Besprozvannykh V.V ., Ermolenko A.V ., Atopkin D.M.
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Transcript of 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 RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii RFE
Skrjabinolecithum spasskii Vietnam
Skrjabinolecithum spasskii Vietnam
Skrjabinolecithum spasskii Vietnam
Skrjabinolecithum spasskii Vietnam
Skrjabinolecithum sp. nov. 1 RFE
Platydidymus flecterotestis RFE
Platydidymus flecterotestis RFE
Platydidymus flecterotestis RFE
Platydidymus flecterotestis RFE
Skrjabinolecithum indicum Vietnam
Skrjabinolecithum indicum Vietnam
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.
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