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

Velkova-Jordanoska L.1, Kostov V.,2 Kostoski G., 1 Stojanovski S.1

1Hydrobiological Institute – Ohrid, R. Macedonia 2Institute of science, University “St. Cyril and Methodius” Skopje

Abstract The spring barbell Barbus peloponnesius today is considered as a 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

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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; leonhardi Bielz, 1853; Barbus rebeli Koller, 1926 and Barbus cyclolepis waleckii Rolik, 1970.

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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 twice up to the point of optimization of the conditions of the reaction and retaining of clear reproductive bands. That is why the total genomic DNA for this analysis has been isolated from blood of the researched fish individuals, so as for the samples for analysis to be with as high quality as possible. After the catch of every individual blood was taken in heparinized test tube and has been transported on ice to the Laboratory for Molecular biology. The DNA has been isolated with sodium chloride - chloroform extraction and ethanol precipitation in accordance to the protocol suggested by Gemmel & Akiyama (1996). For the generation of the RAPD profile of DNA of every individual, ten oligonucleotide primers were used: C-02, C-04, C-05, C-06, C-07, C-08, C-09, C-11, C-15, and C-16 (OPERON). The DNA amplification has been undertaken under the conditions suggested by Callejas & Ochanado (2001). The RAPD-PCR analyses were conducted in 10 µl total reaction volume, which comprised 25 mM MgCl2, 2, 5 mM dNTP, 50 pmoles primer, 1 µl Stoffel Fragment buffer 10×, 10 U/µl of Stoffel Fragment DNA polymerase and 1 µl of the extracted DNA.. The amplification was conducted in accordance to the following protocol: (first denaturation: 2 min. at 94˚C; 35 cycles of amplification: 15 sec at 94˚C; 20 sec at 36˚C; 60 sec at 72˚C; in the end – 1 cycle: 5 min at 94˚C). The products of the amplification were electrophoreticly divided on 2% agarose gels with TBE baffer (0,089 M Tris, 0, 089 M boric acid, 2 mM EDTA, pH – 8,3) which comprised ethidium bromid. As a molecular standard was used 100 bp Ladder marker. The gels were photographed with digital camera under UV light.

Statistic Procession of the Data

The RAPD fragments were marked in accordance to their presence in the gel (1), or if they were absent with (0). The index of similarity (SI) of the common bands between any two individuals within one population (Si) was calculated according to Nei & Li (1976), and between populations (Sij) for all possible combinations, according to Lynch (1990): SI = 2Nab / (Na + Nb) whereby: Nab is total number of common fragments between individuals a and b. Na and Nb are number of fragments in each individual, respectively. Sij = 1+ S'ij - 0.5 (Si + Sj) whereby: Si is average similarity of the individuals in the population i. Sj is average similarity of the individuals in the population j. Sij is average similarity between random pairs of individuals between the populations i and j. When S'ij is equal to the average similarity of two populations, Sij=1, and that indicates that the populations in question are homogeneous.

RESULTS AND DISCUSSION

In the molecular analysis of the samples there have been used total of 10 primers, six of which provided clear amplification products (C-02, C-07, C-08, C-11, C-15 and C-16). With the mentioned primers 56 fragments were obtained with size of 305 to 1459 basic pairs (Table 1). The length of the obtained fragments was determined with software, that is, with the assistance of the program Gel-Pro. Every fragment has been marked with the mark of the primer and with its respective length. For instance, the fragment C-02-539 represents a fragment with a size of 539 bp obtained with amplification with the primer C-02.

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Table 1: Fragments obtained with RAPD analysis of the samples of Barbus peloponnesius Val.

Barbus peloponnesius Val. Lake Ohrid. River Vardar River Crna C02-1458 + C02-1324 + C02-1200 + C02-1122 + + C02-995 + + + C02-942 + + C02-858 + + + C02-843 + C02-786 + C02-764 + + + C02-692 + C02-659 + + C02-539 + C02-496 + C02-461 + + + C02-336 + C07-1378 + C07-1101 + + C07-1000 + + C07-722 + + C07-667 + + C07-576 + + C07-535 + + C07-393 + C08-951 + C08-742 + C08-640 + + C08-579 + + C08-398 + C08-369 + C08-305 + + C11-1024 + + C11-977 + + C11-911 + + + C11-825 + + + C11-629 + + + C11-546 + C11-524 + C11-503 + + C11-357 + C15-1222 + C15-1054 + + + C15-979 + C15-812 + C15-706 + + + C15-665 + + + C15-613 + + C15-579 + + C16-1130 + C16-936 + + C16-746 + + C16-643 + + C16-607 + C16-535 + C16-529 + + C16-484 +

The amplification with the primer C-02 indicated interspecies differences between the researched samples. There have been recorded differences within one same species Barbus peloponnesius, caught

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from different localities, i.e. differences have been registered between the populations of Lake Ohrid and River Vardar (Figure 1).

M 1 2 3 4 5 6 7 8

Figure 1: Differences between RAPD profiles of Barbus peloponnesius from Lake Ohrid and River Vardar obtained with the amplification with the primer C-02: 1-4 Barbus cyclolepis (River Strumica); 5, 6 Barbus peloponnesius (Lake Ohrid); 7,8 Barbus peloponnesius (River Vardar)

The RAPD profile of the species Barbus peloponnesius obtained with amplification with the primer C-16 is represented at Figure 2. Differences between the researched samples from Lake Ohrid and the River Vardar can be seen.

M 1 2 3 4 5

Figure 2: Differences between the RAPD profiles of Barbus peloponnesius from Lake Ohrid and River Vardar obtained with amplification with the primer C-16: 1 Barbus cyclolepis (River Strumica); 2, 3 Barbus peloponnesius (Lake Ohrid); 4, 5 Barbus peloponnesius (River Vardar)

The data retained with the molecular (RAPD) analysis indicate to interspecies and intraspecies differences between the samples of the researched populations of barbell caught from the rivers and lakes in the Republic of Macedonia. The Index of Similarity (SI) of the common fragments between any two individuals within one population (Nei & Li, 1976) is represented by Table 2.

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Table 2: Index of Similarity (SI) between individuals of every researched population

Barbus peloponnesius

Lake Ohrid River Vardar River Crna

C02 0,64 0,635 1 C07 1 0,887 - C08 1 1 - C11 1 1 1 C15 1 0,95 1 C16 1 0,689 -

From the data presented in Table 2 it can be seen that there is an noticeable no homogeneity of the population of Barbus peloponnesius caught from the River Vardar. The similarity between populations (Sij) from all researched localities was calculated in accordance to Lynch (1990). The highest similarity has been detected between the populations of Barbus peloponnesius from the River Vardar and River Crna. Unlike them, the similarity between the populations of Barbus peloponnesius from Lake Ohrid and the River Vardar represented rather high differences. The RAPD method has numerous advantages and has been applied for analysis of the genetic structure and phylogenies of the species and populations from the genus Barbus present on the Iberian Peninsula. Studies on genetic structure of the populations are crucial for the planning of conservative programs and strategies with which the genetic variability can be efficiently sustained (Callejas & Ochando, 2002). By the RAPD-PCR amplification with the primers C-02, C-11 and C-15 some differences between the populations of Barbus peloponnesius from Lake Ohrid and River Vardar have been recognized. The statistical analysis indicate that the intrapopulation similarity between these two populations with the primer C-02 is estimated at 0, 536, with the primer C-11 is 0,5 and with the primer C-15 is 0,57. According to Tsigenopoulos et al. (2002) the species Barbus peloponnesius from the River Vardar belongs to the Danube group. It is assumed that during the Quarter from central Danube through the outflow of the Rivers Morava and Vardar this species has been dispersed into the Rivers of the watershed of the Aegean Sea. On the basis of the data attained from the mtDNA analysis (Tsigenopoulos et al. 2002), the hypothesis has been approved. In accordance to the researches of the same author, reofil barbell populations of the River Soca, i.e. Barbus caninus and from the River systems to the East, including the basin of the River Danube (excluding the Albanian and Greek Rivers), which have been names as Barbus petenyi are genetically relatively similar to one another and all of them, apparently originate from the Danube group. In that group, the sample from the River Belka (tributary to the River Sava, from the Danube watershed) is considered as Barbus petenyi, but it has been characterized as genetically more similar to the reofilic taxon from the River Soca entitled as Barbus caninus and less similar to the samples from the River Danube (Barbus petenyi). The small genetic distance based on allozymes and mtDNA data between these populations has been as a result of dispersion of Barbus petenyi from the upper flow of Danube near the basin of the Rivers Belka and Idria. For quite some time the species Barbus peloponnsius was considered as a synonym or as a sub- species of Barbus meridionalis Risso, 1826 from northeastern Spain, southern France and northwestern Italy (Almaca, 1984). According to Karaman (1971), Barbus peloponnsius is a separate species while Barbus petenyi Heckel and Kner, 1858 which is a reofilic species of barbell from Central Europe is its sub-species. The multivariate analysis of the morphological similarity between the populations of Barbus peloponnsius, Barbus meridionalis and Barbus petenyi indicated that Barbus peloponnsius and Barbus petenyi significantly differ in morphological regard from Barbus meridionalis from south France (Karakousis et al. 1993). In the same study a great similarity between Barbus petenyi from Central Europe and Barbus peloponnesius from Northern Greece has been registered. Furthermore, morphological differences have been recognized among the populations of Barbus peloponnesius from southern and northern parts of Greece (Karakousis et al., 1995).

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In regards to the systematic status of Barbus peloponnesius the electrophoretic analysis of Karakousis et al. (1995) indicated that the group Barbus peloponnesius, Barbus petenyi and Barbus rebeli are showing genetic differences as separate species of Barbus meridionalis and Barbus haasi. The population of Barbus petenyi from Central Europe (or the River Danube) indicated some genetic similarity to Barbus peloponnesius from the northern parts of Greece. That is why the authors Karakousis et al. (1995) classified Barbus petenyi as a subspecies of Barbus peloponnesius instead as a subspecies of Barbus meridionalis as suggested by Almaca (1984) or as an independent species. The same results have been attained with the morphological analysis of Karakousis et al. (1993). The populations of Barbus peloponnesius in Greece have been classified into three different groups by Karakousis et al. (1995): the populations from Peloponnesus were analyzed as a subspecies Barbus peloponesius peloponesius, the populations from the western regions of Greece as the subspecies Barbus peloponnesius rebeli and the populations from the northern areas of Greece as the subspecies Barbus peloponnesius petenyi. All mentioned populations are described as synonyms of one unique species Barbus peloponnesius by Kottelat (1997). The same author, latter (Kottelat, 2007) as an area of distribution of Barbus peloponesius considers the watershed of the River Kalamas up to Pamisos in Western Greece while for the population which inhabited the region from the River Drim to Aoos (Vjosa), including the Lake Scutari and Ohrid, he suggest it as Barbus rebeli. Likewise, the populations which inhabited the Rivers Vardar, Gallikos, Ludias and Aliakmon from the Aegean Sea watershed, the author classified as Barbus balcanicus. The similarity between the populations from the northern regions of Greece which inhabits the watershed of the River Vardar and Aliakmon and the barbel populations from the River Danube indicate to the dispersion of the species of Danube through Morava to the River Vardar and in the opposite direction (Karaman, 1971) and to the fact that Northern Greece is the areal of speciation of the species Barbus peloponnesius. According to Karaman (1971) in the area of the southwestern part of the Aegean coast, which linked the southern part of the Balkan Peninsula with Asia Minor, a new species Barbus peloponnesius has been formed as the highest achievement of that adaptive evolution. The distribution was from Peloponnese in north, through Western Greece and Albania to the Ohrid-Drim-Scutari system, and from there it has been transferred to the Danube and Vardar system. Thereafter, the species was not able to be distributed due to the unsuitable hydrographic connections during the time of its expansion.

CONCLUSIONS

The results of our molecular analyses indicated obvious no homogeneity of the populations of Barbus peloponnesius sampled from the River Vardar. The largest similarity has been detected between the populations of Barbus peloponnesius from the River Vardar and Crna Reka. There have been recorded some differences between the populations which inhabits the River Vardar and its tributaries and the Lake Ohrid, however we believe that the data is insufficient for determination of these populations as representatives from two different species.

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