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

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Highlights

  • DNA sequencing and cDNA sequencing uncovered different patterns of ciliate diversity in the hydrothermal vent sediments.

  • A novel group of ciliates was detected in the hydrothermal vent sediments.

  • Abundant ciliate taxa have a wider range of distribution than rare taxa.

  • Differences in the ciliate community structure between hydrothermal vents and adjacent sea areas are mainly due to rare taxa.

Abstract

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.

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 (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 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 (López-García et al., 2003, López-García et al., 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.

Section snippets

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 and HV2

Data overview

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

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, 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

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

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

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