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Carassius auratus gibelio (Bloch, 1782)

1. 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. 2. Carassius auratus gibelio (Bloch, 1782). diploid gonochoristic form ( 2n ≈ 100).

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Carassius auratus gibelio (Bloch, 1782)

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

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

  3. 3 Goal 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.

  4. 4 Tasks • 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. • 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). • To collate ploidy data and intraspecious mtDNA variability of silver crucian carp. • To study distribution of bisexual diploid and gynogenetic polyploid forms of silver crucian carp on the area.

  5. 5 Methods 1. Amplification of mtDNA: ND3/ND4L/ND4 , 12S/16SrRNA. 2. RFLP–analysis ofamplified 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 REAPclustering 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 χ2 (Zaykin, Pudovkin,1993) • stability of the topology was examined through 1000 bootstrap replications(Felsenstein 1993). 4. Morphometry for determination of ploidy

  6. Staining by AzocarmineG 6 2n 3n 3n

  7. 7 Staining by AgNO3 2n 3n

  8. 8 № Sampling site number 1 Razdol'naya River52 2 Karasik River 64 3 Lebedinka River 29 4 Lake of Bol'shoi Pelis Island26 5 Amur River29 6 Lake Khanka 20 7 Sakhalin 50 8 Kamchatka River57 9 р. Syr Darya River basin20 10 Rybinskoye water basin 33 Totally 380 The map of sampling sites

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

  10. Phylogenetic relationship between haplotypes of silver crucian carp in the Razdol'naya River 10 1рС1 4р С4 9р С5 7р 10р 11р44р С2 45р 38р 13р 6р 14р 31р 33р 36р 37р 19р15р 16р 3n 4n:♀♀: 19 (I) ♀♀: 6 (I) 3 (II) ♂♂: 2 (I) 3 (II) Values of divergency between haplotypes 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 - 2n I 95 89 С163р 40р 2р 29р 5р 8р 17р II 99 42р 0 0.5 1 1.5 2 Nucleotide substitutions, % The UPGMA tree of mtDNA haplotypes.

  11. 11 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. A C I H D E F G B 40 59 61 61 I 81 75 59 II 0 2 3 4 6 Nucleotide substitutions, % The UPGMA tree of mtDNA haplotypes.

  12. 12 GeographicdistributionofmtDNA phylogroups and ploidy frequencies in the silver crucian carp populations ? ? 3n ? ? 2n 3n

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

  14. GeographicdistributionofmtDNA phylogroups and ploidy frequencies in the silver crucian carp populations 14 2n 3n

  15. 15 Conclusions • 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. • 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. • 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.

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