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

Parus bucharensis Pseudopodoces humilus Podoces biddulphi

J.Avise et al 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 the principles and processes governing the geographical distributions of genealogical lineages, especially those within and among closely related taxa”

Step 1 DNA - extraction sampling  PCR Sequencing

Step 2 alignment Tree construction Phylogenetic analysis and

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

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

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

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

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

“Pitfalls” 1.Poor statistical support 2.Misrepresentative and unequal sampling in relation to species range 3.Inadequate molecular marker At the stage of tree construction 1. Introgression 2.Ancestral polymorphism and incomplete lineage sorting At the stage of tree interpretation

Clusters with high support кластеры Unsupported clusters ML cyt b tree for the genus Microtus (after Jaarola et al, 2004)

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

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

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

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).

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

Saturation Genetic divergence frequency

Cyt b tree for the closely related species in Clethrionomyini With an account of all substitutions АБ Without transitions ti3

Saturation Genetic divergence frequency

16 new species in the g.Plecotus 106 specimens, control region of mtDNA, 150 bp; Spitzenberger et al. Revision of the genus Plecotus Zoologica Scripta, 35,3, 2006

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

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

Correlation between gene and population/species genealogies

Introgression Cl.glareolus Cl.rutilus

Cyt b; NJ Cl.glareolus Cl.rutilus

Cl.rutilus Cl. glareolus 4 Median-joining net of haplotypes of Cl.glareolus and Cl.rutilus

The scale and geographic zone of introgression

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.

Вaker & Bradley, 2006: Genetic species concept and speciation in mammals speciesNphylogroups  5% Cryptic species Geomys bursarius 1043 G.personatus1354 Peromyscus pectoralis 1021 Reithrodontomy s microdon species

Tree inferred from mtDNA cytb Between populations M.gregalis – 14% Between subgenera С.ruf. - C. rutilus – 10%

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

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.

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

Thank you for attention =)

Acknowledgements Petrova T.V. Bodrov S.Yu. Rodchenkova E.N. Support from BOE and RFBR