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Applied and Environmental Microbiology, April 2009, p. 1801-1810, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01811-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Diversity and Stratification of Archaea in a Hypersaline Microbial Mat ^,

Charles E. Robertson,¹ John R. Spear,² J. Kirk Harris,³ and Norman R. Pace^1*

Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347,¹ Division of Environmental Science and Engineering, Colorado School of Mines, Golden, Colorado 80401,² Department of Pediatrics, University of Colorado Denver, Aurora, Colorado 80045³

Received 5 August 2008/ Accepted 16 December 2008

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The Guerrero Negro (GN) hypersaline microbial mats have become one focus for biogeochemical studies of stratified ecosystems. The GN mats are found beneath several of a series of ponds of increasing salinity that make up a solar saltern fed from Pacific Ocean water pumped from the Laguna Ojo de Liebre near GN, Baja California Sur, Mexico. Molecular surveys of the laminated photosynthetic microbial mat below the fourth pond in the series identified an enormous diversity of bacteria in the mat, but archaea have received little attention. To determine the bulk contribution of archaeal phylotypes to the pond 4 study site, we determined the phylogenetic distribution of archaeal rRNA gene sequences in PCR libraries based on nominally universal primers. The ratios of bacterial/archaeal/eukaryotic rRNA genes, 90%/9%/1%, suggest that the archaeal contribution to the metabolic activities of the mat may be significant. To explore the distribution of archaea in the mat, sequences derived using archaeon-specific PCR primers were surveyed in 10 strata of the 6-cm-thick mat. The diversity of archaea overall was substantial albeit less than the diversity observed previously for bacteria. Archaeal diversity, mainly euryarchaeotes, was highest in the uppermost 2 to 3 mm of the mat and decreased rapidly with depth, where crenarchaeotes dominated. Only 3% of the sequences were specifically related to known organisms including methanogens. While some mat archaeal clades corresponded with known chemical gradients, others did not, which is likely explained by heretofore-unrecognized gradients. Some clades did not segregate by depth in the mat, indicating broad metabolic repertoires, undersampling, or both.

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Photosynthetic microbial mats occur worldwide and serve as models for microbial community interactions. Fossil microbial mats in the form of stromatolites are one of the earliest distinguishable life forms in the rock record (3.4 billion years) (53) and are often studied to gain insights into the development of life on Earth (3, 11) and the influence that early life may have had on the development of the planet's atmosphere, geosphere, and hydrosphere (20).

A large contemporary microbial mat system that has received the attention of microbiological studies is found in the solar saltern operated by the Exportadora de Sal SA in Guerrero Negro (GN), Mexico. The GN saltern, fed from Pacific Ocean seawater pumped from the Laguna Ojo de Liebre, occupies 100 km² and consists of 12 precrystallizer ponds with increasing salinity due to evaporation. Many of the ponds in this saltern contain persistent microbial mats. More than a million metric tons of biomass (17 km² by 6 cm by 1.2 gm/cm³) covers the floor of hypersaline pond 4 in the form of a laminated photosynthetic microbial mat that is 4 to 6 cm thick. The subjective, macroscopic appearance of the pond 4 mat is stable from year to year, and the mat has been a subject of numerous microbiological and biogeochemical studies (3, 8, 15, 19, 24, 29, 30, 38, 47, 49, 56). Chemical measurements of the mat (8, 29) show that the top 2 to 3 mm forms an oxic zone in daylight due to oxygenic photosynthesis. A zone of low hydrogen sulfide (H₂S) (HS^–, <1.6 µM) occurs between 3 and 6 mm, and below that is high hydrogen sulfide (>2 µM) formed by the reduction of seawater sulfate. At night, with the cessation of photosynthesis, the entire mat becomes anoxic.

These steep variations in chemistry with depth in the mat are expected to foster the development of a diversity of microorganisms, and indeed, molecular surveys based on small-subunit rRNA (16S rRNA) gene sequences have identified a spectacular diversity of bacteria among the microbial constituents of the mat, with thousands of novel species, few with described close relatives (29). In contrast to the diversity of bacteria, studies of eukaryotes using a similar approach encountered only a simple composition, a few diverse sequences dominated by two kinds indicative of nematodes. (15).

The archaeal diversity in pond 4 mat and similar settings has received little attention. The usual focus of saltern investigations has been on crystallizer ponds rather than concentrator ponds such as pond 4, and numerous extremely halophilic archaea have been discovered in saturated brines (2, 44, 48). The pond 4 mat is distinct from the crystallizer environment, covered by brine 1 m deep with only 80 salinity, <3x seawater. Studies of archaea in the pond 4 mat have usually been related to methanogenesis, which is generally considered a minor metabolic theme of the mat (3, 47). Consequently, the aims of this study were to describe the archaeal diversity in the pond 4 mat and to determine how that diversity is distributed with respect to depth in the mat and previously measured chemical gradients (16, 28, 29). The results uncover substantial unrecognized archaeal diversity and very likely indicate the occurrence of so-far-undetected chemical gradients in the mat. This study completes the first survey of the three domains of life in the GN pond 4 mat.

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Sample collection.
Bulk core samples were obtained from pond 4 (near pond 5) (Global Positioning System location, N27 41.245 W113 55.027) in October 2001 as described previously (29). Core samples for sectioning were taken from the same pond 4 location in November 2005. Samples were immediately frozen in liquid nitrogen for transport to the laboratory.

DNA extraction, PCR, cloning, and sequencing.
DNA extraction using a bead-beating protocol, PCR, cloning, and sequencing were performed as described previously (29). DNAs were amplified with universal primers (515F/1391R) and archaeon-specific primers (333FA/1391R and 4FA/1391R). The 2005 mat core samples were sectioned into 23 layers, with 1-mm slices for 10 mm and 3-mm slices in deeper strata. The 10 strata analyzed consisted of the first 6-mm samples and four DNA pools constructed from equimolar mixtures of DNA extracted from multiple layers (see Table S1 in the supplemental material). Amplification was conducted with the archaeon-specific primer 23FA (5'-ATT CCG GTT GAT CCT GC-3') and primer 1391R.

Sequence, statistical, and phylogenetic analyses.
Sequences were assembled with XplorSeq (17). Bellerophon (23) and Mallard (1) were used to screen all sequences for potential chimeras. Sequences judged to be potentially chimeric by either software package were discarded. The phylogenetic relationships of the sequences were determined by maximum-parsimony insertion into ARB (31) dendrograms provided by the Greengenes (9) (public sequences of 1,250 nucleotides [nt]) and SILVA (39) (900 nt) internet portals after sequence alignment with NAST (10) (for Greengenes) or the SINA Web aligner (for SILVA). (We find that the SILVA database, which includes sequences up to 900 nt, provides a broader representation of reported archaeal sequences than does the Greengenes database.) Hypervariable regions in the sequences were not considered in the parsimony insertion calculations by masking them with lanemaskPH (included with the Greengenes database, January 2008) or the SILVA 93 pos_var_Archaea_93 mask. The validities of the resulting phylogenetic relationships were verified by generating 1,000 maximum-likelihood bootstrap trees with RAxML (50) on a distance-based subsampling (3% minimum distance between sequences) of the archaeal branch of the SILVA 93 ARB dendrogram after parsimony insertion of the sequences from this study. The distance-based subsampling was accomplished with SeqClusterX (C. E. Robertson, B. Holland, and D. Knox, unpublished), which selected cluster-representative sequences after complete linkage clustering. The bootstrap trees were evaluated and annotated, and branches with less than 70% support were collapsed with Phylovis (C. E. Robertson and D. Knox, unpublished). Phylovis was also used to prepare the rough drafts of the phylogenetic-tree figures, which were fully annotated and finalized with Adobe Illustrator. In order to obtain a rough estimate of the phylogenetic distance within a cluster of sequences from this study, we defined a metric termed the maximum intraclade distance (ICD). The ICD was obtained by identifying the largest distance in the uncorrected hypervariability-masked distance matrix produced by ARB's neighbor-joining distance matrix computation function invoked on the sequences of interest. Operational taxonomic units (OTUs) were computed with the sortx module of XplorSeq (17), which uses a radial sorting algorithm with average-neighbor linkage to assign sequences to clusters. Species diversity, richness, coverage, and evenness statistics were computed through rarefaction of the resampled sortx 99% OTU clustering data using the biodiv module of XplorSeq (17).

Nucleotide sequence accession numbers.
All nonchimeric sequences were submitted to GenBank, which assigned accession numbers EU731010 to EU732045. The mapping of accession numbers (including accession numbers for a few archaeal sequences described previously by Ley et al. [29]) to sampling date, clone name, stratum in the mat, and phylogenetic classification is provided in Tables S2 and S3 of the supplemental material.

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Archaeal abundance and distribution in the pond 4 mat.
In order to assess the bulk relative abundances of representatives of the three phylogenetic domains in this environment, we determined rRNA gene sequences derived from mat cores taken in 2001 and amplified with the (nominally) universal primer set 515F/1391R by PCR. These primers, in principle, capture rRNA genes from all archaea, bacteria, and eukaryotes, although some refractory examples are known (13, 14). Collectively, 557 sequences were determined and sorted phylogenetically into domain-level groups. The phylogenetic distribution among the sequences is summarized in Fig. 1A. Since the genomes of different organisms can contain different numbers of rRNA genes, these ratios do not necessarily correspond directly to the specific numbers of individual cells in this environment. The results do, however, provide an overall perspective on the phylogenetic composition of archaeal rRNA genes in the pond 4 mat. The ratios of the rRNA sequences in the mat may also provide a rough bulk assessment of the relative contributions of representatives of the three phylogenetic domains to the metabolic functions of the mat.

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FIG. 1. Relative proportions of the three domains of life seen in the GN pond 4 mat and a comparison of bacterial and archaeal species richnesses. (A) The pie chart shows the relative proportions of each domain as detected in 557 16S rRNA sequences obtained with universal primers from core samples of the pond 4 mat taken in 2001. The archaea are further subdivided into the two main phylogenetic clades, the Euryarchaeota and the Crenarchaeota. (B) Measured (Sobs) and estimated (SChao1) (32) species richnesses versus sequence identity are shown for bacterial and archaeal sequences found with all primer pairs in the 2001 mat bulk samples. Rarefaction was used to compensate for the differences in numbers of observed bacteria (1,584) and archaea (282).

Although few identical sequences were encountered in libraries from 2001 and 2005, similar phylogenetic kinds of organisms were encountered (see Table S2 in the supplemental material), consistent with previous observations of the relative stability of the mat. However, too few archaeal sequences (only 50) were examined in the libraries made with universal PCR primers to warrant more than general comparisons.

To gain a more-detailed perspective on the specific distribution of archaeal sequences in the mat, PCR-based archaeon-specific clone libraries were prepared from each of 10 strata of the mat (see Materials and Methods). An additional 817 archaeal rRNA sequences were determined, approximately equally distributed among the strata. Although still undersampled (see below), we could use these sequences with previously determined bacterial sequences (29) to estimate statistically the comparative diversities of archaea and bacteria in the mat and the relative numbers of OTUs at different degrees of sequence identity. We define species-level diversity at 99% sequence identity over the alignment masked to exclude highly variable sequences (lanemaskPH or pos_var_Archaea_93). This corresponds to approximately 97% identity in raw rRNA sequence alignments, which include hypervariable regions, approximately to the level of intraspecies sequence identity (18). As shown in the observed and estimated (Chao 1) (6) species richness curves in Fig. 1B, while the archaea in the bulk mat represent substantial species-level diversity, they are less diverse than the resident bacteria, with severalfold-lower numbers of OTUs at all levels of sequence similarities. The Chao 1 predictions of diversity based on the observations show that both bacteria and archaea are undersampled by severalfold in these analyses, the usual situation with complex microbial communities. Nonetheless, we have presumably sampled the most abundant populations.

The archaeal sequences are not distributed uniformly in the mat. As shown in Fig. 2A, the observed and projected species richness of archaeal sequences decreases severalfold from the top of the mat into the deeper, highly reduced zones. This is accompanied by differentiation in the kinds of organisms that are detected. In Fig. 2B, we compare the distribution in the mat of members of the Crenarchaeota and Euryarchaeota, generally considered the two broad-relatedness groups that constitute the Archaea. Figure 2B shows that euryarchaeote rRNA genes are most abundant in the upper few mm, the oxic zone during the day, where rRNA genes of the crenarchaeota are comparatively rare. In contrast, crenarchaeote rRNA genes increase in diversity and numbers with depth. Calculations of archaeal species diversity and the predicted coverage of the analysis with depth in the mat are shown in Fig. 2C. Diversity declines precipitously at 3 to 4 mm into the mat, which approximately coincides with the oxic-anoxic boundary. Good's coverage is approximately inversely proportional to diversity in this case, so substantial archaeal diversity remains to be documented throughout the mat.

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FIG. 2. Species richness and diversity of archaea vary by depth in the mat. (A) The observed (Sobs) and estimated (SChao1) (32) species richnesses of archaea decrease with depth in the mat. Rarefaction was used to compensate for differences in the numbers of sequences per layer (see Table S3 in the supplemental material) in the 817 archaeal sequences from the 2005 mat core samples. (B) The number of 99% OTUs versus depth for the Euryarchaeota and Crenarchaeota. Euryarchaeota dominate the diversity of archaeal species in the mat and are almost the exclusive archaea in the top 2 mm. Crenarchaeota are consistently present in the mat below 3 mm and begin to dominate diversity below 27 mm. (C) Simpson's reciprocal diversity index (32) indicates the highest diversity in the top 3 mm of the mat, at which point it decreases rapidly to a depth of 5 mm and remains relatively constant through to the deepest sample. Good's coverage index (32) shows approximate inverse proportionality to diversity and indicates that all layers of the mat may benefit from sequencing beyond what was attempted in this study. Indices are shown for 99% OTUs computed on the 817 archaeal sequences from the 2005 mat core samples.

Phylogenetic analysis of mat constituents.
We aligned the sequences with sequences drawn from public databases and used the program RAxML to perform likelihood phylogenetic analyses with large data sets, all as detailed in Materials and Methods. The results of the phylogenetic analysis of euryarchaeal and crenarchaeal sequences are compiled in the two dendrograms of Fig. 3, respectively. Only a few of the sequences have specific relationships to described organisms; most are only remotely related to cultured examples of archaea. As identified in Fig. 3, however, many of the main groups of sequences are more or less closely related to archaeal sequences previously detected in other environmental rRNA sequence surveys. Tables 1 and 2 summarize the environments and studies where sequences representative of the main GN groups were encountered previously and their relationships with the GN sequences. Sediments and other stringently anoxic environments seem to be primary sources of sequences similar to those analyzed here.

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FIG. 3. Phylogenetic trees of members of the Euryarchaeota and Crenarchaeota of the pond 4 mat. Bootstrap support values (percent) are indicated at the bases of branches. Branches with less than 70% bootstrap support were collapsed to better reveal the statistically supported structure of the trees. GenBank sequences reported elsewhere that are present in each clade are indicated with accession numbers along with the distance to the closest sequence in this study. Clones show total numbers of clones from all samples and primer pairs, numbers of clones from primers 4FA/515F/333FA/1391R, and numbers of clones from primer 23FA/1391R. The ICDs were obtained by the identification of the largest uncorrected distance in the sequence distance matrix calculated by the ARB neighbor-joining distance matrix calculation function applied to the sequences of interest, masked for hypervariability, within a specific clade. (A) The Euryarchaeota. The Euryarchaeota clade names were selected in order of analysis except for GNMethanos (cluster with classic methanogens), GNHalos (cluster with the halobacteria), and GNThrm, which cluster with sequences in the Greengenes taxonomy Thermoplasmata/E2/terrestrial and Thermoplasmata/E2/aquatic group (III). (B) The Crenarchaeota. The Crenarchaeota clade names were selected based upon similarity to previously published sequences and are prefixed as follows: DV, deep-sea hydrothermal vent; MB, marine benthic; MV, mud volcano. MBCrenUn is a synthetic clade formed from all of the nonclade sequence singletons and doubletons found within the MBCren branch of the tree shown in B.

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TABLE 1. GN archaeal clades represented in previous studies of diverse environmentsa

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TABLE 2. Environmental survey studies which report clusters of archaea similar to the clusters found in this study

Currently, there is no recognized classification scheme for clades, relatedness groups, of uncultured archaea. The designations that we use are specified in the dendrograms (Fig. 3), which also identify the maximum ICD of the GN component of the particular relatedness groups. This provides some view of the extent of variation among the GN sequences that fall into the particular group. An ICD of only a few percent indicates little phylogenetic diversity among the GN organisms detected, perhaps species-level variation, organisms expected to have generally similar physiologies. A total of >10 to 15% ICD indicates substantial phylogenetic depth among the organisms detected by the sequences. Most (>90%) of the mat sequences are representatives of only four large phylogenetic groups, designated with the prefixes Eury4, Eury5, Eury6, and MBCren in Fig. 3. All these groups have large ICD values, 19% to 33%, which is indicative of the potential in each of these groups for substantial physiological as well as phylogenetic diversity.

Figure 4 illustrates the distribution in the mat of the specific euryarchaeal and crenarchaeal sequence clusters identified in Fig. 3. The marked stratification of many of the sequence groups suggests the occurrence of microenvironmental conditions that enrich for the particular clades. For instance, the most abundant euryarchaeal rRNA sequences in the mat, representing the Eury5J clade, are stratigraphically tightly confined to the uppermost few mm, suggesting a dependence on chemistry or perhaps some syntrophic relationship restricted to the oxic or photic layer. On the other hand, sequences representative of the Eury4AA group are the most abundant euryarchaeal sequences in the middle layers of the mat, well below the oxic stratum. The Eury4AA phylogenetic group includes sequences from other diverse sources, including hydrothermal vents, e.g., GenBank accession number AB019747 (52), and Kalahari Shield subsurface water, e.g., accession number DQ336956 (4).

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FIG. 4. Phylogenetic clades versus depth in the mat for the sectioned core samples from 2005. (A) The Euryarchaeota. The height of the three-dimensional cones represents the percentage of total sequences (817) for each depth sample point for each euryarchaeal clade (solid filled circles represent 0%). The order of the clades was selected to minimize visual interference among the data points. Although most numerous and diverse at the top of the mat, the Euryarchaeota are present in all layers sampled. (B) The Crenarchaeota. As in A, the height of the cones is the percentage of total sequences from the 2005 sectioned core samples. The Crenarchaeota show the highest abundance in the deeper layers of the mat. The largest crenarchaeal clade, MBCrenUn, is composed of many species but does not readily segregate by depth distribution or phylogenetic bootstrap support. (C) Additional detail showing minority euryarchaeal clades found in the top 5 mm of the mat, including the mat methanogen clade GNMethanos.

Additional fine structures in the distributions of the different major phylogenetic groups in the strata of the mat are evident in Fig. 4A and B. Figure 4C shows the distribution of some of the minor euryarchaeotes, including the GNMethanos group. The GNMethanos sequences, although not abundant in the mat, are sufficiently closely related to characterized methanogens (97 to 99% identity in rRNA sequences) that we can infer the potential for methanogenesis in these environmental organisms.

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Archaeal diversity in the GN mat.
The ecology of the GN pond 4 mat is driven by diurnal photosynthesis in the upper mm of the mat and by the reduction of seawater sulfate and fermentations in the deeper mat. Based on microscopy and culture studies, the GN and similar microbial mats had been considered relatively "simple ecosystems" (7, 33, 34, 36, 37, 40, 51). However, rRNA sequence surveys have revealed instead that the pond 4 mat teems with complex microbial diversity (28-30). In this study, we explore the distribution and phylogenetic diversity of archaea in the mat.

To assess the overall genetic contribution of archaea to the pond 4 mat biota, we analyzed rRNA sequences derived from PCR libraries prepared using nominally universal primers. The phylogenetic distribution of the sequences for bacteria/archaea/eukaryotes in the mat, 90/9/1, indicates that the functions of the mat, although dominated by bacteria, are likely influenced by archaeal metabolisms, an example of which is the detection of small amounts of methane in gases collected from the mat (3). Although bacterial diversity is much greater than archaeal diversity, archaeal and bacterial diversities are approximately proportional to depth. Maximum archaeal diversity occurs within the top few mm (the oxic zone), consisting mainly of members of the Euryarchaeota with no cultured representation. Euryarchaeotes dominate the archaeal diversity to a depth of 26 mm, far into the anoxic, high-hydrogen sulfide zone of the mat. In contrast, few crenarchaeal sequences are found above 2 mm, but numbers and prevalences increase with depth to become the most numerous and diverse archaeal sequences in the mat below 27 mm.

Phylogenetic stratification and chemical gradients.
The archaeal constituents of the pond 4 mat are stratified, as long observed for the bacterial constituents (7). The basis for the stratifications of phyla is presumably metabolic, the requirement of organisms for particular conditions or nutrients. Different phyla tend to occupy different strata, which indicates the occurrence of many such specifically favorable chemical gradients, although little is known about the nature of such microenvironments. The mat community overall is driven by photosynthesis, but light penetrates only a few mm into the mat. Consequently, most of the mat volume is supported by fermentations of photosynthetic products or by the metabolism of hydrogen derived from fermentations, such as in methanogenesis or sulfate reduction. The photic zone is expected to have the highest metabolic rate, and indeed, previous measurements of ATP concentrations throughout the mat found the highest concentrations in the top few mm, indicating that it is the most biochemically active stratum of the mat (29).

One clear chemical boundary in the mat is the daily interface between the oxic and anoxic zones, the low-sulfide zone 3 to 5 mm into the mat. This interface is populated by a rich diversity of euryarchaeal phyla, individually tightly stratified across this interface (Fig. 4A and C). The metabolic properties of these euryarchaeotes are mainly not known, although a few members of the GNMethanos group, are specifically related to known methanogens (Fig. 4A) and can probably conduct methanogenesis (below). In contrast to the prevalence of euryarchaeota in the upper mat, crenarchaeotes apparently avoid the high-energy upper layers of the mat and are found primarily in the deep anoxic zone. The main occurrence of crenarchaeotes at depth in the mat may reflect the energy-poor state of that portion of the mat. ATP concentrations in the deeper zones of the mat strata are <10% of the concentration at the highly metabolically active mat surface, indicating relatively low metabolic activity in the deep zones of the mat (29). The crenarchaeotes found in the deepest layer of the mat are phylogenetically nearest to crenarchaeotes that were previously reported to prevail in other extremely low-nutrient environments, in the deep sea and benthic sediments (5, 25, 46, 52, 55). The prevalence of crenarchaeotes in the deepest, most-energy-poor region of the mat is consistent with the model for archaeal dominance of energy-poor environments suggested previously by Valentine (54).

rRNA phylogenetics and metabolism.
Extrapolation from an rRNA-based phylogeny of environmental sequences to physiological properties of organisms that correspond to the sequences is possible only if the environmental organisms are closely related to characterized microbes. The GN sequences are only distantly related to previously described organisms, so few specific properties of the GN archaea can be inferred. Thus, this survey and others show the need for a more-comprehensive study of the properties of archaea in general; our understanding of the phylogenetic and biochemical diversity among these organisms is abysmally poor. Environmental sequence surveys point to a vast reservoir of untapped biodiversity capable of important metabolisms that likely play significant roles in elemental cycling.

None of the crenarchaeal groups and only two clades of the Euryarchaeota, which collectively represent less than 3% of all sequences, contain close cultured representatives. The GBHalos clade contains just three such sequences, which are 98 to 99% identical to those of Halorubrum sp., Halobacterium sp., or Haloplanus sp., all classic salt-tolerant euryarchaeaotes. The GNMethanos relatedness group contains sequences that are 98 to 99% identical to Methanohalophilus sp., a well-characterized halotolerant methanogen that produces methane from H₂ and CO₂ using methanol or methylamine as a substrate (22, 41-43). This high level of rRNA sequence identity predicts that the environmental organisms can also conduct methanogenesis. Although representatives of this group occur throughout the mat, they are found predominantly in the surface mm, concentrated particularly at the oxic-anoxic boundary (Fig. 4C). The restriction of methanogens to the layers just below the surface of the mat indicates a balance between the presence of oxygen during the day, which poisons methanogenesis, and bacterial sulfate reduction in the deeper layers, which can outcompete methanogenesis for hydrogen and acetate substrates (21). Recognizable methanogens represent only a small proportion of the archaea detected, but it is possible that some of the GN archaea with no characterized close relatives also conduct methanogenesis. However, the relative rarity of methanogens as observed by sequences in the mat is consistent with data from studies that showed that the pond 4 mat produces only small amounts of methane under field conditions (3, 47). The depth distribution of some of the archaeal phylogenetic clades tends to be narrowly defined despite the fact that these same clades have relatively large ICDs, which might predict broad biochemical potential and thereby wider distribution in the mat. The Eury5J group, for instance, has an ICD of 15%, far more diverse than the genus level, but is restricted to the upper mm of the mat, avoiding even the oxic-anoxic boundary where most of the euryarchaeal species reside. On the other hand, other clades with ICDs of >10 to 15% (e.g., Eury5K) show more than one depth maximum or are broadly distributed throughout the mat (e.g., MBCrenUn) (see below), consistent with the occurrence of many species with differing biochemistries in these clades. Despite efforts, diverse clades with broad distributions in the mat could not be resolved into stratified phyla. This indicates that some of the clades detected have representatives with multiple metabolic repertoires, which allow occupancy in different strata of the mat. It is also possible that the mat composition is sufficiently undersampled that adequate bootstrap support (70% bootstrap support for in-clade) for some stratified phyla does not emerge from phylogenetic analyses.

An example of a clade that may fall into this last category is the large MBCrenUn (marine benthic crenarchaeota, unaffiliated) clade shown in Fig. 4B. MBCrenUn is composed of numerous sequence singletons and doubletons that are all on the same branch of the tree in Fig. 3B as clades DVCrenA and MBCrenB through MBCrenI with 77% bootstrap support but are not specifically associated with those groups. Thus, the MBCrenUn group is part of a larger phylogenetic radiation that also includes clades that dominate the lower levels of the mat, in some cases in a stratified manner. Representatives of the MBCrenI, MBCrenD, and MBCrenH clades are abundant only in the lowest layer of the mat. This would be consistent with a previous proposal that the MBCrenB group contains new forms of sulfate reducers (27).

Conclusion.
This study provides perspective on the diversity of archaea that occur in moderately hypersaline, stratified microbial mats in general and builds a phylogenetic framework for the GN pond 4 mat in particular. We document substantial novel phylogenetic diversity of archaea, placed in specific, although little-understood, ecological contexts. In addition to the utility in the identification of novel environmental organisms, the sequences are also the basis of tools, such as hybridization probes and PCR primers, with which to explore these main constituents of this complex and highly productive community further.

 
This research was supported by funds to N.R.P. from the Astrobiology Institute of NASA and by an Agouron postdoctoral fellowship to J.R.S.

We thank representatives of Exportadora de Sal of Guerrero Negro, Baja California Sur, Mexico, for access to sites and assistance. We also thank the GN mat researchers at the NASA Ames Research Center for field support, permitting, and logistical assistance.

 
* Corresponding author. Mailing address: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347. Phone: (303) 735-1864. Fax: (303) 492-7744. E-mail: Norman.Pace{at}colorado.edu

Published ahead of print on 29 December 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, April 2009, p. 1801-1810, Vol. 75, No. 7
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