Preliminary taxonomic review of wrasses species (labridae) from vietnam with an integration of morphological and molecular data

Journal of Fisheries science and Technology Special issue - 2015 16 • NHA TRANG UNIVERSITY PRELIMINARY TAXONOMIC REVIEW OF WRASSES SPECIES (LABRIDAE) FROM VIETNAM WITH AN INTEGRATION OF MORPHOLOGICAL AND MOLECULAR DATA Dang Thuy Binh1, Le Phan Khanh Hung1, Truong Thi Oanh1, Luong Thi Tuong Vi1 ABSTRACT The family Labridae (the wrasses) containing 82 genera and about 600 species of fish – is the fifth largest fish family and second largest marine fish family. The Labridae is one of the most morphologically and ecologically diversified families of fish in size, shape and color. They eat algae and organic detritus, having an important role in helping to restore coral reefs after impact of environmental perturbations. The classification of Labridae in Vietnam is based primarily on morphological characteristics. The purpose of this study is a taxonomic review of the family Labridae distributed in Vietnam. Currently, 11 species belong to Cheilinus, Halichoeres, Iniistius, Scarus, and Oxycheilinus are described based on morphological characters. Combined with Genbank sequences, a phylogenetic tree was constructed based on 16S gene of mitochondrial DNA. A comprehensive survey and checklist update of Labridae in Vietnam is necessary. The research results can be used as data sources for the study of biodiversity and management of reef fishes resources in Vietnam. Keywords: Labridae, Reef fishe Morphology, Phylogeny, Mitochondrial DNA 1 Institute for Biotechnology and Environment, Nha Trang University (Email: binhdangthuy@gmail.com) I. INTRODUCTION The family Labridae (the wrasses) are a diverse group of fishes that vary in body shape, size and coloration (Mark et al., 2005). Labrids inhabit tropical marine and temperate waters around the world, and are most common in shallow waters in a variety of habitats such as coral reefs, rocky reefs, sand, grass and algae (Carpenter et al., 2001). Fishes of the family Labridae have an important role for general marine ecosystems and coral reefs in particular. They eat algae and organic detritus, helping to restore coral reefs after impact of environmental perturbations. The family Labridae currently include 600 species in 82 genera (Mark et al., 2005, Parenti and Randall, 2011). In terms morphological, Allen et al. (2005) used body shape, body color, stripes, spines and the rays of the dorsal fin as primary distinguishing characteristics and classified 185 species in 32 genera of family Labridae. Nguyen Nhat Thi and Nguyen Van Quan (2004) reported 94 species, 28 genera belong to Labridae in Truong Sa Archipelago, Vietnam. Nguyen Van Quan (2009) reported 38 species belong to Labridae from marine water of Nha Trang Bay, Vietnam. Nguyen Van Long (2009) studies on coral reef fishes in the coastal waters of South Central Vietnam from 2006 – 2007, and combined with available data of species composition from previous studies, a total of 73 species belong to Labridae was recorded. Above information is relatively fragmented and not entirely reflect the classification system of fish species in Vietnam. According to Mark (1993) the tribe Cheilinini is monophyletic, including the five genera Cheilinus. Doratonotus, Epibulus, Oxycheilinus and Wetmorella that form the “cheiline” lineage, Journal of Fisheries science and Technology Special issue - 2015 NHA TRANG UNIVERSITY • 17 and the five genera Cirrhilabrus. Paracheilinus. Pseudocheilinus. Pseudocheilinops, and Pteragogus that constitute the “pseudocheiline” lineage. Mark et al. (2005) analyzed DNA sequence data of 12S rRNA, 16S rRNA from the mitochondrial genome, and the protein-coding genes (RAG2 and Tmo4C4) from the nuclear genome for 98 fish species, including 84 labrid fish and 14 outgroups collected in Chicago. Combined tree topology confirmed the monophyly of a family Labridae that includes the parrotfishes and butterfishes. John et al. (2012) studied phylogeny of 61 Labridae species using the 16S rRNA gene, of which twenty-two reciprocally monophyletic sister species pair were identified: 64% were allopatric, and the remainder were sympatric. In the present study, species identification was confirmed by morphological and genetic characters; phylogeny of the genus Cheilinus, Halichoeres, Iniistius, Oxycheilinus, Scarus and Thalassoma was clarify based on 16S gene of mitochondrial DNA. These data contribute to the DNA barcoding of reef fish species, as data sources for the study of biodiversity and management of reef fishes resources in Vietnam. II. MATERIALS AND METHODS 1. Sampling and identification Wrasses fishes were collected at Nha Trang and Cam Ranh Bays in Khanh Hoa Province, Vietnam. The samples were transferred to the laboratory, keeping in 95% ethanol or on ice, and then store at – 40oC before analysis. For morphological analysis, all specimens were identified based on taxonomic characters such as body and fins colour, the presence or absence of spot, bar or stripe on body and fins, number of spine and soft rays belong to dorsal, pectoral, ventral, anal and caudal fin. Species was identified following Carpenter and Niem (2001), Allen et al. (2005). 2. DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from approximately 25 mg of tissue sample using GeneJET Genomic DNA Purification Kit (Thermo Scientific) following to the manufacturer’s instructions. DNA was then stored at - 200C. The lysate was used as template for Polymerase Chain Reaction (PCR) to amplify a fragment of the 16S gene of mitochondrial DNA. Each PCR had a reaction volume of 25 µl and contained 10ng DNA template; 2.5 µl 10X Dream Taq buffer (Fermentat); 0.125 nM each dNTP; 0.1 pM each primer; 0,5 unit of Taq polymerase (5U/1 µl) and distilled water to the final volume. Primers used for amplification and sequencing are 16Sar 5’-CGCCTGTTTATCAAAAACAT-3’ and 16Sbr 5 ’ -CCGGTCTGAACTCAGATCACGT-3 ’ (Palumbi et al., 2002). Biorad thermocyclers (Icycler) were used under the following temperature program: initial denaturation step at 940C for 3 min, followed by 38 cycles of denaturation step at 940C for 30s, annealing temperature was at 480C for 30s and final extension step at 720C for 5 minutes. All PCR products were loaded and run on 1,5% agarose gel stained with ethidium bromide, and bands were visualized under a UV transilluminator. The PCR products were purified using a PCR clean up system kit (Promega), and pre-sequenced using dye – labels dideoxy terminator (Big Dye Terminator v. 3.1, Applied Biosystems) with the same primer as the PCR reaction at the following temperatures: 960C for 30s, 500C for 30s and 600C for 4 min. Products were sequenced in ABI Prism 3.700 DNA Analyser (Applied Biosystems). Journal of Fisheries science and Technology Special issue - 2015 18 • NHA TRANG UNIVERSITY Sequence contigs were assembled using Geneious v. 7 ( The resulting sequences were confirmed by the Basic Logical Alignment Search Tool (BLAST, Sequences were initially aligned by eye using the sequence editor BioEdit 7.0 (Hall, 1999). 3. Phylogenetic analysis 11 sequences in this study together with 16 sequences of other Labrid species available from Genbank were used in the phylogenetic analysis. Data were analysed using 3 approaches, i.e., Neighbour Joining (NJ), Maximum Parsimony (MP) and Bayesian Inference (BI). NJ analyses were conducted from MEGA 6 under 1000 replicate. MP analysis were conducted using PAUP* 4.0 (Swofford, 2002). Bootstrap support values of the MP analysis were used to assess the robustness of the findings. Bootstrap support values were computed from 1,000 replicates randomized 10 times with tree-bisection-reconnection (TBR) addition sequence. Prior to BI analyses, best-fit models of nucleotide substitution were selected by the Akalike Information Criterion as implemented by and MrModeltest 2.2 (Nylander, 2004). Bayesian analyses were conducted in MrBayes 3.1.2 under the selected best-fit models and parameters. Numbers at the interior branches of the majority-rule consensus tree present posterior probability (PP). Tree display and editing were performed in TreeView 1.6.6 (Page, 1996). III. RESULTS AND DISCUSSION 1. Species identification This study currently classifies 11 labrid species in 6 genera. Wrasses vary greatly in both body shape and size, whether small or large, slender or deep-bodied, all wrasses have terminal mouths, usually displaying prominent canines, thick lips, and a single, continuous dorsal fin. Morphologic characters of wrasses collected in Khanh Hoa Province, Vietnam were listed in Table 1, and fish image were presented in Figure 1. Table 1. Morphological characteristics of 11 labrids in Khanh Hoa province, Vietnam (main taxonomic characters were highlight) Genus Species Morphological characteristics Cheilinus Cheilinus oxycephalus (Bleeker, 1853) Body colour is brownish red, with small whitish spots; dark brown spot anteriorly on each side of upper lip. All fins are red, black spot on front of dorsal fin; often a trio of small black spots on tail base (Fig. 1A). Cheilinus fasciatus (Bloch, 1791) Body and tail has alternating white and blackish vertical stripes, with a splendid yellow-orange area in pectoral region (Fig. 1B). Oxycheilinus Oxycheilinus digramma (Lacepède, 1801) Body colour is red. The side of Oxycheilinus digramma’s head has horizontal stripes, while the front of the head has red spots; diagonal lines on lower gill cover (Fig. 1C). Iniistius Iniistius pavo (Valenci- ennes, 1840) Body has grey dorsally, yellow-white ventrally. Usually with 5 dark vertical bars below the eye and a black spot on the upper side of the body. First two dorsal spines form a separate fin (Fig. 1D). Journal of Fisheries science and Technology Special issue - 2015 NHA TRANG UNIVERSITY • 19 Halichoeres Halichoeres hartzfeldii (Bleeker, 1852) Body colour is orange with two orange-yelllow stripe; bright yellow marking around base of pectoral fin. Three blackish spot at base of caudal fin; a small black spot at upper base of pectoral fin (Fig. 1E). Halichoeres hortulanus (Lacepède, 1801) Body colour is bluish white with blue bar on each scale, it becomes a distinctive checkerboard pattern on the body. Head is light green with orange bands. Two large yellow spots on back, a large black patch encircled with yellow at centre of dorsal fin (Fig. 1F). Halichoeres miniatus (Valenciennes, 1839) Body distinguished by irregular dusky bars on lower side; pinkish bands on head; dark blotch on middle of dorsal fin (Fig. 1G). Halichoeres melanochir (Fowler & Bean, 1928) Body colour is purple with black spots and black scale margins on side. The pectoral and pelvic fins are yellow; prominent black spot covering pectoral fin base. The dorsal, anal and caudal fins are orange (Fig. 1H). Thalassoma Thalassoma lunare (Linnaeus, 1758) Body colour is dark green with vertical black lines; purple bands on head. Purple pectoral fins with blue margin, deep lunate tail with yellow center (Fig. 1I). Scarus Scarus ghobban (Forsskal, 1775) Body colour is yellow to yellow-brown with 5 blue bars; blue bands around mouth, eye and tail. The dorsal, anal and caudal fins are orange with a blue borders (Fig. 1K). Scarus prasiognathos (Valenciennes, 1840) Body colour is dark reddish, becoming paler posteriorly on caudal peduncle, with numerous small whitish spots of unequal size. Head brownish orange red with a few scattered deep blue dots and a narrow deep blue band to orbit, dental plates blue (Fig. 1L). Fig.1. Wrasses of the family Labridae from Khanh Hoa Province, Viet Nam (Arrow show taxonomic characters mentioned in Table 1.) Journal of Fisheries science and Technology Special issue - 2015 20 • NHA TRANG UNIVERSITY 2. Phylogenetic analysis The 16S RNA dataset consists of more than 600 bp, of which 546 bp were unambiguously aligned. Tree topology from the MP method was similar to that of the best NJ tree and showed only minor differences from the BI tree. The phylogenetic tree was divided into four main groups with high BT support. Group I was subdivided into four groups with high node support (MP 100%, PP 100%, MJ 100%). Subgroup I.1 include species of two different genera Thalassoma, (Thalassoma lunare, T. lutescens), and Halichoeres (Halichoeres hartzfeldii). This indicates unsuitability between morphological and genetic classification. H. hartzfeldii is more closely related to T. lunare than to other Halichoeres. Mark and Michael (2005) reported the similar phylogenetic relationship between H. hartzfeldii and T. lunare. In subgroup I.2, Halichoeres hortulanus was clustered with H. scapularis. Subgroup I.3 includes H. nebulosus and H. miniatus. With these species, body moderately deep, shade of green, pink diagonal band on cheek, jaws prominent though mouth small, caudal fin slightly rounded. Subroup I.4 includes H. melanochir and H. prosopeion. Fig.2. Phylogenetic relationships of wrasses based on 16S RNA. Bootstrap value from NJ analysis along the branch. Scolopsis bilineata were used as out group. Fish image were displayed Journal of Fisheries science and Technology Special issue - 2015 NHA TRANG UNIVERSITY • 21 Group II consist of Scarus ghobban and S. prasiognathos. Group III includes 2 species of genus Cheilinus (Cheilinus fasciatus, C. oxycephalus) and 2 species of genus Oxycheilinus (Oxycheilinus unifasciatus, O. digramma). Cheilinus oxycephalus belongs to genus Cheilinus, but it shows the close relationship with Oxycheilinus unifasciatus and O. digramma belong to genus Oxycheilinus. This relationship is strongly supported in morphologically characters: Dorsal fin continuous, with IX spines and 10 soft rays; anal fin with III spines and 8 soft rays; pectoral fins with ii unbranched and 10 branched rays. Group IV include Iniistius pavo and I. aneitensis. Both of two species have the same morphological characteristics; such as steep blunt head, large white patch on lower forebody, may display dusky bars. The Labridae has long been recognized as a remarkably diverse family, which highly vary in shape, size colour of the body, and structural changes in the skull, in particular the jaws reflected difference feeding behavior (Mark et al. 2005). Despite variation of taxonomic characters, labrid species showed monophyly at high taxonomic level (Order, tribe and family). However, non-monophly was detected at the genus level. Current study showed unresolved groups of Thalassoma and Halichoeres as well as Cheilinus and Oxycheilinus. Bernardi et al. (2004) also detected the non-monophyly of Thalassoma as Thalassoma and Gomphosus species (G. varius and G. caerulaeus) were clustered in the same clade. Research also discovered the separate group of Atlantic Ocean and Indo-Pacific Ocean caused probably by closing of the Isthmus of Panama. Mark (1993) report the tribe Cheilinini is monophyletic, including the five genera Cheilinus. Doratonotus. Epibulus. Oxycheilinus, and Wetmorella that form the “cheiline“ lineage. However, Cheilinus and Oxycheilinus were not strongly supported as distinct clade based on current genetic characters (16S rRNA makers). Current research provides key identification for Wrasses in Vietnam. Formal description, voucher preservation and DNA barcoding could help update species checklist in Vietnam. Additional sampling and more relevant molecular makers should be applied for Labridae DNA barcoding data and resource management. IV. CONCLUSION 11 labrids species of 6 genera in Khanh Hoa, Vietnam were morphological identified. Phylogenetic tree constructed based on 16S RNA sequences polymorphisms indicates the monophyly of family Labridae, however, phylogenetic relationships of Thalassoma, Halichoeres, Cheilinus and Oxycheilinus remain unclear groups. ACKNOWLEDGMENT This study was partly supported by projects “Parasite risk assesment with integrated tools in EU fish production value chains”. We also thank Prof. Kent Carpenter (Old Dominion University) for assistance in identity of fish species. REFERENCES 1. Allen G., Steene R., Humann P. & DeLoach N., 2005. Reef Fish Identification: Tropical Pacific, New World Publications. Journal of Fisheries science and Technology Special issue - 2015 22 • NHA TRANG UNIVERSITY 2. Bernardi G., Bucciarelli G., Costagliola D., Robertson D. R., Heiser J. B., 2004. Evolution of coral reef fish Thalassoma spp. (Labridae). 1. Molecular phylogeny and biogeography. Marine Biology 144: 369-375 3. Carpenter K. E. and Niem V. H., 2001. FAO species identification guide for fishery purposes. The living marine resources of the western central Pacific, Volume 6, Bony fishes part 4 (Labridae to Latimeriidae), pp. 3381–3468. 4. Hall T. A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT, Nucleic Acids Symp. Ser., vol. 41, pp. 95–98. 5. John C., Oya K., Lynne H., Ross R., Kendall C., 2012. Patterns and processes in the evolutionary history of parrotfishes (Family Labridae) 6. Mark W. W., 1993. Phylogenetic relationships of the tribe cheilinini (Labridae: Perciformes). Bulletin of Marine Science, 52(1): 351-394. 7. Mark W. W., Michael E. A., Peter C. W., David R. B., Justin R. G., Jennifer L. F., Kendall D. C. and Lydia L. S., 2005. Local phylogenetic divergence and global evolutionary convergence of skull function in reef fishes of the family Labridae. Proc. R. Soc. B 272, 993-1000 8. Mark W.W., Michael E. A., 2005. Phylogenetic relationships and evolutionary history of the reef fish family Labridae. no. 36, pp. 370–390. 9. Nguyen Nhat Thi and Nguyen Van Quan, 2004. Biology diverse and reef fishes resource potentiality of the Truong Sa (Spratly) Archipelago. Journal of Marine Science and Technology, no. 4, pp. 47 – 64. 10. Nguyen Van Long, 2009. Coral reef fishes in the coastal waters of south-central Vietnam. Journal of Marine Science and Technology, no. 3, pp. 38 – 66. 11. Nguyen Van Quan, 2009. Supplement study on ichthyofauna of coral reef fishes in Nha Trang Bay marine protected area, Khanh Hoa provice. Journal of Marine Science and Technology, no. 1, pp. 46 – 54. 12. Nylander J. A., 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University. 13. Page R. D., 1996. TreeView: an application to display phylogenetic trees on personal computers, Bioinformatics/ computer Appl. Biosci., vol. 12, no. 4, pp. 357–358. 14. Palumbi S., Romano S., Mcmillan W. O., Grabowski G., 2002. The simple fool’s guide to PCR, vol. 96822, no. 808, pp. 1–45. 15. Parenti P., Randall J. E., 2011. Checklist of the species of the families Labridae and Scaridae: an update. Smithiana Bulletin 13: 29–44. 16. Swofford D., 2002 “PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4.” Sinauer Associates, Sunderland, Massachussets.