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Genetic Diversity and Taxonomy of Sabanejewia Balcanica (Osteichthyes: Cobitidae) in the Waters of the Czech Republic and Slovakia

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Genetic Diversity and Taxonomy of Sabanejewia Balcanica (Osteichthyes: Cobitidae) in the Waters of the Czech Republic and Slovakia Folia Zool. – 57(1–2): 60–70 (2008) Genetic diversity and taxonomy of Sabanejewia balcanica (Osteichthyes: Cobitidae) in the waters of the Czech Republic and Slovakia Eva Bartoňová1, Ivo PAPoušEK1, věra Lusková1, Ján koščo2, Stanislav LUSK1, karel HaLačka1, Miroslav ŠVÁTORA3 and Lukáš vetešník1 1 Institute of Vertebrate Biology of the AS CR, v.v.i., Květná 8, 603 65 Brno, Czech Republic; e-mail: [email protected] 2 Department of Ecology, University of Prešov, str. 17. Novembra 1, 081 16 Prešov, Slovakia; e-mail: [email protected] 3 Department of Zoology, Faculty of Science, Charles University, Viničná 7, 128 44 Praha 2, Czech Republic; e-mail: [email protected] received 15 november 2006; accepted 21 March 2008 abstract. seven populations (oľšava/Hornád r., oľšava/topľa r., ublianka r., ondava r., Ipeľ r., Malý Dunaj r. and vlára r.) of Balcan spined loach were subjected to phylogenetic analysis based on variability of the cytochrome b gene and mitochondrial specific marker. they were separated in to five groups but these groups were represented by specimens from different populations. Genetic distances among populations and among groups were quite low. subsequently, analysis of relations among our groups and sublineages of Danubian-Balkanian complex of Sabanejewia was made. the first four groups were genetically close to sublineage III, while the fifth one to the sublineage Iv. these findings agree with the conclusion about conspecificity of analysed Danubian-Dniester populations belonged to monotypic S. balcanica. Phylogenetic analysis showed that the most suitable populations for the restoration of this species in the Bečva river are populations dwelling the Ipeľ and vlára rivers. Key words: Balcan spined loach, conservation, cytochrome b, mitochondrial marker Introduction Sabanejewia balcanica (karaman, 1922) is a small economically insignificant species and therefore it has been reflected by ichthyologists` a long time. But in the last 10–15 years it has attracted greater attention. the reason was wide expansion of molecular-genetic methods which has been helped by division and description of new species within the larger Sabanejewia taxon. another significant fact is recent emphasis on biodiversity conservation, which enforces identification of actual populations of endangered species. the genus Sabanejewia was separated from the genus Cobitis by vladykov (1929) but the genus name Sabanejewia started to be used generally after revalidation by nalbant (1963). However, in 1928 vladykov already indicated description of new genus based on study of sexual dimorphism of european spined loaches (vladykov 1928). By this time, two species were included within scope of new genus, those being S. aurata (Filippi, 1865) and S. balcanica (karaman, 1922). Berg (1949) considers only one species (S. aurata) to be a valid but he still includes it in the genus Cobitis. the validity of Sabanejewia as a separate genus was later confirmed by Grossu et al. (1971) on the grounds of electrophoretic analysis of muscular proteins and serum proteins. according to recent data this genus includes several species and subspecies described by 60 different authors based on morphological characters (nalbant 1957, karaman 1963, vasiľeva & vasiľev 1988, Witkowski 1994, economidis & nalbant 1996, etc.). after critical evaluation of european ichthyofauna, kottelat (1997) considered S. balcanica (karaman, 1922), S. bulgarica (Drensky, 1928), S. larvata (Filippi, 1859) and S. romanica (Bacescu, 1943) as valid species of the genus Sabanejewia in europe (exclusive of the former ussr). Further advances in species structure of genus the Sabanejewia was brought by application of karyological (vasiľeva & ráb 1992) and genetic methods (Ludwig et al. 2000, Perdices et al. 2003). Based on analyses of mtDna, Perdices et al. (2003) defined inside the genus Sabanejewia six main evolution lineages, namely: S. larvata, S. romanica, S. aurata/S. caucasica, S. kubanica, S. baltica and the Danubian-Balkanian complex, that was further divided into six sublineages: I. s. vallachica/S. balcanica from a syntopic population from romania, II. S.balcanica/S.doiranica from western Greece, III. S. bulgarica/S. balcanica/S. montana from the middle part of the Danube basin, Iv. S. radnensis/S. balcanica from the Mures basin, v. S. thrakica/S. balcanica from Greece and vI. S. balcanica from the Mur basin. the taxonomic status of Sabanejewia populations in the Danube river basin and in territory of Balkan Peninsula is still ambiguous. Iftime (2002) confirmed this from morphometric analyses. He expressed the opinion that it is not necessary to keep current division to subspecies in the Danube and Dniester river basins. It is evident that relations among Sabanejewia populations in drainages of western Carpathian Bow (left tributaries of the slovakian Danube, tisza and Bodrog river basins) as well as in other parts of the Danube river basin are insufficient or totally missing. It is necessary to clear this situation with regards to european conservation legislature, namely Council Directive 92/43/EEC. In supplement II two species S. aurata and S. larvata are presented. For member countries of eu it is obligatory to define special areas of conservation (saC) for Sabanejewia species. thus, for national legislature it is needful to clarify species taxonomy of the genus Sabanejewia, because in national legislature of the Czech republic and slovakia only Sabanejewia aurata is mentioned. actually, S. balcanica occurs in the Czech republic only in a short lap of the vlára river near state boundary with slovakia (Lusk et al. 2002). In the 1950s, this species also dwelt the Bečva river basin, but now it is absent there (Lusk et al. 2000). In territory of Slovakia, S. balcanica is a common species in the tisza river basin, while in western part of slovakia, which belongs to the Danube river basin, its distribution is restricted to several rivers (koščo et al. 2006). Main goals of our study were 1) to define genetic diversity and relations among Sabanejewia populations in territories of the Czech republic and slovakia, 2) to evaluate relations among these populations and sublineages revealed in the Danubian-Balkanian complex and subsequently to define the taxonomic status of Sabanejewia loaches in the Czech republic and slovakia, and 3) to identify vanished population from Bečva river and, if possible, to find the most related population(s) based on relationship or similarity for the restoration of this species in the Bečva river. Materials and Methods Individuals of S. balcanica were caught by electrofishing in 2002–2006. a total number of 77 specimens (table 1) from seven populations from the Danube and tisza river basins were 61 Table 1. analysed specimens from seven populations from the Danube and tisza river basins. specimen no. no. of Drainage Collection locality accession no. specimens system 2727-2730 4 tisza r. oľšava/Hornád (slovakia) DQ996468-471 2732 1 tisza r. oľšava/Hornád (slovakia) DQ996472 2734-2736 3 tisza r. oľšava/Hornád (slovakia) DQ996473-475 eu400433-434 2949 1 Danube r. vlára (Czech republic) DQ996517 2951-2952 2 Danube r. vlára (Czech republic) DQ996518-519 3570-3574 5 tisza r. ublianka (slovakia) DQ996476-480 eu400439-442 4526-4527 2 tisza R. Ondava (Slovakia) DQ996481-482 eu400443-444 4889-4893 5 tisza r. oľšava/topľa (slovakia) DQ996483-487 eu400435-437 4910-4914 5 tisza r. oľšava/topľa (slovakia) DQ996488-492 eu400438 5022-5023 2 Danube r. vlára (Czech republic) eu400418-419 5039-5040 2 Danube r. vlára (Czech republic) eu400420-421 5604-5616 13 Danube r. Ipeľ (slovakia) DQ996493-505 eu400426-428 5621-5624 4 Danube r. Ipeľ (slovakia) DQ996506-509 eu400429 5655-5661 7 Danube r. Ipeľ (slovakia) DQ996510-516 eu400430-432 5662 1 Danube r. Ipeľ (slovakia) eF447286 5667 1 Danube r. vlára (Czech republic) DQ996520 5668 1 Danube r. vlára (Czech republic) eu400422 5669-5679 11 Danube r. vlára (Czech republic) DQ996521-531 eu400423-425 6162-6163 2 Danube r. Bečva (Czech republic) DQ996534-535 6165 1 Danube r. Bečva (Czech republic) eu400445 6166 1 Danube r. Bečva (Czech republic) DQ996536 eu400446 6167-6168 2 Danube r. Bečva (Czech republic) eu400447-448 6600-6601 2 Danube r. vlára (Czech republic) eF447287-88 6602-6603 2 Danube r. vlára (Czech republic) DQ996532-533 6964-6965 2 Danube r. Malý Dunaj (Czech republic) eF447289-90 6967-6968 2 Danube r. Malý Dunaj (Czech republic) eF447291-92 6972 1 Danube r. Malý Dunaj (Czech republic) eF447293 analysed on the cytochrome b (cyt b) gene (Fig. 1). three individuals from the Bečva river were analysed with partial success. these individuals were caught in 1952 and preserved probably in ethanol. Furthermore, 23 selected individuals and newly eight individuals (table 1) were analysed on the mitochondrial specific (mt specific) marker (Fig. 1). Complete genomic Dna was isolated from fin clippings (preserved in 95% ethanol) according to standard phenol-chloroform-isoamylalcohol method (sambrook et al. 1989) with minor modifications. entire cyt b gene was amplified (1140 bp) with primers and under conditions previously described by Perdices & Doadrio (2001), mt specific 62 Fig. 1. the map of sample localities in the Czech republic and slovakia. Localities are follows: 1-Bečva r., 2-vlára r., 3-Malý Dunaj r., 4-Ipeľ r., 5-oľšava/Hornád r., 6-oľšava/topľa r., 7-ondava r., 8-ublianka r. marker (448 bp) was amplified with primers and under conditions according to Ludwig et al. 2000. In the case of samples of S. balcanica from the Bečva r. we had to divide cyt b gene in two parts. the first part was PCr amplified with primers L15267 (5‘-AAT GaC ttG aaG aaC CaC CGt-3‘) and H15891 (5‘-Gtt tGa tCC CGt ttC GtG TA-3‘) (Briolay et al. 1998), the second part with primers L15840 (5‘-GAT tCt tCG CAT tCC aCt t-3‘) (Briolay et al. 1998) and H15918r (5‘-CtC CAT CtC CGG ttt aCa aGa C -3‘) (song et al. 1998). amplification of these samples proceeded according to the following protocol: denaturation 95 °C 3 min, 35 cycles 90 °C 1 min/46 resp.
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