Genetic Divergence Among the Populations of B. petenyi from Eleshnitza River (Bulgaria) and Some Tributaries of Vardar River (Macedonia)

Transcription

1 29 Bulgarian Journal of Agricultural Science, 12 (26), National Centre for Agrarian Sciences Genetic Divergence Among the Populations of B. petenyi from Eleshnitza River (Bulgaria) and Some Tributaries of Vardar River (Macedonia) P. IVANOVA 1, L. VELKOVA-JORDANOSKA 2 and I. DOBROVOLOV 1 1 Institute of Fisheries and Aquaculture, BG - 9 Varna, Bulgaria 2 SI Hydrobiological Institute, MC - 6 Ochrid, Macedonia Abstract IVANOVA, P., L. VELKOVA-JORDANOSKA and I. DOBROVOLOV, 26. Genetic divergence among the populations of B. petenyi from Eleshnitza River (Bulgaria) and some tributaries of Vardar River (Macedonia). Bulg. J. Agric. Sci.,12: Genetic-biochemical study on barbus (B. petenyi) from Eleshnitza River (Bulgaria) and some tributaries of Vardar River (Macedonia) are carried out. Thirteen loci are analyzed and polymorphism on three of them was found. On the base of genetic distances (D Nei ) calculated barbus species from Eleshnitza River and some tributaritiеs of Vardar River (Treska, Bregalnitza and Orizarska) belong to two different subspecies. Comparative genetical analyses on B. petenyi from Bulgarian and Macedonian Rivers with B. meridionalis and B. pelopenesius are needed to specify while in the investigated area B. petenyi exactly exist or it is presented form some hybrid population. Key words : barbus, electrophoresis, isoelectric focusing, genetic distances Introduction The Genus Barbus in Europe is composed of more than 25 species and subspecies, displaying different ecological preferences (Berrebi, 1995; Berrebi et al., 1996). Karaman (1924) described the subspecies B. meridionalis meridionalis for Ochrid Lake, while for Vardar River, Doirinsko Lake and their tributaries B. m. petenyi. Apostolski et al. (1956), Poljakov et al. (1958) and Vukovic and Ivanovich (1971) recognized the B. 1 pavl_petya@yahoo.com 2 lidvejo@yahoo.com m. petenyi existence in Macedonia. According to Karaman (1971) and Dimovski and Grupce (1987) B. meridionalis inhabited Vardar River and Prespa Lake. Black (balkan) barbus from Bulgarian rivers is determined as Barbus meridionalis petenyi Heckel 1847 (Drensky, 1951; Marinov, 1989; Karapetkova and Zhivkov, 1995). According to Karaman (1971) B. meridionalis is not distributed in the Central and East Europe, while B. peloponesius is widely presented in Bulgaria. Krupka and Holcik (1976, cit after

2 Genetic Divergence Among the Populations of B. petenyi from Eleshnitza River Мarinov (1989)) are not agree with Karaman opinion. Marinov (1989) considered that the evidence for Karaman (1971) opinion did not exist. Sorič (1987) considered that B. m. petenyi inhabited Black Sea and Aegean Basins. Economidis (1989) and Georgiev (1998) stress on the fact that the black barbel in the Macedonian waters belongs to the species B. peloponnesius. Kotlic and Berrebi (22) and Kotlik et al. (22) on the base of DNA analyses proved that Barbus petenyi occurs in the tributaries of the Lower Danube River in the Stara Planina (Balkan) in Bulgaria. The only known occurrence outside the Danube River basin is in the Kamtchiya River basin of the Black Sea drainage in the Eastern Stara Planina mts in Bulgaria. Kotlik et al. (24) considered that in Kamtchiya River the subspecies Barbus tauricus esherichii is presented. Kotlik and Berrebi (21) described Barbus barbus samples from Kamtchiya and Dvojnitca River. Kotlik et al. (22) described B. balcanicus population in the Vardar River. Tsigenopoulus et al. (1999) and Tsigenopoulus and Berrebi (2) described B. petenyi from Vardar River. On the base of allozyme analyses Dobrovolov (1986; 1993; 1996 and 1999) found diagnostic interspecies differences of barbel (Genus Barbus) from the Bulgaran Rivers. According to Karakousis et al. (1995), B. peloponnesius и B. m. petenyi are one and the same species. Dobrovolov (1999) considered that comparative electrophoretical investigations should be done for clarifying of its taxonomical status in Bulgarian Rivers. The problem with the taxonomic status of the barbel in the Bulgarian and Macedonian Rivers is still discussional. The present working is trying to clear out this status, in the mentioned rivers, on the basis of the determined gene markers. Material and Methods Ten specimens from the tributaries of Vardar River (Macedonia) the rivers Treska, Orizarska, and Bregalnica and twelve specimens from Eleshnitza River, Bulgaria, are electrophoretical analysed for the period May - September 24. For analysis of the enzyme and nonenzyme protein systems, a homogenate of white dorsal muscle was used. The proteins were separated by horizontal starch gel electrophoresis according to Smithies (1955), modified by Dobrovolov (1973). Isoelectric focusing (IEF) on thin polyacrilamide Ampholine gel with ph gradients between with the equipment of LKB (Stockholm, Sweden) was used as well. The muscle proteins were stained with Amido Black 1B or Commassie Brilliant Blue R-25. Esterase (EC EST), lactate dehydrogenase (EC LDH), malate dehydrogenase (EC MDH), malic enzyme (EC MEP), phosphoglucomutase (EC PGM) and в glicerophosphate dehydrogenase (EC в-gpdh) and superoxide dismutase (EC SOD) were analyzed. The staining of different enzymes was performed according to Shaw and Prasad (197). The buffer systems of Dobrovolov (1976) and Clayton and Gee (1969) were used for the electrophoresis. The nomenclature of loci and alleles used here followed essentially the recommendation of Shaklee et al. (199). Results and Discussion On the starch gel electroforegrams, differences among the specimens from the four compared rivers are not observed

3 292 P. Ivanova, L. Velkova-Jordanoska and I. Dobrovolov І ІІ ІІІ ІV Fig. 2. Isoelectric focusing (IEF) of general muscle proteins (PROT) on polyacrilamide Ampholine (ðí- gradient 3.5-1) of B. petenyi: Eleshnitza River (1-2), Bregalnitza River (3-4), Orizarska River (5-6) and Treska River (7-8). ² V² - zones of general muscle proteins, origin V VІ Fig. 1. Electrophoregrams of general muscle proteins (PROT) on starch gel by barbus: Eleshnitza River (1-3), Bregalnitza River (4-5), Orizarska River (6-7) and Treska River (8-9), origin (Figure 1). Using isoelectric focusing (IEF) of PROT on polyacrilamide Ampholine gel plates with ph gradient differences in the electrophoretic mobility of the fractions between samples from Eleshnitza River and from the three tributarities of Varder River were determined (found) (Figure 2). In the examined specimens from the rivers Orizarska, Treska and Brigalnitza, the characteristic for B. petenyi (Eleshnitza, River) fraction III is not observed. Probably, the further analyses of a larger number of specimens from the Vardar River tributaries, this fraction could, also, be found in the heterozygote individuals. The existence of a zero allele in the mentioned zone III is suppositional. On the starch gel electrophoregrams two esterase loci (EST-1 * and EST-2 * ) are visualized. The electroforetic mobility of EST-1 * fractions (with more close to the start position) is common for the specimens from the four compared rivers. As for EST-2 * locus, a different position on the fractions of the specimens from the Eleshnitza River and those from the three tributaries of Vardar River are observed. In the last mentioned rivers the fractions are common and slower than the fractions in the individuals from Eleshnitza River (Figure 3). The polymorphism in the esterase loci was not observed. In the malate dehydrogenase two loci (MDH-1 * and MDH-2 * ) are visualized (Figure 4).The first locus is monomorphic. In the second one (MDH- 2 * ) a polymorphism with three-allele type of codominant inherit is observed. Allelic frequencies are presented on Table 1. The MDH 2 * fractions of the samples from Bregalitza and Eleshnitza Rivers (samp-

4 Genetic Divergence Among the Populations of B. petenyi from Eleshnitza River Fig. 3. Scheme of esterase zymogram on starch gel: Eleshnitza River (1-2), Bregalnitza River (3-4), Orizarska River (5-6) and Treska River (7-8), origin les 5 and 8) belongs to the heterozygote individuals. On the Figure 4 (6 and 7) the typical for the triploids expression was visualized. The fractions situated between the two MDH * loci are midlocus hybrid fractions. Regarding the phosphoglucomutase two monomorphic loci (PGM-1 * and PGM-2 * ) are visualized in the specimens from the all Rivers analyzed. Differences in the electroforetic mobility of the fractions from the Rivers compared is not observed. Polymorphism on LDH-A * locus only in the specimens from the tributaries of Vardar River is observed. Two LDH-B * loci (LDH-B1 * and LDH-B2 * ) were found. LDH-B2 * polymorphism is determined only in the examined specimens from the Rivers Eleshnitza and Orizarska (Table 1, Figure 5). MDH-1* LDH-A* LDH-B1* MDH-2* LDH-B2* Fig. 4. Zymograms of malate dehydrogenase (MDH) on starch gel: Treska River (-2), Orizarska River (3-4), Bregalnitza River (5-6) and Eleshnitza River (7-8), origin Fig. 5. Zymîgrams of lactate dehydrogenase (LDH) on barbus: Eleshnitza River (1-2), Bregalnitza River (3-4), Orizarska River (5-6) and Treska River (7). Polimorphism in the LDH-Â2* locus is observed, origin

5 294 P. Ivanova, L. Velkova-Jordanoska and I. Dobrovolov Table 1 Аlellic frequencies in the barbus (B. petenyi ) populations: Treska River (1), Orizarska River (2), Bregalnitza River (3) and Eleshnitza (4). Genetic identity (INei) and genetic distances (DNei) Loci Аllele Treska R. (1) Orizarska R. (2) Bregalnitza R (3) analysed and genetic distance calculated (D Nei ) (Table 1) barbus from the Eleshnitza River and those from the Rivers Treska, Bregalnitza and Orizarska (tributaries of Vardar River) belong to two different subspecies. Eleshnitza R. (4) EST-1* EST-2* β-gpdh-1* β-gpdh-2* LDH-A* LDH-B1* LDH-B2* smdh-1* smdh-2* MEP* PGM-1* PGM-2* SOD* D Nei INei The decoding of the SOD polymorphism in the barbus from the Bulgarian Rivers was done from Dobrovolov (1999). Four SOD * loci were found. The population of B. petenyi from Eleshnitza River is definitely polymorphic. Such a polymorphism for the barbus from the tributaries of Vardar River are not established. в GPDH fractions were situated on the anode part of the electrophoregrams. Two monomorphic loci, common for the samples from the four Rivers compared are visualized. On the basis of the data Conclusions After genetic-biochemical analyses on barbus (B. petenyi) from Eleshnitza River (Bulgaria) and some tributaries of Vardar River (Macedonia) and genetic distances

6 Genetic Divergence Among the Populations of B. petenyi from Eleshnitza River (D Nei ) calculated the subspecies differences were proved. Comparative genetical analyses on B. petenyi from Bulgarian and Macedonian Rivers with B. meridionalis and B. pelopenesius are needed to specify while in the investigated areas B. petenyi exactly exist or it is presented form some hybrid population. References Apostolski, K., Н. Petrovski, О. Pojska and М. Sidorovski, The fishes of Macedonia. Institut of Fishery of HPM, Scopie. Berrebi, P., Speciation of the genus Barbus in the north Mediterranean basin: resent advances from biochemical genetics. Biological Conservation, 72: Berrebi, P., P. Kotlik, P. Skelton and P. Rab, Systematics of Barbus: state of the art and heuristic comments. Folia Zoologica, 45: Clayton, J. W. and G. H. Gee, Lactate dehydrogenase isozymes in longnose and blacknose Dace (Rhinichthys cataractae and R. atratulus) and their hybrid. J. Fish. Res. Bd. Canada, 26 (11): Dimovski, А. and R. Grupche, Contribution to taxonomy of Genus Barbus (Pisces, Cyprinide) in Macedonia. Fragm. Balc. Mus. Maced. Sci. Nat., 13 (1/288): Dobrovolov, I. S., Micro starch gel electrophoresis. Proceeding of the Institute of Oceanography and Fisheries, Varna, 12: (Bg). Dobrovolov, I. S., Multiple forms of lactate-dehydrogenase in anchovy (Engaulis encrasiholus L.) from the Black sea, the sea of Azov and the Atlantic ocean. Comptes rendus de l Academie bulgare des Sciences, 29 (6): Dobrovolov, I. S., Isoelectric focusing of barbels muscle myogens in Bulgaria in connection with their systematics. Comptes rendus de l Academie bulgare des Sciences, 39 (5): Dobrovolov, I. S., Taxonomie des barbaux du genere Barbus en Bulgarie etude biochimique. Cah. d Etol., 2: Dobrovolov, I. S., Biochemical genetic characteristics of Barbel (Barbus Cuvier Genus) from bulgarian rivers. Folia Zoologica, 45 (1): Dobrovolov, I. S., Superoxide dismutase polymorphism of Barbus meridionalis petenyi (Pisces, Barbus Cuvier) from Bulgarian rivers. Proceedings of the Institute of Fisheries, Varna, 25: Drensky, P. S., Fishes in Bulgaria, Fauna of Bulgaria, BAS, 2: 269 pp. (Bg). Economidis, S. P., Distribution pattern of the genus Barbus (Pisces, Cyprinidae) in the freshwaters of Greece. Extrait des Travaux du Museum d Histoire naturelle Grigore Antipa, Bucharest, 3: Georgiev, S., A key for the fish (Osteichthyes) and ells (Cephalaspidomorpha) determination in Macedonia, 177 pp (Mc). Karakoisis, Y., A. Machordom, I. Doadrio and P. S. Economidis, Phylogenetic relationships of Barbus peloponnesius Valenciennes, 1842 (Osteichthyes: Cyprinidae) from Greece and other species if Barbus as revealed by allozyme electrophoresis. Biochemical Systematic and Ecology, 23 (4): Karaman, S., Cobitis balcanica Kar., die vierte europaische Schmerlenart. Blatter fur Aquarienund Terrarienkunde, Stuttgart, 35 pp. Karaman, M., Zoogeografski odnosi

7 296 P. Ivanova, L. Velkova-Jordanoska and I. Dobrovolov Prespanskog I Ohridskog jezara. IZDANI- JA. Zavod za ribarstvo SRM, IV, 5 (Sr). Karapetkova, Ě. and Ě. Zhivkov, Bulgarian fish species, 247 pp. (Bg). Kotlik, P. and P. Berrebi, 21. Phylogeography of the barbel (Barbus barbus) assessed by mitochondrial DNA variation. Molecular Ecology, 1: Kotlik, P. and P. Berrebi, 22. Genetic subdivision and biodiversity of the Danubian rheophilic barb Barbus petenyi inferred from phylogenetic analyses of mitochondrial DNA variation. Molecular Phylogenetic and Evolution, 24: Kotlik, P., C. Tsingenopoulos, P. Rab and P. Berrebi, 22. Two new Barbus species from the Danube River basin with redescription of B. petenyi (Teleostei: Cyprinidae), Folia Zool., 51 (3): Kotlik, P., G. Bogutskaya and G. Ekmekçi, 24. Circum Black Sea phylogeography of Barbus freshwater fishes: divergence in the Pontic glacial refugium. Molecular Ecology, 13: Marinov, B. T., Ňŕksonomy, faunistic and bionomy of some Genera from Family Cyprinidae and Cottidae (Pisces) in Bulgaria. Ph.D. thesis (Bg). Poljakov, G. D., Nd. Filipi, K. Bacho and A. Hysenaj, Peshqit e Shqiperise. Tirana. Shaklee, J., F. W. Allendorf, D. C. Morizot and G. S Whitt, 199. Gene Nomenclature for protein-coding loci in fish. Trans. Amer. Fisheries Soc., 119: Sorič, V. M., Barbus meridionalis petenyi Heckel, 1847 and Barbus meridionalis rebeli Koller, 1925 in Jugoslaviya. Biosistematica, 13 (2): Shaw, C. R. and R. Prasad., 197. Starch gel electrophoresis of enzymes a compilation of recipes, Biochem. Genet., 4: Smithies, O., Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults. Biochem. J., 61: Tsigenopoulus, C. S. and P. Berrebi, 2. Molecular phylogeny of North Mediterranean freshwater barbs (genus Barbus, Cyprinidae) inferred from cytochrome b sequences: biogeographic and syastematic implicationjs. Mol. Phylogenet. Evol., 14: Tsigenopoulus, C. S., Y. Karakousis and P., Berrebi, The North Mediterranean Barbus lineage: Phylogenetic hypotheses and taxonomic implications based on allozyme data, J. Fish Biol., 54: Vukovic, T. and B. Ivanovic, Slatkovodne ribe Jugoslavije. Zemaljski muzej BiH. Sarajevo (Sr).