PHYLOGENETIC ANALYSIS AND SPECIES IDENTIFICATION OF 11 GORGONIAN CORALS (OCTOCORALLIA: ALCYONACEA) IN THE NORTH CENTRAL COAST OF VIETNAM BASED ON MSH1 mtDNA AND 28S rDNA MARKERS

Vietnam contains diverse marine ecosystems with the high biodiversity of marine organisms, including gorgonian corals of Alcyonacea order. In order to support traditional classification of these corals, in this study mitochondrial barcoding markers msh1 and nuclear 28S rDNA were developed for analysis of 11 specimens collected in 2015 and 2016 from different islands and bays along the North Central coast of Vietnam. Phylogenetic analyses based on msh1 and 28S sequence polymorphism showed that all specimens belonged to Anthozoa class, Octocorallia sub-class and Alcyonacea order. At lower taxa levels, they were divided into 4 sub-orders, 7 families and 7 genera according to 7 distinct clades with bootstrap values from 99-100%.The identifications of 7 out of 11 specimens including Sinularia brassica (2 specimens)and Sinularialeptoclados , Dichotellagemmacea , Annella reticulata, S. conferta and S. nanolobata were in concordance between morphological and molecular methods. The other 4 specimens were only identified at genus levels of Astrogorgia sp ., Melithaea sp . Scleronephthya sp . and Muricella sp . by either msh1 -morphology or msh1 - 28S markers. These results highlight the importance of molecular markers to elucidate patterns of biodiversity and species identification of soft coral. inferences using Neighbor Joining (NJ) method from Mega 3.1 with Bootstrap test of 5000 replicates. For species determination, homology level after aligning DNA sequences of the studied samples and references in the same clades on NJ trees obtained from phylogeny reconstruction analysis was estimated using Multiple Sequence Alignment method of DNAMAN 4.15 (LynnonBioSoft). Specific point mutations (SNPs) within msh1 and 28S sequences were also indicated using Align of ClustalW and Sequence Data Explorer methods from Mega3.1.

Vietnam contains diverse marine ecosystems with the high biodiversity of marine organisms, including gorgonian corals of Alcyonacea order. In order to support traditional classification of these corals, in this study mitochondrial barcoding markers msh1 and nuclear 28S rDNA were developed for analysis of 11 specimens collected in 2015 and 2016 from different islands and bays along the North Central coast of Vietnam. Phylogenetic analyses based on msh1 and 28S sequence polymorphism showed that all specimens belonged to Anthozoa class, Octocorallia sub-class and Alcyonacea order. At lower taxa levels, they were divided into 4 sub-orders, 7 families and 7 genera according to 7 distinct clades with bootstrap values from 99-100%.The identifications of 7 out of 11 specimens including Sinularia brassica (2 specimens)and Sinularialeptoclados, Dichotellagemmacea, Annella reticulata, S. conferta and S. nanolobata were in concordance between morphological and molecular methods. The other 4 specimens were only identified at genus levels of Astrogorgia sp., Melithaea sp. Scleronephthya sp. and Muricella sp. by either msh1-morphology or msh1-28S markers. These results highlight the importance of molecular markers to elucidate patterns of biodiversity and species identification of soft coral.   Lien et al. (2015) analyzed phylogenetic relationship of Sinularia, Sarcophyton andLobophytum genera in Nha Trang Bay (South Central coast of Vietnam) using 696bp sequences of msh1 genes and 866bp fragment of irg-cox1 gene. There has not been, however, research on this approach of soft corals in the North Central coast of Vietnam. Accurate identification of soft coral species is fundamentalfor genetics, physiology, ecology, particularly applied pharmacology. Molecular identification has been used in biodiversity and conservation, but it also has great potential for applications in taxonomy. Recently, based on nucleotide sequences, several coral species-specific molecular markersfromribosomal subunits 16S, 18S, 28S and genes coding for proteins on mitochondrial DNA (mtDNA) have been found (France et al., 1996;McFadden et al., 2006aMcFadden et al., , 2006bShearer et al., 2002;Herrera et al., 2009). Among molecular markers on mtDNA, msh1 gen encoded for MSH1 protein, has been used widely in molecular taxonomy of soft coral. It is known that the genetic diversity of msh1 gene is as twice as other coding genes on mtDNA. Moreover, msh1 gene is present in mtDNA of almost all soft corals published in the present GenBank (van der Ham et al., 2009). The diversity of the msh1 gene also occurs among species in a genus and has been used to phylogenetic analysis of soft coral species in the Asia-Pacific (Thoma et al., 2009).
In the present study, phylogenetic analysis and species identification of 11 soft coral specimens collected in the North Central region of Vietnam were investigated based on the 639bp on their msh1mtDNA and the 704bp fragment on 28S rDNA gene.The congruence between morphological analysis and molecular markers in species identification could offer an alternative taxonomic method for further biodiversity studies of soft corals in Vietnam.

Materials
Soft coral specimens were collected in different islands and bays of the North Central region of Vietnam in 2015-2016using SCUBA diving equipment. A map of sampling location and specimen name are shown in Fig 1. The specimens were washed several times with fresh water to remove sand, algae and other surface organisms and then 5 times with distilled water.One portion of each sample was stored in 70% ethanol for morphological analysis and the smaller portion was kept at -80 o C for DNA extraction. All vouchers were deposited at Institute for Marine Biochemistry, Vietnam Academy of Science and Technology (VAST).Morphological characters of specimens were identified by Prof. Do Cong Thung (Institute of Marine Resources and Environment, VAST) right after sampling and cleaning.

PCR and DNA Sequencing:
Genomic DNA of each specimen was isolated using DNeasy® Tissue Kit (Qiagen) according to the manufacturer's instruction. The concentration of DNA was quantified using a NanoDrop 1000 instrument (Thermo Scientific, USA) and by electrophoresis in 0.8% agarose.

DNA Analysis:
Phylogenetic relationship of the 11 soft coral samples (Table 1) was analyzed based on referred sequences from Gene Bank using ClustalW method of Mega 3.1 (Kumar et al., 2004). Phylogeny reconstruction was done with tree 507 inferences using Neighbor Joining (NJ) method from Mega 3.1 with Bootstrap test of 5000 replicates. For species determination, homology level after aligning DNA sequences of the studied samples and references in the same clades on NJ trees obtained from phylogeny reconstruction analysis was estimated using Multiple Sequence Alignment method of DNAMAN 4.15 (LynnonBioSoft). Specific point mutations (SNPs) within msh1 and 28S sequences were also indicated using Align of ClustalW and Sequence Data Explorer methods from Mega3.1.

Phylogenetic analysis
After sequencing and extracting, partial msh1mtDNA of 11 specimens and 28S rDNA of 10 specimens respectively ranged from 707-719bp and 739-753bp, of which fragments of 627-639bp msh1 and 695-709bp 28S of the specimens were used for analysis due to the available length of related referred sequences from GenBank. Phylogenetic analyses based on msh1mtDNA sequence polymorphism of 11 specimens and referred taxa (Fig2) showed that all samples of this research belonged toAnthozoa class, Octocorallia Sub-class and Alcyonacea order. At lower taxa levels, they were divided into 4 sub-orders, 7 families and 7 genera according to 7 distinct clades (1 to 7) with bootstrap values from 99-100%. Phylogenetic tree of 28S rDNA (Fig3) were similar to that of msh1mtDNA, but only with 6 genera (without clade 4) because sequence of 28S fragment ofNL-07 was not available. Of 11 specimens, 5 were clustered into clades 2a and 2b with referred taxa of Sinularia genus. The results of phylogenetic analyses indicated the high taxonomical diversity of Alcyonaceancorals in sampling regions. Clade order between msh1 and 28Swas not the same possibly because of difference in evolution rate between mtDNA and nuclear rDNA. Basically, mitochondrial genes evolve 50-100 times slower than nuclear genes in anthozoans (Hellberg, 2006 On phylogenetic trees instructed by msh1and28S markers, taxa of 6 genera, including Annella, Astrogorgia, Dichotella, Melithaea, Muricella and Scleronephthya were monophyletic, while those of Sinulariawere found polyphyletic with 2 separate clades 2a and 2b (Fig 2 and 3). The polyphyletic sub-clades of 2a and 2bmay be resulted fromthe hybridization among taxa in this genus, which was mentioned in some previous studies. Chemical markers of five new terpenoids isolated from Sinularia maxima and S. polydactylaelucidated the hybridization between these two soft coral species (Kamel et al., 2009). More recently, DNA barcode markers, mtMutS and 28S rDNA, proved the hybridization occurring within Sinularia genus (Quattrini et al., 2019). Analyses of Sinulariahybridization for clade 2b (Fig 3) based on 695-719bp 28S rDNA sequences of 10 soft coral specimens and related reference sequences resulted in seven SNPs along the aligned sequences. These SNPs could, therefore, add more evidence of hybridization of soft corals, which was one of the important motivations for evolution of Sinularia genus and Anthozoa class (Willis et al., 2006).

Species determination: Analysis of homology levels
Alignments between sequences of msh1mtDNA and 28S rDNA fragments of 11 specimens and reference taxa in each clade on Fig 2 and Fig 3 showed the high homology level (99.7-100.0%) for both markers (Table 1). Five specimenswere classified as Sinularia brassica (HV-03, NL-06), S.leptoclados (SLE-05), Dichotellagemmacea (NL-03) and Annella reticulata (VM-02) based on msh1 marker with 99.7-100% of identity and 28S marker with 99.9-100% of identity), which was also in agreement with previously morphological identification. In case of unclassified by morphological criteria due to samples damaged, NL-08 and NL-05 specimens were still diagnostic at the genus level as Astrogorgiasp. and Melithaea sp., respectively. Msh1 markers with identity to referred taxa from 99.8-100% were in accordance with morphological analysis to identify specimens of SN-04 and SCO-05 to beSinulariananolobata and Sinulariaconferta, while 28S rDNA sequences of these two species were not available from GenBank. Though 28S fragments of HM-09 and Scleronephthyacorymbosawas aligned with 100% of identity, this specimen was still decided to be Scleronephthya sp. because morphological diagnosis classified this specimen only at genus level and msh1 sequence of S. corymbosawas unavailable in GenBank. Similarly, NL-07 was unclassified at species level in Muricella genus due to morphological identification to be Muricella sp. and identity of msh1 sequence of this specimen and Muricella sp. referred in GenBankto be 100%. In short, among 11 specimens, seven of HV-03 andNL-06(Sinularia brassica), SLE-05(S.leptoclados), NL-03 (Dichotellagemmacea), VM-02 (Annella reticulata), SN-04(S. conferta)and SCO-05(S. nanolobata) were classified at species level, of which the first five wereby three markers (msh1, 28S and morphology) and the other two by msh1 and morphology. The other four of NL-08, NL-05, HM-09 and NL-07 were only identified at genus level to be Astrogorgiasp., Melithaea sp., Scleronephthya sp. and Muricella sp. by either msh1-morphology or msh1-28S markers.   Fig. 2

and 3; **) H: homology level (%); ***) By Prof. Do Cong Thung
Single nucleotide polymorphism analysis Separate alignment of msh1 fragments (Fig 2) and 28S fragments (Fig3) of soft coral taxa respectively resulted in 174 and 188 single nucleotide polymorphisms (Fig4 and 5). These diagnostic SNPs distinguished each of 7 specimens to be classified at species level and each of 4 being identified at genus level ( Table 1). Number of SNPs along msh1 fragments of 11 taxa ranged from 0-50 (Fig 4) while 28S fragments of 10 taxa varied from 1-85 along them (Fig 5), revealing lower substitution rates of msh1mtDNA than those of 28S rDNA in corals as estimated by Hellberg (2006).Moreover, nucleotide substitution rate of the same coral taxon also varied depending on particular genomes. In this study, Melithaea sp. hasthe highest number of 50SNPs on msh1 fragment,while the taxon is the second position with18 SNPs on 28S fragment. In contrast, D. gemmacea had the highest number of 84 SNPson its 28S fragments but was the second of 38 SNPs on msh1 fragments of the species (Fig4 and 5). The lowest number of SNPs on msh1 (0-9) and 28S fragments of Sinularia taxa in clade 2b again implied their hybridization. Difference in nucleotide substitution rate between msh1mtDNA and 28S rDNA could interpret the difference in order of 7 clades between msh1 and 28S phylogenetic trees of 11 specimen and related referred sequences (Fig 2 and 3).

Msh1 and 28S markers supporting morphological clasification in determination of soft coral taxa:
Recently, classification of corals still has been based on morphological characters of colony morphology and/or sclerites comparison. In fact, the first approach was unable to determine at species level in some genera for mixture of morphological characters among them, while their sclerite were so varied that many species were not identified if applied skeleton composition comparison (van Ofwegen and Groenenberg, 2007). Similarly, both approaches were unable to identify some octocoral taxa or were incongruent with morphospecie identification due to high variation, especially taxa in such genus as Sinularia,within which the hybridization between closely related taxa happened . They also concluded that a single DNA marker, e.g.,mtMutSwas not powerful enough to discriminate most closely related species such 512 asSinularia spp. Loci from rDNA genome, i.e., 18S, ITS, 5.8S and 28S were shown to be more powerful than those from mtDNA to resolve relationships of corals below genuslevel (van Ofwegen and Groenenberg, 2007). Therefore, markers from mtDNA could be combined with those from rDNA to discriminate coral taxa at species level, especially closely-related species within most coral genera (Benayahu et al., 2018). In this study, results of determination of 11 soft corals by msh1 and 28S markers were in agreement to those by morphological classification (Table 1), again indicating the strong support of the two DNA markers to morphospecies of these soft coral species. In spite of powerfulness in discrimination of coral taxa, including closely-related species in a genus, molecular markers including msh1 and 28S loci showed some disadvantages. Of all specimens, five were identified at species level by all msh1, 28Sand morphological markers. However, the other 6 specimens were determined at species level or only genus level with the pair marker of msh1-morphospecies, msh1-28S due to unavailability of related msh1mtDNA and 28S rDNA in GenBank. When related sequences of these two markers are available, these genus-level taxa will be resolved.

Divergence of gorgonian corals:
The molecular analysis in this study indicated the high diversity of gorgonian corals in Alcyonaceaorder along the North Central coast of Vietnam (Fig 1). All 11 specimens were classified to be 10 species (Table1) in 7 genera, 7 families and 4 suborders ( , in which a total of 45 taxa belonging to 12 genera and 7 familieswere identified. Among them, Sinularia genus was the highest diversity group with 19 species, followed bySarcophyton with 8 species and Lobophytum with 6 species.However, it is the fact thatthe above findings have been only spotty trying. Recently, molecular markers, especially DNA barcodes have been proved really powerful to resolve various problematic aspects of marine animals. An overall project on application of these tools to deal with phylogenetic patterns, species determination and diversity research on marine animalsincluding soft coralsalong 3000km coastal length of Vietnam, could be, therefore, programmed in order to effective conservation and sustainable development of these resources in the country.

Conclusion:-
In this study, the developed molecular markers clearly supported the classification of soft corals at species level.The identifications of 9 out of 11 soft coral specimens based on msh1mtDNA and 28S rDNA polymorphism of the soft coral specimens and referred taxa werein concordance with the traditional taxonomy. There were 7 specimens to be classified at species level, of which 5 were identifiedby msh, 28S and morphological markers and the other two by msh1 and morphospecies. The other four were only identified at genus level by either msh1-morphology or msh1-28S marker pairs. All of the taxa belonged toAnthozoa class, Octocorallia sub-class and Alcyonacea order, from which they were divided into 4 sub-orders, 7 families and 7genera according to 7 distinct clades on phylogenetic trees with high confidence intervals from 99-100%.