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RAPD Analysis of Genetic Variations in Barbus Peloponnesius (Pisces, Cyprinidae) from River Vardar

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RAPD Analysis of Genetic Variations in Barbus Peloponnesius (Pisces, Cyprinidae) from River Vardar 1 RAPD Analysis of Genetic Variations in Barbus Peloponnesius (Pisces, Cyprinidae) from River Vardar Velkova-Jordanoska L.1, Kostov V.,2 Kostoski G., 1 Stojanovski S.1 1Hydrobiological Institute – Ohrid, R. Macedonia 2Institute of animal science, University “St. Cyril and Methodius” Skopje Abstract The spring barbell Barbus peloponnesius today is considered as a species which inhabits the waters in the Balkan Peninsula. The name differs from its older name Barbus meridionalis petenyi (for spring barbell of the Danube watershed) and Barbus meridionalis rebeli (for spring barbell of the watershed of the Adriatic and Ionic Sea). In this paper the molecular RAPD method was used for investigation of the population structure of the species Barbus peloponnesius from River Vardar and its tributaries. A total of ten oligonucleotide primers were used to obtain various RAPD profiles. Certain interspecies differences among the investigated population were evidenced. KEY WORDS: RAPD-PCR, genetic variations, Barbus peloponnesius INTRODUCTION The present ichthyofauna of the fresh waters in the Western and Central Balkan Peninsula was developed in the current shape and composition during the last Pleistocene glaciations (before approximately 10 000 years). However, because the largest part of the Balkan Peninsula, during the ice age of the Pleistocene, was left outside of the ice coverage (except some ice masses with higher altitudes), resulted in the possibility for the numerous fish species from the region to form own shelters, out of which they went back to the North, mainly through the River Danube, after the end of the Virm glaciations. According to Karaman (1971), the centre of origin and development of the genus Barbus is unknown until present time. In the northern regions of Front Asia two expansive species were present which thereafter have formed two filetic lines, Barbus barbus and Barbus capito, and those afterwards have populated the regions of Asia Minor, Europe and Africa. The representatives of these two lines were described together in these areas, thus it is assumed that they have been distributed simultaneously. The two filetic lines have had many similar directions of speciation; hence they have formed convergent same shapes. For instance, the species Barbus meridionalis and Barbus peloponesius (Syn. Barbus petenyi) were considered as sub-species of one same species, even though they are classified under different filetic lines. According to the researches of Karaman (1971), the sub-species ssp. pergamonensis from the region of Asia Minor, which is rather similar to Barbus peloponnesius, moved to Greece while the continental link was still present and inhabited the island Euboea and the River Sperhios in Attica. Out of the populations which inhabited the southern parts of the Balkan Peninsula, with time, the species Barbus peloponnesius was formed, most probably in Greece. After its formation, the species inhabited the Ohrid- Drim-Scutari system in the northern areas and to east the Vardar system and from there it reached the River Danube. According to Karaman (1971), the European Continent has been inhabited by Barbus from two directions, i.e. through Asia Minor and the Balkan Peninsula and through North Africa and the Iberian Peninsula. The most complex relationships have been formed in the Balkans. In the waters of the Danube and Vardar systems, there are species which belong to the same filetic line (Barbus barbus and Barbus peloponnesius). These species are ecologically differenced, i.e. Barbus barbus in the valley slow-flowing waters while Barbus peloponnesius in the fast-flowing waters. Both species originate from different directions. The systematic status and description of the populations of Barbus barbus is limited and not clear. In the literature, there are confuse and illogical data concerning the distribution of these taxons, which BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010 1 2 indicate that in the same systems (Danube, Drim and Vardar) there are living even two sub-species of this fish. According to Stephanidis (1971), Barbus meridionalis peloponnesius is to be found in the waters on Peloponnese and in regards to some morphological traits it differs of the typical form of Barbus meridionalis Risso and from the sub-species Barbus meridionalis petenyi. Barbus meridionalis Valenciennes, 1842 was described on the basis of samples taken from the River Alpheios (Karytaena) on Peloponnesus. For quite some time this species was considered as synonymous of the Barbus meridionalis Risso from Southern France and Northwestern Italy. According to Dimovski and Grupce (1987), Barbus meridionalis petenyi Heck is sub-species of the south-Mediterranean species Barbus meridionalis Riso, which is to be found in the Rivers of the Danube watershed, Dniester, Vistula, Oder and the Balkan Peninsula. In the River Bregalnica (tributary of River Vardar), this is the most common fish which is to be found in the middle and upper flow of the River. Usually, it inhabits the parts with fast-flowing water and jackal bottom (Dimovski and Grupce 1971). This species was widely distributed in the River Treska and was located in the area of the springs up until the region where this River inflows in Vardar (Dimovski and Grupce 1972). Barbus meridionalis petenyi Heck is a common fish in the River Vardar and has been located from the town of Gevgelija to the town of Gostivar. Karaman S. (1924) marks this species as one of the most common in all smaller tributaries of Vardar, and it has been found in some points in Vardar itself, especially in its upper part (Dimovski and Grupce 1973). In Macedonia, besides Barbus meridionalis petenyi Heck some authors are noting down the sub-species Barbus meridionalis rebeli Koller in the waters of Lake Ohrid (Dimovski and Grupce, 1973; Naumoski, 1995). According to Economidis (1989) Barbus peloponnesius can be considered as a different species from Barbus meridionalis Risso. In Greece Barbus meridionalis Risso has been described in the entire Western part, i.e. from Peloponnese up to Epirus, as well as in Albania, southwestern Yugoslavia, and in Central and Western Macedonia. On the basis of discriminative analysis of the morphological characteristics undertaken by Karakousis et al. (1993), the sample from Southern France which has been classified as Barbus meridionalis meridionalis indicated differences in regards to Barbus meridionalis petenyi and Barbus meridionalis peloponnesius. This supported the opinion of Karaman (1971) that Barbus peloponnesius is different species from Barbus meridionalis. The sample from the River Danube was classified within the “peloponesius” group. The similarities of that sample with samples from the River Vardar and its tributaries indicated on possible flow of gens between those populations through the River Morava. These results, on the other hand, represent the need of more information for the genetic structure of Barbus peloponnesius so as for a clarification of the relationships with the other species (Karakousis et al., 1993). The stream barbell, Barbus peloponnesius today is considered as a species that inhabits the Balkan Peninsula, unlike the previous name Barbus meridionalis petenyi (for the barbel from the Danube watershed), and Barbus meridionalis rebeli (for stream barbel from the watershed of Adriatic and Ionic Sea). The Mediterranean barbell Barbus meridionalis Risso is present in the western part of the Mediterranean, i.e. in Northern Italy and the fresh waters of France. Up to present time the status of numerous species from the genus Barbus is a subject of many intensive researches (Simonovic & Nikolic, 1996). According to the current researches of Kotlik et al., (2002) and re-description of the species, Barbus petenyi Heckel, 1852, “…the newly described species is Barbus balcanicus which has been evidenced in the mountainous springs and Rivers and less often in lakes and accumulations, in the Mountain Dinara in the Balkan Peninsula…” Authors are assuming that presumably Barbus balcanicus is to be found in the basin of the River Vardar in the Republic of Macedonia. The population of this species which has been evidenced in the most distant western regions has been located in the River Soca in Slovenia, which is a part of the watershed of the Adriatic Sea. According to Kottelat (1997), synonyms for the name of the species Barbus peloponnesius Valenciennes, 1842 are: Barbus peloponnesius Valenciennes, in Cuvier & Valenciennes, 1842; Barbus petenyi Heckel, 1848; Barbus petenyi Heckel, 1952; Pseudobarbus leonhardi Bielz, 1853; Barbus rebeli Koller, 1926 and Barbus cyclolepis waleckii Rolik, 1970. BALWOIS 2010 - Ohrid, Republic of Macedonia - 25, 29 May 2010 2 3 MATERIAL AND METHODS In the scope of this study there have been analyzed total of 21 individual fish which have been sampled from the River Vardar and its tributary Crna Reka. These samples were compared with samples from Lake Ohrid. The samples are from the species of barbell Barbus peloponnesius Val. (=Syn. Barbus meridionalis petenyi Heck.) described within the territory of the Republic of Macedonia by Kottelat (1997). Since the RAPD-PCR method is extremely sensitive to the quality of the isolated DNA, the conditions of the reaction and in some cases the appearance of non-reproductive amplified products, these issues were resolved with the use of genomic DNA with high quality, strict follow of the protocol under standardized conditions and repetition of the amplified reactions
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