Sponge-Specific Unknown Bacterial Groups Detected in Marine

Home , Cliona, Clionaidae, Cliona celata

J. Microbiol. Biotechnol. (2015), 25(1), 1–10 http://dx.doi.org/10.4014/jmb.1406.06041 Research Article Review jmb

Sponge-Specific Unknown Bacterial Groups Detected in Marine Sponges Collected from Korea Through Barcoded Pyrosequencing S Jong-Bin Jeong1†, Kyoung-Ho Kim2†, and Jin-Sook Park1*

1Department of Biological Science and Biotechnology, Hannam University, Daejeon 305-811, Republic of Korea 2Department of Microbiology, Pukyong National University, Busan 608-737, Republic of Korea

Received: June 13, 2014 Revised: September 29, 2014 The bacterial diversity of 10 marine sponges belonging to the species Cliona celata, an Accepted: October 13, 2014 unidentified Cliona species, Haliclona cinerea, Halichondria okadai, Hymeniacidon sinapium, Lissodendoryx isodictyalis, Penares incrustans, Spirastrella abata, and Spirastrella panis collected from Jeju Island and Chuja Island was investigated using amplicon pyrosequencing of the 16S

First published online rRNA genes. The microbial diversity of these sponges has as of yet rarely or never been October 15, 2014 investigated. All sponges, except Cliona celata, Lissodendoryx isodictyalis, and Penares incrustans,

*Corresponding author showed simple bacterial diversity, in which one or two bacterial OTUs occupied more than Phone: +82-42-629-8771; 50% of the pyrosequencing reads and their OTU rank abundance curves saturated quickly. Fax: +82-42-629-8769; Most of the predominant OTUs belonged to Alpha-, Beta-, or Gammaproteobacteria. Some of the E-mail: [email protected] OTUs from the sponges with low diversity were distantly (88%~89%) or moderately † These authors contributed (93%~97%) related to known sequences in the GenBank nucleotide database. Phylogenetic equally to this work. analysis showed that many of the representative sequences of the OTUs were related to the S upplementary data for this sequences originating from sponges and corals, and formed sponge-specific or -related clades. paper are available on-line only at The marine sponges investigated herein harbored unexplored bacterial diversity, and further http://jmb.or.kr. studies should be done to understand the microbes present in sponges. pISSN 1017-7825, eISSN 1738-8872

Introduction reveal so-called unidentified majorities [1] and unknown bacterial diversity in sponges [13, 36]. The massive parallel Sponges are well known as reservoirs of microbes; they 454 pyrosequencing, a next-generation sequencing method, are inhabited by high numbers of bacteria, which were has been applied to investigate the bacterial diversity in estimated to be up to 109 cells per milliliter of sponge tissue various environments [7, 30, 34] as well as in marine [14], accounting for as much as 50% of the biomass of the sponges [17, 18, 24, 33, 34, 45, 46]. sponges [31]. The microbes in sponges have various roles, Marine sponges harbor various kinds of bacteria, including as food sources [29], pathogenic agents [2], comprising a total of 25 bacterial phyla [34]. The most symbiotic residents [49], sponge skeleton stabilizers, diverse phyla contained in sponges are Proteobacteria, metabolic waste consumers, secondary metabolite producers Chloroflexi, and Poribacteria, but many less diverse phyla such [11], and nutrient cycle mediators [40]. as Acidobacteria, Actinobacteria, Cyanobacteria, Gemmatimonadetes, Numerous studies have been performed to reveal the Bacteroidetes, Spirochaetes, Firmicutes, Nitrospirae, TM7, diversity in sponges. Culture-dependent studies based on SBR1093, and OS-K have also been observed [34]. the isolation of pure cultures revealed various kinds of However, some sponges, known as low microbial bacteria, [44, 48] and novel strains were isolated and abundance (LMA) sponges, have low microbial content of proposed as new taxa [28, 51]. Many studies based on only about 105-106 cells per gram or milliliter [12]. LMA culture-independent methods have also been performed to sponges also have lower taxonomic diversity (less number

January 2015 ⎪ Vol. 25⎪ No. 1 2Jeong et al.

of taxa) than high microbial abundance (HMA) sponges, command of the Mothur package [32]. The QIIME package ver. and were reported to contain only up to five phyla in a 1.7.0 was used for to determine the OTUs and for their taxonomic study of five specimens of three different LMA sponges, assignment [22]. Sequence names were marked in accordance Callyspongia vaginalis, Niphates digitalis, and Raspailia topsenti with the samples and merged into one file. The uclust approach in [10]. the QIIME package was used for clustering the OTUs and the determination of representative sequences. A sequence similarity In the present study, the bacterial diversity in 10 sponges of 97% was used as the criterion for taxonomic assignments. The collected from Jeju Island and Chuja Island, Korea, was representative sequences of OTUs were compared with the investigated using pyrosequencing to reveal the unidentified 97_otus and the 97_otu_taxonomy files of the gg_13_05 version of bacterial diversity of sponges, including Cliona celata, the Greengenes database [27] and taxonomy was assigned using Haliclona cinerea, Lissodendoryx isodictyalis, Spirastrella abata, the RDP classifier [23]. Sequences related to chloroplasts and Spirastrella panis, and Penares incrustans, which have rarely mitochondria were discarded from further analyses. Diversity been explored to date. Most of the sponges, except Cliona estimators, including Chao1, Shannon, and Simpson indices, were celata, Lissodendoryx isodictyalis, and Penares incrustans, were determined for subsamples made with sequences of the same found to contain very simple diversity comparable to that numbers. Unifrac analysis [26] was performed to compare the of LMA sponges, and some of the predominant OTUs were samples. For the Unifrac analysis, similarities of 97%, 94%, 91%, found to be unrelated to known sequences. 88%, and 85% were used as criteria for OTU clustering. The PyNAST program [3] was used for the alignment of representative Materials and Methods sequences obtained from the clustering, after which the FastTree program [25] was used for construction of a phylogenetic tree using the aligned sequences. UPGMA trees were made from the Sample Collection and DNA Extraction Unifrac analysis. Some representative sequences were compared Sponge specimens were collected from Mueung-ri, Daejeong- against the nucleotide database of GenBank and related sequences eup, Seogwipo City, Jeju Province (Jeju Island), Korea (n =6) in were used for the construction of a phylogenetic tree. A neighbor- Feb. 2011 by scuba diving, and from around Chuja Island in the joining tree was constructed with the MEGA5 program [39], in South Sea of Korea (n = 4) in Nov. 2009 by scuba diving or from which the Kimura 2-parameter method [21] was used to calculate the intertidal region. Samples were collected aseptically and a distance matrix. delivered to the laboratory. Small pieces (about 1 cm3 in volume) of sponge tissues were washed with sterilized seawater, frozen at Results and Discussion -70oC for 24 h, and then lysophilized at -50oC, 0.033 Mbar for 24 h. The freeze-dried tissues were aseptically homogenized in a mortar. The G-spin genomic DNA extraction kit (Intron, South Sponges and Their Bacterial Communities Korea) was used for DNA extraction. All 10 specimens were so-called siliceous sponges (the class Demospongiae), which have spicules made out of silicon Barcoded Pyrosequencing dioxide [41]. Cliona celata is an ecologically/biotechnologically The V1 to V3 region of the 16S rRNA genes were amplified important species that is well known as an excavating and using primer sets (V1-9F: 5’-X-AC-GAGTTTGATCMTGGCTCAG-3’ cosmopolitan sponge [50]. The sterol composition and and V3-541R: 5’-X-AC-WTTACCGCGGCTGCTGG-3’; X indicates novel fatty acids were discovered from Haliclona cinerea the barcode sequences that were composed of various combinations sponges from the Black Sea [8, 19]. Lissodendoryx isodictyalis of six nucleotides to tag different samples). The Genome is an encrusting sponge with blue-gray color and a strong Sequencer FLX titanium (Roche, Germany) system was used for odor [35]. Lipids with cytotoxicity and other bioactivities pyrosequencing, according to the method in the manufacturer’s were isolated from Spirastrella abata sponges from Korea manuals (Macrogen, Korea). [15]. The diversity of cultured and uncultured bacteria of Bioinformatic Analysis of Reads Spirastrella abata and Spirastrella panis have been investigated After pyrosequencing, reads were sorted according to tags, and with restriction fragment length polymorphism and then the primer and tag sequences were cut out. Sequences less denaturing gradient gel electrophoresis (DGGE) [4, 16]. than 300 bp in size and with any ambiguous bases, “N”, were Bioactive triterpenoids and penaramides were discovered discarded from further analyses. Sequences with reverse from Penares incrustans sponges [37, 42]. However, the complement orientation were adjusted into the same orientation. latter five species have not yet been studied extensively. Sequences were aligned and the front and rear parts of sequences As far as we know, bacterial diversity (especially uncultured) that did not match with the other sequences were cut. Putative of sponges such as Cliona celata, Haliclona cinerea, Lissodendoryx chimera sequences were detected with the chimer.uchime [6] isodictyalis, Spirastrella abata, Spirastrella panis, and Penares

J. Microbiol. Biotechnol. Sponge-Specific Unknown Bacterial Groups in Marine Sponges from Korea 3

incrustans have not yet been or have rarely been published as of yet. Nucleotide records of the GenBank database were surveyed using three or four keywords: (16S) AND (rRNA) AND (“genus name”), or (16S) AND (rRNA) AND (“genus name”) AND (“species name”) of each sponge. The numbers of records that came up for each sponge species or genus name were as follows: Cliona celata, 0; Cliona, 6; Haliclona cinerea, 0; Haliclona, 1839; Halichondria okadai, 122; Halichondria, 608; Hymeniacidon sinapium, 131; Hymeniacidon, 1,245; Lissodendoryx isodictyalis, 0; Lissodendoryx, 1,598; Penares incrustans, 3; Penares, 6; Spirastrella abata, 0; Spirastrella panis, Fig. 1. Weighted UPGMA tree from Unifrac analysis showing 1; Spirastrella, 5. When species names were used, six sponge relationships between samples according to bacterial profile. species among eight showed no or no more than three OTUs were determined based on 97% similarity. Numbers at the records. Even when genus names were used, three sponge branched points indicate percentages of frequency of each cluster genera including Cliona, Penares, and Spirastrella showed no detected in the five UPGMA trees, which were constructed based on more than six records. This shows that the bacterial different 16S rRNA gene similarities (97%, 94%, 91%, 88%, and 85%). diversity of most of the sponges in this study was rarely The scale bar represents Unifrac distance. investigated. Therefore, this study may provide the first observation of the comprehensive bacterial diversity of the highly different from each other. A previous study with 32 sponges examined. sponges showed the bacterial patterns to have no correlation with sponge phylogeny. Sponges from different genera Unifrac Analysis of Sponges According to Bacterial Diversity were grouped closer than those from the same genus based A total of 28,913 sequences were assigned taxonomically on their bacterial compositions [34]. However, Sppa2S, after non-bacterial reads including chloroplasts and Spab1S, and Spab1M belonged to the same genus and mitochondria-related sequences were discarded. UPGMA showed a closely related relationship in a clade. Although trees from Unifrac analyses showed somewhat different Spab1S and Spab1M were collected from different sample tree topologies in accordance with 16S rRNA gene similarities sites, their bacterial profiles were very similar, which (91%~85%). One group composed of Sppa2S, Spab1S, and implies that taxon-specific bacterial communities exist in Spab1M showed the same topology in all trees, and one some sponges. specimen, Pein1M, was always distinguished from the others (Fig. 1). The relationships among other specimens Diversity Estimation of Sponges was not robust in the tree topologies that were made based The sponges used in this study generally showed low on different criteria of 16S rRNA gene similarities, because and moderate diversity, except for the specimen Pein1M. the bacterial communities from each of the sponges were Several sponges showed extremely low diversity, which

Table 1. Description of sponge specimens. No. Sample ID Sponge species Taxonomy (Class; Order; Family) Sitea Longitude, Latitude Depth (m) 1 Clce1S Cliona celata Demospongiae; Hadromerida; Clionaidae C 126.2488228E, 33.9876219N 20 2 Clsp1S Cliona sp. Demospongiae; Hadromerida; Clionaidae C 126.3303845E, 33.9522348N 0 3 Haci1M Haliclona cinerea Demospongiae; Haplosclerida; Chalinidae M 126.1957111E, 33.2510333N 20 4 Haok1M Halichondria okadai Demospongiae; Halichondrida; Halichondriidae M 126.1957111E, 33.2510333N 20 5 Hysi1M Hymeniacidon sinapium Demospongiae; Halichondrida; Halichondriidae M 126.1957111E, 33.2510333N 20 6 Liis2M Lissodendoryx isodictyalis Demospongiae; Poecilosclerida; Coelosphaeridae M 126.1957111E, 33.2510333N 20 7 Pein1M Penares incrustans Demospongiae; Astrophorida; Geodiidae M 126.1957111E, 33.2510333N 20 8 Spab1M Spirastrella abata Demospongiae; Hadromerida; Spirastrellidae M 126.1957111E, 33.2510333N 20 9 Spab1S Spirastrella abata Demospongiae; Hadromerida; Spirastrellidae C 126.3132397E, 33.8670804N 14-17 10 Sppa2S Spirastrella panis Demospongiae; Hadromerida; Spirastrellidae C 126.3303845E, 33.9522348N 0 aC, Chuja Island; M, Mureung-ri.

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Table 2. Diversity estimation results from pyrosequencing reads. Sample ID Total reads Total OTUs No. reads subsampled a OTUs Chao1 Shannon Simpson Clce1S 3,952 214 730 85 234 3.32 0.701 Clsp1S 5,378 258 730 72 214 1.98 0.405 Haci1M 3,300 57 730 31 58 0.81 0.164 Haok1M 266 7 - - - - - Hysi1M 1,290 48 730 38 49 2.66 0.688 Liis2M 739 45 730 44 60 3.65 0.866 Pein1M 1,868 285 730 187 310 6.35 0.971 Spab1M 1,868 30 730 23 33 1.16 0.270 Spab1S 4,833 77 730 28 59 1.43 0.350 Sppa2S 4,935 165 730 59 117 2.31 0.491 aReads were subsampled from each sample and used to calculate diversity estimators. Data from Haok1M were not used for diversity index calculation because the number of reads was too small. OTUs were determined based on 16S rRNA gene similarity of 97%. could be expressed by Shannon indexes below 3.00 and genus Spirastrella was reported in a previous study [16], even down to 0.81 (Table 2). The simplest bacterial where only five bands were observed in the DDGE patterns. community was that of the specimen Haok1M, identified The specimen Clsp1S showed much lower diversity than as Halichondria okadai, which contained only seven OTUs. Clce1S, another specimen of the same genus, Cliona. Rank- In a previous study, specimens of the sponge Halichondria accumulated relative abundance curves showed distinguished panacea in the same genus also showed simpler diversity patterns among the sponge specimens (Fig. 2). The sponges than seawater, with some specimens showing less than five with low diversity had single predominant OTUs with bands in the DGGE patterns [47]. Another sponge showing high proportions from 50.8% to 92.6%. Some of them had extremely low diversity was Haci1M, a specimen of the only a few or dozens of OTUs, but few OTUs showed up in sponge Haliclona cinerea. Two species in the same genus, high proportions. The sponges with relatively high diversity H. heliophila and H. tubifera, were also reported to have low such as Liis2M, Clce1S, and Pein1M showed different curve diversity and species-specific bacterial communities [9]. shapes. Pein1M showed evenly distributed abundances Spab1M and Spab1S also had low levels of diversity with a among the OTUs. Clce1S also showed a similar moderate single highly abundant OTU. Low diversity in the sponge slope, except for the OTU ranking first. Liis2M showed evenly distributed abundances among OTUs with several highly abundant OTUs, but the curve saturated quickly. The bacterial diversities of marine sponges examined in previous studies were much higher than those of the present study [17, 18], and many more bacterial phyla were detected. In the present study, however, several sponges had very low diversity and were composed of very few OTUs, with one or two OTUs sometimes occupying almost all reads. This might be derived from several factors, such as different sampling sites, experimental procedure, number of reads, and sponge condition. There are reports of low diversity in sponges in which very few OTUs were found in many sponges [34], so it may not seem to be a rare phenomenon. Fig. 2. Rank-cumulative relative abundance curve in which relative abundances of OTUs were accumulated sequentially in order of abundance from the largest. Bacterial Diversity at Phylum and Class Levels The first 19 OTUs are shown. OTUs were determined based on 97% Pein1M was clearly distinguished from the other sponges similarity. at the phylum level. It had a bacterial community spanning

J. Microbiol. Biotechnol. Sponge-Specific Unknown Bacterial Groups in Marine Sponges from Korea 5

Fig. 3. Taxonomic assignment results at the class level. Only phyla with an abundance of more than 1% in at least one sample are shown. “Unclassified” means that the sequence was related to reads of which the position has not been verified taxonomically in that level. Square brackets indicate that the name was not verified taxonomically. to 14 phyla, in which the bacteria were evenly distributed were related to other marine invertebrates such as coral (9 phyla were more than 1% in proportion). Clce1S also and octacoral, and to marine environments such as seawater, contained many phyla (16 phyla), but only five of the phyla marine sediments, and microbialites. Most of the sequences were more than 1%, and Proteobacteria occupied up to were taxonomically unidentified from genus even to class 83.0%. A much higher proportion of Proteobacteria was level based on QIIME analysis. observed in all specimens except Pein1M (12.5%), ranging For example, the sequence of denovo103, which was from 70.7% to 100.0%. These high proportions were highly predominant in the clade Spirastrella, was related to unusual, but not impossible according to previous studies. a clone (Accession No. JQ515708) reported from the coral A high proportion of Proteobacteria (up to 72.6%) was also Montastraea faveolata [20]. Denovo103 was distantly related observed in a previous study [18]. In the low microbial (93% similarity) to the known sequence JQ515708 and abundance sponges Crella cyathophora, Stylissa carteri, and was taxonomically identified only at the class level as Niphates digitalis, 78%, 87%, and 93% abundances of Alphaproteobacteria. The representative sequence of denovo694, Proteobacteria, respectively, were roughly calculated from the most abundance OTU from Haok1M, showed 100% data on the investigation of bacterial communities [10]. similarity with a sequence (AB054177) obtained from the Diversity at the class level of bacteria detected herein is same sponge species Halichondria okadai from an unpublished shown in Fig. 3. Reads related to Alphaproteobacteria were study. Interestingly, the second most abundant OTU from predominant in Spab1S, Spab1M, Sppa2S, Clsp1S, and Haok1M (denovo197) also showed similarity with a sequence Haok1M. In contrast, Gammaproteobacteria was the major (AB054180, 98%) from the same unpublished study. The class in Hysi1M and Liis2M, and they also contained a two sequences, denovo694 and denovo197, showed high significant amount of Alphaproteobacteria. Clce1S and Haci1M similarity with clones in public databases, but they were contained Betaproteobacteria as the major class, and Clce1S very different from strains that have been cultivated also contained Gammaproteobacteria as a significant member. and identified taxonomically (in Alphaproteobacteria and Gammaproteobacteria with 90% and 86% similarity, respectively). BLASTN and Phylogenetic Analysis of Predominant OTUs Denovo624, the representative sequence of the most Some representative sequences of highly abundant OTUs abundant OTU of the sponge HaciM, showed the best hit were compared against the nucleotide database in GenBank with a sequence (JF824778, 98% similarity) obtained from (Table 3). The criterion of 94% similarity was used for the same sponge genus, Halichondria tubifera, and belonged OTU determination to reduce the sequence number for to the Betaproteobacteria. Another sequence (KC492704) comparison. Above all, representative sequences were belonging to Betaproteobacteria was reported to be important related to sequences mainly from sponges. Some sequences in a different sponge, Crambe crambe, although the similarity

January 2015 ⎪ Vol. 25⎪ No. 1 6Jeong et al.

sp.

okadai faveolata faveolata

nolitangere

stellata

Host tubifera cauliformis

Montastraea Halichondria Haliclona Erythropodium caribaeorum Hymeniacidon heliophila Lissodendoryx Hymeniacidon heliophila Montastraea faveolata Aplysina Siderastrea Neofibularia Montastraea sediment r Coastal seawater Site Best hit from BLASTN BLASTN from hit Best Similarity Accn no. EU315451 99% Sponge JF824773 99% Sponge FJ215374 98% Sponge JQ515708 93%AB054177 100% Coral Sponge EU802464 90%JF835683 Seawaterseawater Deep 97% Coral HM134528 99% Seawate JF824778 98% Sponge DQ889898 88% Octocoral KF688134 99% Soil Soil FJ203517 96%KF286090 Coral 100% Sponge FJ358860 98% Sediment sandy reef Marine ;

a PAUC34f JQ516302 99% Coral

HTCC2188

assignment EC94 EC94

unclassified genus unclassified unclassified family unclassified family unclassified unclassified family unclassified ;

Gammaproteobacteria Psychrobacter ; ; unclassified family unclassified unclassified class SAR202 class unclassified EU816842 99% Sponge from QIIME analysis Taxonomic Proteobacteria ;Alphaproteobacteria; order unclassified Proteobacteria; Alphaproteobacteria; order unclassified Proteobacteria; Betaproteobacteria; Proteobacteria; Alphaproteobacteria; Kiloniellales; Proteobacteria; Betaproteobacteria; order unclassified Proteobacteria; Gammaproteobacteria; Oceanospirillales; Actinobacteria; Acidimicrobiia; Acidimicrobiales; Proteobacteria; Gammaproteobacteria; Oceanospirillales; Proteobacteria; Gammaproteobacteria; Alteromonadales; [Rhodothermi]; Bacteroidetes; [Rhodothermales]; Rhodothermaceae; Proteobacteria; Gammaproteobacteria; order unclassified Proteobacteria; Gammaproteobacteria; order unclassified Proteobacteria Pseudomonadales Moraxellaceae Proteobacteria; Alphaproteobacteria; Rhodobacteraceae; Rhodobacterales; genus unclassified Chloroflexi; unclassified order unclassified unclassified family unclassified 1M Pein 1M Haok 1S Clsp 2S Sppa 1M Spab 1S Spab 2M Liis Ratio in sponge specimen sponge in Ratio 1M Hysi 1M Haci 1S Clce Characteristics of predominant OTUs. of predominant Characteristics OTU denovo500 0.0 0.0 23.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Table 3. denovo694 0.0denovo624 0.0 0.0 0.9 92.2denovo613 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0denovo659 0.0 0.0 0.0 0.0 0.0 94.8denovo441 0.0 0.0 0.0 0.0 51.0 0.0 0.0 0.0 0.0 0.0 0.0 79.4 0.0 0.0 0.0 0.0 27.1denovo366 0.0 0.0 0.0 0.0 0.0 0.0 0.0denovo4 0.0 0.0 0.0 20.7 0.0 0.0 0.0 0.0 0.0 0.0denovo228 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 10.5denovo394 0.0 0.0 0.0 9.0 0.0 0.0 0.0 0.0 0.0 0.0 11.5 0.0 0.0 0.0 0.1 0.0 0.0denovo652 2.6 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.3 phylum Unclassified denovo450 0.0 0.0 0.0 10.4 0.0 0.0 0.0denovo540 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.8 denovo103 0.1 0.0 0.0 0.0 98.0 96.7 84.0 0.0 0.0 0.0 denovo424 0.2 0.0 0.0 8.4 0.0 0.0 0.1 0.0 0.0 0.0 denovo205 56.4 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0

J. Microbiol. Biotechnol. Sponge-Specific Unknown Bacterial Groups in Marine Sponges from Korea 7 sp. sp. heliophila heliophila heliophila

okadai faveolata

Host sp. californiana strobilina

Artificial microbialite Tethya Hymeniacidon Hymeniacidon Montastraea Holoxea Halichondria Acanthostrongylophora Acanthostrongylophora Hymeniacidon Ircinia Artificial microbialite Site Best hit from BLASTN 98% Sponge Similarity Accn no. JF824772 99%EU917666 Sponge 98% Microbialite EF513720 99% Sponge KF585199 99% Sponge JQ199855 100% Seawater Seawater AF025554 100% Seawater bacterioplankton Marine EU917666 96% Microbialite JF824767 99% Sponge JF824776 99% Sponge EF629744 97% Sponge summarized. has not been verified taxonomically in that level. Square brackets indicate that the ; ; ss EU290440 89% Sponge ss AB054180 98% Sponge ; t from QIIME t from QIIME a AncK6 EF513716 ; Synechococcaceae Flavobacteriaceae analysis ; ; unclassified genus unclassified cla classunclassified FJ203517unclassified cla 96%Alphaproteobacteria Coral unclassified genus unclassified unclassified family unclassified Flavobacteriia ; ; unclassified family unclassified ; Taxonomic assignmen Proteobacteria; Proteobacteria; Alphaproteobacteria; Kiloniellales; Cyanobacteria Synechococcophycideae Synechococcales Synechococcus Bacteroidetes Flavobacteriales Mesonia Proteobacteria; Alphaproteobacteria; Sphingomonadales, family unclassified Proteobacteria; Alphaproteobacteria; order unclassified Proteobacteria; Nitrospirae; Nitrospira; Nitrospirales; Nitrospiraceae; Proteobacteria; Proteobacteria Actinobacteria; Acidimicrobiia; Acidimicrobiales; TK06 family unclassified Proteobacteria; Gammaproteobacteria; Chromatiales; Chloroflexi; Caldilineales; Anaerolineae; Caldilineaceae; 4 Unclassified phylum 1M Pein 1M Haok 1S Clsp 2S Sppa 1M Spab ge specimen 1S Spab 2M Liis Ratio in spon 1M Hysi 1M Haci Clce1S Continued. OTU Classification was shown at the genus level. “Unclassified” means that the sequence was related to reads of which the position name was not verified taxonomically. OTU was determined based ona 16S rRNA gene similarity of 94%. denovo631 0.0 0.0 0.0 4.3 0.0 0.0 0.0 0.0 0.0 0.0 denovo197 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.1 0.0 denovo448 6.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 denovo360 0.0 0.0 0.0denovo516 0.0 0.0denovo462 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.6 0.0 0.0 0.0 0.0 0.1 3. 0.0 0.0 Table 3. denovo26 0.0 0.0denovo548 2.2 4.9 0.0 0.0 0.0 0.0 0.0denovo245 0.0 4.5 0.7 0.0 0.0 0.0 0.0 0.0denovo110 0.4 0.0 0.0 0.0 4.7 0.0 0.0 0.0 0.0 4.4 0.0denovo439 0.0 0.0 0.0 1.0 0.0 0.0 0.2 0.0 0.0 0.0denovo160 4.3 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0denovo442 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.9 0.0 0.0 0.0 0.0 4.2 0.0denovo499 0.0 0.0 0.0 0.0Ratios in each specimen, taxonomicassignment, and results of theBLASTN comparisonwith the nucleotide database of GenBank are 0.0 a 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8

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Fig. 4. Neighbor-joining tree of predominant OTUs. A 16S rRNA gene similarity of 94% was used for OTU determination to reduce the number of OTUs. Bootstrap values above 70% are shown at the branches. was low (88%). The betaproteobacterial cells (KC492704) study. The low similarity of denovo205 with other sequences were proven to predominantly inhabit the sponge through (<88%) implies that new sponge specimens can be reservoirs cloning-sequencing, pyrosequencing, catalyzed reporter of novel bacteria that have been unexplored so far. deposition fluorescent in situ hybridization, and transmission Many monophyletic and sponge-specific phylogenetic electron microscopy, implying that they have potentially clusters in Bacteria and Archaea have been described in important roles in sponges [5]. Denovo205 was the most previous papers [38, 40]. Among them, the cluster within abundant OTU in Clce1S and had the closest similarity with Betaproteobacteria is one of the largest sponge-specific a sequence (DQ889898, 88%) that had been detected from clusters that branched deeply from cultivated strains and the octocoral Erythropodium caribaeorum in an unpublished is composed only of sequences obtained directly from

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environments [38]. Denovo205 and denovo624 were also 8. Elenkov I, Popov S, Andreev S. 1999. Sterols from two Black included in this sponge-specific cluster, as shown in Fig. 4. Sea sponges (Haliclona sp.). Comp. Biochem. Physiol. B 123: Through this study, it was revealed that the bacterial 357-360. communities of some sponges were very simple but differed 9. Erwin PM, Olson JB, Thacker RW. 2011. Phylogenetic diversity, from one another, and that some predominant OTUs of host-specificity and community profiling of sponge-associated bacteria in the northern Gulf of Mexico. PLoS One 6: e26806. those sponges showed high dissimilarity with known 10. Giles EC, Kamke J, Moitinho-Silva L, Taylor MW, Hentschel sequences, phylogenetically forming a sponge-specific clade. U, Ravasi T, Schmitt S. 2013. Bacterial community profiles Considering that about 8,500 species of sponges have been in low microbial abundance sponges. FEMS Microbiol. 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