Abramson N.I., Kostygov A.Yu . Zoological Institute RAS, Sankt—Petersburg, Russia

1. APPLICATION OF MOLECULAR MARKERS IN THE STUDIES OF PHYLOGENY AND PHYLOGEOGRAPHY: ADVANCES, PITFALLS AND PERSPECTIVES OF DEVELOPMENT Abramson N.I., Kostygov A.Yu. Zoological Institute RAS, Sankt—Petersburg, Russia

2. Parus bucharensis Pseudopodoces humilus Podoces biddulphi

4. J.Avise et al.1987. Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics • Molecular Ecology, 1998, vol.7, No 4 “Phylogeography is a field of study concerned with theprinciples and processes governing the geographical distributionsof genealogical lineages, especially those withinand among closely related taxa”

6. Step 1 sampling DNA - extraction • PCR Sequencing

7. Step 2 alignment Phylogenetic analysis and Tree construction

8. The topology of the tree serves to deduce phylogeny taxonomy Evolutionary scenario Reconstruction of refugia and colonization history

9. Isolation of populations in different refugia andintraspecific differentiation periodical shifts of range borders and mixing of formerly isolated populations - Pleistocene glaciations Some key phenomena are: speciation by vicariance and hybridization, the roles of dispersal, founder effects, range expansions and secondary contacts in structuring population genetics

10. Evolutionary scenario • Reconstruction of refugia and colonization history • Reconstruction of demographic histories Haplotype diversity – Hd Nucleotide diversity - 

11. Mismatch distribution of pairwise differences Nucleotide diversity is lowhaplotype -high Recent population growth and expansion after botlleneck Nucleotide diversity is high Haplotype - high Relatively stable population number over time, no signs of “Founder effects”

12. The topology of the tree serves to deduce phylogeny taxonomy Evolutionary scenario Reconstruction of refugia and colonization history

13. “Pitfalls” At the stage of tree construction • Poor statistical support • Misrepresentative and unequal sampling in relation to species range • Inadequate molecular marker At the stage of tree interpretation • Introgression • Ancestral polymorphism and incomplete lineage sorting

14. MLcyt b tree for the genus Microtus(afterJaarola et al, 2004) Unsupported clusters Clusters with high support кластеры

15. ML cyt b tree for tribe Clethrionomyini (red-backed voles) Lebedev V.S., Banniikova A.A., Tesakov A.S. & Abramson N.I., 2007

16. 2. Misrepresentative and unequal sampling in relation to species range Cook et al., 2004

17. Cyt b tree for the species of the g. Clethrionomys s. stricto Triangle square is proportional to the number of sequenced specimens Support of species clusters highly increased but!!! The phylogenetic structure totally unresolved

18. A case of phylogeographic study with extremely unequal sampling in relation to species range (Brunhoff et al.,.2003.) Grey color designate the range of a tundra vole (Microtus oeconomus).

19. 3.Inadequate molecular marker • 18 rRNA; ITSI & ITS2 • mtDNA (cyt b; COXI; control region) applicability of molecular marker to the recovery of phylogeny at a particular taxonomic level

20. Saturation frequency Genetic divergence

21. Cyt b tree for the closely related species in Clethrionomyini А Б Without transitions ti3 With an account of all substitutions

22. Saturation frequency Genetic divergence

23. Spitzenberger et al. Revision of the genus Plecotus Zoologica Scripta, 35,3, 2006 16 new species in the g.Plecotus106 specimens, control region of mtDNA, 150 bp;

24. 106 specimens, CR-150 bp Extremely high variability plus short fragment of analysed sequence and small samples oversized values of divergence

25. Criteria for choice of molecular marker • Enough number of informative sites in relation to the taxonomic level • Low level of homoplasy (saturation) • Relatively equal rate of evolution within the group under study

26. Correlation between gene and population/speciesgenealogies

27. Introgression Cl.rutilus Cl.glareolus

28. Cyt b; NJ Cl.glareolus Cl.rutilus

29. Median-joining net of haplotypes of Cl.glareolusandCl.rutilus 4 15 7 3 2 2 5 13 6 3 2 4 2 4 59 2 2 2 2 3 2 3 2 Cl.rutilus Cl. glareolus Cl.glareolus

30. The scale and geographic zone of introgression

31. Universal species criteria? • Genetic distance –universal and operational • Independent from assumptions on speciation and species concept (Ayala, 1975 ) Avise, Johns, 1999: - cyt b –in vertebrates evolves approximately with an equal rate; the divergence in 13% correspond to interspecies level; below 13% - to intraspecies. Thus, universal criteria and universal tool for the identification of species boundaries.

32. Вaker & Bradley, 2006: Genetic species concept and speciation in mammals

33. Tree inferred from mtDNAcytb Between populationsM.gregalis – 14% Between subgeneraС.ruf. - C. rutilus – 10%

34. Tree inferred from nuclear geneLCAT (ML) Between populations ofM.gregalis – 0,7 – 0,1% M.gregalis –Ch. nivalis -3.3% C. ruf. - C. glar. 3.7%

35. General conclusions • The major pitfalls in application of molecular markers to phylogenetic reconstructions are very similar to those that classical morphology and phylogenetics dealt with earlier.

36. «there is no unerring criteria permitting one to distinguish species and well pronounced varieties

37. Thank you for attention =)

38. Acknowledgements • Petrova T.V. • Bodrov S.Yu. • Rodchenkova E.N. • Support from BOE and RFBR 06-04-49294