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1 Mitochondrial DNA Part A DNA Mapping, Sequencing, and Analysis ISSN: (Print) (Online) Journal homepage: Molecular phylogenetic relationships of some common sweetlips (Haemulidae: Plectorhynchinae) and the synonyms controversy of two Plectorhinchus species Rishen Liang, Chao Wang, Qing Zou, Aiguo Zhou & Meng Zhou To cite this article: Rishen Liang, Chao Wang, Qing Zou, Aiguo Zhou & Meng Zhou (2016) Molecular phylogenetic relationships of some common sweetlips (Haemulidae: Plectorhynchinae) and the synonyms controversy of two Plectorhinchus species, Mitochondrial DNA Part A, 27:3, , DOI: / To link to this article: Published online: 08 Dec Submit your article to this journal Article views: 13 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at Download by: [Sun Yat-Sen University] Date: 04 June 2016, At: 05:28

2 ISSN: (print), (electronic) Mitochondrial DNA Part A, 2016; 27(3): ! 2014 Informa UK Ltd. DOI: / SHORT COMMUNICATION Molecular phylogenetic relationships of some common sweetlips (Haemulidae: Plectorhynchinae) and the synonyms controversy of two Plectorhinchus species Rishen Liang 1, Chao Wang 2, Qing Zou 3, Aiguo Zhou 3, and Meng Zhou 1 1 College of Life Science, Zhongkai University of Agriculture and Engineering, Guangzhou, People s Republic of China, 2 Qingyuan Animal Husbandry and Fishery Technology Extension Station, Guangzhou, People s Republic of China, and 3 College of Animal Science, South China Agricultural University, Guangzhou, People s Republic of China Downloaded by [Sun Yat-Sen University] at 05:28 04 June 2016 Abstract Molecular phylogenetic topologies from 40 individuals of 17 sweetlips were constructed based on the mitochondrial cytochrome b (cyt b) and cytochrome c oxidase subunit I (COI) genes. All phylogenetic results strongly revealed the division of the sweetlips into three morphological distinct groups. Group I: sweetlips with colorful patterns, Group II and Group III: species with uniformly dark patterns. The relationships of those morphologically confused Plectorhinchus species were well-resolved in the phylogenetic results. We also confirmed that the genus Diagramma was placed inside the colorful Plectorhinchus groups, suggesting close relationship of Diagramma within Plectorhinchus. Additionally, we found that two species, P. orientalis and P. vittatus, which were traditionally considered as synonyms for oriental sweetlips, which were suggested to be two distinct species. Sequence divergence also revealed a great genetic variation between P. orientalis and P. vittatus (6.0% in Cyt b and 7.4% in COI), which was largely greater than the species diagnosis divergence value (2%) suggested by Hebert et al. This new finding suggested P. orientalis and P. vittatus might be two distinct species and should not be placed as synonyms. Introduction Fishes of the genus Plectorhinchus, commonly known as sweetlips, belong to the family Haemulidae of order Perciformes. Sweetlips are mainly found in the oceanic waters of tropical, subtropical areas throughout the Indo-Western Pacific (Carpenter & Niem, 2001; McKay, 1984). Many of them are of commercial importance to fisheries worldwide. Morphologically, classification and identification within Plectorhinchus remains problematic because of their spectacular morphological diversification. Closely related species share common overlapping color patterns. Moreover, colorations and patterns in many species change greatly throughout the lives, thus making the juveniles and adolescents quite distinct from their adult forms. Despite abundant morphological data focused on the genus Plectorhinchus, some interrelationships within Plectorhinchus group remained unclear. Until now, no comprehensive molecular study has concentrated on the Plectorhinchus group and resolved the confused classificational issues. Some current molecular studies have made preliminary investigations on the Haemulidae phylogeny (Ren & Zhang, 2007; Sanciangco et al., 2011; Tavera et al., 2012; Zhu et al., 2005), but the number of Plectorhinchus species were very limited and most of their analyses just focused on the subfamily Haemulinae. Detailed Correspondence: Rishen Liang, College of Life Science, Zhongkai University of Agriculture and Engineering, Guangzhou , People s Republic of China. Tel: cheetahliang@ 126.com Keywords Diagrammus, mitochondrial DNA, molecular phylogeney, Plectorhinchus, synonyms History Received 26 July 2014 Revised 6 October 2014 Accepted 18 October 2014 Published online 8 December 2014 inference on relationships within Plectorhinchus was not the major goal in their studies, and the results relating to Plectorhinchus were not thoroughly enlightening. In this study, we performed molecular phylogenetic analyses of 17 representative sweetlips species distributed along the Indo- Western Pacific based on two mitochondrial genes (cyt b and COI). Our results were compared to previous morphology based relationship hypotheses. All of these genes have been widely used in resolving the phylogenetic relationships of Perciformes fish. Outgroup taxa were chosen from the species in the subfamily Haemulinae, as they were closely related to Plectorhynchinae. We aimed to utilize the available molecular evidence to resolve the interrelationships of morphologically debated sweetlips, providing molecular information for furthering the understanding and identification of this diverse reef fish group. Materials and methods Sample collection and DNA extraction Specimens used in this study were collected in their natural habitats using hooks and commercial trawls (Table 1). A total of 40 individuals from 17 sweetlips species were sampled for this analysis. Four grunt species from four genera of the subfamily Haemulinae were selected as outgroup taxa Total genomic DNAs were extracted using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Each DNA extract was visualized on a 1% ethidium bromide-stained agarose gel and then stored at 20 C.

3 2210 R. Liang et al. Mitochondrial DNA Part A, 2016; 27(3): Table 1. Names, locations, and sequence information of specimens used in this study. Downloaded by [Sun Yat-Sen University] at 05:28 04 June 2016 GenBank Accession No. Species Location Catalog number Label cyt b COI Ingroup species Plectorhinchus albovittatus New Caledonia: Noumea JNC 2131 NCN JX JX Plectorhinchus chaetodonoides China: Paracel Islands SCAUA 117 CPI1 JX JX China: Paracel Islands SCAUA 208 CPI2 JX JX New Caledonia: Noumea JNC 1558 NCN1 JX JX New Caledonia: Noumea JNC 1559 NCN2 JX JX Plectorhinchus chubbi South Africa: KwaZulu-Natal, SAIAB SAK JX JX Park Rynie Plectorhinchus cinctus China: Guangdong, Shenzhen SCAUA 115 CGS JX JX China: Guangdong, Yangjiang SCAUA 198 CGY1 JX JX China: Guangdong, Yangjiang SCAUA 199 CGY2 JX JX Plectorhinchus diagrammus China: Paracel Islands SCAUA 118 CPI1 JX JX China: Paracel Islands SCAUA 206 CPI2 JX JX China: Paracel Islands SCAUA 207 CPI3 JX JX Plectorhinchus gaterinus Tanzania:Nyama Reef SAIAB TNR1 JX JX Tanzania: Nyama Reef SAIAB TNR2 JX JX Plectorhinchus gibbosus Seychelles: Maha, Port Launay KUI: KUIT 6832 SMP1 JX JX Seychelles: Maha, Port Launay SAIAB SMP2 JX JX Plectorhinchus lineatus China: Paracel Islands SCAUA 120 CPI1 JX JX China: Paracel Islands SCAUA 201 CPI2 JX JX New Caledonia: Noumea JNC 2731 NCN JX JX Plectorhinchus orientalis China: Paracel Islands SCAUA 121 CPI1 JX JX China: Paracel Islands SCAUA 203 CPI2 JX JX China: Paracel Islands SCAUA 204 CPI3 JX JX Plectorhinchus picus China: Paracel Islands SCAUA 122 CPI JX JX USA: CNMI, Saipan KU: KUI 5732 UCS JX JX Plectorhinchus plagiodesmus Mozambique: Inhambane, tidal SAIAB MI JX JX bay in front of lodge Plectorhinchus playfairi Mozambique: Maputo, Wreck at SAIAB MMI JX JX point, Inhaca Island Plectorhinchus schotaf Mozambique: Maputo, Wreck at SAIAB MMI JX JX point, Inhaca Island Plectorhinchus sordidus Kenya: Diani Beach SAIAB KDB JX JX Plectorhinchus sp. Fiji: Viti Levu, Charybdis reef KU: KUI 4337 FVL JX JX Plectorhinchus vittatus Seychelles: Maha, North Point of KU: KUIT 6921 SMC1 JX JX Cenception Island Seychelles: Maha, North Point of SAIAB SMC2 JX JX Cenception Island Seychelles: Maha, North Point of SAIAB SMC3 JX JX Cenception Island Diagramma pictum China: Guangdong, Shenzhen SCAUA 114 CGS JX JX China: Paracel Islands SCAUA 205 CPI JX JX New Caledonia: Noumea JNC 3239 NCN1 JX JX New Caledonia: Noumea JNC 3246 NCN2 JX JX Diagramma centurio Seychelles: Mahe, Victoria KU: KUIT 6868 SMV JX JX Mascarene Islands SAIAB MI JX JX Parapristipoma trilineatum China: Guangdong, Yangjiang SCAUA 112 CGY1 JX JX China: Guangdong, Yangjiang SCAUA 113 CGY2 JX JX Outgroup species Pomadasys maculatus China: Guangdong, Yangjiang SCAUA 123 CGY JX JX Haemulon aurolineatum Belize: Carrie Bow Cay KU: KUIT 209 BCBC JX JX Anisotremus surinamensis Puerto Rico: Playa Sardinera KU: KUI 2422 PPS JX JX Conodon nobilis USA: Texas, Gulf of Mexico KU: KUI 5135 UTG JX JX KU: University of Kansas, Natural History Museum and Biodiversity Research Center; JNC: Collections of Professor Jean-Lou Justine (MNHN, Paris) in Noumea, New Caledonia; SAIAB: South African Institute for Aquatic Biodiversity; SCAUA: South China Agricultural University, College of Animal Science. PCR amplification and sequencing Primers of the two genes (cyt b and COI) used for amplification referred to the studies (Marko et al., 2004; Ward et al., 2005). The PCR mixture was 50ml in volume, with 37 ml sterile distilled H 2 O, 2 ml dntps (10 mm), 5 ml reaction buffer (TaKaRa, Dalian, China), 1 ml each primer (10 mm), 2 ml Taq polymerase and 2 ml DNA template. The thermocycling profiles were carried out under the following protocol:: Initial denaturation step at 94 C for 5 min, followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 54 C for 45 s, and extension at 72 C for 60 s, and then a final extension at 72 C for 10 min. All PCR products were isolated on 1.5% agarose gels and sent to Shanghai Sangon Biological Company (Shanghai, China) for sequencing. Sequence alignment and phylogenetic analyses Alignments of cytb and COI sequences were straightforward and performed in Clustal W (Thompson et al., 1994) with default parameters. All sequences have been deposited in GenBank

4 DOI: / Molecular phylogenetic relationships of sweetlips 2211 Downloaded by [Sun Yat-Sen University] at 05:28 04 June 2016 (Table 1). Base composition, number of variable sites and sequence divergences were calculated by the program Mega 4.0 (Tamura et al., 2007). Sequence divergences were calculated based on the Kumira s two-parameter model. In order to reveal more phylogenetic information from the two genes, the partition homogeneity test (Farris et al., 1994, 1995) was detected in PAUP version 4.0b10 (Swofford, 2003) to examine the utility and feasibility of combining the two dataset. Phylogenetic analyses were conducted on the two markers and the concatenated datasets using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) methods. MP and ML analyses were conducted by PAUP version 4.0b10 with heuristic searches using tree bisection reconnection (TBR). Nodal support was estimated by 1000 total bootstrap pseudo replicates. BI method was performed with MrBayes3.0 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) and run for 1,000,000 generations in two independent runs of four simultaneous Markov Chain Monte Carlo (MCMC) chains, with trees sampled every 100 generations. The majorityrule 50% consensus tree was computed from the remaining sampled trees after discarding the burn-in samples. Results Gene variations, saturation effects, and sequence divergences The mitochondrial cytb and COI were 789 bp and 651 bp, respectively. The partition homogeneity test value was p ¼ 0.44, revealing no significant difference between Cyt b and COI genes. Therefore, two genes could be combined for all further phylogenetic analyses. After alignment, the combined dataset were 1440 bp in length, there were 900 sites conservative, 540 sites variable, and 495 sites parsimony informative. The overall base composition was T: 27.7%, C: 31.8%, A: 23.3%, and G: 17.2% and the Ts/Tv ratio was Sequence divergence among species was calculated using Kumira s two-parameter model. The mean uncorrected sequence divergence for all ingroup Plectorhinchus taxa was 15.0% in cytb and 13.8% in COI. A relatively low divergence was revealed between the two genera Plectorhinchus and Diagramma. The average values were 14.3% for cytb and 13.4% for COI, lower than the mean ingroup Plectorhinchus divergences above. In addition, most inter-genera (Diagramma and Plectorhinchus) species divergence values were even lower than the inter-plectorhinchus species. For example, the divergence value was 11.6% between P. chaetodonoides and Diagramm species in Cyt b, but more than 12% between P. chaetodonoides and P. gibbosus, and P. cinctus, and P. plagiodesmus. This was also observed for COI. Phylogeny analysis Three methods MP, ML and BI, came to almost identical wellresolved tree topologies from the concatenated dataset with most nodes receiving high bootstrap values and posterior probabilities (Figure 1). Three trees strongly supported the division of Plectorhinchus into three groups. Group I consisted of sweetlips of shared marked coloration and patterns, Group II and Group III were both comprised of sweetlips with drab colors and single patterns. The genus Plectorhinchus was found to be nonmonophyletic, for two Diagramma species, group within the genus Plectorhinchus, and was a sister lineage to a cluster P. chaetodonoides + P. picus in Group I. Within the interrelationships of the Plectorhinchus group, certain pairs of sister species and clusters were well recovered: (1) Group I: P. chaetodonoides + P. picus, P. lineatus + P. diagrammus, P. orientalis + P. vittatus were tight sister species. (2) Group II: P. c cplayfari was sister to the cluster P. chubbi and P sordidus, P. schotaf was sister to the above 3 species. (3) Group III: P. gibbosus and P. plagiodesmus were grouped together and P. cinctus was nested with them. P. trilineatum was basal to the big Plectorhinchus tribe. Analyses of the individual mitochondrial cyt b, COI partitions based on the ML method revealed similar topologies, with only slight differences in some small arrangements of interspecies relationships, such as the placement of some colorful sweetlips like P. chaetodonoides and P. picus (Figures not shown). In the cyt b tree, P. chaetodonoides and P. picus were grouped as sister species and were sisters to the cluster of the pair: P. lineatus + P. diagrammus and P. vittatus + P. orientalis. As for the COI trees, they were not clustered as sister species but found to be nested within the Diagramma clusters. Other differences were observed in the relationships within P. cinctus, P. gibbosus, and P. plagiodesmus, but two trees consistently supported the fact that they were clustered in one group. Discussion Phylogenetic relationships of sweetlips groups Sweetlips (Plectorhinchus and Diagramma) is the second largest group in the family Haemulidae. The phylogenetic results were morphologically consistent, dividing the Plectorhinchus taxa into three morphologically distinct groups. Plectorhinchus Group I included species that shared remarkable coloration and characteristics. Group II and Group III included species with uniformly dark colors and single patterns. Geographically, the colorful Plectorhinchus species were highly abundant in the Western Pacific, from Japan to Australia. The non-colorful taxa were generally only found in the Indian Ocean. Phylogenetic relationships within the colorful reef Plectorhinchus Morphologically, identification and classification of the colorful coral reef Plectorhinchus were difficult because of their confused color patterns and drastic appearance changes from juvenile to adult phases. Interrelationships within Plectorhinchus remain uncertain based on the early morphological taxonomic diagnoses (Cheng & Zheng, 1987; McKay, 1984; Shen, 1993). In this initial phylogenetic study, certain sister relationships of Plectorhinchus in Group I: P. diagrammus + P. lineatus, P. vittatus + P. orientalis, P. picus + P. chaetodonoides were confirmed and well-resolved. By analyzing the comparison of morphological differentiation and developmental transformations in each cluster, we found that sister species in each cluster showed great resemblance in appearance and underwent identical processes from juvenile to adult phases by morphological diagnosis. As an example, the pair P. diagrammus + P. lineatus shared common straight black stripes along bodies in their juvenile stages; the stripes in adults were more subdivided, becoming diagonal in P. lineatus while remaining horizontal in P. diagrammus. The pair P. picus + P. chaetodonoides shared similar blotches on their juvenile bodies. The blotches morphed into numerous small black spots as they grew to adulthood. The two species P. vittatus + P. orientalis were covered with connected black blotches and spots in juveniles; the spots gradually broke into horizontal stripes in adulthood (Carpenter & Niem, 2001; McKay, 1984). The phylogenetic relationships of Plectorhinchus revealed in this study were in accordance with their morphological variations, which provided the molecular demonstration for the morphological contentious taxonomy of Plectorhinchus.

5 Downloaded by [Sun Yat-Sen University] at 05:28 04 June R. Liang et al. Mitochondrial DNA Part A, 2016; 27(3): Figure 1 The majority rule consensus trees of sweetlips (Plectorhinchus, Diagramma) obtained from partitioned Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) analysis of the combined cyt b and COI dataset. Numbers at the nodes are Bayesian posterior probabilities, and bootstrap values of ML, MP, respectively. Asterisks indicate 100% support for the node. Support values less than 50% or nonsupportive values for the node are indicated by a dash. Pictures of species were the Fishbase website provided by Randall JE, Cornish, A, FAO and website of eol.org.

6 DOI: / Molecular phylogenetic relationships of sweetlips 2213 Downloaded by [Sun Yat-Sen University] at 05:28 04 June 2016 Phylogenetic relationships of Diagramma and Plectorhinchus Genus Diagramma were morphologically differed from Plectorhinchus in that Diagramma had a lower number of dorsal spines. Generally, there were 9 10 dorsal spines in Diagramma and in Plectorhinchus. Morphology-based studies suggested the Diagramma was distinct from Plectorhinchus based on this major differentiation (Carpenter & Niem, 2001; Cheng & Zheng, 1987; McKay, 1984; Shen, 1993). But some molecular studies suggested close phylogenetic relationships of Diagramma within Plectorhinchus (Ren & Zhang, 2007; Sanciangco et al., 2011). Diagramma species in this study were monophyletic and strongly grouped inside the colorful Plectorhinchus taxa in Group I, sister to a cluster P. picus + P. chaetodontoides, which was in accordance with previous molecular evidence. Morphologically, Diagramma was also colorful and presented a remarkable developmental transformation. Juveniles Diagramma species were yellowish with broad black longitudinal stripes, the stripes would eventually broke into numerous small dusky spots as they grow up. These molecular and morphological data might reinforce the demonstration of the intensely close relationship between Diagramma and Plectorhinchus. Phylogenetic identification of Plectorhinchus vittatus and Plectorhinchus orientalis Morphologically, Plectorhinchus vittatus (Linnaeus, 1758) and Plectorhinchus orientalis (Bloch, 1793) have long been considered as synonyms and referred to the same species: Oriental sweetlips (Randall & Johnson, 2000; Satapoomin & Randall, 2000). Most morphological reports and fish checklists have used either P. vittatus or P. orientalis to represent the species of oriental sweetlips (Froese & Pauly, 2014; Gell & Whittington 2002; Gillibrand et al., 2007; Unsworth, 2010). In this study, we sampled 4 individuals of P. orientalis and 3 individuals of P. vittatus. The phylogenetic constructions show that individuals of P. vittatus and P. orientalis each formed a separate group, then the two specific groups were clustered as sister lineages, clearly distinguished by the longer internal branch length, suggested their independent evolutionary status in the Plectorhinchus clade. Sequence divergence also revealed great averagely divergence value between them, which were 6.0% in cytb and 7.4% in COI. However, the value among individuals of P. vittatus and individuals of P. orientalis were all less than 0.5% in cytb and COI. Hebert (2003a,b) studied the COI sequences of species from 11 phyla and suggested that inter-specific divergences value of COI were rarely greater than 2% and most were less than 1% (Avise, 2000; Hebert, 2003b). It was also found in cytb (Avise & Walker, 1999). The divergence values between P. orientalis and P. vittatus were largely exceeding the threshold of species diagnosis values, strongly revealing that P. vittatus and P. orientalis might be two distinct species and should not be placed as synonyms. This phylogenetic result suggested that the fundamental taxonomic status of P. vittatus and P. orientalis might potentially be redefined. Acknowledgements We wish to thank the following colleagues for their kind assistance in providing samples for this study: Dr Bernard Mackenzie (South African Institute for Aquatic Biodiversity), Dr Andrew Bentley (University of Kansas) and Dr Jean-Lou Justine (Aquarium des Lagons, New Caledonia). Declaration of interest The study was supported by the Natural Science Foundation of China (No ) and National Undergraduate Scientific and Technological Innovation Project (No.2014A23). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References Avise JC, Walker D. (1999). Species realities and numbers in sexual vertebrates: Perspectives from an asexually transmitted genome. Proc Natl Acad Sci USA 96: Avise JC. (2000). Phylogeography: The history and formation of species. Cambridge, MA: Harvard University Press. Carpenter KE, Niem VH. (2001). FAO species identification guide for fishery purposes. The living marine resources of the western central Pacific. Volume 5: Bony fishes part 3 (Menidae to Pomacentridae). Rome: FAO. p Cheng QT, Zheng BS. (1987). Systematic synopsis of Chinese fishes. Beijing: Science Press. Farris JS, Kallersjo M, Kluge AG, Bult C. (1994). Testing significance of incongruence. Cladistics 10: Farris JS, Kallersjo M, Kluge AG, Bult C. (1995). Constructing a significance test for incongruence. Syst Biol 44: Froese R, Pauly D. (2014). FishBase. Available at: version (Accessed April 2014). Gell FR, Whittington B. (2002). Diversity of fishes in seagrass beds in the Quirimba Archipelago, northern Mozambique. Mar Freshwater Res 53: Gillibrand CJ, Harris AR, Mara E. (2007). Inventory and Spatial Assemblage Study of Reef Fish in the Area of Andavadoaka, South- West Madagascar (Western Indian Ocean). Western Indian Ocean J Mar Sci 6: Hebert, PDN, Ratnasingham S, dewaard JR. (2003b). Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc Lond B 270: Hebert PDN, Cywinska A, Ball SL, dewaard JR. (2003a). Biological identifications through DNA barcodes. Proc R Soc Lond B 270: Huelsenbeck JP, Ronquist F. (2001). MrBayes: Bayesian inference of Phylogeny. Bioinformatics 17: Marko PB, Lee SC, Rice AM, Gramling JM, Fitzhenry TM, McAlister JS, Harper GR, Moran AL. (2004). Mislabelling of a depleted reef fish. Nature 430: McKay RJ. (1984). Haemulidae. In: Fischer W and Bianchi G, editors. FAO species identification sheets for fishery purposes. Western Indian Ocean (Fishing Area 51). Vol. 2. Rome: FAO. p Randall, JE, Johnson JW. (2000). Perca lineata and P. vittata established as valid species of Plectorhinchus (Perciformes: Haemulidae). Mem Queensland Mus 45: Ren G, Zhang Q. (2007). Phylogeny of haemulid with discussion on systematic position of the genus Hapalogenys. Acta Zootaxon Sin 32: Ronquist F, Huelsenbeck JP. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: Sanciangco MD, Rocha LA, Carpenter KE. (2011). A molecular phylogeny of the Grunts (Perciformes: Haemulidae) inferred using mitochondrial and nuclear genes. Zootaxa 2966: Satapoomin U, Randall JE. (2000). Plectorhinchus macrospilus, a new species of thicklip (Perciformes Haemulidae) from the Andaman Sea off southestern Thailand. Phuket Mar Biol Cent Res Bull 63:9 16. Shen SJ. (1993). Fishes of Taiwan. Taipei: Department of Zoology, National Taiwan University. Swofford DP. (2003). Phylogenetic analysis using parsimony (and other methods) Version 10. Sunderland, MA: Sinauer. Tamura K, Dudley J, Nei M, Kumar S. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: Tavera J, Acero A, Balart E F, Bernardi G. (2012). Molecular phylogeny of grunts (Teleostei, Haemulidae), with an emphasis on the ecology, evolution, and speciation history of New World species. BMC Evol Biol 12:57. Thompson JD, Higgins DG, Gibson TJ. (1994). CLUSIAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22: Unsworth, RKF. (2010). Seagrass meadows of the Wakatobi National Park. In: Clifton J, Unsworth RKF, Smith DJ, editors. Marine

7 Downloaded by [Sun Yat-Sen University] at 05:28 04 June R. Liang et al. Mitochondrial DNA Part A, 2016; 27(3): conservation and research in the Coral Triangle: The Wakatobi National Park. New York: Nova Publishers. p Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN. (2005). DNA barcoding Australia s fish species. Phil Trans R Soc B 360: Zhu SH, Zheng WJ, Zou JX, Yang YC, Shen XQ, Liu BQ. (2005). Molecular phylogenetic analysis of five Pomadasyidae fish based on mitochondrial cytochrome b sequences. J Tropical Oceanography 25: 42 5.

8 胡椒鲷鱼类分子系统进化关系研究及两种胡椒鲷同种异名争议分析 摘要 : 本研究基于线粒体细胞色素 b (Cyt b) 及细胞色素氧化酶辅酶 I(COI) 基因构建了胡椒鲷鱼类 17 种 40 个个体之间系统进化关系 系统进化树上, 胡椒鲷鱼类主要分成三大类群 : 类群 I 的种类具有色彩鲜艳的外部条纹 ; 类群 II 和类群 III 包含的种类大部分颜色比较单一, 灰暗, 无鲜艳条纹 形态分类混乱的胡椒鲷种类的分类关系在本研究的进化树上得以清晰呈现 同时, 我们发现少棘胡椒鲷属的种类都位于胡椒鲷属的类群 I 内部, 显示出少棘胡椒鲷属与胡椒鲷属具有非常近的亲缘关系 此外, 本研究发现, 在形态分类上被认为是同种异名的两种胡椒鲷 : 条纹胡椒鲷与东方胡椒鲷, 两者之间的 Cyt b 及 COI 基因具有较大的差异 (Cyt b 为 6%,COI 为 7.4%), 该变异值已大大超过物种鉴定最小种间遗传差异值 (2%), 显示两者有可能是两个独立的物种, 而不应该被认为是同种异名 前言胡椒鲷鱼类 (Plectorhynchus) 隶属鲈形目 Perciformes 仿石鲈科 Haemulidae, 包括胡椒鲷属 (Plectorhinchus), 广泛分布于印度洋及太平洋西部温带, 亚热带及热带沿海及岛礁海域, 是重要的海洋经济食用及观赏性鱼类 形态上, 胡椒鲷鱼类近缘物种十分相似, 许多种类都具有近似的体色和斑纹, 且体色与斑纹会随着个体的逐步发育发生巨大的变化, 使幼鱼与成鱼的形态截然不同 胡椒鲷属的形态差异多样性给传统基于外部性状的分类鉴定带来很大的困扰, 许多种类的分类与命名相互混淆 在分子水平鉴定上, 关于胡椒鲷亚科鱼类的分子系统分类研究还没见报道, 仅在部分石鲈科鱼类系统进化分析涉及零星的胡椒鲷亚科鱼类, 其重点分析内容均放在其他类群, 对于胡椒鲷亚科物种分类的数据分析十分缺乏, 关于胡椒鲷亚科鱼类的系统性的分类与鉴定研究依然存在巨大空白 本研究选择两个线粒体 DNA 基因 (Cyt b 及 COI) 作为分子标记, 以石鲈亚科的鱼类作为外类群, 构建了采集于印度 - 西太平洋区域的 17 种胡椒鲷鱼类分子系统进化关系 以期为形态分类混乱的胡椒鲷属鱼类提供分子水平的分类依据, 并为今后的相关模糊物种的分类鉴定提供快速便捷鉴定依据 材料与方法样品采集与 DNA 提取本研究样品主要在其分布海区进行采集 ( 表 1), 共采集胡椒鲷鱼类 17 种 40 个个体, 另外采集了 4 种石鲈属鱼类作为外类群 DNA 提取利用 Puregene DNA 提取试剂盒进行提取, 提取的 DNA 溶解于 50μL 灭菌水中, 经 1% 琼脂糖凝胶电泳检测, 20 保存备用 参考 Marko et al.,2004 及 Ward et al., 2005 的研究设计扩增 Cyt b 及 COI 基因的引物 PCR 反应体系为 50 μl, 其中灭菌蒸馏水 37μL,dNTPs ( 各 10 mm) 2 μl,10 buffer 5 μl, 上下游引物各 1 μl, Ex Taq 酶 (1 U/μL) 2 μl PCR 反应条件为 : 94 预变性 5min,94 变性 30s,54 退火 45s,72 延伸 60s,35 个循环, 最后 72 延伸 10min PCR 产物经 1.5% 琼脂糖凝胶电泳分离, 纯化后送到上海生工生物技术有限公司进行双向测序

9 表 1. 本研究样品物种名 采集地及序列信息 物种 采集地 标本号 标签 GenBank Accession No. cyt b COI 内类群白带胡椒鲷 New Caledonia: Noumea JNC 2131 NCN JX JX 斑胡椒鲷 China: Paracel Islands SCAUA 117 CPI1 JX JX China: Paracel Islands SCAUA 208 CPI2 JX JX New Caledonia: Noumea JNC 1558 NCN1 JX JX New Caledonia: Noumea JNC 1559 NCN2 JX JX 查氏胡椒鲷 South Africa: KwaZulu-Natal, Park SAIAB SAK JX JX Rynie 花尾胡椒鲷 China: Guangdong, Shenzhen SCAUA 115 CGS JX JX China: Guangdong, Yangjiang SCAUA 198 CGY1 JX JX China: Guangdong, Yangjiang SCAUA 199 CGY2 JX JX 四带胡椒鲷 China: Paracel Islands SCAUA 118 CPI1 JX JX China: Paracel Islands SCAUA 206 CPI2 JX JX China: Paracel Islands SCAUA 207 CPI3 JX JX 密点胡椒鲷 Tanzania:Nyama Reef SAIAB TNR1 JX JX Tanzania: Nyama Reef SAIAB TNR2 JX JX 黑胡椒鲷 Seychelles: Maha, Port Launay KUI: KUIT 6832 SMP1 JX JX Seychelles: Maha, Port Launay SAIAB SMP2 JX JX 斜纹胡椒鲷 China: Paracel Islands SCAUA 120 CPI1 JX JX China: Paracel Islands SCAUA 201 CPI2 JX JX New Caledonia: Noumea JNC 2731 NCN JX JX 东方胡椒鲷 China: Paracel Islands SCAUA 121 CPI1 JX JX China: Paracel Islands SCAUA 203 CPI2 JX JX China: Paracel Islands SCAUA 204 CPI3 JX JX 暗点胡椒鲷 China: Paracel Islands SCAUA 122 CPI JX JX USA: CNMI, Saipan KU: KUI 5732 UCS JX JX 桔唇胡椒鲷 Mozambique: Inhambane, tidal bay SAIAB MI JX JX in front of lodge 白条胡椒鲷 Mozambique: Maputo, Wreck at SAIAB MMI JX JX point, Inhaca Island 灰胡椒鲷 Mozambique: Maputo, Wreck at SAIAB MMI JX JX point, Inhaca Island 红唇胡椒鲷 Kenya: Diani Beach SAIAB KDB JX JX 胡椒鲷 Fiji: Viti Levu, Charybdis reef KU: KUI 4337 FVL JX JX 条纹胡椒鲷 Seychelles: Maha, North Point of KU: KUIT 6921 SMC1 JX JX Cenception Island Seychelles: Maha, North Point of SAIAB SMC2 JX JX Cenception Island Seychelles: Maha, North Point of SAIAB SMC3 JX JX Cenception Island

10 少棘胡椒鲷 China: Guangdong, Shenzhen SCAUA 114 CGS JX JX China: Paracel Islands SCAUA 205 CPI JX JX New Caledonia: Noumea JNC 3239 NCN1 JX JX New Caledonia: Noumea JNC 3246 NCN2 JX JX 南非少棘胡椒鲷 Seychelles: Mahe, Victoria KU: KUIT 6868 SMV JX JX Mascarene Islands SAIAB MI JX JX 三线矶鲈 China: Guangdong, Yangjiang SCAUA 112 CGY1 JX JX China: Guangdong, Yangjiang SCAUA 113 CGY2 JX JX 外类群大斑石鲈 China: Guangdong, Yangjiang SCAUA 123 CGY JX JX 西仿石鲈 Belize: Carrie Bow Cay KU: KUIT 209 BCBC JX JX 黑异孔石鲈 Puerto Rico: Playa Sardinera KU: KUI 2422 PPS JX JX 八带石鲈 USA: Texas, Gulf of Mexico KU: KUI 5135 UTG JX JX 所得利用 Cyt b 及 COI 基因利用 Clustal W 进行序列排列比对, 所有序列已上传 GenBank 利用 MEGA 5.0 计算序列的碱基组成 序列间的碱基变异频率和转换颠换频率, 基于 Kimura s 2-parameter 模型计算各物种的遗传距离 为了获得更多的系统进化信息, 对 Cyt b 及 COI 基因利用 PAUP4.0b10 软件进行同质性检测, 确定两基因是否可以合并分析 分子系统进化树利用最大简约法 (maximum parsimony,mp), 最大似然法 (Maximum likelihood,ml) 和贝叶斯法 (Bayesian inference,bi)) 进行构建 最大简约法在 PAUP Version 4.0b 10 软件包中完成, 使用启发式搜寻, 树二等分再连接 tree-bisection-reconnection (TBR) 参数构树, 所有数据未加权 系统进化树各分支的支持率采用 1000 次重复抽样分析 (Bootstrap analysis) 进行检验贝叶斯法分析在 MrBayes 软件中完成, 随即选取起始树, 计算 代, 取样代数 100, 系统进化树节点置信度由后验概率 (Posterior probabilities) 提供 结果基因差异, 散点分析及序列分歧度测序所得 Cyt b 及 COI 序列分别为 789bp 及 651bp 同质性检测 p=0.44, 表明两序列之间不存在同质性差异, 可以合并进行下一步的分析 合并序列总共为 1440bp, 其中保守位点 900 个, 变异位点 540 个, 简约性信息位点 495 个 碱基平均含量分别 T:27.7%,C: 31.8%,A:23.3%,G:17.2%, 转换 / 颠换值为 种胡椒鲷遗传距离利用 MEGA 5.0 基于 Kimura s 2-parameter 模型进行计算 在内类群中,17 种胡椒鲷鱼类两基因平均遗传距离分别为 Cyt b:15% 及 COI:13.8% 我们发现胡椒鲷属 (Plectorhinchus) 与少棘胡椒鲷属 (Diagramma) 之间的遗传距离相对较小, 平均遗传距离为 Cyt b:14.3% 及 COI:13.4%, 低于胡椒鲷属内部的平均遗传距离 此外, 部分种类属间遗传距离 ( 胡椒鲷属与少棘胡椒鲷属 ) 遗传距离比胡椒鲷属内部种类的遗传距离更大, 如, 斑胡椒鲷与少棘胡椒鲷 Cyt b 之间的遗传距离为 11.6%, 低于斑胡椒鲷与黑胡椒鲷 (12%), 花尾胡椒鲷与桔唇胡椒鲷 (12%) 之间的遗传距离 在 COI 基因之间也存在同样情况

11 系统进化分析基于所得结合序列, 采用 MP, ML 和 BI 法三种方法构建的系统树具有相似的拓扑结构, 并且都具有较高的节点支持率 ( 图 1) 在进化树上, 系统进化树上, 胡椒鲷鱼类主要分成三大类群 : 类群 I 的种类具有色彩鲜艳的外部条纹 ; 类群 II 和类群 III 包含的种类大部分颜色比较单一, 灰暗, 无鲜艳条纹 系统进化树上, 胡椒鲷属鱼类并没有形成单系, 因为两种少棘胡椒鲷的鱼类位于胡椒鲷属类群 I 中, 并于斑胡椒鲷 + 暗点胡椒鲷形成姐妹分支 而在胡椒鲷属属内种间关系中, 下列鱼类的分类关系得到体现 :(1) 类群 I: 斑胡椒鲷 + 暗点胡椒鲷, 斜纹胡椒鲷 + 四带胡椒鲷, 条纹胡椒鲷与东方胡椒鲷形成紧密的姐妹关系 (2) 类群 II 种, 白条胡椒鲷与查氏胡椒鲷 + 红唇胡椒鲷形成姐妹分支, 灰胡椒鲷再与其相聚 (3) 类群 III 中, 黑胡椒鲷与桔唇胡椒鲷紧密聚成一支, 花尾胡椒鲷再与其相聚 三线矶鲈位于整个胡椒鲷属大类群基部 基于单独的 Cyt b 及 COI 基因构建的 ML 系统数也得到相似的拓扑结构, 除了少数种间关系的聚类有差别 如斑胡椒鲷与暗点胡椒鲷的分类地位 在 Cyt b 系统树上, 斑胡椒鲷与暗点胡椒鲷形成姐妹种, 然后与斜纹胡椒鲷 + 四带胡椒鲷及条纹胡椒鲷 + 东方胡椒鲷形成的分支聚在一起 而在 COI 树上, 两者并没有形成姐妹种, 而是位于少棘胡椒鲷属的分支中 其他的差别主要花尾胡椒鲷 黑胡椒鲷和桔唇胡椒鲷之间的关系 讨论胡椒鲷鱼类大类群系统进化分析胡椒鲷鱼类是仿石鲈科鱼类中的第二大类群 系统进化树所得出的物种聚类关系与形态学分类资料一致, 主要把胡椒鲷鱼类分成三大类群 类群 Ⅰ 的胡椒鲷种类体表均具有各种鲜艳的色彩和斑纹, 色彩斑纹随着个体发育会产生变化 类群 Ⅱ 及类群 III 的胡椒鲷种类体色暗淡单一, 无鲜艳色彩与斑纹, 在地理分布上, 类群 Ⅰ 的胡椒鲷种类主要分布在西太平洋地区, 类群 Ⅱ 的种类大多数仅分布于印度洋, 少数在西太平洋也有分布 具鲜艳色彩的胡椒鲷类群的系统分类关系形态学对于类群 Ⅰ 中胡椒鲷的分类存在较大争议, 皆因该类群近缘种类形态相似, 体色与斑纹还会随着个体发育产生巨大变化, 如前言所述 这种形态差异多样性给传统形态分类鉴定带来很大的困扰, 许多物种分类不清 本研究构建的聚类结果对上述胡椒鲷鱼类的分类关系有较为清晰的展现 进化树上斑胡椒鲷与暗点胡椒鲷, 四带胡椒鲷与斜纹胡椒鲷, 条纹胡椒鲷与东方胡椒鲷, 以较高的支持率两两聚成姐妹分支 形态上, 它们均拥有相似的形态特征, 并且在个体发育过程中两两还具有几乎一致的变化过程 如斑胡椒鲷与暗点胡椒鲷, 两者在幼鱼期体表具有棕色或黑色的斑纹, 而成鱼体表的斑纹逐渐断裂成无数的小黑点 四带胡椒鲷与斜纹胡椒鲷在幼鱼期体表均有数条黑色水平纵带, 成鱼后, 四带胡椒鲷体表保留为四条水平条带, 而斜纹胡椒鲷发育为许多斜行波状条纹 对于条纹胡椒鲷与东方胡椒鲷, 这两种胡椒鲷幼鱼体表都覆盖着不规则断裂状的黑色斑纹, 成鱼后斑纹逐渐变成笔直的水平纵带 结果支持胡椒鲷鱼类的分类关系需要结合幼鱼, 成鱼形态及发育过程才能真实反应, 研究为部分形态分类混乱的胡椒鲷种类提供了有效的分子水平的分类证据

12 图 1. 基于 Cyt b + COⅠ 序列利用最大简约法, 最大似然法与贝叶斯法构建的分子系统进化树 少棘胡椒鲷属与胡椒鲷属之间的关系少棘胡椒鲷属与胡椒鲷属之间的差异主要在于少棘胡椒鲷属具有相对较少的鳍棘数, 一般情况下, 少棘胡椒鲷属背鳍鳍棘数为 9~10, 而胡椒鲷属为 11~14 大部分分类资料根据背鳍鳍棘数把少棘胡椒鲷从胡椒鲷属中划分出来 而近期也有部分研究认为少棘胡椒鲷属与胡

13 椒鲷属在分子水平上有较近的亲缘关系 本研究结果显示两种少棘胡椒鲷紧密聚在一起, 位于胡椒鲷属的内部, 与斑胡椒鲷 + 暗点胡椒鲷形成的分支聚在一起, 与近期的分子水平相关研究一致 形态上, 少棘胡椒鲷与其他色彩鲜艳的胡椒鲷种类一样, 体色和斑纹会随着个体发育逐渐变化 两种少棘胡椒鲷的幼鱼体侧有 3 条黑色纵带, 发育成鱼后纵带逐渐破裂成无数深色小斑点 研究结果结合分形态学分析进一步确定少棘胡椒鲷属与胡椒鲷属之间存在非常近的亲缘关系条纹胡椒鲷 P. vittatus 与东方胡椒鲷 P. orientalis 分类鉴定目前绝大多数分类资料认为条纹胡椒鲷 P. vittatus (Linnaeus, 1758) 与东方胡椒鲷 P. orientalis (Bloch, 1793) 均代表同一个物种 : 东方胡椒鲷 Oriental sweetlips 大部分形态分类资料及物种名录要么使用 P. vittatus 要么使用 P. orientalis 代表东方胡椒鲷 Oriental sweetlips 这个种类 本研究分别获得鉴定为条纹胡椒鲷与东方胡椒鲷的样本材料, 其中条纹胡椒鲷样本 3 尾, 东方胡椒鲷样本 4 尾 系统进化树上我们发现 P. vittatus 与 P. orientalis 的个体分别形成独立的分支, 两大分支再相聚, 显示两者在胡椒鲷属类群中具有独立的进化地位 Cyt b 及 COI 的遗传距离分析同样揭示了两者之间存在较大的差异, 其值分别为 :Cyt b (6%) 及 COI (7.4%), 而两胡椒鲷内部不同个体间两基因的遗传距离均不超过 0.5% Hebert 等 (2003a,b) 对 11 个门 个物种的 COⅠ 基因序列进行分析, 得出物种内的 COⅠ 遗传距离很少有大于 2%, 大部分的种内距离是小于 1% 在 Cyt b 基因也有同样的研究 而本研究两胡椒鲷之间的遗传距离差异值已大大超过物种鉴定最小种间遗传差异值 (2%), 显示两者有可能是两个独立的物种, 而不应该被认为是同种异名 致谢感谢以下单位及人员对本研究的样品的提供与支持 :Dr Bernard Mackenzie (S 南非水生生物多样性研究院 ), Dr Andrew Bentley ( 堪萨斯大学 ) and Dr Jean-Lou Justine ( 新喀里多尼亚泻湖水族馆 ).