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

Step 1 60

40

20

0

60 100 140180

Владивосток

Южно-Сахалинск

Магадан

Анадырь

Екатеринбург

Эльген

DNA - extractionDNA - extraction

sampling

PCRPCR

SequencingSequencing

Step 2alignment

Tree construction

Phylogenetic analysis and

The topology of the tree serves to deduce

phylogeny

taxonomy

Evolutionary scenarioReconstruction of

refugiaand 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 – HdNucleotide diversity -

Nucleotide diversity is lowNucleotide diversity is low haplotype -highhaplotype -high

Mismatch distribution of pairwise differences

Recent population growth and expansion after botlleneck

Nucleotide diversity is highNucleotide diversity is high Haplotype - highHaplotype - high

Relatively stable population number over time, no signs of

“Founder effects”

The topology of the tree serves to deduce

phylogeny

taxonomy

Evolutionary scenarioReconstruction of

refugiaand colonization

history

“Pitfalls”

1. Poor statistical support2. Misrepresentative and unequal sampling in relation

to species range3. Inadequate molecular marker

At the stage of tree construction

1. Introgression2. Ancestral polymorphism and incomplete lineage

sorting

At the stage of tree interpretation

Clusters with high supportкластеры

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

freq

uenc

y

Cyt b tree for the closely related species in Clethrionomyini

With an account of all substitutions

А Б

Without transitions ti3

Saturation

Genetic divergence

freq

uenc

y

16 new species in the g.Plecotus106 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

303 Cl.gl. Nov Uz 277 Cl.gl. Sverdlov Shigaevo 125 Cl Gl Komi Shej Pech Cl gl isolate CG16H Novosib Chulymskii Cl gl UAM30029 Finland Sotkomo Cl gl isolate CG216 Omsk Cl gl isolate CG315 Omsk 118 Cl gl Arch Koslan 296 Cl.gl. Sverdl Visim. Cl gl isolate CG467 Omsk 200 Cl gl Valaam Cl gl isolate CG215 Omsk 67 Cl.gl. Vlad Tas 67 Cl.gl. Vlad Tas(2) Cl gl AJ639708 Bialowieza 180 Cl.Gl. Kursh 169 Cl.gl. Tver Cl gl isolate CG45NN NNovg 73 Cl gl Komi Dan 116 Cl gl Arch Koslan 124 Cl gl Komi Stor 70 Clgl Komi Pri 131 Cl Gl Komi Stor 174 Cl.gl. Len-Prim 307 Cl.gl. Pul 416 Cl gl Len Volos Milod 4 Cl.gl. Len Lug Zel 198 Cl.gl. Pskov 191 Cl.gl. Len Box 206 Cl gl Pskov Cl gl isolate CG16S Samara 456 Cl gl Len Tich 425 Cl gl Len Volos 352 Cl.gl. Len Vol 411 Cl gl Vologod 193 Cl.gl. Len-Lug Kur Cl gl UAM24464 Wales Cl gl Ger Konstanz Cl rut IK55 96 Magadan 288 Cl rut Sverd Hom30 07 - UCBU Cl rut Cl rut NVBS417 01 Nobosibirsk 143 Cl rut Arch Kob Cl gl isolate CG40B Baskortostan Blagov Cl gl AJ639673 Ural 333 Cl gl Sverd Hom1 336 Cl gl Sverd Hom30 134 Cl.rut. Komi Dan sol4 Cl.gl. Solov 60 Cl.rut. Arch Chadroma Cl rut isolate CRT481 Omsk Cl rut AF3151 Finl Kilpis 189 Cl.gl. Orenb. 149 Cl.rut. Arch Podg. 347 Cl rut Sverd Visim 218 Cl.rut. Komi Storoch. 300 Cl gl Sverd Visim 228 Cl.gl. Kar Pes 256 Cl.gl. Kar Ker P5 Cl gl White sea 255 Cl.gl. Kar Sred 251 Cl.gl. Kar Cher 71 Cl.gl. Arkh Vosh 84 Cl.gl Arkh Podg 82-Cl.gl. Arkh-Kob 221 Cl.gl. Kar Kar 155 Cl gl Murm Zap 152 Cl Gl Murm Kar 2 46 Cl ruf Mur Kol

64

56

63

51

93

100

8794

100

62

55

79

62

62

58

Cyt b; NJ

Cl.glareolus

Cl.rutilus

7

13

22

3

59

2

2

2

3

3

2

6

15

2

2

22

44

5

3

Cl.rutilus

Cl. glareolus

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

species N phylogroups

5%

Cryptic species

Geomys bursarius

10 4 3

G.personatus 13 5 4

Peromyscus

pectoralis

10 2 1

Reithrodontomys

microdon

7 3 2

61 species

718 87 55

Tree inferred from mtDNA cytb

rClethrionomys utilus

C.gapperi

C.californicus

C.glareolus

C.rex

C.rufocanus

M kikuchiicrotus i

M. Оecon Netomus

M.oeconCan

M.oeconRus

M.fortis

ChitaM.gregalis

NovM.gregalis

M.gregalis Nov

M.gregalis Yamal

MgAF163895

M.greg h 1

AltaiM.gregalis

MongM.gregalis olia

M.gregalis Buryatia

M.hyperboreus

M.middendorfi

M.agr Rusestis

M.guentheri

M.socialis

M.kirgisorum

M.rossiaemeridionalis

M.arvalis

M.thomasi

M.gerbei

M.subterraneus

0.05

Between populations M.gregalis – 14%

Between subgenera С.ruf. - C. rutilus – 10%

Tree inferred from nuclear gene LCAT (ML) Ak torquesodon

P manicueromiscus latus

Chionomys nivalis

A.macrotis Kazachstan

0.01

ChitaM.gregalis

MongM.gregalis olia

M.gregalis Buryatia

NovM.gregalis

NovM.gregalis

AltaiM.gregalis

M.gregalis Yamal

M.оeconomus

M.оeconomus

M.оeconomus

M.оeconomus

Clethrionomys rufocanus

C.glareolus

A.macrotis Altai

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

Thank you for

attention

=)

Acknowledgements

• Petrova T.V.

• Bodrov S.Yu.

• Rodchenkova E.N.

• Support from BOE and RFBR 06-04-49294