1. Diversity of two forms of silver crucian carp Carassius auratus gibelio based on mitochondrial DNA analysis Diversity of two forms of silver crucian carp Carassius auratus gibelio based on mitochondrial DNA analysis O. Apalikova, A. Podlesnikh, Vl. Brykov A.V. Zhirmunsky Institute of Marine Biology FEB RAS 1

2. Carassius auratus gibelio (Bloch, 1782) diploid gonochoristic form (2n ≈ 100) gynogenetic polyploid form (3n ≈ 150, 4n ≈ 200) phylogenetic relations of these two forms remain unclear. Probably FAR EAST is a territory of silver crucian carp origin. 2

3. Сollation of phylogeography and ploidy level of silver crucian carp in the samples, covering much of its area to clarify mechanism of possible genetic exchange between the forms, as well as revision of the place of their divergency. 3Goal

4. Tasks 1. To define ploidy level of specimens from various samples of silver crucian carp, received from waters of the Far East, Central Asia and European part to Russia. 2. To define the level an intraspecious variability of silver crucian carp on the data of restrictase fragment length polymorphism (RFLP) of mitochondrial DNA area, encoding 3-rd and 4-th subunits of NADH dehydrogenase and two subunits (12S/16S) of ribosomal RNA, amplified in polymerase chain reaction (PCR). 3. To collate ploidy data and intraspecious mtDNA variability of silver crucian carp. 4. To study distribution of bisexual diploid and gynogenetic polyploid forms of silver crucian carp on the area. 4

5. Methods 1. Amplification of mtDNA: ND3/ND4L/ND4, 12S/16S rRNA. 2. RFLP –analysis of amplified mtDNA- fragments. 3. Statistical analysis of mtDNA variety. mitochondrial DNA polymorphism within groups was estimated as haplotype diversity (h) (Nei & Tajima 1981; Nei 1987)and nucleotide diversity (p) using REAP clustering and constructing phenogram by the UPGMA with the use of the NTSYS software package (Rohlf, 1990) significance of differences in the frequencies of haplotype variants between samples of diploid and triploid fish was estimated using the pseudoprobability test for heterogeneity χ 2 (Zaykin, Pudovkin,1993) stability of the topology was examined through 1000 bootstrap replications (Felsenstein 1993). 5 4. Morphometry for determination of ploidy

6. Staining by Azocarmine G Ploidy Square of nucleus Standard Deviation Number of estimation 2n153.868 (1)± 20.50890 - 100 3n205.565 (1.3)± 26.79390 - 100 3n-4n277.349 (1.8)± 44.38090 - 100 2n2n2n2n 3n3n 6

7. Staining by AgNO 3 Number of nucleoli per cell01234 2n species106030 3n species 3040 4n species 10304020 7 2n2n2n2n 3n

8. The map of sampling sites № Sampling site number 1 Razdol'naya River 52 2 Karasik River 64 3 Lebedinka River 29 4 Lake of Bol'shoi Pelis Island 26 5 Amur River 29 6 Lake Khanka 20 7 Sakhalin 50 8 Kamchatka River 57 9 р. Syr Darya River basin 20 10 Rybinskoye water basin 33 Totally 380 8

9. Ploidy of Carassius auratus gibelio of Razdol'naya River : 14 (5 ♂♂ and 9 ♀♀) – 2n, 19 ♀♀ – 3n and 4n PCR-RFLP –analysis ND3/ND4L/ND4 – MspI, BsuRI (HaeIII), RsaI, MvaI, Hin6I (HhaI); 12S/16S rRNA – MspI, FnuDII, HaeIII, RsaI, MvaI, HhaI, BstDEI 9

10. 1р С1 4р С4 9р С5 7р 10р 11р 44р С2 45р 38р 13р 6р 14р 31р 33р 36р 37р 19р 15р 16р С16 3р 40р 2р 29р 5р 8р 17р 0 0.5 1 1.5 2 Nucleotide substitutions, % 42р I II A C D B B1 - 0.0021 - 0.0132 0.0154 - 0.0254 0.0275 0.0156 - 0.0228 0.0249 0.0133 0.0022 - Values of divergency between haplotypes The UPGMA tree of mtDNA haplotypes. 10 Phylogenetic relationship between haplotypes of silver crucian carp in the Razdol'naya River 3n 4n: ♀♀: 19 (I) ♀♀: 6 (I) 3 (II) ♂♂: 2 (I) 3 (II) 2n 99 89 95

11. ACIHDEFGBACIHDEFGB 0 2 3 4 6 Nucleotide substitutions, % 40 61 75 59 81 59 The UPGMA tree of mtDNA haplotypes. II I Values of divergency between haplotypes А B C D E F G H I - 0.0582 - 0.0034 0.0615 - 0.0140 0.0500 0.0174 - 0.0353 0.0603 0.0385 0.0428 - 0.0416 0.0584 0.0447 0.0491 0.0076 - 0.0345 0.0409 0.0378 0.0421 0.0265 0.0257 - 0.0076 0.0697 0.0108 0.0216 0.0178 0.0246 0.0460 - 0.0035 0.0649 0.0068 0.0173 0.0430 0.0489 0.0416 0.0117 - 10 n.s. 11

12. 2n2n 3n Geographic distribution of mtDNA phylogroups and ploidy frequencies in the silver crucian carp populations 12 ? ? ? ?

13. I Phylogroup 3n2n → по: Liu S. J. et al., 2006 13 2n II Phylogroup possible mechanism of genetic exchange between triploid and diploid forms

16. 2n2n 3n 14 Geographic distribution of mtDNA phylogroups and ploidy frequencies in the silver crucian carp populations

17. 1.The existence of only two substantially differing from each other mtDNA phylogroups indicates that the gynogenetic form has emerged from the diploid form only once and evolved independently for a long time after that. 2.The absence of haplotypes transitional between the two mtDNA phylogroups suggests that the secondary contact between the gynogenetic and bisexual forms in continental populations occurred within recent historical time. 3.The obtained data confirm that genetic (asymmetric) exchange between the two forms is possible, and can be explanation of high morphological and, probably, genetic similarity between them. Conclusions 15