Infrequent Transitions between Saline and Fresh Waters in
One of the Most Abundant Microbial Lineages (SAR11)
Ramiro Logares,*,1 Jon Bråte,2 Friederike Heinrich,1 Kamran Shalchian-Tabrizi,2 and Stefan Bertilsson1
1
Limnology/Department of Ecology and Evolution, Uppsala University, Uppsala, Sweden
Microbial Evolution Research Group, Department of Biology, University of Oslo, Oslo, Norway
*Corresponding author: E-mail: [email protected].
Associate editor: Douglas Crawford
2
Abstract
Key words: environmental transitions, prokaryotes, saline, freshwater, SAR11, 16S rDNA.
Introduction
The SAR11 lineage within the alpha-Proteobacteria has received great attention from the scientific community since
it was discovered in the early 1990s (Giovannoni et al.
1990). This interest is certainly justified as they appear
to be the dominant bacterioplankton in the surface of
the oceans, where they can contribute to as much as
50% of the total microbial community (Morris et al.
2002). With an estimated global population size of
2.4 1028 cells, the SAR11 group is among the most successful organisms on the planet, but many questions still
remain unanswered concerning its ecology and physiology.
Several subgroups have been proposed within the SAR11
cluster, which in some cases seem to be correlated with
specific environments and therefore suggest the occurrence of adaptive evolution (Field et al. 1997; Brown and
Fuhrman 2005; Kan et al. 2008). The difficulties to cultivate
SAR11 strains have been one of the main obstacles for understanding their ecological roles in the ecosystem. The
first SAR11 culture (Candidatus Pelagibacter ubique) was
isolated off the coast of Oregon (Rappe et al. 2002). The
small vibroid cells formed by this strain were capable of
heterotrophic growth, but more recent studies point to
a rather complex metabolism and ecology. For instance,
it contains a functional gene coding for the light-dependent proton pump ‘‘Proteorhodopsin,’’ suggesting a capacity for phototrophic or at least photoheterotrophic energy
acquisition (Giovannoni, Bibbs, et al. 2005). Such complexities contrast with the fact that SAR11 members have one
of the smallest genomes known for any free-living microorganism (;1.3 Mb; Giovannoni, Tripp, et al. 2005). Recent
evidence also showed that the strain C. P. ubique is dependent on exogenous sources of reduced sulfur for growth
(Tripp et al. 2008), and this may explain to a certain extent
the low success in isolating members of this group. This
dependency also implies strong ecological links to other
planktonic organisms capable of synthesizing these
compounds.
In addition to marine environments, members of SAR11
have also been found in brackish (Kan et al. 2008) and fresh
waters (LD12; Zwart et al. 2002), but their numerical significance in communities from these environments is still
uncertain. Besides, there are so far no comprehensive and
synoptic studies on the evolutionary relationships between
marine, brackish, and freshwater SAR11 lineages. Nevertheless, previous studies have suggested that distinct evolutionary lineages may be present in marine, brackish, and
fresh waters (Zwart et al. 2002; Kan et al. 2008). This
may seem counterintuitive, as the high abundance of
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Mol. Biol. Evol. 27(2):347–357. 2010 doi:10.1093/molbev/msp239
Advance Access publication October 6, 2009
347
Research article
The aquatic bacterial group SAR11 is one of the most abundant organisms on Earth, with an estimated global population
size of 2.4 1028 cells in the oceans. Members of SAR11 have also been detected in brackish and fresh waters, but the
evolutionary relationships between the species present in the different environments have been ambiguous. In particular, it
was not clear how frequently this lineage has crossed the saline–freshwater boundary during its evolutionary
diversification. Due to the huge population size of SAR11 and the potential of microbes for long-distance dispersal, we
hypothesized that environmental transitions could have occurred repeatedly during the evolutionary diversification of this
group. Here, we have constructed extensive 16S rDNA–based molecular phylogenies and undertaken metagenomic data
analyses to assess the frequency of saline–freshwater transitions in SAR11 and to investigate the evolutionary implications
of this process. Our analyses indicated that very few saline–freshwater transitions occurred during the evolutionary
diversification of SAR11, generating genetically distinct saline and freshwater lineages that do not appear to exchange
genes extensively via horizontal gene transfer. In contrast to lineages from saline environments, extant freshwater taxa
from diverse, and sometimes distant, geographic locations were very closely related. This points to a rapid diversification
and dispersal in fresh waters or to slower evolutionary rates in fresh water SAR11 when compared with marine
counterparts. In addition, the colonization of both saline and fresh waters appears to have occurred early in the evolution
of SAR11. We conclude that the different biogeochemical conditions that prevail in saline and fresh waters have likely
prevented the environmental transitions in SAR11, promoting the evolution of clearly distinct lineages in each
environment.
Logares et al. · doi:10.1093/molbev/msp239
SAR11 in the oceans would have promoted repeated colonizations of brackish and fresh waters. The rationale is that
large populations can contribute many individuals and genotypes for colonizing new environments (many genotypes
increase the chances that at least one of them will succeed
in the new environment). However, it should also be considered that genome streamlining in SAR11 (Giovannoni,
Tripp, et al. 2005) may reduce the adaptability of the group
to diverse environmental conditions, although diverse
genes in hypervariable genome regions (Wilhelm et al.
2007; Gilbert et al. 2008) may counterbalance such effect.
Here, we investigate the transitions between marine,
brackish, and fresh waters during the evolution of SAR11
by using extensive 16S rDNA–based molecular phylogenies
containing publicly available sequences from various geographic locations. In addition, we have analyzed metagenomic libraries from environments distributed along
a wide salinity range in order to determine if SAR11 members in marine, brackish, and fresh waters are different at
the whole-genome level. Overall, our results indicate that
historical transitions between saline and fresh waters have
been uncommon events during the evolutionary diversification of SAR11, which promoted the emergence and
persistence of genetically distinct lineages in each
environment.
Materials and Methods
Phylogenetic Analyses
Preliminary Bayesian and maximum likelihood (ML) phylogenetic analyses using a reduced data set indicated the existence of strongly supported marine and freshwater SAR11
clusters, as well as a putative brackish one (results not
shown). Sequences from these clusters were subsequently
used for extensive Blast data-mining searches in Genbank
against the NCBI-nr database (searches finalized in December
2008). In total, about 700 sequences were retrieved, several
originating from the GOS (Global Ocean Sampling; Rusch
et al. 2007) data set (the GOS-associated sequences have
already been curated (i.e., checked for chimeras and errors)
and used in previous works, see Shaw et al. (2008); additionally, we have rechecked all used sequences for the presence of chimeras). The brackish and marine SAR11 query
sequences matched several GOS sequences from estuaries
(Delaware and Chesapeake bays, US and Bay of Fundy, Canada) and various marine environments, respectively. In
contrast, the freshwater sequences retrieved mostly GOS
sequences from the freshwater Lake Gatun in Panama
(the only freshwater lake included in the GOS data set
[GS20], which drains into the Panama Canal) as well as
some from estuaries. In all cases, the sequences retrieved
via Blast were highly similar to our query sequences. After
eliminating short and potentially erroneous sequences, the
retrieved sequences were used to construct an alignment
(using MAFFT [v6.240] with the algorithm E-INS-I; Katoh
and Toh 2008). This alignment consisted of 604 sequences
and 1,530 characters (AL1). The sequences in AL1 originated from several different geographical locations, some348
MBE
times separated by large distances. An additional more
stringent alignment was constructed by removing ambiguously aligned sites using GBLOCKS (Castresana 2000) as well
as visual examination. This alignment consisted in 604 sequences and 1,351 characters (AL2). A third alignment was
generated (AL3) using Nearest Alignment Space Termination (DeSantis, Hugenholtz, Keller, et al. 2006) in Greengenes (http://greengenes.lbl.gov/; DeSantis, Hugenholtz,
Larsen et al. 2006). AL3 consisted of 524 sequences and
1,268 characters and included 12 SAR11 sequences retrieved from metagenomic libraries from the Antarctic
Ace Lake. Several sites were removed from this alignment
using GBLOCKS as well as visual examination. All sequences
that became identical after the exclusion of sites were
removed from this alignment, which was used strictly for
investigating phylogenetic patterns and not for environment–phylogeny comparisons. An extra alignment (AL4)
based on AL3 was constructed by removing eight sequences
that branched at the base of the marine–brackish cluster
(EU800530, EU801112, EU800189, EU801347, EU800751,
EU801707, EU800662, and EU800327). This was carried
out in order to test for the effects that these deep branching
sequences could have had in the overall tree-topology reconstructions. AL4 consisted of 516 sequences and 1,268
characters. All alignments are summarized in supplementary table S1, Supplementary Material online.
Phylogenies were constructed using ML as implemented
in RaxML (v.7.0.4; Stamatakis 2006). The analyses in RaxML
were run with a rapid hill-climbing mode under the general
time reversible (GTR) þ MIX þ I option using 25 rate categories. The MIX þ I option in RaxML performs a tree
search under the GTR þ CAT approximation, and when
the analysis is finished, it changes the model to GTR þ
G þ I to evaluate the final topology of the tree and calculate likelihood values under this model. The GTR þ CAT is
an approximation for the GTR þ G model that accelerates
the computation of trees and is recommended for large
data sets (see RaxML user manual). We have carried out
additional analyses in RaxML under the GTR þ G þ I model
(with four rate categories; best topology and bootstrap), in
order to compare with the results given by GTR þ MIX þ I.
Overall, no significant differences (topology and bootstrap)
were found when using GTR þ MIX þ I and GTR þ G þ I;
hence, the two approaches will be considered as analogous
for the purpose of this work. In the analyses with RaxML,
the shape parameter (a) of the Gamma (G) distribution
and the proportion of invariable sites (I) were estimated
from the data set using default options. A total of 100 heuristic tree searches per alignment were carried out from
random starting trees, and the topology with the highest
likelihood was selected. Bootstrapping was carried out with
1,000 pseudoreplicates under the GTR þ MIX þ I/GTR þ
G þ I models, with one heuristic search per replicate. All
phylogenetic analyses were run at the University of Oslo
Bioportal (http://www.bioportal.uio.no/) computer cluster.
The trees generated were visualized and edited in FigTree
(v1.2.2; Rambaut 2009) and Interactive Tree Of Life (Letunic
and Bork 2007). Alignments and trees are available on request.
Saline–Freshwater Transitions in Prokaryotes · doi:10.1093/molbev/msp239
Unifrac Analyses
We have used the Unifrac and P value approach (Martin
2002; Lozupone and Knight 2005) to test statistically
whether there is an evolutionary separation between marine, brackish, and freshwater groups. The microbial communities in given environments are considered
evolutionary different if the Unifrac value for the real tree
is greater than a random distribution of the sequences
among the environments (see Martin 2002; Lozupone
and Knight 2005 for a detailed description of Unifrac
and the P value). To carry out the Unifrac analyses, the habitats of the sequences included in the analyses were determined according to the ‘‘Features’’ information in the
GenBank records or by referring to the publication of
the sequence. Each site was classified as 1 5 Marine
(30–40 PSU), 2 5 Brackish (0.5–30 PSU), and 3 5 Freshwater (,0.5 PSU). This classification is used in aquatic sciences and derives from ‘‘The Venice System for the
Classification of Marine Waters According to Salinity’’
(see Anonymous 1958; Hammer 1986). Note that in contrast to marine and fresh waters, brackish environments
may experience wide salinity changes in diverse spatiotemporal scales (see Dahl 1956; Lampert and Sommer 2007).
The analyses were carried out considering different combinations of environmental categories. In one analysis, 1 and
2 were considered as one single ‘‘marine–brackish’’ category, and in another analysis, 2 and 3 were considered
as the single ‘‘brackish–freshwater’’ category. We also carried out analyses considering marine, brackish, and freshwater as independent environmental categories. The used
input tree was the best tree (highest likelihood) obtained
after 100 heuristic searches in RaxML using the AL2 alignment. Unifrac tests were run with 100 permutations and P
values were corrected for multiple comparisons using the
Bonferroni correction. Phylogenetic outgroups were not
considered in the Unifrac tests.
Phylogenetic Diversity within Marine–Brackish
and Freshwater Clades
In order to estimate the amount of evolutionary diversification (i.e., amount of molecular evolution) that has occurred within both the marine–brackish and freshwater
clusters (phylogenetic diversity), we have added the branch
lengths within each of them (F 5 sum of branch lengths
within freshwater cluster; M 5 sum of branch lengths
within marine–brackish cluster). Subsequently, in order
to normalize these values, we divided F and M by the
number of sequences contained by the freshwater and
marine–brackish clusters correspondingly (F1 5 F/127
and M1 5 M/461). Afterward, we calculated the ratio
F1/M1, which estimates the proportion of evolutionary
diversification between the freshwater and marine–
brackish clusters.
Fragment Recruitment Analyses (FRAs)
FRA uses the genome of a given strain to detect homologous fragments (sequences) in metagenomic libraries (see
Rusch et al. 2007). Here, we have used the genome of the
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marine SAR11 strain C. P. ubique HTCC1062 (accession
number CP000084) to check for the presence of evolutionary-related strains in metagenomic libraries from environments distributed along a wide salinity range (0–175 PSU).
This approach can shed light on marine–freshwater transitions. For example, if close relatives of the marine strain
HTCC1062 are present in fresh waters, then various reads
matching with high similarity different areas of the query
genome should be detected in freshwater metagenomic libraries. This analysis can also provide an indication of
whether or not the differences or similarities in the 16S between different groups are reflected in the rest of their genomes. This is particularly relevant, as substantial horizontal
gene transfer could still occur between genetically differentiated saline and freshwater lineages, specially in SAR11 (see
Vergin et al. 2007; Gilbert et al. 2008). The strain C. P. ubique
HTCC1062 was the first member of SAR11 to be isolated
(Rappe et al. 2002). This isolate has been collected from
a depth of 10 m, at a location 27.6 km off from the coast
of Oregon (44°39.1#N, 124°24.7#W). The salinity of the water from where HTCC1062 was isolated was about 33 PSU
(Grantham et al. 2004). FRA was carried out in both CAMERA (http://camera.calit2.net/; Seshadri et al. 2007) and
Meta Genome Rapid Annotation using Subsystem Technology (MG-RAST) (http://metagenomics.nmpdr.org/; Meyer
et al. 2008). FRA was performed in 13 publicly available
metagenomes from diverse aquatic environments (see table
1). These metagenomic data sets were selected in order to
encompass a wide salinity gradient and different environmental conditions (e.g., lakes vs. marine). Other metagenomic libraries have also been investigated, but they are
not included in the final analyses because they do not provide additional information (data not shown). All metagenomic libraries originated from water samples taken at one
depth (normally surface) at each sampled site. However,
metagenomic libraries from different depths were available
for Ace Lake, and we have included them in this study as
this lake presents strong vertical gradients in several physicochemical variables (Rankin et al. 1999) that can affect
microbial community composition.
16S Survey of Metagenomic Libraries
Large metagenomic libraries can potentially contain genome fragments from rare populations that are not normally detected in regular clone libraries of amplified 16S
rDNA genes. Thus, for example, if there were freshwater
SAR11 strains present in low abundances in marine environments, then those strains could be detected by Blastsearching known fresh waters sequences against marine
metagenomic data sets. Using this approach, we aimed
at detecting relatives of marine, brackish, and freshwater
SAR11 sequences in the 13 analyzed metagenomes. Representative 16S rDNA sequences belonging to freshwater
(Z99997, LD12), Brackish (U70686, putative group), and
Marine (AF510191, HTCC1062) groups were used as
queries for Blast searches in MG-RAST (Meyer et al.
2008) against the investigated metagenomic libraries. Only
those retrieved sequences with an E-value , 0.0001 and
349
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350
Only recruited fragments with .50% similarity were considered in the analyses. Metagenomic data sets were obtained from CAMERA and follow the nomenclature given in CAMERA or GOS (see Rusch et al. 2007). The size fraction from
which each sample was obtained ranged between 0.1 and 0.8 lm (except GS00C, which was obtained from the fraction 0.22–0.8 lm).
Size Data set Number of Mean of
(Number of Recruited Fragment
Min Bit Score/Max
Sequences) Fragments Identity
SD E-value/Min align.
95,521
21,174
83.3
11.9
34/1.26E202/17
103,058
14,710
82.3
12.5
32/7.94E203/15
368,835
46,208
73
11.2
35/1.00E202/18
59,679
16,770
86.6
12.2
35/7.94E203/19
124,435
16,172
74.5
12.4
45/5.01E203/23
126,162
15,561
71.8
12.0
44/7.94E203/25
28,481
0
0
0
N/A
281,490
15,301
82.6
12.4
36/1.00E202/19
54,446
2
74
7.1 149/3.16E238/91
10,042
3
63
1.7 167/5.01E240/134
96,72
9
74
15.7 190/6.31E247/146
100,085
29
65.4
15.1
57/7.94E207/51
296,355
8,419
64.3
9.5
45/5.01E03/29
Salinity
Library
Sample Location
Environment
Latitude/Longitude
Depth (m)
(PSU)
1 (S9)
Open Ocean, Antarctica
Marine
63°53#28$S–112°4#23$E
2
34.0
2 (S8)
Newcomb Bay, Antarctica
Marine
66°16#12$S–110°31#59$E
2
34.0
3 (GS00C) Sargasso Sea, Atlantic Ocean
Marine
32°12#29$N–64°00#36$W
5
36.7
4 (GS006) Bay of Fundy, Canada
Estuary
45°6#42$N–64°56#48$W
1
25.0-31.0
5 (GS011) Delaware Bay, United States
Estuary
39°25#4$N–75°30#15$W
1
3.5
6 (GS012) Chesapeake Bay, United States Estuary
38°56#49$N–76°25#2$W
13.2
3.5
7 (S7)
Organic Lake, Antarctica
Hypersaline, marine derived 68°25#58$S–78°17#59$E
0.1
175.0
8 (S6)
Ace Lake, Antarctica
Saline, marine derived
68°24#0$S–78°10#58$E
5
22.0
9 (S4)
Ace Lake, Antarctica
Saline, marine derived
68°24#0$S–78°10#58$E
12.7
22.0
10 (S3)
Ace Lake, Antarctica
Saline, marine derived
68°24#0$S–78°10#58$E
14
32.0
11 (S2)
Ace Lake, Antarctica
Saline, marine derived
68°24#0$S–78°10#58$E
18
34.0
12 (S1)
Ace Lake, Antarctica
Saline, marine derived
68°24#0$S–78°10#58$E
23
N/D
13 (GS020) Lake Gatun, Panama
Freshwater
9°9#52$N–79°50#10$W
2
0.1
Table 1. Metagenomic Libraries Used for Fragment Recruitment Analysis Using the Genome of the Marine SAR11 Strain Candidatus Pelagibacter ubique HTCC1062 as Query and a General
Description of the Results.
Logares et al. · doi:10.1093/molbev/msp239
a Bit score . 1,000 were further analyzed, as they have reasonable chances of being SAR11 members. The percentage
of similarity between the query and the retrieved sequences
was analyzed.
Results
Deep Evolutionary Divergence between Marine–
Brackish and Freshwater SAR11 Lineages
Our phylogenetic analyses (based on AL2/4) gave significant support for the SAR11 cluster (100% bootstrap support [BS]), as well as for the marine–brackish (.70% BS)
and freshwater (.70% BS; so-called LD12, Zwart et al.
2002) subclusters (fig. 1, see summary of the phylogenetic
results in supplementary table S1, Supplementary Material
online). In the trees based on AL2, the marine–brackish
cluster contained 461 sequences, whereas the freshwater
cluster contained 127. Within the marine–brackish cluster,
there were 220 marine (48%) and 241 brackish (52%) sequences (fig. 2). No freshwater sequences fell into the
marine–brackish cluster. The freshwater cluster (LD12)
was composed by 119 freshwater (94%) and 8 brackish
(6%) sequences (fig. 2). Marine sequences were absent in
the freshwater cluster. Due to the low proportion of
brackish sequences falling into the freshwater cluster (that
probably do not represent indigenous brackish lineages
that have derived from freshwater ancestors), we have
named this cluster simply ‘‘freshwater’’ (instead of
‘‘freshwater–brackish’’).
Overall, the phylogenetic diversity (i.e., amount of molecular evolution) within the marine–brackish cluster was
at least 7.5 times higher than the diversity within the freshwater cluster (fig. 1; the normalized phylogenetic diversity
within the freshwater clade was 0.0014, whereas the diversity within the marine–brackish cluster was 0.0111; calculations based on AL2). Despite the significant statistical
support for the backbone of the SAR11 phylogeny, most
of the branching patterns within the marine–brackish
and freshwater subclusters were unsupported (fig. 2).
Within the marine–brackish SAR11, the general clustering
patterns obtained in our phylogenetic reconstructions
(marine–brackish phylogeny backbone; AL2) agreed to
a certain extent with previous work, and therefore, we will
follow the cluster nomenclature already proposed (see, e.g.,
Kan et al. 2008). Specifically, the subclusters SAR11-I (66%
BS) and SAR11-II (97% BS) were recovered in our phylogenetic reconstructions (fig. 2). However, the previously proposed cluster SAR11-III (normally sister to the freshwater
cluster) was not supported by our analyses (fig. 2). We constructed the more stringent alignment AL4, excluding sequences branching at the base of the marine–brackish
cluster (see fig. 2 ‘‘A’’) in order to test if these sequences
could have affected the tree-topology reconstruction
and if SAR11-III would be recovered under these new conditions. However, the removal of those sequences increased
the BS for the previously observed topology.
Overall, SAR11-I was recovered as a mosaic of marine
and brackish sequences (fig. 2) with no clear internal
Saline–Freshwater Transitions in Prokaryotes · doi:10.1093/molbev/msp239
FIG. 1. Distinct saline and freshwater lineages in SAR11. SAR11 ML
phylogenetic tree (phylogram) based on 604 sequences (AL2)
calculated under the GTR þ MIX þ I model in RaxML. The best
topology was obtained after 100 heuristic searches. BS values
presented over the nodes derive from 1,000 pseudoreplicates. (A)
phylogram showing the backbone of the phylogeny, with the
marine–brackish (red) and freshwater (blue) subgroups collapsed.
Note the reduced phylogenetic diversity (i.e., amount of molecular
evolution) within the freshwater clade when compared with the
marine–brackish. (B) Radial phylogram illustrating the amount of
genetic divergence within the marine–brackish and freshwater
clusters. The name of some major clusters is indicated over the
branches. The arrows indicate nodes where some subclusters start.
The scale bar indicates substitutions/site.
clustering pattern. Most of the brackish sequences originated from the Chesapeake and Delaware bays. In addition,
SAR11-I contained sequences from various locations, such
as the Arctic, Southern Ocean, deep sea, and the coast of
Oregon, United States. The strain C. P. ubique (HTCC1062),
which was genome sequenced and used in this work for
FRAs, fell within this cluster.
SAR11-II was predominantly composed of marine sequences (fig. 2). Within this group, we have recovered
the clusters SAR11-IIA (91% BS; only represented by two
sequences), SAR11-IIB (74% BS), and SAR11-IIC (79% BS)
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(fig. 2). SAR11-IIB contained 154 sequences from diverse
geographic origins, such as the Arctic and Southern Ocean,
Newport Harbor (US), and the sea near Costa Rica and
Panama. SAR11-IIC was composed of sequences originating
from different locations in the Pacific Ocean, the Sargasso
Sea, Caribbean Sea, and Arctic Ocean. Overall, most of the
recovered branching patterns within SAR11-II subclusters
were unsupported.
Several sequences, most of them brackish, had a basal
position in relation to SAR11-I and II (fig. 2). Some of these
sequences were previously assigned to SAR11-III (e.g.,
U70686; Kan et al. 2008 and AF353226; Stingl et al.
2007), a group that, as mentioned before, was not
supported in our analyses. Many of the basal sequences
originated from Chesapeake and Delaware bays. Other
sequences from diverse locations such as Arctic Ocean,
vicinity of Panama and Cocos Island were also present.
The freshwater cluster (LD12) included sequences originating from various freshwater environments around the
world, sometimes separated by large geographical distances
(see supplementary file S1, Supplementary Material online).
In particular, several sequences within LD12 originate from
Lake Gatun in Panama and were obtained during the GOS
expedition (Lake Gatun was the only freshwater body included in GOS). There was low support for the internal
branching patterns within LD12. The eight brackish sequences within LD12 did not present any obvious clustering pattern among each other or with other freshwater
sequences.
Within SAR11-I and II, among the basal sequences of the
marine–brackish cluster and within LD12, a number of statistically supported subclusters were present. These were
usually near the tip of the branches and included very similar sequences. Such clusters may correspond to ecotypes
or species, but because their analysis is not comprehended
within the main focus of our study, they will not be considered further.
Test of Environment-Specific Evolution
The first Unifrac analysis considered marine, brackish, and
fresh waters as three distinct environments. When all the
three environments were analyzed together, our results
indicated that at least one of them contained environment-specific lineages (P , 0.01; Unifrac significant). In
the pairwise comparison of environments, we observed
a marginally significant separation between marine, brackish, and fresh waters (P , 0.03 in all combinations; Unifrac
marginally significant), suggesting different lineages in the
different environments. In the second analysis, we considered marine–brackish as one environment and freshwater
as another, and we obtained a significant difference between them (P , 0.01; Unifrac significant). In the third
analysis, we considered marine as one environment and
brackish–freshwater as another, and a significant difference
was also obtained (P , 0.01; Unifrac significant). Overall,
the fact that the last two comparisons were both statistically significant suggests that environment-specific
evolution has occurred within each environment (i.e., that
351
Logares et al. · doi:10.1093/molbev/msp239
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FIG. 2. Distribution of marine, brackish, and freshwater sequences across SAR11 phylogeny. Cladogram of the same tree shown in figure 1
indicating the distribution of marine (red), brackish (green), and freshwater (blue) sequences across the phylogeny. Major clusters or groups of
sequences are delineated with solid black lines and subclusters with discontinuous lines. BS values . 50% are presented over the nodes (circles;
size proportional to the support) and derive from 1,000 pseudoreplicates. A 5 group of basal sequences that were removed in some analyses to
test the tree topology. B 5 marine subclade basal to SAR11-I and II. Note that the proportion of marine, brackish, and freshwater sequences in
the tree most likely does not represent the relative diversity of these groups in nature.
there seem to be endemic marine, brackish, and freshwater
lineages as the pairwise comparison between these three
environments previously indicated). Otherwise, if strains
in brackish waters were closely related to several marine
or freshwater ones, one of the previous combinations would
have been either not significant or marginally significant.
FRAs Support Phylogenetic Patterns
Overall, our FRAs indicated that saline and freshwater
SAR11 are clearly distinct at the whole-genome level. Thus,
352
the 16S rDNA divergence between saline and freshwater
lineages reflects genomewide divergence in SAR11. Using
the genome of the marine SAR11 strain C. P. ubique
(HTCC1062), we carried out FRA in metagenomic libraries
from environments distributed along a wide salinity spectrum (see Materials and Methods). The most similar fragments (average similarity . 80%) were recruited from the
Southern Ocean in Antarctica (S8/9), Bay of Fundy
(Canada, GS006), and the saline surface waters of the
Antarctic Ace Lake (S6, table 1; fig. 3). Slightly less similar
Saline–Freshwater Transitions in Prokaryotes · doi:10.1093/molbev/msp239
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FIG. 3. FRA results using Candidatus Pelagibacter ubique HTCC1062 as query genome. The gray columns show the average percentage of
similarity of the recruited fragments and its standard deviation. Black columns indicate the percentage of the corresponding metagenomic
library that was recruited. Note that the gray and black columns should not be compared. The used metagenomic libraries are indicated in the
x-axis and described in table 1. Only recruited fragments with at least 50% similarity were considered in the analyses. Other FRA results are
indicated in table 1.
fragments (but still with relatively high average similarities;
70–80%) were recovered from the Sargasso Sea (GS00C)
and the brackish estuaries Chesapeake (GS012) and Delaware (GS011) bays. The most dissimilar fragments recovered originated from the freshwater Lake Gatun
(Panama), indicating that at least no close relatives of
the marine HTCC1062 strains are present in fresh waters
(still, the average similarity of these fragments fell between
60% and 70%, which suggests that a fraction of them belong to LD12). In other metagenomic libraries, the amount
of recruited fragments was very low or null, indicating that
SAR11 members are absent or in very low densities in those
environments. Such were the cases of the Antarctic marine-derived lakes Organic (hypersaline), as well as the deep
layers of Ace Lake (saline, 12- to 23-m depth; see table 1).
We have also calculated the percentage of the total
metagenomic library represented by the recruited fragments in each analysis (fig. 3), as this can provide an insight
into the relative abundance of SAR11 in different environments. The largest amounts of fragments (20–29%) were
recruited from the Southern Ocean near Antarctica (S9)
and from the Bay of Fundy (GS006) (fig. 3). The amount
of fragments recruited from coastal Antarctic areas (S8),
the Sargasso Sea (GS00C), and the brackish estuaries Delaware (GS011) and Chesapeake (GS012) bays represented
in all cases between 11% and 15% of the corresponding
metagenomic libraries. The lowest amounts of fragments
(,5%) were recruited from the surface waters of the saline
Antarctic Ace Lake (S6) and the freshwater Lake Gatun
(Panama, GS020) (see fig. 3).
The Blast searches against all the analyzed metagenomic
libraries using representative 16S rDNA sequences from
marine, brackish, and fresh waters supported the previously
observed patterns (table 2). The freshwater sequence
Z99997 retrieved very similar sequences only from the
freshwater Lake Gatun in Panama and from the brackish
Delaware Bay, United States (table 2). This supports that
the somewhat different fragments recruited by the marine
genome HTCC1062 in Lake Gatun originate from freshwater SAR11 members. The brackish–coastal sequence
U70686 (assigned to this category by Kan et al. 2008) retrieved very similar sequences from the brackish Delaware
Bay. The marine sequence AF510191 (strain HTCC1062;
C. P. ubique) recruited very similar sequences from Antarctic marine waters (S8/S9), Bay of Fundy, Delaware and
Chesapeake bays, and the surface of Ace Lake (table 2).
The sequences retrieved from Ace Lake were included in
our phylogenetic analyses (AL3/4), and all of them clustered within the marine–brackish group SAR11-I. As expected from the FRA, none of the queries retrieved
sequences from Organic Lake or from the deeper layers
(.12 m) of Ace Lake.
Discussion
Distinct Marine–Brackish and Freshwater
Phylogenetic Clusters within SAR11
Our phylogenetic and statistical analyses based on the 16
rDNA strongly supported an evolutionary separation between freshwater and saline SAR11 groups (see also Zwart
et al. 2002; Rappe and Giovannoni 2003; Atamna-Ismaeel
et al. 2008). Within the marine–brackish cluster, we have
recovered with moderate-high support the formerly recognized clusters SAR11-I and SAR11-II (Stingl et al. 2007; Kan
353
MBE
354
Only those sequences with E-value , 0.0001 and bit-score . 1000 were considered in the analysis. The values in the last three columns indicate average percentage of similarity of the recruited sequences to the reference, standard deviation
(SD), and number of sequences recruited using the format X % (X SD; X sequences).
a
This sequence belongs to a putative brackish cluster (see Kan et al. 2008).
b
In GS011, sequences belonging to apparently two groups were recruited by the three references, therefore resulting values are presented independently.
Marine (AF510191,
HTCC1062)
97.9 (1.9; 30)
97.9 (1.3; 18)
96.4 (1.4; 63)
98.9 (0.7; 18)
98.0 (0.3; 25)/94.2 (2.5; 6)
98.1 (4.5; 15)
0
98.7 (0.7; 19)
0
0
0
0
89.4 (0.8; 11)
Brackish/Coastal
(U70686)a
0
0
89.1 (0.5; 15)
0
89.7 (1.0; 10)/98.4 (1.1; 5)
95.5 (4.7; 6)
0
96.0 (0; 2)
0
0
0
0
91.3 (0.5; 17)
Freshwater
(Z99997, LD12)
89.3 (1.1; 3)
88.0 (0; 1)
89.2 (0.8; 15)
89.4 (0.5; 8)
89.8 (1.2; 11)/98.5 (0.7; 2)b
91.3 (1.1; 3)
0
89.3 (1.4; 6)
0
0
0
0
98.5 (1.1; 26)
Salinity
(PSU)
34.0
34.0
36.7
25.0–31.0
3.5
3.5
175.0
22.0
22.0
32.0
34.0
N/D
0.1
Depth (m)
2
2
5
1
1
13.2
0.1
5
12.7
14
18
23
2
Sample Location
Open Ocean, Antarctica
Newcomb Bay, Antarctica
Sargasso Sea, Atlantic Ocean
Bay of Fundy, Canada
Delaware Bay, United States
Chesapeake Bay, United States
Organic Lake, Antarctica
Ace Lake, Antarctica
Ace Lake, Antarctica
Ace Lake, Antarctica
Ace Lake, Antarctica
Ace Lake, Antarctica
Lake Gatun, Panama
Metagenomic
Libraries
1 (S9)
2 (S8)
3 (GS00C)
4 (GS006)
5 (GS011)
6 (GS012)
7 (S7)
8 (S6)
9 (S4)
10 (S3)
11 (S2)
12 (S1)
13 (GS020)
Table 2. Average Percentage of Similarity of the 16S rDNA Sequences Recruited from the Metagenomic Libraries to Marine, Brackish, and Freshwater SAR11 Reference Sequences.
Logares et al. · doi:10.1093/molbev/msp239
et al. 2008). In contrast, the previously defined cluster
SAR11-III was not recovered in our taxon richer trees. Usually, SAR11-III has been recovered in phylogenies including
fewer taxa (e.g., Stingl et al. 2007; Kan et al. 2008; Carlson
et al. 2009; unpublished results), and hence, its presence
could have been the outcome of reduced taxon sampling.
The variable position of the SAR11-III cluster in other phylogenetic trees (compare, e.g., Kan et al. 2008; Carlson et al.
2009) supports the latter hypothesis. Further work including more sequences and other genes is needed to determine whether SAR11-III is a natural group.
In contrast to earlier publications (see Stingl et al. 2007;
Kan et al. 2008), our analyses did not recover the subclusters IA and IB within SAR11-I. In our results, sequences previously assigned to cluster IB grouped adjacent to each
other but did not constitute a principal subcluster. Thus,
SAR11-IA and IB may have appeared as main subclusters in
previous phylogenies also as a product of reduced taxon
sampling.
SAR11-I encompassed a mix of intercalated brackish and
marine sequences, suggesting that members of this group
could have moved repeatedly (i.e., adapted back and forth)
between brackish and marine waters. Alternatively, this
pattern might reflect a wide salinity tolerance of an originally marine or brackish group. For example, in several of
the cases where marine and brackish sequences were
closely related (e.g. AF510192 and EU919824), the brackish
sequences originated from either Newport Harbor
(26.5 PSU) or Kongsfjorden in the Arctic (in the latter, salinity can be reduced to less than 28 PSU due to runoff of
freshwater from the glaciers; see Piwosz et al. 2009). Thus,
several of the brackish sequences that were closely related
to marine sequences could actually represent marine
strains living in waters with salinity slightly lower than
in marine environments.
Within SAR11-II, the previously defined clusters SAR11IIA/B/C (Kan et al. 2008) were recovered by our analyses
with significant statistical support. Overall, the cluster
SAR11-II seems to contain mostly marine sequences from
various locations around the world.
Several sequences were basal to the groups SAR11-I and
II (hereafter BASAL; fig. 2). Sequences assigned to SAR11-III
in other works (see Stingl et al. 2007; Kan et al. 2008;
Carlson et al. 2009; the latter work named this group
SAR11-IV) were included within this set of basal sequences
in our phylogenies. Basal sequences were mostly retrieved
from brackish waters, except a number of marine sequences that clustered together (fig. 2 ‘‘B’’). This marine basal
cluster could represent an early marine ancestor of the
saline SAR11.
Within LD12, we observed some subclusters featuring
moderate to significant BS that may correspond to different ecotypes, comparable with the ecotypes suggested for
marine SAR11 counterparts (Rappe and Giovannoni 2003;
Brown and Fuhrman 2005; Carlson et al. 2009). Notably, we
found that the phylogenetic diversity (i.e., amount of
molecular evolution) within the freshwater SAR11 group
appears to be around 7.5 times lower than within the
Saline–Freshwater Transitions in Prokaryotes · doi:10.1093/molbev/msp239
marine–brackish cluster. In particular, freshwater SAR11
strains obtained from distant geographic locations were
very closely related, a pattern also observed in phylogenies
based on SAR11 proteorhodopsins (Atamna-Ismaeel et al.
2008). Overall, this points to a rapid diversification and dispersal in fresh waters and/or to different evolutionary rates
in saline and freshwater SAR11 lineages. Slower evolutionary rates in diverse genetic markers have been found in
freshwater crustaceans when compared with halophilic
counterparts (Hebert et al. 2002). The negative effects of
salt in the fidelity of DNA replication seem to have accelerated the rate of molecular evolution in the halophilic
taxa, despite their osmoregulatory capabilities (Hebert
et al. 2002), and analogous effects could have also affected
the evolution of saline SAR11. However, similar evolutionary rates have been observed in diverse saline and freshwater prokaryotes and microeukaryotes (Logares et al. 2009),
suggesting that the potential effects of salinity in molecular
evolution are not generalized.
The moderate–low BS for the internal topology of the
marine–brackish and freshwater clusters may be a consequence of a relatively rapid diversification, where the divergence time has been too short for the 16S rDNA to reflect
the diversification process. A comparable lack of support
appears to be present even in trees based on the fastevolving internal transcribed spacer rDNA (Brown and
Fuhrman 2005). Nevertheless, other processes may account
for the blurry diversification patterns. For example, high intraspecific recombination rates have been found in SAR11
(Vergin et al. 2007), which could decrease the phylogenetic
signal in the 16S through the generation of mosaic molecules with diverse evolutionary origins (Dewhirst et al. 2005).
Even though most environmental sequences used in our
analyses have been used (and checked) in other works (see,
e.g., Shaw et al. 2008 regarding the curation of the used
GOS sequences) and even though we have reassessed their
quality, it could still be possible that some erroneous sequences could have remained in our analyses. The solitary
environmental sequences branching at the base of the
marine–brackish cluster (fig. 2 ‘‘A’’) could represent such
an example. As mentioned earlier, the removal of those
sequences did not generate changes in the general tree topology; on the contrary, it increased its BS. Overall, the consistency in the general topologies and BS values that we
have obtained across different phylogenetic reconstructions make us confident that the potential effect of undetected erroneous sequences in our conclusions is most
likely minimal.
Exclusive Brackish Groups?
Our phylogenetic and statistical analyses suggested that
there might be exclusive SAR11 brackish lineages, as already
suggested by Kan et al. (2008) based on more reduced data
sets. Here, we have analyzed a relatively large number of
brackish sequences, and almost all of them (94%) grouped
with marine sequences, constituting the marine–brackish
cluster. It is important to note that within the marine–
MBE
brackish cluster, marine and brackish sequences were
not closely related for the most part, pointing to environment-specific evolution. The basal position of several
brackish sequences suggests that at least some of them
diverged early in the evolution of SAR11. Despite several
of the analyzed brackish sequences came from environments with salinities close to freshwater, the transitions between brackish and freshwater environments seem to have
been uncommon events. Overall, only 6% of the included
brackish sequences grouped within the freshwater clade,
and these brackish sequences probably represent freshwater strains that ended up in brackish surface waters as
a product of riverine freshwater discharge or runoff. The
absence of freshwater SAR11 from environments that
present even a slight increase in salinity mirrors early
results from studies on macroorganisms by Remane
(1934), suggesting that the distributions of freshwater
microbes and multicellular organisms may be similarly
affected by salinity.
Metagenomic Analyses Support the Observed
Phylogenetic Patterns and Give Insight into
Distributions along the Salinity Gradient
Our metagenomic analyses indicated a significant amount
of divergence at the genome level between SAR11 lineages
present in saline and fresh waters, which agrees with our
16S-based phylogenies. This suggests that the rate of horizontal gene transfer between saline and freshwater SAR11
lineages is probably not very high. We have used the genome of the marine SAR11 strain C. P. ubique HTCC1062
to perform FRA against selected metagenomic libraries
obtained from environments distributed along a wide salinity gradient (0–175 PSU). As initially expected, the
most similar fragments were recruited from saline environments (marine samples or the saline Ace Lake) and the
most dissimilar ones from the freshwater Lake Gatun. From
brackish environments, very similar and somewhat more
dissimilar fragments were recruited, suggesting that
a mix of marine, brackish, and freshwater SAR11 lineages
may populate these environments. The results of the
16S recruitment analyses pointed in the same direction
as our FRA results.
The presence of 16S sequences belonging to the marine–
brackish group SAR11-I in the surface waters of the marinederived Antarctic Ace Lake indicates that marine SAR11
strains have the potential to colonize and survive in saline
continental waterbodies. This is also supported by other
SAR11 sequences (affiliating to a putative coastal-brackish
group) that have been retrieved from saline lakes in China
(Wu et al. 2006; Kan et al. 2008). Overall, it is surprising that
members of SAR11-I manage to survive in Antarctic lakes,
where environmental conditions are considered to be
extreme (Laybourn-Parry and Pearce 2007).
Infrequent Saline–Freshwater Transitions in
SAR11
If transitions between environments with different salinity
would have been common during the evolutionary
355
MBE
Logares et al. · doi:10.1093/molbev/msp239
diversification of SAR11, intercalated marine, brackish and
freshwater sequences across the phylogenies would have
been expected (see Logares et al. 2009). However, the presence of statistically supported and distinct saline and freshwater clusters clearly indicates that transitions between
these environments have been rare during the diversification of the group. Freshwater–saline transitions seem to
have been rare also in other microbial groups (Logares
et al. 2009). However, the huge population size of SAR11
led us to hypothesize that at least a few more transitions
could have occurred within this lineage, as has been observed in other microbial groups (e.g., Alverson et al.
2007; Shalchian-Tabrizi et al. 2008).
Environmental transitions between marine and brackish
waters seem to have occurred more often during the evolution of SAR11 than transitions between freshwater and
brackish environments, which mirrors patterns from multicellular organisms (Remane 1934). This is probably related
to intrinsic genetic, physiologic, or ecologic limitations
in SAR11. In particular, it has been shown that marine
SAR11 require exogenous reduced sulfur originating from
other plankton for growth (Tripp et al. 2008), and the absence of such accompanying taxa or specific compounds in
new environments may preclude transitions. In addition,
genome streamlining in SAR11 (Giovannoni, Tripp et al.
2005) may reduce the plasticity of the group to switch between environments. Overall, more research is needed to
determine what mechanisms prevent saline–freshwater
transitions.
Concluding Remarks
Our phylo-metagenomic analyses indicated that few
saline–freshwater transitions occurred during the evolutionary diversification of SAR11, generating genetically distinct lineages in each environment. In addition, the rate of
horizontal gene transfer between saline and freshwater
SAR11 does not appear to have been very high during
the evolution of the group. The divergence between saline
and freshwater SAR11 probably involved several adaptive
changes at the genome level. Future genomic studies are
needed to unveil the mechanisms of adaptation of
SAR11 to these environments and answer the question
‘‘what makes it a bacterium saline or freshwater’’?
Supplementary Material
Supplementary file S1 and supplementary table S1 are
available at Molecular Biology and Evolution online
(http://www.mbe.oxfordjournals.org/).
Acknowledgments
Financial support was provided by the Swedish Research
Council for Environment, Agricultural Sciences and Spatial
Planning (Formas) via Uppsala Microbiomics Centre
(UMC), and the Swedish Research Council (grants to
S.B.). Phylogenetic analyses were run at the University of
Oslo Bioportal (http://www.bioportal.uio.no/). We thank
N. Rodriguez Ezpeleta for the help provided with some phy356
logenetic analyses and the staff of CAMERA and MG-RAST
projects for support/advice with the analyses. We thank
the comments and suggestions of the four reviewers
and the editor that helped to improve this manuscript.
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