Molecular diversity and distribution pattern of ciliates in sediments from deep-sea hydrothermal vents in the Okinawa Trough and adjacent sea areas

Deep-Sea Research I 116 (2016) 22–32

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Deep-Sea Research I journal homepage: www.elsevier.com/locate/dsri

Molecular diversity and distribution pattern of ciliates in sediments from deep-sea hydrothermal vents in the Okinawa Trough and adjacent sea areas Feng Zhao a, Kuidong Xu a,b,n a b

Institute of Oceanology, Chinese Academy of Sciences, 266071 Qingdao, China Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, 266071 Qingdao, China

art ic l e i nf o

a b s t r a c t

Article history: Received 26 October 2015 Received in revised form 5 July 2016 Accepted 15 July 2016 Available online 16 July 2016

In comparison with the macrobenthos and prokaryotes, patterns of diversity and distribution of microbial eukaryotes in deep-sea hydrothermal vents are poorly known. The widely used high-throughput sequencing of 18S rDNA has revealed a high diversity of microeukaryotes yielded from both living organisms and buried DNA in marine sediments. More recently, cDNA surveys have been utilized to uncover the diversity of active organisms. However, both methods have never been used to evaluate the diversity of ciliates in hydrothermal vents. By using high-throughput DNA and cDNA sequencing of 18S rDNA, we evaluated the molecular diversity of ciliates, a representative group of microbial eukaryotes, from the sediments of deep-sea hydrothermal vents in the Okinawa Trough and compared it with that of an adjacent deep-sea area about 15 km away and that of an offshore area of the Yellow Sea about 500 km away. The results of DNA sequencing showed that Spirotrichea and Oligohymenophorea were the most diverse and abundant groups in all the three habitats. The proportion of sequences of Oligohymenophorea was the highest in the hydrothermal vents whereas Spirotrichea was the most diverse group at all three habitats. Plagiopyleans were found only in the hydrothermal vents but with low diversity and abundance. By contrast, the cDNA sequencing showed that Plagiopylea was the most diverse and most abundant group in the hydrothermal vents, followed by Spirotrichea in terms of diversity and Oligohymenophorea in terms of relative abundance. A novel group of ciliates, distinctly separate from the 12 known classes, was detected in the hydrothermal vents, indicating undescribed, possibly highly divergent ciliates may inhabit this environment. Statistical analyses showed that: (i) the three habitats differed significantly from one another in terms of diversity of both the rare and the total ciliate taxa, and; (ii) the adjacent deep sea was more similar to the offshore area than to the hydrothermal vents. In terms of the diversity of abundant taxa, however, there was no significant difference between the hydrothermal vents and the adjacent deep sea, both of which differed significantly from the offshore area. As abundant ciliate taxa can be found in several sampling sites, they are likely adapted to large environmental variations, while rare taxa are found in specific habitat and thus are potentially more sensitive to varying environmental conditions. & 2016 Elsevier Ltd. All rights reserved.

Keywords: Ciliate diversity Marine sediments Hydrothermal vent Illumina MiSeq platform Biogeography Abundant taxa

1. Introduction Deep-sea hydrothermal vents are characterized by their extreme environmental conditions. Taking the hydrothermal vents in the Okinawa Trough as an example, the measured temperature in the vents ranged from 100 °C to 350 °C, with high pressure over 10 MPa, and high hydrogen sulfide ranging from 1.3 to 13.7 mM/kg n Corresponding author at: Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China. E-mail address: [email protected] (K. Xu).

http://dx.doi.org/10.1016/j.dsr.2016.07.007 0967-0637/& 2016 Elsevier Ltd. All rights reserved.

(Glasby and Notsu, 2003). Hydrothermal vents are inhabited by a specific macrobenthic community that differs significantly from that of adjacent sea areas (Tyler and Young, 2003; Levin et al., 2009). Previous studies also indicate that deep-sea hydrothermal vents encompass very specific and diverse habitats for prokaryotic microorganisms (Kato et al., 2009; Kim and Hammerstrom, 2012). A recent study by Anderson et al. (2015) indicates that abundant and rare bacterial lineages are both geographically restricted in deep-sea hydrothermal vents, whereas archaeal lineages are comparatively different. By contrast, the abundant archaeal lineages are cosmopolitan and similar to those in adjacent sea

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Fig. 1. Location of the six sites and the images of the hydrothermal vent sites HV1 and HV2.

areas, whereas the rare lineages are geographically restricted (Anderson et al., 2015). In comparison with the macrobenthos and prokaryotes, microbial eukaryotes in deep-sea hydrothermal vents have received little attention. Whether microbial eukaryotes have similar patterns of diversity and distribution as bacteria or archaea in hydrothermal vents is still not known. Among the microbial eukaryotes, ciliates are thought to be a predominant group in the hydrothermal vents of the East Pacific Rise, where at least 20 ciliate species have been recorded following observations by microscopy (Small and Gross, 1985). Subsequent studies based on cultivation led to the descriptions of a limited number of ciliates from hydrothermal vents as well as reports on their physiological characterization (Baumgartner et al., 2002). Due to the extreme environmental conditions in which they live, most ciliates in deep-sea hydrothermal vents are difficult to cultivate resulting in underestimation of their diversity using classical methods. In recent years, molecular techniques based on the 18S rDNA have revealed a high diversity of microbial eukaryotes in deep-sea hydrothermal vent sediments (Edgcomb et al., 2002; Coyne et al., 2013). Ciliates have often been reported as the most diverse group of microbial eukaryotes in different hydrothermal vents (LópezGarcía et al., 2003, 2007; Sauvadet et al., 2010). Seven classes of ciliates, as well as a novel deep-sea group, were detected in the Guaymas Basin hydrothermal vent sediments by 18S rDNA clone library (Coyne et al., 2013). So far, all studies on the molecular diversity of ciliates and other microbial eukaryotes in deep-sea hydrothermal vents have been based on clone library methods. The clone library approach, however, has inherent methodological limitations, including biases in the plasmid ligation step, the relatively high expense and labour-intensity of sequencing sufficient numbers of clones using the Sanger method (Stoeck et al., 2010). The high-throughput sequencing techniques overcome the limitations caused by the ligation steps of the clone library method enabling hundreds of thousands of sequences to be processed simultaneously. It has

been shown that DNA can be preserved in marine sediments over time (Coolen et al., 2009). Thus, the microeukaryotic diversity detected by DNA sequencing likely include not only active organisms, but also extracellular DNA, dead cells and resting stages of organisms. By contrast, cDNA surveys have been used to identify the active protist communities and may provide a different picture of biodiversity from that of DNA surveys (Massana et al., 2015). Therefore, diversity estimation based on a combination of DNA and cDNA high-throughput sequencing will help to better understand the diversity of microbial eukaryotes. In this paper, we combined the high-throughput DNA and cDNA sequencing of 18S rDNA for the first time to reveal the diversity and group composition of ciliates in sediments from hydrothermal vents. Meanwhile, the ciliate diversity in the hydrothermal vents was compared with those from adjacent deep-sea and offshore sea areas, based on the analysis of high-throughput DNA sequencing data. We aim to (i) evaluate the molecular diversity of ciliates in sediments from deep-sea hydrothermal vents, and (ii) to compare the community composition and distribution of benthic ciliates among the offshore, hydrothermal vent and surrounding deep-sea area.

2. Material and methods 2.1. Study sites and sample collection The Okinawa Trough is a back-arc basin located behind the Ryukyu trench and Ryukyu Islands, and contains several active hydrothermal vents. Two sites in the immediate vicinity of the hydrothermal vents in the Okinawa Trough were chosen for sample collection. Site HV1 (27°47.4487′N, 126°53.8253′E) was about 5 m away from the nearest hydrothermal vent, and site HV2 (27°47.4404′N, 126°53.8223′E) was about 20 m away (Fig. 1). The water depth in this area was 1008 m. Sediment samples of HV1

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and HV2 were collected using a push-core operated by the remotely operated vehicle (ROV) “Discovery” equipped by the R/V KEXUE on 12 and 17 April 2014, respectively. For comparison, two adjacent deep-sea sites in the surrounding area of the hydrothermal vents were selected for sample collection. Sites D1 (27°34.0199′N, 126°55.9865′E, 1598 m depth) and D2 (27°40.5768′N, 126°59.1798′E, 1527 m depth) are 13.3 km and 15.5 km away from the hydrothermal vents, respectively. Sediment samples were taken using a 0.1-m2 modified Gray-O′Hara box corer on 23 April 2014 (Fig. 1). In addition, offshore sediment samples were collected from two sites (O1 and O2) in the shallow Yellow Sea in August 2014 using a 0.1-m2 modified Gray-O′Hara box corer. Sites O1 (32°59′N, 122°56′E, 35 m depth) and O2 (31°31′ N, 124°28′E, 50 m depth) are about 580 km and 480 km away from the hydrothermal vent, respectively (Fig. 1). At each site, three replicate samples were taken from the surface 0–1 cm of sediments. Sediment samples were stored at
80 °C for the DNA extraction. Only the samples from the two sites of the hydrothermal vents were available for both the DNA and RNA extraction. 2.2. DNA/RNA extraction, cDNA synthesis and PCR amplification of ciliate 18 S rDNA A 0.3 g subsample from each replicate sample was taken to extract the total DNA with the Power Soil DNA isolation kit (MoBio Laboratories, USA). The total RNA of samples from sites HV1 and HV2 was extracted with the PowerSoil Total RNA isolation kit (MoBio Laboratories, USA). At each site, 2 g of sediment which consisted of 0.6–0.7 g each from the three replicate samples, was used to extract the total RNA. Three subsamples of the total RNA were reverse-transcribed into cDNA using 1st strand cDNA synthesis kit (TAKARA BIO INC., Japan) using random primers provided with the kit according to the manufacturer's instructions. After transcription of each individual sample, the transcribed products from each site were subjected to SSU cDNA amplification. Ciliate 18S rRNA gene was amplified with the PrimeSTAR GXL DNA High Fidelity Polymerase (TAKARA BIO INC., Japan) by nested PCR (Stock et al., 2013). First, amplification with the ciliate specific primers CilF, CilR I, CilR II and CilR III was performed in order to amplify specifically the ciliate 18S rDNA (Lara et al., 2007). Ciliate 18S rDNA was amplified using GeneAmps PCR System 9700 (PE Applied Biosystems, USA) with the following program: 95 °C for 5 min; 35 cycles consisting of 94 °C for 45 s, 58 °C for 60 s and 72 °C for 60 s; followed by 72 °C for 10 min. Subsequently, the purified PCR products from the first reaction were subjected to a second PCR, which adopted eukaryote-specific primers for the amplification of the hyper-variable V4 region (Stoeck et al., 2010). The PCR protocol started with 10 identical amplification cycles at an annealing temperature of 57 °C, followed by 25 cycles with primer annealing at 49 °C (Stoeck et al., 2010). Three subsamples of PCR products for each sample were mixed and used for high-throughput sequencing as described below.

Technology Co., Ltd. Sequence libraries were generated using NEB Next Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit 2.0 Fluorometer (Thermo Scientific, USA) and Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina MiSeq platform and 300 bp paired-end reads were generated. Sequences were submitted to GenBank-SRA with accession number SPR064020. 2.4. Sequence data processing Sequence analysis was performed by UPARSE pipeline (Edgar, 2013). The procedure included the following steps: merging of paired reads, read quality filtering, dereplication, discarding singletons, OTU-clustering within the identify threshold of 97%, mapping the sequences to the OTUs and generating the OTU table. The taxonomic information for each representative sequence for each OTU was annotated using QIIME (Caporaso et al., 2010). The assignation of a taxonomic category here belongs necessarily to the group of ciliates. Only unique sequences with a best BLAST hit of at least 80% sequence similarity were assigned to a taxonomic category. Remaining sequences were assigned to “Others”. In each sample, OTUs were defined as abundant when representing equal to or above 1% of the total sequences in a sample, and rare when representing equal to or below 0.1% of the total sequences in a sample (Pedrós-Alió, 2006; Fuhrman, 2009). Maximum-likelihood (ML) trees of abundant OTUs and the novel group of ciliates were performed online with 1000 replicates bootstrapping using RAxML-HPC2 on XSEDE (7.3.2) on the CIPRES Science Gateway (Stamatakis et al., 2008; CIPRES, 2014). 2.5. Statistical analysis In order to compute alpha diversity, we rarified all data sets to 20,000 sequences and calculated two metrics by using the QIIME (Caporaso et al., 2010): ACE, which is a non-parametric richness estimator (Chao et al., 1992), and Observed Species, which counts the total number of OTUs found in each sample. UPGMA cluster analysis of OTUs (all the OTUs, abundant OTUs and rare OTUs) was carried out using the Bray-Curtis similarity coefficient. The presence/absence transformation and log(x þ1) transformation were applied to the OTU tables prior to analysis, respectively. Results based on the presence/absence and log(x þ1) transformations were virtually identical, therefore only the results based on the presence/absence transformation are shown. Similarity profile (SIMPROF) tests were used to find statistically significant evidence of genuine clusters of samples (p set at 0.05) (Clarke and Gorley, 2006). RALATE analysis was applied to calculate the correlation coefficients (Spearman's ρ) between the ciliate community and water depth, and to make a significance test. The statistical analyses were performed by the PRIMER v6 (Plymouth Marine Laboratory, UK).

3. Results 2.3. Illumina high-throughput sequencing 3.1. Data overview Equal volumes of 1  loading buffer (contained SYB green) and PCR products were mixed and operate electrophoresis on 2% agarose gel for detection. Samples with a bright main band of about 480 bp were chosen for further experiments. The quantification of PCR products was assessed by the Qubit 2.0 Fluorometer (Thermo Scientific), and PCR products were mixed in equidensity ratios. The mixed PCR products were purified with GeneJET Gel Extraction Kit (Thermo Scientific, USA). Sequencing was performed at the Novogene Bioinformatics

In total, 315,009 sequences from DNA (six sites: O1, O2, D1, D2, HV1 and HV2) and cDNA (two sites: HV1 and HV2, and marked as HV1R and HV2R) analyses were obtained after quality filtering (Table 1). The number of sequences for each sample varied from 27,100 at the offshore site O2 to 66,866 at the deep-sea site D1, with an average of 39,376 sequences (Table 1). After mapping total sequences to ciliate-related OTUs, a total of 280,536 sequences, varying from 22,627 at site O2 to 55,616 at site D1, with an average

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Table 1 Summary of the sequences and OTUs data for each sample. DNA sequencing

No. of total filtered sequences No. of ciliate sequences No. of ciliate OTUs (97%) No. of abundant ciliate OTUs Proportion of abundant ciliate OTUs No. of rare ciliate OTUs Proportion of rare ciliate OTUs

cDNA sequencing

O1

O2

D1

D2

HV1

HV2

HV1R

HV2R

41,939 35,011 108 9 9% 61 58%

27,100 22,624 115 15 13% 67 60%

66,866 55,499 69 18 26% 24 35%

27,847 23,021 58 17 30% 11 19%

39,971 37,781 57 12 21% 21 37%

37,866 37,125 28 10 36% 4 14%

35,288 32,229 140 15 11% 60 43%

38,132 35,714 124 12 10% 56 45%

of 35,067 sequences, remained for further analyses. Based on a similarity of 97%, a total of 353 different ciliate OTUs were obtained (Table S1). The total number of OTUs yielded from DNA sequencing from each site varied from 28 to 112, with an average of 72 OTUs; that from cDNA sequencing varied from 124 (HV2R) to 140 OTUs (HV1R) (Table 1). 3.2. Ciliate diversity detected from DNA samples The indexes of ACE and observed-species based on DNA sequencing indicated that the offshore sites had the highest ciliate diversity, followed by the deep-sea sites, the diversity in the hydrothermal vents being the lowest (Table 2). The proportions of locally rare or abundant OTUs varied with habitats. Higher proportions of abundant OTUs were observed both for the hydrothermal vent samples (28.5% on average at sites HV1 and HV2) and the adjacent deep-sea samples (28% on average at sites D1 and D2). By contrast, the proportion of abundant OTUs was only about 11% on average at the two offshore sites. On average 89% of the total OTUs in the offshore area, 72% in the deep sea and 71.5% in the hydrothermal vents were rare or infrequent. The proportions of rare OTUs were similar to those of abundant OTUs in the hydrothermal vent and adjacent deep-sea sediments (26% and 27% on average, respectively), while those in the offshore sediments accounted for 59% on average (Table 1). More than 87% of the total OTUs detected by DNA sequencing were unique to one or two sites (Fig. 2E). Among the 353 OTUs identified, a total of 13 OTUs were common in all the three habitats and OTU 1 was the most abundant and detected from all the six sites (Fig. 2E; Table S2). The abundant OTUs detected at the deep-sea sites could either be found at the offshore sites or the hydrothermal vent sites, while no abundant OTU occurred in both the offshore area and hydrothermal vents, except the most abundant OTU 1 (Fig. 2F). Four abundant OTUs (OTUs 3, 4, 11 and 58) were found in both the hydrothermal vents and adjacent deep-sea area, but only two (OTUs 22 and 362) were present in both the offshore and deep-sea areas (Figs. 2F and 3). The overlap of individual OTUs between sites in the same habitat was higher than that among different habitats (Fig. 2). UPGMA clustering based on total OTUs showed that the two sites of each habitat clustered together and the six sites formed three major groups: the deep-sea cluster (D1, D2) was grouped Table 2 Alpha diversity indices of ciliate communities in different samples. DNA sequencing O1 ACE index Observed species

O2

cDNA sequencing D1

D2

HV1

HV2

HV1R

117.64 126.44 85.69 78.72 59.00 29.92 146.92 110 116 72 61 57 28 142

HV2R 142.58 126

with the offshore cluster (O1, O2) first, and then grouped with the hydrothermal vent cluster (HV1, HV2) (Fig. 4A). The SIMPROF analysis based on total OTUs showed the three clusters were significantly different (p ¼0.001; Fig. 4A). RELATE analysis indicate that the ciliate communities in three habitats had no significant correlation with the water depth (R ¼0.435, p ¼0.13). By contrast, UPGMA clustering based on abundant OTUs showed that the hydrothermal vent sites formed a cluster and was grouped with cluster formed by the deep-sea sites, and then grouped with the offshore cluster (Fig. 4B). The SIMPROF analysis based on abundant OTUs showed that there was no significant difference in the diversity of abundant ciliates between the hydrothermal vent and adjacent deep-sea sites, while the difference was significant (p ¼0.001) between the offshore cluster and hydrothermal vent-deep-sea cluster (Fig. 4B). UPGMA clustering based on rare OTUs showed that the offshore sites formed a cluster which was grouped with the deep-sea site D1 first, and then grouped with the deep-sea site D2, and finally grouped with the hydrothermal vent cluster (Fig. 4C). The SIMPROF analysis based on rare OTUs showed that the diversity of rare ciliates was significantly different between the offshore sites and the deep-sea site D1 as well as D2 (both p¼ 0.001) and between the offshore-deep-sea cluster and the hydrothermal vent cluster (p ¼ 0.001) (Fig. 4C). 3.3. Phylogenetic analysis of ciliate 18S rRNA gene detected from DNA samples The OTUs obtained by DNA sequencing were related to 11 of the 12 known classes of ciliates, i.e. excluding the newly established class Cariacotrichea from an anoxic environment. These are: Spirotrichea, Oligohymenophorea, Litostomatea, Plagiopylea, Phyllopharyngea, Karyorelictea, Prostomatea, Nassophorea, Heterotrichea, Armophorea and Colpodea. In addition, a novel ciliate group independent of the known classes was detected from the hydrothermal vent sites (Figs. 5 and 6). All the OTUs analyzed in this study were related to a total of 109 identified genera and six unclassified ciliate assemblages (Table S3). The sequences of Spirotrichea were most abundant at the offshore sites in particular at site O1, where they accounted for 98% of the total sequences. By contrast, the relative abundance was low at the hydrothermal vent sites, with a sequence contribution only about 2% at site HV2. The sequences related to the genera Tintinnopsis, Sinistrostrombidium and Strombidium were most abundant at the offshore sites, the sequences related to the genera Trachelostyla, Anteholosticha and Tunicothrix were dominant at the deep-sea sites, and the sequences related to the genera Trachelostyla, Aspidisca and Novistrombidium were most abundant at the hydrothermal vent sites. Like sequence abundance, the number of OTUs affiliated to Spirotrichea was the highest at the offshore sites (54–55 OTUs), followed by that at the deep-sea sites (26–33 OTUs), and only 8–17 OTUs were found at the hydrothermal vent sites (Fig. 5B). Among the total Spirotrichea OTUs,

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Fig. 2. Number of shared and unique OTUs between the two sites in the offshore (A), deep-sea (B) and hydrothermal vent sediments (C detected by DNA sequencing and D detected by cDNA sequencing) and among different habitats based on the total (E), abundant (F) and rare OTUs (G), and those detected by DNA and cDNA sequencing in the hydrothermal vent sediments (H-J).

seven were common in the three habitats and nine were unique to the hydrothermal vent habitat (Table S2 and S4). The Spirotrichea OTUs were affiliated to 42 known genera and three unclassified taxa (Table S3). The OTUs related to the Strombidiida and Euplotida genera were commonly found in all three habitats, and those related to the Tintinnida and Choreotrichida genera were only detected from the offshore sites. The ML tree of abundant OTUs related to Spirotrichea showed that most of the deep-sea OTUs formed a clade related to the subclass Hypotrichia, which was separated from the clade formed by the offshore OTUs. Among the three abundant Spirotrichea OTUs in the hydrothermal vent habitat, two were shared with the deep-sea OTUs and one was nested within a clade comprising deep-sea OTUs affiliated to the subclass Euplotia (Fig. 3). In contrast to those of Spirotrichea, the sequences of Oligohymenophorea were very abundant at the hydrothermal vent as well as deep-sea sites, accounting for 89% of the total sequences at site HV2, but contributed to no more than 5% of the total sequences at the offshore sites (Fig. 5A). Among the Oligohymenophorea sequences, the sequences related to Trichodina, Pleuronema and Homalogastra were most abundant at the offshore sites, but their sequence proportion was less than 1.5%. The sequences related to the genera Ancistrum, Homalogastra and Trichodina were most abundant at the deep-sea sites with a high sequence proportion (more than 10%). The sequences related to the genera Trichodina, Ancistrum and Pleuronema were most abundant at the hydrothermal vent sites, where those relate to Trichodina accounted for more than 30% of the total sequences at site HV1. Unlike the

pattern of sequences, the number of OTUs affiliated to Oligohymenophorea was the highest at the offshore sites (21–28 OTUs), followed by the deep-sea sites (16–17 OTUs), and only 12–16 OTUs were found from the hydrothermal vent sites (Fig. 5B). Among the Oligohymenophorea OTUs, five were common in the three habitats and nine were unique to the hydrothermal vent habitat (Table S2 and S4). The Oligohymenophorea OTUs were affiliated to 30 known genera and an unclassified taxon (Table S3). The OTUs related to the Sessilida genera were found only in the deep-sea and hydrothermal vent area, while the OTUs relate to parasitic genera of Astomatida and Apostomatida were present only in the offshore and deep-sea areas. The ML tree of abundant OTUs showed that the most abundant and widely distributed OTU 1 was affiliated to mobilid Peritrichia and separated from the clade formed by the deep-sea and hydrothermal vent OTUs (Fig. 3). Litostomatea ciliates were also detected from all the sites, but less abundant and contributed from 0.01% to 7% to the total sequences (Fig. 5A). The sequences related to Balantidion were most abundant at the offshore sites, and those related to Litonotus were most abundant at the deep-sea sites. No abundant Litostomatea sequences were found in the hydrothermal vents. Among the OTUs affiliated to Litostomatea, 12 OTUs were obtained from the offshore site O1 and six from O2, seven OTUs from the deep-sea site D1 and two from D2, and only two OTUs from the hydrothermal vent site HV1 and one from HV2 (Fig. 5B). All the Litostomatea OTUs were affiliated to 9 known genera (Table S3). DNA sequencing detected a low number of sequences of

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Fig. 3. Maximum likelihood (ML) tree of the ciliate 18S rDNA sequences representing abundant OTUs in different habitats revealed by DNA and cDNA sequencing (marked as different shapes).

Fig. 4. UPGMA clustering and SIMPROF analyses of samples based on the total OTUs (A), the abundant OTUs (B) and the rare OTUs (C) revealed by both DNA and cDNA sequencing. Significant groups (p o0.05) are shown by red bars.

Nassophorea from the offshore sites, and more sequences from the deep-sea sites, but none in the hydrothermal vents (Fig. 5A). The number of Nassophorea OTUs was comparatively stable at the offshore and deep-sea sites, ranging from two to three (Fig. 5B).

The Nassophorea OTUs were affiliated to five known genera (Table S3). Representatives of Plagiopylea were found only at the hydrothermal vent sites, contributing only about 1% to the total

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Fig. 5. Relative abundance of sequences (A) and the number of OTUs (B) related to the major taxonomic groups of ciliates detected by DNA or cDNA sequencing at different sites.

sequences and comprising four OTUs at site HV1 and three at HV2 (Fig. 5B). Only a single sequence of Plagiopylea was detected from an offshore site and none from the deep-sea sites (Fig. 5B). All the Plagiopylea OTUs were affiliated to the anaerobic genus Epalxella. Like Plagiopylea, the novel ciliate group was also abundant in the hydrothermal vents, where the solely abundant OTU 15 accounted for 7% and 6% of the total sequences at sites HV1 and HV2, respectively (Table 3). A total of eight OTUs were found from site HV1 and two from site HV2. Two OTUs different from those in the hydrothermal vent were also detected from the deep-sea site D2 (Table 3). Phyllopharyngeans were abundant in the deep-sea area, contributing to about 13% of the total sequences at site D2 (Fig. 5A). Four OTUs affiliated to Phyllopharyngea were obtained from the offshore site O1, and five OTUs each were detected from sites O2, D1 and D2. No sequence was detected from the hydrothermal vent site HV2, and only 0.08% of the total sequences with one OTU were found from HV1. The Phyllopharyngea OTUs were affiliated to five known genera. Sequences related to Acineta were more abundant than other genera, in particular at the deep-sea sites (Table S3). Prostomatea were abundant at sites O2 (55.1%) and HV1 (19.5%), where two abundant OTUs similar to the parasite Cryptocaryon irritans were detected from both sites (Fig. 5A). The other two abundant OTUs detected from the offshore sites were most closely related to Pinacocoleps (Table S3). The highest number of OTUs was obtained from the offshore sites (8–14 OTUs), and only one or two OTUs were detected from the other sites (Fig. 5B). Karyorelictea sequences accounted for 7% of the total sequences with two OTUs at site D2, less than 1% of the total abundance with one OTU at the offshore site O1 and two at O2, and no sequence was obtained from sites D1 and HV2 (Fig. 5A). The Karyorelictea OTUs were affiliated to the genera Trachelocerca and Kentrophoros (Table S3). Two OTUs affiliated to the genus Kentrophoros were only detected from the hydrothermal vent sites (Table S4). Heterotrichea sequences accounted for 3% of the total sequences with two OTUs at site HV1, less than 1% with one OTU at sites O1, D1 and HV2, and no sequences were obtained from sites O2 and D2 (Fig. 5A). Three OTUs were unique to the hydrothermal vent sites (Table S4). All the Heterotrichea OTUs were affiliated to four genera (Table S3). A few sequences belonging to Colpodea were detected only from the deep-sea site D1, accounting for 0.4% of the total sequences. A handful of sequences of Armophorea (ciliates in this class occupy anoxic habitats) were detected also from sites D2 and HV1, accounting for merely 0.06% and 0.2% of the total sequences, respectively (Fig. 5A).

3.4. Diversity of active ciliates in the hydrothermal vents detected from cDNA samples Compared with DNA sequencing, cDNA sequencing of the samples from the hydrothermal vents (HV1R and HV2R) detected a higher number of OTUs, representing a higher diversity of active ciliates (Fig. 2). In contrast to DNA sequencing, cDNA sequencing showed that Plagiopylea, which prefer anaerobic to microaerophilic environments, were the most diverse and abundant ciliate group in the hydrothermal vents, contributing to 26% of the total sequences at site HV1 and 66% at site HV2 (Fig. 5). More than 30 Plagiopylea OTUs were detected by cDNA sequencing. The Plagiopylea OTUs were affiliated to the ciliate genera Trimyema and Epalxella (Fig. 3; Table S3). The cDNA sequences related to Epalxella was most abundant at the hydrothermal vent sites. The most abundant OTU 1 detected by DNA sequencing from all the six sites was not obtained by cDNA sequencing. The cDNA sequencing detected a distinctly lower proportion of Spirotrichea sequences at site HV1 (9% vs. 45%) than DNA sequencing, while the two techniques detected the same proportion of 2% at site HV2. Although the proportion of Spirotrichea sequences was comparatively low, the number of Spirotrichea OTUs was the second highest at sites HV1 (30) and HV2 (27). Likewise, cDNA sequencing detected higher proportions of Oligohymenophorea sequences, accounting for 42% at HV1R and 22% at HV2R. The number of OTUs affiliated to Oligohymenophorea ranged from 17 at HV1R to 19 at HV2R. The cDNA sequences related to the genera Homalogastra and Pleuronema were most abundant in Oligohymenophorea, and those affiliated to Homalogastra accounted for 23% of the total cDNA sequences at the hydrothermal vent sites (Table S3). Compared with DNA sequencing, cDNA sequencing detected a higher proportion of Karyorelictea sequences from sites HV1 (5.6% vs. 0.3%) and HV2 (5.9% vs. none), and a higher number of OTUs from sites HV1 (13 vs. 3) and HV2 (12 vs. 0) (Fig. 5B). The Karyorelictea OTUs were affiliated to three genera, the sequences related to Trachelocerca and Kentrophoros accounting for 4.1% and 1.5% of the total cDNA sequences at the hydrothermal vent sites (Table S3). UPGMA clustering based on the total OTUs showed that the hydrothermal vent ciliate diversity detected by DNA (HV1 and HV2) and cDNA (HV1R and HV2R) sequencing formed a cluster, which was separated from the cluster formed by the deep-sea and offshore sites, as revealed by DNA sequencing (Fig. 4A). When analysis was restricted to the abundant OTUs, the active ciliate communities in the hydrothermal vents (HV1R and HV2R) formed an independent cluster separated from the ciliate communities detected by DNA sequencing (Fig. 4B). UPGMA clustering based on

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Fig. 6. Maximum likelihood (ML) tree of the ciliate 18S rDNA sequences representing the novel group.

the rare OTUs showed that the active ciliate communities in the hydrothermal vents (HV1R and HV2R) formed a cluster that then grouped with HV1, and was separated from the cluster formed by the deep-sea and offshore sites (Fig. 4C).

4. Discussion 4.1. Distribution pattern of ciliates in the hydrothermal vents and adjacent areas The diversity and geographical distribution of microorganisms

have long been debated (Finlay, 2002; Foissner, 2008). Hydrothermal vents are characterized by their extreme environmental conditions, which make these habitats significantly different from adjacent sea areas. Bachraty et al. (2009) and Ruff et al. (2015) indicated a high degree of endemism of prokaryotes in methane seeps and hydrothermal vents and suggested a high local diversification in these heterogeneous ecosystems. The specific and stable benthic environmental conditions of hydrothermal vents could provide suitable habitats for isolated evolution and species sorting. By using high-throughput sequencing, Anderson et al. (2015) reported that both the abundant and rare bacteria lineages, as well as rare archaeal lineages, in deep-sea hydrothermal vents

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F. Zhao, K. Xu / Deep-Sea Research I 116 (2016) 22–32

Table 3 Distribution of Novel Group of ciliates in the hydrothermal vent and adjacent area. Sequence abundance of each OTUs is indicated for a given sample. Sites

Best hit

OTU ID OTU15

D2 0

HV1 2628

HV2 2200

HV1R 36

HV2R 330

OTU63

0

115

336

9

0

OTU72

0

790

0

22

4

OTU110 OTU152 OTU195 OTU203 OTU207

150 0 0 0 0

0 117 40 50 68

0 0 0 0 0

0 5 3 5 12

0 4 3 0 4

OTU224 OTU275 OTU286 OTU341 OTU349

40 0 0 0 0

0 16 0 0 0

0 0 0 0 0

0 21 16 11 10

0 5 6 4 0

are geographically restricted, whereas the abundant archaeal lineages are cosmopolitan, similar to those in adjacent sea areas. However, data on the molecular diversity of ciliates and other microbial eukaryotes in deep-sea hydrothermal vents are scant and all are based on clone library methods. Our study based on high-throughput sequencing indicates that the diversities of both the total and rare ciliate taxa in the Okinawa Trough hydrothermal vent sediments are significantly different from those in the adjacent deep sea and the offshore region. The results showed that about 89% of the total OTUs in the offshore area, 72% in the deep sea, and 71.5% in the hydrothermal vents were rare or infrequent. This is generally consistent with the estimation of Foissner (1999, 2008), who indicated that the vast majority of protists (possibly 490%) have a moderate, low, or very low abundance. The rare and moderate species, many of which have yet to be discovered, may be endemic (Foissner, 2008). Due to their limited dispersal ability, rare species might be responsible for the differences between the ciliate communities in hydrothermal vents and the surrounding deep sea. As concerns the diversity of abundant taxa, however, no significant difference was observed between the hydrothermal vents and adjacent deep sea, while the abundant taxa in both habitats were significantly different from that in the offshore area. The data indicate that the diversity of abundant ciliates in the hydrothermal vents was more similar to that in the surrounding deep-sea area than in the offshore area. The hydrothermal vents had four abundant OTUs that were shared with the surrounding deep sea, while the deep sea had only two OTUs shared with the offshore area. No abundant OTU occurred in both the offshore area and hydrothermal vents, except the most abundant OTU 1. It is likely that abundant taxa have greater dispersal ability and larger adaptive range than rare taxa and some can overcome the obstacle of environmental factors that influence the spread of ciliate in hydrothermal vents. This partly supports the view of Finlay (2002), who strongly indicated that because of their high abundance, the dispersal of microbial eukaryotes is rarely restricted by geographic barriers. However, only the most abundant OTU 1 could be detected from all the studied sites. Both rare and abundant ciliates showed limited distribution in the three habitats. Including factors intrinsic to organisms (e.g. dispersal ability, reproductive behavior and habitat specificity), geographical distance and physical-chemical factors (e.g. water depth and currents and chemical components) may also play important roles in the distribution of ciliates in the seas. Among these factors, water depth has usually been suggested to be one of the important

Sequences Gy-08–84 RM1-SGM13 Gy-08–84 RM2-SGM09 Gy-08–3 RM2-SGM09 Gy-08–49 Gy-08–90 Gy-08–49 Gy-08–84 Gy-08–67 RM1-SGM12 Gy-08–49 Gy-08–84 Gy-08–49 Gy-08–28 Gy-08–49

Sequence ID JX268887 AB505470 JX268887 AB505517 JX268807 AB505517 JX268853 JX268893 JX268853 JX268887 JX268871 AB505519 JX268853 JX268887 JX268853 JX268832 JX268853

Habitats hydrothermal vent Cold seep hydrothermal vent Cold seep sediment hydrothermal vent Cold seep hydrothermal vent hydrothermal vent hydrothermal vent hydrothermal vent hydrothermal vent Cold seep hydrothermal vent hydrothermal vent hydrothermal vent hydrothermal vent hydrothermal vent

Similarity 93% 92% 91% 90% 99% 99% 90% 89% 90% 87% 99% 99% 90% 99% 96% 98% 96%

factors affecting the distribution of microorganisms in both planktonic and benthic marine ecosystems (Bik et al., 2012; Gong et al., 2015). The water depth differed significantly among the three habitats (  1000 m,4 1500 m, and r50 m). RELATE analysis, however, indicated that the ciliate communities in three habitats had no significant correlation with the water depth. Nonetheless, we could not conclude that the differences in ciliate communities among the three habits were not related to water depth due to the limited depth gradients and the low number of study sites. 4.2. Major ciliate community composition in the hydrothermal vents and adjacent areas Ciliates have been reported as a diverse and dominant group in hydrothermal vents by using both microscopy and molecular survey data (Small and Gross, 1985; Coyne et al., 2013). However, there is still no comprehensive estimation of ciliate diversity in hydrothermal vents and a comparison of the ciliate community composition between hydrothermal vents and adjacent sea areas. Within the phylum Ciliophora, Spirotrichea and Oligohymenophorea are the two most diverse classes and each has a broad distribution in natural environments (Lynn, 2008). Our investigation indicates that these two classes were the most dominant groups in the three habitats. Among the 13 overlapped OTUs in the three habitats, seven were affiliated to Spirotrichea and five to Oligohymenophorea. Our study further revealed that the proportion of Spirotrichea OTUs and sequences decreased distinctly from the offshore area towards the hydrothermal vents, while that of Oligohymenophorea increased. This likely indicates that Oligohymenophorea have higher tolerance than Spirotrichea to the extreme environmental conditions of hydrothermal vents. Of particular interest are the abundant Oligohymenophorea sequences related to the parasitic genera Trichodina and Ancistrum detected in sediments from the hydrothermal vents. Ancistrum is a genus of ectocommensal or ectoparasitic ciliates found in the mantle cavity and on the gills of certain marine and freshwater molluscs (Xu et al., 2015). Trichodina is also a genus of ciliates parasitizing aquatic animals, particularly on the gills and/or skin of fish and in the mantle cavity and on the gills of molluscs. All members of these ciliates are mobile and are occasionally found in surrounding water, where they may survive several days outside their hosts (Fenchel, 1965). In the hydrothermal vent area of the Iheya Ridge, mid Okinawa Trough, where the sediment samples were collected, the bivalves Bathymodiolus and Calyptogena were

F. Zhao, K. Xu / Deep-Sea Research I 116 (2016) 22–32

very abundant (Ohta and Kim, 2001; Tokeshi, 2011). It may be assumed that the sequences related to Trichodina and Ancistrum likely originated from these bivalves. Although no host records have been reported for either Bathymodiolus or Calyptogena, relative bivalves such as those of Modiolus and Mactra have been found as hosts of Trichodina and Ancistrum (Xu and Song, 2003). The presence of Trichodina and Ancistrum in the hydrothermal vents is not an exception. High proportion of parasitic protists affiliated to the Alveolata groups Perkinsozoa, Apicomplexia, dinoflagellates and ciliates have also been detected from hydrothermal vents in both the Pacific and Atlantic Oceans (Edgcomb et al., 2002; López-García et al., 2003 and 2007). Moreira and López-García (2003) regarded the deep-sea hydrothermal vents as oases for parasites and a high proportion of parasitic protists may add a new factor that has to be considered to understand the hydrothermal vent ecology and population dynamics. Nonetheless, further investigation is needed to confirm the existence of these parasites in the hydrothermal vents and to understand the role they play. Plagiopylea ciliates have generally been found in high sulfide, anoxic sediments (Esteban et al., 1993). Previous studies indicate that these ciliates are abundant in sediments from the Guaymas Basin hydrothermal vents (Edgcomb et al., 2002; Coyne et al., 2013). Our study confirmed that Plagiopylea, in particular the genera Epalxella and Trimyema, were diverse and abundant in the Okinawa Trough hydrothermal vent sediments, where they were probably one of the main components of active ciliate community. By contrast, these ciliates were almost absent from the surface sediments in the offshore region as well as the deep sea adjacent to the hydrothermal vents. Karyorelictea ciliates have been confirmed as another active and diverse group in the hydrothermal vent sediments. These ciliates are also thought to be active components in sediments of deep-sea cold seeps (Takishita et al., 2010). Coyne et al. (2013) showed that most of Karyorelictea ciliates in the Guaymas Basin hydrothermal vent were related to the family Trachelocercidae. These ciliates were also abundant in the deep-sea sediments of the Okinawa Trough, but had very low in abundance in the offshore sediments from which however, a high abundance and biomass of Karyorelictea are frequently obtained using morphological methods (Meng et al., 2012). Likewise, the usually highly diverse and abundant Heterotrichea and Litostomatea in the offshore sediments, as revealed by morphological methods, were not fully detected by both DNA and cDNA sequencing. As such, further investigations are needed to bridge the gap between morphological and molecular techniques for accurate estimation of ciliate diversity. DNA and cDNA sequencing uncovered markedly different ciliate community compositions in the hydrothermal vents. This is largely due to the differences inherent to the DNA/cDNA-based techniques. The DNA can be buried and preserved in marine sediments over time (Coolen et al., 2009). Thus, the ciliate diversity detected by DNA sequencing include not only active organisms, but also extracellular DNA, dead cells and resting stages of certain species (Vlassov et al., 2007; Zinger et al., 2012). Generally, OTUs found in the DNA surveys indicate species present, while OTUs found in cDNA surveys represent active species (Massana et al., 2015). The large variation in the rDNA copy number among species may also severely affect DNA/cDNA comparisons in eukaryote diversity (Prokopowich et al., 2003; Gong et al., 2013). In the present study, for instance, DNA sequencing detected the second most abundant sequences as being affiliated to Trachelostyla, but cDNA sequencing yielded only a small proportion of such sequences. By contrast, the sequences affiliated to Epalxella accounted for about 50% of the total cDNA sequences, but no more than 1% of the total DNA sequences. The most plausible explanation is that

31

Trachelostyla might have a high number of rDNA copies, while Epalxella might have a low rDNA copy number. 4.3. The novel ciliate group The environmental conditions of hydrothermal vents are significantly distinct from their surrounding environments and novel, possibly highly divergent, ciliates may exist in such habitats. By using DNA and cDNA sequencing, we detected a novel ciliate group, which is separated from 12 known classes but close to the environmental sequences from the sediments of cold seeps (Takishita et al., 2010) and hydrothermal vents (Coyne et al., 2013). This group was relatively abundant in the Okinawa Trough hydrothermal vent sediments, with sequences accounting for 7–10% of the total sequences, similar to sequence proportion of 8.3% obtained from the Guaymas Basin hydrothermal vent sediments (Coyne et al., 2013). The high sequence contribution implies that they might be an important component of the ciliate community in the sediments of hydrothermal vents. The data indicate that the novel group is well adapted to the hydrothermal vent environment and could exist in far distant locations, such as the western and eastern Pacific. Whether this group is also widely distributed in cold seeps needs further investigation. In the future it will be necessary to study the taxonomy and phylogeny of this novel group ciliates by combining the rDNA detection method, fluorescent in situ hybridization (FISH), light microscopy and scanning electron microscopy (SEM), similar to that conducted by Orsi et al. (2012) in their description of the Class Cariacotrichea.

5. Conclusions DNA and cDNA sequencing revealed a different community composition of ciliates in sediments from the hydrothermal vents. A novel group of ciliates distinctly separated from the 12 known classes was detected in the hydrothermal vents, indicating undescribed, possibly highly divergent ciliates may inhabit such environments. The three habitats were significantly different in terms of the diversity of both the rare and total ciliate taxa, with the adjacent deep sea more similar to the offshore area than to the hydrothermal vents. By contrast, no significant difference was present in the diversity of abundant taxa between the hydrothermal vents and adjacent deep sea, while both habitats were differed significantly from the offshore area.

Acknowledgments This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA11030201) and the National Natural Science Foundation of China (No. 41476144). We thank the WPOS sample center of Institute of Oceanology, Chinese Academy of Sciences and the R/V KEXUE for providing the sediment samples. Special thanks are due to Dr. Alan Warren (The Natural History Museum, London) for kindly refining the English. Thanks are extended to two anonymous reviewers for their thoughtful comments and suggestions on the manuscript.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.dsr.2016.07.007.

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References Anderson, R.E., Sogin, M.L., Baross, J.A., 2015. Biogeography and ecology of the rare and abundant microbial lineages in deep-sea hydrothermal vents. FEMS Microbiol. Ecol. 91, 1–11. Bachraty, C., Legendre, P., Desbruyeres, D., 2009. Biogeographic relationships among deep-sea hydrothermal vent faunas at global scale. Deep-SEA Res. I 56, 1371–1378. Baumgartner, M., Stetter, K.O., Foissner, W., 2002. Morphological, Small Subunit rRNA, and physiological Characterization Of Trimyema minutum (Kahl, 1931), an anaerobic ciliate from submarine hydrothermal vents growing from 28 °C to 52 °C. J. Eukaryot. Microbiol. 49, 227–238. Bik, H.M., Sung, W., De Ley, P., Baldwin, J.G., Sharma, J., Rocha-Olivares, A., Thomas, W.K., 2012. Metagenetic community analysis of microbial eukaryotes illuminates biogeographic patterns in deep-sea and shallow water sediments. Mol. Ecol. 21, 1048–1059. Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T., Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D., Pirrung, M., Reeder, J., Sevinsky, J.R., Tumbaugh, P.J., Walters, W.A., Widmann, J., Yatsunenko, T., Zaneveld, J., Knight, R., 2010. QIIME allows analysis of highthroughput community sequencing data. Nat. Methods 7, 335–336. Chao, A., Lee, S.M., Jeng, S.L., 1992. Estimating population size for capture-recapture data when capture probabilities vary by time and individual animal. Biometrics 48, 201–216. CIPRES (Cyberinfrastructure for Phylogenetic Research), 2014. CIPRES Science Gateway. Avaliable: 〈http://www.phylo.org〉. K.R., Clarke, R.N., Gorley, 2006. PRIMER v6: User Manual/Tutorial. PRIMER-E Ltd, Plymouth, UK. Coolen, M.J.L., Saenz, J.P., Giosan, L., Trowbridge, N.Y., Dimitrov, P., Dimitrov, D., Eglinton, T.I., 2009. DNA and lipid molecular stratigraphic records of haptophyte succession in the Black Sea during the Holocene. Earth Planet. Sci. Lett. 284, 610–621. Coyne, K.J., Countway, P.D., Pilditch, C.A., Lee, C.K., Caron, D.A., Cary, S.C., 2013. Diversity and distributional patterns of ciliates in Guaymas Basin hydrothermal vent sediments. J. Eukaryot. Microbiol. 60, 433–447. Edgar, R.C., 2013. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996–998. Edgcomb, V.P., Kysela, D.T., Teske, A., Gomez, A.D., Sogin, M.L., 2002. Benthic eukaryotic diversity in the Guaymas Basin hydrothermal vent environment. Proc. Natl. Acad. Sci. USA 99, 7658–7662. Esteban, G., Finlay, B.J., Embley, T.M., 1993. New species double the diversity of anaerobic ciliates in a Spanish lake. FEMS Microbiol. Lett. 109, 93–100. Fenchel, T., 1965. Ciliates from Scandinavian molluscs. Ophelia 2, 71–174. Finlay, B.J., 2002. Global dispersal of free-living microbial eukaryote species. Science 296, 1061–1063. Foissner, W., 1999. Protist diversity and distribution: some basic considerations. Biodivers. Conserv. 150, 363–368. Foissner, W., 2008. Protist diversity and distribution: some basic considerations. Biodivers. Conserv. 17, 235–242. Fuhrman, J.A., 2009. Microbial community structure and its functional implications. Nature 459, 193–199. Glasby, G.P., Notsu, K., 2003. Submarine hydrothermal mineralization in the Okinawa Trough, SW of Japan: an overview. Ore Geol. Rev. 23, 299–339. Gong, J., Dong, J., Liu, X., Massana, R., 2013. Extremely high copy numbers and polymorphisms of the rDNA operon estimated from single cell analysis of oligotrich and peritrich ciliates. Protist 164, 369–379. Gong, J., Shi, F., Ma, B., Dong, J., Pachiadaki, M., Zhang, X., Edgcomb, V.P., 2015. Depth shapes alpha- and beta-diversities of microbial eukaryotes in surficial sediments of coastal ecosystems. Environ. Microbiol. 17, 3722–3737. Kato, S., Hara, K., Kasai, H., Teramura, T., Sunamura, M., Ishibashi, J.I., Kakegawa, T., Yamanaka, T., Kimura, H., Marumo, K., Urabe, T., Yamagishi, A., 2009. Spatial distribution, diversity and composition of bacterial communities in sub-seafloor fluids at a deep-sea hydrothermal field of the Suiyo Seamount. Deep-Sea Res. I 56, 1844–1855. Kim, S., Hammerstrom, K., 2012. Hydrothermal vent community zonation along environmental gradients at the Lau back-arc spreading center. Deep-Sea Res. I 62, 10–19. Lara, E., Berney, C., Harms, H., Chatzinotas, A., 2007. Cultivation-independent analysis reveals a shift in ciliate 18S rRNA gene diversity in a polycyclic aromatic hydrocarbon-polluted soil. FEMS Microbiol. Ecol. 62, 365–373. Levin, L.A., Mendoza, G.F., Konotchick, T., Lee, R., 2009. Macrobenthos community

structure and trophic relationships within active and inactive Pacific hydrothermal sediments. Deep-Sea Res. II 56, 1632–1648. López-García, P., Philippe, H., Gail, F., Moreira, D., 2003. Autochthonous eukaryotic diversity in hydrothermal sediment and experimental microcolonizers at the Mid-Atlantic Ridge. Proc. Natl. Acad. Sci. USA 100, 697–702. López-García, P., Vereshchaka, A., Moreira, D., 2007. Eukaryotic diversity associated with carbonates and fluid-seawater interface in Lost City hydrothermal field. Environ. Microbiol. 9, 546–554. Lynn, D.H., 2008. The ciliated protozoa: Characterization, classification, and guide to the literature, third ed. Springer Science, Dordrecht, Netherlands. Massana, R., Gobet, A., Audic, S., Bass, D., Bittner, L., Boutte, C., Chambouvet, A., Christen, R., Claverie, J.M., Decelle, J., Dolan, J.R., Dunthorn, M., Edvardsen, B., Forn, I., Forster, D., Guillou, L., Jaillon, O., Kooistra, W.H.C.F., Logares, R., Mahe, F., Not, F., Ogata, H., Pawlowski, J., Pernice, M.C., Probert, I., Romac, S., Richards, T., Santini, S., Shalchian-Tabrizi, K., Siano, R., Simon, N., Stoeck, T., Vaulot, D., Zingone, A., de Vargas, C., 2015. Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing. Environ. Microbiol. 17, 4035–4049. Meng, Z., Xu, K., Dai, R., Lei, Y., 2012. Ciliate community structure, diversity and trophic role in offshore sediments from the Yellow Sea. Eur. J. Protistol. 48, 73–84. Moreira, D., López-García, P., 2003. Are hydrothermal vents oases for parasitic protists? Trends Parasitol. 19, 556–558. Ohta, S., Kim, D., 2001. Submersible observations of the hydrothermal vent communities on the Iheya Ridge, Mid Okinawa Trough, Japan. J. Oceanogr. 57, 663–677. Orsi, W., Edgcomb, V., Faria, J., Foissner, W., Fowle, W.H., Hohmann, T., Suarez, P., Taylor, C., Taylor, G.T., Vd’acny, P., Epstein, S.S., 2012. Class Cariacotrichea, a novel ciliate taxon from the anoxic Cariaco Basin, Venezuela. Int. J. Syst. Evol. Microbiol. 62, 1425–1433. Pedrós-Alió, C., 2006. Marine microbial diversity: can it be determined? Trends Microbiol. 14, 257–263. Prokopowich, C.D., Gregory, T.R., Crease, T.J., 2003. The correlation between rDNA copy number and genome size in eukaryotes. Cell 50, 48–50. Ruff, S.E., Biddle, J.F., Teske, A.P., Knittel, K., Boetius, A., Ramette, A., 2015. Global dispersion and local diversification of the methane seep microbiome. Proc. Natl. Acad. Sci. USA 112, 4015–4020. Sauvadet, A.L., Gobet, A., Guillou, L., 2010. Comparative analysis between protist communities from the deep-sea pelagic ecosystem and specific deep hydrothermal habitats. Environ. Microbiol. 12, 2946–2964. Small, E.B., Gross, M.E., 1985. Preliminary observations of protistan organisms, especially ciliates from the 21°N hydrothermal vent site. Bull. Biol. Soc. Wash. 6, 401–410. Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the RAxML web servers. Syst. Biol. 57, 758–771. Stock, A., Edgcomb, V., Orsi, W., Filker, S., Breiner, H.W., Yakimov, M.M., Stoeck, T., 2013. Evidence for isolated evolution of deep-sea ciliate communities through geological separation and environmental selection. BMC Microbiol. 13, 150. Stoeck, T., Bass, D., Nebel, M., Christen, R., Jones, M.D.M., Breiner, H.W., Richards, T. A., 2010. Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Mol. Ecol. 19, 21–31. Takishita, K., Kakizoe, N., Yoshida, T., Maruyama, T., 2010. Molecular evidence that phylogenetically diverged ciliates are active in microbial mats of deep-sea coldseep sediment. J. Eukaryot. Microbiol. 57, 76–86. Tokeshi, M., 2011. Spatial structures of hydrothermal vents and vent-associated megafauna in the back-arc basin system of the Okinawa Trough, western Pacific. J. Oceanogr. 67, 651–665. Tyler, P.A., Young, C.M., 2003. Dispersal at hydrothermal vents: a summary of recent progress. Hydrobiologia 503, 9–19. Vlassov, V.V., Laktionov, P.P., Rykova, E.Y., 2007. Extracellular nucleic acids. Bioessays 29, 654–667. Xu, K., Song, W., 2003. Parasitic ciliates of cultured molluscs. In: Song, W., Zhao, Y., Xu, J., Hu, X., Gong, J. (Eds.), Pathogenic Protozoa in Mariculture. Science Press, Beijing, pp. 115–145 (in Chinese). Xu, K., Song, W., Warren, A., 2015. Two new and two poorly known species of Ancistrum (Ciliophora, Scuticociliatia, Thigmotrichida) parasitizing marine molluscs from Chinese coastal waters of the Yellow Sea. Acta Protozool. 54, 195–208. Zinger, L., Gobet, A., Pommier, T., 2012. Two decades of describing the unseen majority of aquatic microbial diversity. Mol. Ecol. 21, 1878–1896.