2 Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China;
3 Department of Life Sciences, Natural History Museum, London SW7 5BD, United K ingdom;
4 College of Life Sciences, Capital Normal University, Beijing 100048, China
Ciliated protists are a major component of microzooplankton (20–200 μm in size). As intermediaries between primary producers and higher consumers, planktonic ciliates play essential roles in the circulation of materials and energy within a microbial loop (Azam et al., 1983; Song et al., 2009). Members of the subclass Oligotrichia have high abundance and episodically dominate marine planktonic ciliate communities (Pierce and Turner, 1992). Previous investigations have revealed that oligotrich ciliates are widely distributed in marine habitats and constitute a relatively high proportion of biomass in the water column (Endo et al., 1983; Martin and Montagnes, 1993; Leakey et al., 1996; Pitta et al., 2001). They also make a significant contribution to trophic interactions, energy flux and nutrient cycling (Worden et al., 2015). Although the importance of oligotrichs in ecological and biogeochemical processes has been revealed in various studies, their diversity is not well documented.
In recent decades, the application of silver-staining methods in taxonomic studies of oligotrichs has significantly increased our knowledge of their systematics and diversity (Modeo et al., 2003; Agatha, 2011; Foissner, 2014). However, due to their small size, rapid locomotion, ability to readily encyst, and susceptibility to sudden extirpation, species identification of oligotrichs based on morphological methods alone is difficult, especially for those that are rare and/or present in low abundance (Agatha et al., 2005; Xu, 2007; Lee et al., 2011). To date, about 100 morphospecies of marine oligotrichs have been reported although many of these are poorly known (Liu et al., 2015). Furthermore, there is increasing evidence for the existence of a high, undiscovered oligotrich diversity, and it has been estimated that about 83%–89% of oligotrich species are still unknown (Knowlton, 1993; McManus and Katz, 2009; Agatha, 2011; Liu et al., 2011, 2013, 2015, 2016; Song et al., 2018; Wang et al., 2018). Over the past decade, several diversity surveys of oligotrichs in northern coastal waters of the South China Sea have been conducted using morphological methods, usually in combination with molecular methods to reveal their phylogenetic relationships (e.g. Gao et al., 2009; Liu et al., 2010, 2011, 2013, 2015; Zhang et al., 2010; Liu, 2011; Zhang, 2012; Li et al., 2013; Song et al., 2013, 2015a; Gao et al., 2016a). Collectively these studies have recorded 46 morphospecies belonging to 15 genera. Nevertheless, even in this intensively studied region, new species (16 out of 46 morphospecies) are continuously being described (Song et al., 2015b; Liu et al., 2016). This suggests the existence of a high diversity of oligotrichs in northern coastal waters of the South China Sea (Song et al., 2018).
Several studies have demonstrated that molecular methods could greatly facilitate the discovery of diversity of low-abundance species (Díez et al., 2001; Countway et al., 2005; Bachy et al., 2013, 2014; Santoferrara et al., 2013; Huang et al., 2018; Sheng et al., 2018; Wang et al., 2019). The small subunit ribosomal RNA gene (SSU rDNA) is the most frequently used marker to study species identification and molecular diversity of ciliates (Edgcomb et al., 2002; Bachy et al., 2012). However, recent studies showed that the SSU rDNA is too conserved to distinguish closely related ciliate species (e.g. Stoeck et al., 2010; Strüder-Kypke and Lynn, 2010; Zhao et al., 2018). Evidence is now accumulating that the large subunit ribosomal RNA gene (LSU rDNA) is more reliable for species separation in various ciliate groups such as Oligotrichia and its sister group Choreotrichia (Santoferrara et al., 2013, 2015, 2017; Stoeck et al., 2014; Zhao et al., 2016, 2018). Additionally, the LSU rDNA sequence is easy to obtain and has potential applicability as a specieslevel marker for a wide range of taxa (Tautz et al., 2003; Stoeck et al., 2014; Zhao et al., 2018). Consequently, LSU rDNA might be better than SSU rDNA for studying the biodiversity of the Oligotrichia.
Here, an investigation based on LSU rDNA sequences was carried out at five widely separated sampling sites in northern coastal waters of the South China Sea. The main aims of this study were to provide specific primers for oligotrich ciliates and to investigate their molecular diversity in an area where their morphospecies diversity is well studied.2 MATERIAL AND METHOD 2.1 Sampling
Surface water samples were collected from five widely separated sites along the coast of southern China: mangrove forests in Zhanjiang (110°26'E, 21°9'N) on 23 July, 2015; mangrove forests in Zhuhai (113°36'E, 21°22'N) on 2 April, 2015; an estuary in Shenzhen (113°58'E, 22°31'N) on 7 May, 2015; an estuary in Guangzhou (113°37'E, 22°45'N) on 19 May, 2015; and nearshore waters in Huizhou (114°42'E, 22°47'N) on 1 June, 2015 (Fig. 1). All samples were collected with HQM-1 sampling bottles during high tides and immediately stored at 4℃. Within one hour of collection, each water sample was pre-filtered through a 200-μm mesh nylon sieve and filtered through a 0.22-μm pore polycarbonate membrane filter (Millipore, Billerica, MA, USA). Membranefi lters were stored at -80℃ before DNA extraction.2.2 DNA extraction and sequencing
Total DNA was extracted from membrane filters using PowerSoil DNA Isolation Kits (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions. Hitherto, there was no LSU rDNA primer designed for oligotrichs. Using Primer Premier 5.0 (Ren et al., 2004), oligotrichspecific primers were designed to target all sequenced oligotrichs. These were based on alignments of LSU rDNA sequences of 32 oligotrichs and 25 nonoligotrich ciliates retrieved from the NCBI database (Supplementary Table S1). Our designed primer set was 80F (5'-GCCARAGCCCAAGATGGWAA-3') and 700-R3 (5'-CKGGAYACTCGCGYGTACGT-3'), covering the 600-bp D1/D2 region of the LSU rDNA. The specificity of the newly designed primers was experimentally evaluated by PCR using DNA samples of 15 oligotrich and 15 non-oligotrich species which were cultivated in the Laboratory of Protozoology at the South China Normal University, Guangzhou, or in the Laboratory of Protozoology, Ocean University of China, Qingdao (Supplementary Table S2). The taxonomy identification and morphological descriptions for most of these species have been published (Liu, 2011; Liu et al., 2012, 2013, 2015, 2016; Shen, 2012; Chen, 2014; Wu et al., 2017). The amplification for primer testing and cloning libraries were performed with 0.8 μL of each primer, 2.8 μL template DNA, 5 μL dNTPs, 5 μL Ex TaqTMbuffer and 0.4 μL TaKaRa Ex Taq DNA Polymerase in a total volume of 50 μL. Cycling conditions were 98℃ for 5 min, followed by 30 cycles of 95℃ for 15 s, 56℃ for 15 s and 72℃ for 90 s, with a final extension at 72℃ for 10 min. PCR products were checked in 1.5% agarose gels stained with GelRed (Biotium). Purified PCR products from each sample were cloned into the pMD-18 vector using TIANgel Mini Purification kit (Tiangen). Positive bacterial clones from each sample were sequenced in both directions on ABI3500 Dx sequencer using M13F and M13R primers.2.3 Sequence alignment and phylogenetic analyses
All sequences were checked using BlastN in NCBI websites. No chimeras were detected and nonoligotrich sequences were deleted. All newly obtained oligotrich sequences were clustered at a 99% threshold using QⅡME (Caporaso et al., 2010). This was based on the prediction that nearly all intra-specific genetic differences were ≤1%, and inter-specific differences were ≥7%, following the calculation of similarities among all available LSU rDNA oligotrich sequences, i.e., 21 isolates of nine species (Supplementary Table S3). All sequences of species in Supplementary Table S3 were derived from genomic DNA extracted from species identified in our laboratory. It is noteworthy that 99% and 99.4% similarity have previously been used to delineate tintinnid and Paramecium species, respectively (Santoferrara et al., 2013; Stoeck et al., 2014). A BLAST search on the NCBI database was conducted in order to check the identity of operational taxonomic units (OTUs) with published sequences.
All available LSU rDNA sequences of oligotrichs, including newly sequenced ones and those downloaded from GenBank, were used for phylogenetic analyses. Oligotrichs were classified following the system of Gao et al. (2016b). Additionally, several sequences from the subclasses Hypotrichia and Choreotrichia were included in our analyses. Certesia quadrinucleata and Aspidisca leptaspis (order Euplotida) were selected as outgroup taxa. Sequences were aligned and manually edited using the multiple-alignment program Clustal in Bioedit 22.214.171.124 (Hall, 1999). A Bayesian tree was constructed with MrBayes 3.2.2, using the GTR+ G+I model selected by MrModeltest (version 2; Nylander, 2004). Markov chain Monte Carlo simulations were run for 1 000 000 generations with four chains. Trees were sampled every 100 generations and the first 2 500 trees were discarded as burn-in. A maximum likelihood tree (ML) was constructed using online software RaxML-HPC2 on XSEDE (http://www.phylo.org/). The branches of the resulting tree were evaluated by GTRGAMMA of nucleotide substitution and a bootstrap analysis based on 1 000 replicates. MEGA7 (Kumar et al., 2016) and Adobe Photoshop CS5 (Demir and Sayood, 1998) were used to visualize tree topologies.3 RESULT 3.1 Specific LSU rDNA primer pairs for subclass Oligotrichia
The specificity of newly designed primers was demonstrated by positive PCR results for all 15 oligotrich species and negative PCR results for all 15 non-oligotrich species (Supplementary Table S2). A total of 168 environmental sequences from all samples was recovered, of which only three were not oligotrichs when checked using BLAST searches. These findings demonstrate that our newly designed LSU rDNA primer pairs are good candidates for detecting oligotrich species in environmental samples. All 165 newly generated oligotrich sequences were submitted to the GenBank with accession numbers MK501384–MK501548.3.2 Community composition of oligotrichs
The three non-oligotrich OTUs were deleted before downstream analyses. Using the 99% similarity threshold, 60 OTUs were obtained from the 165 remaining sequences (Supplementary Table S4). Among 60 OTUs, three (OTU1, OTU3, and OTU12) had 99% similarities with oligotrich sequences in GenBank, and 30 and 27 OTUs had < 93%, 93%–99% similarities, respectively (Supplementary Table S4). OTU14 and OTU30 had best hits to Halteria grandinella. The remaining 58 OTUs all had best hits or second-best hits, if the first one is unidentified, to one of 11 oligotrich morphospecies, i.e., Strombidium sp., S. guangdongense, S. basimorphum, S. paracalkinsi, S. crassulum, Spirotontonia turbinata, Varistrombidium kielum, Cyrtostrombidium paralongisomum, Apostrombidium parakielum, Sinistrostrombidium cupiformum, and Williophrya maedai (Fig. 2, Supplementary Table S4).
At Huizhou (HZ), eight OTUs were obtained (Table 1, Fig. 3). Among these, OTU12 was the most frequently detected with six sequences, followed by OTU30 with four sequences. Two sequences each of OTU1 and OTU49, and one sequence each of OTU14, OTU15, OTU27, and OTU40, were also detected. At Guangzhou (GZ), nine OTUs were detected. OTU41, OTU46, OTU51, and OTU57 were the most frequently detected with 5, 13, 10, and 10 sequences, respectively. Shenzhen (SZ) had the largest number of OTUs among the five sampling sites (27 out of 60), most of which (26 out of 27) had only one or two sequences, although 23 sequences were detected for OTU49. Fewest OTUs (six out of 60) were present at Zhuhai (ZH), i.e., OTU6, OTU8, OTU9, OTU10, OTU24, and OTU25, all of which were ≤99% similar to sequences in GenBank. OTU24 was the most frequently encountered with 12 sequences at this site. At Zhanjiang (ZJ), 12 OTUs were detected, ten of which were < 93% similar, and two 93%–99% similar, to sequences in GenBank.
OTUs with one or two sequences accounted for about 77% of total OTUs (46 out of 60). OTU49 (HZ, SZ) and OTU10 (GZ, ZH) were present at two sites whereas each of the other 58 OTUs was detected only at one site. OTU49 was the most frequently encountered (20 at SZ and two at HZ). Among the five sampling sites, HZ had most OTUs (n=2) with high similarity (99%) to sequences in GenBank. Two OTUs (OTU1, OTU12) out of eight at this site had a similarity of 99% to oligotrich sequences in GenBank. Other sites had few OTUs with high similarity to sequences in GenBank, e.g., only OTU3 at GZ. All OTUs at SZ, ZJ, and ZH had < 99% similarities to oligotrich sequences in GenBank (Table 1).3.3 Phylogenetic trees of oligotrich ciliates
In the LSU rDNA tree, the subclass Oligotrichia formed a large clade that was sister to the subclass Choreotrichia (Fig. 4). Support for deep nodes was generally low whereas that for crown clades was variable. Most of the 36 identified oligotrich species (Fig. 4, green shaded) did not group with newly sequenced OTUs. Fifty-one OTUs could be divided into 12 clades (Clades 1–12) each containing 2–9 OTUs with variable support. Of the remaining nine OTUs, OTU4 was sister to Strombidium species (92% ML, 1.00 BI) and OTU40 was sister to another clade (51% ML, 0.83 BI). The other OTUs (i.e., OTU42, OTU3, OTU36, OTU5, OTU28, OTU59, and OTU19) did not group with any sequence with more than 50% support. Among Clades 1–12, only Clades 1, 4, 6, 8, and 12 were nested within, or sister to, clades containing identified species. Furthermore, branch lengths of Williophorya maedai in Clade 4 and Strombidium triquetrum in Clade 6 were very long. Seven of the 12 clades comprised OTUs from one site only. All OTUs in Clades 3–5 were from ZJ, all those in Clades 10–11 were from SZ, all those in Clade 6 were from HZ, and all those in Clade 2 were from GZ.4 DISCUSSION 4.1 Strombidium-related OTUs are abundant in the northern coastal waters of the South China Sea
Based on the findings of the present study, Strombidium is the most abundant and widely distributed oligotrich taxon in northern coastal waters of the South China Sea during late spring and early summer. Community composition analyses indicated that 35 out of 60 OTUs were most closely related to Strombidium, i.e., best hit or second-best hit if the first one is unidentified (Supplementary Table S4). Distribution analyses revealed that: these Strombidium-related OTUs were present at all five sampling sites; one of the two OTUs that were detected at two sites, i.e., OTU49 (HZ, SZ) was also Strombidium-related; and, most of the high-abundant OTUs were closely related to Strombidium, for example, seven out of 10 OTUs with more than five sequences, and 15 out of 20 OTUs with more than two sequences. Furthermore, analyses of the phylogenetic trees showed that many OTU clades, i.e., OTU1 and OTU12 in Clade 6, OTU25, OTU41, OTU9 and OTU24 in Clade 8, and OTU29 and OTU49 in Clade 9, were sister to Strombidium sequences (Fig. 4). These findings are consistent with morphological studies which concluded that Strombidium is the most frequently encountered oligotrich genus in coastal waters of the South China Sea with about 65 reported morphospecies (e.g. Li et al., 2013). It should be noted, however, that Strombidium was found to be non-monophyletic in our phylogenetic trees (Fig. 4), which was consistent with previous molecular phylogenetic studies (e.g. Doherty et al., 2007; McManus et al., 2010; Song et al., 2015a, b; Gao et al., 2016a; Liu et al., 2016).4.2 Diversity of oligotrich species is still underestimated
The potential for improving understanding of ciliate diversity and distribution by using groupspecific primers has been previously reported (e.g. Doherty et al., 2010; Bachy et al., 2013; Su et al., 2018). Interestingly, our phylogenetic trees showed that more than 50% of identified species did not have a close relationship with any newly sequenced OTUs in the LSU rDNA trees (Fig. 4, green shaded), and 27 out of 60 OTUs had a low similarity with sequences of identified species (Supplementary Table S4). A possible reason for this is the lack of LSU rDNA sequence data in reference databases for certain common morphospecies compared, for example, to SSU rDNA sequences (Zhao et al., 2018). Based on morphological methods, 47 species of 15 oligotrich genera (Strombidium, Novistrombidium, Williophrya, Spirostrombidium, Parallelostrombidium, Omegastrombidium, Varistrombidium, Spirotontonia, Pseudotontonia, Cyrtostrombidium, Pseudostrombidium, Antestrombidium, Laboea, Limnostrombidium, and Sinistrostrombidium) have previously been reported in northern coastal waters of the South China Sea, but LSU rDNA sequences for only 27 species are available in the GenBank (Gao et al., 2016a, b). Given the proximity of the present sampling sites to those of previous morphology-based studies, it is reasonable to expect that some OTUs will be affiliated unsequenced taxa among these 47 morphospecies. Undersampling in this study might be another reason for the low proportion of OTUs affiliated to known oligotrichs (Fig. 4). Morphologically, most of these identified species (e.g. Parallelostrombidium and Spirostrombidium species) possess thigmotactic membranelles and thus have a tendency to temporarily attach to substrates (Agatha, 2004). All our samples, however, came from surface water, so species attached to benthic substrates and/or located in other layers of the water column were unlikely to have been collected. Furthermore, samples from only five sites, one sampling season, and at a single depth per site, were analyzed. In order to mitigate these problems, future studies should include more sampling sites at different spatial and temporal scales. Nevertheless, the fact that most OTUs could not be assigned to identified species indicates that oligotrich species diversity might be much richer than expected.4.3 Utility of LSU rDNA for investigating ciliate diversity
Previous studies using various ciliate groups indicated that the D1–D2 region of LSU rDNA can be used for distinguishing ciliate species and is, therefore, a good candidate gene marker for studying ciliate diversity (Santoferrara et al., 2013, 2015, 2017; Stoeck et al., 2014; Zhao et al., 2016, 2018). Our newly designed oligotrich-specific primes covering the 600-bp D1-D2 region of the LSU rDNA are very reliable as demonstrated by their application both to environmental and to individual morphospecific DNA samples (Supplementary Tables S2, S3). The only significant constraint is that, although the fragment length produced by the new primers can be sequenced with a single Sanger read, it is too long for most current high-throughput sequencing (HTS) methods (e.g. around 300–500 bp for Miseq/Hiseq). Nevertheless, it has been reported that the Ion S5 sequencing system could sequence 600bp fragments, which means that our primers should be useful for HTS in the future (Dinauer et al., 2014).
A major constraint on the utility of LSU rDNA for investigating ciliate diversity is the paucity of sequences in publicly accessible databases compared, for example, to SSU rDNA (Zhao et al., 2018). As a consequence, the bulk of the phylogenetic diversity of oligotrichs based on LSU rDNA sequence data corresponds to undescribed genera or families (Santoferrara et al., 2017). Therefore, it is likely the LSU rDNA database is under-populated. Another reason for the lack of matches with reference sequences might be that population sizes of the OTUs in the present study were tiny, representing lowabundant species that were probably overlooked in morphology-based studies (Yi et al., 2010). For example, 17 out 27 OTUs with < 93% similarity to sequences of oligotrich species in the GenBank database was each represented by only one sequence. There is increasing evidence that low-abundant ciliate species are usually undetected in traditional morphological studies but their presence can be revealed using molecular methods (e.g. Jung et al., 2012; Bachy et al., 2013; Grattepanche et al., 2013; Dong et al., 2014; Dunthorn et al., 2014; Santoferrara et al., 2016; Zhao and Xu, 2016; Tucker et al., 2017). This is consistent with previous reports that a large number of oligotrich morphospecies are still undescribed (Agatha, 2011; Santoferrara et al., 2017). Therefore, more effort should be focused on oligotrichs in respect of populating LSU rDNA sequence databases and documenting morphospecies if we want to determine the extent of their local and global species diversity.5 CONCLUSION
This study represents one of the first attempts to investigate oligotrich diversity using group-specific LSU rDNA primers. The main limitations were that of undersampling and the paucity of LSU rDNA oligotrich sequences in publicly accessible databases. Nevertheless, significant advancements were made in terms of designing a pair of oligotrich-specific LSU rDNA primers for future diversity studies, and providing more information for oligotrich diversity in northern coastal waters of the South China Sea. In accordance with previous morphology-based studies, Strombidium-related OTUs are the most widely distributed and abundant among oligotrichs in northern coastal waters of the South China Sea. Future studies should focus more efforts on populating LSU rDNA sequence databases and describing morphospecies, in order to increase knowledge and understanding of oligotrich diversity.6 DATA AVAILABILITY STATEMENT
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Electronic supplementary material
Supplementary material (Supplementary Tables S1–S4) is available in the online version of this article at https://doi.org/10.1007/s00343-019-9021-0.
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