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 Type Platyhelminthes Class Trematoda Subclass Digenea Family Haploporidae Subfamily Waretrematinae Genus Skrjabinolecithum Belous, 1954 Species: – S. spasskii Belous, 1954 – S. indicum (Zhukov, 1972) – S. vitellosum (Martin, 1973) – S. lobolecithum (Martin, 1973) Type species from Liza haematocheila, Razdolnaya River, Primorye region

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

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 – from Zhukov (1971), C – from Overstreet & Curran (2005). Platydidymus flecterotestis Overstreet & Curran (2005). Skrjabinolecithum flecterotestis А C B Skrjabinolecithum sp. nov. 1 – new species, described from Liza haematocheila Skrjabinolecithum indicum – from Vietnam (Cat Ba Island) from Valamugil seheli.

5. The aim of this study is to 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 SpeciesNHostLocation P. flecterotestis4Liza haematocheilaRussian Far East, Razdolnaya Riv. S. spasskii18Liza haematocheilaRussian Far East, Razdolnaya Riv., Kievka Riv. S. spasskii7Liza haematocheilaVietnam, Cat Ba Island S. sp.nov.11Liza haematocheilaRussian Far East, Utinoe Lake S. indicum3Valamugil seheliVietnam, Cat Ba Island N – sample size;

7. 18SITS1ITS228S 5.8S IGS Amplification and sequencing of partial 28S rRNA gene and ITS1- 5.8S-ITS2 rDNA fragment D1-D3 D2D1 D3D3 5.8S ITS1 ITS2

8. RESULTS Sequence lenght, bp Nucleotide sites VPi SG+C, % Platydidymus flecterotestis, n=4 104700153.8 Skrjabinolecithum spasskii, RFE, n=18 104754153.7 Skrjabinolecithum spasskii, Vietnam, n=4 104700053.6 Skrjabinolecithum sp.nov.1, n=1 1047---53.7 Skrjabinolecithum indicum, n=3 104151452.7 Characterization of 28S rDNA sequences of different species of 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. 12345 1. P. flecterotestis 12 10 2 138 2. S. spasskii RFE0.88 6 10 139 3. S. spasskii Vietnam0.820.50 8 138 4. S. sp.nov.10.120.750.69 138 5. S. indicum12.7

10. I II III IV Maximum likelihood phylogenetic tree of the genus Skrjabinolecithum based on 28S rRNA gene sequence data. Nodal numbers – statistical support for ML/BI algorithms.

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

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

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

14. 0.1 Skr3_12 KC206500_Spiritestis herveyensis SkrV2 SkrP2 Skr3_18 SkrV3 Skr34_1 SkrP4 Skr3_17 KC430096_Intromugil mugilicolus Skr4_11 Skr3_11 Skr34_4 SkrV1 Skr4_12 SkrP3 Skr4_13 Skr4_9 Skr4_15 Skr4_10 Skr34_2 SkrP1 Skr34_3 82/1.0 100/1.0 99/1.0 Genotype 1 (RFE) Genotype 2 (RFE) Genotype 3 (Vietnam) 0.3% 0.7% 0.9% 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. d = 2.1 – 10.9% (genus Saccocoelium, from Blasco-Costa et al., 2009)

15. Genetic differentiation of S. spasskii Nucleotide position530543564569662 S. spasskii RFECCTCT S. spasskii VietnamTTCTA Variable nucleotide sites with fixed substitutions of partial 28S rRNA gene (1047 bp) sequences of S. spasskii from Russian Far East and Vietnam. Gray colored cells – variable nucleotide sites for Genotype 2 (RFE). Nucleotid e position 140145199242301347348355357120612551256 S. spasskii RFE CGTCTCCTATTC S. spasskii Vietnam TACTGATAGCCT Variable nucleotide sites with fixed substitutions of ITS1-5.8S-ITS2 (1511 bp) sequences of S. spasskii from Russian Far East and Vietnam. Gray colored cells – variable nucleotide sites for Genotype 2 (RFE).

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

17. Platydidymus flecterotestis is 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 Platydidymus flecterotestis and 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. spasskii revealed by these data. Genetic differentiation of Far Eastern and Vietnamese S. spasskii was most likely due to geographic isolation. Mixed variants of S. spasskii ribosomal 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 haematocheila from Vietnam to the Russian Far East; The question about taxonomical status of S. sp.n.1 and S. indicum is need to be investigated.

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