ORIGINAL RESEARCH Pyrosequencing analysis of a bacterial community associated with lava-formed soil from the Gotjawal forest in Jeju, Korea Jong-Shik Kim1, Keun Chul Lee2, Dae-Shin Kim3, Suk-Hyung Ko3, Man-Young Jung4, Sung-Keun Rhee4 & Jung-Sook Lee2 1Gyeongbuk Institute for Marine Bioindustry, Uljin 767-813, Korea 2Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea 3Research Institute for Hallasan, Jeju Special Self-Governing Province 690-816, Korea 4Department of Microbiology, Chungbuk National University, Cheongju 361-763, Korea

Keywords Abstract 16S rRNA gene, bacterial diversity, Gotjawal forest soil, pyrosequencing. In this study, we analyzed the bacterial diversity in soils collected from Gyorae Gotjawal forest, where globally unique topography, geology, and ecological fea- Correspondence tures support a forest grown on basalt flows from 110,000 to 120,000 years ago Jong-Shik Kim, Gyeongbuk Institute for and 40,000 to 50,000 years ago. The soils at the site are fertile, with rocky areas, Marine Bioindustry, Uljin 767-813, Korea. and are home to endangered species of plants and animals. Rainwater pene- Tel: +82-54-780-3451; Fax: +82-54-780- trates to the groundwater aquifer, which is composed of 34% organic matter 3469; E-mail: [email protected] containing rare types of soil and no soil profile. We determined the bacterial community composition using 116,475 reads from a 454-pyrosequencing analy- Funding Information sis. This dataset included 12,621 operational taxonomic units at 3% dissimilar- This work was supported by the National ity, distributed among the following groups: Proteobacteria (56.2%) with 45.7% Research Foundation of Korea(NRF) grant of a-Proteobacteria, Actinobacteria (25%), Acidobacteria (10.9%), Chloroflexi funded by the Korea government(MSIP) (2.4%), and Bacteroidetes (0.9%). In addition, 16S rRNA gene sequences were (No.NRF-2014R1A2A1A11052888). amplified using polymerase chain reaction and domain-specific primers to con- struct a clone library based on 142 bacterial clones. These clones were affiliated Received: 8 July 2014; Revised: 4 December a 2014; Accepted: 12 December 2014 with the following groups: Proteobacteria (56%) with 51% of -Proteobacteria, Acidobacteria (7.8%), Actinobacteria (17.6%), Chloroflexi (2.1%), Bacilli (1.4%), MicrobiologyOpen 2015; 4(2): 301–312 Cyanobacteria (2.8%), and Planctomycetes (1.4%). Within the phylum Proteo- bacteria, 56 of 80 clones were tentatively identified as 12 unclassified genera. doi: 10.1002/mbo3.238 Several new genera and a new family were discovered within the Actinobacteria clones. Results from 454-pyrosequencing revealed that 57% and 34% of the sequences belonged to undescribed genera and families, respectively. The char- acteristics of Gotjawal soil, which are determined by lava morphology, vegeta- tion, and groundwater penetration, might be reflected in the bacterial community composition.

Introduction ecological features shaped by volcanic activity occurring 110,000–120,000 and 40,000–50,000 years ago (Park On Jeju Island in Korea, the word “Gotjawal” refers to 2010). Overall, the Gotjawal forest represents a species- any naturally formed forest that grows on basalt-flow rich ecosystem of coexisting plant species, such as ferns rocky terrain and presents a virtually impassable mixture and broad-leaved trees, at both the northern and southern of trees and undergrowth. In addition, these forests func- distributional limits (Yang et al. 2006; Kim et al. 2010), tion as the main source of water for Jeju’s population; harboring a total of 506 plant and 784 insect species iden- rainwater is purified and recharged by the porous rocks tified to date (Jung 2010; Kim et al. 2010). The forest and groundwater aquifers within the forest. Gotjawal for- occurs on a highly irregular substrate of a’a lava flows ests are characterized by lava domes, microclimates, and (Fig. 1). Jeju’s lava forests may be a globally unique area

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(A) these microbial communities (Kim et al. 2014a). Gener- ally, soil microbial communities are highly diverse, and estimates of unclassified species may reach 99% of 16S rRNA gene sequence databases, since most soil bacteria are difficult to cultivate (Torsvik et al. 1990; Amann et al. 1995). Microbial communities associated with volcanoes have been studied in the lava flow soils, rocks, and/or glass found at Kilauea volcano, Hawaii (King 2003; Dunfield and King 2004; Nanba et al. 2004; Gomez-Alvarez et al. 2007; King and Weber 2008; Nacke et al. 2011; King and King 2012); Mauna Loa, Hawaii (Crews et al. 2001); (B) Miyake Island, Japan (Ohta et al. 2003); Llaima volcano, Chile (Hernandez et al. 2014); and Mt. Hekla, Iceland (Kelly et al. 2010). Cutler et al. (2014) suggested that plant community composition is a significant determinant for fungal communities, but is less relevant for bacterial community composition during long-term changes in soil microbial communities. Bacteria are able to colonize recent volcanic deposits, which can contain numerous unknown bacterial species (Gomez-Alvarez et al. 2007). Various aspects of the structure and function of microbial communities have been studied in recent Hawaiian volca- nic deposits (Dunfield and King 2004), and these deposits in particular have been shown to harbor very distinct microbial assemblages. Three hundred-year-old lava- derived forest soils have been shown to exhibit substantial Figure 1. (A) A map of Jeju Island showing the Gotjawal distribution (blue line) and the sampling site (blue triangle symbol). (B) diversity (Nusslein and Tiedje 1998). Photographs showing the Gyorae Gotjawal forest in Jeju (N 33° 260 In this study, we analyzed the composition and diver- 24.9″ and E 126° 390 44.5″, Korea). Pahoehoe lava and a’a lava were sity of bacteria in Gotjawal soils using 454-pyrosequenc- mixed when the lava flowed. The lava-formed forest was developed, ing and polymerase chain reaction (PCR) cloning-based with characteristic topography including lava domes and depressions. approaches. The results of our study will provide impor- Vegetation in this area is mainly deciduous broadleaf trees, Styrax tant insights into the understanding of the soil microbial japonicus, Zelkova serrata, Thunb Makino, and Acer palmatum Thunb. community in lava forest soils. ex Murray (Maple). Within the herb layer, Arachniodes standishii (T. Moore) Ohwi is the predominant species. Materials and Methods and critical for understanding lava-formed forests. How- Collection of soil samples in Gotjawal ever, many regions of the Gotjawal forest have been deforested, used for charcoal and edible mushroom pro- The geographic coordinates of the sample collection site duction, and grazed by horses, cows, and other herbi- were 33° 26.0230 N and 26° 39.460 W (Gyorae Gotjawal; vores. In response to these insults, secondary forests have Fig. 1A and B). In May 2009, samples of soil from behind developed on disturbed sites. Nonetheless, these forests or between the lava and trees were collected aseptically have been gradually disappearing in recent decades, and using ethanol-disinfected spatulas. Samples were placed in approximately 50% of these forests have been destroyed; clean, sealable plastic bags. These samples were stored in currently, only about 6% of the original forest area a cooler during transfer to the laboratory and were then remains due to unregulated construction and urbaniza- stored at 5°C for 1 week until further processing. After tion (Jung 2009). sieving (using a 2-mm sieve), subsamples were frozen at Until recently, few studies had characterized the 70°C. Soil DNA extraction was conducted within microorganisms in Gotjawal forest soils; therefore, 1 week of collection. The soil samples had a pH of 4.5, microbial analyses are necessary to understand the electrical conductivity of 3.44 dS/m, organic matter con- microbial communities within these soils and for eluci- tent of 34%, and NO3 concentration of 300.48 mg/kg dating the characteristics that permit the formation of dry soil (Kim et al. 2014a).

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95°C for 45 sec, 55°C for 45 sec, and 72°C for 90 sec, Soil DNA extraction with a final extension step for 5 min at 72°C. PCR prod- DNA was directly extracted from three subsamples using ucts were purified with a QIAquick PCR purification kit a FastDNA SPIN kit for soil (QBiogene Inc., Vista, CA) (Qiagen, Valencia, CA) and ligated into a pUC118 Hin- according to the manufacturer’s protocol. The extracted cII/BAP vector (Takara Bio Inc.), which was transformed DNA was purified using a FastPure DNA kit (Takara into competent Escherichia coli DH5a cells (Invitrogen Bio Inc., Shiga, Japan) and concentrated using a Zymo- Corp., Carlsbad, CA) using heat shock. Plasmids from clean gel DNA recovery kit (Zymo Research Corp., E. coli DH5a transformants were isolated using the Orange, CA). The purified DNA from 20 subsamples PureLink Quick Plasmid Miniprep kit (Invitrogen Corp.). was then combined and used to generate amplicons for The 16S rRNA genes from the bacterial clones were 454-pyrosequencing and for construction of the clone sequenced using an Applied BioSystems model 3730xl library. automated DNA sequencing system (Foster City, CA).

454-Pyrosequence analysis Data analysis Detailed information for the 454-pyrosequencing has been Pyrosequencing described previously (Finkel et al. 2012). Bacterial 16S rRNA gene amplicons were generated using the universal For the determination of operational taxonomic units primers 27F (50-GAG TTT GAT CMT GGC TCA G-30) (OTUs), we defined species, genus, family, and phylum lev- and 800R (50-TAC CAG GGT ATC TAA TCC-30) flanking els at 3%, 5%, 10%, and 20% dissimilarity, respectively, fol- V1–V3 hypervariable regions in the small-subunit rRNA lowing the procedures used by Schloss and Handelsman gene. These amplicons were pyrosequenced using a Roche (2005). For taxonomy-based analysis, the SILVA database 454 GS-FLX pyrosequencer with Titanium reagents (Pruesse et al. 2007) was used with the unique sequences at (Macrogen, Seoul, Korea) according to the manufacturer’s an 80% confidence threshold cutoff. Rarefaction curves instructions. were analyzed using the R VEGAN package (Oksanen et al. 2011; Fig. 2), and richness estimates were analyzed using MOTHUR (Schloss et al. 2009). Short reads (26,382 reads) Clone library analysis less than 289 bp and reads with ambiguous nucleotides Detail information for the cloning and transformation has (17,730 reads) were removed from analysis. Long reads been previously published (Kim et al. 2012). The primers more than 724 bp were trimmed using CD-Hit-OTU (Li used to amplify the 16S rRNA genes for bacteria were 27F et al. 2012). Chimeric sequences (1091 reads) were and 1492R (Lane 1991). The amplification conditions for removed using MOTHUR. Reads from all datasets were the PCR were 95°C for 5 min, followed by 30 cycles of quality filtered using a Q20 quality cutoff.

97%

95% No of phylo types 90%

80% 0 2000 4000 6000 8000 10,000 12,000

0 20,000 40,000 60,000 80,000 100,000 120,000 No of 16S rRNA gene suquences

Figure 2. Rarefaction curves of 454-pyrosequences indicating the observed number of OTUs in the Gyorae Gotjawal forest soil.

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Clone library analysis 60 Pyrosequencing Putative chimeric sequences were identified using Bellero- 50 Cloning phon (Huber et al. 2004). The 16S rRNA gene sequences 40 were aligned using the Nearest Alignment Space Termina- tion (NAST) aligner (DeSantis et al. 2006a), and the 30 aligned sequences were compared to the Lane mask using 20 the Greengenes website (DeSantis et al. 2006b). The Sequence Match feature of RDPII (Cole et al. 2009) was 10 used to find GenBank sequences representing the most clo- 0 sely related type strain for each clone, which were then (%) included as references in the phylogeny. Using the Greeng- enes Automatic Taxonomic Classification algorithm (DeSantis et al. 2006a) and GenBank, a set of related sequences was interpreted as a novel genus (or species) if they were classified as the same genus (or species). Taxa Phylogenetic trees were constructed using neighbor- joining with MEGA version 5.0 for Windows (Tamura Figure 3. Phylogenetic distribution of operational taxonomic units (OTUs) and 454-pyrosequencing/clones observed in Gotjawal soil. et al. 2011). Evolutionary distances were calculated using the Kimura 2-parameter method (Kimura 1980). Boot- strap analyses of the neighbor-joining data were con- ria (3.5%), b-Proteobacteria (2.9%), Chloroflexi (2.4%), and ducted based on 1000 samples to assess the support for Bacilli (1.5%). In the clone library, the least abundant inferred phylogenetic relationships. DOTUR (Schloss and sequences were represented by b-Proteobacteria (2.8%), Handelsman 2005) was used to calculate taxon richness Chloroflexi (2.1%), Cyanobacteria (2.8%), Planctomycetes and diversity estimates. A distance matrix was obtained (1.4%), Bacilli (0.7%), and d-Proteobacteria (0.7%; Fig. 3). using the Calculate Distance Matrix algorithm from the At a cutoff of 3% dissimilarity (i.e., 97% sequence Greengenes website (DeSantis et al. 2006a,b). identity) among the pyrosequencing reads, 12,621 OTUs were obtained from the 85,324 unique sequences (Table 2). To estimate species richness, the ACE (abun- Nucleotide sequence accession numbers dance-based coverage), Boot, and Chao1 estimators were All pyrosequencing reads were deposited in the DDBJ used. At the OTU cutoff of 3%, 12,621 OTUs were Sequence Read Archive (SAR) under the study accession obtained from the 116,475 reads. The respective total number DRP002233. Bacterial clone sequences were numbers of species were estimated to be 23,489, 14,920, deposited in the DDBJ under the following accession and 19,871 (Table 2). At 3% dissimilarity, 73 OTUs were numbers: AB821051–AB821192. obtained from the 142 bacterial clone sequences. ACE, Boot, and Chao1 estimators for this dataset were 199, 92, Results and Discussion and 189, respectively (Table 2). To determine richness based on pyrosequencing data- sets and bacterial clone sequences, we identified 12,621, Analysis of bacterial sequences 7500, 3126, and 807 OTUs and 73, 60, 40, and 18 OTUs In this study, we examined a total of 116,475 reads and based on 3% (species level), 5% (genus level), 10% (fam- 142 clones representing 10 and 7 phyla, respectively, from ily level), and 20% (phylum level) dissimilarity, respec- Gotjawal forest soil. Figure 3 and Table 1 summarize the tively (Table 2). Bacterial community composition based phylogenetic distribution of the 454-pyrosequences and on 454-pyrosequences (3% dissimilarity) revealed that clone sequences of the 16S rRNA gene. The class a-Prote- 12,621 OTUs were represented in the soil (Fig. 2). The obacteria, and phyla Actinobacteria and Acidobacteria Shannon–Wiener (H) and reciprocal Simpson’s (1/D) dominated the bacterial community in the soil, represent- indices based on the clone library were 3.95 and 3.52 (H) ing 45.6%, 25.2%, and 10.9% of the 454-pyrosequences, and 48.1 and 20.8 (1/D) at 3% and 5% dissimilarity, respectively. The same phyla or classes dominated the while the H and 1/D indices of pyrosequences were 7.75, clone library, representing 51.2%, 21%, and 10% of 7.03 (H) and 526, 306 (1/D) at 3% and 5% dissimilarity, sequences, respectively. respectively. Chao1, based on the clone library and pyro- The least abundant sequences of 454-pyrosequencing sequences of the Gotjawal soil, was also higher than the were represented by c-Proteobacteria (4.1%), d-Proteobacte- samples (189, 130 and 19,871, 10,833) at the same

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Table 1. Relative abundances of the phylogenetic groups in Gotjawal soil.

Phylum/Class Order Family Genus

Proteobacteria 56.4 Alpha 45.7 Rhizobiales 30.2 Hyphomicrobiaceae 3.2 Pedomicrobium 1.7 Rhodomicrobium 0.3 Blastochloris 0.5 Rhodoplanes 0.3 Prosthecomicrobium 0.2 Rhodomicrobium 0.3 Hyphomicrobium 0.2 Xanthobacteraceae 16.3 Pseudolabrys 0.9 Labrys 0.4 Rhodobiaceae 1.0 Rhodobium 0.6 Bradyrhizobiaceae 4.8 Bradyrhizobium 4.2 Afipia 0.1 Rhizobiaceae 0.6 Rhizobium 0.6 Rhodocyclales 0.2 Rhodocyclaceae 0.2 Azospira 0.2 Sphingomonadales 0.1 Sphingomonadaceae 0.1 Sphingomonas 0.0 Caulobacterales 0.6 Caulobacteraceae 0.6 Phenylobacterium 0.1 Beta 2.9 Burkholderiales 1.8 Burkholderiaceae 1.2 Burkholderia 1.2 Comamonadaceae 0.4 Variovorax 0.2 Gamma 4.1 Pseudomonadales 0.1 Pseudomonadaceae 0.1 Pseudomonas 0.1 Xanthomonadales 3.4 Xanthomonadaceae 0.6 Dyella 0.2 Legionellales 0.4 Coxiellaceae 0.4 Aquicella 0.3 Delta 3.5 Myxococcales 1.8 Kofleriaceae Haliangium 0.7 Polyangiaceae 0.4 Sorangium 0.3 Acidobacteria 10.9 Acidobacteriales 4.0 Acidobacteriaceae 4.0 Candidatus Solibacter 2.6 Edaphobacter 0.6 Acidobacterium 0.4 Unclassified Bryobacter 0.8 Candidatus Koribacter 0.3 Actinobacteria 25.2 Actinomycetales 17.5 Acidothermaceae 11.5 Acidothermus 11.5 Mycobacteriaceae 2.0 Mycobacterium 2.0 Streptomycetaceae 1.0 Streptomyces 0.5 Streptacidiphilus 0.3 Kitasatospora 0.2 Micromonosporaceae 1.1 Micromonospora 0.3 Actinoallomurus 0.2 Luedemannella 0.3 Microbacteriaceae 0.3 Agromyces 0.0 Thermomonosporaceae 0.3 Actinomadura 0.1 Frankiaceae 0.3 Frankia 0.3 Pseudonocardiaceae 0.3 Pseudonocardia 0.2 Nocardiaceae 0.1 Marmoricola 0.0 Solirubrobacterales 2.5 Solirubrobacteraceae Solirubrobacter 0.0 Patulibacteraceae 0.1 Patulibacter 0.1 Bacilli 1.5 Bacillaceae 0.7 Bacillus 0.7 Paenibacillaceae 0.6 Paenibacillus 0.5 Gemmatimonadetes 0.6 Gemmatimonadaceae 0.6 Gemmatimonas 0.3 Chloroflexi 2.4 Ktedonobacteraceae Ktedonobacter 2.2 Clostridia 0.2 Thermoanaerobacteriales 0.1 Thermodesulfobiaceae 0.1 Coprothermobacter 0.1 Nitrospirae 0.2 Nitrospirales 0.2 Nitrospiraceae 0.2 Nitrospira 0.2 Unknown 0.4 15.6 37.6 57.5 Bacteroidetes 0.9 Sphingobacteriales 0.8 Chitinophaga 0.0 Planctomycetes 0.2 Planctomycetales 0.1 Planctomyces 0.0

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Table 2. Estimates of taxon richness and diversity indices for 16S rRNA gene-454-pyrosequencing and the clone library based on various evolu- tionary distance criteria for demarcating operational taxonomic units.

Richness Diversity index

Evolutionary distance No. of OTUs ACE Boot Chao1 Shannon Simpson

Pyro seqs 0.03 12,621 23,489 14,920 19,871 7.75 526 0.05 7500 10,718 8687 10,833 7.03 307 0.1 3126 4060 3535 4096 5.77 79 0.2 807 1016 899 1024 4.02 14 Clones 0.03 73 199 92 189 3.95 48 0.05 60 165 75 130 3.52 21 0.1 40 80 49 69 2.72 7 0.2 18 29 21 28 1.64 3

OTU, operational taxonomic unit; ACE, abundance-based coverage estimator. dissimilarity. The diversity indices and richness of the The results of clone library analysis showed that a-Pro- Gotjawal soil bacteria were higher than Hawaiian volcanic teobacteria represented a high percentage of the total bac- deposits (Gomez-Alvarez et al. 2007) (Table 2). teria in the Gotjawal soil, which may be a key factor in Moreover, based on the comparison of the obtained the maintenance of the forest ecosystem (Table 1). Differ- 16S rRNA gene sequences to their closest known relatives, ent results were obtained with a forest soil, where a-Prote- we discovered several new taxa at the species and genus obacteria made up only approximately 11.4% of the total levels. Figure 4A shows the discovery of two novel genera bacteria (Nacke et al. 2011). Recently, our research group within d-Proteobacteria and 10 novel genera within a-Pro- reported a new genus, Variibacter gotjawalensis gen. nov., teobacteria. Unexpectedly, in 454-pyrosequencing, sp. nov., isolated from the soil of Aewol Gotjawal forest, b- (2.9% of OTUs), c- (4.1% of OTUs), and d-Proteobac- one of four Gotjawal sites on Jeju Island (Kim et al. teria (3.5% of OTUs) accounted for only low percentages 2014b); this isolate belongs to a-Proteobacteria and shows of the total OTUs. Other orders were affiliated with high similarity with Bradyrhizobium oligotrophicum LMG Caulobacterales (0.6% of OTUs), Rhodocyclales (0.2%), 10732 (93.6%). and Sphingomonadales (0.1%; Table 1). Acidobacteria The order Rhizobiales of a-Proteobacteria Among the Acidobacteria identified by 454-pyrosequencing, Among the a-Proteobacteria (45.7% of OTUs) represented Acidobacteriales (4.0% of OTUs), Acidobacterium (0.4% of in the 454-sequence dataset, the order Rhizobiales (30.2% OTUs), Edaphobacter (0.6% of OTUs), and Candidatus of OTUs) was represented by the families Hyphomicrobia- Koribacter (0.3% of OTUs) were affiliated with subdivision ceae, Xanthobacteraceae, Rhodobiaceae, Bradyrhizobiaceae, 1. Bryobacter (0.8% of OTUs) and Candidatus Solibacter and Rhizobiaceae, with Xanthobacteriaceae as the core (2.6% of OTUs) were affiliated with subdivision 3. group (16.3%; Fig. 4A). Overall, however, the phylotypes In a clone library analysis, the phylum Acidobacteria identified at the genus level were unknown. Within the has been shown to occur in diverse environments as a family Bradyrhizobiaceae (4.8% of OTUs), the genus dominant (up to 73%) bacterial group (Chan et al. 2006; Bradyrhizobium accounted for 4.2% of the total OTUs, Janssen 2006). However, in forest soil, Acidobacteria rep- suggesting that this genus, and nitrogen fixers in general, resent only 20.4% of total bacteria (35). The branched may be major contributors to the Gotjawal nitrogen cycle. patterns of subdivisions 1, 2, and 3 were similar. From However, further careful studies are required to support the 10 clones of the phylum Acidobacteria, five clades rep- this hypothesis. resented previously discovered subdivisions that were

Figure 4. Phylogenetic tree of 142 bacterial clone sequences. (A) Proteobacteria from Gotjawal soils (80 clone sequences), with reference sequences. Twelve previously unknown clades are indicated as novel genera within the phylum. (B) Acidobacteria (13 clone sequences), with reference sequences. (C) Actinobacteria (28 clone sequences). (D) Chloroflexi, Bacilli, Cyanobacteria, and Planctomycetes (10 clone sequences). Bold letters refer to clone sequences. Each clade that appears to be a previously discovered taxon, based on the 16S rRNA divergence from the nearest previously known relative, is indicated.

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(A) 88 GJ16S1 C09 100 GJ16S1 B09 GJ16S1 A07 GJ16S2 H07 GJ16S1 E05 GJ16S1 B11 GJ16s4 B02 GJ16S2 G10 GJ16S2 D11 GJ16S2 D02 GJ16S2 C04 New Genus (19) GJ16S2 C08 GJ16s4 H02 84 GJ16S1 B02 GJ16S1 H10 GJ16S2 G01 99 GJ16S1 G10 100 GJ16S1 A06 99GJ16s3 F01 75 GJ16s3 A10 GJ16S2 D04 Xanthobacteraceae (39) Pseudolabrys taiwanensis (DQ062742) GJ16s3 D11 GJ16S2 D10 82 GJ16S2 G08 GJ16S1 F03 100 GJ16S2 G09 80 GJ16S2 F02 GJ16s3 D04 GJ16S2 F10 GJ16S2 B11 GJ16S1 B03 GJ16S1 F12 GJ16S1 A11 New Genus (14) GJ16S2 C06 Rhizobiales (57) GJ16S1 E08 GJ16S1 H04 99 GJ16S2 A09 GJ16s3 B09 GJ16s3 F07 GJ16S2 A10 GJ16S1 A05 New Genus (3) 93 GJ16S1 G12 Afipia broomeae F186 (U87759) 82 Rhodopseudomonas boonkerdii NBRC106595 (AB682462) Agromonas oligotrophica (D78366) 99 GJ16S1 G05 Bradyrhizobiaceae (7) Bradyrhizobium elkanii (EU481825) Nitrobacter winogradskyi Nb-255 (CP000115) GJ16S1 D08 GJ16S2 H09 85 GJ16S1 D01 99Methylosinus sporium (Y18946) GJ16s3 G05 Beijerinckia indica (AJ563931) Beijerinckiaceae (1) GJ16S1 E06 GJ16S2 E12 97 GJ16S2 C12 New Genus (4) 100 GJ16S1 E09 99Prosthecomicrobium enhydrum NBRC102409 (AB681765) Hyphomicrobium aestuarii DSM1564 (Y14304) 77 GJ16S2 E10 Pedomicrobium australicum (X97693) 100 GJ16S2 D05 Hyphomicrobiaceae (4) 99 GJ16S1 B07 100 GJ16S2 D08 100GJ16s3 B04 GJ16S2 H08 New Genus (2) GJ16S1 G03 GJ16S2 E06 100 Phenylobacterium lituiforme (AY534887) Caulobacteraceae (2) Caulobacterales (2) 98 100 GJ16S1 D03 GJ16S1 H11 New Genus (2) 99 GJ16S1 D10 100 GJ16S1 C12 Reyranella massiliensis 3B26 (HM048834) GJ16S1 D12 100 GJ16S1 E07 New Genus (1) Rhodopila globiformis (D86513) Acetobacteraceae (1) 86 89 Azospirillum rugosum (AM419042) Skermanella xinjiangensis (EU586202) Rhodospirillaceae GJ16S1 F01 Rhodospirillales (14) GJ16s3 B01 New Genus (2) Stella vacuolata (AJ535711) GJ16s3 G09 GJ16S1 F06 94 GJ16s3 D12 98 GJ16S2 C09 New Genus (6) 84 GJ16S1 E10 91 GJ16S1 E02 100 GJ16S1 F08 New Genus (1) 100 GJ16s3 B02 84 New Genus (1) 89 Sorangium cellulosum (EU240497) Polyangiaceae (1) Myxococcales (1) Cystobacter armeniaca DSM14710 (NR043939) Geobacter grbiciae (AF335183) Desulfohalobium retbaense (U48244) GJ16S2 C05 New Genus (1) Bdellovibrio bacteriovorus DSM50701 (AJ292759) Methylonatrum kenyense (DQ789390) 100 GJ16S2 E11 GJ16S1 H08 96 Derxia gummosa (AB089482) Alcaligenaceae (2) GJ16S1 G01 Burkholderiales (4) 100 Ramlibacter tataouinensis (AF144383) 100 GJ16S2 A07 Comamonadaceae (2) 89 Variovorax soli (DQ432053) Bacillus sonorensis NRRLB-23154 (AF302118) Pyrococcus horikoshii DSM12428 (AY099169)

0.05

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(B) Subdivision

100 GJ16S2 D09 100 GJ16S2 A04 GJ16S2 G04 76 Ellin7137 (AY673303) 1 Acidobacterium capsulatum ATCC51196 (3) 100 98 Ellin337 (AF498719) 100 Edaphobacter modestus Jbg-1 Ellin345 (AF498727) GJ16s3 H03 87 GJ16S1 B04 100 GJ16S1 E03 2 (5) 100 soilclone DA052 (Y07646) GJ16S2 E05 97 94 GJ16S1 A01 soilclone C002 (AF013515) GJ16S1 A03 100 GJ16s3 D09 99 3 agricultural soil clone SC-I-40 (AJ252634) (3) GJ16S2 H03 95 91 Ellin6076 (AY234728) soilclone S111 (AF013560) 99 soilclone 11-25 (Z95709) soilclone 11-14 (Z95707) sponge symbiont PAUC37f (AF186413) 78 GJ16S2 E07 100 11 (1) 91 Uncultured bacterium isolate ALT39 (AY703463) Geothrix fermentans (U41563) 100 H. foetida DSM6591-T (X77215) soilclone DA008 (Y12597) seaclone KM3-25-B4 (EF597701) 100 GJ16s3 E10 6 (1) 81 98 soilclone kb 2426 (Z95732) soilclone 32-20 (Z95713) 83 isolate ALT6 (AY703471) clone BPC015 (AF154085) 100 isolate ALT5 (AY703470) Pyrococcus horikoshii DSM12428 (AY099169)

0.05

Figure 4. Continued most closely related to subdivisions 1, 2, 3, 6, and 11 Functional bacterial genera (Fig. 4B). As Roesch et al. (2007) and Uroz et al. (2010) have sug- Actinobacteria gested, and as is shown in Table 3, the nitrogen-fixing bacteria primarily detected by 16S rRNA gene pyrose- Among the Actinobacteria identified by 454-pyrosequenc- quencing belonged to Bradyrhizobium (4.2%) and Rhizo- ing, Actinomycetales (17.5% of OTUs) and Solirubrobacte- bium (0.6%). A large number of sequences from the rales (2.5%) were the most abundant families, while cellulolytic bacterial genus Acidothermus (Barabote et al. Acidothermus was the largest genus at 11.5% of OTUs. 2009) were also detected in the forest soil (Table 1). In Mycobacterium (2.0%) and Micromonospora (0.3%) were addition, few anaerobic AOB (0.002%) and methane-oxi- also readily detected. dizing bacteria (0.002–0.04%, Methylocella genus) were Among the cloned sequences, Actinobacteria were dom- detected. However, 16S rRNA gene taxonomies were only inant (21%). A phylogenetic analysis of these sequences loosely correlated with function. revealed that the Gotjawal soil contained a variety of pre- This study has some limitations. First, the dataset was viously undiscovered genera and species of Actinobacteria small, and no replications were performed. This makes (Fig. 4C). We discovered six new clades at the genus level both the analysis and comparison with other studies diffi- and one new clade at the family level. cult. Additionally, many of the identified sequences were Other phyla observed in the pyrosequencing dataset not affiliated with known taxa. While this is a common included Ktedonobacter (a genus in Chloroflexi,2.2%)and occurrence, further studies are necessary to determine the Gemmatimonas (a genus in Planctomyces,0.3%;Table1). importance of these results with regard to the ecology and Cyanobacteria (four clones) and Chloroflexi (two clones) were characteristics of the Gotjawal forest. Analysis of the extent observed at low abundances in the clone library (Fig. 4D). to which sequences from Gotjawal are completely novel,

308 ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. J.-S. Kim et al. Pyrosequencing of Lava-Formed Forest Soil

Family Order/Subclass (C) GJ16S2 B04 GJBac3 H01 GJ16s3 C03 GJ16S2 E01 GJ16S1 D09 78 94 GJ16S1 A04 GJ16S1 C07 GJ16S2 C10 New Genus 96 78 GJ16s3 E12 (14) Thermomonosporaceae GJ16s3 E06 (14) GJ16S1 C06 GJ16S2 C01 90 96 GJ16S2 A06 Actinomycetales (18) GJ16S1 B12 Actinoallomurus purpureus (AB364588) Actinocorallia longicatena (AJ293707) 88 Actinomadura atramentaria (AJ420138) Luedemannella flava (NR041339) Asanoa ishikariensis (NR025429) 100 100 GJ16s3 H05 96 Micromonosporaceae 100 GJ16s3 C08 (2) Streptomyces flavoviridis (GQ985452) GJ16s4 F01 84 90 New Genus (2) Streptomycetaceae (2) 100 GJ16s3 E04

100 GJ16s3 C10 New Genus (2) GJ16S1 A12 Iamiaceae (2) Iamia majanohamensis (AB360448) 100 Acidimicrobium ferrooxidans (AFU75647) Acidimicrobiales (3) GJ16S2 E03 92 Acidimicrobiaceae (1) Aciditerrimonas ferrireducens (AB517669) GJ16S1 D07 New Genus Rubrobacter radiotolerans (X98372)

100 GJ16S2 F03 GJ16S2 B07 New Genus (2) Rubrobacteridae (4) 100 GJ16S1 C10 GJ16S2 C07 100 New Genus (2) 98 GJ16S1 F09 GJ16s3 B05 New Family Pyrococcus horikoshii (AY099169)

0.05

GJ16S1 A09 (D) 100 New Genus (2) 100 GJ16S2 F09 chloroflexi clone Tik 06(AM749753) Chloroflexi (3) GJ16S1 H01 New Genus (1) 100 Ktedonobacter racemifer DSM44963(NR042472) Bacillus sonorensis NRRLB-23154(AF302118) GJ16S2 G11 100 Bacilli (1) 100 Paenibacillus pectinilyticus (EU391157) GJ16s3 D03 New Genus (2) GJ16s3 B06 89 GJ16s3-D08 100 New Genus (1) Cyanobacteria (4) 86 cyanobacterium clone SBNB5C11(FJ228289) GJ16s3 H04

99 Planktothricoides raciborskii INBaOR(AB045953)

100 GJ16S1 H12 New Genus (1) planctomycetaceae clone117(JF500994) Planctomycetes (2) 100 GJ16S2 G03 New Genus (1) 98 Pirellula staleyi DSM 6068(AJ231183) Pyrococcus horikoshii DSM12428(AY099169)

0.05

Figure 4. Continued

ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 309 Pyrosequencing of Lava-Formed Forest Soil J.-S. Kim et al.

Table 3. Number and percent of sequences classified to known func- tional bacterial genera based on the data derived from 454-pyrose- Conflict of Interest quences. The authors declare no conflict of interest. Count Percent

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