#379
Survey of Protist Genetic Diversity in the
Hydrothermal Environments of Lassen Volcanic National Park N-063
[email protected]
Patricia B. Brown*; Gordon V. Wolfe**
California State University, Chico
[email protected]
Table 2 a-c: Blast Identity from clone libraries
Introduction
6
no D N A re c ove re d
DNA
Source
pH
T
#
clones
BH100
1.7
30
2
+ e uk PCR
5
- e uk PCR (also BH 100, 101, 115)
4
pH
Hydrothermal environments support diverse prokaryote
assemblages, but less is known about eukaryotes, or
hydrothermal food webs. Culture-based studies suggest
protists might possibly be found up to 60-70 C (Tansey
and Brock, 1978), and many acidophilic taxa are well
known (Packrof and Woelfl, 2000).
Recent reports of novel protist rRNA genes in extreme
environments, such as acidic or anoxic sites (Amaral
Zettler et al., 2002, Edgcomb et al., 2002, Dawson and
Pace, 2002, Baker et al., 2003) suggest that diversity
might be higher than previously thought.
Here, we present data of a survey of eukaryal rRNA
diversity in the Lassen Volcanic National Park (LVNP)
hydrothermal environments.
Parauronema longum (ciliate)
1
Bodo edax (ciliate)
BH105
B H116
3.2
15
BH109 BH107a/b
BH113BH108
1
BH106
0
0
20
40
60
80
3.2
15
100
Chlorella (chlorophyte
1
Dunaliella (chlorophyte
4
Uncultured Rio Tinto Chlorphyte
6
Aulacoseira ambigua (stramenopile)
1
Pinnularia (stamenopile)
1
Microspora (chlorophyte)
6
Pinnularia (stramenopile)
4
Aulcoseira baicalensis (stramenopile)
5
T (C)
BH107
1.8
68
Staurastrum punctulatum (desmid)
12
Aulacoseira ambigua (stramenopile)
1
Powellomyces variabilis (chytrid)
1
Cercozoa (unculture Rio Tinto)
1
Pinnularia (stramenopile)
1
USW126
USW124
5
USW 127
BH118
pH
4
USW 123
3
USW125
USW122
+ e uk PCR
1
0
20
40
60
80
pH
T
#
clones
mat
12
DK105
mat
7
DK107
mat
DK110
4.5
3
45
DK116
4
22
6
pH
DNA extraction and quantification: Sediment and mat
samples were dewatered and resuspended in Tris buffer, pH
8. We compared a modified CTAB/chloroform extraction
with Wizard (Promega) purification, and several soil DNA kits
(UltraClean kit MoBio Labs, FastDNA, BIO101). Denaturing
Gradient Gel Electrophoresis (DGGE) was used to compare
extraction methods. For most samples, we combined
extracted DNA by multiple methods to minimize extraction
bias. DNA was quantified fluorimetrically with PicoGreen
(Molecular Probes).
D K110
DK111
D K116
D K112
3
DK109 DK114
DK113
DK101
DK104
DK106
DK103
DK100
2
Chlamydomonas (chlorophyte)
Hexapod
1
Echinamoeba thermarum (amoeba)
1
Pichia (chytrid)
Hexapod
1
Acanthamoeba castellanii
1
Chlorella (chlorophyte)
2
Sclerotium (basidiomycete)
6
Podura (hexapod)
4
Pinnularia (stramenopile)
5
Pinnularia (stramenopile)
1
Kleb sormidium nitens (chlorophyte)
2
Chlorella (chlorophyte)
4
Pinnularia (stramenopile)
1
Chlorella (chlorophyte)
1
hexapod
pH
T
#
clones
5.1
15
1
Monosiga ovata (choanoflagellate)
SW117
2.2
34
2
Chlamydomonas (chlorophyte)
USW123
3
25
1
Rio Tinto clone
6
Stenostomum leucops
(platyhelminth)
1
mite
USW124
5
44
BLAST Identity
6
no DNA re covere d
0
20
40
60
80
100
Pinnularia (stramenopile)
2
Scenedesmus sub spicatus
(chlorophyte)
USW125
3
44
6
Pinnularia (stramenopile)
USW126
6
45
2
Chlamydomonad (chlorophyte)
2
nemotode
USW127
5
36
1
Rhizophydium (fungi)
T (C)
1
PCR Amplification: We amplified eukaryal SSU rDNA with
several primer combinations (table 1) using standard PCR
conditions.
Bacillaria paxillifer (stramenopile)
14
Pinnularia (stramenopile)
1
Archaeal
16S
751/1204
Metopus palaeformis (alveolate)
Podura
DK105.3
Eukaryote 18S
4/516
82/516
615/1200
82/1390
DNA
Source
pH
T
DNA
µg/ml*
Eubact
16S
27/534
BH107
1.8
68
7.5
+++**
BH100
1.7
30
152
+++
+
BH101
1.7
30
33
+++
+++
BH118
2.2
19
40
+++
+
++
+++
++
BH105
3.2
15
423/26
+++
++
+++
+++
+++
BH106
3.2
15
304/22
++
++
+++
+++
DK108
3
85
13
+++
+++
+/-
+/-
DK109
3
85
1.4/20
+++
+++
+
DK110
4.5
60
2.4
+++
+++
+
+++
DK112
3
45
297/10
0
+++
+++
+
++
++
++
+++
+/-
+
BH107.17
unculturedeukaryote
BH107.11
pichia
saitoella
USW127.15
BH107.10
Fungi
+
chlorella
DK116.2
BH100.9
USW124.2a
BH100.4
DK112.7
BH100.7
BH100.6
BH100.7a
USW124.6
chloromonas
100
57
76
62
Chlorphytes
BH100.15
dunaliella
100
55
89
69
22
312/58
+
98
94
++
94
91
++
4
+++
+
DK102
mat
13
++
+++
DK105
mat
454/58
+++
+/-
DK107
mat
8/10.7
+++
+
+++
+++
+
+++
+++
++
++
+++
+++
DK112.15
klebsormidium
100
++
+
+++
BH107.13
chytrid
cercomonas
88
83
Amoeba
BH107.14
gymnophrys
91
a.alpigena
BH105.9
BH107.1
aularcoseira
BH105.1
100
70
USW119
2
85
6/32
+
USW126
6
45
66/7
+++
USW124
5
44
37/35
USW125
3
44
76/2.6
USW127
5
36
USW123
3
25
+++
96
+/+++
+++
+++
+
+
+++
+++
+++
++
+
+++
+++
+++
303/4
+++
++
+++
+++
+++
94/34
++
+/-
++
+
+++
+
pinnularia
100
99
75
55
98
66
100
*DNA extractions for CTAB/UltraClean DNA kit shown where both used
78
**+++ = strong PCR product, etc
79
100
100
100
Figure 1: Location of LVNP (red square) and Bumpass
Hell site.
BH105.10
microspora
USW124.3
USW124.7
USW124.8
100
72
Table 1: PCR results with different primer sets. Blank=primer not used. +++ =
relative amount of PCR product.
RioTintoCloneRT1n1
BH118.1
DK102.2
DK102.3
61
99
76
DK116
USW124.5
USW126.6
BSL101.2
BSL101.3
BSL101.1
SW117.1
SW117.2
chlamydomonad
100
56
+/-
2. Protist communities appear largely photosynthetic,
typically mats and benthic assemblages in shallow
streams consisting of acidophilic diatoms and
chlorophytes. Interestingly, Cyanidium, a dominant
acidophilic rhotophyte found at other hydrothermal sites
(Tansey and Brock, 1978), was only found in Boiling
Springs Lake. Heterotrophic protists, especially aveolates
and ciliates, were found, but we suspect that some
delicate cells may have ruptured during pH neutralization.
Chlorella (chlorophyte)
8
Figure 2c: PCR success vs. pH and temperature for
Devil’s Kitchen
1. Diverse eukaryal rRNA genes were found in many LVNP
hydrothermal environments. Protist taxa dominate
sequences, with fungi and some metazoa appearing in
cooler environments and mats.
Hartmannella (amoeba)
1
+ e uk PCR (also mats: DK 102, 107)
0
5. Based on the phylogeny, the clones cluster in groups of
chlorophytes, stramenopiles, ciliates and fungi (fig. 3).
Conclusions
DNA
Source
SW105
- euk PCR
1
4. Based on clones, acidophilic protists dominate eukaryotes in
LVNP hydrothermal environments. Many gave 90-98%
homology to known acidophilic taxa, or environmental clones
from other acidic sites (Rio Tinto, Spain). Most sites showed
phototrophic assemblages, dominated by stramenopiles,
chlorophytes, and some chrysophytes. Heterotrophic taxa
included diverse alveolates, amoebae, and fungi. Occasional
metazoa (hexapods, nematodes, platyhelminths) were
detected in low temperature, less acidic environments,
especially in mats.
BLAST Identity
1
60
Figure 2b: PCR success vs. pH and temperature for
Upper Sulfur Works
4
Chlamydomonas (chlorophyte)
DK102
100
5
Chysophyte
DNA
Source
DK112
Study Sites: We sampled mud pots, springs, and mats from
sites at Upper Sulfur Works, Bumpass Hell, Boiling Springs
Lake and Devil’s Kitchen (figs. 1, 2).
Englemanniella (amoeba)
2
1
T (C)
Methods
Chloromonas (chlorophyte)
1
19
- e uk PCR
0
3. Eukaryal 18S rDNA was amplified successfully from most
samples except those above 65 C (fig. 2, table 1). We found
primer combinations 82/516 worked better than 4/516 (table
1).
Poterioochromonas (stramenopile)
1
2.2
USW119
2
2. DNA extractions ranged from 0 to 454 mg/ml. Many mud
pots gave low or no DNA recoveries (table 1). Bacterial 16S
rDNA was amplified successfully from most sites, while
Archaeal 16S rDNA was amplified mostly at Devil’s Kitchen
Pinnularia (stramenopile)
1
Figure 2a: PCR success vs. pH and temperature for
Bumpass Hell
6
1. Bumpass Hell environments are more acidic and cooler than
Devil’s Kitchen and Upper Sulfur Works, which show similar
ranges (fig. 2).
Philasterides (ciliate)
2
B H118, 119
BH117
BH111
BH112
BH110
2
Questions:
1. What eukaryotes reside in the hydrothermal features of
LVNP? Do protists dominate diveristy?
2. Are the sites dominated by heterotrophic or autotrophic
organisms?
3. How does pH and temperature influence what protists
are found in the hydrothermal features?
Cloning and Sequencing: PCR products were cloned using
the TOPO-TA cloning kit (Invitrogen). Inserts were
reamplified directly from colonies, screened by RFLP (RspI
and HaeIII), and unique restriction patterns sequenced using
ABI Big Dye 3.1 on an ABI 310 sequencer.
Uncultured Rio Tinto Chlorphyte
1
6
BH106
BH105
3
Results
BLAST Identity
Platyophyra vorax (ciliate)
3
DK112.10
DK112.12
DK116.3
BH107.16
DK110.1
DK112.11
DK112.14
BH107.1a
Stramenopiles
BH107.14a
chrysocapsa
poterioochromonas
BH107.3
BH100.10
BH100.2
BH100.3
60
philasterides
100
BH107.20
engelmanniella
hartmannella
USW124.2
10
Figure 3: 18S rRNA phylogenetic tree
Aveolates
BH100.5
BH100.13
Ameoba
References
1. Amaral Zettler, L. A., F. Gómez, E. Zettler, B. G. Keenan, R. Amils, and M. L. Sogin.
2002. Eukaryotic diversity in Spain'
s river of fire. Nature 417:137.
2. Baker, B. J., P. Hugenholtz, S. C. Dawson, and J. F. Banfield. 2003. Extremely
acidophilic protists from acid mine drainage host Rickettsiales-lineage endosymbionts
that have intervening sequences in their 16S rRNA genes. Appl. Environ. Microbiol.
69:5512-5518.
3. Dawson, S. C., and N. R. Pace. 2002. Novel kingdom-level eukaryotic diversity in
anoxic environments. Proceedings of the National Academy of Sciences, USA
99:8324-8329.
4. Edgcomb, V. P., D. T. Kysela, A. Teske, A. de Vera Gomez, and M. L. Sogin. 2002.
Benthic eukaryotic diversity in the Guaymas Basin hydrothermal vent environment.
Proceedings of the National Academy of Sciences 99:7658-7662.
5. Packrof, G., and S. Woelfl. 2000. A review on the occurrence and taxonomy of
heterotrophic protists in extreme acidic environments of pH values <3. Hydrobiologia
433:153-156.
6. Tansey, M. R., and T. D. Brock. 1978. Microbial life at high temperatures: ecological
aspects. Pages 159-216 in D. J. Kushner, editor. Microbial Life in Extreme
Environments. Academic Press, London.
Acknowledgements
Sampling help and advice:
National Park Service
Patty Siering, Humboldt State
Rachel Whitaker, UC Berkeley
Financial Support:
CSUC Research Foundation
Sigma Xi Grants-in-Aid
NSF CCLI program