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Besprozvannykh V.V ., Ermolenko A.V ., Atopkin D.M.

Trematodes of the family Haploporidae Nicoll, 1914 from mugilid fishes of Primorye region: ribosomal genes sequence data of the genus Skrjabinolecithum Belous, 1954. Besprozvannykh V.V ., Ermolenko A.V ., Atopkin D.M. Institute of Biology and Soil Sciences FEB RAS Vladivostok.

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Besprozvannykh V.V ., Ermolenko A.V ., Atopkin D.M.

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  1. Trematodes of the family Haploporidae Nicoll, 1914 from mugilid fishes of Primorye region: ribosomal genes sequence data of the genus Skrjabinolecithum Belous, 1954 Besprozvannykh V.V., Ermolenko A.V., Atopkin D.M. Institute of Biology and Soil Sciences FEB RAS Vladivostok

  2. Systematic position of the genus Skrjabinolecithum • TypePlatyhelminthes • ClassTrematoda • SubclassDigenea • FamilyHaploporidae • SubfamilyWaretrematinae • GenusSkrjabinolecithum Belous, 1954 • Species: • S. spasskii Belous, 1954 • S. indicum (Zhukov, 1972) • S. vitellosum (Martin, 1973) • S. lobolecithum (Martin, 1973) Type species fromLiza haematocheila, Razdolnaya River, Primorye region

  3. A B Skrjabinolecithum spasskii: А – mature specimen (original data), B – mature specimen from Belous (1954).

  4. Hapalotrema flecterotestis Zhukov, 1971 Trematodes of Skrjabinolecithum: S. flecterotestis (= H. flecterotestis Zhukov, 1971; P. flecterotestis (Zhukov, 1971) Overstreet et Curran, 2005; Carassotrema flecterotestis (Zhukov, 1971) Yiu, Peng et Liu, 2005: А – original data, B – fromZhukov (1971), C – fromOverstreet & Curran (2005). PlatydidymusflecterotestisOverstreet & Curran (2005). Skrjabinolecithum flecterotestis А B C Skrjabinolecithum sp. nov. 1 – new species, described from Liza haematocheila Skrjabinolecithumindicum– from Vietnam (Cat Ba Island) from Valamugilseheli.

  5. The aim of this study isto confirm the validity of the species Skrjabinolecithum from Russian Far East and Vietnam using molecular data. • Sequencing of partial 28S rRNA gene and ITS1-5.8S-ITS2 rDNA fragment of investigated Skrjabinolecithum species; • Estimation of intra- and interspecific genetic differntiation of Skrjabinolecithum species using p-distances. • Reconstruction of the phylogenetic relationships of the genus Skrjabinolecithum.

  6. List of investigated species N – sample size;

  7. 18S ITS1 ITS2 28S IGS 5.8S Amplification and sequencing of partial 28S rRNA gene and ITS1-5.8S-ITS2 rDNA fragment D1-D3 5.8S D3 ITS2 ITS1 D1 D2

  8. RESULTS Characterization of 28S rDNA sequences of different speciesof the genus Skrjabinolecithum V – variable sites; Pi – parsimony-informative sites; S – singleton sites; RFE – Russian Far East

  9. Genetic differentiation of Skrjabinolecithum species by 28S rRNA gene sequences (amount of variable sites – above diagonal, p-distances, % - below diagonal). RFE – Russian Far East.

  10. Maximum likelihood phylogenetic tree of the genus Skrjabinolecithum based on 28S rRNA gene sequence data. Nodal numbers – statistical support for ML/BI algorithms. Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE I Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE 96/0.97 Skrjabinolecithum spasskii RFE Skrjabinolecithum spasskii RFE 93/0.87 Skrjabinolecithum spasskiiRFE Skrjabinolecithum spasskiiRFE Skrjabinolecithum spasskiiRFE II 87/0.60 Skrjabinolecithum spasskiiRFE Skrjabinolecithum spasskii Vietnam 99/1.00 Skrjabinolecithum spasskii Vietnam 66/0.93 Skrjabinolecithum spasskii Vietnam Skrjabinolecithum spasskii Vietnam Skrjabinolecithum sp. nov. 1RFE 69/0.31 III Platydidymus flecterotestis RFE 70/1.00 Platydidymus flecterotestis RFE 61/0.53 Platydidymus flecterotestis RFE Platydidymus flecterotestis RFE IV Skrjabinolecithum indicum Vietnam 99/1.00 Skrjabinolecithum indicum Vietnam 81/0.96 Skrjabinolecithum indicum Vietnam AY222266 Pseudomegasolena ishigakiense 99/1.00 AY222267 Atractotrema signai

  11. 100 S. spassskii RFE 100 S. spasskii RFE+Vietnam 100 100 S.sp.nov.1 100 P. flecterotestis RFE 61 64 72 S. indicum Vietnam 97 100 EF032696 Saccocoelioides sp. FJ211239 Forticulcita gibsoni FJ211237 Haploporus benedeni 100 100 FJ211236 Lecithobotrys putrescens FJ211262 Dicrogaster contracta 100 100 FJ211261 Dicrogaster contracta 100 FJ211238 Dicrogaster perpusilla FJ211260 Saccocoelium obesum 99 100 100 FJ211259 Saccocoelium obesum 63 FJ211234 Saccocoelium brayi FJ211233 Saccocoelium cephali 100 73 FJ211258 Saccocoelium tensum 99 FJ211257 Saccocoelium tensum 100 AY222265 Hapladena nasonis AY222266 Pseudomegasolena ishigakiense AY222267 Atractotrema signai DQ836244 Paragonimus westermani Waretrematinae Chalcinotrematinae Forticulcitinae Haploporinae Megasoleninae Maximum likelihood phylogenetic tree of the family Haploporidae based on 28S rRNA gene sequence data.

  12. d = 0.9 – 4.8% (genus Saccocoelium, from Blasco-Costa et al., 2009) d = 9.6 – 15.8% (familyHaploporidae, from Blasco-Costa et al., 2009) 12.7% ? P. flecterotestis 12.7% 0.12% S. indicum S. sp. nov. 1 0.8% 0.7% 12.7% S. spasskii

  13. Maximum likelihood phylogenetic tree of S. spasskii based on 28S sequence data. Statistical support showed for ML/BI algorithms. RFE – Russian Far East Skr3_12 Skr3_13 Skr3_15 Skr3_16 Skr3_18 Skr4_1 100/0.63 Skr4_10 Skr4_11 Genotype 1 RFE Skr4_12 Skr4_13 P1 100/1.0 P2 P3 P4 0.4% Skr3_17 0.5% Skr4_2 Skr4_9 100/0.62 Skr4_15 100/1.0 Genotype 2 (RFE) S.spas34_1 S.spas34_2 S.spas34_3 S.spas34_4 100/0.95 0.1% SkrV1 SkrV2 SkrV3 Genotype 3 (Vietnam) KC206500_Spiritestis herveyensis 100/1.0 KC430096_Intromugil mugilicolus KC430095_Intromugil alachuaensis 0.02 d = 0.9 – 4.8% (genusSaccocoelium, from Blasco-Costa et al., 2009) d = 2.7 – 2.8% (genus Intromugil, from Pulish & Overstreet, 2013)

  14. Maximum likelihood phylogenetic tree of S. spasskii based on ITS1-5.8-ITS2 sequence data. Statistical support showed for ML/BI algorithms. RFE – Russian Far East. Skr3_12 Skr4_10 SkrP1 Skr4_12 SkrP2 82/1.0 Skr3_18 SkrP4 SkrP3 Skr4_13 Genotype 1 (RFE) 99/1.0 Skr4_11 Skr3_17 Skr3_11 Skr4_9 100/1.0 0.7% Skr4_15 Skr34_4 0.9% Skr34_2 SkrV3 Genotype 2 (RFE) SkrV1 SkrV2 Skr34_1 0.3% Skr34_3 KC430096_Intromugil mugilicolus 100/1.0 KC206500_Spiritestis herveyensis Genotype 3 (Vietnam) 0.1 d = 2.1 – 10.9% (genusSaccocoelium, from Blasco-Costa et al., 2009)

  15. Genetic differentiation ofS. spasskii Variable nucleotide sites with fixed substitutions of partial 28S rRNA gene (1047 bp) sequences ofS. spasskii from Russian Far East and Vietnam. Gray colored cells – variable nucleotide sites for Genotype 2 (RFE). Variable nucleotide sites with fixed substitutions of ITS1-5.8S-ITS2 (1511 bp) sequences ofS. spasskii from Russian Far East and Vietnam. Gray colored cells – variable nucleotide sites for Genotype 2 (RFE).

  16. Hypothesis of genetic differentiation events of S. spasskii Primorye Region Razdolnaya River China Primorye Region Mixed variants of S. spasskii rDNA sequences Kievka River S. spasskii d=0.5/0.9% Vietnam, Cat Ba Liza haematocheila

  17. Platydidymusflecterotestisis closely related to S. spasskii by 28S rDNA with genetic differentiation 0.8% that represents minimal interspecific divergence level (Blasco-Costa et al., 2009). These data confirmed the validity of Platydidymusflecterotestisand support its membership to the genus Skrjabinolecithum. • Molecular variation of ribosomal DNA sequences with fixed point substitutions was revealed within species S. spasskii (from Liza haematocheila) obtained from different geographical locations. Three genotypes of S. spasskiirevealed by these data. Genetic differentiation of Far Eastern and Vietnamese S. spasskiiwas most likely due to geographic isolation. • Mixed variants of S. spasskiiribosomal DNA sequences from the same definitive host specimen can be explained by two main events: 1 - geographic isolation of the first intermediate host species (mollusks) in the past and 2 - migration or introduction of infected Liza haematocheilafrom Vietnam to the Russian Far East; • The question about taxonomical status of S. sp.n.1 and S. indicum is need to be investigated.

  18. THANKS FOR YOUR ATTENTION!

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