Population Structure and Systematics of Opsariichthys Bidens (Osteichthyes: Cyprinidae) in South-East China Using a New Nuclear Marker: the Introns (EPIC-PCR)
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Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2004? 2006 871 155166 Original Article OPSARIICHTHYS ( CYPRINID) PHYLOGEOGRAPHY IN CHINA P. BERREBI ET AL. Biological Journal of the Linnean Society, 2006, 87, 155–166. With 3 figures Population structure and systematics of Opsariichthys bidens (Osteichthyes: Cyprinidae) in south-east China using a new nuclear marker: the introns (EPIC-PCR) PATRICK BERREBI*1, XAVIER RETIF1, FANG FANG2 and CHUN-GUANG ZHANG3 1Laboratoire Ecosystèmes Lagunaires, UMR 5119, cc093, University Montpellier 2, place E. Bataillon, 34095 Montpellier, cedex 05, France 2Department of Vertebrate Zoology, Swedish Museum of Natural History, POB 50007, SE 104 05 Stockholm, Sweden 3Research Center of Animal Evolution and Systematics, Institute of Zoology, Chinese Academy of Sciences. 25 Beisihuanxi Road, Haidian, Beijing, 100080, China Received 28 February 2004; accepted for publication 1 February 2005 Chinese fish farming is the oldest aquaculture in the word. The present pressure on the wild ichthyofauna and its diversity is threatening aquaculture because potential genitors are often caught in the wild. One of the possible responses to this threat is to provide new natural fish taxa for aquaculture. The objective of this study was to analyse the genetic structure of populations of Opsariichthys bidens and to describe its subdivisions, if any, using nuclear markers, to serve as a guideline for stock selection and management in the potential aquaculture of this species. In 2002 and 2003, two collecting trips were made, one in the middle Chang Jiang basin in Hunan Province, and another in the Xi Jiang basin in Guangxi Province, China. Length polymorphisms of the intron amplification (EPIC-PCR) were analysed on 24 systems, only five of which gave easily interpretable and polymorphic patterns, corresponding to 11 presumptive loci. According to the multidimensional statistics, the genetic analysis demonstrated the existence of four clearly independent geographical taxa included in O. bidens, characterized by unambiguous diagnostic intron loci. The distribution of these taxa confirms a similarity between both catchment populations: the middle Chang Jiang and the Xi Jiang samples of the Zhu Jiang basin. An additional output of the study was the choice of the population of each group to be first tested in Chinese fish farms. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87, 155–166. ADDITIONAL KEYWORDS: China – fish farming – introns – Opsariichthys – phylogeography. INTRODUCTION Gorges Dam, especially in the upper and middle reaches. Reconstruction of connectivity seems to be The Chang Jiang (Yangtze River), the longest river in the most urgent necessary improvement, to maintain China and the third largest in the world, harbours 361 the lakes of the middle Chang Jiang in contact with fish species, of which 177 are endemic (Fu et al., 2003). the river network, especially for spawning migrations The basin is usually divided into three ecological parts (Fu et al., 2003). (upper, middle and lower basins), or into five (headwa- Xi Jiang, one of the major tributaries of Zhu Jiang ters, upper, middle, lower and mouth of the river), but (Pearl River), drains mainly through Guangxi Prov- these divisions are not supported by the fish faunal ince, and is the longest river in southern China. Very compositions (Fu et al., 2003). little is known about the ichthyofauna of Xi Jiang The ichthyofauna is threatened by hydrological River, except for a book on fishes from Guangxi Prov- alterations, mainly the Gezhou Ba Dam and the Three ince, which recorded some 133 freshwater fish species, of which about 33 are endemic (Anonymous, 1981). *Corresponding author. China has been exposed to pollution, overfishing E-mail: [email protected] and all kinds of environmental destruction for © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87, 155–166 155 156 P. BERREBI ET AL. decades, and the overall result has been a drastic In 2002 and 2003, two collecting trips were made, decrease both in number of fish species and in the size one in the middle Chang Jiang basin in Hunan Prov- of populations (Li, 2001). ince, and the other in the Xi Jiang basin in Guangxi Chinese fish farming is the oldest aquaculture in Province and large series of O. bidens were obtained the word. Traditional methods are applied to a few (see Table 1). The objective of this study was to anal- species, mostly cyprinids. The present pressure on the yse the genetic structure of populations of O. bidens wild ichthyofauna and its diversity also threatens and to describe its subdivisions, if any, using nuclear aquaculture because potential genitors are often markers, to serve as a guideline for stock selection and captured in the wild. One of the possible reactions management for the potential aquaculture of the spe- to this threat is to provide new natural fish taxa for cies. The aim was to obtain population data such as aquaculture. genetic diversity and level of panmixia. In a recent collaboration between Europe and Because keeping samples frozen in the field was China, new feral species were analysed with a view to nearly impossible in southern China (lacking the proposing some of them for breeding in Chinese fish infrastructure to provide liquid nitrogen), allozyme farms. The genus Opsariichthys and especially O. analysis was abandoned and the exon-primed intron- bidens were targeted. Information about the genetic crossing (EPIC-PCR) technique was used instead. structure of O. bidens populations is scarce. Phylogeo- Introns can be considered as the new universal graphical data is important for evaluating the suit- nuclear markers, applicable to all species without any ability of the species for fish farming, and for prerequisite genetic knowledge. detecting possible conservation needs at the popula- tion level. O. bidens is one of the most widespread cyprinid MATERIAL AND METHODS species of eastern Asia. The genus Opsariichthys is considered to be most closely related to Opsaridium SAMPLING and Raiamas, but also to Zacco, Parazacco or Can- Sampling was conducted in two regions, the middle didia. However, Howes (1980) demonstrated that Chang Jiang (Yangtze) basin in Hunan Province in Opsariichthys and Zacco are more distantly related March–April 2002 (sites A6–A32) and in the Xi Jiang than was expected. Both genera are well established (Pearl River) in Guangxi Province in March 2003 and morphologically distinct. Z. platypus was chosen (sites B04–B54). Figure 1 and Table 1 provide geo- as the outgroup. graphical and biological data on these samples. A total Chang Jiang 30°N A16 A11 +A14 A06 A32 A29 B04 B09 B822 B1 B10 B54 Xi Jiang B46 B34 20°N 100°E 110°E 120°E Figure 1. Map of south-eastern China showing location of the sample sites. See Table 1 for site names and descriptions. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87, 155–166 OPSARIICHTHYS (CYPRINID) PHYLOGEOGRAPHY IN CHINA 157 of 198 specimens of O. bidens and nine specimens of Z. platypus (outgroup) were collected from 14 locali- ties. Table 1 indicates the method of collecting sam- Sampling gear ples. Samples collected from natural waters by our field team were considered to represent part of a nat- ural population; samples bought at the fish markets were probably derived from a single natural popula- tion but the exact origin remains unknown, and the possibility cannot be excluded that occasional market samples could have been composites with specimens March 2003March 2003March 2003 – – Seine April 2002 – 2 April 2002 – captured from different localities. 26–27 March 2003 – 22 March 2003 – 16 March 2003 Seine 11 March 2003 Seine Collection date 18 March 200221 March 200221 March 2002 Seine 24 March 2002 Cast net Cast net Seine 10 March 2003 – The fish were first observed in the field for rapid identification, using mainly the mouth characters to separate Opasriichthys and Zacco species (Opsari- 2 7 1 0 4 0 03 03 06 46 ichys has much longer premaxilla and maxilla bones, Sample size with a deep notch on the premaxilla, which meets a strong extension from the dentary. Zacco has a smaller E1 E8 E1 E6 E2 E2 E2 E1 E7 E1 E2 E1 E2 E9 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ mouth, shorter premaxilla and maxilla, with a smooth 53 57 34 34 56 57 20 12 00 59 01 38 51 16 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ lateral edge both on the premaxilla and on the den- 17 32 58 58 28 09 19 53 34 11 23 18 04 06 B22). ° ° ° ° ° ° ° ° ° ° ° ° ° ° = tary). Identifications were confirmed at the Swedish Museum of Natural History (both genera are well sep- N; 111 N; N; 110 N; N; 107 N; N; 107 N; N; 111 N; 111 N; 111 N; 110 N; 110 N; 111 N; 110 N; 109 N; 110 N; 108 N; ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ arated by many osteological characters, with several 40 24 01 36 58 15 26 45 00 00 12 02 50 27 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ synapomorphies supporting the monophyly of each 31 51 06 49 48 59 58 47 04 00 28 59 50 55 ° ° ° ° ° ° ° ° ° ° ° ° ° ° genus, according to Howes, 1980), except for the 24 specimens of O. bidens from site EC-2003–010 (= B10). With the exception of the nine outgroup specimens of Z. platypus, all individuals analysed were identified as O. bidens. Station number Coordinates MOLECULAR ANALYSES DNA was extracted from fin tissue using the phenol : chloroform method. PCR reactions were car- ried out in an Eppendorf Mastercycler. The 10-µL were obtained from locality EC-2003–022 ( reaction mixture consisted of 1 µL 10× reaction buffer (Promega), 2.5 mM MgCl2, 0.2 mM each dNTP (Invitrogen), 0.5 µM each primer (MWG-Biotech AG, labelled with CY5 or fluoresceine), 0.3 U Taq poly- merase (Sigma) and 1 µL DNA template.