Zoological Studies 48(4): 569-583 (2009) Speciation and Phylogeography of Opsariichthys bidens (Pisces: Cypriniformes: Cyprinidae) in China: Analysis of the Cytochrome b Gene of mtDNA from Diverse Populations Gao-Yan Li1,2, Xu-Zhen Wang3, Ya-Hui Zhao1, Jie Zhang1, Chun-Guang Zhang1,*, and Shun-Ping He3 1Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 2Graduate University of Chinese Academy of Sciences, Beijing 100049, China 3Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China (Accepted December 29, 2008) Gao-Yan Li, Xu-Zhen Wang, Ya-Hui Zhao, Jie Zhang, Chun-Guang Zhang, and Shun-Ping He (2009) Speciation and phylogeography of Opsariichthys bidens (Pisces: Cypriniformes: Cyprinidae) in China: analysis of the cytochrome b gene of mtDNA from diverse populations. Zoological Studies 48(4): 569-583. The cyprinid fish Opsariichthys bidens Günther is distributed in all major river systems of continental East Asia, and represents an attractive model for phylogeographic studies among cyprinid species or within a given species. In this study, we investigated the phylogeographic and demographic history of this species, using partial sequences of the cytochrome (cyt) b gene in mitochondrial (mt)DNA. Fish samples were collected from almost all major river systems where O. bidens is distributed in China. Sequence analysis showed remarkably high polymorphism, with 125 haplotypes in the 234 specimens examined, and with 89.8% of haplotypes occurring in only 1 specimen. A neutrality test indicated that some groups were not at mutation-drift equilibrium, suggesting a past population expansion. These results were supported by a mismatch distribution analysis. Based on our analysis, O. bidens consists of 4 groups belonging to 2 clades. The divergence time of the 2 clades was estimated to be 11.06-8.04 my. This value corresponds to the time of the 2nd uplift of the Qinghai-Tibet Plateau, the emergence of the East Asian monsoon, and the Epoch-6 Event. A two species scheme is proposed. http://zoolstud.sinica.edu.tw/Journals/48.4/569.pdf Key words: Demographic history, Genetic divergence, Mismatch distribution analysis, Molecular clock, Neutrality test. Cyprinid fishes represent the most extensive subspecies (Chen 1998). Opsariichthys bidens freshwater fish family, the Cyprinidae, which consist is one of the most widespread cyprinid species in of more than 340 genera and more than 2000 East Asia. It is distributed in all major river systems species (Banarescu et al. 1991, Nelson 2006). in Mainland China (except for the Qinghai-Tibet They naturally occur in almost all types of aquatic Plateau and Xinjiang Uygur Autonomous Region), habitats on all continents except for Australia and as well as in the Korean Peninsula and Vietnam’s South America (Banarescu et al. 1991, Howes Red River (Chen 1998). It was once divided 1991). These fish species are a major component into O. uncirostris amurensis Berg,1932 and O. of the primary freshwater fish fauna of Eurasia. In uncirostris bidens Günther, 1873 according to the China, this family is found in almost all freshwater numbers of lateral line scales and gill rakers (Wu habitats and is the largest freshwater fish family, et al. 1964). But a statistical analysis of lateral with more than 132 genera, and 532 species and line scales revealed that their numbers decrease * To whom correspondence and reprint requests should be addressed. Shung-Ping He and Chun-Guang Zhang contribute equally to this work. Tel: 86-10-64807116; 86-27-68780430. Fax: 86-10-64807116; 86-27-68780430. E-mail:[email protected]; [email protected] 569 570 Zoological Studies 48(4): 569-583 (2009) , with the fish s distribution from north to south, analyses would also be helpful in understanding which was considered to be related to temperature the evolutionary history of other cyprinid fishes. gradients. Hence, they were then considered The aims of the present study were to clarify the to be a monotypic species (Chen 1982). This longstanding debate on the taxonomy of O. bidens treatment has been widely accepted. But recent and to study the phylogeography and demographic molecular biological approaches have revealed history among its diverse populations. The cyt a more-complicated scenario. Perdices et al. b gene is a useful marker for these purposes. It (2005) showed that populations of O. bidens in the is probably the best-known mitochondrial gene Yangtze, Pearl, and Hai River systems seemed with respect to structure and function of its protein to be mixtures of 5 species. Berrebi et al. (2006) product (Esposti et al. 1993). Some work has suggested that there were at least 3 subspecies been done on the O. bidens cyt b gene (Perdices in O. bidens or even 3 species within the complex et al. 2005). We used the cyt b gene as a genetic in the Yangtze and Pearl River systems. Similar marker for our comprehensive study. situations also occur for other cyprinid species. As pointed out by Su et al. (2001), such broadly distributed species might simply not represent MATERIALS AND METHODS single taxa. Moreover, phylogeographic studies have revealed multiple mitochondrial lineages Sampling inside different cyprinid taxa from China’s major drainages (Xiao et al. 2001, Perdices et al. 2004). Samples were collected from all major However, there is also other molecular systematics habitats of O. bidens in Mainland China. In total, research of the Opsariichthines (Wang et al. 2007), 98 specimens were collected from 26 localities which tentatively concluded that there was only from 2000 to 2006 (Table 1, Fig 1). All specimens 1 species within the genus Opsariichthys, with were first identified in the field, and then confirmed O. bidens and O. uncirostris retained as 2 local in the laboratory. populations of O. uncirostris. Obviously, some important relations within O. Selection of outgroups bidens remain unclear. A further study is required for a robust phylogeography encompassing more Opsariichthys is considered to be most samples distributed over the river systems of East closely related to the genera Zacco, Parazacco, Asia. Due to its wide distribution in East Asia, and Candidia in the family Cyprinidae (Chen analyses of the phylogenetic and demographic 1982). Opsariichthys bidens lives in sympatry history of O. bidens across the major river with Z. platypus in almost all localities, and these systems of China could shed light on historical 2 species are very similar in gross morphology, connections of major river systems in Asia. Such except for the shape of the mouth (O. bidens has Table 1. Summary of sample information. GenBank accession numbers of the cytochrome b genes are provided Locality (no.) Haplotype Counts Group (or subgroup) Species-voucher GenBank accession no. Amur R. (1) H42 1 B2 132593 FJ602011 H46 1 B2 132602 FJ602014 H48 1 B2 132601 FJ602013 H50 1 B2 132600 FJ602012 Tumen R. (2) H41 1 B2 132591 FJ601948 Liao R. (3) H46 1 B2 132612 FJ602015 H49 1 B2 132613 FJ602016 Hai R. (4) H39 1 B2 BE68-1 AY646619a Hai R. (5) H46 1 B2 73121 FJ601924 H47 1 B2 73124 FJ601925 Hai R. (6) H40 1 B2 BE66-1 AY646618*a Li et al. – Speciation and Phylogeography of Opsariichthys bidens in China 571 Table 1. (Cont) Locality (no.) Haplotype Counts Group (or subgroup) Species-voucher GenBank accession no. Yalu R. (7) H43 1 B2 60020 FJ602010 H44 1 B2 60022 FJ601950 H45 1 B2 60023 FJ601951 H58 1 B2 60018 FJ601949 H59 1 B2 60024 FJ601952 H62 1 B2 60025 FJ601953 Biliu R. (8) H55 1 B2 40093 FJ601919 H56 1 B2 40095 FJ601921 H57 1 B2 40094 FJ601920 H60 1 B2 40096 FJ601922 H61 1 B2 40099 FJ601923 Huai R. (9) H51 7 B2 139547, 139548, 139549, FJ601926, FJ601927, FJ601928, 139550, 139553, 1395554, FJ601929, FJ601930, FJ601931, 139556 FJ601932 H52 1 B2 139558 FJ601933 Yellow R. (10) H63 4 B1 40336, 40338, 4033839, FJ601954, FJ601955, FJ601956, 4033840 FJ601957 Qing R. (11) H116 2 A1 E12, E14 FJ601967, FJ601968 H118 1 A1 E13 FJ601969 Han Shui (12) H53 1 B2 X11 FJ601997 H110 1 A1 X12 FJ601998 Li Shui (13) H79 1 A2 LI16-3 AY646530a H103 1 A1 LI16-2 AY646529a H105 1 A1 LI16-5 AY646532a H111 1 A1 LI16-1 AY646528a H113 1 A1 LI16-4 AY646531a H114 1 A1 LI16-6 AY646533a Li Shui (14) H78 1 A2 LI20-5 AY646538a H103 6 A1 LI20-1, -3, -4, -6, -7, -8 AY646534, AY646536, Y646537, AY646539, AY646540, AY646541a H104 1 A1 LI20-2 AY646535a Yuan R. (15) H103 1 A1 YU11-5 AY646559a H113 3 A1 YU11-1, -2, -4 AY646555, AY646556, AY646558a H117 1 A1 YU11-3 AY646557a Yuan R. (16) H81 2 A1 YU14-5, -6 AY646564, AY646565a H98 1 A1 YU14-2 AY646561a H99 1 A1 YU14-7 AY646566a H100 1 A1 YU14-3 AY646562a H102 1 A1 YU14-1 AY646560a H103 1 A1 YU14-4 AY646563a H106 1 A1 YU14-9 AY646568a H113 1 A1 YU14-8 AY646567a Yuan R. (17) H54 1 B2 T23 FJ601993 H103 1 A1 T26 FJ601996 H113 1 A1 T25 FJ601994 H115 1 A1 T22 FJ601995 Yuan R. (18) H95 1 A1 YU2-4 AY646545a H103 2 A1 YU2-1, -2 AY646542, AY646543a H113 2 A1 YU2-3, -5 AY646544, AY646546a H119 1 A1 YU2-6 AY646547a Yuan R. (19) H94 1 A1 YU6-1 AY646548a H107 1 A1 YU6-4 AY646551a 572 Zoological Studies 48(4): 569-583 (2009) Table 1. (Cont) Locality (no.) Haplotype Counts Group (or subgroup) Species-voucher GenBank accession no. H108 1 A1 YU6-3 AY646550a H113 3 A1 YU6-5, -6, -7 AY646552, AY646553, Y646554a H117 1 A1 YU6-2 AY646549a Yongning R. (20) H80 1 A2 YU54-1 AY646569a Zi Shui (21) H97 1 A1 ZI29-2 AY646513a H101 4 A1 ZI29-1, -4, -5, -6 AY646512, AY646515, Y646516, AY646517a H113 1 A1 ZI29-3 AY646514a Zi Shui (22) H88 1 A1 ZI32-5 AY646522a H93 1 A1 ZI32-2 AY646519a H112 4 A1 ZI32-7, -8, -9, -10 AY646524, AY646525, Y646526, AY64652-7a H113 4 A1 ZI32-1, -3, - 4, -6 AY646518, AY646520, Y646521, AY646523a Xiang R.