Bacterial and Archaeal Diversities in Yunnan and Tibetan Hot Springs, China

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Bacterial and archaeal diversities in Yunnan and Tibetan hot springs, China

ARTICLE in ENVIRONMENTAL MICROBIOLOGY · OCTOBER 2012 Impact Factor: 6.2 · DOI: 10.1111/1462-2920.12025 · Source: PubMed

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Environmental Microbiology (2013) 15(4), 1160–1175 doi:10.1111/1462-2920.12025

Bacterial and archaeal diversities in Yunnan and Tibetan hot springs, China

Zhao-Qi Song,1,2† Feng-Ping Wang,3† Xiao-Yang Zhi,1 springs (pH 3.2–8.6; temperature 47–96°C) in Yunnan Jin-Quan Chen,1,3 En-Min Zhou,1 Feng Liang,2 Province and Tibet, China by using a barcoded 16S Xiang Xiao,3 Shu-Kun Tang,1 Hong-Chen Jiang,4** rRNA gene-pyrosequencing approach. Aquificae, Chuanlun L. Zhang,5,6 Hailiang Dong4,7 and Proteobacteria, Firmicutes, Deinococcus-Thermus Wen-Jun Li1,8* and Bacteroidetes comprised the large portion of the 1Key Laboratory of Microbial Diversity in Southwest bacterial communities in acidic hot springs. Non- China, Ministry of Education, and Laboratory for acidic hot springs harboured more and variable bac- Conservation and Utilization of Bio-resources, Yunnan terial phyla than acidic springs. Desulfurococcales Institute of Microbiology, Yunnan University, Kunming and unclassified Crenarchaeota were the dominated 650091, China. groups in archaeal populations from most of the non- 2Engineering Technology Research Center of Biomass acidic hot springs; whereas, the archaeal community Degradation and Gasification, University of Henan structure in acidic hot springs was simpler and char- Province, Shangqiu Normal University, Shangqiu acterized by Sulfolobales and Thermoplasmata. The 476000, China. phylogenetic analyses showed that Aquificae and 3Key Laboratory of MOE for Microbial metabolism and Crenarchaeota were predominant in the investigated School of Life Sciences and Biotechnology, State Key springs and possessed many phylogenetic lineages Laboratory of Ocean Engineering, Shanghai Jiao Tong that have never been detected in other hot springs in University, Shanghai 200240, China. the world. Thus findings from this study significantly 4State Key Laboratory of Biogeology and Environmental improve our understanding of microbial diversity in Geology, China University of Geosciences, Wuhan terrestrial hot springs. 430074, China. 5Department of Marine Sciences, University of Georgia, Athens, GA 30602, USA. Introduction 6 State Key Laboratory of Marine Geology, Tongji Hydrothermal systems may have existed on Earth for University, Shanghai 200092, China billions of years (Gold, 1992). They are well-isolated habi- 7 Department of Geology and Environmental Earth tats distributed globally in different regions, and the micro- Science, Miami University, Oxford, OH 45056, USA. organisms inhabiting therein are extremophiles adapted 8 Key Laboratory of Biogeography and Bioresource in to conditions quite different from the surrounding environ- Arid Land, Xinjiang Institute of Ecology and Geography, ments. Because the unique physical, chemical and Chinese Academy of Sciences, Üru˝mqi 830011, China. geographical characteristics of hot springs make extremophiles physiologically distinct from other microorganisms Summary in other environments, they become model ecosystems for research on the origin and evolution of life, biogeo- Thousands of hot springs are located in the north- chemistry and biogeography (Pace, 1997; Whitaker et al., eastern part of the Yunnan–Tibet geothermal zone, 2003). Since the Brock’s pioneering work in the 1960s which is one of the most active geothermal areas in and 1970s, extensive microbial investigations have been the world. However, a comprehensive and detailed performed in hot springs of Yellowstone National Park understanding of microbial diversity in these hot (Barns et al., 1994; 1996; Ferris et al., 1996; Ward and springs is still lacking. In this study, bacterial and Castenholz, 2000; Spear et al., 2005; de la Torre et al., archaeal diversities were investigated in 16 hot 2008; Kan et al., 2011), the Great Basin in the USA (Huang et al., 2007; Zhang et al., 2008; Costa et al., Received 18 November, 2011; revised 17 September, 2012; accepted 2009), Iceland (Hjorleifsdottir et al., 2001; Marteinsson 9 October, 2012. For correspondence. *E-mail [email protected]; et al., 2001; Huber et al., 2002; Kvist et al., 2007; Mirete Tel. (+86) 871 5033335; Fax (+86) 871 5033335; **E-mail [email protected]; Tel. (+86) 27 67883061; Fax (+86) 27 et al., 2011), Kamchatka in Russia (Perevalova et al., 67883451. †These authors contributed equally to this work. 2008; Kublanov et al., 2009; Reigstad et al., 2010;

Kochetkova et al., 2011) and north-eastern Australia throughput 454 pyrosequencing in conjunction with (Kimura et al., 2005; Weidler et al., 2007; 2008). detailed geochemical analyses. The north-eastern edge of the Yunnan–Tibet geothermal zone is one of the most active geothermal areas in the world and hosts thousands of hot springs. These hot Results springs possess a variety of hydrothermal features, such Environmental characteristics as hydrothermal explosion craters, geysers, fumaroles and boiling springs (Kearey and Wei, 1993). Previously, A total of 16 hot spring sites from five thermal fields several cultivation-dependent and -independent studies (Tengchong, Longling and Eryuan in Yunnan Province, were performed in Yunnan hot springs. For example, elevations at ~ 1800 m; Gulu and Qucai in Tibet, eleva- some thermopiles were isolated and their practical appli- tions at ~ 4700 m) were selected for field measurements cations were evaluated (Xue et al., 2001; Lin et al., 2002; and sample collections. Hot springs investigated in this Xiang et al., 2003). Diversities of Actinobacteria, Crenar- study possess a range of temperature (47–96°C) and chaeota and ammonia-oxidizing Archaea were also pH (3.2–8.6) conditions (Table 1). Generally speaking, studied (Pearson et al., 2008; Zhang et al., 2008; Song Yunnan springs possess higher concentrations of nitrate et al., 2009; Jiang et al., 2010; Song et al., 2010). These and nitrite than Tibetan springs. The four acidic springs studies suggested that the Yunnan hot springs host many Sx1, Drty14, Drty4 and Zzq from Yunnan Province had new taxonomic and functional lineages. However, a com- higher concentration of ferrous iron than non-acidic prehensive investigation on the community diversity and springs (Table 2). Chemistry-based principal component composition in Yunnan hot springs is still lacking. In addi- analysis (PCA) showed that Tibetan hot springs were tion, microbial communities in several Tibetan hot springs grouped together and were separated from those of were studied by using clone library and denaturing gradi- Yunnan Province, indicating that chemistry of Tibetan hot ent gel electrophoresis (DGGE) (Lau et al., 2006; 2009; springs is more similar to each other than to the ones of Huang et al., 2011). However, these two conventional Yunnan Province (Fig. 1). The acidic springs (Drty4, Zzq techniques only produced limited sequencing information and Drty14; pH: 3.2–4.5) fell into the same sector, indi- in comparison with high-throughput sequencing tech- cating that their chemistry is more similar to each other niques (e.g. 454 pyrosequencing). The objectives of this than to other hot springs (Fig. 1). study were: (i) to fully understand the bacterial and archaeal diversity in the selected hot springs (with a range Pyrosequencing data of pH and temperature conditions) of Yunnan Province and Tibet, and (ii) to examine microbial difference A total of 41141 bacterial and 30651 archaeal sequences between hot springs of Yunnan Province and Tibet and were identified. The coverage calculation (Tables 3 and 4) Yellowstone. To fulfil these two purposes, the 16S rRNA and rarefaction analysis (data not shown) indicated that gene phylogenetic analysis was conducted by using high- the analysed sequences covered the diversity of bacterial

Table 1. Description of hot spring samples investigated in this study.

Sample code Pinyin name – English name Sample description Temperature (°C) pH GPS location (N/E)

Yunnan Province Zzq Zhenzhuquan – Pearl Spring Brown sandy sediment 96 4.3 24o57′03″/98o26′09.5″ Dgg Dagunguo – Great Boiling Pot Ashen geyserite 94 8.1 24o57′12.7″/98o26′17.4″ Hmz2 Hamazui 2 – Frog Mouth Spring 2 Black sediment 82 7.8 24o57′12.6″/98o26′17.5″ Eynj2 Eryuanniujie 2 – Eryuan Niujie Spring 2 Black mat 73 7.3 26o15′01.2″/99o59′22.2″ Hmz1 Hamazui 1 – Frog Mouth Spring 1 Grey mat 77 7.8 24o57′12.6″/98o26′17.5″ Eynj3 Eryuanniujie 3 – Eryuan Niujie Spring 3 Black sandy sediment 78 7.4 26o15′01.2″/99o59′22.2″ Drty4 Diretiyanqu 4 – Experimental Site 4 Brown sediment 67 3.2 24o57′12.7″/98o26′17.4″ Sx4 Shangxiao 4 – Shangxiao 4 Black sandy sediment 66 8.0 24o39′23.3″/98o40′03.4″ Sx1 Shangxiao 1 – Shangxiao 1 Black and green sandy sediment 53 6.0 24o39′23.3″/98o40′03.4″ Drty14 Diretiyanqu 14 – Experimental Site 14 Brown sediment 47 4.5 24o57′12.7″/98o26′17.4″ Tibet Gl15 Gulu 15 – Gulu 15 Ashen geyserite 84 8.6 30o52′34.1″/91o36′38.8″ Gl16 Gulu 16 – Gulu 16 Grey sandy sediment 78 7.0 30o52′34.8″/91o36′40.6″ Qc4 Quca i4 – Qucai 4 White sandy sediment 76 7.0 30o39′59.2″/91o35′28.2″ Qc8 Quca i8 – Qucai 8 Brown sandy sediment 74 7.0 30o39′57.7″/91o35′29.3″ Qc1 Quca i1– Qucai 1 Grey sandy sediment 71 7.0 30o40′0.4″/91o35′27.9″ Qc3 Quca i3 – Qucai 3 Brown and green mat 57 6.7 30o39′59.4″/91o35′28.4″

and archaeal populations in the investigated hot springs.

1111 Both bacterial and archaeal richness is markedly different among the investigated hot springs. For example, the numbers of bacterial and archaeal operation taxonomic units (OTUs) (cut-off: 0.03) ranged from 180 to 637 and from 109 to 420 respectively (Tables 3 and 4). Bacteria were more diverse than Archaea in each spring (Tables 3 and 4). UniFrac analysis clearly showed a signiﬁcant (P-value < 0.0001) difference in the archaeal and bacterial compositions among the investigated springs. Inter- estingly, more than a half of the springs showed a high relative abundance (> 20%) of unique OTUs to total OTUs (Table 5).

Bacterial diversity

All the obtained bacterial sequences fell into the following phyla: Acidobacteria, Actinobacteria, Aquiﬁcae, Bacter- oidetes, Chlamydiae, Chlorobi, Chloroﬂexi, Cyanobacte- ria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitros- pira, Candidate division OD1, Candidate division OP10, Planctomycetes, Proteobacteria, Spirochaetes, Candidate division SR1, Thermodesulfobacteria, Ther- momicrobia, Thermotogae, Candidate division TM7, Ver-

Al Ca K Mg Mn Na Prucomicrobia TDS and unclassiﬁed bacteria. Sequences affiliated with Proteobacteria, Firmicutes, Aquiﬁcae, - 2

4 Deinococcus-Thermus and Thermotogae dominated the

SiO springs (Fig. 2), and these major groups varied in relative abundance among them. UniFrac analysis showed signiﬁ- <

- cant (P-value 0.0001) differences in the archaeal and 2 S

); ND, not determined. bacterial compositions among the springs. 1 - - 2 4 SO 0.6 + 2 Fe a 0.4 Tibetan hot springs - 3 0.2 NO Eynj2 Dgg Hmz 1 & 2 - 2 Sx1 NO Sx4 PC2 (32%) + ); BD, below the detection limit (0.001 mg l

1 NH4 - -0.2 Zzq

+ Eynj3

4 Drty4 PO 3- NH - 4 NO2 -0.4 Fe2+ - NO3 -

3 Drty14 4 4.99 0.56 0.67 3.9 BD ND ND 679.22 0.0537 1.975 69.32 0.1429 BD 299.3 0.034 ND -0.6-0.6 -0.4 -0.2 0.0 0.0 0.2 0.4 0.6 PC1 (39%) b Water chemistry of the investigated hot springs.

Fig. 1. The ﬁrst two principal coordinate axes (P1 and P2) for PCA The Hmz2 and Hmz1 are connected with each other and thus have same water. Values are reported as milligrams per litre. and the distributions of sampling locations in response to these Table 2. Sample PO a. b. TDS, total dissolved solids) (mg l Eynj2Eynj3Drty4Sx4Sx1 1.06 19.65Drty14Gl15 BD 1.94 441.85Gl16 8.2Qc4 BD BD 5.83 3.71Qc8 0.48Qc1 382.08 0.91 BDQc3 1.22 2.11 0.76 2.34 0.02 1.88 1.49 0.13 0.67 BD 1.13 0.49 BD 1.82 0.23 2.57 2.34 0.008 0.29 1.63 3.688 0.016 27.88 3.88 ND 0.31 BD ND 0.052 0.03 0.29 ND 1.913 1.017 0.067 0.06 0.03 ND 0.01 0.114 ND ND 0.04 ND ND BD 0.055 ND 0.01 677.57 0.01 643.7 0.04 4 ND 0.01 ND ND 0.04 680.59 80 0.0324 0.03 10 0.0253 629.74 0.02 7.301 12 681.15 172.33 39.61 0.8 37.06 0.25 0.3423 6 0.6235 2.05 0.02 11 23.95 ND 47.12 0.02 ND 28.09 38.51 84.4 ND 0.05 31.03 ND 18.58 BD 18.07 ND 14.04 ND ND 8.213 24.51 ND ND 20.3 0.8083 0.1135 ND 1.371 0.034 ND ND ND 0.0409 9.223 235.2 ND ND 2.318 0.0524 206.3 ND 20.49 0.9865 0.0511 ND ND ND 0.1603 73.94 ND 0.0356 0.4202 ND ND 47.05 ND 176.5 0.0467 ND ND ND ND 0.0685 ND ND ND ND 0.0923 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 2600 1337 ND 1085 ND 1620 1037 ZzqDggHmz2 & 1 1.96 1.74 164.27 BD 2.21 0.36 5.49 0.201 3.35 ND BD ND ND 645.68 ND 0.5373axes. 677.39 3.901Rectangles 0.134 25.32 1.75 represent 0.5995 116.4 0.521 chemical 0.1197 59.64 BD 0.0155 factors. ND 387.4 0.1272 ND

Table 3. Observed bacterial richness and diversity estimates based on 97% and 95% OTU clusters respectively.

Coverage Richness Shannon’s index Simpson index No. OTUs (%) (ACE) (Chao & Shen) (MLE) No. Community sequences 97% 95% 97% 95% 97% 95% 97% 95% 93% 95%

Zzq 1 723 431 324 87 90 851 625 5.34 4.87 0.014 0.020 Dgg 10 628 396 269 99 99 658 502 3.83 3.09 0.058 0.108 Hmz2 1 817 196 131 95 97 367 253 4.07 3.31 0.038 0.095 Eynj2 2 078 435 330 89 92 882 639 5.31 4.82 0.011 0.019 Hmz1 2 055 416 297 89 93 832 593 5.16 4.64 0.015 0.024 Eynj3 1 835 325 237 91 94 633 432 4.58 4.02 0.045 0.067 Drty4 2 263 180 102 97 98 290 176 4.04 3.31 0.034 0.062 Sx4 2 498 337 223 94 96 613 396 4.72 4.11 0.023 0.037 Sx1 1 533 257 189 92 94 486 384 4.69 4.20 0.020 0.032 Drty14 1 272 185 110 93 97 318 177 4.21 3.61 0.035 0.049 Gl15 1 942 339 254 91 94 656 475 4.69 4.12 0.037 0.060 Gl16 1 861 224 146 95 96 388 271 4.26 3.68 0.032 0.049 Qc4 2 593 364 271 93 95 715 542 4.73 4.23 0.021 0.032 Qc8 1 964 327 228 92 94 625 445 4.94 4.27 0.016 0.030 Qc1 3 152 637 454 90 93 1223 864 5.52 4.92 0.012 0.022 Qc3 1 927 428 332 88 91 837 661 5.24 4.80 0.015 0.021 Total 41 141 3582 2302 – – – – – – – –

Proteobacteria, Aquificae, Deinococcus-Thermus, the Thermothrix and Thiomonas. The Sx1 hot spring had Bacteroidetes and Firmicutes dominated bacterial com- the highest abundance of deltaproteobacterial sequences munities in the four acidic samples (Zzq, Drty4, Drty14 (70% of proteobacterial sequencing reads), which fell into and Sx1). Within the Proteobacteria, Alphaproteobacteria several subgroups: Desulfobacca, Desulfomonile, Desul- dominated the acidic springs Zzq and Drty14 (34% and furella, Desulfatibacillum and Geobacter. The Drty14 and 75% of proteobacterial reads respectively). Most of these Drty4 hot springs also harboured a certain amount of sequencing reads fell into the genus Acidicaldus, which deltaproteobacterial sequences (36% and 19% of proteo- had been detected in acid-sulfate-chloride and acid- bacterial sequencing reads respectively) and most of sulfate hot springs in Joseph’s Coat and Rainbow Springs these sequences were affiliated with the Desulfurella. The in Yellowstone National Park (Kozubal et al., 2012). A Zzq, Drty4 and Drty14 samples contained high abun- large number of betaproteobacterial sequences (14% and dance of Aquificae sequences, which accounted for 20% of proteobacterial sequencing reads respectively) 24–52% of the total sequences in each sample. Above were detected in the Drty14 and Sx1 hot springs, and 99% of these sequences were affiliated with Hydrogeno- most of them belonged to the Burkholderiales, specifically baculum, the only acidophilic genus within the Aquificae.

Table 4. Observed archaeal richness and diversity estimates based on 97% and 95% OTU clusters respectively.

Coverage Richness Shannon’s index Simpson index No. OTUs (%) (Chao1) (Chao & Shen) (MLE) No. Community sequence 97% 95% 97% 95% 97% 95% 97% 95% 97% 95%

Zzq 3 939 293 107 0.98 0.99 416 156 4.60 3.17 0.020 0.073 Dgg 3 605 288 148 0.97 0.98 466 247 4.26 3.24 0.034 0.088 Hmz2 1 690 285 155 0.92 0.96 481 252 4.77 3.92 0.022 0.041 Eynj2 1 016 229 152 0.88 0.92 448 297 4.54 3.67 0.035 0.082 Hmz1 1 781 228 138 0.94 0.96 442 243 4.29 3.50 0.036 0.066 Eynj3 1 079 301 191 0.84 0.90 660 380 5.14 4.34 0.014 0.033 Drty4 2 389 259 105 0.96 0.98 360 169 4.57 3.30 0.025 0.066 Sx4 2 015 178 107 0.95 0.97 386 252 3.54 2.47 0.076 0.176 Sx1 716 130 91 0.90 0.93 267 184 3.75 3.01 0.088 0.181 Drty14 2 091 113 62 0.98 0.98 212 187 3.05 2.36 0.094 0.154 Gl15 1 387 109 61 0.97 0.98 164 106 3.25 2.08 0.097 0.289 Gl16 2 165 172 97 0.97 0.98 290 198 3.71 3.08 0.059 0.088 Qc4 1 735 217 134 0.94 0.96 433 265 3.96 3.11 0.062 0.121 Qc8 1 004 284 175 0.85 0.92 574 319 5.12 4.37 0.017 0.030 Qc1 2 253 374 214 0.92 0.96 637 327 5.01 4.19 0.018 0.036 Qc3 1 786 420 259 0.88 0.94 825 409 5.36 4.69 0.013 0.020 Total 30 651 2587 1245 ––––– – – –

Table 5. The relative abundance of unique (present in a single hot spring) OTUs to total OTUs and the relative abundance of sequences represented by unique OTUs to total sequences of each investigated sample.

Archaea Bacteria

Relative abundance of Relative abundance of Relative abundance sequences represented Relative abundance sequences represented of unique OTUs to by unique OTUs to of unique OTUs to by unique OTUs to total OTUs (%) total sequences (%) total OTUs (%) total sequences (%)

Sample 97% 95% 97% 95% 97% 95% 97% 95%

Zzq 48 32 20 6 27 23 18 13 Dgg 48 33 13 4 66 54 47 23 Hmz2 43 33 16 9 39 28 12 6 Eynj2 47 32 20 11 48 41 27 17 Hmz1 36 30 9 4 44 43 21 16 Eynj3 46 34 17 9 33 27 10 6 Drty4 37 31 16 4 36 30 5 3 Sx4 43 26 24 3 49 37 21 10 Sx1 54 42 33 20 55 44 22 13 Drty14 65 50 38 25 50 48 24 18 Gl15 65 46 20 7 60 48 34 18 Gl16 32 26 7 2 31 27 6 3 Qc4 34 26 9 5 39 34 10 6 Qc8 24 15 9 4 23 21 7 4 Qc1 49 35 20 8 48 42 18 12 Qc3 75 65 62 45 71 63 55 42

Some other phyla were also recovered from acidic hot Rhodobacteraceae, Caulobacteraceae, Beijerinckiaceae, springs such as Firmicutes, Deinococcus-Thermus, Syntrophaceae, Moraxellaceae, Thiotrichaceae and Xan- Bacteroidetes and Actinobacteria (Fig. 2). thomonadaceae The Firmicutes comprised 0.2–5.5% of In comparison, non-acidic hot springs in Yunnan prov- the total sequences in each non-acidic spring, and can be ince harboured more diverse bacterial population than classiﬁed into two classes: Clostridia and Bacilli among acidic hot springs (Fig. 2), and they were dominated by which Clostridia sequences were predominant (67%). In Proteobacteria, Aquiﬁcae, Thermotogae, Bacteroidetes, addition, 16% of the Firmicutes sequences could not be Firmicutes, Deinococcus-Thermus and Thermodesulfo- assigned to any descried classes. The Bacteroidetes and bacteria. Proteobacteria comprised 3–49% of the total Deinococcus-Thermus were also recovered from all non- sequences in each non-acidic spring. Alpha-, bata-, delta- acidic samples, with the former being mainly represented and gamaprotebacterial sequences were detected in by the class Sphingobacteria and the later being mainly all non-acidic samples but with variable abundances, represented by the genus Thermus. The Thermotogae respectively, and the related subgroups included: sequences were detected from Sx4, Eynj2, Eynj3 Hmz2

Unclassified bacteria Fig. 2. Bacterial diversity in the different hot springs. Stacked bar graph represents the Actinobacteria relative distribution of major phyla in the Aquificae different samples. Bacteroidetes Chloroflexi Cyanobacteria Deinococcus-Thermus Dictyoglomi Firmicutes Gemmatimonadetes Planctomycetes Proteobacteria Thermodesulfobacteria Thermotogae Sx4 Sx1

Zzq Verrucomicrobia Qc8 Qc4 Qc1 Qc3 Dgg Gl16 Gl15 Drty4 Hmz2 Hmz1 Eynj2 Eynj3 Drty14 Other known phyla acidic (Yunnan) non-acidic (Yunnan) non-acidic (Tibet)

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 15, 1160–1175 Microbial diversities in hot springs 1165 and Hmz1 with abundances ranging from 3% to 30%, and Archaeal diversity the Fervidobacterium represented the predominant genus of this phylum. Some other phyla (e.g. Actinobacteria, All the obtained archaeal sequences can be clustered into Dictyoglomi, Acidobacteria and Planctomycetes) were 2587 OTUs (cut-off: 97%). These archaeal OTUs were also detected from non-acidic hot springs in Yunnan but affiliated with the following taxa: Crenarchaeota (including with low abundances (Fig. 2). Sulfolobales, Thermoproteales, Desulfurococcales and All the investigated Tibetan hot springs were non-acidic. Caldisphaerales), Euryarchaeota (including Thermo- The major phyla of these Tibetan hot springs were similar plasmata, Methanobacteria, Halobacteria, Archaeoglobi, to the Yunnan non-acidic samples, but showed different Methanomicrobia and Methanococci), Nanoarchaeota relative abundances (Fig. 2). For example, Bacteroidetes and Korarchaeota. The Nanoarchaeota were only constituted 0.4–5% of the total sequences obtained detected in three springs and Korarchaeota from seven from each of the Tibetan hot springs, versus 2–21% of the hot springs with low relative abundances (< 2.3%). The total sequences in each Yunnan non-acidic spring; Euryarchaeota and Crenarchaeota were the dominant Deinococcus-Thermus sequences constituted 30–57% of phyla in the investigated hot springs. the total sequences obtained from three Tibetan hot The three acidic hot springs (Zzq, Drty4 and Drty14) springs (Gl15, Qc4 and Qc8), versus 4–15% of the total had simpler communities than non-acidic ones. The sequences obtained from each Yunnan non-acidic hot majority (98%) of total archaeal sequences recovered spring. from the high temperature spring Zzq fell into the single genus Sulfolobus, belonging to the class Sulfolobales of the Crenarchaeota. The archaeal community of low- Phylogenetic analysis of Aquificae and Proteobacteria temperature spring Drty14 was dominated by class Ther- All the obtained Aquificae-related sequences were distrib- moplasmata of the Euryarchaeota (97% of archaeal uted among 15 hot springs with the relative abundances sequences from this sample), and 84% of the Thermo- of 0.2–52% of the total sequences, and can be clustered plasmata sequences belonged to the genus Picrophilus.It into 176 OTUs. The majority (154 out of 176) of the appeared that the Drty4 hot spring had an archaeal com- Aquificae OTUs were clustered into five known genera munity structure somewhere between Drty14 and Zzq: of Thermocrinis, Sulfurihydrogenibium, Hydrogenobacter, 20% of the archaeal sequences of Drty4 fell into Thermo- Hydrogenobaculum and Persephonella (23, 3, 34, 78 and plasmata (Fig. 5), and 79% were affiliated with Sulfolo- 16 OTUs respectively). The phylogentic tree showed bales. Different from Zzq, Drty4 harboured a small portion that the majority of the representative Aquificae-related (6%) of sequences belonging to genus Metallosphaera.In OTUs from this study formed four groups (Fig. 3). Group 1 contrast to the above acidic hot springs, the slightly acid was composed of the Persephonella-related sequences hot spring Sx1 had more diverse and different phyloge- (BLAST similarities ranging from 90% to 97%) and were netic groups: Sulfolobales, Thermoproteales, Desulfuro- present in springs Hmz2, Hmz1, Eynj2, Eynj3 and Sx4. coccales, unclassified Crenarchaeota and Euryarchaeota Group 2 was composed of Hydrogenobaculum-related (Fig. 5). sequences and dominated the Aquificae taxa (94–97% of Class Desulfurococcales and unclassified Crenar- Aquificae-related sequences and 24–52% of total bacte- chaeota were the two dominant (50–90%) groups in non- rial sequences) in three acidic hot springs (Zzq, Drty4 and acidic hot springs in Yunnan (Fig. 5). Thermoproteales Drty14; pH ranged from 3.2 to 4.5). Groups 3 and 4 had was also detected in almost all these non-acidic springs close relationship, comparing with other two clades. but with a low relative abundance (except Eynj3 and Group 3 was discovered in eight neutral and/or alkaline Hmz2). For Euryarchaeota, most of the obtained hot springs and was mainly related to Hydrogenobacter sequences could not be assigned to any known sub- and Thermocrinis, which were dominant in Gl15 and groups. The unclassified Euryarchaeota were recovered Hmz2 respectively. Group 4 was composed of OTUs that from all non-acidic samples, and were especially domi- could not be assigned to any described classes (BLAST nant in the Sx4 hot spring that constituted 47% of total similarities ranging from 91% to 94%). archaeal species. In addition, a large number of methano- The representative alpha-, bata-, delta- and gamapro- bacterial sequences (29% of the total sequences) were tebacterial OTUs, on the other hand, did not cluster recovered from one sample (Hmz1). Similar to those from together into some tight clades in the proteobacterial phy- non-acidic Yunnan hot springs, Desulfurococcales and logentic tree (Fig. 4). Many of them were similar to those unclassified Crenarchaeota were the predominant (repre- detected from Yellowstone especially for betaproteo- senting 16–89% and 0.4–48% of the obtained total bacterial sequences, or clustered with the sequences archaeal sequences respectively) lineages in Tibetan hot detected from thermophilic microbial mats of hot spring in springs. In addition, some other groups were abundantly the central Tibet (Lau et al., 2009). distributed in certain Tibetan springs. For example, the

56 AB282756 Thermosulfidibacter takaii Hot springs in this study AM712345 uncultured Thermodesulfobacterium sp DQ413023 Desulfurobacterium sp. Hot springs from Yellowstone AJ507320 Desulfurobacterium crinifex AJ001049 Desulfurobacterium 76 AF068793 uncultured bacterium AJ316619 Thermovibrio ruber CP002444 Thermovibrio ammonific Desulfurobacteriaceae AY268937 Thermovibrio guaymasen 77 AB278143 uncultured bacterium AY268936 Desulfurobacterium pac AB105049 Balnearium lithotrophi 93 AJ132734 uncultured eubacterium AY862073 Hydrogenothermus WB3 AY861787 Hydrogenothermus OPP AY862025 Hydrogenothermus WB1 FJ434217uncultured bacterium DQ645250 uncultured Aquificales AY862028 uncultured Hydrogenothermus WB1 AY862072 Hydrogenothermus WB3B062 AY686713 Sulfurihydrogenibium yellowstonense AY861844 uncultured HydrogenothermusOPPB118 DQ906017 uncultured Sulfurihydrogenibium sp. DQ243782 Aquificales SSp B60 AY861801 uncultured Hydrogenothermus OPP AY862077 uncultured Hydrogenothermus WB3 AF445697 uncultured Aquificales AB 073126 uncultured Aquificales 56 AY861855 Hydrogenothermus OPP AY862074 uncultured Hydrogenothermus WB3 69 AJ292525 Hydrogenothermus marinus VM1 AB086419 Persephonella hydrogeniphila 29W JF935227uncultured bacterium AB071324 Sulfurihydrogenibium subterraneum HGM-K1 Hydrogenothermaceae 74 AY672501uncultured bacterium AF528192 Sulfurihydrogenibium azorense Az-Fu1 Fc8A70 YN_TB_B3315 DQ989208 Venenivibrio stagnispumantis CP001230 Persephonella marina 83 YN_TB_B5308 57 YN_TB_B5452 60 YN_TB_B1723 YN_TB_B5291 89 YN_TB_B5270 67 YN_TB_B5435 YN_TB_B1336 Group 1 YN_TB_B4178 63 YN_TB_B13913 YN_TB_B1751 YN_TB_B1692 YN_TB_B1745 FJ791957uncultured bacterium HM448270 Aquificales SBC BP4 B AY861719 uncultured Hydrogenobaculum CPC AJ320225Hydrogenobaculum sp. EF156514 uncultured Hydrogenoba EU545990 YNP Hydrogenobaculum 96 DQ924717uncultured Hydrogenobaculum sp. EU545991 YNP Hydrogenobaculum EU545989 YNP Hydrogenobaculum YN_TB_B4385 YN_TB_B9106 YN_TB_B17435 51 YN_TB_B17495 65 YN_TB_B20204 YN_TB_B1085 YN_TB_B10250 YN_TB_B26211 YN_TB_B12791 YN_TB_B1091 99 YN_TB_B124 Group 2 YN_TB_B9156 YN_TB_B1104 YN_TB_B1222 52 YN_TB_B12841 YN_TB_B25051 YN_TB_B4455 YN_TB_B11837 YN_TB_B10253 YN_TB_B9994 65 98 YN_TB_B17415 YN_TB_B25103 YN_TB_B20408 99 AJ320223 Aquifex sp. Gri14L3B AB120294 Hydrogenivirga caldilitoris HM448211 Thermocrinis SBC BP2A 61 62 DQ243738 uncultured Thermocrini AY862031 uncultured Hydrogenobacter WB1A Aquificaceae DQ324897uncultured Thermocrini HM448205 Thermocrinis SBC BP2A HM448363 uncultured Thermocrinis 60 EU635954 uncultured bacterium AJ320221Thermocrinis sp. G3L1B GU233840uncultured Thermocrinis 83 CP001931Thermocrinis albus EF100635uncultured Aquificacea YN_TB_B12527 AY251061uncultured Hydrogenobacter sp. L77876 Thermothrix thiopara GU233444 Thermothrix azorensis YN_TB_B16613 YN_TB_B1921 YN_TB_B16077 YN_TB_B25606 60 YN_TB_B1901 YN_TB_B15017 YN_TB_B2372 YN_TB_B16945 YN_TB_B2482 Group 3 YN_TB_B682 YN_TB_B3526 YN_TB_B681 YN_TB_B841 YN_TB_ B15269 YN_TB_B22307 YN_TB_B20607 YN_TB_B729 YN_TB_B7571 Group 4 83 YN_TB_B5096 YN_TB_B20999 96 YN_TB_B5230 AY861781 uncultured Hydrogenobacter OPPB YN_TB_B6508 72 100 HM448341 Aquificales SBC QL1 HM448250 Aquificales SBC BP4 Unidentified Group 79 HM448376 Aquificales SBC QL2 HM448247 Aquificales SBC BP4 100 HM448254 Aquificales SBC BP4 100 DQ243737 Aquificales BP B68

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Fig. 3. Phylogenetic relationships between the Aquiﬁcae-related 16S rRNA gene sequences obtained in this study and those from Yellowstone hot springs and other geothermal features.

YN_TB_B24266 YN_TB_B10667 YN_TB_B8232 YN_TB_B3755 Hot springs in this study AF513446 Rhizobiaceaebacterium LA52 AY862042 Alpha proteobacterium clone WB1B012 YN_TB_B11583 Hot springs from Yellowstone AY862013 Alpha proteobacterium clone QLBB012 YN_TB_B14284 D32234 Methylobacteriumsp. F37 AY882707 Methylobacteriumsp. clone SK149 YN_TB_B13525 EF447047 Alpha proteobacteriumTibetan thermophilicmat clone YN_TB_B991 YN_TB_B14984 YN_TB_B10974 U88041 Amaricoccuskaplicensis YN_TB_B11472 YN_TB_B8991 YN_TB_B6555 Y13244 Roseobactergallaeciensis FJ885016 Proteobacteriumclone F1AD11 YN_TB_B2336 YN_TB_B17696 YN_TB_B23515 YN_TB_B14057 YN_TB_B16954 YN_TB_B4837 Alphaproteobacteria FJ886040 Proteobacteriumclone F1AG09 YN_TB_B15653 AB110635 Sphingomonassp. MD-1 DQ243762 Proteobacteriumclone YNP_ObP_B118 YN_TB_B14842 AB021370 Sphingomonasechinoides EF205453 Alpha proteobacteriumTibetan thermophilicmat clone YCB25 AY861735 Alpha proteobacteriumclone OPPA012 YN_TB_B17236 AJ227814 Hyphomonasjannaschiana YN_TB_B11396 M59061 Azospirillumlipoferum EU156155 Alpha proteobacterium clone pCOF_65.7 YN_TB_B15315 YN_TB_B13712 YN_TB_B18950 YN_TB_B11177 FJ886329 Proteobacteriumclone F1AB02 YN_TB_B10953 YN_TB_B4388 YN_TB_B6214 YN_TB_B5542 JQ247723 Acidicaldussp. MK6 YN_TB_B1151 AY140238 Acidicaldus organivorans strain Y008 YN_TB_B1523 DQ834024 Desulfurellasp. clone SK663 EF192881 Bacterium Rap1 22D AF254389 Bacterium UASB TL9 AF148938 BdellovibriobacteriovorusstrainBEP2 M34127 Rhodomicrobiumvannielii YN_TB_B249 EF205525 Delta proteobacterium Tibetan thermophilicmat clone DTB134 YN_TB_B7596 YN_TB_B19926 YN_TB_B15410 EF205515 Delta proteobacterium Tibetan thermophilicmat clone DTM76 YN_TB_B16166 YN_TB_B3833 YN_TB_B11054 YN_TB_B23056 YN_TB_B2876 YN_TB_B4767 AY861756 Delta proteobacterium clone OPPB013 Deltaproteobacteria AF026997 Unidentified delta proteobacteriumOPT47 DQ834048 Desulfurellasp. clone SK688 YN_TB_B273 EF393274 Bacterium clone ORSFAM_e02 AF027000 Unidentified delta proteobacteriumOPT56 YN_TB_B3357 AY861742 Delta proteobacteriumclone OPPA021 AF026994 Unidentified delta proteobacteriumOPS22 AY862012 Delta proteobacterium clone QLBB011 AF026998 Unidentified delta proteobacterium OPB16 AY862067 Delta proteobacterium clone WB3B047 AF026991 Unidentified delta proteobacteriumOPS105 YN_TB_B6938 DQ006288 Desulfatimicrobiummahresensisstrain SA1 AM110971 Halomonas sp. FJ885534 Proteobacteriumclone F3BB06 DQ834047 Thiomonassp. clone SK687 DQ243741 Proteobacteriumclone YNP_BP_B75 DQ243781 Proteobacteriumclone YNP_SSp_B57 YN_TB_B5781 AJ012228 Luteimonasmephitis FJ828370 Gamma proteobacteriumclone YN_TB_B11710 YN_TB_B13681 YN_TB_B6723 YN_TB_B6302 YN_TB_B1272 AF039167 Rhodanobacter lindanoclasticus EF127600 Organism EME044 NR_025815 Silanimonaslentastrain YN_TB_B21566 YN_TB_B4387 DQ834035 Acidithiobacillussp. clone SK675 YN_TB_B2015 HM163482 Acinetobacter lwo ffiistrain 2DT134M Gammaproteobacteria YN_TB_B5101 YN_TB_B8688 AB304258 Thiofabate pidiphila YN_TB_B13414 L40994 Beggiatoa alba YN_TB_B5008 YN_TB_B14280 GU003816 Klebsiella clone A10 YN_TB_B2976 YN_TB_B2347 FJ886171 Proteobacterium clone F2BC04.1 EF205513 Gamma proteobacteriumTibetan thermophilicmat clone DTM41 EF205512 Gamma proteobacteriumTibetan thermophilicmat clone DTM21 YN_TB_B23037 YN_TB_B8733 FJ348430 Serratia sp. YN_TB_B3425 YN_TB_B16296 YN_TB_B14261 EF205489 Gamma proteobacterium Tibetan thermophilicmat clone YCC51 YN_TB_B27211 YN_TB_B19569 YN_TB_B10720 EF205566 Beta proteobacterium Tibetan thermophilicmat clone TP70 AY861832 Beta proteobacterium clone OPPB105 YN_TB_B23327 AF084947 Rhodoferax antarcticus AY882778 Acidovorax sp. clone SK224 YN_TB_B1510 YN_TB_B4383 FJ439052 Burkholderiales bacterium clone PII2E YN_TB_B1593 AB009828 Hydrogenophilus thermoluteolus AF026983 Unidentified beta proteobacterium OPS140 AF037106 Nitrosomonas europaea Betaproteobacteria YN_TB_B247 AJ243144 Thiobacillus denitrificans AY861797 Beta proteobacterium clone OPPB064 FJ886036 Proteobacterium clone F2BG03 EU156142 Beta proteobacterium clone pCOF_65.7_B2 AY966001 Massilia lutea YN_TB_B14221 EF205487 Beta proteobacterium Tibetan thermophilicmat clone YCC80 YN_TB_B3311 YN_TB_B13094 EF205485 Beta proteobacterium Tibetan thermophilicmat clone YCC30 YN_TB_B25693 FJ885029 Uncultured proteobacteriumclone F1BA08 YN_TB_B11119 EF205449 Beta proteobacterium Tibetan thermophilicmat clone YCB6 NR_044685 Chlorobaculumtep idum

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Fig. 4. Phylogenetic relationships between the Proteobacteria-related 16S rRNA gene sequences obtained in this study and those from Yellowstone hot springs and other geothermal features.

Fig. 5. Archaeal diversity in the different hot Sulfolobales Crenarchaeota springs. Stacked bar graph represents the Desulfurococcales relative distribution of the mainly phyla in the different stations. Thermoproteales Caldisphaerales Unclassified Crenarchaeota Archaeoglobi Euryarchaeota Halobacteria Methanobacteria Thermoplasmata Other Euryarchaeota Unclassified Euryarchaeota Korarchaeota Nanoarchaeota Unclassified Archaea Sx1 Sx4 Zzq Dgg Qc4 Qc8 Qc1 Qc3 Gl15 Gl16 Drty4 Hmz1 Hmz2 Eynj3 Eynj2 Drty14 acidic (Yunnan) non-acidic (Yunnan) non-acidic (Tibet) class Archaeoglobi of Euryarchaeota represented 31% Discussion and 20% of archaeal population in Gl16 and Qc8 hot springs respectively. Halobacteria were also detected in High-throughput 454 pyrosequencing is a robust tool in Qc1 and Qc8 with 6% and 7% of total archaeal species analysing the microbial communities from diverse envi- respectively. ronments and can detect rare members of the microbial community while providing several thousand sequences of sufficient length to accurately identify consortia Phylogenetic analysis of Desulfurococcale and members and to estimate microbial diversity and richness unclassified Crenarchaeota (Youssef et al., 2009). With the use of the high-throughput The Desulfurococcales and unclassified Crenarchaeota 454 pyrosequencing technique, we recovered community represented a large portion of archaeal community in all structures from Yunnan and Tibetan hot springs with non-acidic hot springs (50–90% of total archaeal species greater detail than previous studies using conventional in each sample) in this study. Previous studies have clone library and DGGE approaches. For example, we recovered many new and representative uncultured phy- previously studied the crenarchaeotal population in the logenetic clades belonging to these two groups (Barns Zzq and Dgg hot springs by using the clone library tech- et al., 1994; 1996; Meyer-Dombard et al., 2005; 2011); nique and only four to six OTUs were obtained (Song however, the automatic classifiers at the RDP level lack et al., 2010). In comparison, more than 200 creanar- the fine classification of these subgroups. Thus, to better chaeotal OTUs were recovered from the Zzq and Dgg hot define the affiliation of Desulfurococcales and unclassified springs in this study. Huang and colleagues (2011) Crenarchaeota taxa in this study, we selected some rep- detected 82 bacterial and 41 archaeal OTUs (cut-off: resentative OTUs and reference sequences to perform 97%) from 10 Tibetan hot springs using the clone library the phylogenetic analysis. The nomenclatures of partial method, and the bacterial OTUs were affiliated with 10 clades were referred to Meyer-Dombard’s work at bacterial phyla (Firmicutes, Proteobacteria, Cyanobacte- Yellowstone (Meyer-Dombard et al., 2005; 2011). Our ria, Chloroflexi, Bacteroidetes, Acidobacteria, Nitrospirae, sequences were distributed in a broad spectrum of Planctomycetes, Thermodesulfobacteria, Aquificae and archaeal phylogenetic lineages in Fig. 6. Few sequences unclassified Bacteria) with Proteobacteria, Cyanobacteria were similar to those detected from Yellowstone, and the and Chloroflexi being the dominant groups. In compari- most clusters were in tight clades within each known son, 1711 bacterial and 1161 archaeal OTUs (cut-off: group, especially for the class Desulfurococcales and 0.03) were obtained from six Tibetan hot springs in this ‘uncultured Crenarchaeota’ (UC) group II and IV. The study, and in addition to the 10 bacterial phyla mentioned latter group harboured more OTUs than the other groups above, 13 other bacterial phyla (with low relative abun- and some of them formed a large subgroup (character dances) were also detected in this study. The dominant A denoted) (Fig. 6). This subgroup, however, was only phylogenetic groups were Proteobacteria, Bacteroidetes, found in five Tibetan hot springs (Qc3, Gl16, Qc1 Qc4 Chloroflexi, Deinococcus-Thermus and unclassified Bac- and Qc8). teria for the Tibetan hot springs in this study.

YN_TB_A24590 Hot springs in this study YN_TB_A23040 YN_TB_A6260 Hot springs from Yellowstone FJ489512 Sulfolobus sp. TYQ13 Sulfolobales Sulfolobus solfataricus P2 AF167083 Metallosphaera sp. YN_TB_A22324 X90480 S.azoricus YN_TB_A7725 HM448065 SBC MS2 A113 DQ243778 Uncultured Thermoproteales DQ243746 Uncultured Thermoproteales HM448063 SBC MS2 A97 DQ243751 Uncultured Thermoproteales L07511 Pyrobaculum.islandicum M35966 T.tenax DQ441496 Uncultured archaeon AB005296 Thermocladium modesti DQ243774 Uncultured Thermoproteales AB063645 Vulcanisaeta souniana Thermoproteales YN_TB_A11107 YN_TB_A11848 YN_TB_A18293 YN_TB_A2911 YN_TB_A798 YN_TB_A1341 YN_TB_A592 YN_TB_A2026 YN_TB_A630 YN_TB_A731 YN_TB_A884 YN_TB_A4711 YN_TB_A1302 DQ441511 Uncultured archaeon EU635920SSE L4 H05 HM448060 SBC MS2 A25 DQ243731 Uncultured Desulfuroc DQ243729 Uncultured Desulfurococcales HM448078 SBC BP2A A40 DQ243725 Uncultured Desulfurococcales DQ243734 Uncultured Desulfurococcales YN_TB_A1292 YN_TB_A15530 X99555 P.fumarius D83259 Aeropyrum.pernix YN_TB_A21458 YN_TB_A4110 YN_TB_A9961 EU427996 Uncultured crenarchaeta YN_TB_A425 YN_TB_A2024 HM448092 SBC BP2B A32 DQ243745 Uncultured Desulfurococcales DQ243728 Uncultured Desulfurococcales Desulfurococcales YN_TB_A11094 YN_TB_A4309 YN_TB_A1995 YN_TB_A13778 YN_TB_A776 YN_TB_A15442 YN_TB_A17945 AJ271794 Ignicoccus pacificus YN_TB_A3973 YN_TB_A4700 YN_TB_A1353 HM448086 SBC BP2B A21 DQ243732 Uncultured Desulfurococcales YN_TB_A73 AB087499 Caldisphaera lagunens AF191225 Acidolobus.aceticus EU635921 SSW L4 A01 HM448083 SBC BP2B A8 DQ243730 Uncultured M21087 Pyrodictium.occultum DQ243773 D3 Uncultured Desulfurococcales DQ243775 Uncultured Desulfurococcales DQ243754 Uncultured Desulfurococcales YN_TB_A5281 DQ243760 Uncultured Desulfurococcales DQ243733 Uncultured Desulfurococcales EU635919SSW L4 A04 DQ886561 Uncultured crenarchae YN_TB_A11231 EU635922SSE L4 F03 DQ886562 Uncultured crenarchaeota DQ243727 Uncultured crenarchaeota YN_TB_A4681 HM448157 SBC OJO A41 DQ243772 Uncultured crenarchaea AY861905 OPPC036 DQ441517 Uncultured archaeon EU635923 SSW L4 H06 EU635924 SSE L4 G03 DQ243777 Uncultured crenarchaeota AY236856 Uncultured archaeon AY861911 OPPC043 pJP41 L25301 DQ441516 Uncultured archaeon YN_TB_A19216 YN_TB_A1454 UC gp. a YN_TB_A7216 YN_TB_A18036 YN_TB_A10985 YN_TB_A574

0.02

Fig. 6. Phylogenetic relationships between the crenarchaeotal 16S rRNA gene sequences obtained in this study and those from Yellowstone hot springs and other geothermal features.

Hot springs in this study YN_TB_A8332 YN_TB_A16241 Hot springs from Yellowstone YN_TB_A5767 YN_TB_A10913 YN_TB_A20865 YN_TB_A16338 YN_TB_A9320 YN_TB_A14205 YN_TB_A5746 YN_TB_A5797 YN_TB_A4381 YN_TB_A5295 YN_TB_A6598 YN_TB_A8330 YN_TB_A8416 YN_TB_A9339 A YN_TB_A5834 YN_TB_A20779 YN_TB_A16294 YN_TB_A11059 YN_TB_A5760 YN_TB_A9345 YN_TB_A1572 YN_TB_A8476 YN_TB_A3286 YN_TB_A9382 YN_TB_A14308 YN_TB_A9290 YN_TB_A2995 YN_TB_A10989 YN_TB_A9340 YN_TB_A5379 EU635905 SSW L4 D05 YN_TB_A543 YN_TB_A6170 EU635929 SSW L4 G03 EU635904 SSW L4 E01 AY861924 OPPC059 DQ243757 Uncultured crenarchaeota pSL123 U63345 DQ441515 Uncultured archaeon YN_TB_A15684 UC gp. EU635928 SSW L4 E05 YN_TB_A15693 YN_TB_A3263 YN_TB_A3315 YN_TB_A2194 YN_TB_A593 YN_TB_A1540 YN_TB_A15687 YN_TB_A4287 YN_TB_A6213 YN_TB_A3345 YN_TB_A2213 YN_TB_A15020 YN_TB_A19148 YN_TB_A177 YN_TB_A10909 AY861940 OPPD002 AY861952 OPPD016 AY861913 OPPC045 pJP89 L25305 DQ243753 Uncultured crenarchaeota YN_TB_A6555 YN_TB_A8367 YN_TB_A9310 YN_TB_A10161 YN_TB_A8325 YN_TB_A9570 YN_TB_A5811 YN_TB_A2216 YN_TB_A10988 YN_TB_A5829 EF203627 Uncultured archaeon YN_TB_A8388 YN_TB_A8517 YN_TB_A11614 AY704372 FS27421A03 YN_TB_A9368 YN_TB_A5795

0.01

Fig. 6. Continued

A recent study revealed the bacterial and archaeal Drty4 and Drty14 (temperature: 47–96°C; pH: 3.2–4.5) in diversities in 11 Fe-rich acidic hot springs (temperature: this study. The acidic Yunnan hot springs (Zzq, Drty4 and 53–88°C; pH 2.4–3.6) located in Joseph’s Coat and Drty14) possess similar preponderant bacterial taxonomic Rainbow Springs in Yellowstone National Park (Kozubal groups (e.g. Hydrogenobaculum, Acidicaldus)tothe et al., 2012), which were similar to acidic hot springs Zzq, acidic hot springs in Joseph’s Coat and Rainbow Springs

Sulfolobales, Thermoproteales, Desulfurococcales and UC gp. a

UC gp.

EU635902 SSE L4 A06 YN_TB_A118 AY861949 OPPD013 U63340 pSL22 Hot springs in this study EU635903 SSW L4 H01 YN_TB_A0 YN_TB_A5543 Hot springs from Yellowstone YN_TB_A13098 YN_TB_A10917 YN_TB_A13847 EU635907 SSE L4 D02 U63339 pSL17 YN_TB_A548 EU635906 SSW L4 G05 AB301869 Uncultured crenarchaeota YN_TB_A65 YN_TB_A1740 YN_TB_A9329 EU635917 SSE L4 B03 DQ490011GBS L2 E07 EU239960 Nitrosocaldus yellowstornis UC gp. b HM448058 SBC MS2 A16 SUBT14 AF361211 DQ490012 GBS L2 E12 EU635908SSW L4 C03 EU635909SSE L4 D01 EU635910SSW L4 F01 EU635915G04b L4 A08 HM448139 SBC QL2 A2 YN_TB_A328 YN_TB_A23863 DQ243726 Uncultured crenarchaeota DQ490010GBS L2 C12 EU635911G04b L4 A09 HM448074 SBC BP2A A17 EU635912SSW L4 E04 EU635914SSE L4 C01 EU635913G04b L4 D08 DQ490009GBS L2 A09 pSL78 U63344 DQ243744 Uncultured crenarchaeota YN_TB_A17438 YN_TB_A6158 YN_TB_A8254 UC gp. AY861957 OPPD023 YN_TB_A1487 YN_TB_A8293 YN_TB_A6022 YN_TB_A8299 YN_TB_A1437 YN_TB_A255 YN_TB_A437 YN_TB_A14266 YN_TB_A682 YN_TB_A5781 YN_TB_A10431 AY861948 OPPD012 YN_TB_A9623 EU635916 SSW L4 G06 DQ243750 Uncultured crenarchaeota DQ243749 Uncultured crenarchaeota pSL4 U63341 AB050221 SAGMAP YN_TB_A6428 SAGMA2 AB050233 AB019727 pIVWA1 DQ085 Nitrosopumilus maritimus U51469 CSU51469 AB050238 SAGMA8 U71109 archae on PM7 PET122 AY278102 SCA1166 U62816 SCA1173 U62818 X96695 Archaeal sp. X96692 Archaeal sp. YN_TB_A441 YN_TB_A9282 FJ716374 Uncultured archaeon X96689 Archaeal sp. AY861944 OPPD004 DQ223192 Uncultured crenarchaeota EU307069 Uncultured crenarchaeota AY861956 OPPD021 DQ243758 Uncultured crenarchaeota UC gp. U63343 pSL12 YN_TB_A199 YN_TB_A13751 c& YN_TB_A16443 YN_TB_A1804 YN_TB_A16356 YN_TB_A6913 YN_TB_A7 YN_TB A9547 YN_TB_A11235 YN_TB_A3754 EU635918 SSE L4 A02 YN_TB_A2214 AY861942 OPPD003 DQ243759 Uncultured crenarchaeota YN_TB_A10868 YN_TB_A15696 YN_TB_A20766 YN_TB_A3358 YN_TB_A1827 AB113623 Uncultured crenarchaeota YN_TB_A3800 YN_TB_A2197 YN_TB_A2235 YN_TB_A1445 YN_TB_A3572 YN_TB_A1566 A119 AY182074 YN_TB_A658 YN_TB_A21040 AJ42037 Halococcus dombrowskii U68538 Halobacterium salinarum Methanoculleus.marisnigri AF02 M59124 Methanobacterium.bryan

0.05

Fig. 6. Continued

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 15, 1160–1175 1172 Z.-Q. Song et al. in Yellowstone (Kozubal et al., 2012). However, some diverse prokaryotic diversity in the investigated hot bacterial taxonomic groups differ between Yunnan and springs than our previous studies (using clone library and Yellowstone hot springs. For example, Thermus, Ther- DGGE techniques) in the same region. The phylogenetic mothrix, Desulfurella and Thiomonas were only detected analysis shows that Yunnan and Tibetan hot springs with relative high abundance from Yunnan hot springs. In possess high abundance of sequences affiliated with contrast, Acidimicrobium, Methylacidiphilum and Meio- Aquificae and Crenarchaeota, which is consistent with thermus were only recovered from Yellowstone springs previous studies in Yellowstone hot springs. However, with high abundance (Kozubal et al., 2012). the detailed community composition of Aquificae and Cre- For Archaea, the above Yellowstone hot springs were narchaeota differed between hot springs of Yellowstone dominated by Crenarchaeota. Generally speaking, Cre- and Yunnan/Tibet: many Aquificae- and Crenarchaeota- narchaeota always dominate hot springs of Yellowstone sequences obtained in the investigated hot springs are and other regions, such as hot springs in Iceland, Kam- phylogenetically separated from those from Yellowstone chatka (Russia), Thailand (Purcell et al., 2007; Wilson hot springs. et al., 2008; Costa et al., 2009; Kublanov et al., 2009). However, in this study the majority (97.6%) of total Experimental procedures sequences in Drty14 (temperature: 47°C; pH: 4.5) fell into the class Thermoplasmata of Euryarchaeota. The Sx1 Field measurement and sample collection and Sx4 hot springs were also dominated by unclassified At each hot spring, water temperature and pH were deter- euryarchaeotal sequences. It is unclear whether geogra- mined using a Hach pH meter equipped with a pH probe and phy plays a role in the disparate distribution of archaeal a temperature probe. Nitrite, nitrate, ammonium, phosphate species from acidic environments between China and and ferrous iron were measured using Hach kits according to other regions mentioned above. the manufacturer’s instructions. After chemical measure- Desulfurococcales and unclassified Crenarchaeota ments, mat-containing sinter or sediments were collected into were the dominant groups in most of the investigated 50 ml Falcon tubes and immediately were stored in liquid non-acidic hot springs from Yunnan and Tibet, which is nitrogen (at the sites in Yunnan Province) or dry ice (at the Tibetan sites). The samples were kept in liquid nitrogen or dry consistent with previous studies in Yellowstone hot ice in the field and during transportation, and then were springs (Meyer-Dombard et al., 2005; 2011; Spear et al., stored at -80°C in the laboratory until further analysis. 2005). However, the hot springs of Yunnan Province and Tibet harboured more diverse unclassified crenarchaeotal (UC) group IV lineages than Yellowstone. Furthermore, Nucleic acid extraction and 454 pyrosequencing the UC group IV contained a large subgroup (character A One to two grams of sinter or sediments were subjected to assigned), which has never been revealed from Yellow- DNA extraction with the use of an E.Z.N.A. Soil DNA Kit stone (Fig. 6). Interestingly, sequences affiliated with this (Omega Bio-Tek, USA) according to the manufacturer’s subgroup were only from the Tibetan hot springs, indicat- instructions. Quadruplicates were performed for each ing this group of Crenarchaeota may not be ubiquitous in sample. The resulting DNA extracts from each sample were terrestrial hot springs. Indeed, sequences of this sub- mixed and were used for downstream PCR experiments. The bacterial and archaeal hypervariable V4 region of the 16S group have also been detected from a hot spring (64°C) rRNA genes were amplified using primer sets of B-forward located in another thermal field in Tibet. Temperature (5′-AYTGGGYDTAAAGNG-3′)/B-reverse (5′-TACNVGGGTA might be a limiting environmental factor since the group TCTAATCC-3′) and A-forward (5′-YMGCCRCGGKAAHACC- denoted by character A dominates the low-temperature 3′)/A-reverse (5′-CTACNSGGGTMTCTAAT-3′) respectively springs and has low abundance in high-temperature (http://pyro.cme.msu.edu/pyro/help.jsp). The employed pri- springs. In addition, GenBank sequences closely related mer sets targeted regions with high classification accuracy to our sequences within the A group are mainly from using the RDP’s naive Bayesian rRNA classifier. To pool multiple samples for one run of 454 sequencing, a sample moderate-temperature hot springs in Uzon Caldera, Kam- tagging approach was used (Meyer et al., 2008). Each tag chatka, Russia and Thailand (Kanokratana et al., 2004; was added to the 5′ end of forward primer (http://pyro.cme. Burgess et al., 2012). Taken together it appears that the A msu.edu/pyro/help.jsp). PCR amplification consisted of an group of Crenarchaeota may prefer low–moderate tem- initial denaturation at 95°C for 10 min, 25 cycles of denatura- perature habitats. tion at 95°C for 45 s, annealing at 42°C and 55°C for bacteria In summary, we investigated the prokaryotic diversity in and archaea respectively) for 1 min, and extension at 72°C hot springs of Yunnan Province and Tibet using 454 pyro- for 1 min, and a final extension at 72°C for 5 min. Individual reagents and their concentrations were as follows: 1¥ PCR sequencing technologies. Our study shows that the 454 buffer with 1.5 mM Mg2Cl, dNTPs (100 M each), 0.25 M pyrosequencing technique is more robust than the clone each primer, 2.5 U of DNA polymerase (Ex-Taq) (TaKaRa, library and DGGE methodologies in characterizing micro- Dalian, China) and ~ 50 ng of total DNA. PCR products were bial diversity in hot springs. This study shows more purified using an E.Z.N.A. Gel Extraction Kit (Omega Bio-Tek,

USA) according to the manufacturer’s instructions. The puri- pyrosequencing data (Hamady et al., 2010). PCA of the fied PCR products were annealed to oligonucleotides that are sulfate, nitrite, nitrate, ammonium, ferrous iron were per- complementary to an adaptor sequence that are tethered formed using R software (version 2.6.0). onto mm-size beads. Subsequently, nucleic acid was pyrose- quenced using Roche Diagnostics GmbH/454 Life Sciences Acknowledgements Corporation (Branford, CT, USA). The raw sequence reads were deposited into the NCBI sequencing read archive under This work was supported by grants from the National Basic Accession No. SRA053606. Research Program of China (Nos. 2010CB833801 & 2012CB822004), the Key Project of International Cooperation of China Ministry of Science & Technology (MOST) (No. Pyrosequencing data processing 2013DFA31980), the National Natural Science Foundation of China (Nos. 31070007 40972211, 41030211 & 41002123), the According to Sogin’s description (Sogin et al., 2006), low- U.S. National Science Foundation (Nos. MCB-0546865, quality reads were removed. All available bacterial and ETBC-1024614 and OISE-0968421), and the Fundamental archaeal sequences were longer than 200 bp. All sequence Research Funds for the Central Universities of China, China reads were compared with a reference database of known 16S University of Geosciences (Wuhan). W-J Li was also sup- rRNA genes [obtained from SILVA and Ribosomal Database ported by ‘Hundred Talents Program’ of the Chinese Academy Project (RDP) databases] and taxonomically assigned with of Sciences. We also thank Chaolei Jiang, Jianxin Fang, rdp_classifier-2.0 at 50% of confidence threshold (Wang et al., Jinchuan Yin, Shaochuan Gong, Jiafu Sheng, and Mr Ma and 2007), then the invalid reads, which cannot be classified in any the entire staff from Yunnan Tengchong Volcano and Spa Archaea or Bacteria, were removed. Subsequently alignment TouristAttraction Development Corporation for strong support. and OTUs cluster of the available sequences were performed through RDP pipeline (http://pyro.cme.msu.edu/). Based on References the results of OTUs cluster and assigned taxonomy of the available sequences, the comprehensive statistics including Barns, S.M., Fundyga, R.E., Jeffries, M.W., and Pace, N.R. the distribution and abundance of each OTU in the investi- (1994) Remarkable archaeal diversity detected in a Yellow- gated hot springs were obtained by using a Python script. stone National Park hot spring environment. 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