) and nitrogen-fixing soil bacteria, or rhizobia. Legumes
colonized by effective rhizobia gain access to nitrogen that would be otherwise unavailable to
them; in turn, rhizobia receive photosynthate from their host plants (Sprent & Sprent, 1990;
Waters et al., 1998; Denison, 2000; Barrett et al., 2014). The legume-rhizobia mutualism plays a
key role in determining plant productivity in native ecosystems (van der Heijden et al., 2008). In
addition, legumes that find suitable symbionts may be more capable of establishment, growth,
and survival than those that do not (Thrall et al., 2000; Thrall et al., 2005). This has been shown
in both revegetation and agricultural settings where legumes inoculated with rhizobial strains
known to be effective symbionts had higher rates of performance than those that were noninoculated (Bullard et al., 2005; Thrall et al., 2005). For legumes introduced to novel regions,
those finding rhizobial symbionts with which they can develop a mutualistic relationship may
have an advantage over species that cannot.
Here we focus on invasive acacias as a model to test the role that specificity in
mutualistic interactions might play in determining invasiveness. Acacia is a genus of legumes
including a diverse range of species introduced around the globe that have become differentially
invasive in novel ranges (Richardson et al., 2011). Acacias are native to Australia, where over
1,000 species occur (Miller et al., 2011); species of Acacia have been introduced abroad for
purposes such as forestry, agriculture, erosion control, and ornamental display (Kull & Rangan,
30
2008; Carruthers et al., 2011). Many of these species have subsequently escaped their intended
use (Rejmánek & Richardson, 2013) outcompeting native species, increasing soil erosion,
altering soil chemistry, and facilitating invasion of non-native grasses (Richardson et al., 2000a;
Richardson & Kluge, 2008). An in-depth investigation of the global introduction status of
Australian Acacia species by Richardson et al. (2011) (later updated by Rejmánek & Richardson
[2013]) found that 386 Acacia species have been introduced to areas outside of Australia. Of
these species, 23 (~6%) have become invasive, 48 (~12%) naturalized with no record of
invasiveness, and 315 (~82%) with no record of naturalization or invasion. Global variation in
invasiveness among Acacia species provides a large-scale opportunity to evaluate mechanisms
that may limit or constrain their establishment when introduced to novel ranges.
Acacias and other legumes often differ in their ability to nodulate with different rhizobial
strains (Thrall et al., 2000; Rodríguez-Echeverría et al., 2012). Thrall et al. (2000) found that
less common acacias within Australia tended to be more specific rhizobial hosts than more
widespread Acacia species. Moreover, population level variation in plant growth within certain
Acacia species for the same rhizobial symbionts has also been observed (Burdon et al., 1999;
Barrett et al., 2012). In a detailed study of two Acacia species (A. salicina and A. stenophylla)
where each was paired with soils collected from its own and the other species’ populations,
Thrall et al. (2007) found considerable variation in host-specificity between the two species, with
A. salicina growing well regardless of soil origin, and A. stenophylla growing best in its own
soils. Such variation in legume-host specificity with rhizobia may play an important role in
determining invasiveness of different host species following introduction to novel ranges.
Introduced Acacia species that are more promiscuous hosts (capable of nodulating with a
wider suite of rhizobial strains) may more readily find suitable symbionts as predicted by the
31
Enhanced Mutualism Hypothesis (Richardson et al., 2000b), or be less selective with regard to
rhizobial strains they form associations with in new environments, as predicted by the Generalist
Host Hypothesis (Rodríguez-Echeverría, 2010; Birnbaum et al., 2012). Acacia species that
become invasive may also be co-introduced to novel ranges with their native symbionts as
predicted by the Accompanying Mutualist Hypothesis (Rodríguez-Echeverría et al., 2012;
Crisóstomo et al., 2013; Ndlovu et al., 2013) and may suffer reduced performance if they do not
encounter familiar symbionts (Wandrag, et al. 2013). Promiscuous hosts may have a competitive
advantage promoting establishment and colonization over native plant species; the latter could
include both non-nitrogen fixing and more selective legume hosts. By investigating a suite of
Acacia species that vary in invasiveness globally, we can assess whether host promiscuity has
potential to differentially influence establishment and colonization of legumes in novel
environments.
We hypothesized that rhizobial associations would influence invasiveness of Acacia
species, with the following predictions: 1) invasive acacias will be more promiscuous hosts,
developing effective symbiotic associations (based on plant growth and nodulation response)
with a wider suite of rhizobial strains than naturalised or non-invasive Acacia species; and 2)
both growth and nodulation (nodule presence/absence, abundance) will be higher in invasive
acacias. To test these predictions, we used the assignations of Richardson et al. (2011) to choose
Acacia species from three categories of global invasiveness (invasive, naturalized, and noninvasive) and then grew them with a genetically diverse set of rhizobial strains ranging in
average symbiotic effectiveness from moderately to highly effective (as defined by Bever et al.,
2013). We then compared symbiotic response (defined below) based on aboveground dry weight
(biomass), as well as nodulation response to assess plant performance with different rhizobial
32
strains. By comparing plant performance across a variety of strains ranging in effectiveness, we
were able to examine host promiscuity of Acacia species varying in invasiveness abroad.
3.2. METHODS
3.2.1. Acacia species
We selected native Australian Acacia species from three categories of invasiveness, as
defined by Richardson et al. (2011): 1) invasive outside Australia (23 species); 2) naturalized
with no record of invasiveness (48 species); 3) recorded as introduced outside Australia with no
record of naturalization (315 species). We chose four Acacia species from each category of
invasiveness; particular Acacia species were selected based on evidence of widespread
introduction and well-documented introduction histories. Invasive species chosen for this study
are designated as some of the worst acacia invaders globally (Richardson & Rejmánek, 2011).
All species selected were those with records of repeated introductions abroad, and included:
(invasive) A. dealbata, A. longifolia, A. mearnsii, A. melanoxylon; (naturalized) A. cultriformis,
A. murrayana, A. pendula, A. redolens; and (non-invasive) A. bivenosa, A. colei, A. hakeoides, A.
stenophylla. These species vary in geographic distribution across Australia (Figure 3.1). In
addition to the criteria noted above, species were selected from each invasive category to
represent both widespread (>1000 herbarium records: A. dealbata, A. longifolia, A. melanoxylon,
A. murrayana, A. hakeoides, A. stenophylla; [AVH, 2014]) and limited distributions (<1000
herbarium records: A. mearnsii, A. cultriformis, A. pendula, A. redolens, A. bivenosa, A. colei;
[AVH, 2014]) within their native range as there is at least some evidence that more widespread
acacias may tend towards greater promiscuity (Thrall et al., 2000).
33
3.2.2. Rhizobial strain preparation
Bever et al. (2013) examined average effectiveness of 40 rhizobial strains with nine
Acacia species native to Australia. Symbiotic effectiveness was based on plant growth response
to inoculation with these different strains. They found extensive variation in symbiotic
Invasive
Acacia dealbata*
Acacia longifolia*
Acacia mearnsii
Acacia melanoxylon*
Naturalized
Acacia cultriformis
Acacia pendula
Acacia murrayana*
Acacia redolens
Non-Invasive
Acacia bivenosa
Acacia colei
Acacia hakeoides*
Acacia stenophylla*
Figure 3.1. Distribution maps of Acacia species used in this experiment within Australia (based
on herbarium records from the Australian National Herbarium, Canberra, Australia [AVH,
2014]). “*” denotes widespread species (i.e. >1000 herbarium records). All other species are
limited in distribution (i.e. <1000 herbarium records).
effectiveness of these strains; some strains were broadly effective across all Acacia species (as
measured by aboveground biomass), some varied in effectiveness depending on Acacia species
with which they were paired, while others were relatively ineffective symbionts with all Acacia
34
species tested (i.e. low plant growth response among all Acacia species tested). We chose a
subset of twelve strains from those evaluated by Bever et al. (2013) to represent variation in
average effectiveness to challenge plants with diverse symbiotic conditions. Strains chosen
represented three genera known to associate with Australian Acacia species: Bradyrhizobium,
Rhizobium, and Sinorhizobium (Lafay & Burdon, 2001; Hoque et al., 2011) and were all
originally isolated from Acacia species root nodules in Australia (Table S3.1 in Supporting
Information).
To prepare inoculants, we streaked samples from cultures stored at -80 °C individually on
yeast mannitol agar plates. Plates were incubated at 28 °C for 7–12 days until colony growth.
Cultures were transferred to 50-ml Falcon tubes containing yeast mannitol broth (YMB) and
grown for 5–7 days until media became cloudy. Tubes were then centrifuged at 5,500 rpm for 60
min until a pellet formed; YMB was decanted and tubes were filled to 50 ml with sterile water.
We resuspended the pellet and measured optical density (OD) of the solution. Inoculants were
adjusted to an OD of 2.0 x 107 cells/ml through addition of sterile water. Identity of rhizobial
strains selected from stored samples was confirmed via re-sequencing the 16S rRNA gene prior
to inoculating plant hosts.
3.2.3. Glasshouse experiment
We conducted a glasshouse inoculation experiment to examine symbiotic selectivity of
Acacia species differing in invasiveness with rhizobia. Glasshouse and laboratory facilities were
located at the CSIRO Agriculture Flagship site in Canberra, Australia. We obtained seeds from
the Australian Seed Company and the Australian Tree Seed Centre in June 2013. All acacia seed
was Australian in origin. Seeds were surface sterilized by soaking in 90% ethanol for ten
seconds, 10% (v/v) sodium hypochloride for 5 minutes, and washed in six changes of sterile
35
water (Somasegaran & Hoben, 1994). Seeds were scarified using a boiling water treatment (i.e.
boiling water was poured over surface sterilized seeds), left to soak overnight at 4 °C, and then
sown in a mixture of autoclaved sand and soil (1:1 by volume). Germination media was
autoclaved to ensure sterility. Seedlings were grown between one to two weeks in a laboratory
growth chamber (25 °C) to avoid contamination by free-living bacteria. Seedlings were
transferred from growth chamber to glasshouse facilities and transplanted in pots filled with
steam-sterilized sand and vermiculite (1:1 by volume).
Seedlings were inoculated with individual rhizobial strains three days after transplanting.
Ten replicates of each Acacia species were treated with 1 ml (OD = 0.2) of inoculant of each
strain. Two controls were also included; a nitrogen-free control in which non-inoculated plants
were watered only with a nitrogen-free solution and sterile water (designated as N–) and a control
in which non-inoculated plants were watered with a nutrient solution containing nitrogen
(designated as N+). Nitrogen was provided to plants in the form of ammonium nitrate (NH4NO3)
(6 mM/plant once a week). We separated rhizobial treatments by bench within the glasshouse to
minimize potential for cross-contamination. Plants were grown for 18 weeks in a temperaturecontrolled glasshouse (~20 °C) under natural light conditions. All plants except for the N+
control were watered with sterile N-free McKnights solution (1949) twice weekly and with
sterile water as necessary. N+ control plants were watered with McKnight’s solution amended
with ammonium nitrate on the same watering schedule as all other plants.
Plants were harvested in October 2013. During harvest, plants were clipped at the soil
surface and aboveground material was placed in paper bags. Roots were scored for nodulation
quantity (0, < 10, 10 – 50, > 50) and quality (none, ineffective [black or very small white
nodules], intermediate [mixture of small to medium white/pink nodules], good [pink nodules],
36
and very good [large nodules with pink/red centres]) (Thrall et al., 2011). Aboveground material
was oven-dried at 70 °C for 48 h and weighed.
3.2.4. Statistical analysis
We measured response of acacias in three invasiveness categories and as individual hosts
to inoculation with 12 rhizobial strains. Although not the explicit focus of this study, we also
examined response of Acacia species that are either widespread or limited in native range
distribution to 12 rhizobial strains. Differences among invasiveness categories and between
range distributions to individual strains was measured by assessing host symbiotic response
(based on aboveground biomass; defined below), presence/absence of nodules, and nodulation
index of effectiveness (based on nodule quantity score categories). There was a low level of
contamination of N– controls (~14%) and any control plants with nodules were excluded from
analyses. Symbiotic response was calculated following methods of Thrall et al. (2011). We
divided biomass of each invasive group/native distribution/acacia host by average biomass of the
non-inoculated control for that group/distribution/host. A value of 1 indicates that biomass for a
particular group/distribution/host x strain combination did not differ from the N– control, a value
of >1 indicates that the group/distribution/host performed better than its N– control for a
particular strain, and a value of <1 indicates that the group/distribution/host performed worse
than its N– control. Using this measure of symbiotic response to analyse plant responses rather
than aboveground biomass allowed us to minimise any bias due to potential differences in
intrinsic growth rates among Acacia species.
We examined these variables for the entire data set using generalized linear mixed
models (GLMM) with Acacia species as a random effect to control for species-specific variation
that may influence growth or nodulation response. We used GLMM because it allowed us to
37
include variability in growth and nodulation response of individual Acacia species in our model,
while using invasiveness category as our main predictor. In our models, symbiotic response and
nodulation index have Gaussian distributions and nodule presence/absence has a binomial
distribution with a logit link function. We also examined symbiotic response and nodule
presence/absence for individual host species to examine species-specific response to rhizobial
strains. We used ANOVA to analyse symbiotic response for individual hosts across rhizobial
strains, and logistic regression to analyse nodule presence/absence. A post-hoc pairwise t-test
with a Bonferroni adjustment was used to compare symbiotic response of different host species
to each rhizobial strain. For invasiveness and native range distribution categories and individual
host species, N– controls were used as a standard to compare symbiotic response across strains.
All analyses were conducted using R statistical programming language version 3.0.2 (R Core
Team 2013).
We used the R statistical package ‘lme4’ (Bates et al., 2012) to examine whether
symbiotic response/nodulation presence/nodule index was affected by an interaction between
invasive group/native range distribution and rhizobial strains, with Acacia species as a random
effect. We used likelihood ratio testing to compare full and reduced models, and to determine
variables for inclusion in the model. Models with the lowest Akaike information criterion (AIC)
score were selected for further analysis unless the difference in AIC values was <10, in which
case models were considered equally likely (Burnham & Anderson, 2002; Bolker et al, 2009).
We used the R statistical package ‘multcomp’ (Hothorn et al., 2008) to examine whether there
were differences in symbiotic response among invasiveness categories/range distributions for
individual strains.
38
3.3. RESULTS
3.3.1. Symbiotic response across invasiveness categories and host species
We found a significant interaction between invasiveness category and strain for
symbiotic response, indicating that growth response of a particular invasive group was dependent
on the strain with which it was grown (Table S3.2). When compared across invasiveness
categories, the invasive group had a significantly higher symbiotic response than naturalized or
non-invasive groups for six of 12 strains (Tables 3.1 & S3.3). Naturalized and non-invasive
groups did not differ significantly in symbiotic response across strains. When examined within
invasiveness categories, the invasive group had a significantly higher symbiotic response than its
N– control for five strains, followed by non-invasive (four strains), and naturalized groups (two
strains) (Figure 3.2).
Table 3.1. Average symbiotic response (i.e. average biomass of each invasive group per
rhizobial strain divided by the average biomass of the non-inoculated control for that group) for
each invasiveness category among rhizobial strains. The invasive category had a significantly
greater symbiotic response (marked with a “*”) than the naturalized and non-invasive categories
for six strains. Naturalized and non-invasive categories did not differ significantly in symbiotic
response among strains. Strains marked as B = Bradyrhizobium, R = Rhizobium, and S =
Sinorhizobium.
Strain
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
Invasive
36.83*
34.38*
61.40*
48.41*
1.76
1.32
37.70*
5.96*
1.49
2.73
2.73
3.19
Naturalized
5.62
4.01
12.87
9.79
1.64
1.41
4.60
1.90
1.16
1.82
1.14
1.65
39
Non-Invasive
8.46
6.79
7.98
8.82
1.50
1.57
5.68
2.43
3.47
3.42
2.88
5.01
We also found a significant interaction between host species and strain for symbiotic
response (Table S3.4), indicating that (as shown in earlier studies) individual hosts have
differential growth responses depending on rhizobial strains with which they are grown. Host
species in different invasiveness categories varied in consistency of their responses to individual
rhizobial strains. For example, hosts within the invasive group responded similarly, with a
significantly higher symbiotic response to the same four to five rhizobial strains than naturalized
and/or non-invasive species. Host species in the naturalized group varied in symbiotic response
across strains, but consistently associated with fewer strains than invasive species. One species,
A. murrayana, responded poorly to all strains with which it was paired. Host species in the noninvasive group varied the most in their symbiotic responses, with one species clearly responding
positively to multiple strains, two species moderately, and one species poorly to all strains with
which it was inoculated (Figure S3.1 & Table S3.5 in Supporting Information).
Symbiotic response
Invasive
Naturalized
Non-Invasive
75
75
75
50
50
50
25
25
25
0
0
01B 02B 03B 04B 05S 06B 07R 08B 09S 10S 11S 12R N- N+
0
01B 02B 03B 04B 05S 06B 07R 08B 09S 10S 11S 12R N- N+
01B 02B 03B 04B 05S 06B 07R 08B 09S 10S 11S 12R N- N+
Strains
Figure 3.2. Symbiotic response of invasiveness categories to different rhizobial strains. The
horizontal solid line at 1 indicates the point at which host species within a given invasiveness
category has the same symbiotic response as the N– control. The dashed line is the average
symbiotic response for all host species within a given invasiveness category combined across all
strains. Points above the solid line indicate a positive symbiotic response, and points below the
solid line indicate a negative symbiotic response. Error bars represent standard errors (SE) of the
means. Strains marked as B = Bradyrhizobium, R = Rhizobium, and S = Sinorhizobium.
40
3.3.2. Symbiotic response across native range distributions
We did not find a significant interaction between native habitat distribution and strain for
symbiotic response (Table S3.6). Acacia species that are widespread in their native range
(>1,000 herbarium records) and more limited in distribution (<1,000 herbarium records) did not
differ in symbiotic response for individual strains (Figure 3.3). The best models based on AIC
values indicated that symbiotic response of acacias with different range distributions varied
based on strains with which they were grown as well as differences in range within their native
continent.
50
Limited
rest
Widespread
wide
Symbiotic response
40
30
20
10
N+
N-
12R
11S
10S
09S
08B
07R
06B
05S
04B
03B
02B
01B
0
Strains
Figure 3.3. Symbiotic response for species in limited versus widespread native range
distributions among rhizobial strains. Strains marked as B = Bradyrhizobium, R = Rhizobium,
and S = Sinorhizobium.
41
3.3.3. Nodulation response across invasiveness categories and host species
We found a significant interaction between invasiveness category and strain for
nodulation presence (Table S3.7) indicating that nodulation presence for an individual
invasiveness category was dependent on strain with which the invasive group was grown. While
nodulation presence differed within individual invasiveness categories, it only differed for one
strain among invasiveness categories. In other words, for each individual rhizobial strain tested
except one, we found no significant difference in nodulation presence among invasiveness
categories. When examined individually, invasive and naturalized groups showed 50% or greater
nodulation for eight strains, and non-invasive group for ten strains (Figure 3.4).
% nodulation presence
Invasive
Naturalized
Non-Invasive
1.00
1.00
1.00
0.75
0.75
0.75
0.50
0.50
0.50
0.25
0.25
0.25
0.00
0.00
01B 02B 03B 04B 05S 06B 07R 08B 09S 10S 11S 12R
0.00
01B 02B 03B 04B 05S 06B 07R 08B 09S 10S 11S 12R
01B 02B 03B 04B 05S 06B 07R 08B 09S 10S 11S 12R
Strains
Figure 3.4. Percent nodulation for each invasiveness category among rhizobial strains. Strains
marked as B = Bradyrhizobium, R = Rhizobium, and S = Sinorhizobium.
Individual host species differed in nodulation presence depending on the strain with
which they were paired. In general, nodulation success was high; all species showed 50% or
greater nodulation for eight or more strains, except for two species: A. colei (only one strain >
50%) and A. murrayana (three strains > 50%) (Figure S2). For species that had a high symbiotic
response, nodulation was consistently high. However, our results also showed that effective
nodulation does not always translate to a high symbiotic response.
42
We found a significant interaction between invasiveness category and strain for
nodulation index (Table S3.8). The invasive group had a higher nodulation index than
naturalized and non-invasive groups for the same two strains. For all other strains, nodulation
index did not differ significantly among invasiveness categories (Figure S3.3).
3.3.4. Nodulation response across native range distributions
We found a significant interaction between native habitat distribution and strain for
nodulation presence (Table S3.9). Models predicting nodulation index that included native
habitat distribution, strain, and an interaction between the two received similar support (Table
S3.10). Acacia species that vary in distribution did not differ in nodule presence for individual
strains (Figure 3.4), and differed in nodule index for only one strain.
3.4. DISCUSSION
The goal of this study was to examine whether invasive acacias are more promiscuous
hosts with regard to their rhizobial symbionts than naturalized and non-invasive acacias. We
found that Acacia species categorized as invasive were able to effectively associate with a
significantly greater number of arbitrarily selected rhizobial strains than species that have so far
proven to be non-invasive. This is important because acacias that are more promiscuous hosts
may more readily find suitable symbionts when they are introduced to novel ranges, hence
facilitating their establishment and subsequent range expansion (Rodríguez-Echeverría et al.,
2009). Fidelity for specific rhizobial partners may thus be an important trait contributing to
variation in species invasiveness.
Invasive Acacia species had more consistent patterns of positive symbiotic response to
rhizobial strains with which they were paired (i.e. all invasive Acacia species had a significantly
greater symbiotic response than their N– control for the same five strains), whereas species
43
within naturalized and non-invasive groups were more variable in their response to different
strains. This indicates that Acacia species that become invasive are more generally able to
associate with a broader set of rhizobial strains, supporting our hypothesis that invasive acacias
are generally more promiscuous hosts.
Our results are consistent with findings of Birnbaum et al. (2012), who examined the
ability of multiple invasive Acacia species to develop effective symbiotic associations with
rhizobia from soils in which hosts do not naturally occur in Australia. Invasive Acacia species
were not limited in plant growth response when grown with biota from non-native soils, although
rhizobial community composition did vary among sites sampled. In contrast, Wandrag et al.
(2013) examined the acacia-rhizobia symbiosis using three host species introduced into New
Zealand that vary in invasiveness, and found that Acacia species growing in unfamiliar soils in
their introduced range suffered reduced growth and nodulation, regardless of invasive status.
Acacias in Australia may be more likely to find compatible rhizobial symbionts (i.e. those that
are more closely related to their native symbiotic strains) on their native continent, even in
unfamiliar soils, than in more geographically distant regions such as New Zealand, which may be
driving the difference in results seen between these two studies. Although not the specific focus
of this study, our results differed from those of Thrall et al. (2000), who found that less common
acacias within Australia tended to be more specific rhizobial hosts than more widespread Acacia
species. Further research is needed to examine the extent to which geographic origin of rhizobial
strains influences Acacia species symbiotic responses.
Other mutualistic factors besides promiscuity may play a role in influencing invasion
success of Acacia species abroad. In particular, recent studies have indicated that some invasive
Acacia species have been introduced to novel ranges concurrent with their native rhizobial
44
symbionts (Rodríguez-Echeverría, 2010; Ndlovu et al., 2013; Crisóstomo et al., 2013).
Widespread distribution of other mutualistic soil organisms may also influence invasive success
of introduced plant species. Schwartz et al. (2006) highlighted the potential unintended
consequences of introduction of mycorrhizal fungi outside their native range as inoculum for
agricultural and restoration purposes. Wide-scale introduction of symbiotic rhizobial and
mycorrhizal organisms may increase probability of non-native species encountering their native
symbionts in their novel range (Schwartz et al., 2006). Co-introduction of symbiotic organisms
bypasses the need for species to develop novel associations with new partners, and may facilitate
rapid establishment and colonization of these species. Additional research is necessary to
examine whether co-introduction of symbionts is a widespread phenomenon in acacia
introduction history. However, we note that even in the case of co-introductions, ability to
effectively associate with a broad range of rhizobia may be important, as studies have shown that
within-species variability in response to different rhizobial strains can limit growth of some
Acacia species (Burdon et al., 1999).
Even though our results support the idea that more invasive species are more
promiscuous hosts, it is possible that reduction in suitable symbionts due, for example, to greater
distance in relatedness between rhizobia from more widely distributed regions, will limit growth
of even the more promiscuous Acacia species. We note that all rhizobial strains used in this
study were Australian in origin. Future work would benefit from examining growth responses of
additional Acacia species to native rhizobial strains, as well as additional strains from regions
where these species are invasive. Acacia species may be more likely to associate and have a
beneficial growth response with rhizobial strains that occur in their native range, even if they do
not normally occur with those strains. Recent evidence has shown that Acacia species that are
45
invasive outside of their native ranges within Australia are still able to find suitable rhizobial
symbionts in regions where they were introduced (Birnbaum et al., 2012), however,
effectiveness of this symbiosis may vary when acacias associate with more distantly related
rhizobial strains (Rodríguez-Echeverría et al., 2012). Bacterial strains that occur in the same
region may be more closely related to one another, hence plant hosts may be more capable of
developing effective symbioses with strains occurring within their native range (but see Barrett
et al., 2012). In a recent examination of fungal communities occurring with Acacia species that
have become invasive within their native continent of Australia, Birnbaum et al. (2014) found
that, of the four Acacia species examined, three species associated with similar soil fungal
communities, including symbiotic mycorrhizal fungi, in their native and invaded populations.
This suggests that soil fungal, and possibly also soil bacterial communities within these hosts’
native continent can be broadly distributed, and plant species may encounter the same or closely
related strains in unfamiliar habitats within their larger continental range (Rodríguez-Echeverría
et al., 2012). Examining growth responses of Acacia species when paired with rhizobial strains
with which they occur in their introduced range may allow us to determine whether patterns of
symbiotic response found here hold true on a larger geographic scale, and provide valuable
insight towards potential differences in symbiotic effectiveness with more distantly related
rhizobial strains.
As in all short-term ecological experiment conducted in controlled environments, it is
useful to consider the extent to which their results are relevant to long-term, real-world
conditions. Our experiment examined plant growth response over a three-month period, covering
early phase growth of different Acacia species. It has been shown that seedling and juvenile
phases are key for establishment and survival of plants in challenging environments (Thrall et
46
al., 2005; Barrett et al., 2012). Therefore, measuring plant performance over this early growth
phase is likely to accurately predict relative establishment success of Acacia species in areas
where they are introduced abroad.
Level of nodulation did not differ greatly among invasiveness categories, nor did
nodulation index, and was generally high for all groups. For specific hosts, however, nodulation
success (as measured by presence of nodules) differed, with certain species nodulating
successfully with a greater number of strains than others. These results, combined with symbiotic
response results indicate that, whereas species in all invasiveness categories are capable of
developing symbiotic associations with different rhizobial strains, those associations may not
translate into an effective symbiotic response. In other words, even though species in different
invasiveness categories can develop nodules with multiple strains, nodulation presence per
se does not necessarily translate to a higher growth response. Effective rhizobial association may
require nodulation, but then additional factors may determine overall effectiveness of the
association, i.e. the extent to which the association confers increased plant performance and thus
potential for population expansion.
Our study supports the hypothesis that host promiscuity with rhizobia, with regard to
symbiotic response, is one mechanism that contributes to Acacia species invasion. Invasive
Acacia species were more consistently able to form effective symbiotic relationships with more
rhizobial strains than naturalized and non-invasive species, suggesting that they are less
constrained in finding suitable symbiotic rhizobial partners when introduced abroad. However,
some non-invasive Acacia species (e.g. A. bivenosa and A. stenophylla) were also promiscuous
hosts. It may be that fewer individuals of these species have been introduced abroad (i.e. lower
propagule pressure), and that limited propagules constrained their colonization and expansion in
47
novel regions (Williamson, 1996; Simberloff, 2009). These species, which are currently less
widespread on a global scale, should be monitored closely for further expansion.
When Acacia species are introduced outside their native range and subsequently escape
intended use they have significant potential for negative impacts in both natural and managed
environments (Gaertner et al., 2009; Yelenik et al., 2004). A relatively small proportion of
Acacia species introduced outside their native range are currently recognized as invasive
(Richardson et al., 2011), however many more species have become naturalized and may have
the potential to become invasive. Our results highlight the importance of monitoring and
stopping intentional movement of naturalized and non-invasive Acacia species, particularly that
are promiscuous hosts, to avoid potential future invasion of these species. Acacia species
continue to be introduced around the world for purposes such as ornamental trade, timber, woodfuel, etc. (Kull et al., 2011). Our research underscores the need to test species before introducing
them to novel regions to determine whether they are promiscuous hosts, thereby circumventing
further spread. In their native range, however, many Acacia species are commonly used to
restore degraded lands (Murray et al., 2001), and promiscuous hosts may be excellent candidates
for use in restoration projects.
48
CHAPTER 4. PROVENANCE OF RHIZOBIAL SYMBIONTS IS SIMILAR FOR
INVASIVE AND NON-INVASIVE ACACIAS IN CALIFORNIA
4.1. INTRODUCTION
Species that are introduced to novel regions around the world often become isolated from
their native mutualists. Those species that are introduced concurrent with their native mutualistic
partners may have a competitive advantage over other non-native or native organisms in their
novel range (Richardson et al., 2000). One category of co-introduced mutualistic organisms is
plants and their microbial mutualistic symbionts (Rodríguez-Echeverría et al., 2011). Bacterial
mutualists may be transported along with plant materials in accompanying soils, on seeds, or as
intentional inoculants to improve establishment of species abroad (Pérez-Ramírez et al., 1998,
Bala et al., 2003). The co-introduction of plant species with their microbial mutualists may
influence their effective establishment, colonization, and subsequent invasion in novel ranges.
Legumes in particular may benefit from co-introduction to a novel range with their
bacterial mutualists (Parker, 2001). Legumes have a beneficial symbiotic relationship with
nitrogen-fixing bacteria (i.e. rhizobia) that influences their establishment success, particularly in
low-nitrogen habitats (Sprent, 2001). Recent research indicates that legumes that have become
invasive in novel ranges have been co-introduced with their native rhizobial mutualists
(Rodríguez-Echeverría, 2010; Rodriguez Echeverria et al., 2011; Crisóstomo et al., 2013;
Ndlovu et al. 2013). Availability of native beneficial symbiotic organisms may promote the
invasive potential of certain legumes introduced abroad.
Acacias are legumes native to Australia that have been introduce widely to regions
outside their native range. They are a model system for examining mechanisms by which suites
of introduced species become invasive (Richardson et al., 2011). Over 1,000 Acacia species
occur in Australia (Miller et al., 2003), 386 of which have been introduced around the world for
49
a variety of purposes, including forestry, fuel wood, erosion control, and for ornamental use
(Richardson et al., 2011). In areas where they are introduced, acacias range from highly invasive
(23 species), naturalized (48 species), to non-invasive (315 species), with some species causing
drastic changes to natural areas, and others having minor impact (Richardson et al., 2011;
Richardson & Rejmánek, 2011). Understanding the driving forces behind why certain Acacia
species are successful invaders and why others are not is important for developing methods to
control their spread, and can elucidate the mechanisms behind legume invasion in general.
One region where multiple Acacia species have been introduced abroad is the state of
California (USA). Acacias were introduced to California beginning in the mid-1800s through the
ornamental trade (Butterfield, 1938). There are currently 16 Acacia species that occur and vary
in invasiveness in this region (two invasive, five naturalized, and nine non-invasive) (Cal-IPC,
2006; Jepson eFlora, 2015; CalFlora, 2015). While Acacia species are differentially invasive
within California, all species introduced to this region except for one have become invasive in at
least one part of the globe (Richardson et al., 2011). The difference in invasiveness of Acacia
species within the localized region of California provides the opportunity to test mechanisms of
invasion by comparing multiple closely related species and examining what drives their
differential establishment and colonization success abroad.
Mutualisms are important in the life history of Acacia species and may have an important
role in their ability to invade novel regions. Acacias are recognized as having mutualistic
relationships with a range of species, including ants that protect them from herbivores (Holmes,
1990) and birds that disperse their seeds (Glyphis, et al. 1981), but it is their interaction with
nitrogen-fixing soil bacteria (i.e. rhizobia), that may be particularly important to their
establishment and colonization success in novel ranges. Rhizobia are gram-positive bacteria that
50
occur in the soil and infect roots of many legumes, creating nodules on the roots in which the
rhizobia are housed (Beringer et al., 1979). Rhizobia fix atmospheric nitrogen and convert it to
amino acids and proteins, which are usable by the plant; in turn, the plant provides carbon
substrates to the bacteria (Sprent & Sprent, 1990; Sprent, 2001). For Acacia species that are
capable of finding effective symbiotic rhizobia, this interaction provides an available source of
nitrogen for the plant. Acacias that can find mutualistic bacterial partners in their novel range
may have a competitive advantage when introduced abroad.
The availability of suitable rhizobia may be essential for legume establishment in novel
ranges. Parker et al. (2001) developed models to predict the likely success of legume invasion in
novel ranges under different rhizobial availability. He found that, without adequate rhizobial
partners, legumes may fail in their ability to colonize novel ranges. While acacias may find
suitable rhizobial partners in their novel range, if they are co-introduced with their native
mutualistic symbionts, they may be able to bypass developing novel rhizobial associations.
Availability of their native mutualists also circumvents constraints that arise if acacias do not
encounter beneficial novel symbiotic partners. Co-introduction of acacias with their native
rhizobia may facilitate their establishment in areas where they are introduced.
The acacia-rhizobia symbiosis is especially useful for testing two contemporary
hypotheses that focus on the role of mutualisms in invasions: the Invasional Meltdown
Hypothesis (Simberloff & Holle, 1999) and the Enhanced Mutualism Hypothesis (Reinhart &
Callaway, 2006). The Invasional Meltdown Hypothesis predicts that exotic species facilitate
each other’s establishment and colonization abroad. Rodríguez-Echeverría (2010) found
evidence for this in Portugal, where invasive Australian Acacia species have been introduced
with their Australian rhizobial mutualists. Ndlovu et al. (2013) and Crisóstomo et al. (2013) also
51
found Acacia species in South Africa and Portugal respectively associating with rhizobia of
Australian origin. The Enhanced Mutualism Hypothesis suggests that exotic species benefit from
mutualisms that originate in their introduced range (Reinhart & Callaway, 2006). If Acacia
species are not introduced abroad with their native rhizobial mutualists, those that become
invasive may be more capable of developing associations with rhizobia native to their novel
range. By studying groups of closely related introduced species that vary in their invasion
success, there is the potential to pinpoint specific mechanisms that enable certain species to
invade.
The ability of acacia species to associate with a wider diversity of rhizobial strains,
hereafter called “host promiscuity”, may also influence the invasiveness of introduced Acacia
species. Those species that can associate with more rhizobial strains may more easily find
compatible rhizobial mutualists when introduced abroad (Thrall et al., 2000). Higher host
promiscuity has been show in acacias that have become invasive in multiple regions of the world
(Klock et al., 2015), as well as acacias that are more widely distributed in their native continent
(Thrall et al., 2000). The co-introduction of non-native acacias species with their native rhizobial
symbionts, paired with the ability to associate with a wider diversity of rhizobial strains should
their native symbionts not be available, may greatly promote the ability of certain Acacia species
to become invasive when introduced abroad.
The aim of this study is to examine the role of rhizobial provenance and diversity of
rhizobial symbionts in the establishment success of Acacia species introduced abroad. I took a
phylogenetic approach, studying three different loci to assess whether invasive acacias were
introduced abroad with their native rhizobial mutualists. I selected four Australian Acacia
species that differ in invasiveness in California (two invasive and two non-invasive), and
52
identified rhizobial strains associating with each species in their native and novel ranges using
three genes, 16S rRNA, nifD, and nodC. The 16S rRNA gene has been commonly used for
identification of bacterial genera associated with legumes, whereas the nifD and nodC genes
have been used to determine biogeographic placement of rhizobial strains (Parker et al., 2002;
Qian et al., 2003; Moulin et al., 2004; Stępkowski et al., 2005; Andam & Parker, 2008). I
hypothesized that provenance and diversity of rhizobia associating with acacias in California
would influence acacia invasiveness with the following predictions: 1) invasive acacias would be
found more commonly associating with rhizobia of Australia origin; 2) non-invasive acacias
would be found more commonly associating with rhizobia of California origin; and 3) invasive
acacias would be more promiscuous hosts, associating with a greater diversity of rhizobial strains
where they are native and where they are introduced abroad.
4.2. METHODS
4.2.1. Study species
I examined bacterial communities in the nodules of four Acacia species native to
Australia: two Acacia species that have shown indication of invasiveness (A. dealbata and A.
melanoxylon) in California (CalFlora, 2015; Cal-IPC, 2006), and two species that have not
become invasive in this region (A. longifolia and A. verticillata) (Jepson eFlora, 2015). In
California, A. dealbata and A. melanoxylon occur along the entire coastal region. Acacia
dealbata has expanded inland as well. Acacia longifolia and A. verticillata occur along the
California coast from the San Francisco Bay Area south to San Diego (Jepson, eFlora, 2015).
Within their native continent, all Acacia species examined here are widely distributed (i.e.
>1,000 herbarium records as reported by the Australian Virtual Herbarium [AVH, 2015]). They
53
are found primarily in southeast Australia, with A. melanoxylon extending further north (AVH,
2015) (Figure 4.1).
4.2.2. Bacterial isolation
I collected nodules from the roots of four Acacia species occurring in northern California
in August 2012 and 2013. Nodules were collected from nine A. dealbata populations, 18 A.
melanoxylon populations, five A. longifolia populations, and two A. verticillata
Figure 4.1. Distribution maps of Acacia species used in this experiment in their native continent
of Australia (based on herbarium records from the Australian National Herbarium, Canberra,
Australia [AVH, 2015]).
populations (Table 4.1). Variation in number of collection sites among species was due to
relative differences in presence of these species in this region. Nodules were obtained from one
to five plants per population by carefully digging ~30 cm around the base of seedlings or adult
plants. When seedlings were present in a population, the entire plant was excavated, attached dirt
was gently removed, and nodules were clipped from the roots, leaving 0.5 cm of root tissue
54
Table 4.1. Collection sites for rhizobial strains isolated from Acacia species in Australia and
California. “-” indicates strains that were collected prior to this study and used in this analysis,
but for which precise GPS coordinates are not available.
Acacia species
longifolia
longifolia
longifolia
longifolia
longifolia
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
longifolia
longifolia
Location
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
AU
CA
CA
CA
CA
CA
CA
CA
CA
CA
CA
CA
Description
Durras Beach, NSW
Bawley Point, NSW
Kioloa, NSW
Lake Tabourie, NSW
Mollymook, NSW
Bemboka, NSW
Ben Lomand, NSW
Captains Flat, NSW
Inglis, TAS
Kandos, NSW
Licola, VIC
Snug, TAS
Highlands, VIC
Huon, TAS
Lileah, TAS
Mt Coree, ACT
Mt Gambier, SA
Mt Lindsay, NSW
Nabowlah, TAS
Otways, VIC
Shelly's Forest Rd., NSW
Green Cape Rd., NSW
Princes Hwy., NSW
Nadgee, NSW
Nadgee, NSW
Nadgee, NSW
Nadgee, NSW
Rocca Rd., Fairfax
Stapleton, San Rafael
Ray Court, San Rafael
San Geronimo
Lagunitas
Inverness
Magnolia Ave., Larkspur
Acacia Ave., Larkspur
Hopland
Civic Center, San Rafael
Headlands, Sausalito
55
Latitude
-35.636056
-35.50005
-35.551244
-35.4178
-35.367419
-37.151361
-37.202172
-37.202386
-37.367672
-37.367669
-37.369161
-37.400769
37.988074
37.975977
37.986147
38.01571
38.01191
37.98676
37.935111
37.930867
38.968628
37.999334
37.825209
Longitude
150.285525
150.384494
150.369128
150.416764
150.46765
149.889083
149.950731
149.833767
149.766894
149.766894
149.767944
149.817433
-122.588088
-122.554143
-122.554928
-122.659583
-122.702268
-122.598402
-122.534847
-122.532236
-123.102528
-122.534717
-122.528488
(Table 4.1 continued)
Acacia species Location
longifolia
CA
longifolia
CA
longifolia
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
melanoxylon
CA
verticillata
CA
verticillata
CA
Description
Mar West, Tiburon
Bolinas Rd., Fairfax
Overlook Rd., Bolinas
Cypress Rd., Fairfax
Concrete Pipe, Fairfax
Salmon bridge, Fairfax
Bolinas Rd. 2, Fairfax
Inverness
Limantour Rd., Inverness
Scenic Rd., Fairfax
Marin Catholic, Kentfield
Acacia Ave., Larkspur
Camino Alto, Mill Valley
Old St. Hilary's, Tiburon
Paradise Cove, Tiburon,
Paradise Cove, Tiburon
Muir Beach, CA
Ft. Baker, Sausalito
MAHE Institute
MAHE Stables
China Camp, San Rafael
Old St. Hilary's, Tiburon
Headlands, Sausalito
Latitude
37.825209
37.978081
37.905064
37.98003
37.974651
37.981691
37.984407
38.088338
38.055023
37.98676
37.952052
37.930867
37.900397
37.879044
37.89542
37.893685
37.878435
37.83452
37.832203
37.83127
38.004596
37.879044
37.828511
Longitude
-122.528488
-122.596856
-122.696508
-122.598659
-122.612193
-122.592959
-122.591351
-122.841948
-122.838058
-122.598402
-122.537707
-122.532236
-122.518592
-122.457269
-122.466078
-122.462559
-122.553632
-122.481454
-122.533457
-122.514941
-122.471212
-122.457269
-122.532267
on either side of the nodule (Somasegaran & Hoben, 1994). For adult trees, nodules were
extracted from roots attached to the base of the plant. Nodules were stored in collection vials
containing silica gel and sterile cotton wool and maintained at room temperature for one month
until use, as recommended by Somasegaran & Hoben (1994).
To culture bacteria, desiccated nodules were first rehydrated by soaking in sterile water
overnight. One to three nodules per plant were individually surface sterilized by immersion in
95% ethanol for less than ten seconds, followed by soaking in 3% NaCl for two to four min, and
then rinsed at least five times with sterile water (Somasegaran & Hoben, 1994). Nodules were
individually crushed using sterile plastic pestles. 100 mL of the suspension was transferred to
56
plates containing yeast mannitol agar (YMA; 5.0 g Mannitol, 0.5 g K2HPO4, 0.2 g MgSO4.7H2O,
0.1 g NaCl, 0.5 g yeast extract, 1 L distilled H2O, 20.0 g agar) (Somasegaran & Hoben, 1994).
Plates were incubated at 25 °C for up to 21 days. Single well-isolated colonies were selected
from original plates and successively subcultured two to three times to obtain pure cultures. Pure
cultures were stored on YMA plates at 4°C and also at -80°C in in YMA broth supplemented
with 20% glycerol for long-term storage.
Root nodules were collected from A. longifolia and A. verticillata in Australia in April
2013. Nodules were collected from five populations of each species, and from four to five plants
per population, stored in plastic bags and kept on ice, then transferred to 4 °C for no longer than
two days before processing. Three nodules per plant were processed and bacterial isolates were
cultured as described above. Bacterial isolates from A. dealbata and A. melanoxylon root nodules
were obtained from cultures stored in the CSIRO permanent culture collection. These isolates
were originally obtained during a collection expedition across the east coast of Australia and
Tasmania from 1993 – 1995, as described by Burdon et al. (1999). I cultured 19 isolates from ten
A. dealbata populations, and 16 isolates from nine A. melanoxylon populations.
To extract DNA from pure cultures, single isolates were suspended in 100 mL TE buffer
and boiled at 95 °C for 5 min. Extractions were examined for concentration and integrity of
DNA using gel electrophoresis in 1% agarose in 1X TBE buffer. Lysed cells were stored at 4 °C
until use but not longer than 4 h.
4.2.3. PCR and sequencing
To determine phylogenetic and biogeographic placement of rhizobial strains associating
with different Acacia species, I sequenced three genes from DNA extractions including 16S
rRNA, nifD, and nodC. The 16S rRNA gene was used for bacterial identification and
57
phylogenetic placement. The 16S rRNA gene (~1,200 bp) was amplified using the primers
forward 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and reverse 1429R (5'TACGGYTACCTTGTTACGACTT-3') (Lane et al. 1985) for cultures obtained in California,
and forward GM3 (5'-AGAGTTTGATCMTGGC-3') and reverse GM4 (5'TACCTTGTTACGACTT-3') (Muyzer et al., 1995) for cultures obtained in Australia. PCR
program was as follows: initial pre-denaturation cycle at 94 °C for 5 min, followed by 35 cycles
of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 2 min,
and a final extension at 72 °C for 10 min. Reactions were carried out in a volume of 50 µL
containing: 50 ng template DNA, 0.2 µM of each primer, 200 µM of each dNTP (Astral
Scientific, Taren Point, Australia), 4.8 mM MgCl2, and 0.3 U of Taq DNA polymerase (New
England Biolabs, Ipswich, MA, USA) in Taq DNA polymerase reaction buffer, using a Hybaid
PCR Express thermocycler (Integrated Sciences, Willoughby, Australia).
I also sequenced the nifD (~620 bp) and nodC (~620 bp) genes, which play a role in
nitrogen fixation and root nodulation respectively (Sprent, 2001). I selected a subset of samples
identified by the 16S rRNA analysis as Bradyrhizobium and Rhizobium to use for the
biogeographic analysis. These genera were predominately identified in our analysis as
associating with acacias in both California and Australia, and primers, as well as existing
sequences are readily available to identify variation in genetic and biogeographic structure of
these rhizobial genera. The gene nifD was amplified using the primers forward nifD2F (5'CATCGGIGACTACAAYATYGGYGG-3'] and reverse nifD1R (5'-CCCAIGARTGCATYT
GICGGAA-3') (Fedorov et al., 2008). PCR touchdown program was as follows: initial predenaturation cycle at 96 °C for 2 min, followed by 7 cycles of denaturation at 96 °C for 30 s,
annealing at 55 °C* for 45 s (*reduced by 1 °C for each cycle), extension at 45 °C for 1 min,
58
followed by 25 cycles of denaturation at 96 °C for 30 s, annealing at 48 °C for 45 s, extension at
72 °C for 90 s, and a final extension at 72 °C for 5 min. The gene nodC was amplified using the
primers forward nodCF540 (5'-TGATYGAYATGGARTAYTGGCT-3') and reverse
nodCR1160 (5'-CGYGACARCCARTCGCTRTTG-3') (Sarita et al., 2005). PCR touchdown
program was as follows: initial pre-denaturation cycle at 96 °C for 2 mins, followed by 2 cycles
of denaturation at 96 °C for 20 s, annealing at 63 °C for 30 s, extension at 72 °C for 30 s; 2
cycles of denaturation at 96 °C for 20 s, annealing at 62 °C for 30 s, extension at 72 °C for 35 s;
3 cycles of denaturation at 96 °C for 20 s, annealing at 59 °C for 30 s, extension at 72 °C for 40
s; 4 cycles of denaturation at 96 °C for 20 s, annealing at 56 °C for 30 s, extension at 72 °C for
30 s; 5 cycles of denaturation at 96 °C for 20 s, annealing at 53 °C for 30 s, extension at 72 °C
for 50 s; 25 cycles of denaturation at 96 °C for 20 s, annealing at 50 °C for 30 s, extension at 72
°C for 60 s, and a final extension at 72 °C for 5 min.
Aliquots of 5 µL of the PCR product were examined for successful amplification and
concentration using gel electrophoresis in 1% agarose in 1X TBE buffer. PCR product for
bacterial isolates obtained in California were sequenced by Beckman Coulter Genomics
(Danvers, MA, USA), and by the John Curtain School of Medical Research for isolates obtained
in Australia (Canberra, Australia).
4.2.4. Phylogenetic analysis
Nucleotide sequences were assembled using Geneious v. 7.1.7 and aligned with ClustalW
(Larkin et al., 2007). I conducted a BLAST search of the 16S rRNA, nifD, and nodC gene
sequences to verify identity as rhizobia and to compare our sequences with those available in
Genbank (http://blast.ncbi.nlm.nih.gov). Cultures that were identified by the 16S rRNA gene as
rhizobia were selected for further biogeographic analysis using nifD and nodC sequences.
59
Sequences with the highest similarity values for each gene respectively were used in
phylogenetic analyses.
I clustered 16S rRNA sequences into Operational Taxonomic Units (OTUs) using the
software program Uclust (Edgar, 2010) at 97% similarity. I then blasted OTU representative
sequences as well as the complete alignment against a curated alignment of 16S rRNA gene
sequences from the Greengenes database (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi). I also
clustered nifD and nodC sequenced into OTUs as above. I conducted a maximum likelihood
(ML) phylogenetic analysis for each gene using RaxML software (Stamatakis, 2014), using the
nucleotide substitution general time reversible (GTR) model for 16S rRNA; for nifD and nodC
analyses I used GTRCAT with two partitions (partition 1, first and second codon; partition 2,
third codon). Support for ML analysis was estimated using 1000 bootstrap replicates. I used
FigTree software v. 1.4.2 to edit the phylogenetic tree.
4.2.5. Rhizobial richness and abundance
I analysed data obtained from the 16S rRNA OTU analysis to determine whether there
was a difference in rhizobial species associating with different Acacia host species between
native and novel continents. I also analysed data from the nifD and nodC phylogenetic analyses
to determine whether there was a difference in the biogeographic structure of rhizobial strains
identifying with Acacia species between continents and among invasiveness categories within
their introduced region of California using Principal Coordinate Analysis (PCoA) with an
unweighted UniFrac distance matrix. I used the R statistical package ‘phyloseq’ (McMurdie &
Holmes, 2013) to conduct ordinations. I tested for differences in the genetic structure of rhizobial
strains between continents and among invasiveness categories using PerManova. Ordination
60
analyses were conducted using R statistical programming language version 3.2.0 (R Core Team,
2015), and PerManova analyses were conducted using QIIME (Caporaso et al., 2010).
4.3. RESULTS
4.3.1 Culture and molecular identification of nodulating bacteria
I obtained a total of 299 isolates identified as nitrogen-fixing root nodule bacteria by the
16S rRNA gene from the four Acacia species examined in this study. Additional reference
sequences were obtained from rhizobia nodulating with other Acacia species native to Australia.
Isolates obtained corresponded to 8 OTUs based on 16S rRNA gene; two OTUs were from
reference rhizobial strains previously collected in Australia, the remaining six were from strains
isolated from Acacia species of interest in this study. Phylogenetic analysis of the 16S rRNA
gene showed the majority of isolates clustering with rhizobia in the genus Bradyrhizobium
(OTU1 and OTU8, 283 isolates) (Figure 4.2). I also found isolates clustering with the genera
Mesorhizobium (OTU4, 3 isolates), Rhizobium (OTU2, OTU5 and OTU7, 29 isolates),
Sinorhizobium (OTU3, 10 isolates), and Phyllobacterium (OTU6, 3 isolates) (Table 4.2,
Supporting Table S4.1).
4.3.2 nifD and nodC
Amplification of the nifD gene was successful in a subset of isolates identified by the 16S
rRNA gene as rhizobia (242 isolates total; 133 from Australia and 109 from California). The
nifD sequences were clustered into 19 OTUs. The majority of isolates were identified as a nifD
gene amplified from Bradyrhizobium sp.1 (~93% total; ~94 % CA; ~92% AU), a slow-growing
rhizobium commonly found associating with acacias in Australia (Lafay & Burdon, 1998, 2001).
The remainder of isolates were identified as Rhizobium (~7% total; ~6% CA; ~8% AU).
61
44
Xanthobacter flavus X94199
Azorhizobium sp. AF288311
Azorhizobium doebereinerae AF391130
Labrys sp. AF008564
Labrys methylaminiphilus AY766152
Afipia genosp. U87762
Bradyrhizobium elkanii X70402
Afipia broomeae U87759
100
67
Afipia sp. M69186
Afipia felis M65248
68
60
94Nitrobacter sp. L35506
74
80 Nitrobacter winogradskyi AM286377
Nitrobacter alkalicus AF069957
44
92
Nitrobacter vulgaris AY055795
Nitrobacter hamburgensis L11663
97 Bradyrhizobium sp. 2; OTU 8
Bradyrhizobium
canariense AF363142
22
64
87 Bradyrhizobium sp. AF230719
36
100 Bradyrhizobium sp. AF338176
59
Blastobacter sp. S46917
62
Bradyrhizobium yuanmingense Z94804
1754
Bradyrhizobium sp. 1; OTU 1
Bradyrhizobium sp. X87273
91 Afipia genosp. U87766
100
Rhodopseudomonas sp. D25312
82
Rhodopseudomonas oryzae AF416660
Balneimonas sp. AB098515
85
Bosea sp. U87773
100
85
Bosea vestrisii AF288306
Afipia genosp. U87775
Kaistia sp. AY039817
Parvibaculum sp. AY007683
37
Chelativorans multitrophicus NZ AAED02000003
Mesorhizobium alhagi DQ319189
97
Nitratireductor sp. DQ659453
Nitratireductor aquibiodomus AF534573
63
45
43
Pseudaminobacter salicylatoxidans AF072542
25
Pseudaminobacter sp. D32248
53
97
Aquamicrobium sp. AF323256
3
Defluvibacter sp. AJ132378
67
Chelativorans sp. EF157205
Aminobacter sp. AJ011759
17 76
Mesorhizobium ciceri X67230
36
Mesorhizobium temperatum AJ417091
75
86 Mesorhizobium sp; OTU 4
86
65
Mesorhizobium sp. X68391
Mesorhizobium huakuii AY225390
Mesorhizobium albiziae DQ100066
Phyllobacterium leguminum AY785323
38
65
55
87Phyllobacterium sp. D12789
96
Phyllobacterium myrsinacearum EU169165
55 Phyllobacterium ifriqiyense AY785325
Phyllobacterium brassicacearum; OTU 6
89 Sinorhizobium arboris AM181746
50 Sinorhizobium sp. 1; OTU 3
Sinorhizobium sp. D12783
Sinorhizobium saheli X68390
88
Sinorhizobium kostiense AM181750
20
Martelella mediterranea AY345411
40
Shinella granuli AB187585
76 Shinella sp. D14255
16
Shinella zoogloeoides AB248361
15
Rhizobium sullae U89823
6 Rhizobium sp. 3; OTU 7
84
33
97
Rhizobium multihospitium AY038615
48
55
Rhizobium etli U28916
44
Rhizobium leguminosarum U29386
13
59
62
Rhizobium mesosinicum DQ310796
Mycoplana sp. DQ419570
47
30
71
Agrobacterium vitis U28505
25
Rhizobium undicola Y17047
70
Rhizobium galegae D11343
3
Mycoplana peli EF125188
Agrobacterium rubi D14503
90
90
55
Agrobacterium larrymoorei Z30542
41
87
14
Rhizobium sp. AJ004859
24
Rhizobium sp. 2; OTU 5
38
25
Rhizobium giardinii AY177365
86
18
Agrobacterium sp. X73041
Rhizobium sp. X73042
Rhizobium sp. 1; OTU 2
Sinorhizobium terangae X68388
Sinorhizobium xinjiangense AF250353
Ensifer sp. D14516
Ahrensia sp. AJ582086
Candidatus Liberibacter americanus AY742824
100
92
Candidatus Liberibacter asiaticus L22532
99
Candidatus Liberibacter africanus L22533
100 Candidatus Liberibacter psyllaurous EU980389
Candidatus Liberibacter sp. EU921626
Ancylobacter sp. M62790
55
Xanthobacter autotrophicus X94201
Xanthobacter sp. AF532187
Azorhizobium caulinodans D13948
100
44
0.05
Figure 4.2. Phylogenetic tree based on the 16S rRNA gene. Isolates are clustered into operational
taxonomic units (OTUs) (sequences with 97% similarity or higher were considered the same
OTU). Species in “blue” indicate rhizobial species collected from nodules in this study. Final
optimized ML = -10820.69.
62
Table 4.2. Rhizobial genera associated with different Acacia species from different locations
based on the 16S rRNA gene. Genera were determined by comparison of sequences with
GenBank online database (http://blast.ncbi.nlm.nih.gov).
Location
AU
Species
A. dealbata
AU
A. melanoxylon
AU
A. longifolia
AU
A. verticillata
CA
A. dealbata
CA
A. melanoxylon
CA
A. longifolia
CA
A. verticillata
Rhizobia genera
Bradyrhizobium
Rhizobium
Uncult. bacterium
Agrobacterium
Bradyrhizobium
Uncult. bacterium
Agrobacterium
Bradyrhizobium
Mesorhizobium
Phyllobacterium
Rhizobium
Uncult. bacterium
Uncult. Rhizobium
Agrobacterium
Bradyrhizobium
Uncult. Agrobacterium
Uncult. bacterium
Bradyrhizobium
Phyllobacterium
Rhizobium
Uncult. bacterium
Agrobacterium
Bradyrhizobium
Mesorhizobium
Phyllobacterium
Rhizobium
Uncult. bacterium
Uncult. Rhizobium
Bradyrhizobium
Mesorhizobium
Bradyrhizobium
Rhizobium
63
No. Samples
17
1
1
1
12
3
1
37
1
1
1
3
1
2
48
2
1
36
1
1
6
1
34
1
1
8
5
1
40
1
27
3
% Sp/Loc
89
5
5
6
75
19
2
82
2
2
2
7
2
4
91
4
2
82
2
2
14
2
67
2
2
16
10
2
98
2
90
10
Amplification of the nodC gene was successful in a subset of rhizobial isolates (240
isolates total; 129 from Australia and 111 from California). Isolates clustered into 26 OTUs. As
with nifD, the majority of isolates were identified as a nodC gene amplified from
Bradyrhizobium sp. 1 (~98% total; ~97% CA; ~98% AU), with the reminder identified as
Rhizobium spp. (~2% total; 3% CA; 2% AU).
4.3.3. Host biogeographical pattern based on 16S, nifD, and nodC loci
I obtained 133 bacterial isolates from root nodules of acacias in Australia and 165 isolates
from root nodules of acacias in California. For A. dealbata I obtained 19 (AU) and 44 (CA)
isolates respectively, A. melanoxylon 16 (AU) and 51 (CA) isolates, A. longifolia 45 (AU) and 41
(CA) isolates, and A. verticillata 53 (AU) and 30 (CA) isolates.
The most common bacterial genus isolated from California and Australia acacia nodules
was Bradyrhizobium sp.1 (OTU1) (Table 4.2). This was true across all Acacia species I collected
nodules from, in both their native and introduced continents. Other rhizobial genera isolated
from acacia nodules included Mesorhizobium, Rhizobium, Phyllobacterium and Sinorhizobium
(only from reference nodules previously collected in Australia), although these genera were
found in lower abundance for all Acacia species in both continents). I did not detect a
geographical difference between rhizobial strains collected from nodules in Australia and
California based on the 16S rRNA phylogeny.
Phylogenetic analysis based on the nifD gene showed a similar pattern as 16S rRNA. Our
analysis showed that for multiple copies of the nifD gene, isolates collected from Australia and
California had the same genetic structure (Figure 4.3). Our analysis also showed that, for
multiple copies of the nifD gene, there were no differences in the genetic structure between
isolates collected from invasive and non-invasive acacias in California.
64
Phylogenetic analysis based on the nodC gene showed similar patterns as 16S rRNA and
nifD analyses. For multiple copies of the nodC gene I did not detect a difference among
continents or invasiveness categories in rhizobial genetic structure (Figure 4.4). This indicates
that there is little geographic difference in rhizobial isolates collected from different Acacia
species in Australia and California.
NIF_2
NIF_15
NIF_13
NIF_12
NIF_7
67
62
NIF_3
Invasive
NIF_17
NIF_1
85
a
Invasive
a
Native
a
Non−Invasive
NIF_10
Continent
NIF_9
Australia
NIF_0
California
NIF_14
Abundance
1
NIF_8
5
NIF_18
NIF_16
NIF_5
NIF_11
56
NIF_4
51
NIF_6
Figure 4.3. Phylogenetic tree based on the nifD gene. Isolates are clustered into operational
taxonomic units (OTUs) (sequences with 97% similarity or higher were considered the same).
Icon shape indicated the continent from which rhizobial isolates were collected (circle =
Australia and triangle = California) and color indicates invasiveness category (red = invasive,
green = native, and blue = non-invasive). Icon size indicated abundance of rhizobial isolates.
Final optimized ML = -4130.15.
4.3.4. Rhizobial richness and abundance in relation to host invasiveness
Acacia dealbata and A. melanoxylon, both of which are invasive in California, associated
with a wider diversity of strains than A. longifolia or A. verticillata in their introduced range
65
(Figure 4.5). In Australia, all species associated with the same number of bacterial genera except
for A. longifolia, which was found associating with a greater diversity of bacterial genera in its
native continent.
NOD_17
NOD_13
NOD_22
NOD_14
NOD_0
NOD_24
92
85
NOD_7
NOD_11
Invasive
66
NOD_18
NOD_12
NOD_10
a
Invasive
a
Native
a
Non−Invasive
NOD_20
Continent
NOD_25
79
Australia
NOD_21
California
NOD_15
84
NOD_1
Abundance
NOD_2
1
71
NOD_5
5
NOD_16
NOD_4
NOD_3
NOD_8
NOD_6
56
NOD_19
NOD_23
NOD_9
Figure 4.4. Phylogenetic tree based on the nodC gene. Isolates are clustered into operational
taxonomic units (OTUs) (sequences with 97% similarity or higher were considered the same
OTU). Icon shape indicated the continent from which rhizobial isolates were collected (circle =
Australia and triangle = California) and color indicates invasiveness category (red = invasive,
green = native, and blue = non-invasive). Icon size indicated abundance of rhizobial isolates.
Final optimized ML = 4716.35.
I did not detect a difference in the biogeographic structure of rhizobial strains collected
from Acacia species between California and Australia, based on the nifD (PerManova, F = 1.279,
P = 0.272; Figure 4.6, a) or nodC (PerManova, F = 1.022, P = 0.394; Figure 4.6, b) PCoA
analyses. In addition, I did not detect a difference in the genetic structure of rhizobial strains
66
Figure 4.5. Diversity of rhizobial strains associating with Acacia species in Australia (native
continent) and California (introduced region).
a) nifD
b) nodC
0.2
0.0
0.0
Continent
Continent
Australia
Axis.2 [16.3%]
Axis.2 [20.5%]
Australia
California
California
Host
Host
−0.1
dealbata
dealbata
longifolia
longifolia
melanoxylon
melanoxylon
−0.2
verticillata
verticillata
−0.4
−0.2
−0.1
0.0
0.1
−0.4
0.2
−0.2
0.0
Axis.1 [51.9%]
Axis.1 [65.4%]
Figure 4.6. Ordination of rhizobial strain identity and abundance for the a) the nifD and b) nodC
genes for rhizobial strains collected from Acacia species in Australia (circles) and California
(triangles) derived from PCoA base on a UniFrac matrix. Rhizobial strains more similar in
identity are closer together in ordination space.
67
collected from different invasiveness categories (i.e. invasive or non-invasive in California, and
native in Australia) for the nifD (PerManova, F = 1.262, P = 0.249) or nodC (PerManova, F =
1.864, P = 0.090) genes.
4.4. DISCUSSION
The goal of this study was to examine whether invasive and non-invasive Acacia species
were co-introduced to California with their native Australian rhizobial symbionts. I predicted
that acacia-rhizobia co-introduction occurred for invasive, but not non-invasive acacias, thereby
providing a competitive advantage for Acacia species that have become invasive in California.
Our results suggest that the acacias examined here, regardless of invasive status, were cointroduced with their Australian rhizobial symbionts, as evidenced by the lack of geographic
distinction for the nifD and nodC genes found among isolates collected across both ranges,
regardless of acacia invasiveness category.
4.4.1. Diversity of nodulating bacteria
Based on the 16S rRNA gene, acacias in California and Australia associate with a
diversity of rhizobia from different genera. In both ranges, however, Acacia species tested were
found primarily associating with rhizobia from the genus Bradyrhizobium. These results are in
line with previous studies showing that, especially in Australia, acacias are primarily nodulated
by Bradyrhizobium spp. (Lafay & Burdon, 1998, 2001). Similar results have been found for
acacias introduced to multiple other regions of the globe such as Portugal (RodríguezEcheverría, 2010) and South Africa (Ndlovu, et al. 2013). I also found Acacia species
associating with Rhizobium spp., a faster-growing rhizobial genera associating with acacias in
their native continent (Barnet & Catt, 1991). Both Bradyrhizobium spp. and Rhizobium spp. were
found associating with acacias in Australia and California.
68
4.4.2. Co-introduction and host promiscuity with rhizobia in native and novel ranges
This study provides evidence for the co-introduction of acacias with their native rhizobial
mutualists to novel ranges. Previous research has shown similar patterns. Rodríguez-Echeverría
(2010) found evidence that A. longifolia, which was introduced to Portugal and has become
invasive in dune ecosystems of this region, was co-introduced with its native Australian rhizobial
mutualists. In addition, she found two native legumes co-occurring with A. longifolia associating
with beneficial rhizobial symbionts that had nifD and nodA genes of Australian origin,
suggesting that native legumes in this region are associating with rhizobia co-introduced with A.
longifolia (Rodríguez-Echeverría, 2010). Crisóstomo et al. (2013) found evidence for the cointroduction of Australian acacias with their native rhizobial mutualists. They showed that A.
saligna was likely co-introduced to Portugal with rhizobia of Australian origin, particularly those
in the genus Bradyrhizobium (Crisóstomo et al., 2013). Co-introductions of acacias with their
native rhizobia appear to be geographically widespread. Ndlovu et al. (2013) found that A.
pycnantha, which has become invasive in South Africa, associated with native rhizobial strains
as well as those of Australian origin. These results, as well as the results found here, provide
evidence for the Invasional Meltdown Hypothesis, in which two invasive species facilitate each
other’s invasion (Simberloff & Holle, 1999; Simberloff, 2006).
Our investigation indicates that acacias introduced to California, whether invasive or noninvasive, were co-introduced with their native rhizobial mutualists. In previous studies, all
Acacia species examined were invasive (Crisóstomo et al., 2013; Rodríguez-Echeverría, 2010;
Ndlovu et al., 2013), whereas my study examined acacias that are both invasive and noninvasive in their introduced region. I found that introduced acacias associated with rhizobia of
Australian origin regardless of invasive status in California. It may also be possible that
69
individual Acacia species were introduced to California with their native symbionts, and that
other species have been able to associate with strains co-introduced with those species. Either
way, the association of acacias with their native rhizobial symbionts, while likely promoting
establishment and colonization of acacias, is not the sole mechanism delineating which plant
species become invasive when introduced abroad. Since both invasive and non-invasive acacias
have the benefit of their native symbionts in California there are likely additional, or perhaps
different mechanisms influencing the invasive success of certain Acacia species in this region.
Host promiscuity with rhizobial symbionts may be a mechanism that differentiates
invasive status of acacias introduced abroad. More promiscuous acacia hosts can effectively
associate with a wider range of beneficial rhizobial symbionts. Within their native continent,
Thrall (2000) showed that Acacia species with wider distributions are more promiscuous hosts,
suggesting that their ability to associate with larger numbers of rhizobial symbionts may
influence their ability to colonize a wider native range. Klock et al. (2015) found that acacias
invasive in multiple regions of the globe are more promiscuous hosts in regards to their rhizobial
symbionts; growth response of invasive Acacia species was higher across a greater number of
rhizobial strains than naturalized or non-invasive acacias. Based on the phylogenetic analysis
conducted here on the 16S rRNA gene, I found a difference in host promiscuity in regards to the
number of rhizobial strains with which acacias introduced to California associate. Acacia
dealbata and A. melanoxylon, both invasive in California, associated with a greater number of
rhizobial strains in their introduced range than A. longifolia and A. verticillata, which are not
invasive in California. In Australia, all Acacia species examined associated with less than or
equal to the number of strains they associated with in California, with the exception of A.
longifolia, which associated with three more rhizobial species in Australia than in California.
70
Our results indicated that the two Acacia species that have become invasive in California
associate with a greater diversity of rhizobia abroad then in their home range. The capacity to
associate with a greater diversity of strains when introduced abroad may influence their ability to
invade when introduced to novel ranges. Our results also show that A. longifolia is one of the
most promiscuous species in its home range, associating with the widest diversity of rhizobial
strains out of all Acacia species examined in Australia. Although not currently invasive in
California, A. longifolia has become invasive in eight regions of the globe, as designated by
Richardson & Rejamánek (2011) (later updated by Rejmánek & Richardson [2013]). I was
limited in sampling due to availability of A. longifolia populations in California. It is therefore
possible that, had our sampling been more widely distributed in California, I would have found
A. longifolia associating with more rhizobial strains. While not currently invasive in California,
the ability of A. longifolia to associate with multiple rhizobial strains in its home range, coupled
with evidence of co-introduction with its native symbionts, as well as records of invasiveness in
multiple other regions of the world, make this species a prime contender for future invasive
status, and one that should be managed before populations expand beyond its current distribution
in California.
I collected nodules from acacia populations within their native and introduced ranges.
Despite limitations in collection breadth, I still found all Acacia species in California to be
nodulated by rhizobia of Australia origin, based on the 16S rRNA, nifD, and nodC genes. Future
studies may benefit from expanding the number of populations as well as the number of Acacia
species collected from, to determine whether all species introduced to California are found
associating with rhizobia of Australian origin.
71
Acacias are important species economically, environmentally, and culturally in their
native range. Many Acacia species are used to restore degraded habitats, and a great deal of
effort has gone into determining the best rhizobial strains to use as seed inoculants, thereby
reducing the need for nitrogenous fertilizers that may be harmful to environment (Thrall et al.,
2005). As important as these species are in their native range, many have become ecologically
detrimental to novel ranges where they are introduced. For example, Australian acacias have
changed the community structure of native habitats (van Wilgen et al., 2011), altered soil
chemistry that has led to the invasion of weedy grasses (Yelenik et al., 2004), and outcompeted
native vegetation in sensitive dune habitats (Rodríguez-Echeverría et al., 2012). In California,
where 16 Australian Acacia species have been introduced, multiple species appear to be
expanding their ranges, two of which are recognized as invasive. From a global perspective, it is
therefore key to determine the mechanisms that enable certain Acacia species to become
invasive, so that we can use this information to better prevent their further introduction, as well
as manage current invasive populations.
To better understand mechanisms driving Acacia invasions it is essential to study aspects
of the biology of these species in their native and introduced ranges (Hierro et al., 2005). Here, I
evaluated an important mutualistic relationship in the life history of acacias, the acacia-rhizobia
symbiosis, in areas where these species are native and introduced. By studying these species in
both their native and introduced ranges I was able to develop a more complete understanding of
the role of the acacia-rhizobia symbiosis in acacia invasiveness. In addition, comparing species
that are closely related, yet differ in their invasive capacity allowed me to assess whether or not
the co-introduction of native rhizobial symbionts was limited to species that have become
invasive, and was therefore truly a mechanism I could claim delineates invasiveness of Acacia
72
species. Although I did not find that association with native rhizobial symbionts differs among
invasive and non-invasive acacia, it may still be an important mechanism contributing to the
current establishment and colonization of invasive species, and the potential future invasion of
species that are currently naturalized or non-invasive. Continued investigation of the mechanisms
that do and do not influence invasiveness of acacias is important for the control and management
of these species abroad.
73
CHAPTER 5. CONCULSIONS
5.1. SUMMARY OF KEY RESULTS
My dissertation provides an in-depth investigation into the role of rhizobial mutualisms in
the global and regional invasiveness of Australian Acacia species abroad. This project takes two
approaches to examining the mechanisms driving species invasions by 1) evaluating the role of
mutualisms in invasions, and 2) conducting field and laboratory experiments that incorporate
data from a suite of invasive species’ native and novel regions.
This dissertation examines the role of host promiscuity in the invasion of Australian
Acacia species on a regional and global scale. Results from my dissertation indicate that, on a
regional scale (i.e. within the introduced range of California), host promiscuity with rhizobia has
not delineated differential invasiveness among Acacia species. I propose that this may be
because all Acacia species that occur in California are promiscuous rhizobial hosts. All acacias
that occur in California except for one are invasive in at least one region of the globe. When
examined on a global scale, I found evidence that invasive acacias are more promiscuous hosts
(i.e. have a higher symbiotic response, based on aboveground biomass, across more rhizobial
strain inoculants than naturalized or non-invasive hosts) (Klock et al., 2015). Results from
Chapters 2 and 3 together suggest that acacias that have become invasive around the world may
have done so in part because they are less selective for rhizobial strains with which they can
form beneficial symbiotic associations. Although most Acacia species introduced to California
have not yet become invasive in this range, their proven capacity to invade other regions of the
globe suggests that they may, if conditions are suitable, also become invasive in California.
Other mechanisms, such as herbivory by novel organisms, competition from other plant species
native to California, absence of other important mutualistic organisms such as seed dispersing
74
birds and/or ants, or differential propagule pressure among Acacia species introduced to
California may also influence differences in invasiveness seen in this region.
Even though I did not observe differences in plant performance among Acacia species
varying in invasiveness in California, I did observe differences in plant performance among
different soil inoculants (Chapter 2). This suggests that it may be the microbial communities
occurring in soils where Acacia species are planted, as well as host promiscuity of the plants
themselves that promote differential establishment of Acacia species introduced abroad. If
Acacia species are planted in areas where generalist rhizobial strains are present they may be
able to successfully establish, colonize, and invade those areas, regardless of host promiscuity.
Future research would benefit from pairing individual Acacia species with generalist and specific
rhizobial strains to determine whether plant performance varies depending on the prior known
effectiveness of mutualistic strains.
I found that Acacia species differing in invasiveness in California appear to have been cointroduced to this region with their native rhizobial symbionts (Chapter 4). The availability of
native symbionts did not differ among Acacia species varying in invasiveness in California.
Whereas co-introduction of acacia hosts with their native rhizobial symbionts did not differ
among invasive and non-invasive species, it is nonetheless important to recognize the crosscontinental introduction of two symbiotic non-native taxa to a novel region. It is possible that the
presence of Australian rhizobia in areas where acacias are introduced has helped them to
establish in specific habitats where they currently occur, and the availability of native rhizobial
symbionts may contribute to the future spread and invasion of these species within California
and other localized ranges where they have been introduced.
75
5.2. IMPLICATIONS FOR CONSERVATION AND HABITAT RESTORATION
Grasping the mechanisms underlying plant species invasions is important not only in
terms of furthering our general understanding of processes associated with invasion, but also
has wide-ranging applied value. Australian Acacia species, such as A. cyclops, A. longifolia, A.
melanoxylon, and A. saligna (all of which are present in California), have invaded thousands of
ha in South Africa, from terrestrial to riparian ecosystems, across agricultural lands to areas of
conservation concern (Richardson et al., 1997; Henderson, 2001; Richardson & Kluge, 2008).
Many Acacia species are also invasive in parts of Europe, including France, Portugal, and
Spain (Lorenzo et al., 2008; Lorenzo et al., 2010). The cross-continental invasiveness of
Australian Acacia species make investigation of mechanisms enabling their invasion a key
focus of governmental programs such as the Working for Water Program in South Africa
(Richardson & van Wilgren 2004). A 2011 special issue of the scientific journal Diversity and
Distributions was dedicated to understanding the mechanisms behind Acacia species invasions,
indicating the acute need to provide insight to control their spread. Investigation of the
mechanisms that allow Acacia species to invade will lead to more efficient and effective
management in the diverse geographic areas they have colonized.
Results from my dissertation have helped discern the specificity of Acacia species for
rhizobial partners, which can assist in informing management of a suite of invasive species,
while also indicating rhizobial strains that could be used to increase growth of leguminous
crops. Rhizobia are important for the growth of key crop species in the United States, such as
soybeans (Glycine max), peas (Pisum sativum), and alfalfa (Medicago sativa) (Stalker et al.,
2004), which are in the same family (Fabaceae) as acacias. Better understanding of the legumerhizobia symbiosis may reduce the need for nitrogenous fertilizers, thereby mitigating release
76
of harmful pollutants to the environment. Our results will help inform restoration efforts in
Australia that aim to reintroduce native Acacia species to disturbed pasturelands, and which
rely upon knowledge of effective rhizobial symbionts to inoculate seeds for increased
restoration success, as well as provide insight for managing these species in areas where they
have become invasive. By understanding the specificity of the link between acacias and
rhizobia, we can accomplish multiple goals, elucidating the mechanisms that enable legumes to
invade novel ranges and informing management of nitrogen-fixing species in areas where they
are desirable or have become invasive.
77
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86
APPENDIX A. SUPPORTING INFORMATION FOR CHAPTER 2
Table S2.1 Soil collection sites for Acacia species inoculants, including soil texture and measures of pH, nitrate nitrogen (NO3),
phosphorus, and organic carbon (%).
Site
Code
Description
Texture
Casurina Sands
Ginninderra
Happy Valley
Namadgi Diverse
Namadgi Grassland
Namadgi Legume
Pine Bramble
Sawyer’s Gully
Snow Gum
Yarrangobilly Caves
CS
GE
HV
ND
NG
NL
PB
SG
SN
YC
Riparian
Agricultural
Agricultural grassland
Diverse native habitat
Undisturbed grassland
Daviesia spp. dominated
Abandoned pine plantation
Eucalyptus understory
Eucalyptus snowy field
Eucalyptus legume understory
Sand
Clay Loam
Clay Loam
Sandy Loam
Clay Loam
Clay Loam
Sandy Loam
Sandy Loam
Clay Loam
Sandy Loam
87
pH (1:5
Water)
6.3
5.8
5.8
5.7
5.9
5.9
5.4
4.6
5.2
5.5
(NO3)
mg/kg
8.7
31
12
1.6
1.4
<1.0
<1.0
<1.0
<1.0
1.9
Phosphorus
(Colwell)
16
24
18
11
8
14
8
8
21
32
Organic
Carbon %
0.99
1.1
3
2.2
1.5
2.2
0.65
4.2
3.9
6.4
Table S2.2 GLMM models predicting difference in aboveground biomass (g) among California
invasive rankings. SO = Soil, CA = California invasive ranking, SP = Species. SP is included in
all models as a random effect. Model 1 tests for the presence of an interaction between SO and
CA. Aikake weights (wi) indicate the model with the highest relative likelihood of being the best
model (the closest wi to 1 is the best model).
Model
number
1
2
3
4
5
Number of model
parameters
3
2
3
1
2
Model
variables
SO*CA; SP
SO; SP
SO+CA; SP
SP
CA; SP
88
AIC
Delta
wi
1812.0
1831.1
1833.4
2145.5
2147.4
0.00
19.1
21.4
333.5
335.4
1.00
7.12 x 10-5
2.25 x 10-5
3.81 x 10-73
1.48 x 10-73
Invasive
Naturalized
YC
SN
SG
NL
PB
NG
HV
NG
NL
PB
SG
SN
YC
NG
NL
PB
SG
SN
YC
vert
Biomass (g)
6
4
2
Soils
CS
GE
YC
SN
SG
PB
NL
NG
HV
ND
CS
0
GE
YC
SN
0
SG
0
PB
2
NL
2
NG
4
HV
4
ND
HV
CS
YC
SN
SG
PB
vert
6
CS
ND
vert
GE
NL
Soils
6
Soils
HV
Soils
NG
HV
ND
YC
CS
0
GE
0
SN
0
SG
2
PB
2
NL
2
NG
4
HV
4
ND
4
CS
6
GE
6
ND
pycn
6
GE
long
ND
CS
YC
SN
SG
NL
PB
NG
HV
ND
YC
CS
0
GE
0
SN
0
SG
2
NL
2
PB
2
NG
4
HV
4
ND
4
CS
6
mela
Biomass (g)
cult
6
GE
bail
6
GE
Biomass (g)
deal
Non-Invasive
Soils
Figure S2.3 Aboveground biomass (g) response of individual host species to different soil
treatments. The first column is invasive species, the second column is naturalized species, and
the third column is non-invasive species. The horizontal solid line indicates the point at which
the host has the same biomass response as its least effective soil. The dashed line is the average
biomass response for each host species across all soils. Error bars represent standard errors (SE)
of the means.
89
Table S2.3 GLMM models predicting difference in survival among California invasive rankings.
SO = Soil, CA = California invasive ranking, SP = Species. SP is included in all models as a
random effect. Aikake weights (wi) indicate the model with the highest relative likelihood of
being the best model (the closest wi to 1 is the best model). * indicates model that won’t
converge. This model still produces an AIC value, indicated in (), but this value is not reliable.
Model
number
*
1
2
3
4
Number of model
parameters
3
2
3
1
2
Model
variables
SO*CA; SP
SO; SP
SO+CA; SP
SP
CA; SP
90
AIC
Delta
wi
(568.37)
543.37
547.24
711.71
715.57
n/a
0.00
3.87
168.34
172.20
n/a
0.87
0.13
2.44 x 10-37
3.54 x 10-38
Invasive
Naturalized
long
SN
YC
SG
NL
PB
HV
NG
NG
NL
PB
SG
SN
YC
NG
NL
PB
SG
SN
YC
0.00
0.00
0.00
Soils
0.75
0.50
CS
YC
SN
SG
PB
NL
NG
HV
ND
CS
% survival
1.00
GE
YC
0.25
SN
0.25
SG
0.25
PB
0.50
NL
0.50
NG
0.75
HV
HV
vert
0.75
ND
ND
FAKE
1.00
GE
FAKE
CS
HV
Soils
1.00
GE
ND
CS
YC
SN
SG
PB
NL
YC
Soils
NG
0.00
HV
0.00
ND
0.00
CS
0.25
GE
0.25
SN
0.25
SG
0.50
PB
0.50
NL
0.50
NG
0.75
HV
0.75
ND
0.75
CS
1.00
GE
1.00
GE
pycn
1.00
Soils
ND
CS
SN
YC
SG
NL
PB
CS
NG
0.00
HV
0.00
ND
0.00
GE
0.25
SN
0.25
YC
0.25
SG
0.50
NL
0.50
PB
0.50
NG
0.75
HV
0.75
ND
0.75
CS
1.00
GE
cult
1.00
mela
% survival
Non-Invasive
bail
1.00
GE
% survival
deal
Soils
Figure S2.2 Percent survival for each host species x soil treatment combination. The first
column is invasive species, the second column is naturalized species, and the third column is
non-invasive species.
91
Table S2.4 GLMM models predicting difference in nodulation presence among California
invasive rankings. SO = Soil, CA = California invasive ranking, SP = Species. SP is included in
all models as a random effect. Aikake weights (wi) indicate the model with the highest relative
likelihood of being the best model (the closest wi to 1 is the best model). * indicates model that
won’t converge. This model still produces an AIC value, indicated in (), but this value is not
reliable.
Model
number
*
1
2
3
4
Number of model
parameters
3
2
3
1
2
Model
variables
SO*CA; SP
SO; SP
SO+CA; SP
SP
CA; SP
92
AIC
Delta
wi
(497.62)
478.34
482.26
576.37
580.31
n/a
0.00
3.92
98.03
101.97
n/a
0.88
0.12
4.53 x 10-22
6.31 x 10-23
Invasive
Naturalized
YC
SN
SG
NL
PB
NG
HV
NG
NL
PB
SG
SN
YC
NG
NL
PB
SG
SN
YC
HV
ND
% nodulation presence
vert
1.00
0.75
0.50
0.25
Soils
CS
GE
YC
SN
SG
PB
NL
NG
HV
ND
0.00
CS
YC
0.00
SN
0.00
SG
0.25
PB
0.25
NL
0.50
NG
0.50
HV
0.75
ND
0.75
Soils
HV
CS
YC
SN
SG
FAKE
1.00
GE
FAKE
CS
PB
Soils
1.00
GE
NL
YC
Soils
NG
0.00
HV
0.00
ND
0.00
CS
0.25
GE
0.25
SN
0.25
SG
0.50
PB
0.50
NL
0.50
NG
0.75
HV
0.75
ND
0.75
CS
1.00
GE
1.00
ND
pycn
1.00
GE
long
ND
CS
YC
SN
SG
NL
PB
CS
NG
0.00
HV
0.00
ND
0.00
GE
0.25
YC
0.25
SN
0.25
SG
0.50
NL
0.50
PB
0.50
NG
0.75
HV
0.75
ND
0.75
CS
1.00
GE
cult
1.00
mela
% nodulation presence
Non-Invasive
bail
1.00
GE
% nodulation presence
deal
Soils
Figure S2.3 Percent nodulation for each host species x soil treatment combination. The first
column is invasive species, the second column is naturalized species, and the third column is
non-invasive species.
93
Table S2.5 GLMM models predicting difference in nodulation index among California invasive
rankings. SO = Soil, CA = California invasive ranking, SP = Species. SP is included in all
models as a random effect. Model 1 tests for the presence of an interaction between SO and CA.
Aikake weights (wi) indicate the model with the highest relative likelihood of being the best
model (the closest wi to 1 is the best model).
Model
number
1
2
3
4
5
Number of model
parameters
3
2
3
1
2
Model
variables
SO*CA; SP
SO; SP
SO+CA; SP
SP
CA; SP
94
AIC
Delta
wi
1339.6
1349.3
1352.2
1611.7
1615.6
0.0
9.7
12.6
272.1
276.0
0.97
0.01
0.01 x 10-1
7.94 x 10-60
1.13 x 10-60
APPENDIX B. SUPPORTING INFORMATION FOR CHAPTER 3
Table S3.1 Original Acacia species hosts for each rhizobial strain used in growth experiments.
“*” indicates that strains were collected from Acacia species used in this study.
Strain
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
Original host
A. melanoxylon*
A. dangarensis
A. leucoclada
A. binervata
A. stenophylla*
A. sophorae
A. sophorae
A. implexa
A. salicina
A. stenophylla*
A. salicina
A. sophorae
95
Table S3.2 GLMM models predicting difference in symbiotic response among invasiveness
categories. ST = Strain, IC = Invasiveness category, SP = Species. SP is included in all models as
a random effect. Model 1 tests for the presence of an interaction between ST and IC. Aikake
weights (wi) indicate the model with the highest relative likelihood of being the best model (the
closest wi to 1 is the best model).
Model
number
1
2
3
4
Number of model
parameters
3
3
2
2
Model variables
AIC
Delta
wi
ST*IC; SP
ST+IC; SP
ST; SP
IC; SP
11794
112490
12502
13400
0.0
696.0
708.0
11606.0
1.00
7.34 x 10-152
1.82 x 10-154
0.00
5
1
SP
13412
1618.0
0.00
96
Table S3.3 Average biomass (g) of each invasiveness category when grown with each rhizobial
strain.
Strain
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
N–
N+
Invasive
0.67
0.67
1.08
0.87
0.04
0.03
0.67
0.12
0.03
0.05
0.05
0.07
0.03
1.49
Naturalized
0.17
0.12
0.30
0.26
0.04
0.04
0.12
0.05
0.03
0.04
0.03
0.05
0.03
0.64
97
Non-Invasive
0.33
0.26
0.34
0.36
0.08
0.08
0.22
0.11
0.23
0.25
0.19
0.26
0.07
1.11
Table S3.4 Summary of Analysis of Variance results testing the effects of host species and
rhizobial strain on the host-symbiotic response.
Source
d.f.
SS
F
P
Host species
Rhizobial strains
Host x Rhizobia
Residual
11
13
143
1328
133930
304211
234282
117109
138.07
265.36
18.58
<0.001
<0.001
<0.001
98
Table S3.5 Average aboveground biomass (g) of each Acacia species when grown with each rhizobial strain.
Strain
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
N–
N+
deal
0.68
0.63
0.91
0.62
0.02
0.03
0.57
0.10
0.02
0.10
0.06
0.03
0.03
1.39
Invasive
long
mear
0.78
0.56
1.07
0.41
1.48
1.19
1.20
0.91
0.08
0.01
0.03
0.01
0.80
0.69
0.23
0.08
0.03
0.01
0.04
0.01
0.05
0.04
0.18
0.02
0.03
0.01
1.52
1.45
mela
0.66
0.56
0.72
0.76
0.05
0.04
0.61
0.06
0.06
0.06
0.05
0.05
0.06
1.6
cult
0.41
0.21
0.65
0.57
0.03
0.04
0.23
0.08
0.05
0.04
0.04
0.04
0.03
0.93
Naturalized
murr pend
0.02
0.18
0.02
0.20
0.08
0.20
0.04
0.23
0.03
0.08
0.02
0.07
0.02
0.12
0.02
0.05
0.01
0.05
0.03
0.05
0.01
0.07
0.01
0.12
0.01
0.06
0.55
0.43
99
redo
0.05
0.04
0.26
0.18
0.01
0.02
0.10
0.04
0.01
0.03
0.01
0.02
0.02
0.63
bive
0.76
0.57
0.68
0.77
0.07
0.06
0.51
0.18
0.14
0.10
0.12
0.35
0.03
0.96
Non-Invasive
cole
hake
0.02
0.36
0.04
0.23
0.02
0.38
0.01
0.41
0.02
0.12
0.02
0.17
0.01
0.21
0.02
0.08
0.02
0.04
0.02
0.13
0.02
0.05
0.01
0.05
0.02
0.13
0.85
0.95
sten
0.18
0.19
0.28
0.26
0.12
0.07
0.15
0.15
0.71
0.75
0.56
0.64
0.10
1.66
Table S3.6 GLMM models predicting difference in symbiotic response among native range
distributions (widespread versus limited). ST = Strain, NR = Native range distribution category,
SP = Species. SP is included in all models as a random effect. Model 3 tests for the presence of
an interaction between ST and NR. Aikake weights (wi) indicate the model with the highest
relative likelihood of being the best model (the closest wi to 1 is the best model).
Model
number
1
2
3
4
Number of model
parameters
2
3
3
1
Model variables
AIC
Delta
wi
ST; SP
ST+NR; SP
ST*NR; SP
SP
12502
12504
12519
113412
0.0
12.0
17.0
910.0
0.73
0.27
0.00
01.82 x 10-198
5
2
NR; SP
13414
912.0
6.69 x 10-199
100
Table S3.7 GLMM models predicting difference in nodulation presence among invasiveness
categories. ST = Strain, IC = Invasiveness category, SP = Species. SP is included in all models as
a random effect. Model 1 tests for the presence of an interaction between ST and IC. Aikake
weights (wi) indicate the model with the highest relative likelihood of being the best model (the
closest wi to 1 is the best model).
Model
number
1
2
3
4
Number of model
parameters
3
2
3
1
Model variables
AIC
Delta
wi
ST*IC; SP
ST; SP
ST+IC; SP
SP
1081.4
1100.5
1102.8
1523.3
0.0
19.1
21.4
441.9
1.00
7.12 x 10-05
2.25 x 10-05
1.10 x 10-96
5
2
IC; SP
1525.9
444.5
3.01 x 10-97
101
Table S3.8 GLMM models predicting difference in nodulation index among invasiveness
categories. ST = Strain, IC = Invasiveness category, SP = Species. SP is included in all models as
a random effect. Model 1 tests for the presence of an interaction between ST and IC. Aikake
weights (wi) indicate the model with the highest relative likelihood of being the best model (the
closest wi to 1 is the best model).
Model
number
1
2
3
4
5
Number of model
parameters
3
2
3
1
2
Model variables
AIC
Delta
wi
ST*IC; SP
ST; SP
ST+IC; SP
SP
IC; SP
2879.6
3027.5
3028.5
3562.9
3564
0
147.9
148.9
683.3
684.4
1.00
7.65 x 10-33
4.64 x 10-33
4.20 x 10-149
2.42 x 10-149
102
Table S3.9 GLMM models predicting difference in nodulation presence among Australian range
distributions. ST = Strain, AU = Australian range, SP = Species. SP is included in all models as a
random effect. Model 1 tests for the presence of an interaction between ST and IC. Aikake
weights (wi) indicate the model with the highest relative likelihood of being the best model (the
closest wi to 1 is the best model).
Model
number
1
2
3
4
5
Number of model
parameters
3
2
3
1
2
Model variables
AIC
Delta
wi
ST*AU; SP
ST; SP
ST+AU; SP
SP
AU; SP
1080
1100.5
1100.9
1523.3
1523.8
0
20.5
20.9
443.3
443.8
1.00
3.54 x 10-05
2.89 x 10-05
5.48 x 10-97
4.27 x 10-97
103
Table S3.10 GLMM models predicting difference in nodulation index among Australian range
distributions. ST = Strain, AU = Australian range, SP = Species. SP is included in all models as a
random effect. Model 1 tests for the presence of an interaction between ST and IC. Aikake
weights (wi) indicate the model with the highest relative likelihood of being the best model (the
closest wi to 1 is the best model).
Model
number
1
2
3
4
5
Number of model
parameters
2
3
3
1
2
Model variables
AIC
Delta
wi
ST; SP
ST+AU; SP
ST*AU; SP
SP
AU; SP
3027.5
3027.6
3035.2
3562.9
3563.0
0
0.1
7.7
535.4
535.5
0.51
0.48
0.01
2.78 x 10-117
2.64 x 10-117
104
120
140
long
120
60
60
40
40
40
20
20
20
0
0
0
140
mear
120
140
pend
120
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0
120
140
mela
120
140
redo
120
100
80
80
60
60
60
40
40
40
20
20
20
0
0
0
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
100
80
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
100
Strains
Strains
hake
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
100
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
100
140
cole
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
80
60
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
100
80
120
Non-Invasive
bive
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
120
100
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
Symbiotic response
140
murr
80
140
Symbiotic response
140
120
100
80
60
40
20
0
100
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
Symbiotic response
140
Naturalized
cult
sten
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
140
120
100
80
60
40
20
0
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
Invasive
deal
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
NN+
Symbiotic response
140
120
100
80
60
40
20
0
Strains
Figure S3.1 Symbiotic response of individual host species to different rhizobial strains. The first
column is invasive species, the second column is naturalized species, and the third column is
non-invasive species. The horizontal solid line at 1 indicates the point at which the host had the
same symbiotic response to a rhizobial strain as the N– control. The dashed line is the average
symbiotic response for each host species across all strains. Note the different y-axes among the
graphs. Points above the solid line indicate a positive symbiotic response, and points below the
solid line indicate a negative symbiotic response. Error bars represent standard errors (SE) of the
means. Strains marked as B = Bradyrhizobium, R = Rhizobium, and S = Sinorhizobium.
105
Naturalized
Non-Invasive
0.50
0.50
0.25
0.25
0.25
0.00
0.00
0.00
long
1.00
murr
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
0.75
0.50
1.00
1.00
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
0.75
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
1.00
0.75
1.00
0.50
0.25
0.25
0.25
0.00
0.00
0.00
mear
1.00
pend
1.00
0.50
0.25
0.25
0.25
0.00
0.00
0.00
mela
1.00
redo
1.00
0.75
0.75
0.50
0.50
0.50
0.25
0.25
0.25
0.00
0.00
0.00
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
0.75
Strains
Strains
hake
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
0.75
0.50
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
0.75
0.50
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
0.75
1.00
cole
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
0.75
0.50
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
0.75
0.50
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
0.75
1.00
bive
sten
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
cult
1.00
01B
02B
03B
04B
05S
06B
07R
08B
09S
10S
11S
12R
% nodulation presence
% nodulation presence
% nodulation presence
% nodulation presence
Invasive
deal
Strains
Figure S3.2 Percent nodulation for each host species x rhizobial strain combination. The first
column is invasive species, the second column is naturalized species, and the third column is
non-invasive species. Strains marked as B = Bradyrhizobium, R = Rhizobium, and S =
Sinorhizobium.
106
2.5
Invasive NI
I
Naturalized
N
Non-Invasive
Average nodulation index
2.0
1.5
1.0
12R
11S
10S
09S
08B
07R
06B
05S
04B
03B
02B
01B
0.5
Strains
Figure S3.3 Average nodulation index for different invasiveness categories among rhizobial
strains. The different shapes depict different invasiveness categories (square = invasive, circle =
naturalized, triangle = non-invasive). Strains marked as B = Bradyrhizobium, R = Rhizobium,
and S = Sinorhizobium.
107
APPENDIX C. SUPPORTING INFORMATION FOR CHAPTER 4
Table S4.1. Identity of rhizobial strains collected from Acacia species in Australian and California. Rhizobial identification based on
BLAST comparison of sequences with archived bacterial sequences from GenBank (http://blast.ncbi.nlm.nih.gov).
16S OTULB
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
Continent
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Species
binervata
dangarensis
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
deanei
deanei
elata
implexa
leucoclada
longifolia
longifolia
longifolia
Status
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Source
soil
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
soil
soil
soil
soil
soil
nodule
nodule
nodule
108
Population
WJ
MD
KA
KA
KA
LI
CF
CF
CF
CF
SN
SN
SN
SN
TAS
TAS
TAS
TAS
KA
BE
BL
WW
EG
GL
SO
INV
LT
LT
BP
Sample ID
CPI_241
CPI_239
acde_cpi234
acde_cpi812
CPI_234
acde_cpi728
acde_cpi740
acde_cpi741
acde_cpi742
acde_cpi743
acde_cpi756
acde_cpi757
acde_cpi758
acde_cpi759
acde_cpi800
acde_cpi801
acde_cpi802
acde_cpi803
acde_cpi814
acde_cpi820
acde_cpi736
CPI_238
CPI_259
CPI_236
CPI_257
CPI_240
aclo_48
aclo_51
aclo_19
Organism
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
16S OTULB
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
Continent
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Species
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
Status
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Source
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
109
Population
DB
DB
DB
BP
BP
BP
DB
BP
BP
BP
BP
DB
KI
KI
KI
KI
LT
LT
LT
LT
LT
LT
LT
MK
MK
MK
MK
MK
MK
MK
MK
MK
DB
DB
Sample ID
aclo_1
aclo_12
aclo_15
aclo_16
aclo_17
aclo_18
aclo_2
aclo_25
aclo_27
aclo_28
aclo_29
aclo_3
aclo_34
aclo_35
aclo_38
aclo_39
aclo_40
aclo_41
aclo_44
aclo_47
aclo_49
aclo_50
aclo_52
aclo_55
aclo_56
aclo_58
aclo_59
aclo_60
aclo_62
aclo_63
aclo_65
aclo_69
aclo_7
aclo_8
Organism
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
16S OTULB
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
Continent
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Species
longifolia
longifolia
longifolia
mearnsii
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
nanodealbata
salicina
sophorae
trachyphloia
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
Status
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Source
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
soil
nodule
nodule
nodule
nodule
soil
soil
soil
soil
soil
nodule
nodule
nodule
nodule
nodule
nodule
nodule
110
Population
LT
MK
MK
BR
LIL
HV
MG
ML
MT
MT
MT
LIL
HU
HU
HU
NA
MG
GE
LIL
HV
ML
NA
BLI
LH
PA
BEE
BB
SH
SH
SH
SH
GC
GC
GC
Sample ID
aclo_43
aclo_61
aclo_64
CPI_235
acme_cpi253
acme_cpi707
acme_cpi709
acme_cpi714
acme_cpi716
acme_cpi717
acme_cpi718
acme_cpi726
acme_cpi787
acme_cpi788
acme_cpi789
acme_cpi795
CPI_232
CPI_233
CPI_253
acme_cpi708
acme_cpi713
acme_cpi796
CPI_489
CPI_237
CPI_248
CPI_246
CPI_258
acve_12
acve_13
acve_14
acve_15
acve_17
acve_18
acve_19
Organism
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
16S OTULB
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
Continent
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Species
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
Status
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Source
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
111
Population
SH
GC
GC
GC
GC
GC
GC
GC
PH
PH
NA38
NA39
NA39
NA39
NA39
NA39
NA39
NA39
NA39
NA40
NA40
NA40
NA40
NA40
NA40
NA41
NA41
NA41
NA41
SH
NA41
NA41
NA41
NA41
Sample ID
acve_2
acve_21
acve_22
acve_23
acve_25
acve_27
acve_28
acve_29
acve_31
acve_32
acve_33
acve_35_3
acve_36
acve_37
acve_41
acve_44
acve_45
acve_46
acve_49
acve_52
acve_55
acve_57
acve_59
acve_61
acve_64
acve_65
acve_65_2
acve_67
acve_69
acve_7
acve_70
acve_71
acve_72
acve_74
Organism
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
16S OTULB
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
Continent
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
Species
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
Status
Native
Native
Native
Native
Native
Native
Native
Native
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Source
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
112
Population
NA41
NA41
NA41
NA41
NA41
SH
SH
NA39
SG
RR
RR
RR
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
AA
ST
SR
AA
ST
RR
ST
SG
WPS
AA
AA
Sample ID
acve_75
acve_76
acve_77
acve_78
acve_79
acve_8
acve_9
acve_42_3
de_061-1
de_042-1
de_034-1
de_036-1
de_03
de_13
de_04
de_15
de_05
de_16
de_07
de_09
de_11
de_01
de_12
de_121-1
de_048-1
de_051-2
de_116-1
de_044-1
de_038-1
de_043-1
de_066-1
de_086-1
de_113-1
de_116-3
Organism
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
16S OTULB
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
Continent
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
Species
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
dealbata
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
Status
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Source
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
113
Population
SG
ST
AA
AA
AA
WPS
AA
AA
SR
RR
RR
RR
SD
ST
SD
SD
OR
MAR_LO
MAR_LO
CC
MAR_LO
OR
FFX
CC
FFX
OR
CC
CC
OR
CC
MH
OR
MH
FFX
Sample ID
de_063-3
de_046-1
de_118-1
de_115-1
de_114-2
de_087-1
de_117-1
de_117-2
de_049-1
de_039-1
de_037-1
de_037-2
de_072-1
de_047-1
de_067-1
de_070-1
lo-70
lo-35
lo-36
lo-07
lo-34
lo-62
lo-48
lo-03
lo-57
lo-67
lo-05
lo-14
lo-68
lo-06
lo-30
lo-69
lo-19
lo-48
Organism
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
16S OTULB
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
Continent
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
Species
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
longifolia
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
Status
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Naturalized
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Source
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
114
Population
OR
CC
MH
MAR_LO
FFX
MAR_LO
OR
OR
CC
MH
OR
FFX
CC
MAR_LO
FFX
OR
FFX
FFX
OR
MH
FFX
OR
CY
711
SC
IN
AA
LIM
CY
SC
CY
MS1
MS2
IN
Sample ID
lo-61
lo-08
lo-25
lo-37
lo-51
lo-39
lo-62
lo-71
lo-10
lo-26
lo-63
lo-55
lo-12
lo-31
lo-56
lo-65
lo-60
lo-52
lo-73
lo-21
lo-49
lo-74
me_016-1
me_030-1
me_098-2
me_077-1
me_130-1
me_009-1
me_017-1
me_099-1
me_014-1
me_206-1
me_213-1
me_079-1
Organism
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
16S OTULB
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
Continent
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
Species
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
Status
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Source
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
115
Population
AA
FB
PC1
MI
SC
MC
IN
SC
CP
AA
AA
LIM
SB
IN
FB
MC
MC
IN
AA
TIB_ME
IN
MV
MV
IN
MAR_VE
MAR_VE
MAR_VE
MF
MAR_VE
MF
MF
MF
MAR_VE
MF
Sample ID
me_128-2
me_181-4
me_161-1
me_202-1
me_102-1
me_104-3
me_080-1
me_098-1
me_020-1
me_128-1
me_129-3
me_008-1
me_023-1
me_076-1
me_181-2
me_106-3
me_103-2
me_077-2
me_125-1
me_141-3.1
me_084-1
me_137-2
me_136-1
me_078-1
ve-10
ve-07
ve-11
ve-29
ve-08
ve-23
ve-189-1
ve-30
ve-09
ve-17
Organism
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
16S OTULB
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_1
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
Continent
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
California
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
California
California
California
California
California
California
California
California
Species
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
verticillata
longifolia
longifolia
melanoxylon
salicina
stenophylla
verticillata
verticillata
verticillata
verticillata
dealbata
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
Status
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Native
Native
Native
Native
Native
Native
Native
Native
Native
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Invasive
Source
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
soil
soil
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
116
Population
MF
MAR_VE
MF
MAR_VE
MF
MF
MAR_VE
MAR_VE
MF
MF
MF
MF
MF
MF
MF
MF
TIB_VE
KI
DB
OT
PA
KE
GC
PH
NA41
NA41
SG
SC
711
MB
MI
PC2
TIB_ME
711
Sample ID
ve-25
ve-01
ve-26
ve-03
ve-19
ve-27
ve-15
ve-12
ve-20
ve-28
ve-21
ve-22
ve-24
ve-192-1
ve_191-1
ve-194-1
ve-154-1
aclo_33
aclo_6
acme_cpi779
CPI_249
CPI_243
acve_30
acve_31_2
acve_68
acve_73
de_063-1
me_097-2
me_028-3
me_173-1
me_203-3
me_166-3
me_141-2.1
me_028-2
Organism
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Bradyrhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
16S OTULB
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_2
OTULB_3
OTULB_3
OTULB_3
OTULB_3
OTULB_3
OTULB_3
OTULB_3
OTULB_3
OTULB_3
OTULB_3
OTULB_4
OTULB_4
OTULB_4
OTULB_5
OTULB_5
OTULB_5
OTULB_6
OTULB_6
OTULB_6
OTULB_7
OTULB_8
Continent
California
California
California
California
California
California
California
California
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
Australia
California
California
Australia
Australia
Australia
Australia
California
California
Australia
California
Species
melanoxylon
melanoxylon
melanoxylon
melanoxylon
melanoxylon
verticillata
verticillata
verticillata
salicina
salicina
salicina
salicina
salicina
salicina
stenophylla
stenophylla
stenophylla
stenophylla
longifolia
longifolia
melanoxylon
dealbata
longifolia
sophorae
longifolia
dealbata
melanoxylon
sophorae
melanoxylon
Status
Invasive
Invasive
Invasive
Invasive
Invasive
Non-Invasive
Non-Invasive
Non-Invasive
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Native
Naturalized
Invasive
Native
Native
Native
Native
Invasive
Invasive
Native
Invasive
Source
nodule
nodule
nodule
nodule
nodule
nodule
nodule
nodule
soil
soil
soil
soil
soil
soil
soil
soil
soil
soil
nodule
nodule
nodule
nodule
nodule
soil
nodule
nodule
nodule
soil
nodule
117
Population
FB
TIB_ME
TIB_ME
TIB_ME
TIB_ME
MAR_VE
MAR_VE
MAR_VE
PA
PA
PA
PA
PA
PA
KE
KE
KE
KE
DB
CC
MC
CF
KI
BEE
DB
SD
MI
BEE
TIB_ME
Sample ID
me_185-1
me_146-2
me_147-3
me_148-3
me_147-1
ve-05
ve-04
ve-06
CPI_261
CPI_262
CPI_263
CPI_264
CPI_311
CPI_313
CPI_244
CPI_251
CPI_265
CPI_266
aclo_4
lo-13
me_103-1
acde_cpi739
aclo_34_2
CPI_247
aclo_5
de_074-1
me_197-3
CPI_314
me_144-1
Organism
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Rhizobium sp. 1
Sinorhizobium sp. 1
Sinorhizobium sp. 1
Sinorhizobium sp. 1
Sinorhizobium sp. 1
Sinorhizobium sp. 1
Sinorhizobium sp. 1
Sinorhizobium sp. 1
Sinorhizobium sp. 1
Sinorhizobium sp. 1
Sinorhizobium sp. 1
Mesorhizobium sp. 1
Mesorhizobium sp. 1
Mesorhizobium sp. 1
Rhizobium sp. 2
Rhizobium sp. 2
Rhizobium sp. 2
Phyllobacterium brassicacearum
Phyllobacterium brassicacearum
Phyllobacterium brassicacearum
Rhizobium sp. 3
Bradyrhizobium sp. 2
APPENDIX D. LETTER OF PERMISSION
118
9/7/2015
RE: DDI 12363, Host-promiscuity in symbiont associations ca... - Metha M Klock
a chapter in my doctoral dissertation in the Department of Biological Sciences at Louisiana State University.
My dissertation is titled “The Role of Rhizobial Mutualisms in the Establishment and Colonization of
Exotic Acacia species in Novel Ranges”. It will be submitted to the Graduate School at Louisiana State
University no later than November 20, 2015.
Citation information for the article is as follows:
Title: Host-promiscuity in symbiont associations can influence exotic legume establishment and
colonization of novel ranges
Authors: Metha M. Klock, Luke G. Barrett, Peter H. Thrall, and Kyle E. Harms
Year: 2015
Volume: Early View
Pages: Early View
For submission as a dissertation chapter I will need to include the entire text, figures, and supplemental
material in my dissertation. I will make no changes to the title or content of the article, however, to follow
dissertation guidelines put forth by Louisiana State University, I will have to adjust article formatting. I will
include a footnote at the start of the dissertation chapter that acknowledges permission by Diversity and
Distributions to use the chapter.
Thank you for reviewing my request. Please feel free to contact me with further questions.
Sincerely,
Metha M. Klock Metha M. Klock
Department of Biological Sciences
Louisiana State University
[email protected]
mmklock.wordpress.com
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VITA
Metha Martine Klock was born in Greenbrae, California in August 1979. She grew up at
the base of Mt. Tamalpais in Fairfax, and was inspired at a very young age by the natural world
around her. Metha completed her undergraduate degree at Sarah Lawrence College in 2001,
receiving a Bachelor of Arts in Liberal Arts. She worked in book publishing for two years, and
decided to make a career shift to focus on native plant restoration. She began an internship with
the Golden Gate National Recreation Area in 2003 at the Marin Headlands Native Plant Nursery,
where she was in charge of volunteer recruitment, seed collection, and native plant propagation.
It was here that she began to understand the influence of non-native, invasive plants on the
natural environment, saw their effects in person, and aimed to develop ways to mitigate their
effects. Metha returned to school to pursue her Master of Science at Louisiana State University
in 2007, joining Dr. Hallie Dozier’s research group in the School of Renewable Natural
Resources. For her master’s thesis, Metha studied the life history of Chinese privet (Ligustrum
sinense), developing demographic models to predict life stages that could be targeted for
effective management of this invasive species. During her master’s degree, she also participated
in an Organization for Tropical Studies course with Duke University in Costa Rica that inspired
her to continue her graduate studies and pursue her doctoral degree in Ecology. Metha joined Dr.
Kyle E. Harms’ research group in 2009 in the Department of Biological Sciences at Louisiana
State University. Her dissertation focuses on the mechanisms driving invasiveness of non-native
legumes, focusing particularly on the acacia-rhizobia symbiosis.
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