PHYLOGENETIC RELATIONSHIPS OF THE GENUS PSILOTREMA (P. oschmarini, P PHYLOGENETIC RELATIONSHIPS OF THE GENUS PSILOTREMA (P. oschmarini, P. acutirostris, P. simillimum) BASED ON 28S rDNA SEQUENCES Atopkin D.M. Institute of Biology and Soil Sciences, Far Eastern Branch of Russian Academy of Sciences

2 Subclasses: Aspidogastrea and Digenea Class Trematoda: 2 Subclasses: Aspidogastrea and Digenea 12 Orders ~ 90 Families ~ 7000 Species

Life cycle of trematodes Metacercaria Parthenogenetic generation Cercaria Adolescaria Adult worm Miracidium Inside snail tissue Adults eggs Hermaphrodite generation sporocyst mother rediae daugher rediae cercaria

Species of the genus Psilotrema, Odhner, 1913 Psilotrema acutirostris, Oshmarin, 1963 Psilotrema simillimum (Muhling, 1989) Psilotrema oschmarini, Besprozvannykh, 2007 Psilotrema breve Oshmarin, 1963 Psilotrema castoris Orlov, 1946 Psilotrema marki Skvorzov, 1934 Psilotrema medioporum Oshmarin, 1963 Psilotrema oligoon (von Linstow, 1887) Psilotrema pharyngeatum Grabda 1954 Psilotrema spiculigerum (Muhling, 1989) Psilotrema tuberculatum, (de Filippi, 1857) Psilotrema zibethicum Varenov, 1965

Molecular genetic investigations, includes species of the family Psilostomatidae Psilochasmus oxyurus - Tkach,V., Pawlowski,J. and Mariaux,J. 2000. Phylogenetic analysis of the suborder plagiorchiata (Platyhelminthes, Digenea) based on partial lsrDNA sequences // Int. J. Parasitol. 30 (1), 83-93 Sphaeridiotrema pseudoglobulus - Moszczynska,A., Locke,S.A., Mclaughlin,J.D., Marcogliese,D.J. and Crease,T.J. Development of primers for the mitochondrial cytochrome c oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths // Unpublished Ribeiroia marini and R. ondatrae - Wilson,W.D., Johnson,P.T., Sutherland,D.R., Mone,H. and Loker,E.S. 2005. A molecular phylogenetic study of the genus Ribeiroia (Digenea): trematodes known to cause limb malformations in amphibians // J. Parasitol. 91 (5), 1040-1045.

Species of the genus Psilotrema Psilotrema acutirostris, Oshmarin, 1963 Psilotrema simillimum (Muhling, 1989) Psilotrema oschmarini, Besprozvannykh, 2007 Psilotrema breve Oshmarin, 1963 Psilotrema castoris Orlov, 1946 Psilotrema marki Skvorzov, 1934 Psilotrema medioporum Oshmarin, 1963 Psilotrema oligoon (von Linstow, 1887) Psilotrema pharyngeatum Grabda 1954 Psilotrema spiculigerum (Muhling, 1989) Psilotrema tuberculatum, (de Filippi, 1857) Psilotrema zibethicum Varenov, 1965

Investigated species of the genus Psilotrema (photo by V. V Investigated species of the genus Psilotrema (photo by V.V.Besprozvannykh) ~ 1,5 mm ~ 0,55 mm ~ 1,25 mm P. oschmarini P. acutirostris P. simillimum

28S rDNA second structure of Gydrodactulus salaris, Monogenea (from Matejusova and Cunningham, 2004) D1-D3 lenght ~ 1200-1500 bp D3 D3 D3 D3 In this study D2 and a part of D3 variable donains of 28S rRNA gene are used. D1 D1 D1 D1 D2 D2 D2 D1 D2

Aim: To investigate phylogenetic relationships of the genus Psilotrema and family Psilostomatidae within order Echinostomida using partial sequence of 28S rRNA gene

Tasks: Optimizing of DNA extraction and PCR technique for trematodes; Sequencing 28S rRNA gene fragment of three Psilotrema species; Reconstruction of phylogenetic relationships of the genus Psilotrema and the family Psilostomatidae using current data and data from GenBank.

Material Methods DNA extraction Snail Phenol-chlorophorm extraction with Proteinase K lysis Proteinase K lysis with following boiling with Chelex-100 Boiling with Chelex-100 DNA extraction with Silica Fast DNA extraction kit, Q-Biogene isolation Cercaria encyst Adolescaria infecting Chicken PCR and DNA sequence performed with ABI GeneAmp 2700 thermal cycler and ABI 3130 genetic analyzer in the IBSS FEB RAS after 8 days Adults

Echinostomatidae (n=5) Families of the order Echinostomida, used in preset study (n – number of sequences) Psilostomatidae (n=11) Fasciolidae (n=7) Echinostomatidae (n=5) Phylpthalmidae (n=2) Cyclocoeliidae (n=1)

28S rRNA gene fragment variation within Echinostomida Overall alignment (with gaps) Overall alignment (without gaps) Variable Parsimony-informative P.oschmarini 759 P.oschmarini+P.simillimum 14 P.oschmarini+P.acutirostris 13 Psilotrema 12 Fasciola 22 2 Psilostomatidae 760 759 – 760 75 Fasciolidae 758 - 759 79 24 Echinostomatidae 758 64 49 Echinostomida 764 757 – 760 264 181

Nucleotide composition of the 28S r RNA gene fragment of the Psilotrema species T(U) G C P.oschmarini 18.5 26.5 33.0 22.0 P.acutirostris 18.4 26.4 33.2 P.simillimum 26.3 22.1 Psilostomatidae 33.1 Echinostomida 18.2 26.2 33.3 22.4

Genetic p-distances between families of Echinostomida P.oschmarini P.acutirostris P.simillimum Psilostomatidae Echinostomatidae Fasciolidae Phylopthalmidae Cyclocoeliidae Dicrocoeliidae 0.017 0.001 0.1 0.119 0.089 0.125 0.12 0.117 0.168 0.154 0.15 0.155 0.177 0.187 0.199 0.193 0.22

Maximum parsimony tree of the order Echinostomida (tree topology with 50% frequency) Fasciolidae - Cyclocoeliidae Echinostomatidae Philopthalmidae Psilostomatidae Outgroup

Neighbor-Joining tree (model TrN) of the order Echinostomida (tree topology with 30% frequency) Fasciolidae Echinostomatidae - Cyclocoeliidae Psilostomatidae Philopthalmidae Outgroup

UPGMA tree (model TrN) of the order Echinostomida (tree topology with 20% frequency) Fasciolidae Echinostomatidae Psilostomatidae Philopthalmidae - Cyclocoeliidae Outgroup

Maximum parsimony tree of the genus Psilotrema P.oschmarini P.acutirostris + P.simillimum Neighbor-Joining tree of the genus Psilotrema P.oschmarini P.acutirostris + P.simillimum

Conclusions The present study shows that given the current data, family Psilostomatidae is monophyletic and possible an ancient taxon for the order Echinostomida; Psilotrema acutirostris and Psilotrema simillimum are likely more closely related to each other, then to P. oschmarini; Psilochasmus oxyurus is an ancient taxon relative to studied species of the genus Psilotrema; There is no intraspecific variation of the 28S rDNA variable domains between specimens of P. oschmarini, which originated from one miracidium of previous generation; however, further studies required using other molecular markers, including mitochondrial DNA.

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