Progress Toward a Global Genealogy of Common Carp (Cyprinus Carpio L.) Strains Using Mitochondrial Nucleotide Sequences Data

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Progress Toward a Global Genealogy of Common Carp (Cyprinus carpio L.) Strains Using Mitochondrial Nucleotide Sequences Data

B.T. Thai, T. A. Pham, U. D. Thai and C.M. Austin

Abstract

As part of a study of genetic variation in the Vietnamese strains of the common carp (Cyprinus carpio L.) using direct DNA sequencing of mitochondrial control and ATPase6/8 gene regions, samples from a number of other countries were analyzed for comparison. Results show that the levels of sequence divergence in common carp is low on a global scale, with the Asian carp having the highest diversity while Koi and European carp are invariant. A genealogical analysis supports a close relationship among Vietnamese, Koi, Chinese Color and, to a lesser extent, European carp. Koi carp appear to have originated from a strain of Chinese red carp. There is considerable scope to extend this research through the analysis of additional samples of carp from around the world, especially from China, in order to generate a comprehensive global genealogy of common carp strains.

Introduction In China, domestication of com- around the world. The outcome mon carp commenced over 4000 demonstrates that a genealogical The sequence divergence of mito- years ago and nearly three million t approach shows significant potential chondrial DNA (mtDNA) has been are produced annually (FAO 2004). for furthering our understanding used to delineate the historical diver- A wide range of molecular marker of the diversity of wild and gence of genetic groups and haplo- systems have been used to investigate domesticated carp populations type frequencies have revealed recent genetic diversity in carp, including including their origins, genetic history animal movements among local microsatellite (Crooijmans et al. 1997; and relationships. populations (Moritz 1994). MtDNA Yue et al. 2004), randomly amplified sequencing is also being increasingly polymorphic DNA (RAPDs) (Bartfai Materials and Methods used to investigate the relationships, et al. 2003), amplified fragment length origins and diversity of domesticated polymorphism (AFLPs ) (David et al. Sampling animal species, including rabbits, pigs, 2001), allozymes (Brody et al.1979; goats, buffalos and horses (Kierstein Kohlmann and Kersten 1999), restric- Tissue samples of common carp were et al. 2004; Kim et al. 1999, 2002; Long tion fragment length polymorphism obtained from populations or strains et al. 2003; Manjunath et al. 2004; (RFLPs) (Jian et al. 2003; Kohlmann et from Vietnam, China, Japan, Indonesia, Wang et al. 2000). In contrast, similar al. 2003; Riho et al. 2002) and direct India, Hungary, Czech Republic, Israel studies of aquaculture species are sequencing of mtDNA fragments and Australia. Strains, sample codes, rare (Nguyen et al. 2004), perhaps re- (Froufe et al. 2002; Wang and Li 2004). and collection details are listed in flecting their more limited relevance However, there have been no com- Table 1. due to the short history of domes- prehensive studies of common carp tication for most species. A notable that utilise nucleotide sequences. DNA extraction and amplification exception to this is the common carp of mitochondrial DNA (Cyprinus carpio L.), the world’s oldest This paper presents the results of domesticated and arguably the most sequencing the mtDNA ATPase6/8 Total DNA was extracted from fin important aquaculture species (Yue et and control region fragments for 89 clip tissue by the protocol described al. 2004). individual common carp representing by Crandall et al. (1999). Two to six 32 strains or populations from individual fish were sequenced from

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Table 1. Sample code, collecting locality and number of individuals sequenced (N) for each strain or population. Strain Sample code 1 Locality N Collector Xingguonensis XI 3 Wananensis WN 3 Jaing Xi, China Li Sifa Wuyuanensis WU 3 Color CL 3 Dor 70 D70 4 K. Ilan Nasice NA 4 White Koi WK 2 Black Koi BAKGan-Shmuel, Israel 2 Kohaku Koi KK 2 U. Lavi Sanke Koi SK 2 Showa Koi SHK 2 Red Koi KO Komaki-shi, Japan 2 Y. Lemura Blatna CZ Czech Republic 5 K. Lukas Hajduboszomeny HA 5 Szeged SZSzarvas, Hungary 5 L. Varadi Tata TAT 5 Wild Amur WA 3

Hungarian P3 P3 2 G. C. Mair Karnataka, India German X Bangkok LF 2 Y.Basavaraju Bhadra River LB 2 Majalaya MA 2 Rajadanu RJSukamandi, Indonesia 2 N. Estu Widan WI 2 Rockland Reservoir AU Victoria, Australia 2 V. Versace Yen Bai YB Van Chan, Yen Bai, Vietnam 2 Tuyen Quang TQ Hoang Khai, Tuyen Quang, Vietnam 4

Bac Kan BK Bach Thong, Bac Kan, Vietnam 4 B. T. Thai Vietnamese white VN RIA 1, Bac Ninh, Vietnam 2 U. D. Thai Hungarian scale H RIA 1, Bac Ninh, Vietnam 2 Indonesian yellow I RIA 1 , Bac Ninh, Vietnam 4 Y.Chang GenBanK (X61010) GB Taiwan 2 1 H. D. Huang Goldﬁsh (out group) GF Japan 1 M. Murakami

1 Unique haplotypes within strains are presented by the number 1 and 2 immediately following the sample code (e.g., BK1 and BK2). 2 Location of strain is unknown. RIA 1: Research Institute for Aquaculture No 1, Dinh Bang, Tu Son, Bac Ninh, Vietnam.

each population or strain. The mito- Carp-Pro (5’ AACTCTCACCCCTG- Data analysis and phylogenetic chondrial ATPase6/8 gene fragment GCTACCAAAG 3’) and Carp-Phe reconstruction was ampliﬁed using primers L8331 (5’ CTAGGACTCATCTTAG- and H9236 (Lovette et al. 1998) and CATCTTCAGTG 3’). PCR conditions Sequences of common carp from also the control region of primers and procedures for visualization, GenBank (Chang and Huang 1994) designed from common carp puriﬁcation and sequencing are given were included in the data set for sequences on GenBank (AC: X61010), in Murphy and Austin (2002). comparative purposes, and Carassius

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auratus sequences (GenBank acces- variation at both gene regions was variability, the two gene regions sion number NC 002079) were used found to be low, although the control possessed significant phylogenetic as the out group. region exhibited more variation signal and were largely consistent in than the ATPase6/8. Excluding the the patterns of variation within and The DNA sequences from the GenBank Cyprinus carpio (which is of between samples and geographic control region were aligned using unknown origin) based on nucleotide groupings and the relationships the program Clustal X (Thompson substitution, the two most divergent amongst the haplotypes. et al.1997). The DNA sequences ATPase6/8 haplotypes differed by The nucleotide and haplotype from ATPase6/8 regions were 12 bp and the two most divergent diversity (range 0 to 1) in various aligned by eye. Aligned sequences control region haplotypes differed geographic groupings is summarized were imported into PAUP* 4.0.b.10 by 14 bp. The average divergence in Figure 1. Although nucleotide (Swofford 2000) for phylogenetic (~ 1 per cent) is low, given that diversity is low overall, differences analysis. It is important to identify the the control region and ATPase6/8 are apparent between the Asian most suitable model of nucleotide genes are two of the fastest evolving carp (0.008) and carp of European evolution for valid phylogenetic regions of the mitochondrial origin (0.000). These differences were inference (Felsenstein 2004). The genome. The average control region most obvious in terms of haplotype Modeltest 3.06 program (Posada divergence (1.24 per cent) was higher diversity (h), which exceeded 0.93 for and Crandall 1998) was used to test than that of the average ATPase6/8 all carp of Southeast Asian origins but an alternative model of evolution. region (0.68 per cent) (data not was 0.00 for European carp. Koi carp The best model was then used to shown). Despite this relatively limited showed no diversity, were identical calculate pair-wise sequence distances for constructing a neighbor-joining (NJ) tree. Confidence levels in the resulting relationship were assessed using the bootstrap procedure with 1000 replications. Nucleotide (π) and haplotype (h) diversity for specific grouping of samples were calculated using DNASP 4.10 (Rozas et al. 2003). Results

A total of 820 bp of the ATPase6/8 genes and 745 bp of the control region were sequenced for 87 individuals. All sequences obtained in this study have been submitted to GenBank (accession number: AY597942-AY597985 and AY 600150- AY600241). The mean nucleotide composition is A=31 per cent, T=30 per cent, C=23 per cent and G= 16 per cent. The most suitable evolution model identiﬁed for the nucleotide data is: Hasegawa-Kishino- Yano (HKY) + invariable site (I) + gamma distribution shape parameter (G); transition/ transversion ratio = 3.8306. The HKY model allows transition and transversion mutation Figure 1. (a) & (b). Nucleotide and haplotype diversity of geographic common carp group derived from combination ATPase6/8 and control regions. The number in occurring at different rates and base bracket is number of individual sequenced. Japanese carp sample (Koi carp) were frequencies of nucleotide to vary obtained from Japan and Israel. Asian carp include: Vietnamese, Chinese, Japanese, (Hasegawa et al. 1985; Page and Indian and Indonesian carp. European group include samples from Israel, Australia, Holmes 1998). The level of nucleotide India, Vietnam, Czech Republic (Czech-R) and Hungarian.

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to the Chinese Color (CL) carp and Genetic relationships the most distinctive common carp showed minimal divergence from haplotype found so far. This suggests Vietnamese carp. The European carp, The origin of a number of carp strains that carp have a longer evolutionary while clearly distinguishable from is uncertain (Balon 1995). Koi carp for history in China, compared to other all other carp, are closely related example, are thought to have been countries or geographic regions (i.e., to the clade containing the Koi and developed directly from common Vietnam and Europe). Vietnamese carp. Significant haplotype carp stocks in Japan, or Chinese diversity is apparent among the Color carp or German pond carp The genetic makeup of Amur river Indonesian and Chinese samples. (Balon 1995; Wang and Li 2004). The carp represents an anomaly in that, The most divergent haplotype results of this study suggest that Koi while they occur over 5000 km from was represented by the Chinese carp have originated from Chinese northern Vietnam and 10 000 km Wuyuanensis strain (WU) that differs Color carp which have a long history from Central Europe, they possess from between 6 to 20 bp from all of domestication that can be traced one haplotype that differs by only other common carp haplotypes. In back over 1200 years (Wang and Li two bases from a haplotype found at addition, the sample of Amur river 2004). This is consistent with the Tuyen Quang in northern Vietnam carp (WA1 and WA2) is interesting as results of Wang and Li (2004) based and another haplotype identical to it contains two haplotypes, one close on COII sequences, but contrary to the European carp. As the Amur river to Chinese Color carp and therefore those of Froufe et al. (2002) who carp examined were originally derived also Vietnamese carp, and the other sequenced 540 bp of the control from a live gene bank line in the Fish that is identical to European carp. region and found that their single Culture Research Institute Szarvas, The relationship of the carp strains is sample of Japanese Koi (N=4) had a Hungary, presently maintained at shown in Figure 2. haplotype identical to European carp. Karnataka in India, this finding might This anomaly could be due to mixing be attributed to accidental stock Discussion of stocks with Koi carp acquirng the mixing in captivity. However, Froufe et European mtDNA through cross al. (2002) found a similar result for a Mitochondrial variation breeding. Despite their substantial sample of Amur river carp obtained color polymorphisms, only a single from the wild, with some haplotypes This study has demonstrated haplotype was detected among the being very close to European carp that direct sequencing of variable 12 individual Koi carp (representing and others to Asian carp. They mtDNA fragments can provide useful 6 strains) sequenced. This result is interpreted this finding to indicate insights into the genetic diversity, consistent with Koi and Chinese an Asian origin for European carp. origin, divergence and genealogy Color carp having passed through However, the converse possibility, that of carp strains and populations. A a substantial bottleneck during the European carp had been introduced surprising result was the low levels domestication process. to China, has also been proposed of divergence observed in what are (Balon 1995) and is also consistent generally considered to be the most Carp from Europe or originating with the data. rapidly evolving vertebrate mtDNA from Europe had the same haplotype, fragments, the ATPase6/8 genes and which was divergent from all other Taxonomic implications the control region (Verspoor 1998), haplotypes detected. A remarkable especially for a freshwater species feature of the European carp samples The taxonomy of common carp is with such an extensive distribution is the complete absence of any highly contentious and confusing across Eurasia. A second surprising variation, which suggests a history of with over 30 synonyms of C. carpio result was the complete absence of founder effects and small population listed on Fish-Base (www.fishbase. variation within two major strains size associated with translocation org/search.cfm) and a proliferation of carp, the European common carp and domestication, like the Koi carp. of named strains. Considering and Koi carp. These findings are In contrast, Chinese carp showed only relatively recent literature, consistent with the long history of considerable haplotype diversity and Kirpichnikov (1967) recognised four domestication of this species which divergence. While no differences subspecies: Cyprinus carpio carpio has undoubtedly involved significant were detected between the (Europe); C. c. aralensis (Central founder and population bottleneck Xingguonensis (XI) and Wananensis Asia); C. c. haematopterus (East Asia); events leading to localized loss of (WN) strains, their haplotypes are and C. c. viridiviolaceus. (Southeast genetic variation (Balon 1995). highly differentiated from Chinese Asia). In contrast, Balon (1995) Color carp (CL) and Wuyuanensis recognized only two subspecies: (WU) carp with the latter showing Cyprinus carpio carpio (Europe) and

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Cyprinus carpio haematopterus (East Asia). Subsequently, Kirpichnikov (1999) questioned the validity of C. c. viridiviolaceus whereas Li et al. (2001) recognised four morphologically distinctive red common carp strains from China which he referred to as C. c. xinggguonensis, C. c. wannanensis, C. c. wuyanensis, C. c. color. In one of the most recent taxonomic treatments of common carp Kottelat (2001) considers the common carp cultured in Southeast Asia to be a distinct species, C. rubrofuscus, although this is disputed by Nguyen and Ngo (2001) who consider this species to be quite rare. The results of genetic studies using allozyme, microsatellite, and RFLP analysis of the mtDNA ND-3/4 and ND-5/6 regions by Kohlman et al. (2003) were interpreted to provide support for Balon’s (1995) position for the existence of two subspecies, C. c. carpio and C. c. haematopterus, as the Asian and European samples form two distinct clades.

MtDNA sequence data, often used for clarifying species boundaries (Avise 2000), provides little support for any of the previous positions. While the results of this study are largely consistent with those of Kohlman et al. (2003) with regard to the distinctiveness of the European carp from Vietnamese/Amur carp/Koi carp, the ﬁnding that samples of carp from China or of Chinese origin are highly divergent fails to support an independent origin for western and eastern common carp lineages and their recognition as distinct subspecies (or species). Indeed, this ﬁnding, combined with the deepest 0.005 substitutions/site branches of the phylogenetic tree represented by samples of Chinese Figure 2. Neighbour-joining tree derived from ATPase6/8 and control region, using origin, suggests that common carp HKY+I+G model of evolution. Bootstrap values are base on 1000 replicates. most probably originated in China. Bootstrap value is given for nodes with at least 50% or more support.

While genetic distance is not always much less in carp compared to that region sequences among closely a reliable guide to taxonomic usually observed among ﬁsh species related species within the genus recognition, the levels of divergence and sub-species. For example, Hurt Acanthopagrus. It is essential that observed for the control and et al. (2001) observed between 15- some stability to the taxonomy of ATPase6/8 regions (~1 per cent) are 18 per cent divergences in control the common carp is achieved and

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we recommended that this study be project. The authors are very grateful and U. Lavi. 2001. Polymorphism extended to include more samples to the referees and editor for their in ornamental and common of carp, especially from China, with helpful and valuable suggestions. This carp strains (Cyprinus carpio L.) parallel studies of morphology, research was supported by funding as revealed by AFLP analysis and genetic variation at rapidly evolving from the Australian Agency for new set of microsatellite mark- nuclear loci (especially intron regions) International Development (AusAID) ers. Molecular Genetic Genomics and reproductive relationships. through its Vietnam Capacity 266:353-362. Building for Agriculture and Rural FAO. 2004. Common carp produc- The Development of a Development (CARD) pilot project tion. http://www.fishbase.org/re- Global Genealogy of program. port/FAO/FAOAquacultureList. Common Carp Strains cfm?scientific=Cyprinus%20carpio. References Felsenstein, J. 2004. 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