GONG Sanqiang, ZHANG Fengli, and LI Zhiyong



Genotype and Phylogenetic Diversity ofITS2 Sequences WithinClade C in Three Typical Coral Species from Luhuitou Fringing Reef of the South China Sea

GONG Sanqiang, ZHANG Fengli, and LI Zhiyong*

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Dinoflagellates in the genus, including nine clades (A–I), mainly form mutualistic symbioses with corals. More than 100molecular types have been identified by the ITS2-based genotype method within any given clade, and specifically withinclade C. However, the genotype identification method using the ITS2 sequence is likely to lead to high diversity estimates due to the intra-genomic variations in the ITS2 space; thus, further validation is essential for a correct identification.In this study, the molecular diversity ofITS2 sequences cloned from two stone corals,sp. SY-01 andsp. SY-05, and one soft coral,sp. SY-07, living in the northern part of South China Sea (SCS), were analyzed and compared using the ITS2-based genotype identification method, coupled with ITS2-based secondary structural and phylogenetic analyses. As the result, 12ITS2 genotypes were identified, while only six and threeITS2 genotypes were supported by ITS2-based secondary structural and phylogenetic analyses, respectively. In addition, no sharedITS2 genotypes were observed among the three coral species, suggesting coral species-dependentgenotypes were within clade C. In summary, the present study provides a theoretical basis for validating the molecular diversity ofITS2 genotypes in corals.

clade C; hard coral; ITS2 genotypes; ITS2 secondary structure; phylogenetic analysis; soft coral

1 Introduction

The unicellular algal symbionts in the genusare keystone components of coral holobionts, which contribute to the coral host by fixing carbon, while the coral host provides inorganic nutrients and a microenvironment for(Falkowski., 1984; Muscatine, 1990; Barott., 2015). The loss of symbioticfrom coral tissues leads to coral bleaching under global climate change, and further collapse of reef ecosystems will occur if thepopulations fail to recover (Brow., 1999). Thus, understanding the diversity of symbioticin coral holobionts is critical to the-coral symbioses and the future fate of coral reefs (Baker, 2003).

Exploringdiversity at the species level is difficult because the pure cultures of symbioticstrains for morphological descriptions are difficult (Baker, 2003). However, increasing success describingdiversity using molecular methods has opened up an exciting avenue.Over the past two decades, nine large clades (A–I) ofhave been suggested as distinct phylogenetic lineages by various relatively conserved molecular markers, including nuclear small subunit ribosomal-(Rowan.,1992), nuclear large subunit ribosomal-(Pawlowski, 2001), chloroplast large subunit ribosomal DNA-(Santos, 2002; Santos and Coffroth, 2003; Santos, 2003; Pochon., 2010), the coding re- gion of the plastid-encoded photosystem II protein D1-(Takishita, 2003), mitochondrial cytochrome oxidase I-(Takabayashi, 2004), and mitochon- drial cytochrome b-(Zhang, 2005). The exis- tence of nine phylogenetically distinctclades has also been supported by phylogenetic reconstructions using three organelles (mitochondrial, chloroplast, and nuclear) molecular markers (Santos, 2002; Takaba- yashi,2004; Pochon, 2012; Pochon, 2014).

Differenttypes within any given clade can be further identified by the most commonly used less- conserved internal transcribed spacer region 2 (ITS2) se- quence (Hunter,1997; LaJeunesse, 2001; LaJeu- nesse, 2010;Wicks, 2010; Silverstein, 2011; Putnam,2012; Tonk,2013; Thomas, 2014). A particularITS2 genotype has been identified by diagnosing a single-base mutation inITS2 sequences using the genotype method (Wicks., 2010; Silverstein, 2011; Putnam,2012). Particularly, clade Cis the most predominant, geographically widespread, and ecologically diverseamong the nine clades. More than 100ITS2 genotypes within clade C have been suggested in different coral reef dwelling animals (., reef building corals, non-reef building corals, flatworms, sponges, foraminifera, and protists) across different geographical scales (Wicks,2010; LaJeunesse, 2010; Silverstein,2011; Putnam, 2012).

However, the major ITS2-based genotype identification method for diversity analysis ofneeds further validation due to the intra-genomic variations in the ITS2 space (Thomas., 2014). In this study, the molecular diversity ofITS2 sequenceswithin the most predominant clade C, cloned from widespread and ecologically dominant stone coralssp. SY-01,sp. SY-05, and one soft coral,sp. SY-07, from the Luhuitou fringing reef, northern part of the SCS, were analyzed and compared by ITS2-based genotyping. In addition,secondary structural and phylogenetic analyses of theITS2 sequences were performed to validate the ITS2-based geno- type identification method.

2 Materials and Methods

2.1 Coral Samples and Tissue Preparation

Two stone coral species and one soft coral species were collected by SCUBA diving at a 5m depth around the Luhuitou fringing reefs (109.470˚E, 18.200˚N) in the northern part of the SCS in July 2014. These coral species were identified assp. SY-01 (KP774812 and KP774804),sp. SY-05 (KP774816 and KP774808), andsp. SY-07 (KP444811 and KP774803) based on the analyses of nuclear ribosomal 18S rDNA and mitochondrial.

Three coral individualsfrom each coral species were selected and cut into tissue pieces<5mm and <2cm3.Then they were rinsed twice in artificial seawater to remove the microbes that loosely attached to the coral surface, transferred immediately (<15min) to clean tubes containing 20mL RNA Later (Qiagen, Hilden, Germany), and stored at −80℃ for further DNA extraction.

2.2 DNA Extraction, Polymerase Chain Reaction (PCR) Amplification, Cloning, and Sequencing

For total DNA extraction, coral tissue samples of each individual coral species were transferred to 1.5mL tubes (Eppendorf, Hamburg, Germany). A 500μL aliquot of 0.5mm sterile glass beads (BioSpec, Bartlesville , UK) was added with 1mL of AP1 buffer and 6μL RNAse (Qiagen). The coral tissue samples were bead-beaten for 90s with a TissueLyser-48 (Jinxin, China). DNA was isolated with the Qiagen DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s protocol. Then, the ITS region of clade Cwas selectively PCR-amplified with pri- mer set SymITSFP (5’-CTC AGC TCT GGA CGT TGY GTT GG-3’) and SymITS-b (5’-GCG GGT TCA CTT GTC TGA CT-3’) (Lien, 2013), and cloned into the pEASY’T5 Zero Cloning vector (Transgene Biotech, Beijing, China). Fifty clones per coral tissue sample were picked up for further Sanger sequencing. All ITS sequences were processed using Mothur v.1.31.2 software (Schloss, 2009). Chimera and singleton sequences were further removed with UCHIME and singleton commands in Mothur. Phylogenetically distinct ITS sequences were submitted to GenBank with accession numbers KP774819–KP774824.

2.3 Genotype Diversity and Secondary Structure of Symbiodinium ITS2 Sequences Within Clade C

Genotyping ofis mainly based on the ITS2 fragment, and no specific primer is available to amplifyITS2 within clade C from corals; thus, the full-lengthITS sequences were obtained firstly using aclade C specific pri- mer (as described earlier). TheITS2 sequences were further retrieved from the full lengthITS sequences by blasting against theITS2 database (Arif., 2014) and GenBank. Then, the retrievedITS2 sequences were used to prepare the haplotype network (Lien, 2013; Thomas, 2014). The genotype network was drawn using minimum spanning networks (http://www.fluxus-engineering.com/sharenet.htm).

The relative abundances of differentgenotypes in each coral species were calculated according to the number ofITS2 sequences from individual coral species. A rarefaction curve was drawn using script in the Qimme package (Costello., 2010) based on relative abundances of differentgenotypes in each coral species.

To obtain information about the secondary structure of all designatedITS2 genotypes, all ITS2sequences of the designatedgenotypes were further blast against homologous sequences with known ITS2 secondary structures in the ITS2 Database (http:// its2.bioapps.biozentrum.uni-wuerzburg.de) according to the method described by Koetschan. (2009).

2.4 Phylogenetic Analysis of Symbiodinium Types Within Clade C

All valid ITS2 sequences of clade Cwere aligned and edited using MEGA 6.06 (Tamura, 2013). A phylogenetic analysis of ITS2 sequences was performed by manually constructing a maximum likelihood tree (ML) using MEGA 6.06. Reconstructions from the ML analysis were carried out with hierarchical likelihood-ratio tests using the Hasegawa-Kishino-Yano model with a gamma distribution shaped parameter of 2.0. The reliability of internal branches was assessed using the bootstrap method with 1000 replicates. The ITS2 (AF499790.1,EU449103.1,AY258487.1, and AY589775.1) se- quences of clade Cwere used as reference sequences. AITS2 sequence (AF396629.1) fromNCBI was used as the out-group.

3 Results

The stable rarefaction curves indicated that the depth of the cloned ITS2 sequences was sufficient to evaluatediversity within clade C in each coral species (Fig.1). DNA alignments using MEGA 6.06 (Tamura, 2013) showed that the length of clade CITS2 was 197bp. As shown in Figs.2 and 4, three sub-clades ofwithin clade C were observed, corresponding to stone coralssp. SY-01 andsp. SY-05, and the soft coralsp. SY-07.Thus coral species-dependentwere included in clade C.

Fig.1 The rarefaction curves of Symbiodinium genotypes hosted by Acropora sp. SY-01 (blue), Pocillopora sp. SY-05 (red), and Sarcophyton sp. SY-07 (green).

TwelveITS2 genotypes (sp. 1–12) were identified based on the commonly used genotype identification method (Wicks, 2010; Silverstein,2011; Putnam,2012) (Fig.2). First, fourITS2 genotypes (sp. 1–4) in sub-clade I from stone coralsp. SY-01 were closely related toC3a. TheITS2 genotype of sp. 1 was derived from C3a with two mutation sites, while sp. 2 and sp. 3 were derived from sp. 1 with one mutation site, and sp. 4 was closely related to sp.3 with one mutation site. Second, fourITS2 genotypes (sp. 5 and 6 and sp. 7 and 8) in sub-clade II harbored by stone coralsp. SY-05 were closely related to homologousC42 and C1c, respectively. TheITS2 genotype of sp. 5 was identical to C42, and sp. 6 was closely related to sp. 5 (C42) with one mutation site. TheITS2 type of sp. 7 was identical to C1c, and sp. 8 was closely related to sp. 7 (C1c) with one mutation site. Third, four heterologousITS2 genotypes (sp. 9–12) in a separate sub-clade III associated with soft coralsp. SY-07 were closely related toC71. TheITS2 genotype of sp. 9 was identical to C71, whereas sp. 10 and sp. 11 were closely related to sp. 9 (C71) with one mutation site accordingly, and sp. 12 was closely related to sp. 11 with one mutation site.

Fig.2 Genotype network ofthe Symbiodinium ITS2 region. The genotype network was drawn using the Symbiodinium ITS2 sequences from Acropora sp. SY-01 (blue), Pocillopora sp. SY-05 (red), and Sarcophyton sp. SY-07 (green)as well as the NCBI database (gray) by minimum spanning networks (http://www.fluxus-engineering.com/sharenet.htm).

The gene sequence variation sites of theITS2 genotypes were further analyzed using the ITS2 secondary structure. As shown in Fig.3, the ITS2 secondary structure ofclade C contained four typical helices, and helix III was the longest. A total of 12 unique ITS2 sequences with eight nucleotide variation sites (a–h) were detected. Among them, two variation sites (sites a and e) were located in single-stranded regions, and the other six variation sites (sites b–d and f–h) were located in helix regions of the ITS2 secondary structure. Four C3-derivedITS2 genotypes (1–4) harbored by the stone coralsp. SY-01 had two nucleotide changes. One change occurred in a single-stranded region of the ITS2 secondary structure (site a) and therefore did not disrupt base pairing. The other occurred in the stem of helix II (site b), resulting in disrupted base pairing and a change in the helix structure. Analogously, the four C1c-and C42-derivedITS2 genotypes (5–8) harbored by stone coralsp. SY-05 also had two nucleotide changes: one occurred in the single-stranded region of the ITS2 secondary structure (site e) and the other occurred in stems of helix II (site c). Notably, fourITS2 genotypes associated with soft coralsp. SY-07 had three sites of variation: one occurred in the single-stranded region of the ITS2 secondary structure (site e) and two occurred in site c of helix II and site h of helix III, respectively. According to the gene sequence variation sites in the ITS2 secondary structure, six genotypes,., C3a, C42, C1c, C71 and two new genotypes, were identified.

Fig.3 ITS2 secondary structure and sequence variation sites of the Symbiodinium ITS2 sequences. The representative secondary structure of the Symbiodinium ITS2 sequence was drawn by blast searching against homologous sequences with known ITS2 secondary structures in the ITS2 Database (http://its2.bioapps.biozentrum.uni-wuerzburg.de). The ITS2 sequences variation sites (a–h) of the Symbiodinium genotypes were blast against closely related ITS2 sequences from the NCBI database.

Three sub-clades (sub-clades I, II, and III, bootstrap values >50) within clade C were also demonstrated based on the topology and bootstrap values of the ITS2-based phylogenetic tree (Fig.4). However, the 12ITS2 genotypes (sp. 1–12) designated by the genotype method in Fig.2 were only grouped into three phylogenetic supported lineages (bootstrap values > 50) with high similarity to C3a, C42b, and C71, respectively. TheITS2 genotypes designated by the genotype method,., sp. 1 and 2 in sub-clade I,sp. 7 and 8 in sub- clade II, sp. 10, and sp. 11, and sp. 12 in sub-clade III, were not well grouped, as the bootstrap value was <50.

Fig.4 Phylogenetic tree analysis of the Symbiodinium ITS2 region. Twelve ITS2 sequences of the Symbiodinium genotypes from Acropora sp. SY-01 (A. sp.SY-01), Pocillopora sp. SY-05 (P. sp. SY-05), and Sarcophyton sp. SY-07(S. sp. SY-07), as well as closely related ITS2 sequences of Symbiodinium genotypes from the Genebank database were used to construct a maximum likelihood phylogenetic tree using MEGA 6.06.

4 Discussion

Exploringdiversity at the species level is difficult because pure cultures of symbioticstrains for morphological descriptions are difficult to achieve (Baker 2003). However,molecular methods over the past two decades have opened up an exciting avenue for identifying the diversity ofin different coral reef dwelling animals (., reef building corals, non-reef building corals, flatworms, sponges, foraminifera, and protists) across different geographical scales (Wicks,2010; LaJeunesse, 2010; Silverstein,2011; Putnam, 2012). At present, nine large phylogenetically supported molecular clades (A–I) ofhave been classified by various relatively conserved molecular markers (Pochon, 2014), and manymolecular genotypes within the nine clades have been identified by the ITS2- based identification method (Hunter,1997; LaJeunesse, 2001; LaJeunesse, 2010; Wicks, 2010; Silverstein, 2011; Putnam,2012; Tonk,2013; Thomas, 2014).

However, the ITS2-based method is likely to lead to a higher diversity estimate due to the high variation in the ITS2 sequences (Thornhill, 2007; Sampayo, 2009; LaJeunesse., 2011). AITS2 sequence with a single-base mutation is typically designated as aITS2 genotype using the ITS2-based genotype method, but a single-base mutation or intragenomic variation in the ITS2 molecular marker does not represent a different phylogenetic ‘species’ or ‘genotype’ according to the phylogenetic ‘species’ concept (Eldredge., 1980). As shown in this study, 12ITS2 genotypes were designated using the traditional ITS2-based genotype identification method in three coral species, whereas only six and threeITS2 genotypes were supported by the ITS2-based secondary structural and phylogenetic analyses, respectively. It is clear that the ITS2-based genotype identification method lead to higher diversity estimates of-ITS2 genotypes. Based on the analysis of-ITS2 secondary structure and its variation sites, the present results show that the high diversity estimate of thegenotype using the ITS2-based genotype identification method was due to intra-genomic variations in the form of pseudo-genes or numerous functional variants in the single-stranded region of the ITS2 secondary structure (such as variation at sites a and e). In summary, the present study provides a theoretical basis for validating high diversity estimates ofITS2 genotypes by further conducting secondary structural and phylogenetic analyses of theITS2 sequences.

The specificity of the coral-symbiont is suggested in the coral speciessp. SY-01,sp. SY-05, andsp. SY-07 from the northern part of the SCS. For example, the stone coralssp. SY-01 andsp. SY-05 hostC3a and C42b, respectively; and the soft coralsp. SY-07 hostsC71.There are no sharedgenotypes among the three corals ofsp. SY-01,sp. SY-05, andsp. SY-07. This kind of specificity of thegenotypes with their associated corals has been reported previously (LaJeunesse., 2008; Thornhill., 2009). Interestingly, our results reveal that the stone coralssp. SY-01 andsp. SY- 05 harbor phylogenetically relatedgenotypes within clade C compared with thegenotypes hosted by the soft coralsp. SY-07. Specifically,genotype C71 in the soft coralsp. SY-07 is more distant fromgenotypes (., C3a and C42b) in the stone coralssp. SY-01 andsp. SY-05. In other words, the specificity of the coral-symbiont is correlated with the phylogenetic relationship of the coral host.

Although the specificity ofgenotypes with coral hosts is suggested amongsp. SY-01,sp. SY-05, andsp. SY-07 in the northern part of the SCS,genotypes C3a and C42b have also been reported in previous studiesfrom the same or different coral hosts across the Pacific and Indian Oceans, including the Great Barrier Reef (LaJeunesse., 2010), Okinawa (LaJeunesse., 2004), and the SCS (Dong.,2009; Zhou., 2012). In contrast,type C71 from the soft coralsp. SY-07 has only been reported in soft corals living in the Western Pacific (LaJeunesse., 2004). All of these results indicate that both the coral host and reef location affect specificity of the-coral symbionts, and this may reflect different mechanisms ofadaption to environmental stresses,includinglight levels, geographic features, and thermal stress (Baker, 2004; van Oppen, 2005; Oliver., 2011; Lien., 2013).

Acknowledgements

This study was supported by the Major National Scientific Research Project, China (No. 2013CB956103) and the Minhang Leading Talent Project.

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January 16, 2018;

July 7, 2018

© Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2018

. E-mail: zyli@sjtu.edu.cn

(Edited by Qiu Yantao)