Proc. Nati. Acad. Sci. USA Vol. 91, pp. 1609-1613, March 1994 Microbiology Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment (archaebacteria/phylogeny/thermophfly/molecular ecology) SUSAN M. BARNS, RUTH E. FUNDYGA, MATTHEW W. JEFFRIES, AND NORMAN R. PACE* Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, IN 47405 Contributed by Norman R. Pace, November 17, 1993

ABSTRACT Ofthe three primary phylogenetic domains to amplify archaeal and eucaryal genes selectively. Amplifi- Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya cation products were then cloned and the nucleotide se- (eukaryotes) -Archaea is the least understood in terms of its quences of the inserts were determined.t rDNA sequences diversity, physiologies, and ecological panorama. Although obtained were aligned with and compared to an extensive many species of Crenarchaeota {one of the two recognized data base of rRNA sequences from cultivated species. This archaeal kingdoms sensu Woese [Woese, C. R., Kandler, 0. & report describes the recovery, from this single hot spring, of Wheelis, M. L. (1990) Proc. Nadl. Acad. Sci. USA 87, 4576- rDNA clones from a remarkable variety of archaeal types, 4579J} have been isolated, they constitute a relatively tight-knit many of them crenarchaeal species with no known close cluster oflineages in phylogenetic analyses of rRNA sequences. relatives. It seemed possible that this limited diversity is merely apparent and reflects only a failure to culture organisms, not their absence. We report here phylogenetic characterization ofmany MATERIALS AND METHODS archaeal small subunit rRNA gene sequences obtained by Biomass Collection and DNA Extraction. Samples of the polymerase chain reaction amplification of mixed population upper 1-10 mm ofsediment were collected, frozen on dry ice, DNA extracted directly from sediment of a hot spring in and stored at -70'C until processed. Nucleic acids were Yellowstone National Park. This approach obviates the need extracted from sediment samples by a direct lysis procedure for cultivation to identify organisms. The analyses document adapted from several methods (2-6) and designed to obtain the existence not only ofspecies belonging to well-characterized DNA from a broad range of cell types. Approximately 5 ml crenarchaeal genera or families but also ofcrenarchaeal species of sediment was resuspended in buffer A (500 mM Tris-HCl, for which no close relatives have so far been found. The large pH 8.0/100 mM NaCl/1 mM sodium citrate) in the presence number of distinct archaeal sequence types retrieved from this of polyadenosine (100 pg/ml) and lysozyme (5 mg/ml) and single hot spring was unexpected and demonstrates that Cre- incubated for 1 hr at 370C with occasional agitation. Protein- narchaeota is a much more diverse group than was previously ase K was then added to 2 mg/ml, and the mixture was suspected. The results have impact on our concepts of the incubated for a further 30 min. At the end of incubation, 8 ml phylogenetic organization of Archaea. of lysis buffer [200 mM Tris HCl, pH 8.0/100 mM NaCl/4% (wt/vol) SDS/10% (wt/vol) 4-aminosalicylate] was added, Microbiologists have long understood the limitations of cul- and the solution was mixed gently by inversion. Three cycles tivation techniques in assessing the diversity of naturally of freezing in a dry ice-ethanol bath and thawing in a 650C occurring microbial communities. Commonly, only a small water bath were conducted to release nucleic acids. The fraction of organisms observed microscopically can be cul- mixture was then extracted with an equal volume of phenol tivated using standard methods. Recently, sequence-based [saturated with 100 mM Tris-HCl (pH 8.0)], followed by phylogenetic techniques have been used to alleviate the extraction with phenol/chloroform/isoamyl alcohol, 24:24:1 requirement for cultivation to identify microorganisms. Such (vol/vol). Four grams of acid-washed polyvinylpolypyrroli- studies have detected the presence of previously unknown done (PVPP) (Sigma) (6) was added to the aqueous phase, and organisms in each instance of their use (for review, see ref. the mixture was incubated 30 min at 37TC. The PVPP was 1). These techniques sample microbial populations directly pelleted from the mixture by centrifugation, and the resultant through isolating and sequencing specific genes from the supernatant was filtered through a 0.45-gm (pore size) filter environment. Phylogenetic comparative analysis of these to remove residual PVPP. Bulk nucleic acids were precipi- sequences is then used to determine evolutionary relation- tated from solution with isopropyl alcohol and centrifugation. ships between members of the community and cultivated The resulting pellet was resuspended in 500 jLd of TE (10 mM species. The results allow inference of some properties of Tris HCl, pH 8.0/1 mM EDTA), 0.1 g of anhydrous ammo- otherwise unknown organisms in the environment, based on nium acetate was added, and the solution was mixed quickly the properties of their studied relatives. In addition, the and then centrifuged immediately for 30 min at 40C in a sequences can be used to design oligonucleotide probes for microcentrifuge. Nucleic acids were precipitated from the determination of morphotype and abundance of particular supernatant by addition of 1 vol of isopropyl alcohol, incu- organisms and for assistance in cultivation efforts. bated on ice for 10 min, and centrifuged for 30 min. After We have employed molecular phylogenetic techniques to resuspension in TE, high molecular weight DNA was isolated investigate the diversity of Archaea in a hot spring in Yel- from the extract by purification on Sephadex G-200 (Phar- lowstone National Park. Small-subunit rRNA genes were amplified by polymerase chain reaction (PCR) from DNA Abbreviation: RDP, Ribosomal Database Project. extracted directly from sediment, by using primers designed *To whom reprint requests should be addressed. tThe sequences reported in this paper have been deposited in GenBank data base (accession nos. pJP 6, L25306; pJP 7, L25307; The publication costs ofthis article were defrayed in part by page charge pJP 8, L25309; pJP 9, L25308; pJP 27, L25852; pJP 33, L25300; pJP payment. This article must therefore be hereby marked "advertisement" 41, L25301; pJP 74, L25302; pJP 78, L25303; pJP 81, L25304; pJP in accordance with 18 U.S.C. §1734 solely to indicate this fact. 89, L25305). 1609 Downloaded by guest on October 4, 2021 1610 Microbiology: Barns et al. Proc. Natd. Acad. Sci. USA 91 (1994) macia) columns as described (2) or by size selection on 1% order was randomized in neighbor joining, parsimony, and low-melting-point agarose gels (SeaPlaque GTG, FMC), ac- maximum likelihood analyses. cording to the manufacturer's protocols. Location and Chemical Analysis ofHot Spring. The location PCR Amplition of rDNA. Bulk DNA from Sephadex of the hot spring analyzed in this study was determined using fractions or in agarose gel slices was titrated in amplification a PYXIS IPS-360 global positioning system receiver (Sony). reaction mixtures to empirically determine the optimal DNA Ten readings at 30-s intervals were taken on 2 days and concentration for maximum synthesis of 1- to 1.5-kb prod- averaged to give position ofsite. Water and sediment samples ucts. rRNA genes were amplified by PCR under conditions as were analyzed for chemical composition and pH by WW described (7), with inclusion ofacetamide to 5% (wt/vol) and Analytical Sciences (Cleveland, TN) and by H. Huber (Uni- the substitution of a Tricine-containing buffer (300 mM versity of Regensberg, Germany). Tricine, pH 8.4/500 mM KCI/15 mM MgCl2) (8) for the lOx reaction buffer. After an initial 5-min denaturation at 94TC, during which the DNA polymerase was added, thermal RESULTS cycling conditions were as follows: denaturation at 940C for The hot spring analyzed in this study, "Jim's Black Pool," is 1.5 min, annealing at 55TC for 1.5 min, and extension at 720C located in the Mud Volcano area of Yellowstone National for 2 min, repeated for a total of 40 cycles. The oligonucle- Park, Wyoming, "-0.75 miles (1 mile = 1584 m) southsouth- otide primer sequences used were 1391R (9) (5'-GACG- west of Black Dragon's Cauldron, at 440 36' 35.4" ± 1.3" N GGCGGTGTGTRCA-3') and 23FPL (5'-GCGGATCCGCG- and 110° 26' 20.6" ± 1.3" W. The pool of the spring is GCCGCTGCAGAYCTGGTYGATYCTGCC-3'), where R is approximately 3 x 9 m in size, with several boiling source a purine and Y is a pyrimidine. areas (930C). The water and sediment of the spring are deep Purifiction and Clning of PCR Products. Amplified DNA black in color due to a fine black particulate material, from 5 to 10 reaction mixtures was pooled, heated to 650C to obsidian sand, and possibly iron sulfide, which also accumu- melt agarose if necessary, then extracted sequentially with lates on the periphery ofthe pool. Chemical analysis ofwater phenol/chloroform/isoamyl alcohol and chloroform/isoamyl and sediment samples taken from this pool indicates that it is alcohol, and precipitated with ethanol. After centrifugation, similar in chemistry to other Yellowstone hot springs (see ref. DNA pellets were resuspended in TE and products of the 17), although the sediment contains an unusually high iron expected size (1.4 kb) were purified on 4% polyacrylamide content (415,600 mg/kg). Sulfide is present. Temperature gels, eluted into 300 mM sodium acetate/0.1% SDS, and varies across the pool and increases rapidly with increasing precipitated with ethanol. Products were then digested with depth through the sediment. The sediment was =740C at the restriction endonuclease Not I (New England Biolabs) (10), site of sampling, while pH was approximately neutral (water purified on low-melting-point agarose gels (1% SeaPlaque, pH = 6.7 at 18TC and sediment pH = 7.6 at 20TC). FMC), and cloned into pBluescript KS+ (Stratagene), all DNA was isolated directly from sediment samples, and according to manufacturers' directions. Clones containing rRNA genes were amplified by PCR and cloned. One of the 1.4-kb inserts were identified by SDS/agarose gel electro- primers for amplification, 23FPL, was chosen to amplify phoresis of overnight cultures (11). archaeal and eucaryal small subunit rRNA genes selectively, Sequencing and Phylogenetic Analysis. DNA from plasmid to the exclusion of bacterial genes. Use of this primer, in preparations of clones (10) was denatured and sequenced by conjunction with the "universal" 1391R primer (9), resulted in the dideoxynucleotide chain-termination method using Se- the recovery of archaeal rDNA clones almost exclusively. A quenase 2.0 (United States Biochemical) by the manufactur- single bacterial-type clone was retrieved (out of98 screened), er's recommendations. Universal rRNA-specific (9) and M13 in addition to 12 clones containing a common sequence that forward and reverse primers (10) were used in sequencing the does not appear to be ribosomal (data not shown). No eucaryal clones. The following four archaeal-biased sequencing prim- rDNAs were detected. The high G+C content ofthe bacterial ers were also designed and used for sequence analysis: and nonribosomal sequences may have allowed their amplifi- 340RA, 5'-CCCCGTAGGGCCYGG-3'; 744RA, 5'-CCS- cation by the 23FPL primer, itself w68 mol % G+C. Second- GGGTATCTAATCC-3'; 765FA, 5'-TAGATACCCSSG- ary structure models ofthe cloned rDNAs used in this analysis TAGTCC-3'; 1017FA, 5'-GAGAGGWGGTGCATGGCC-3' indicate that they derive from functional rRNA genes. (primer numbering corresponds to the Sulfolobus acidocal- Initially, m250 nt of sequence was obtained from each of darius nucleotide to which primer 3' nucleotide is comple- the 98 insert-containing clones. This information was used to mentary). identify unique sequence types. Clone types pJP6, pJP27, and Sequences were manually aligned with rRNA sequence pJP8 were the most common (23, 8, and 7 occurrences, data from the Ribosomal Database Project (RDP) (12) based respectively), while the remaining types were represented on primary and secondary structural considerations, by using 1-4 times in the collection. This distribution may reflect the the GDE multiple sequence editor distributed by the RDP (12). population present in the original sample (other investigators Sequences were submitted to the CHECK-CHIMERA program have suggested correlation between prevalence of rDNA of RDP to detect the presence of possible chimeric artifacts clone types and population structure; refs. 1 and 18); how- (12, 13). Phylogenetic analyses were restricted to nucleotide ever, the potential selectivity of DNA recovery, amplifica- positions that were unambiguously alignable in all sequences. tion, and cloning prohibits confidence in such quantitation. Least-squares distance matrix analyses were performed us- An additional six clones contained inserts highly similar in ing the algorithm ofDeSoete (14), with correction for multiple sequence to that of pJP27, but differing in 1-4 positions (out undetected mutations (15). Neighbor joining analysis was of =250 nt sequenced) from that clone. Similarly, a sequence accomplished using the PHYLIP package (version 3.5; ob- -98% similar (over =250 nt ofsequence) to that ofclone pJP9 tained from J. Felsenstein, University of Washington, Seat- was found in 4 clones of the library (data not shown). Such tle), while parsimony trees were constructed using PAUP microheterogeneity in rRNA gene clones obtained from (version 3.1.1; obtained from D. L. Swofford, Smithsonian pure-cultured organisms as well as from mixed natural pop- Institution). Maximum likelihood analyses were performed ulations has been observed previously (for review, see ref. 1) using FASTDNAML (version 1.0; distributed by RDP; ref. 12). and may be attributable to allelic variation within or between Bootstrap methods (16) were used to provide confidence members of the population, incorporation errors by polymer- estimates for tree topologies in neighbor joining, parsimony, ases, and/or cloning and sequencing errors. That such het- and maximum likelihood methods. To avoid potential bias erogeneity was not observed in the duplicates of the other introduced by order of sequence addition, taxon addition clone types suggests that these pJP27- and pJP9-related Downloaded by guest on October 4, 2021 Nficrobiology: Bams et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1611

sequences were not generated by PCR, cloning, or sequenc- cultured (similarity values less than 0.87). To infer more ing errors and probably arose from distinct but closely related accurately the phylogenetic affiliation of the organisms rep- template types in the original population. resented by these rDNAs, nearly full-length sequences The 5'-terminal 450 nt of sequence from each unique clone (.1330 nt) were determined. Analyses of these data by all type was then determined and used for approximate phylo- three phylogenetic methods showed that the sequences clus- genetic analyses. Sequences of clones pJP6, pJP7, pJP8, ter specifically with known Crenarchaeota (Fig. 1B). pJP9, pJP74, and pJP81 showed high similarities with 16S The phylogenetic placement of the sequences represented rRNA sequences of cultivated Archaea (similiarity values of in Fig. 1B was further assessed by nucleotide signature 0.89-0.98 to closest cultured relatives) but were not identical analysis and determination of the sensitivity of tree topology to any. Phylogenetic analysis of these sequences by maxi- to sequence selection and base composition. An intradomain mum likelihood, neighbor joining, and maximum parsimony nucleotide signature analysis (19) ofthese sequences is given all resulted in trees with similar topologies (Fig. 1A). The in Table 1. In agreement with the inferred phylogenetic trees, specific relationships of the sequences of pJP7 and pJP74 pJP33, pJP41, and pJP89 all share a majority of sequence with those of Desulfurococcus mobilis, Pyrodictium occul- signature features with Crenarchaeota. The rDNA sequences tum, and Sulfolobus acidocaldarius were not resolved in this of clones pJP27 and pJP78, however, have about as many analysis, as indicated by low bootstrap values for these signature features in common with Euryarchaeota as with branches. The sequences of clones pJP27, pJP41, pJP78, and Crenarchaeota (8 vs. 6 features). This suggests that these pJP89 bore no close similarity to those of any Archaea yet lineages branch deeply in the archaeal tree, perhaps suffi- A Desulfurococcus mobilis pJP74 Sulfolobus acidocaldarius pJP7 occultum pNp 8 89, Pyrobaculum islandicum P 7Pyrobaculum aerophilum Thermoproteus tenax 99-pJP6 NP1 6Thermofilum pendens -pJP 81

dleri ,occus celer __L10~0~ Archaeoglobus fulgidus Npp 9 .10

B - Pyrodictium occultum 55 671 Sulfolobus acidocaldarius 88 Thermoproteus tenax Thermofilum pendens pJP 33 pJP 41 pJP89 pJP 78 pJP27 Methanopyrus kandleri 69 | Thermococcus celer 66 Methanococcus vannielii 80 Methanobacterium thermoautotrophicum 82 901 Methanosarcina barkeri Haloferax volcanli '°°I Aquifex pyrophilus 100 Thermotoga maritima .10

FIG. 1. (A) Phylogenetic tree of archaeal rDNA gene clones obtained from Jim's Black Pool, illustrating close affiliations of these sequences (designated pJP) with those of cultivated Archaea. Tree was inferred by maximum likelihood analysis of 397 homologous positions of sequence from each organism or clone. Scale bar represents 10 mutations per 100-nt sequence positions. The percentage of 100 bootstrap resamplings that support each topological element in maximum likelihood (above line) and maximum parsimony (below line) analyses is indicated. No values are given for groups with bootstrap values less than 50%. [Note: The sequence of Sulfolobus acidocaldarius rRNA used in this analysis was originally reported as having derived from Sulfolobus solfataricus. Recent DNA hybridization and sequence data suggest that the sequence is correctly attributed to Sulfolobus acidocaldarius (P. Dennis, personal communication).] (B) Phylogenetic tree of archaeal rDNA gene clones inferred from maximum likelihood analysis of 1170 homologous positions of sequence from each organism or clone. Scale bar represents 10 mutations per 100-nt sequence positions. The percentage of 100 bootstrap resamplings that support each topological element in maximum likelihood (above line) and maximum parsimony (below line) analyses is indicated. Downloaded by guest on October 4, 2021 1612 Microbiology: Barns et al. Proc. Natl. Acad. Sci. USA 91 (1994) Table 1. Intradomain nucleotide signature analysis for that the sequences analyzed in Fig. 1 are free of chimeric pJP sequences artifacts (data not shown). Position(s) Cren Eury Gp. I pJP33 pJP41 pJP89 pJP27 pJP78 27 556 C.G GC Cren Cren Cren Cren Eury Eury DISCUSSION 28 555 C-G G-y Cren Cren Cren Cren Eury Eury The great phylogenetic diversity of archaeal rDNA clones 30553 GC Y-R Cren Cren Cren Eury A.U* A.U* recovered from this single hot spring was unexpected and is 34 550 COG U.G Eury Cren Cren Eury Cren Cren without precedent in previous studies (1). Moreover, it is 289-311 G-C C.G Eury Eury Eury Eury Eury Eury unlikely that the 98 clones inspected exhaust the diversity of 501-544 CG R-Y Eury Cren Cren Eury Eury Eury this archaeal community, since many of the sequence types 503 542 GC C-G Cren Cren Cren Cren Cren Cren were recovered only once. Gene sequences were obtained 504-541 G-y Y-R Eury Cren Cren Eury Eury Eury here that indicate the presence of both close evolutionary 513-538 U-A C.G Cren Cren Cren GC Cren Cren relatives ofcultivated species and several organisms without 518 U C Cren Cren Cren Cren Eury G known close relatives. Although phylogenetic placement of 658-747 GC Y-R Cren Cren Cren Cren Cren Cren some of the sequences in Fig. 1A is inexact in detail (as 692 C U Eury Cren Cren Eury Eury Eury indicated by low bootstrap values), representatives ofmost of 965 G y Cren Cren Cren Cren Eury Eury the major groups of cultivated Crenarchaeota are evidently 1074*1083 GNU AC Cren Cren Cren Cren Cren Cren present in this environment. Sequences affiliated with the 1244-1293 R-Y Y-R Cren Cren Cren Cren Eury Eury Desulfurococcus/Pyrodictium clade (pJP7 and pJP74), Py- 1252 C U Eury ND Cren Eury Cren Cren robaculum sp. (pJP8), and Thermofilum pendens (pJP6 and Intradomain nucleotide signature analysis of the rDNA sequences pJP81) were recovered. In addition, an rDNA sequence of clones pJP33, pJP41, pJP89, pJP27, and pJP78. Sequence signa- (pJP9) highly similar to that ofArchaeoglobusfulgidus (97% tures for the two archaeal kingdoms, Crenarchaeota (Cren) and similarity) was obtained. The first four ofthese named genera Euryarchaeota (Eury), are taken from Winker and Woese (19). Gp. have been isolated from environments having temperature I, marine Crenarchaeota signature features, is as described by and pH ranges that overlap those of this hot spring, whereas DeLong (20) and Fuhrman et al. (18). Eucaryal signatures are all denoted by an asterisk, and undetermined nucleotides are denoted as prior isolates of Archaeoglobus sp. have been obtained ND. Numbering (nucleotide position) is based on the Escherichia coli from marine environments (23). 16S rRNA sequence. Several of the cloned rDNAs show no close phylogenetic affinity to cultivated species (pJP33, pJP41, pJP89, pJP27, ciently deeply that they should not be defined as Crenar- and pJP78). Each of these lineages diverges from the cren- chaeota or Euryarchaeota. archaeal stem closer to its root than do any previously The correct topology of a phylogenetic tree should be characterized crenarchaeal rDNA sequences. Because ofthe insensitive to the sequences used and the method of analysis great evolutionary distance separating these sequences from (15). Phylogenetic trees differing in composition of archaeal those of cultivated Crenarchaeota, these lineages could rep- taxa and rooted with various sets of bacterial or eucaryal resent organisms having fundamentally distinct physiologies. sequences, or unrooted, were constructed by maximum The rDNA sequences of one group in particular, the pJP27/ pJP78 clade, suggest both early divergence from other ar- likelihood, distance matrix, and maximum parsimony meth- chaeal groups and relatively rapid evolution after that diver- ods. With a single exception (sequences from marine Ar- gence. chaea, see below), no perturbation of the topology shown in Recently, two other archaeal lineages have been detected Fig. 1B was observed for the sequences of clones pJP33, by phylogenetic analyses of rDNA clones obtained from pJP41, and pJP89 (analyses not shown). Parsimony analysis marine bacterioplankton populations (18, 20, 24). The se- placed the divergence of the pJP27/78 lineage prior to the quences ofone ofthese groups are crenarchaeal in affiliation, branching ofthe Crenarchaeota from the Euryarchaeota with although their rapid evolution and relatively low G+C con- some selections of taxa (data not shown). However, boot- tent make their phylogenetic placement problematic. When strap support for this topology was low (<50%) and may be included in the present analysis, these sequences most often a result of systematic error due to a relatively high rate of grouped with those of clones pJP27 and pJP78 in distance- nucleotide substitution in the pJP27/78 lineage (21). matrix, maximum parsimony, maximum likelihood, and Base composition disparities between sequences have transversion analyses. However, bootstrap values for this been shown to promote artifacts in phylogenetic analyses grouping were '55% in all cases, and the marine sequences (22). The high G+C content of the sequences of these clones frequently affiliated with the pJP41 and pJP89 sequences as (0.60-0.70) is comparable to that of the rRNAs of cultivated well, depending on which sequences were used in the anal- Crenarchaeota and extremely thermophilic Euryarchaeota ysis (data not shown). The apparent rapid evolution ofthese (0.62-0.67; ref. 22). Transversion distance-matrix and trans- marine sequences may account for their erratic behavior in version parsimony analyses (22) (data not shown), however, phylogenetic analysis. Table 1 confirms that the marine support the branching order of the clone sequences given in organisms are Crenarchaeota in that they have 10 of 16 the trees of Fig. 1, suggesting that base compositional bias crenarchaeal signature features (20). However, the pJP27 and pJP78 sequences have only 6 of 16 crenarchaeal signatures, has little, if any, influence on this topology. consistent with the hypothesis that they constitute a lineage Hybrid rDNA clones, composed of rDNA from different distinct from that of the marine species and diverge closer to organisms, can arise during PCR amplification of mixed- the root of the crenarchaeal branch than do the marine population DNAs (13). One possible source of such chimeric sequences. Further phylogenetic and phenotypic analyses rDNAs is that partially elongated DNA products formed will be necessary to determine the precise evolutionary during one round of amplification may serve as primers in relationships among these organisms. another round of amplification with a template rDNA from a The ubiquity of thermophilic lineages and their predomi- different organism. Inspection of predicted secondary struc- nence as the deepest and most slowly evolving branches of tures, phylogenetic analyses of different portions of the the archaeal tree led to the proposal that thermophily may be sequences, and evaluation by the CHECK-CHIMERA program an ancestral character ofArchaea (25). The G+C contents of of the RDP indicated that four of the original 98 clones the sequences analyzed in this study are largely within the contained such chimeric sequences. These tests all indicate range of those previously reported for the rRNAs of culti- Downloaded by guest on October 4, 2021 Microbiology: Barns et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1613 vated thermophilic Archaea, supporting this hypothesis (22). National Park for their enthusiastic cooperation. This material is In addition, studies ofbacterial phylogeny suggest an inverse based upon work supported by a National Science Foundation correlation between rate of rRNA sequence evolution and Graduate Fellowship to S.M.B. and a Department of Energy grant retention ofancestral phenotypic characters (25, 26). Several (DE-FG02-92ER-20088) to N.R.P. of the organisms identified in this hot spring are among the deepest and most slowly evolving of known lineages and 1. Ward, D. M., Bateson, M. M., Weller, R. & Ruff-Roberts, A. L. (1992) Adv. Microbial Ecol. 12, 219-286. may, therefore, provide additional perspective on the ances- 2. Tsai, Y.-L. & Olson, B. H. (1992) Appl. Environ. Microbiol. 58, tral phenotype of Archaea. 2292-2295. rRNA gene sequences recovered in this study document 3. Steffan, R. J., Goksoyr, J., Bej, A. K. & Atlas, R. M. (1988) the occurrence ofmany more crenarchaeal lineages than have Appl. Environ. Microbiol. 54, 2908-2915. been recognized previously through cultivation. The results 4. Tsai, Y.-L. & Olson, B. H. (1991)Appl. Environ. Microbiol. 57, alter our understanding of the phylogenetic organization of 1070-1074. Archaea in a fundamental way. Previous analyses of rRNA 5. Torsvik, V., Goksoyr, J. & Daae, F. L. (1990) Appl. Environ. sequences led to the conception of the archaeal domain as Microbiol. 56, 782-787. two distinct evolutionary lineages, Crenarchaeota and Eur- 6. Holben, W. E., Jansson, J. K., Chelm, B. K. & Tiedje, J. M. yarchaeota (27). A relatively large evolutionary distance was (1988) Appl. Environ. Microbiol. 54, 703-711. seen to separate known Euryarchaeota from the phylogenetic 7. Reysenbach, A.-L., Giver, L. J., Wickham, G. S. & Pace, cluster ofcultivated Crenarchaeota, enhancing their apparent N. R. (1992) Appl. Environ. Microbiol. 58, 3417-3418. 8. Ponce, M. R. & Micol, J. L. (1992) Nucleic Acids Res. 20, 623. distinction. Such discrete separation of the two groups has 9. Lane, D. J. (1991) in Nucleic Acid Techniques in Bacterial contributed to speculation that Crenarchaeota might be spe- Systematics, eds. Stackebrandt, E. & Goodfellow, M. (Wiley, cifically related to Eucarya rather than to Euryarchaeota, New York), pp. 115-175. while Euryarchaeota are specifically related to Bacteria (28). 10. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Although all but one of the archaeal lineages discovered in Cloning: A Laboratory Manual (Cold Spring Harbor Lab. this study appear to be Crenarchaeota, some of them branch Press, Plainview, NY). from near the root of the crenarchaeal stem. This, together 11. Sekar, V. (1987) BioTechniques 5, 11-13. with the recognition of similarly deeply diverging Euryar- 12. Larsen, N., Olsen, G. J., Maidak, B. L., McCaughey, M. J., chaeota (e.g., Methanopyrus; ref. 29), blurs the prior sharp Overbeek, R., Macke, T. J., Marsh, T. L. & Woese, C. R. distinction between the two archaeal groups. The expanded (1993) Nucleic Acids Res. 21, 3021-3023. archaeal tree lends further support to the phylogenetic co- 13. Liesack, W., Weyland, H. & Stackebrandt, E. (1991) Micro- herence ofArchaea as a whole. Ifthe wealth ofdiversity seen bial. Ecol. 21, 191-198. in the extends to other environments, it seems 14. DeSoete, G. (1983) Psychometrika 48, 621-626. present study 15. Olsen, G. J. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, likely that additional major lineages of Archaea will be 825-833. discovered. What has been taken to be two distinct archaeal 16. Felsenstein, H. (1985) Evolution 39, 783-791. lineages may even become a bush oflineages arising from the 17. Brock, T. D. (1978) Thermophilic Microorganisms and Life at root of the archaeal tree. High Temperatures (Springer, New York). Molecular methods such as described here allow study of 18. Fuhrman, J. A., McCallum, K. & Davis, A. A. (1993) Appl. microbial ecosystems without the requirement for cultivation Environ. Microbiol. 59, 1294-1302. and description of specific organisms. There is a genuine 19. Winker, S. & Woese, C. R. (1991) Syst. Appl. Microbiol. 14, need for sequence-based surveys of microbial communities: 305-310. our knowledge of the types and distributions of microorga- 20. DeLong, E. F. (1992) Proc. Natl. Acad. Sci. USA 89, 5685- nisms in the environment is rudimentary. Studies of natural 5689. microbial populations undoubtedly will continue to reveal 21. Felsenstein, J. (1978) Syst. Zool. 20, 401-410. 22. Woese, C. R., Achenbach, L., Rouviere, P. & Mandelco, L. organisms and fields for further investigation. Environmental (1991) System. Appl. Microbiol. 14, 364-371. surveys of microorganisms also are likely to expand further 23. Burggraf, S., Jannasch, H. W., Nicolaus, B. & Stetter, K. 0. our understanding of biological diversity and the evolution- (1990) System. Appl. Microbiol. 13, 24-28. ary processes that have led to it. 24. Fuhrman, J. A., McCallum, K. & Davis, A. A. (1992) Nature (London) 356, 148-149. We gratefully acknowledge Steve Koch, Anna-Louise Reysen- 25. Woese, C. R. (1987) Microbiol. Rev. 51, 221-271. bach, and Claire Woodman for their excellent field assistance; Carl 26. Achenbach-Richter, L., Stetter, K. 0. & Woese, C. R. (1987) Woese, Ed DeLong, and Mitchell Sogin for unpublished sequence Nature (London) 327, 348-349. data; Robert Barns for generously lending the global positioning 27. Woese, C. R., Kandler, 0. & Wheelis, M. L. (1990) Proc. Natl. system receiver; Chuck Delwiche, Niels Larsen, and Gene Wickham Acad. Sci. USA 87, 4576-4579. for computer assistance; and Jim Brown, Gene Wickham, and 28. Lake, J. A. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, particularly Carl Woese for extensive helpful comments on the 839-846. manuscript. We also thank Dr. Bernadette Pace for supplying 29. Burggraf, S., Stetter, K. O., Rouviere, P. & Woese, C. R. Thermus aquaticus DNA polymerase, and the staff of Yellowstone (1991) System. Appl. Microbiol. 14, 346-351. Downloaded by guest on October 4, 2021