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Genetic characterization of wild common carp (Cyprinus carpio L.) from Turkey
Aquaculture 258 (2006) 257 – 262 www.elsevier.com/locate/aqua-online
Genetic characterization of wild common carp (Cyprinus carpio L.) from Turkey Devrim Memiş a , Klaus Kohlmann b,⁎ a b
Istanbul University, Fisheries Faculty, Aquaculture Department, Ordu St. No. 200, 34470, Laleli, Istanbul, Turkey Department of Inland Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 310, P.O. Box 850119, 12561 Berlin, Germany Received 4 January 2006; received in revised form 17 March 2006; accepted 28 March 2006
Abstract Wild common carp from three lakes in Turkey were genetically characterized by examining the variability of four microsatellite loci and the restriction fragment length polymorphisms (RFLPs) of the mitochondrial ND-3/4 and ND-5/6 gene regions. Microsatellite variability did not differ significantly among the three populations and was only slightly lower than that of other wild-caught populations but remarkably higher than that of domesticated/captive stocks found in preceding studies. On the other hand, genetic differentiation between Turkish wild carp was significant and high (FST values ranged from 0.21 to 0.27). PCR-RFLP analysis revealed a total of five composite haplotypes. One of them was the typical European/Central Asian haplotype H1 shared by two of the Turkish populations from Sapanca and Iznik Lakes, respectively. The remaining four haplotypes were very similar to H1 differing in fragment patterns of only one or two restriction enzymes. Thus, present data further support the hypothesis of a single origin of present day European domesticated and wild/feral carp from a common ancestor with Central Asian carp. Considering that wild common carp are extremely endangered or already extinct in many areas of their natural distribution range, the examined Turkish populations represent valuable genetic resources of European carp that should be protected. © 2006 Elsevier B.V. All rights reserved. Keywords: Common carp; Cyprinus carpio; Microsatellites; Mitochondrial DNA; PCR-RFLP; Population genetics
1. Introduction The common carp, Cyprinus carpio L., has a long history of domestication in Asia (China) as well as in Europe, and numerous strains and breeds have been developed from its wild ancestor. The domesticated/ cultured forms attained worldwide distribution: Today,
⁎ Corresponding author. Tel.: +49 30 64 181 634; fax: +49 30 64 181 799. E-mail addresses: [email protected] (D. Memiş), [email protected] (K. Kohlmann). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.03.041
carp is among the most important species in freshwater fish culture mainly raised for human consumption with an annual production currently exceeding 3.2 million tons (FAO Fishery Statistics, 2003). Ornamental colour varieties known as “Koi” carp are reared in many garden ponds and tanks. Sport fishing of large carp individuals often based on stocking material from carp farms is becoming more and more popular in many countries. In contrast, wild common carp are extremely endangered or already extinct in many areas of their natural distribution range, partly due to losses of habitats, but mainly because of displacement by and/or hybridisation with domesticated pond carp that are preferred for
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farming due to their faster growth. Since it has been shown that domesticated carp stocks often suffer from a reduced genetic variability (Kohlmann et al., 2003, 2005) wild populations and the preservation of their genetic purity are of utmost importance for the conservation of common carp genetic resources. As a first step, still existing wild populations need to be identified and genetically characterized. This has not been done for wild carp from Turkey so far and is therefore the first major aim of the present study. In order to allow comparison with already existing data published by Kohlmann et al. (2005) the same set of four microsatellite loci (MFW1, MFW6, MFW7, MFW28) has been examined. The natural distribution range of wild carp in Eurasia is nowadays divided into disjunct western (Caspian, Aral and Black Sea basins) and eastern (East and Southeast Asia) areas, where numerous subspecies, races and varieties have been distinguished based on differences in morphological and eco-physiological traits (Kirpitchnikov, 1999). Recent genetic studies demonstrated that European and Central Asian carp are closer related to each other than to East Asian carp (Murakaeva et al., 2003; Gross et al., 2005) and that there is evidence for a single origin of present day European domesticated and wild/feral carp from a common ancestor with Central Asian carp (Kohlmann et al., 2003). However, a large gap between sampling locations exists in these studies: the easternmost European populations were collected in Hungary and the westernmost Central Asian populations came from Uzbekistan. Thus, wild carp from Turkey could provide valuable information to further clarify the origin of European carp. This was the second major aim of the present study. Again, in order to enable direct comparison of results, the same method of PCRRFLP analysis of mtDNA as described by Gross et al. (2002) and Kohlmann et al. (2003) was applied.
2. Materials and methods 2.1. Sampling locations Common carp classified as wild according to their body shape (fully and regularly scaled, elongated, torpedo-like) were collected from three lakes (Fig. 1). Sapanca Lake (30 individuals sampled) and Iznik Lake (48 individuals sampled) are located in the north-west of the Marmara region of Turkey. The surface area of Sapanca Lake is 46.8 km2 with a maximum depth of 55 m. The lake is used as a recreational area and its water is a source of drinking water for the city and district of Adapazari. Iznik Lake is one of the largest (308 km2) and deepest (maximum 65 m) lakes in the Marmara region and is mainly used for fishing, recreation and irrigation (Albay et al., 2003; Okgerman, 2005). This lake is surrounded by orchard and agricultural land. Bafra Cernek Lake (33 individuals sampled) has a surface area of 400 ha and is located in the Kizilirmak Delta, central Black Sea region in northern Turkey. The Kizilirmak Delta covers an area of 519 km2 that includes agricultural land, freshwater marshes and swamps, coastal lakes and lagoons at both sides of the river (DSI, 1988). It is the only remaining large tract of wetland along the Turkish Black Sea coast. 2.2. Genetic analyses Common carp DNA was isolated either from muscle tissue or fin clips using the E.Z.N.A. Tissue DNA Mini Kit (Peqlab Biotechnologie). The four microsatellite loci MFW1, MFW6, MFW7, and MFW28 were amplified by PCR using the primer sequences published by Crooijmans et al. (1997). The forward primer of each pair was labelled with WellRed fluorescence dyes (Proligo) to enable the determination of
Black Sea Bafra Cernek Lake Sapanca Lake
Turkey
Mediterranean Sea
Fig. 1. Map of Turkey showing the sampling locations of wild common carp.
D. Memiş, K. Kohlmann / Aquaculture 258 (2006) 257–262
allele sizes on a CEQ 8000 (Beckman Coulter) using the 400 bp internal size standard. Each PCR reaction mix was composed of 1.5 μl of 10 × PCR buffer (Fermentas), 1.2 μl of 25 mM MgCl2, 1.2 μl of BSA (20 μg/μl), 1.2 μl of 1.25 mM dNTPs, 0.3 μl of each primer (10 pmol/μl), 3 μl genomic DNA, 0.09 μl of Taq DNA-polymerase (5 U/μl; Fermentas) and sterile water up to a final volume of 15 μl. The PCR reaction amplification consisted of an initial denaturation at 95 °C for 5 min, followed by 5 cycles consisting of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min and another 30 cycles consisting of denaturation at 90 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min. A final extension at 72 °C lasted for 7 min. The recorded microsatellite genotypes were used as input data for the GENEPOP software package (Raymond and Rousset, 1995) in order to calculate allele and genotype frequencies, observed and expected heterozygosities, to test for deviations from Hardy–Weinberg equilibrium (probability test: estimation of exact P-values by the Markov chain method) and to assess genic as well as genotypic differentiation between populations. Genetic differentiation between populations was also evaluated by the calculation of pairwise estimates of FST values and testing their significance using the FSTAT software (Goudet, 2002). The restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNAwas performed on two PCR amplified segments encompassing the NADH-3,4 dehydrogenase (ND-3/4) and NADH-5,6 dehydrogenase (ND5/6) gene regions. The primer pairs for amplification of these genes were designed and published by Gross et al. (2002) based on the complete mtDNA sequence of common carp (Chang et al., 1994). Each PCR reaction mix was composed of 5 μl of 10× PCR buffer (Fermentas), 5 μl of 25 mM MgCl2, 4 μl of BSA (20 μg/μl), 2.5 μl of 1.25 mM dNTPs, 1 μl of each primer (10 pmol/μl), 2.5 μl genomic DNA, 0.2 μl of Taq DNA-polymerase (5 U/μl; Fermentas) and sterile water up to a final volume of 50 μl. The PCR amplification consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 40 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2.5 min. A final extension at 72 °C lasted for 10 min. The PCR products of approx. 2300 (ND-3/4) and 2500 bp (ND-5/6) were digested with ten restriction enzymes: Eco47I, BsuRI, HinfI, RsaI, AluI, MboI, HpaII, Hin6I, TaqI and XbaI (Fermentas). The reaction mixes consisted of 10 μl PCR product, 1.5 μl enzyme specific buffer, 0.2 μl restriction enzyme and 3.3 μl sterile water. The resulting fragments were resolved on 1.7% agarose gels using a 0.5 × TBE buffer system, visualised by ethidium bromide staining and sized by comparison with a GeneRuler 100 bp Ladder Plus (Fermentas) using the
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BIO-1D Analysis Software for Electrophoresis Images (Vilber Lourmat). Then, composite haplotypes were designated on the basis of combinations of restriction fragments resulting from the different restriction enzymes. Observed composite haplotypes were compared with already published ones (Gross et al., 2002; Kohlmann et al., 2003) covering common carp populations from a wide range of geographic areas. 3. Results 3.1. Microsatellite-based variability within and differentiation between populations The total number of alleles detected at the four microsatellite loci was 11 at MFW1, 14 at MFW6 and MFW28, and 15 at MFW7. The average number of alleles per locus was high and identical in the Sapanca and Iznik Lake populations (8.0) but slightly lower (6.25) in the Bafra Cernek Lake population (Table 1). Average observed heterozygosities ranged from 0.575 (Iznik Lake population) to 0.817 (Sapanca Lake population). Thus, the wild carp from Sapanca Lake showed the highest overall microsatellite variability. However, the differences between all Table 1 Variability of four microsatellite loci in three wild carp populations from Turkey (n = sample size; A = number of alleles; HO = observed heterozygosity; HE = expected heterozygosity; PHW = estimated exact P-values of Hardy–Weinberg probability tests) Locus MFW1
Parameter Sapanca Lake Iznik Lake Bafra Cernek Lake
n A HO HE PHW MFW6 n A HO HE PHW MFW7 n A HO HE PHW MFW28 n A HO HE PHW Average number of alleles per locus Average HO Average HE PHW
30 8 0.867 0.816 0.354 30 9 0.967 0.853 0.246 30 8 0.933 0.824 0.444 30 7 0.500 0.593 0.180 8.00
47 5 0.426 0.423 0.736 48 10 0.625 0.820 0.000 48 8 0.479 0.414 1.000 48 9 0.771 0.783 0.815 8.00
33 6 0.727 0.687 0.418 33 6 0.606 0.689 0.024 33 5 0.697 0.690 0.895 31 8 0.677 0.706 0.202 6.25
0.817 0.772 0.270
0.575 0.610 0.000
0.677 0.693 0.124
A A A A A A A C A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A B B B B B A A A A A A A A A A A A A A A Variant restriction patterns are designated by capital letters.
A A A A D A A A A A A A A A A A A A A A A B A D D A A A A A H1 H1c H1d H1e H1f
TaqI Hin6I HpaII MboI AluI RsaI HinfI BsuRI ND-5/6
Eco47I BsuRI
HinfI
RsaI
AluI
MboI
HpaII
Hin6I
TaqI
XbaI Eco47I
The digestion of the mitochondrial ND-3/4 and ND-5/6 gene regions with the ten restriction enzymes resulted in a total of five different composite haplotypes detectable on agarose gels (Table 2). One of these five haplotypes was identical to haplotype H1 previously described by Gross et al. (2002) and Kohlmann et al. (2003). The remaining four composite haplotypes were not observed before but were very similar to H1. They showed deviating fragment patterns for only one or two restriction enzymes (Table 2). Therefore, these new haplotypes were designated as varieties of H1, i.e. H1c to H1f [H1b has already been used by Kohlmann et al. (2003) to name a composite haplotype found in Uzbek wild carp]. The B pattern observed at the ND-3/4 gene digested by BsuRI (composite haplotype H1c) had already previously been found in River Amur wild carp, Russia, and domesticated carp from Wuhan, China, due to a gain of one restriction site compared to the A pattern (Kohlmann et al., 2003). Gains of one restriction site compared to the A patterns were also responsible for the new fragment patterns D observed at the ND-3/4 gene after digestion with BsuRI (composite haplotypes H1e and H1f) and MboI (composite haplotype H1f), respectively. In contrast, the new fragment pattern C found at the ND-5/6 gene digested by TaqI (composite haplotype H1d) was caused by a loss of one restriction site compared to the A pattern. Each of the three populations expressed two composite haplotypes (Table 3). Wild carp from Sapanca Lake and
ND-3/4
3.2. RFLPs of mitochondrial DNA
Composite haplotype
three populations were not statistically significant (P N 0.05), neither for average number of alleles per locus nor for observed heterozygosities. Significant deviations from Hardy–Weinberg equilibrium at the locus level were only found at MFW6 in the Iznik and Bafra Cernek Lake populations. At the population level, only wild carp from Iznik Lake showed a highly significant deviation from Hardy–Weinberg equilibrium (Table 1). Genetic differentiation between the three wild carp populations was high. All pairwise comparisons for genic as well as genotypic differentiation revealed highly significant probability values at all four microsatellite loci. Additionally, all FST estimates from pairwise comparisons were also statistically significant and ranged from 0.21 (between Sapanca and Bafra Cernek Lake populations) to 0.27 (between Iznik and Bafra Cernek Lake populations). Although Sapanca and Iznik Lakes are geographically neighbouring, the wild carp populations inhabiting these lakes were genetically highly differentiated as indicated by a FST value of 0.26.
XbaI
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Table 2 Composite mtDNA haplotypes in three wild carp populations from Turkey resulting from the digestion of ND-3/4 and ND-5/6 coding regions by ten restriction enzymes
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D. Memiş, K. Kohlmann / Aquaculture 258 (2006) 257–262 Table 3 Distribution of composite mtDNA haplotypes in three wild carp populations from Turkey Population
H1
H1c
Sapanca Lake Iznik Lake Bafra Cernek Lake
11 46
19
H1d
H1e
H1f
Total n
20
30 48 33
2 13
Iznik Lake shared haplotype H1 but also showed unique haplotypes (H1c in Sapanca Lake and rare H1d in Iznik Lake). The two composite haplotypes H1e and H1f found in Bafra Cernek Lake wild carp did not occur anywhere else. 4. Discussion The genetic variability of the three wild carp populations from Turkey at the four microsatellite loci (average number of alleles per locus of 6.25 and 8.0; average observed heterozygosity of 0.690) is close to the variability reported for the same set of four loci by Kohlmann et al. (2005) for nine wild-caught populations from Europe, Central Asia and East/South-east Asia (average allelic richness of 8.221; average observed heterozygosity of 0.799) but slightly lower than that reported by Lehoczky et al. (2005) for wild carp of river Tisza and Danube origin kept at a live gene bank in Szarvas, Hungary (mean number of alleles per locus: 11.25 in the Tisza carp and 8.75 in the Danube carp; average observed heterozygosity of 0.874 in the Tisza carp and 0.942 in the Danube carp). However, compared to domesticated/captive stocks, the variability of Turkish wild carp is still much higher, in particular for the number of alleles per locus: Kohlmann et al. (2005) reported an average allelic richness of only 4.436 for 13 populations from Europe and East/South-east Asia. In addition to the observed morphological characteristics typical for wild carp (fully and regularly scaled, elongated, torpedo-shaped body) the much higher average number of alleles at microsatellite loci compared to domesticated/captive stocks further indicates that the three Turkish populations studied represent true wild carp. The alternative scenario that these carp should in fact be feral, i.e. descendants of escaped or stocked domesticated carp, seems to be unlikely because then there should be no difference in this feature between them and domesticated carp. The significant deviations from Hardy–Weinberg equilibrium found at locus MFW6 in wild carp from Iznik and Bafra Cernek Lakes could be explained either by sample bias or the presence of null alleles in these two populations. In the presence of null alleles, heterozygotes
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possessing a null allele would be erroneously recorded as homozygotes for the variant allele leading to a deficiency of heterozygotes in the respective population. In fact, observed heterozygosity was lower than the expected one for both instances (Table 1). However, additional statistical tests for heterozygote deficiency implemented in GENEPOP revealed a significant probability value only for the Iznik Lake wild carp. Thus, sample bias might be the reason for the deviation from Hardy–Weinberg equilibrium in Bafra Cernek Lake wild carp. Moreover, none of the carp samples from both lakes failed to amplify the locus MFW6 that would have been an indication for null allele homozygotes. Although the lack of null allele homozygotes could be the result of a low null allele frequency and/or reduced viability of such homozygous individuals, sample bias seems to be the more likely explanation for the deviation from Hardy–Weinberg equilibrium also in the Iznik Lake wild carp population. Interestingly, the genetic differentiation between all of the three Turkish wild carp populations was significant as was the differentiation of the two Hungarian wild carp populations examined by Lehoczky et al. (2005). In contrast, four pairwise comparisons between four Uzbek wild carp populations revealed nonsignificant differentiation (Kohlmann et al., 2005). The discovery of composite haplotype H1 in two out of the three Turkish wild carp populations further supports the hypothesis of a single origin of present day European domesticated and wild/feral carp from a common ancestor with Central Asian carp. All but one of the 11 European domesticated and wild/feral carp populations studied by Kohlmann et al. (2003) were found to be fixed for haplotype H1. Moreover, this haplotype also predominated among the six wild carp populations sampled in Central Asia (Uzbekistan) but was completely missing in the four wild and domesticated stocks from East and South-east Asia. The exception among the European populations was the wild caught carp from the upper river Danube near Straubing, Germany, for which mtDNA and allozyme data suggested not only mixture but also hybridisation with Asian carp (Kohlmann et al., 2003). The endemic composite haplotypes H1c to H1f detected in wild carp from Turkey could be explained as varieties of haplotype H1 resulting from mutational events after colonization of the studied lakes by common carp from an eastern direction and their further migration to the west as far as the Danube river. Possible migration routes of wild carp would not have been restricted to freshwater habitats. In the Caspian Sea, for example, wild carp populations can be found in brackish, littoral habitats near estuaries and bays, which are often overgrown by reeds (Berg, 1949, cited in Baruš et al., 2001). The further distribution of
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common carp into Europe west and north of the Danube's piedmont zone was then clearly caused by humans (Balon, 1995). The major findings of the present study – a much higher genetic variability of the three Turkish wild carp populations in comparison to domesticated/captive stocks and their affiliation to the European phylogeographic lineage – emphasize that these populations represent valuable genetic resources of European carp. This is of particular importance on the background of the global threat to wild carp populations. Because of the significant differentiation between the three populations studied all of them are worth to be protected. Acknowledgements We would like to thank Petra Kersten for her technical assistance in the laboratory and also Dr. Hamdi Aydin and Mr. İsmail Mutlu (Major of Doğanca city) that helped to provide the fish samples from Iznik and Bafra Cernek Lakes. This study was supported by the Research Fund of University of Istanbul, Project Number: UPD-712/ 18042006. References Albay, M., Akcaalan, R., Tüfekci, H., Metcalf, J.S., Beattie, K.A., Codd, G. A., 2003. Depth profiles of cyanobacterial hepatotoxins (microcystins) in three Turkish freshwater lakes. Hydrobiologia 505, 85–95. Balon, E.K., 1995. Origin and domestication of the wild carp, Cyprinus carpio: from Roman gourmets to the swimming flowers. Aquaculture 129, 3–48. Baruš, V., Peňáz, M., Kohlmann, K., 2001. Cyprinus carpio (Linnaeus, 1758). In: Bănărescu, P.M., Paepke, H.-J. (Eds.), The Freshwater Fishes of Europe. Vol. 5/III, Cyprinidae 2, Part III: Carassius to Cyprinus, Gasterosteide. AULA-Verlag GmbH, Wiebelsheim, p. 127. Berg, L.S., 1949. (Freshwater Fishes in the U.S.S.R. and neighbouring countries). Vol. 2, Izd. AN SSSR, Moscow-Leningrad (in Russian). Chang, Y.S., Huang, F.L., Lo, T.B., 1994. The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome. J. Mol. Evol. 38, 138–155.
Crooijmans, R.P.M.A., Bierbooms, V.A.F., Komen, J., Van der Poel, J.J., Groenen, M.A.M., 1997. Microsatellite markers in common carp (Cyprinus carpio L.). Anim. Genet. 28, 129–134. DSI, 1988. Bafra Ovasi Hidrojeolojik Etüd Raporu. (Report on the Hydrogeological Study of the Bafra Plain). DSI Raporu, Samsun, Turkey, p. 44 (in Turkish). FAO Fishery Statistics, 2003: available at www.fao.org/fi/statist/statist.asp. Goudet, J., 2002. FSTAT, a Program to Estimate and Test Gene Diversities and Fixation Indices. (version 2.9.3.2) http://www.unil.ch/izea/ softwares/fstat.html. Gross, R., Kohlmann, K., Kersten, P., 2002. PCR-RFLP analysis of the mitochondrial ND-3/4 and ND-5/6 gene polymorphisms in the European and East Asian subspecies of common carp (Cyprinus carpio L.). Aquaculture 204, 507–516. Gross, R., Kohlmann, K., Kersten, P., Murakaeva, A., 2005. Phylogenetic relationships of wild and farmed common carp (Cyprinus carpio) stocks based on the mitochondrial DNA polymorphisms: implications for taxonomy and conservation. Aquaculture 247 (16–17) (abstract only). Kirpitchnikov, V.S., 1999. Genetics and Breeding of Common Carp. INRA, Paris. (revised by Billard, R., Repérant, J., Rio, J.P., Ward, R.) 97 pp. Kohlmann, K., Gross, R., Murakaeva, A., Kersten, P., 2003. Genetic variability and structure of common carp (Cyprinus carpio) populations throughout the distribution range inferred from allozyme, microsatellite and mitochondrial DNA markers. Aquat. Living Resour. 16, 421–431. Kohlmann, K., Kersten, P., Flajšhans, M., 2005. Microsatellite-based genetic variability and differentiation of domesticated, wild and feral common carp (Cyprinus carpio L.) populations. Aquaculture 247, 253–266. Lehoczky, I., Jeney, Z., Magyary, I., Hancz, C., Kohlmann, K., 2005. Preliminary data on genetic variability and purity of common carp (Cyprinus carpio L.) strains kept at the live gene bank at Research Institute for Fisheries, Aquaculture and Irrigation (HAKI) Szarvas, Hungary. Aquaculture 247, 45–49. Murakaeva, A., Kohlmann, K., Kersten, P., Kamilov, B., Khabibullin, D., 2003. Genetic characterization of wild and domesticated common carp (Cyprinus carpio L.) populations from Uzbekistan. Aquaculture 218, 153–166. Okgerman, H., 2005. Seasonal variations in the length–weight relationship and condition factor of rudd (Scardinus eryhtrophthalmus L.) in Sapanca Lake. Int. J. Zool. Res. 1, 6–10. Raymond, M., Rousset, F., 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Heredity 86, 248–249.
Genetic characterization of wild common carp (Cyprinus carpio L.) from Turkey Devrim Memiş a , Klaus Kohlmann b,⁎ a b
Istanbul University, Fisheries Faculty, Aquaculture Department, Ordu St. No. 200, 34470, Laleli, Istanbul, Turkey Department of Inland Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 310, P.O. Box 850119, 12561 Berlin, Germany Received 4 January 2006; received in revised form 17 March 2006; accepted 28 March 2006
Abstract Wild common carp from three lakes in Turkey were genetically characterized by examining the variability of four microsatellite loci and the restriction fragment length polymorphisms (RFLPs) of the mitochondrial ND-3/4 and ND-5/6 gene regions. Microsatellite variability did not differ significantly among the three populations and was only slightly lower than that of other wild-caught populations but remarkably higher than that of domesticated/captive stocks found in preceding studies. On the other hand, genetic differentiation between Turkish wild carp was significant and high (FST values ranged from 0.21 to 0.27). PCR-RFLP analysis revealed a total of five composite haplotypes. One of them was the typical European/Central Asian haplotype H1 shared by two of the Turkish populations from Sapanca and Iznik Lakes, respectively. The remaining four haplotypes were very similar to H1 differing in fragment patterns of only one or two restriction enzymes. Thus, present data further support the hypothesis of a single origin of present day European domesticated and wild/feral carp from a common ancestor with Central Asian carp. Considering that wild common carp are extremely endangered or already extinct in many areas of their natural distribution range, the examined Turkish populations represent valuable genetic resources of European carp that should be protected. © 2006 Elsevier B.V. All rights reserved. Keywords: Common carp; Cyprinus carpio; Microsatellites; Mitochondrial DNA; PCR-RFLP; Population genetics
1. Introduction The common carp, Cyprinus carpio L., has a long history of domestication in Asia (China) as well as in Europe, and numerous strains and breeds have been developed from its wild ancestor. The domesticated/ cultured forms attained worldwide distribution: Today,
⁎ Corresponding author. Tel.: +49 30 64 181 634; fax: +49 30 64 181 799. E-mail addresses: [email protected] (D. Memiş), [email protected] (K. Kohlmann). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.03.041
carp is among the most important species in freshwater fish culture mainly raised for human consumption with an annual production currently exceeding 3.2 million tons (FAO Fishery Statistics, 2003). Ornamental colour varieties known as “Koi” carp are reared in many garden ponds and tanks. Sport fishing of large carp individuals often based on stocking material from carp farms is becoming more and more popular in many countries. In contrast, wild common carp are extremely endangered or already extinct in many areas of their natural distribution range, partly due to losses of habitats, but mainly because of displacement by and/or hybridisation with domesticated pond carp that are preferred for
258
D. Memiş, K. Kohlmann / Aquaculture 258 (2006) 257–262
farming due to their faster growth. Since it has been shown that domesticated carp stocks often suffer from a reduced genetic variability (Kohlmann et al., 2003, 2005) wild populations and the preservation of their genetic purity are of utmost importance for the conservation of common carp genetic resources. As a first step, still existing wild populations need to be identified and genetically characterized. This has not been done for wild carp from Turkey so far and is therefore the first major aim of the present study. In order to allow comparison with already existing data published by Kohlmann et al. (2005) the same set of four microsatellite loci (MFW1, MFW6, MFW7, MFW28) has been examined. The natural distribution range of wild carp in Eurasia is nowadays divided into disjunct western (Caspian, Aral and Black Sea basins) and eastern (East and Southeast Asia) areas, where numerous subspecies, races and varieties have been distinguished based on differences in morphological and eco-physiological traits (Kirpitchnikov, 1999). Recent genetic studies demonstrated that European and Central Asian carp are closer related to each other than to East Asian carp (Murakaeva et al., 2003; Gross et al., 2005) and that there is evidence for a single origin of present day European domesticated and wild/feral carp from a common ancestor with Central Asian carp (Kohlmann et al., 2003). However, a large gap between sampling locations exists in these studies: the easternmost European populations were collected in Hungary and the westernmost Central Asian populations came from Uzbekistan. Thus, wild carp from Turkey could provide valuable information to further clarify the origin of European carp. This was the second major aim of the present study. Again, in order to enable direct comparison of results, the same method of PCRRFLP analysis of mtDNA as described by Gross et al. (2002) and Kohlmann et al. (2003) was applied.
2. Materials and methods 2.1. Sampling locations Common carp classified as wild according to their body shape (fully and regularly scaled, elongated, torpedo-like) were collected from three lakes (Fig. 1). Sapanca Lake (30 individuals sampled) and Iznik Lake (48 individuals sampled) are located in the north-west of the Marmara region of Turkey. The surface area of Sapanca Lake is 46.8 km2 with a maximum depth of 55 m. The lake is used as a recreational area and its water is a source of drinking water for the city and district of Adapazari. Iznik Lake is one of the largest (308 km2) and deepest (maximum 65 m) lakes in the Marmara region and is mainly used for fishing, recreation and irrigation (Albay et al., 2003; Okgerman, 2005). This lake is surrounded by orchard and agricultural land. Bafra Cernek Lake (33 individuals sampled) has a surface area of 400 ha and is located in the Kizilirmak Delta, central Black Sea region in northern Turkey. The Kizilirmak Delta covers an area of 519 km2 that includes agricultural land, freshwater marshes and swamps, coastal lakes and lagoons at both sides of the river (DSI, 1988). It is the only remaining large tract of wetland along the Turkish Black Sea coast. 2.2. Genetic analyses Common carp DNA was isolated either from muscle tissue or fin clips using the E.Z.N.A. Tissue DNA Mini Kit (Peqlab Biotechnologie). The four microsatellite loci MFW1, MFW6, MFW7, and MFW28 were amplified by PCR using the primer sequences published by Crooijmans et al. (1997). The forward primer of each pair was labelled with WellRed fluorescence dyes (Proligo) to enable the determination of
Black Sea Bafra Cernek Lake Sapanca Lake
Turkey
Mediterranean Sea
Fig. 1. Map of Turkey showing the sampling locations of wild common carp.
D. Memiş, K. Kohlmann / Aquaculture 258 (2006) 257–262
allele sizes on a CEQ 8000 (Beckman Coulter) using the 400 bp internal size standard. Each PCR reaction mix was composed of 1.5 μl of 10 × PCR buffer (Fermentas), 1.2 μl of 25 mM MgCl2, 1.2 μl of BSA (20 μg/μl), 1.2 μl of 1.25 mM dNTPs, 0.3 μl of each primer (10 pmol/μl), 3 μl genomic DNA, 0.09 μl of Taq DNA-polymerase (5 U/μl; Fermentas) and sterile water up to a final volume of 15 μl. The PCR reaction amplification consisted of an initial denaturation at 95 °C for 5 min, followed by 5 cycles consisting of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min and another 30 cycles consisting of denaturation at 90 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min. A final extension at 72 °C lasted for 7 min. The recorded microsatellite genotypes were used as input data for the GENEPOP software package (Raymond and Rousset, 1995) in order to calculate allele and genotype frequencies, observed and expected heterozygosities, to test for deviations from Hardy–Weinberg equilibrium (probability test: estimation of exact P-values by the Markov chain method) and to assess genic as well as genotypic differentiation between populations. Genetic differentiation between populations was also evaluated by the calculation of pairwise estimates of FST values and testing their significance using the FSTAT software (Goudet, 2002). The restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNAwas performed on two PCR amplified segments encompassing the NADH-3,4 dehydrogenase (ND-3/4) and NADH-5,6 dehydrogenase (ND5/6) gene regions. The primer pairs for amplification of these genes were designed and published by Gross et al. (2002) based on the complete mtDNA sequence of common carp (Chang et al., 1994). Each PCR reaction mix was composed of 5 μl of 10× PCR buffer (Fermentas), 5 μl of 25 mM MgCl2, 4 μl of BSA (20 μg/μl), 2.5 μl of 1.25 mM dNTPs, 1 μl of each primer (10 pmol/μl), 2.5 μl genomic DNA, 0.2 μl of Taq DNA-polymerase (5 U/μl; Fermentas) and sterile water up to a final volume of 50 μl. The PCR amplification consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 40 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2.5 min. A final extension at 72 °C lasted for 10 min. The PCR products of approx. 2300 (ND-3/4) and 2500 bp (ND-5/6) were digested with ten restriction enzymes: Eco47I, BsuRI, HinfI, RsaI, AluI, MboI, HpaII, Hin6I, TaqI and XbaI (Fermentas). The reaction mixes consisted of 10 μl PCR product, 1.5 μl enzyme specific buffer, 0.2 μl restriction enzyme and 3.3 μl sterile water. The resulting fragments were resolved on 1.7% agarose gels using a 0.5 × TBE buffer system, visualised by ethidium bromide staining and sized by comparison with a GeneRuler 100 bp Ladder Plus (Fermentas) using the
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BIO-1D Analysis Software for Electrophoresis Images (Vilber Lourmat). Then, composite haplotypes were designated on the basis of combinations of restriction fragments resulting from the different restriction enzymes. Observed composite haplotypes were compared with already published ones (Gross et al., 2002; Kohlmann et al., 2003) covering common carp populations from a wide range of geographic areas. 3. Results 3.1. Microsatellite-based variability within and differentiation between populations The total number of alleles detected at the four microsatellite loci was 11 at MFW1, 14 at MFW6 and MFW28, and 15 at MFW7. The average number of alleles per locus was high and identical in the Sapanca and Iznik Lake populations (8.0) but slightly lower (6.25) in the Bafra Cernek Lake population (Table 1). Average observed heterozygosities ranged from 0.575 (Iznik Lake population) to 0.817 (Sapanca Lake population). Thus, the wild carp from Sapanca Lake showed the highest overall microsatellite variability. However, the differences between all Table 1 Variability of four microsatellite loci in three wild carp populations from Turkey (n = sample size; A = number of alleles; HO = observed heterozygosity; HE = expected heterozygosity; PHW = estimated exact P-values of Hardy–Weinberg probability tests) Locus MFW1
Parameter Sapanca Lake Iznik Lake Bafra Cernek Lake
n A HO HE PHW MFW6 n A HO HE PHW MFW7 n A HO HE PHW MFW28 n A HO HE PHW Average number of alleles per locus Average HO Average HE PHW
30 8 0.867 0.816 0.354 30 9 0.967 0.853 0.246 30 8 0.933 0.824 0.444 30 7 0.500 0.593 0.180 8.00
47 5 0.426 0.423 0.736 48 10 0.625 0.820 0.000 48 8 0.479 0.414 1.000 48 9 0.771 0.783 0.815 8.00
33 6 0.727 0.687 0.418 33 6 0.606 0.689 0.024 33 5 0.697 0.690 0.895 31 8 0.677 0.706 0.202 6.25
0.817 0.772 0.270
0.575 0.610 0.000
0.677 0.693 0.124
A A A A A A A C A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A B B B B B A A A A A A A A A A A A A A A Variant restriction patterns are designated by capital letters.
A A A A D A A A A A A A A A A A A A A A A B A D D A A A A A H1 H1c H1d H1e H1f
TaqI Hin6I HpaII MboI AluI RsaI HinfI BsuRI ND-5/6
Eco47I BsuRI
HinfI
RsaI
AluI
MboI
HpaII
Hin6I
TaqI
XbaI Eco47I
The digestion of the mitochondrial ND-3/4 and ND-5/6 gene regions with the ten restriction enzymes resulted in a total of five different composite haplotypes detectable on agarose gels (Table 2). One of these five haplotypes was identical to haplotype H1 previously described by Gross et al. (2002) and Kohlmann et al. (2003). The remaining four composite haplotypes were not observed before but were very similar to H1. They showed deviating fragment patterns for only one or two restriction enzymes (Table 2). Therefore, these new haplotypes were designated as varieties of H1, i.e. H1c to H1f [H1b has already been used by Kohlmann et al. (2003) to name a composite haplotype found in Uzbek wild carp]. The B pattern observed at the ND-3/4 gene digested by BsuRI (composite haplotype H1c) had already previously been found in River Amur wild carp, Russia, and domesticated carp from Wuhan, China, due to a gain of one restriction site compared to the A pattern (Kohlmann et al., 2003). Gains of one restriction site compared to the A patterns were also responsible for the new fragment patterns D observed at the ND-3/4 gene after digestion with BsuRI (composite haplotypes H1e and H1f) and MboI (composite haplotype H1f), respectively. In contrast, the new fragment pattern C found at the ND-5/6 gene digested by TaqI (composite haplotype H1d) was caused by a loss of one restriction site compared to the A pattern. Each of the three populations expressed two composite haplotypes (Table 3). Wild carp from Sapanca Lake and
ND-3/4
3.2. RFLPs of mitochondrial DNA
Composite haplotype
three populations were not statistically significant (P N 0.05), neither for average number of alleles per locus nor for observed heterozygosities. Significant deviations from Hardy–Weinberg equilibrium at the locus level were only found at MFW6 in the Iznik and Bafra Cernek Lake populations. At the population level, only wild carp from Iznik Lake showed a highly significant deviation from Hardy–Weinberg equilibrium (Table 1). Genetic differentiation between the three wild carp populations was high. All pairwise comparisons for genic as well as genotypic differentiation revealed highly significant probability values at all four microsatellite loci. Additionally, all FST estimates from pairwise comparisons were also statistically significant and ranged from 0.21 (between Sapanca and Bafra Cernek Lake populations) to 0.27 (between Iznik and Bafra Cernek Lake populations). Although Sapanca and Iznik Lakes are geographically neighbouring, the wild carp populations inhabiting these lakes were genetically highly differentiated as indicated by a FST value of 0.26.
XbaI
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Table 2 Composite mtDNA haplotypes in three wild carp populations from Turkey resulting from the digestion of ND-3/4 and ND-5/6 coding regions by ten restriction enzymes
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D. Memiş, K. Kohlmann / Aquaculture 258 (2006) 257–262 Table 3 Distribution of composite mtDNA haplotypes in three wild carp populations from Turkey Population
H1
H1c
Sapanca Lake Iznik Lake Bafra Cernek Lake
11 46
19
H1d
H1e
H1f
Total n
20
30 48 33
2 13
Iznik Lake shared haplotype H1 but also showed unique haplotypes (H1c in Sapanca Lake and rare H1d in Iznik Lake). The two composite haplotypes H1e and H1f found in Bafra Cernek Lake wild carp did not occur anywhere else. 4. Discussion The genetic variability of the three wild carp populations from Turkey at the four microsatellite loci (average number of alleles per locus of 6.25 and 8.0; average observed heterozygosity of 0.690) is close to the variability reported for the same set of four loci by Kohlmann et al. (2005) for nine wild-caught populations from Europe, Central Asia and East/South-east Asia (average allelic richness of 8.221; average observed heterozygosity of 0.799) but slightly lower than that reported by Lehoczky et al. (2005) for wild carp of river Tisza and Danube origin kept at a live gene bank in Szarvas, Hungary (mean number of alleles per locus: 11.25 in the Tisza carp and 8.75 in the Danube carp; average observed heterozygosity of 0.874 in the Tisza carp and 0.942 in the Danube carp). However, compared to domesticated/captive stocks, the variability of Turkish wild carp is still much higher, in particular for the number of alleles per locus: Kohlmann et al. (2005) reported an average allelic richness of only 4.436 for 13 populations from Europe and East/South-east Asia. In addition to the observed morphological characteristics typical for wild carp (fully and regularly scaled, elongated, torpedo-shaped body) the much higher average number of alleles at microsatellite loci compared to domesticated/captive stocks further indicates that the three Turkish populations studied represent true wild carp. The alternative scenario that these carp should in fact be feral, i.e. descendants of escaped or stocked domesticated carp, seems to be unlikely because then there should be no difference in this feature between them and domesticated carp. The significant deviations from Hardy–Weinberg equilibrium found at locus MFW6 in wild carp from Iznik and Bafra Cernek Lakes could be explained either by sample bias or the presence of null alleles in these two populations. In the presence of null alleles, heterozygotes
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possessing a null allele would be erroneously recorded as homozygotes for the variant allele leading to a deficiency of heterozygotes in the respective population. In fact, observed heterozygosity was lower than the expected one for both instances (Table 1). However, additional statistical tests for heterozygote deficiency implemented in GENEPOP revealed a significant probability value only for the Iznik Lake wild carp. Thus, sample bias might be the reason for the deviation from Hardy–Weinberg equilibrium in Bafra Cernek Lake wild carp. Moreover, none of the carp samples from both lakes failed to amplify the locus MFW6 that would have been an indication for null allele homozygotes. Although the lack of null allele homozygotes could be the result of a low null allele frequency and/or reduced viability of such homozygous individuals, sample bias seems to be the more likely explanation for the deviation from Hardy–Weinberg equilibrium also in the Iznik Lake wild carp population. Interestingly, the genetic differentiation between all of the three Turkish wild carp populations was significant as was the differentiation of the two Hungarian wild carp populations examined by Lehoczky et al. (2005). In contrast, four pairwise comparisons between four Uzbek wild carp populations revealed nonsignificant differentiation (Kohlmann et al., 2005). The discovery of composite haplotype H1 in two out of the three Turkish wild carp populations further supports the hypothesis of a single origin of present day European domesticated and wild/feral carp from a common ancestor with Central Asian carp. All but one of the 11 European domesticated and wild/feral carp populations studied by Kohlmann et al. (2003) were found to be fixed for haplotype H1. Moreover, this haplotype also predominated among the six wild carp populations sampled in Central Asia (Uzbekistan) but was completely missing in the four wild and domesticated stocks from East and South-east Asia. The exception among the European populations was the wild caught carp from the upper river Danube near Straubing, Germany, for which mtDNA and allozyme data suggested not only mixture but also hybridisation with Asian carp (Kohlmann et al., 2003). The endemic composite haplotypes H1c to H1f detected in wild carp from Turkey could be explained as varieties of haplotype H1 resulting from mutational events after colonization of the studied lakes by common carp from an eastern direction and their further migration to the west as far as the Danube river. Possible migration routes of wild carp would not have been restricted to freshwater habitats. In the Caspian Sea, for example, wild carp populations can be found in brackish, littoral habitats near estuaries and bays, which are often overgrown by reeds (Berg, 1949, cited in Baruš et al., 2001). The further distribution of
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common carp into Europe west and north of the Danube's piedmont zone was then clearly caused by humans (Balon, 1995). The major findings of the present study – a much higher genetic variability of the three Turkish wild carp populations in comparison to domesticated/captive stocks and their affiliation to the European phylogeographic lineage – emphasize that these populations represent valuable genetic resources of European carp. This is of particular importance on the background of the global threat to wild carp populations. Because of the significant differentiation between the three populations studied all of them are worth to be protected. Acknowledgements We would like to thank Petra Kersten for her technical assistance in the laboratory and also Dr. Hamdi Aydin and Mr. İsmail Mutlu (Major of Doğanca city) that helped to provide the fish samples from Iznik and Bafra Cernek Lakes. This study was supported by the Research Fund of University of Istanbul, Project Number: UPD-712/ 18042006. References Albay, M., Akcaalan, R., Tüfekci, H., Metcalf, J.S., Beattie, K.A., Codd, G. A., 2003. Depth profiles of cyanobacterial hepatotoxins (microcystins) in three Turkish freshwater lakes. Hydrobiologia 505, 85–95. Balon, E.K., 1995. Origin and domestication of the wild carp, Cyprinus carpio: from Roman gourmets to the swimming flowers. Aquaculture 129, 3–48. Baruš, V., Peňáz, M., Kohlmann, K., 2001. Cyprinus carpio (Linnaeus, 1758). In: Bănărescu, P.M., Paepke, H.-J. (Eds.), The Freshwater Fishes of Europe. Vol. 5/III, Cyprinidae 2, Part III: Carassius to Cyprinus, Gasterosteide. AULA-Verlag GmbH, Wiebelsheim, p. 127. Berg, L.S., 1949. (Freshwater Fishes in the U.S.S.R. and neighbouring countries). Vol. 2, Izd. AN SSSR, Moscow-Leningrad (in Russian). Chang, Y.S., Huang, F.L., Lo, T.B., 1994. The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome. J. Mol. Evol. 38, 138–155.
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