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Natural hybridization of two species of barbels in the Western Carpathians

Zdenk Lajbner1, Vra lechtov1, Vlastimil lechta1, Miroslav vtora2, Patrick Berrebi3 & Petr Kotlk1
1 Department of Vertebrate Evolutionary Biology and Genetics, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumbursk 89, 277 21 Libchov, Czech Republic 2 Department of Zoology, Faculty of Sciences, Charles University, 128 44 Prague, Czech Republic 3 Institut des Sciences de lEvolution, UMR 5554 UM2/CNRS, Universite Montpellier 2, Place E. Bataillon, 34095 Montpellier, Cedex 5, France

The work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (LC06073, FRVS 2264/2002 and MSMT 21620828) and by the Academy of Sciences of the Czech Republic (IRP IAPG AV0Z50450515, GA206/09/1154 and IGA IAPG 06/03).

Phylogeny of barbels

A. Machordom, I. Doadrio / Proceedings of the Royal Society London B 268 (2001) 1297-1306. P. Berrebi, C.S. Tsigenopoulos / The Freshwater Fishes of Europe 5/II: Cyprinidae 2/II (2003) 1122.

Danubian rheophilic barbels


4 6
A) Barbus balcanicus Kotlk,

Tsigenopoulos, Rb et Berrebi, 2002

B) Barbus carpathicus Kotlk,

Tsigenopoulos, Rb et Berrebi, 2002

C) Barbus petenyi Heckel, 1852

P. Kotlk, P. Berrebi / Molecular Phylogenetics and Evolution 24 (2002) 1018. P. Kotlk, C. Tsigenopoulos, P. Rb, P. Berrebi / Folia Zoologica 51 (2002) 227240.

Documented natural hybridization of european Barbus species


1. Barbus caninus Bonaparte, 1839 x Barbus plebejus Bonaparte, 1839 2. Barbus haasi Mertens, 1924 x Barbus meridionalis Risso, 1826 3. Barbus meridionalis Risso,1826 x Barbus barbus (Linnaeus, 1758) 4. Barbus peloponnesius Valenciennes, 1842 x Luciobarbus albanicus (Steindacher, 1870) 5. Barbus barbus (Linnaeus, 1758) x Barbus petenyi Heckel, 1852

Barbus barbus (Linnaeus, 1758) x Barbus carpathicus Kotlk, Tsigenopoulos, Rb et Berrebi, 2002
6.

Barbus barbus B. carpathicus

Aims
1. To examine in detail the genotypic composition of the Poprad River barbel population. 2. To compare the results with the Tizsa drainage barbel populations.

Sampling
drainage river coordinates (E / N) 2053 / 4916 Vistula Poprad Orlov Andrejovka 2054 / 4916 Kurn 2055 / 4915 Plave 2050 / 4917 ubotinka 2051 / 4917 Star ubova 2042 / 4919 Hromo 2048 / 4916 2157 / 4902 Danube Laborec Hankovce Kokovce 2158 / 4903 2054 / 4916 Torysa Sabinov locality elevation 479 m.s.l. 474 m.s.l. 470 m.s.l. 488 m.s.l. 485 m.s.l. 510 m.s.l. 490 m.s.l. 180 m.s.l. 188 m.s.l. 320 m.s.l. collection B. barbus B. carpathicus hybrids date (n) (n) (n) 21.10.1997 1 38 0 21.10.1997 0 37 0 21.10.1997 0 1 0 22.10.1997 2 6 0 22.10.1997 0 9 0 23.10.1997 46 31 4 23.10.1997 0 55 0 11.10.1998 1 13 3 12.10.1998 2 1 3 13.10.1998 6 10 4

Diagnostic protein loci:


1. MALATEDEHYDROGENASA: sMdh1*, sMdh2*, sMdh3*, sMdh4* 2. LACTATEDEHYDROGENASA: LdhA1* 3. GLUCOSAPHOSPHATEISOMERASA: Gpi2* 4. PHOSPHOGLUCOMUTASA: Pgm1*, Pgm2* 5. SUPEROXIDISMUTASA: Sod2*

Nuclear DNA analysis: microsatellite locus Barb79 (Chenuil et al., 1997) Mitochondrial DNA analysis: Cytochrome b gene diagnostic endonucleases: HaeIII MboI MspI
Barbus carpathicus RE HaeIII MboI MspI sequence N site fragments N Barbus barbus site fragments

GG!CC 2 142/378 143/236/215 2 142/406 143/264/187 !GATC 2 234/351 117/233/244 1 C!CGG 2 140/210 70/140/384 1 234 140 233/361 144/454

Results of digestion by diagnostic restriction enzyme MspI

Genotypes of hybrid specimens


in ninediagnostic izoenzyme and one microsatelitte loci
All hybrids contained mtDNA characteristic for the common barbel (Barbus barbus).
Locality Star ubova (Poprad) Hankovce (Laborec) Kokovce (Laborec) Hybrid BC1 BC2 F1 F1 F1 F1 BC1 F1 F1 F1 F1 F1 F1 F1 Izoenzyme loci LDH-A1* sMDH-1* sMDH-2* sMDH-3* sMDH-4* GPI-2* P.D. P.D. P.D. P.D. 100/100 85/85 98/98 106/106 100/102 2F2S 100/100 85/85 98/98 106/106 102/102 2F2S 100/200 85/100 98/100 100/106 100/102 2F2S 100/200 85/100 98/100 100/106 100/102 2F2Ff 100/200 85/100 98/100 100/106 100/102 1S3F 100/100 85/100 98/100 100/106 100/102 1S3F 100/100 85/100 98/98 100/106 102/102 1S3F 100/100 85/100 98/100 100/106 100/102 1Sf3F 100/100 85/100 98/100 100/106 100/102 4F 100/100 85/100 98/100 100/106 100/102 1S3F 100/100 85/100 98/100 100/106 100/102 1S3F 100/100 85/100 98/100 100/106 100/102 1S3F 100/100 85/100 98/100 100/106 100/102 4F 100/100 85/100 98/100 100/106 100/102 1S3F PGM-1* 94/100 96/100 100/100 100/100 96/100 100/100 94/94 96/100 94/100 96/100 100/100 96/100 94/100 96/100 PGM-2* P.D. 90/100 90/100 90/100 90/100 90/100 90/100 90/100 90/100 90/100 90/100 90/100 90/100 90/100 90/100 SOD-2* P.D. 50/50 50/50 50/100 50/100 50/100 50/100 50/100 50/100 50/100 50/100 50/100 50/100 50/100 50/100 Microsat. Barb79 P.D. 203/212 275/283 208/275 212/284 212/223 203/208 212/223 203/208 212/223 216/255 203/208 204/247 204/259 208/259

Sabinov (Torysa)

Alelles specific for the carpathian barbel are in bold. P.D. remarks on fully diagnostic locus.

Cytonuclear disequilibria in 3x2 table (according to Avise 2001)1 in all fully diagnostic loci.

M m
DAAM; DAaM; DaaM; DAM 0 ++ 0

AA ++ 0

Aa ++ 0

aa 0 ++

observed genotypes are much more common than expected (significant positive D) absence of the genotype

Avise J.C., 2001: Cytonuclear genetic signatures of hybridization phenomena: Rationale, utility, and empirical examples from fishes and other aquatic animals. Reviews in Fish Biology and Fisheries 10, 253-263.

Pie charts show the expected proportions of genotype categories as identified by the software NewHybrids. These values were obtained by summing probabilities for each of the six categories at each locality.

Main results summary


(population-genetic)

1. Populations of Barbus carpathicus and B. barbus from the Poprad River are probably in a state close to the Hardy-Weinberg equilibrium. 2. No significant diferences between subpopulations within the Poprad River. 3. Both barbel species populations in the Poprad river are significantly diferenciated from conspecific populations of the Danube basin.

Main results summary


(hybridization)

1. Fertile hybrids in both drainages. Detectable individuals of hybrid origin in the Poprad River population are rare (1,74%). 2. Unidiretional hybridization. F1 allways: B. carpathicus + B. barbus . 3. Significantly restricted nuclear and cytoplasmatic gene flow between the species.

Main results summary


(hybridization)

4. Only backcrosses toB. barbus. No introgression to the B. carpathicus genome. 5. Absence of F2 = one sex of F1 probably sterile. 6. Combination of ethological, ecological and physiological characters is most probably responsible for the hybridzation architecture.

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