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

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

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

A B

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.

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.

List of investigated speciesSpecies N Host LocationP. flecterotestis 4 Liza haematocheila Russian Far East, Razdolnaya

Riv.S. spasskii 18 Liza haematocheila Russian Far East, Razdolnaya

Riv., Kievka Riv.

S. spasskii 7 Liza haematocheila Vietnam, Cat Ba Island

S. sp.nov.1 1 Liza haematocheila Russian Far East, Utinoe Lake

S. indicum 3 Valamugil seheli Vietnam, Cat Ba Island

N – sample size;

18S ITS1 ITS2 28S

5.8S

IGS

Amplification and sequencing of partial 28S rRNA gene and ITS1-5.8S-ITS2 rDNA fragment

D1-D3

D2D1 D3

5.8S

ITS1 ITS2

RESULTS

Sequence lenght, bp

Nucleotide sites

V Pi S G+C, %

Platydidymus flecterotestis, n=4 1047 0 0 1 53.8Skrjabinolecithum spasskii, RFE, n=18 1047 5 4 1 53.7Skrjabinolecithum spasskii, Vietnam, n=4 1047 0 0 0 53.6Skrjabinolecithum sp.nov.1, n=1 1047 - - - 53.7Skrjabinolecithum indicum, n=3 1041 5 1 4 52.7

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

Genetic differentiation of Skrjabinolecithum species by 28S rRNA gene sequences (amount of variable sites – above diagonal, p-

distances, % - below diagonal). RFE – Russian Far East.

1 2 3 4 5

1. P. flecterotestis 12 10 2 138

2. S. spasskii RFE 0.88 6 10 139

3. S. spasskii Vietnam 0.82 0.50 8 138

4. S. sp.nov.1 0.12 0.75 0.69 138

5. S. indicum 12.7 12.7 12.7 12.7

Skrjabinolecithum spasskii RFE


















Skrjabinolecithum spasskii Vietnam




Skrjabinolecithum sp. nov. 1 RFE

Platydidymus flecterotestis RFE




Skrjabinolecithum indicum Vietnam



AY222266 Pseudomegasolena ishigakiense

AY222267 Atractotrema signai

81/0.96

99/1.00

99/1.00

99/1.00

87/0.60

93/0.87

96/0.97

66/0.93

69/0.31

61/0.5370/1.00

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.

Waretrematinae

ChalcinotrematinaeForticulcitinae

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

100

100100

100

99100

99

10073

10063

100

100

9764

100

7261

100100

100100

Megasoleninae

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. 1S. indicum0.8%

12.7%

12.7% 0.12%

0.7%

12.7%

?

0.02

Skr3_15

Skr4_11

S.spas34_3S.spas34_4

SkrV3

Skr3_17

Skr3_13

Skr4_10Skr4_1

SkrV1

S.spas34_2

P3

Skr3_12

P1Skr4_13

KC430095_Intromugil alachuaensis

Skr3_16Skr3_18

Skr4_15

P2

SkrV2

KC430096_Intromugil mugilicolus

Skr4_12

Skr4_2Skr4_9

S.spas34_1

P4

KC206500_Spiritestis herveyensis

100/1.0

100/1.0

100/0.63

100/1.0

100/0.62

100/0.95

Genotype 1RFE

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

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

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)

Genetic differentiation of S. spasskii

Nucleotide position 530 543 564 569 662S. spasskii RFE C C T C T

S. spasskii Vietnam T T C T A

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).

Nucleotide position

140 145 199 242 301 347 348 355 357 1206 1255 1256

S. spasskii RFE C G T C T C C T A T T C

S. spasskii Vietnam T A C T G A T A G C C T

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).

Primorye Region

Vietnam, Cat Ba

S. spasskii d=0.5/0.9%

Primorye RegionChina Mixed variants of

S. spasskii rDNA sequences

Kievka River

Razdolnaya River

Liza haematocheila

Hypothesis of genetic differentiation events of S. spasskii

• 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.

THANKS FOR YOUR ATTENTION!

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

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