Contemporary international research efforts have illustrated the ubiquitous nature of the Archaea
, and thus their presence in the hydrothermally active Yellowstone Lake was anticipated. The two vents were selected for microbial community analysis based on their different aqueous chemistry profiles, and two near-surface photic zones were selected because they differed with respect to their relative proximity to lake floor vent activity and to the lake's largest tributary, the Yellowstone River ( and Supplementary Table S1). Vent chemistry differed significantly in pH and in potentially important microbial nutrients such as CO2
( and Supplementary Table S1), all of which would be expected to exert selective effects on the associated microbial physiology types. Gas compositions of vent waters studied in the current project are consistent with previous reports (Remsen et al., 1990
; Cuhel et al., 2002
; Remsen et al., 2002
; Spear et al., 2005
), and significantly greater than that reported for Yellowstone's terrestrial hot springs (for example, Langner et al., 2001
; Macur et al., 2004
; Spear et al., 2005
; D'Imperio et al., 2008
). The H2
measurements () represent a new addition to our understanding of the chemistry in this lake ( and Supplementary Table S1), which is important as H2
is an important energy source for microbial metabolisms in Yellowstone high-temperature ecosystems (Spear et al., 2005
). A more comprehensive assessment and discussion of the lake chemistry can be found in Clingenpeel et al. (2011)
As judged by the strength of normalized and optimized PCRs, the archaeal amplicons for each lake sample were noticeably weaker than those generated with bacterial primers, and thus while not quantitative, the Archaea
were concluded to represent a smaller component of the microbial communities in the various lake environments. Consequently, the archaeal amplicons were combined as a minority component (~15%) of an amplicon pool submitted for pyrosequencing. The minority estimate for Archaea
in these lake environments is consistent with reports of other lakes (although with no thermal inputs) in which a variety of more quantitative techniques were used (Pernthaler et al., 1998
; Glockner et al., 1999
; Keough et al., 2003
; Urbach et al., 2007
). And while PCR biases (Suzuki and Giovannoni, 1996
; von Wintzingerode et al., 1997
) presumably occurred to some extent, we assumed that amplicon strength provides a reasonable comparison of relative occurrence and abundance of specific phylotypes in the four lake environments sampled. Based on the shape of the collector's curves for each site (), coverage appeared somewhat similar for the four environmental samples, although sampling intensity was much greater for the vent samples. This suggests that the estimates of archaeal proportional abundance in the microbial communities associated with different lake environments were reasonable (although still only approximate) and that archaeal diversity was greater in the vent waters. Future efforts will focus on quantitative assessments of targeted taxa to more precisely estimate their abundance, population dynamics and functional roles.
Quality trimming (as per Kunin et al. (2010)
) of the pyrosequences resulted in a 26.8% cull rate. Among the 37
361 remaining reads, classification was complicated by lack of agreement among the automated classifiers. The difficulty experienced in this particular study may stem from the smaller Archaea
training set (compared with that for Bacteria
) available for classifier use. More specifically, the RDP classifier taxonomy base does not contain representative sequences for the Crenarchaeota and Thaumarchaeota clades recovered in this study and hence the difficulty in their classification (James Cole, Ribosome Database Project, personal communication). We were unable to ascertain the basis for the severe and inconsistent classifier problems encountered with the Greengenes classifier.
Although BLAST match comparisons of the 454-FLX reads against the near-full-length clones proved to be the most reliable way of classifying the pyrosequencing data, not all full-length clones could be accounted for in the 454-FLX data sets. Examples include YLA044, YLA030 and YLA026 among several Crenarchaeota phylotypes (), and YLA087, YLA073 and YLA104 among a number of Euryarchaeota full-length clones (). This implies that additional pyrosequencing would likely reveal additional diversity, which is not surprising. Where perfect matches were not found, there were closely related near-full-length clones that could be matched with the pyrosequencing data, which allowed for general assessments of the represented organism's distribution within the lake. We note that not all 454-FLX reads could be matched with the full-length clones (), resulting in 10.9–28.0% of the pyrosequencing reads remaining unclassified using the matching criteria imposed ().
Combining clone distribution patterns with the geochemical data ( and Supplementary Table S1) allows for speculation about ecological context. In spite of the near daily strong winds that generate surface currents contributing to lake mixing (Benson, 1961
), roughly 78% of the OTUs (97% identity) were only found associated with vent emissions (). Exclusive vent associations that could be linked to characterized thermophiles involved the presence of organisms represented by the Crenarchaeota group YLCG-2.2 that are closely related to the thermophilic nitrifier Candidatus
Nitrosocaldus yellowstonii ( and Supplementary Figure S5) found exclusively in the Otter Vent. Distribution patterns and close phylogenetic relatedness to another known nitrifier also putatively identify apparent novel thermophiles. Within the YLCG-1.1 group ( and Supplementary Figure S3), the consistent distribution pattern of the N. maritimus
-like reads suggests that there are YLCG-1.1 organisms (in particular YLA046; Supplementary Figure S2) associated with the West Thumb Deep Vent as well as the near-surface photic zone samples taken in the Inflated Plain and Southeast Arm ( and Supplementary Figure S2). This implies that phylogenetically closely related archaeal organisms have adapted to very different environments, and also reflects the broad environmental distribution of the Marine Group 1 Thaumarchaeota that includes mines, freshwater, saltwater, drinking water plants, soils and sponge symbionts (), an observation noted previously (Nicol and Schleper, 2006
; Schleper, 2010
). Inferring dual thermophilic and mesophilic habitats for the YLCG-1.1 organisms is strengthened by contrasting distribution patterns, for example, YLCG-1.2d, another dominant 454-FLX phylotype ( and Supplementary Figure S4), was abundant in the West Thumb Deep Vent, yet was undetected in the near-surface waters. Additional comparative contrasts add further momentum to such inferences; the near equal distribution of minor signatures such as YLA067, YLA097 and YLA098 () throughout the lake implies that more rare phylotypes could be detected at the level of pyrosequencing used and thus lack of detection of YLCG-1.2d is not necessarily due to shallow sampling. When viewed in sum and in combination, 454-FLX read distribution patterns likely represent real spatial distribution of the Archaea
within this lake.
The above notwithstanding, we also note that low abundance of some predominantly photic zone clones in vent emissions could nevertheless have resulted from low-level proportional mixing of lake water with vent water. Rocks surrounding the various vents made it difficult (or impossible in some cases) for the ROV sampling cup to form a tight seal around the vent orifice (Clingenpeel et al., 2011
). This provides opportunity for surrounding lake water to mix to some degree with vent water during sampling. Geochemical evidence of this comes from the oxygen content in the vent water samples (), which would otherwise be expected to be anaerobic.
Crenarchaeota (Thaumarchaeota) dominated the 454-FLX data, in line with other reports of archaeal diversity for several of the great lakes around the world (Keough et al., 2003
). However, we note with interest the complete absence of Thermoprotei, a common crenarchaeal class found in thermophilic environments, including Yellowstone's hot springs (Barns et al., 1994
; Meyer-Dombard et al., 2005
). One of the primer sets used was the same as in Meyer-Dombard et al. (2005)
, and thus a lack of detection due to primer bias cannot explain the absence of the Thermoprotei in the data. The majority of the Crenarchaeota 454-FLX reads grouped with Marine Group 1 phylotypes (), which was recently proposed as a newly designated phylum, and Thaumarchaeota, which contains the orders Nirosopumilales and Cenarchaeales (Brochier-Armanet et al., 2008
). Molecular surveys have revealed several lineages that are related to this mesophilic archaeal phylum, such as SAGMCG-1, FFS, marine benthic groups B and C, YNPFFA and THSC1 (Schleper et al., 2005
), suggesting the potential expansion of this new phylum. However, given the lack of genomic information and poor resolution of archaeal phylogeny/taxonomy, in this report we cautiously only included Marine Group 1 in the Thaumarchaeota clade. Within this clade, phylotype groups YLCG-1.2b, YLCG-1.2c and YLCG-1.2d were most closely related to a 16S rRNA gene sequence observed in a marine metagenome contig (Rusch et al., 2007
). They were absent in the photic zone waters of Inflated Plain and the Southeast Arm, but comprised 27 to ~45% of the reads in the West Thumb Deep Vent emissions ( and Supplementary Figures S3 and S4), suggesting that the represented organisms are thermophiles. Also, group YLCG-1.2a represented approximately a third of the clones in the Otter Vent waters, again implying thermophily.
Although physiological inference was limited for most of the full-length and 454-FLX sequences, there were two group designations for which specific lake function might be inferred. The relatedness of phylogroup YLCG-1.1 to the marine nitrifier N. maritimus
(Könneke et al., 2005
) is a potentially significant observation in this regard. The represented organism(s) appeared to be an important lake archaeal picoplankton component, comprising ~69 and 84% of the archaeal pyrosequencing reads in the Southeast Arm and Inflated Plain surface photic zone waters, respectively (). The association of this organism(s) with the surface waters is consistent with a recent study that examined high mountain lakes in the Pyrenees, where archaeal nitrifier signatures (N. maritimus accC
gene) were found in the lake neuston environment (Auguet et al., 2008
Another strong nitrifier signature was evident in Yellowstone Lake crenarachaeota groups YLCG-2.1 and YLCG-2.2, found almost exclusively in the Otter Vent waters ( and Supplementary Figure S5), sharing 96–99% identity with Cadidatus
Nitrosocaldus yellowstonii, first isolated from alkaline (pH 8.3) sediments from hot springs in the Heart Lake region in YNP (de la Torre et al., 2008
). Among the lake sites studied, the pH of the Otter Vent (pH 8.4) most closely matched that of the Heart Lake location, even though nitrifier-relevant concentrations of carbon (CO2
) and energy source (NH3
) were greater in the West Thumb Deep Vent ( and Supplementary Table S1).
Although the Euryarchaeota were a minor component of the pyrosequencing libraries, there were two phylotypes that appeared particularly abundant in specific environments. Clone groups YLA099 and YLEG-1.4b comprised ~8–12% of the 454-FLX clones derived from the West Thumb Deep Vent ( and Supplementary Figure S6). Their association with the vent waters is likely not coincidental as they grouped with the marine Deep Sea Hydrothermal Euryarchaeal Group 6 (). Euryarchaeota phylotype group YLEG-2 was also almost exclusively found in the West Thumb Deep Vent emissions ( and Supplementary Figure S7).
Finally, we comment on the striking phylogenetic similarity of Yellowstone Lake's microbial community to that described for marine environments and draw attention to a recent review by Logares et al. (2009)
. A majority of Thaumarchaeota appear related to the marine nitrifier N. maritimus
(), and most of the Euryarchaeota clones grouped within the Deep Sea Hydrothermal Vent Euryarchaeal Group 6 () or the Deep Sea Euryarchaeal Group (). This pattern is consistent with our finding a Prochlorococcus
phylotype in this lake (Clingenpeel et al., 2011
), and presents opportunities to assess interesting evolutionary relationships between marine and freshwater microorganisms.