1. 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. 2 Subclasses: Aspidogastrea and Digenea
Class Trematoda:
2 Subclasses: Aspidogastrea and Digenea
12 Orders
~ 90 Families
~ 7000 Species

3. 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

4. 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

5. Molecular genetic investigations, includes species of the family Psilostomatidae
Psilochasmus oxyurus - Tkach,V., Pawlowski,J. and Mariaux,J 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oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths // Unpublished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 A molecular phylogenetic study of the genus Ribeiroia (Digenea): trematodes known to cause limb malformations in amphibians // J. Parasitol. 91 (5),

6. 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

7. 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

8. 28S rDNA second structure of Gydrodactulus salaris, Monogenea (from Matejusova and Cunningham, 2004)
D1-D3 lenght ~ 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

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

10. 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.

11. 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

12. 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)

13. 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 79 24
Echinostomatidae
758 64 49
Echinostomida
764
757 – 760
264
181

14. 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

15. 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

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

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

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

19. 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

20. 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.

21. THANKS FOR YOUR ATTENTION!