This article was downloaded by: [University of Patras]
On: 12 September 2012, At: 01:52
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Italian Journal of Zoology
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tizo20
Comparative phylogeography of the five Greek vole
species infers the existence of multiple South Balkan
subrefugia
a
a
a
E. Thanou , G. Tryfonopoulos , B. Chondropoulos & S. Fraguedakis-Tsolis
a
a
Section of Animal Biology, Department of Biology, University of Patras, Patras, Greece
Version of record first published: 07 Feb 2012.
To cite this article: E. Thanou, G. Tryfonopoulos, B. Chondropoulos & S. Fraguedakis-Tsolis (2012): Comparative
phylogeography of the five Greek vole species infers the existence of multiple South Balkan subrefugia, Italian Journal of
Zoology, 79:3, 363-376
To link to this article: http://dx.doi.org/10.1080/11250003.2011.651163
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to
anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contents
will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should
be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,
proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in
connection with or arising out of the use of this material.
Italian Journal of Zoology, September 2012; 79(3): 363–376
Comparative phylogeography of the five Greek vole species infers
the existence of multiple South Balkan subrefugia
E. THANOU*, G. TRYFONOPOULOS, B. CHONDROPOULOS,
& S. FRAGUEDAKIS-TSOLIS
Section of Animal Biology, Department of Biology, University of Patras, Patras, Greece
Downloaded by [University of Patras] at 01:52 12 September 2012
(Received 15 May 2011; accepted 6 December 2011)
Abstract
Despite extensive phylogenetic studies in the genus Microtus, several of its species have not been thoroughly evaluated.
The present study focuses on a cytb molecular analysis of five vole species occurring in the southern Balkan Peninsula
(M. felteni, M. thomasi, M. subterraneus, M. levis and M. guentheri), aiming to demonstrate the importance of thorough
intraspecific sampling when the phylogeny of closely related taxa is tested. As a result, the Balkan populations of these
voles showed significant intraspecific variation that distinguishes them from other European and Asian conspecifics and in
some cases reveals distinct lineages even within the Balkan region. Their complex phylogeography suggest the existence of
multiple subrefugia, located within the southern Balkan region, which promoted diversification of these voles during the
Middle and Late Pleistocene glacial periods. The significant role of the southern Balkan Peninsula’s paleogeographical and
paleoclimatical characteristics in these small mammals’ evolution is discussed.
Keywords: Mediterranean Peninsula, Balkan subrefugia, Microtus species, glacial/interglacial cycles, paleogeography
Introduction
Populations distributed at the borders of a species’
distributional range, as those of the Mediterranean
Peninsulas, can easily be isolated or decreased in
abundance, and might show morphological and ecological adaptations because of genetic isolation and
local climatic conditions. Many of the species currently distributed in these peninsulas have experienced severe events (population fragmentation, bottleneck, etc) during the Pleistocene climatic cycles.
Their intraspecific phylogenies imply these events but
also reveal the existence of several southern refugia in the Mediterranean area from which isolated
populations recolonized northern Europe (Hewitt
2001). The Balkan Peninsula, being at the southeastern margin of Europe, is a natural border for
European populations but also a biogeographical
bridge between Asia and Europe, and bears specific
paleogeographical and paleoclimatical characteristics that could play an important role to species’
evolution. Especially during the Pleistocene, the geomorphology of this area was repeatedly altered by
eustatic movements of the sea level that were driven
by climatic change, from cold/dry to warm/humid
conditions, through the glacial/interglacial periods
(Perissoratis & Conispoliatis 2003).
The Mediterranean Peninsulas have acted as
“speciation traps”, concentrating endemics, but also
promoted intraspecific divergence. For example,
Mediterranean populations of the pygmy shrew and
the bank vole (Bilton et al. 1998; Deffontaine et al.
2005; Vega et al. 2010) show little similarity with
their conspecifics further north as well as among
each other, suggesting that the current phylogeographical profile of these species may be explained
by the assumption of multiple Mediterranean refugia. The Italian and the Balkan Peninsulas have
provided many examples of genetically differentiated
lineages within several widely distributed species,
such as the yellow-necked fieldmouse (Michaux et al.
2005), the lesser white-toothed shrew (Dubey et al.
2007a), the European ground squirrel (Kryštufek
et al. 2009a) and the common vole (Bužan et al.
2010). In the Balkan Peninsula and particularly its
*Correspondence: E. Thanou, Section of Animal Biology, Department of Biology, University of Patras, GR 26501 Patras, Greece. Tel: +30 2610 969237. Fax:
+30 2610 969218. Email: [email protected]
ISSN 1125-0003 print/ISSN 1748-5851 online © 2012 Unione Zoologica Italiana
http://dx.doi.org/10.1080/11250003.2011.651163
Downloaded by [University of Patras] at 01:52 12 September 2012
364
E. Thanou et al.
southernmost areas, such as Greece, many rodents
show higher morphological and/or genetic variation, when Greek populations are compared to
their northern European conspecifics. Such examples have been presented in immunological studies on Spermophilus citellus (Fraguedakis-Tsolis &
Ondrias 1985) and molecular ones on Mus spicilegus
(Mitsainas et al. 2009).
A recent study (Fraguedakis-Tsolis et al. 2009)
revealed distinct morphological profiles in Greek
voles: Greek M. guentheri, M. levis and M. thomasi
are distinctively smaller in size and differ in skull
measures from other European populations, while
high morphological variation exists among Greek
populations of M. thomasi, as well as M. subterraneus. Moreover, a molecular analysis on Greek
M. thomasi populations (Tryfonopoulos et al. 2008)
showed a high degree of intraspecific differentiation
and argued in favour of a new cryptic subspecies distributed in SE Central Greece, namely M. thomasi
atticus.
Recently, the phylogenetic relationships between
all currently recognized European Microtus species
were inferred, with the analysis of the cytochrome
b (cytb) mitochondrial marker (Jaarola et al. 2004).
This analysis focused mainly on the relationships
between subgenera and species, including adequate
specimens to represent all the respective taxa but with
relatively few individuals and populations per species.
This resulted in a poor representation of geographical
intraspecific variation, especially from the Balkans.
Even so, their results also revealed a wide range of
molecular variation within European species of the
genus, some of them being much more polymorphic
than others, in concordance with previous studies of American Microtus species (Conroy & Cook
2000).
It is widely accepted that the genus Microtus,
despite its recent evolutionary history and morphological homogeneity (Musser & Carleton 2005)
probably includes many hitherto unidentified cryptic
species (Graf 1982; Hellborg et al. 2005) that only
detailed within-species sampling could uncover. This
is shown in extensive phylogeographic surveys on
several vole species that revealed intraspecific diversification and complicated phylogenetic relationships
between isolated populations (Hellborg et al. 2005;
Piertney et al. 2005; Tougard et al. 2008). Detailed
within-species sampling and subsequent molecular
analyses have also unraveled a complex pattern of
intraspecific divergence that seems to be related
to the role of Mediterranean Peninsulas as glacial
refugia, but mostly to the existence of subrefugia
within them (Castiglia et al. 2008; Centeno-Cuadros
et al. 2009). Such phylogeographic patterns within
refugia are only recently addressed in Iberia and Italy,
whether respective data for the Balkans are still very
limited and refer to the northern parts of the Balkan
Peninsula (Bužan et al. 2010).
In this context, we focused on the five vole
species living in Greece: Microtus guentheri (Danford
& Alston 1880), M. levis Miller 1908 (previously
named M. rossiaemeridionalis Ognev 1924, see Wilson
& Reeder (2005) for nomenclature review of the
species), M. subterraneus (de Sélys Longchamps
1836), M. felteni (Malec & Storch 1963) and
M. thomasi Barrett-Hamilton 1903. The latter two
species are endemics to the SW Balkans, while all of
them show their southernmost European distribution
limit in Greece (Mitchell-Jones et al. 1999).
Cytb analysis was used in this study to measure the
genetic variation among Greek populations of each
of the studied species. Our data were subsequently
compared to those available in the literature, aiming
to investigate the differentiation of Greek voles compared to previously studied populations from other
areas of each species’ distributional range. In this
sense, we aim to explore the role of the Balkan
Peninsula as a refugium for ancestral vole populations and the possible existence of subrefugia from
which subsequent colonization of northern parts of
Europe has occurred. During this study, extensive
sampling was considered as a priority in order to
reveal within-species cryptic variation and provide
new data on each species’ geographical distribution
in Greece, which is not yet sufficiently known.
Materials and methods
Sampling
A total of 99 specimens, belonging to the five
Microtus species distributed in Greece, were used
(Figure 1, Table I). M. thomasi, was represented in
this study both as the nominal subspecies and as the
subpecies M. thomasi atticus. Twenty one previously
published sequences of this species (Tryfonopoulos
et al. 2008) were updated by adding one M. thomasi
thomasi from Albania, and five M. thomasi atticus from new localities which extend its previously known distribution. Moreover, five M. felteni,
16 M. subterraneus, 20 M. levis and 31 M. guentheri were sequenced. Additional sequences retrieved
from GenBank were also included in the analyses (Table I), representing populations of these five
Microtus species from other European locations, as
well as 19 other Microtus species (Table I), in order
to fully represent all the Eurasian voles that are considered closely related to the five species studied
(Jaarola et al. 2004; Kryštufek et al. 2009b). In this
Downloaded by [University of Patras] at 01:52 12 September 2012
Vole phylogeography infers multiple Balkan subrefugia
365
Figure 1. Approximate distribution of the five Microtus species living in Greece. Collection sites of individuals sequenced in this study and
their respective numbers are given in Table I.
sense, our analysis includes all recognized species
and all distinct mtDNA lineages placed within the
“arvalis” (M. arvalis “obscurus”, M. arvalis “arvalis”
and M. levis) and the “socialis” species groups
(M. socialis, M. irani, M. anatolicus, M. dogramacii,
and the “west” and “south” lineages of M. guentheri) and most of the European endemics, according to these previous studies. Chionomis nivalis was
considered to be the best fitting outgroup since
it represents the basal clade to all the Palearctic
species belonging to the genus Microtus (Jaarola et al.
2004).
DNA extraction, PCR amplification and sequencing
DNA was extracted from liver tissue, preserved
®
at -80 ◦ C, using the Invisorb Spin Tissue kit
(Invitek, Germany). A segment of the mitochondrial
cytb gene was PCR-amplified using the set of
primers and PCR conditions previously described in
Tryfonopoulos et al. (2008).
PCR products were visualized via agarose gel
®
electrophoresis, purified with the Invisorb Spin
PCRapid kit (Invitek, Germany) and the light strand
®
was sequenced on an ABI PRISM 3100 capillary
sequencer (VBC Biotech, Austria) using the primers
of the amplification procedure.
All sequences obtained in this study have been
deposited in GenBank and the respective Accession
Numbers are given in Table I.
Sequence data analyses
Sequences were aligned using CLUSTAL-W v1.4
(Thompson et al. 1994) in the BIOEDIT Sequence
Alignment Editor v5.0.9 (Hall 1999). Genetic divergence (uncorrected p-distances) was estimated in
MEGA v3.1 (Kumar et al. 2004), as the number of nucleotide substitutions per site between and
within populations, as well as net between populations’ genetic distances, after excluding the respective within - values of each of the two populations compared. Populations of each species were
geographically grouped, according to their Greek,
European or Asian origin, and net p-distances among
these groups were also calculated. The saturation
of phylogenetic information was examined using the
Xia’s test employed in DAmBE v4.5.40 (Xia 2000;
Xia & Xie 2001).
MODELTEST v3.06 (Posada & Crandall 1998)
was used to determine the best substitution model
for the Bayesian Inference (BI) analysis and
Maximum Likelihood (ML) analysis, under the
Akaike Information Criterion (AIC). Subsequently,
BI analysis was implemented using MrBayes
v3.1 (Ronquist & Huelsenbeck 2003), under the
GTR+I+G model. Four chains were run for three
million generations, sampling trees every 100th generation and “burn-in” data representing 25% of
early generations (7.5x104 trees) were discarded.
Three simultaneous and independent runs were conducted and inspected for consistency to check for
366
E. Thanou et al.
Table I. Microtus species, number of specimens, locations (site and country), codes corresponding to all sequences included in the present
study as they appear in the respective tree (Figure 2) and GenBank accession numbers. Sequences retrieved from GenBank, are marked
with an asterisk (∗ ). Localities in bold refer to new findings for the respective species.
Species
Site (as in Figure 1)
Country
Code
Ass. Nos
3
Agios Stefanos (1)
Greece
2
Afidnes (2)
Greece
1
1
1
1
1
3
Marathonas (3)
Fyli (4)
Oropos (5)
Avlona (6)
Skourta (7)
Styra, Evvoia (8)
Greece
Greece
Greece
Greece
Greece
Greece
2
Pogontas, Evvoia (9)
Greece
3
Lake Dystos, Evvoia (10)
Greece
2
Oraioi, Evvoia (11)
Greece
M. thomasi thomasi
1
1
1
1
1
1
1
M. felteni
1
3
Mt. Panachaiko (12)
Astakos (13)
Elati (14)
Balbouma (15)
Mikrokastro (16)
Mt Pieria (17)
Sarantë
Trebinje
Seli (31)
Pisoderi (32)
Greece
Greece
Greece
Greece
Greece
Greece
Albania
Bosnia
Greece
Greece
1
1
4
Nymfaio (38)
Begova Češma, Mt Pelister
Seli (31)
Greece
FYROM
Greece
1
4
Pisoderi (32)
Kato Vermio (33)
Greece
Greece
1
3
Dobro Pole, Mt Vorras (34)
Mt Pinovo (35)
Greece
Greece
1
2
Lailias, Mt Vrontous (36)
Mt Silo (37)
Greece
Greece
1
1
1
1
4
Val Piora
Pilatus
Çiğlikara
Güzyurdu
Kato Nevrokopi (27)
Switzerland
Switzerland
Turkey
Turkey
Greece
GR1.1
GR1.2
GR1.3
GR2.1
GR2.2
GR3
GR4
GR5
GR6
GR7
GR8.1
GR8.2
GR8.3
GR9.1
GR9.2
GR10.1
GR10.2
GR10.3
GR11.1
GR11.2
GR12
GR13
GR14
GR15
GR16
GR17
ALBANIA
BOSNIA
GR31.1
GR32.1
GR32.2
GR32.3
GR38
FYROM
GR31.2
GR31.3
GR31.4
GR31.5
GR32.4
GR33.1
GR33.2
GR33.3
GR33.4
GR34
GR35.1
GR35.2
GR35.3
GR36
GR37.1
GR37.2
SWITZERLAND1
SWITZERLAND2
TURKEY1
TURKEY2
GR27.1
GR27.2
GR27.3
GR27.4
EF666504∗
EF666505∗
EF666506∗
EF666510∗
EF666511∗
JN650051
JN650052
JN650053
JN650054
JN674457
EF666590∗
EF666591∗
EF666592∗
EF666588∗
EF666589∗
EF666585∗
EF666586∗
EF666587∗
EF666583∗
EF666584∗
EF666524∗
EF666536∗
EF666522∗
EF666497∗
EF666518∗
EF666949∗
JN650055
AY513844∗
FJ641181
FJ641178
FJ641179
FJ641180
JN650050
AY513798∗
FJ641162
FJ641163
FJ641164
FJ641165
FJ641173
FJ641166
FJ641167
FJ641168
FJ641169
FJ641174
FJ641179
FJ641171
FJ641172
FJ641175
FJ641176
FJ641177
AJ717745∗
AY332714∗
AY513834∗
AY513836∗
FJ641158
FJ641159
FJ641160
FJ641161
Downloaded by [University of Patras] at 01:52 12 September 2012
Microtus thomasi
atticus
M. subterraneus
M. levis
No
(Continued)
Vole phylogeography infers multiple Balkan subrefugia
367
Table I. (Continued).
Downloaded by [University of Patras] at 01:52 12 September 2012
Species
M. guentheri “west”
No
Site (as in Figure 1)
Country
Code
Ass. Nos
1
4
Olynthos (28)
Sevastiana (29)
Greece
Greece
4
Arnissa (30)
Greece
2
Seli (31)
Greece
3
Pisoderi (32)
Greece
2
Kato Vermio (33)
Greece
1
1
1
1
1
1
1
4
Kiev
Chernobyl
Kauhava
Svalbard
Istanbul
Mt Erciyes
Kangal-Sivas
Lake Yliki (18)
Ukraine
Bellarus
Finland
Norway
Turkey
Turkey
Turkey
Greece
4
Gravia (19)
Greece
2
Karyes (20)
Greece
4
Neo Monastiri (21)
Greece
4
Sykourio (22)
Greece
3
Amyntaio (23)
Greece
3
Paranesti (24)
Greece
4
Dadia (25)
Greece
3
Mikro Dereio (26)
Greece
1
2
Bitola
Kirşehir
FYROM
Turkey
Elmah
Turkey
GR28
GR29.1
GR29.2
GR29.3
GR29.4
GR30.1
GR30.2
GR30.3
GR30.4
GR31.6
GR31.7
GR32.5
GR32.6
GR32.7
GR33.5
GR33.6
UKRAINE
BELLARUS
FINLAND
NORWAY
TURKEY3
TURKEY4
TURKEY5
GR18.1
GR18.2
GR18.3
GR18.4
GR19.1
GR19.2
GR19.3
GR19.4
GR20.1
GR20.2
GR21.1
GR21.2
GR21.3
GR21.4
GR22.1
GR22.2
GR22.3
GR22.4
GR23.1
GR23.2
GR23.3
GR24.1
GR24.2
GR24.3
GR25.1
GR25.2
GR25.3
GR25.4
GR26.1
GR26.2
GR26.3
FYROM
Turkey GW3
Turkey GW4
Turkey GW5
FJ641157
FJ641142
FJ641143
FJ641144
FJ641145
FJ641146
FJ641147
FJ641148
FJ641149
FJ641150
FJ641153
FJ641154
FJ641155
FJ641156
FJ641151
FJ641152
DQ015676∗
U54495∗
AY513819∗
AY513820∗
AY513821∗
AY513822∗
AY513823∗
FJ641117
FJ641118
FJ641119
FJ641120
FJ641129
FJ641130
FJ641131
FJ641132
FJ641137
FJ641138
FJ641121
FJ641122
FJ641123
FJ641124
FJ641110
FJ641111
FJ641112
FJ641113
FJ641114
FJ641115
FJ641116
FJ641133
FJ641134
FJ641136
FJ641125
FJ641126
FJ641127
FJ641128
FJ641139
FJ641140
FJ641141
FJ767744∗
FJ767745∗
FJ767746∗
FJ767747∗
(Continued)
368
E. Thanou et al.
Table I. (Continued).
Species
Site (as in Figure 1)
Country
Code
Ass. Nos
2
Kortuteli
Turkey
Turkey GW6
Turkey GW7
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
Radi’ah
Aqrabat
unknown
unknown
Brandenburg
Mazarsay
Balkash
Brandenburg
Trendo
Schötz
Lauwersee
Hjerl Hede
Bialystok
Nuijamaa
Lagny
Mantet, Pyrenees
Crimea
Kavka River, Serov
Sisian
Ninotsminda
Iori River Valley
Syria
Syria
Israel
Israel
Germany
Kyrgyzstan
Kyrgyzstan
Germany
Italy
Switzerland
Netherlands
Denmark
Poland
Finland
France
Spain
Ukraine
Russia
Armenia
Georgia
Georgia
M. anatolicus
1
1
1
1
3
Reine
Ortaköy - Aksaray
Amasya
Boyali Köyü - Amasya
Cihanbeyli
Iran
Turkey
Turkey
Turkey
Turkey
M. irani
3
Balkusan
Turkey
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Shiraz
Damar
Beniani
Bağdaşan
Trendo
Fusio
Tretie Roháčske
Smutná dolina valley
Steinberg am Rofan
unknown
Viterbo
Torino, Piedmont
Cerano, Piedmont
Fiume, Freddo
Burgos
Melgar de Fernamental
Setùbal
Algarve
Arrós, Vall d’Aran
Riba
Hecho
Trento
Pilatus
Iran
Turkey
Georgia
Turkey
Italy
Switzerland
Slovakia
Slovakia
Austria
Croatia
Italy
Italy
Italy
Italy
Spain
Spain
Portugal
Portugal
Spain
Spain
Spain
Italy
Switzerland
FJ767751∗
FJ767752∗
FJ767743∗
AY513805∗
AY513806∗
AY513807∗
DQ768147∗
AY513808∗
AY513809∗
DQ768131∗
AY220766∗
AY332711∗
AY220778∗
AY220776∗
EU439457∗
AY220770∗
AY220787∗
AY220789∗
AY220762∗
AY220764∗
AY220761∗
AY220760∗
AY513829∗
AY513830∗
AY513831∗
AY513793∗
AY513794∗
AY513795∗
FJ767740∗
FJ767741∗
FJ767742∗
FJ767748∗
FJ767749∗
FJ767750∗
FJ767739∗
DQ841703∗
AY513790∗
AY513791∗
DQ663663∗
AY332713∗
DQ841701∗
AY513838∗
DQ841693∗
EF379100∗
AY513824∗
AY513825∗
AY513826∗
AY513827∗
AY513812∗
AY513813∗
AY513796∗
AY513797∗
AY513799∗
AY513800∗
AY513801∗
DQ663668∗
AY332716∗
M. guentheri “south”
Downloaded by [University of Patras] at 01:52 12 September 2012
M. agrestis
M. juldaschi
M kirgisorum
M. arvalis “arvalis”
M. arvalis “obscurus”
M. socialis
M. dogramacii
M. majori
M. daghestanicus
M. multiplex
M. tatricus
M. bavaricus
M. liechtensteini
M. savii
M. lusitanicus
M. duodecimcostatus
M. gerbei
Chionomys nivalis
No
Vole phylogeography infers multiple Balkan subrefugia
local optima (Huelsenbeck & Bollback 2001). All
trees collected at stationarity were used to estimate
the topology of a 50% majority rule tree and its
posterior nodal probabilities. For the ML analysis
we used the PHYML online web server (Guindon
et al. 2005) under the nearest neighbour interchange (NNI) method, with 1000 bootstrap replicates and the fixed values of gamma distributed shape
parameter (1.04) and proportion of invariable sites
(0.50) that were estimated by MODELTEST.
Results
Downloaded by [University of Patras] at 01:52 12 September 2012
Sequence composition and variation
The length of the mtDNA segment analysed was
710 bp including 286 variable and 249 parsimony
informative characters (40% and 35% of total length,
respectively). Most substitutions were transitions
(72%) and the transition to transversion ratio (R) was
0.9 for the 1st codon, 2.3 for the 2nd codon, 8.8 for
the 3rd codon, while the overall ratio was 2.5. The
majority of polymorphic sites in this segment were at
the 3rd position (27.5%), followed by 1st (5.3%) and
2nd position (0.9%).
Net uncorrected p-distances among populations of
each species, geographically grouped according to
their Greek, European or Asian origin (Table II),
ranged from 0.6% (Greek M. guentheri “west” –
Turkish M. guentheri “west”) to 1.6%, the latter estimated for the pair M. thomasi thomasi M. thomasi atticus. Values twice as high (3.0–3.3%)
were observed between Greek populations of M. subterraneus and their European or Turkish conspecifics.
Phylogenetic results
The Xia’s test for the saturation of phylogenetic
information showed that the substitution saturation
index of our sequences (Iss) is significant lower
than the critical value (Iss<Iss.c, p < 0.001). Thus
our sequences can be used for phylogenetic analyses, although some saturation occurred at the third
codon position of cytb (value of 0.160 combared to
0.148 for all three codons).
The same tree topology (Figure 2), was recovered by the BI and ML analyses regarding the major
clades, with few minor differences in some terminal branches. This tree depicted the phylogenetic
relationships between most Eurasian Microtus species
in agreement with previous studies (Jaarola et al.
2004; Kryštufek et al. 2009b). The five species this
study focuses on are placed in three distinct clades.
All species of the subgenus Terricola formed one
clade further divided in two subclades: one clustered
369
M. subterraneus together with two other species
widely distributed in Asia (M. daghestanicus and
M. majori), and the other the European endemics
of the Mediterranean peninsulas, such as M. thomasi
(including M. thomasi atticus) and M. felteni. The second major clade included the arvalis group species
(M. arvalis “arvalis”, M. arvalis “obscurus” and
M. levis), and the third major clade the socialis
group species (M. socialis, M. anatolicus, M. irani,
M. dogramacii, and the west and south clades of
M. guentheri). Both M. subterraneus and M. levis were
subdivided in three subclades that separated Greek,
other European and Turkish populations, while
within the west clade of M. guentheri, Greek populations were clustered distinctly from their Turkish
conspecifics.
Discussion
The recent application of molecular analyses to several species across Europe has revealed a similar pattern of postglacial colonization from southern refugia. In many cases, hybrid zones have been described
in the Pyrenees and the Alps and DNA sequence
analysis has revealed that these were formed by
secondary contact of distinct subspecific genetic lineages emanating from refugia in Iberia, Italy and the
Balkans. The Balkan expansion came to recolonize
most of Europe, probably due to an early start in the
east after the ice age, and because the Pyrenees and
Alps hindered the spread of Iberian and Italian lineages. Nevertheless, other lineages may be also found
to occur in Greece and Turkey, and possibly further
east in the Caucasus (Hewitt 2001).
In this sense, most small mammals in Greece
are expected to share the same genome with their
northern European conspecifics, as Greek populations probably survived during glacial periods and
expanded to the north during postglacial ones.
Indeed, all Greek populations of Crocidura leucodon
belong to the European mtDNA lineage without
exceptions (Dudey et al. 2007b). Crocidura suaveolens, on the other hand, follows the same pattern,
in general, but also includes a unique mtDNA lineage from Crete Island and another discrete common lineage distributed in the NE Aegean Island
of Lesbos and W Turkey (Dudey et al. 2007a).
Another small mammal, the bank vole, shows a
distinct phylogenetic lineage distributed in the SE
Balkans and W Turkey, while central and northern
European populations descend from refugia situated
outside the Mediterranean Peninsulas (Deffontaine
et al. 2005). These examples provide evidence of
a complex history of mammalian species expansion and colonization in the southern parts of the
[1] M. guentheri “west” – Greek
[2] M. guentheri “west” – Turkish
[3] M. guentheri “south”
[4] M. dogramacii
[5] M. anatolicus
[6] M. irani
[7] M. socialis
[8] M. levis – Greek
[9] M. levis – European
[10] M. levis – Turkish
[11] M. arvalis ‘arvalis’
[12] M. arvalis ‘obscurus’
[13] M. subterraneus – Greek
[14] M. subterraneus – European
[15] M. subterraneus – Turkish
[16] M. felteni – Greek
[17] M. felteni – European
[18] M. thomasi – Greek
[19] M. thomasi – European
[20] M. thomasi atticus
0.6
6.3
3.9
9.3
6.3
6.4
11.4
11.4
11.8
11.0
11.6
13.2
13.6
14.2
13.1
12.6
10.5
10.9
11.1
[1]
5.8
3.7
8.4
6.0
6.0
11.1
11.1
11.6
10.4
11.4
13.5
13.4
13.9
13.3
12.3
10.4
10.9
10.8
[2]
5.1
7.7
7.2
6.8
11.6
11.1
11.6
9.7
10.6
15.1
15.2
14.9
14.1
13.1
11.2
11.7
12.2
[3]
7.8
5.2
5.3
10.2
10.7
10.3
9.3
10.0
13.6
12.6
12.7
11.4
10.4
9.1
9.3
9.8
[4]
5.2
6.0
12.8
13.2
12.8
10.6
11.9
15.3
15.1
15.1
13.6
12.5
11.9
12.5
12.1
[5]
3.8
10.5
10.2
10.3
8.7
8.6
12.3
11.8
12.1
11.3
9.7
9.2
9.8
9.5
[6]
11.4
11.7
11.6
10.1
11.3
13.2
12.5
12.4
11.9
10.7
9.8
10.4
10.1
[7]
1.2
1.3
5.3
4.8
13.5
13.6
13.0
14.1
13.0
11.0
11.4
11.2
[8]
1.5
5.5
4.6
14.3
14.1
13.6
14.5
13.0
11.8
12.2
11.4
[9]
5.8
4.8
13.8
13.0
12.4
13.7
12.3
10.7
11.1
11.0
[10]
2.9
12.9
12.4
11.6
13.1
11.8
10.9
11.2
10.1
[11]
11.9
12.2
11.8
13.1
12.1
11.0
11.4
11.0
[12]
3.0
3.3
13.0
12.7
11.3
12.0
11.1
[13]
2.8
13.4
12.0
10.3
11.0
10.4
[14]
14.5
13.0
11.0
11.6
9.9
[15]
1.5
8.7
9.3
9.5
[16]
7.0
7.6
7.8
[17]
0.2
1.6
[18]
2.0
[20]
Table II. The net uncorrected p-distances (%), calculated between groups of Greek, other European and Asian populations of the five vole species studied. Genetic distances of relative
species belonging to the “socialis” and the “arvalis” group are also presented.
Downloaded by [University of Patras] at 01:52 12 September 2012
370
E. Thanou et al.
371
Downloaded by [University of Patras] at 01:52 12 September 2012
Vole phylogeography infers multiple Balkan subrefugia
Figure 2. BI and ML tree topology and respective statistical support based on the cytb dataset (posterior probabilities >0.95 followed
by ML bootstrap values >75% are shown above branches). Codes for the sequences included in the analysis are listed in Table I. Maps
show the approximate worldwide range of distribution for the five vole species studied (based on Mitchell-Jones et al. 1999; Kryštufek &
Vohralik 2005; Kryštufek et al. 2009b; Wilson & Reeder 2005; IUCN 2007 European Mammal Assessment Website). Darker-shaded areas
correspond to (a) the 52-chromosome karyotype of M. subterraneus and (e) the approximate distribution of the M. guentheri “south” clade.
Balkan Peninsula. Especially Greece, may have been
colonized through W Turkey, N Balkans (connected
either with NW Europe or the Caucasus) or from
subrefugia within the S Balkan area, where isolated
populations survived and remain in recent times as
unique phylogenetic lineages.
In this study, the cytb analysis of several Greek
populations from the five species of Microtus voles, in
comparison to other European and Asiatic conspecific populations has provided a different phylogeographical pattern for each species, which is discussed
separately, under the light of possible subrefugia that
372
E. Thanou et al.
may have played a role in the colonization of the S
Balkan area.
Downloaded by [University of Patras] at 01:52 12 September 2012
M. levis
M. levis is the only representative of the “arvalis”
group in Greece and European Turkey (Kefelioğlu
1995; Mitchell-Jones et al. 1999). The present
species’ distribution in Europe and W Asia is given
in Figure 2d.
Greek M. levis voles are differentiated from
their European and Turkish conspecifics, although
phylogenetic relationships between the three resulting clades are unresolved. Genetic distances between
Greek and either European or Turkish lineages were
almost equal (Table II), and their split seems to have
occurred simultaneously. Interestingly, the genetic
variation within each of these lineages is low, despite
the fact that both European and Turkish ones include
populations from very distant geographic locations.
The other representative of the “arvalis” group,
M. arvalis, is known to have a complex evolutionary history, which results in the current existence
of many distinct phylogenetic lineages corresponding
to multiple European refugia (Tougard et al. 2008).
Our data also imply a respective phylogenetic pattern
for M. levis, but lack of paleontological and molecular data that fully represent the intraspecific variation
of European and Asian populations does not permit
safe conclusions. So far, the distinct lineage represented by Greek voles, may suggest that N Europe
was colonized through different routes, other than
the Balkans or Minor Asia. A Balkan refugium might
have acted as the gene pool for the Greek lineage and
possibly other populations in adjacent regions.
M. guentheri
As with many other species of social voles,
M. guentheri is mainly distributed in a wide area
from SE Balkans eastwards to W Turkey and southwards to parts of the Middle East (Figure 2e). In a
recent study (Kryštufek et al. 2009b), molecular
data revealed two distinct monophyletic lineages that
divided Guenther’s voles in a “west” and a “south”
clade, while the second clade shared a sister-species
relationship with M. dogramacii. Additionally, the
genetic distance between these two clades were high
enough to suggest that they may be independent
species.
In our study, we covered most of the species’
European distribution (Figure 1e), in order to provide more data on the support of the “west” and
“south” clades’ phylogeny. Our molecular results
support the placement of Greek social voles from
this study and one previously published sequence
from FYROM (Former Yugoslavian Republic of
Macedonia) in a new taxon, M. hartingi BarrettHamilton, 1903 (type loc.: Larissa, Greece), in
agreement with Kryštufek et al. (2009b). All Balkan
social voles are probably included in this taxon.
It should be noted that these voles occupy cultivated lowlands and seem to be favored rather than
disturbed by expanding agricultures. The species distribution is continuous, at least in Greece, and their
populations may easily retain gene flow, throughout
the SE Balkans.
Moreover, our data revealed further diversification
between Turkish and Balkan M. hartingi voles,
although the respective clades show moderate statistical support (0.69/65 and 0.89/72, respectively).
Divergence between these groups is low (0.6%,
Table II), presenting the lowest genetic distance
between “Greek” and “non-Greek” populations
of all studied species. Thus it is probable that
present diversification within M. hartingi resulted
from recent isolation events and the disconnection
between Greece and Anatolia, due to sea level rise
after glacial retreat (Perissoratis & Conispoliatis
2003), rather than the existence of a Balkan
refugium.
M. subterraneus
This species is distributed in a wide area, which
includes most of eastern and central Europe (Çolak
et al. 1998; Mitchell-Jones et al. 1999) (Figure 2a).
Many previous studies have reported differences in
size, allozymic variation and morphological differentiation, when M. subterraneus from the Balkans
and other European populations were compared
(Neithammer 1982b; Kryštufek et al. 1994 and
references therein; Fraguedakis-Tsolis et al. 2009).
Significant intraspecific cytb variation has also been
reported for this species (Jaarola et al. 2004),
although few specimens were tested. Nevertheless,
its phylogeographic history has only been discussed
under the light of karyological studies (Macholán
et al. 2001; Zima 2004), since central and eastern
European populations display a chromosomal number of 2n = 52, in contrast to the typical karyotype
of 2n = 54 found mainly in NW Europe and Turkey
(Figure 2a).
Although Greek and European voles studied here
belong to the 52-chromosome race (Macholan et al.
2001; Mitsainas et al. 2010), our cytb analysis placed
them in two separated and well supported mtDNA
lineages (Figure 2). According to our data, M. subterraneus populations presently distributed in Greece
are highly differentiated from those living in central
Downloaded by [University of Patras] at 01:52 12 September 2012
Vole phylogeography infers multiple Balkan subrefugia
Europe, but still more closely related to them than to
Turkish ones. The genetic distances among Greek,
European and Turkish pine voles are considerably
high (2.8–3.3%, Table II). Genetic distances of such
high values were calculated between pairs of closely
related but distinct species (M. irani – M. socialis,
M. dogramacii – M. guentheri “south”, Table II).
Sufficient representatives from northern European
and Turkish populations should be tested prior to any
suggestions regarding phylogenetic and taxonomic
relationships within M. subterraneus. Yet, our molecular analysis suggest the existence of a Balkan
refugium that sheltered a Balkan lineage currently
distributed in Greece, while Central Europe was
probably not colonized by the dispersal of this lineage.
On the other hand, Greek populations showed a
significant intraspecific variation that distinguished
three subgroups (Figure 2). The first includes Mts
Vrontous and Silo (Figure 1: locations 36, 37) which
correspond to the western and eastern parts of
the Rodopi mountain range along the Greece –
Bulgaria borders. The second include Mts Vorras
and Pinovo (Figure 1: locations 34, 35), the highest
ridges of Tzena mountain range that descends from
NW to SE between FYROM and Greece. The third
includes the remaining populations (Figure 1: locations 31, 32, 33) distributed along the eastern slopes
of Mt Pindos. This pattern implies the existence of
several subrefugia within the southern Balkans and
possibly in the numerous mountain ranges of N
Greece. All specimens of this study were found in
high elevations (over 1500 m). Although common
pine voles may be found in lower altitudes in northern Europe, in the Balkans they occur in mountainous areas, mainly in coniferous and broadleaf forests.
Their habitats are restricted and have suffered severe
fragmentation, which may promote isolation, at least
in the Southern Balkan Peninsula.
M. thomasi and M. felteni
These two species, which retain primitive dental
characters, are considered archaic species of the
European Microtus species group (Brunet-Lecompte
& Chaline 1992). A previous study that included
only one M. felteni specimen and four M. thomasi
ones (Jaarola et al. 2004), suggested a sister-species
relationship between them, which might indicate
common phylogenetic histories, as probably reflected
in their present distribution (Figures 2b, 2c). In our
study we evaluate this conclusion analyzing additional specimens representing more populations from
Greece, covering a major part of both species extant
distribution.
373
Our results demonstrate a polytomy between
clades corresponding to the European endemics,
namely the “multiplex” group, the Italian Peninsula
endemic (M. savii), the Iberian Peninsula endemics
(M. lusitanicus/M. duodecicomstatus) and the Balkan
Peninsula endemics (M. thomasi and M. felteni)
(Figure 2). Our data do not seem to support a sisterspecies relationship between M. thomasi and M. felteni (Figure 2, Table II). On the contrary, the genetic
distance between them is the greatest one for any
pair of the Mediterranean endemic species (7.9%),
while the lowest values for M. felteni and M. thomasi
are the ones exhibited between each of them and
the M. multiplex+M. bavaricus+M. liechtensteini clade
(6.5% and 4.2% respectively). Although this study
does not aim to conclude on the phylogeny between
Microtus species, our results provide evidence against
the sister-species relationship of M. thomasi and
M. felteni and indications of a possibly more complex role of the Balkan refugium on the speciation of
Balkan endemics.
Tree polytomies may result from a lack of information and can be resolved into sequential bifurcations
given additional data (“soft polytomies”) or they may
depict multiple simultaneous speciation events that
generated three or more lineages (“hard” polytomies)
(Maddison 1989; Walsh et al. 1999). Regarding the
polytomy discussed here, also observed in Jaarola
et al. (2004), the scenario of rapid radiation and
simultaneous diversification of many Microtus lineages resulting to the speciation of many European
endemics is very plausible. The two Balkan endemics
may not share a common ancestor but a common Balkan refugium. Both species survived there
through the ice ages and subsequently expanded.
This expansion was blocked to the north by the
Alps and the Dinaric Mountains, where the species
presently forming the “multiplex” group were probably isolated (Martínková et al. 2007). At the intraspecific level, M. felteni showed significant genetic differentiation (1.5%) considering its restricted geographic
distribution (Figures 1c, 2b) and in comparison to
M. thomasi (excluding M. thomasi atticus) which covers a much wider area (Figures 1b, 2c) and exhibits
much lower intraspecific differentiation (see also
Tryfonopoulos et al. 2008). High genetic differentiation implies limited gene-flow between M. felteni
populations and could be attributed to a patched
distribution within the species’ range.
Discontinued distribution of this Balkan endemic
species is also corroborated by our field observations;
thorough efforts and repeated visits during the past
decades were unsuccessful in capturing M. felteni
specimens, from most of its reported expansion
area in Greece (Ondrias 1966; Niethammer 1982a).
Downloaded by [University of Patras] at 01:52 12 September 2012
374
E. Thanou et al.
Felten’s voles were only found in a much smaller
area close to the Greek borders with Albania and
FYROM, in agreement with other recent studies
(Andera 1991; Mitsainas et al. 2010). All sampling
locations (Figure 1c) were at high altitudes, on the
east side of Mt. Pindos. Moreover, it seems that
these voles form small, discontinuous populations,
usually in sympatry with much larger populations
of M. thomasi, in Greece (authors’ personal observations) and probably in other Balkan areas (Bego
et al. 2008). Unexpectedly, M. felteni has recently
been found (Mitsainas et al. 2010; present study)
in a location outside previously known Greek distribution (Figure 1, location 31) suggesting the need
to update the biogeographical information regarding this species. Although preliminary and including
only few M. felteni populations, the present study is
the first molecular approach for this species’ phylogeny and biogeography. Our results demonstrate an
intraspecific genetic diversification probably due to
parapatric isolation in subrefugia within the mountainous S Balkan area
On the other hand, M. thomasi populations are
divided into two well supported groups, as all
populations from Evvoia and SE Sterea Ellada
were highly differentiated from Thomas’ voles living in the remaining parts of Greece and other
Balkan areas (Figures 1, 2c). These Greek populations retain a distinct phylogenetic profile, as they
form a monophyletic clade when several Microtus
species are included in the phylogenetic analyses.
Additionally, they exhibit a sister-clade relationship
with M. thomasi. Thus, our present data further support the validity of the atticus clade as a distinct taxon
and confirm its attribution to the subspecific level, as
was discussed in Tryfonopoulos et al. (2008) or even
specific level as was proposed by Rovatsos & Giagia
Athanasopoulou (2011). Moreover, new data suggest
that the distributional range of the new taxon in the
SE Sterea Ellada is wider than previously supposed,
although populations are small and discontinuous.
Despite thorough field work, we were unable to
identify a possible contact zone between the two subspecies. It is possible that the two lineages are not in
contact thus maintaining the isolation of the atticus
populations, possibly due to either the expansion and
competitive behavior of M. guentheri (Tryfonopoulos
et al. 2008) and/or to the geographical features of the
area. During the Late Pleistocene, flooded masses
of water, which resulted from melting ice in the
north, isolated SE Sterea Ellada from neighboring
areas (Palyvos 2001). Presently, major rivers with a
west-east direction, flowing from east Mt. Pindos,
still divide SE Sterea Ellada from adjoining
regions.
Conclusions
Our results have shown that all species of Greek
voles are genetically differentiated compared to their
European and/or Asiatic conspecifics, in consistency
with previous morphological studies (FraguedakisTsolis et al. 2009). The factors promoting vole
differentiation in this region probably acted during
the Middle and Late Pleistocene glacial periods, due
to the paleogeographical and paleoclimatical characteristics prevailing at that time. Yet, the pattern of
differentiation is characteristic for each of the studied
species. In the case of M. hartingi, the W Turkey and
the Balkan clade were differentiated due to parapatric
isolation when the Bosporus Trait was submerged
under sea-level disconnecting these two areas. On the
other hand, the genetic lineages found within M. levis
may suggest the existence of a Balkan refugium which
provided the genetic stock for a distinct Balkan lineage but did not expand further north to recolonize
Europe. Finally, the phylogeographic history of the
two Balkan endemics, M. thomasi and M. felteni as
well as M. subterraneus suggests that their respective intraspecific variation can be assigned to the
existence of multiple Balkan subrefugia.
Although preliminary, this study provides evidence
which imply that along with the Italian (Castiglia
et al. 2008), and the Iberian (Centeno-Cuadros
et al. 2009), the southern Balkan Peninsula not
only acted as a refugium, but it most probably
included multiple subrefugia, probably situated at
the southern mountainous parts of the Peninsula.
Moreover, it raises several interesting issues regarding the phylogeography of Eurasian vole species
(M. felteni, M. subterraneus and M. levis), which may
hold cryptic variation.
Acknowledgements
The authors wish to thank Dr. C. Stamatopoulos,
Mrs. Ch. Valassaki, Mr. A. Goulios and Mrs. Irena
Gjoni for their assistance in field work and James
Howlett for linguistic advice. This study was partly
funded by the European Social Fund (ESF),
Operational Program for Educational and Vocational
Training II (EPEAEK II, Greece), and particularly
the Program PYTHAGORAS I.
References
Andera M. 1991. First records of Microtus (Pitymys) felteni in
Albania. Mammalian Biology 56:380–387.
Bego F, Kryštufek B, Paspali G, Rogoz E. 2008. Small terrestrial
mammals of Albania: annotated list and distribution. Italian
Journal of Mammalogy 19:3–21.
Downloaded by [University of Patras] at 01:52 12 September 2012
Vole phylogeography infers multiple Balkan subrefugia
Bilton DT, Mirol PM, Mascheretti S, Fredga K, Zima J, Searle JB.
1998. Mediterranean Europe as an area of endemism for small
mammals rather than a source for northwards postglacial colonization. Proceedings of the Royal Society of London Series
B-Biological Sciences 265:1219–1226.
Brunet-Lecompte P, Chaline J. 1992. Morphological convergence
versus biochemical divergence in the holarctic ground voles:
Terricola and Pitymys (Arvicolidae, Rodentia). Neues Jahrbuch
fur Geologie und Palaontologie, Munchen 12:721–734.
Bužan EV, Förster DW, Searle JB, Kryštufek B. 2010. A new
cytochrome b phylogroup of the common vole (Microtus
arvalis) endemic to the Balkans and its implications for the
evolutionary history of the species. Biological Journal of the
Linnean Society 100:788–796.
Castiglia R, Annesia F, Aloise G, Amori G. 2008. Systematics
of the Microtus savii complex (Rodentia, Cricetidae) via
mitochondrial DNA analyses: Paraphyly and pattern of
sex chromosome evolution. Molecular Phylogenetics and
Evolution 46:1157–1164.
Centeno-Cuadros A, Delibes M, Godoy JA. 2009.
Phylogeography of Southern Water Vole (Arvicola sapidus):
evidence for refugia within the Iberian glacial refugium?
Molecular Ecology 18:3652–3667.
Çolak E, Yigit N, Sözen M, Özkurt Ş. 1998. A study on taxonomic
status of Microtus subterraneus de Selys Longchamps, 1836 and
Microtus majori Thomas, 1906 (Mammalia, Rodentia) in
Turkey. Turkish Journal of Zoology 22:119–129.
Conroy CJ, Cook JA. 2000. Molecular systematics of a holarctic rodent (Microtus Muridae). Journal of Mammalogy
81:344–359.
Deffontaine V, Libois R, Kotlík P, Sommer R, Nieberding
C, Paradis E, Searle JB, Michaux JR. 2005. Beyond the
Mediterranean peninsulas: evidence of central European
glacial refugia for a temperate forest mammal species,
the bank vole (Clethrionomys glareolus). Molecular Ecology
14:1727–1739.
Dubey S, Cosson J-F, Magnanou E, Vohralík V, Benda P,
Frynta D, Hutterer R, Vogel V, Vogel P. 2007a. Mediterranean
populations of the lesser white-toothed shrew (Crocidura suaveolens group): an unexpected puzzle of Pleistocene survivors and
prehistoric introductions. Molecular Ecology 16:3438–3452.
Dubey S, Cosson J-F, Vohralík V, Kryštufek B, Diker E,
Vogel P. 2007b. Molecular evidence of Pleistocene bidirectional faunal exchange between Europe and the Near East: the
case of the bicoloured shrew (Crocidura leucodon, Soricidae).
Journal of Evolutionary Biology 20:1799–1808.
Fraguedakis-Tsolis SE, Chondropoulos BP, Stamatopoulos CV,
Giokas S. 2009. Morphological variation of the five vole species
of the genus Microtus (Mammalia, Rodentia, Arvicolinae)
occurring in Greece. Acta Zoologica 90:254–264.
Fraguedakis-Tsolis SE, Ondrias JC. 1985. Geographic variation of the ground squirrel Citellus citellus (Mammalia,
Rodentia) in Greece with the description of a new subspecies.
Säugetierkundliche Mitteilungen 32:185–198.
Graf JD. 1982. Biochemical genetics, zoogeography and taxonomy
in the family Arvicolidae (Mammalia, Rodentia). Revue Suisse
De Zoologie 89:749–787.
Guindon S, Lethiec F, Duroux P, Gascuel O. 2005. PHYML
Online: a web server for fast maximum likelihood-based
phylogenetic inference. Nucleic Acids Research (Web Server
issue):W557–W559.
Hall TA. 1999. BioEdit: a user-friendly biological sequence
alignment program for Windows 95/98/NT. Nucleic Acids
Symposium Series 41:85–98.
375
Hellborg L, Gunduz I, Jaarola M. 2005. Analysis of sexlinked sequences supports a new mammal species in Europe.
Molecular Ecology 14:2025–2031.
Hewitt GM. 2001. Speciation, hybrid zones and phylogeography
– or seeing genes in space and time. Molecular Ecology
10:537–549.
Huelsenbeck JP, Bollback JP. 2001. Empirical and hierarchical
Bayesian estimation of ancestral states. Systematic Biology
50:351–366.
IUCN 2007. Microtus felteni. Microtus guentheri. Microtus
levis. Microtus subterraneus. Microtus thomasi. In: IUCN
2007. European Mammal Assessment. hppt://ec.europa.eu/
environment/nature/conservation/species/ema/. Accessed Nov
2010 20.
Jaarola M, Martínkova N, Gündüz I, Brunhoff C, Zima J,
Nadachowski A, Amori G, Bulatova NS, Chondropoulos B,
Fraguedakis-Tsolis S, González-Esteban J, López-Fuster MJ,
Kandaurov AS, Kefelioğlu H, Mathias MD, Villate I, Searle
JB. 2004. Molecular phylogeny of the speciose vole genus
Microtus (Arvicolinae, Rodentia) inferred from mitochondrial
DNA sequences. Molecular Phylogenetics and Evolution
33:647–663.
Kefelioğlu H. 1995. The taxonomy of the genus Microtus
(Mammalia, Rodentia) and its distribution in Turkey. Turkish
Journal of Zoology 19:35–63.
Kryštufek B, Bryja J, Bužan EV. 2009a. Mitochondrial
phylogeography of the European ground squirrel, Spermophilus
citellus, yields evidence on refugia for steppic taxa in the
southern Balkans. Heredity 103:129–135.
Kryštufek B, Bužan EV, Vohralík V, Zareie R, Özkan B. 2009b.
Mitochondrial cytochrome b sequence yields new insight into
the speciation of social voles in south-west Asia. Biological
Journal of the Linnean Society 98:121–128.
Kryštufek B, Filipucci MG, Macholán M, Zima J, Vujošević
M, Simson S. 1994. Does Microtus majori occur in
Europe? Zeitschrift Fur Saugetierkunde-International Journal
of Mammalian Biology 559:349–357.
Kryštufek B, Vohralík V, editors. 2005. Mammals of Turkey and
Cyprus Volume 2. Rodentia I: Sciuridae, Dipodidae, Gliridae,
Arvicolidae. Koper: University of Primorska. pp. 149–209.
Kumar S, Tamura K, Nei M. 2004. MEGA3: Integrated Software
for Molecular Evolutionary Genetics Analysis and Sequence
Alignment. Briefings in Bioinformatic 5:150–163.
Macholàn M, Filippucci MG, Zima J. 2001. Genetic variation and
zoogeography of pine voles of the Microtus subterraneus/majori
group in Europe and Asia Minor. Journal of the Zoological
Society of London 255:31–42.
Maddison W. 1989 Reconstructing character evolution on polytomous cladograms. Cladistics 5:365–377.
Martínková N, Zima J, Jaarola M, Macholán M, Spitzenberger F.
2007. The origin and phylogenetic relationships of Microtus
bavaricus based on karyotype and mitochondrial DNA
sequences. Folia Zoologica 56:39–49.
Michaux JR, Libois M, Filippucci M-G. 2005. So close and so
different: comparative phylogeography of two small mammal
species, the Yellow-necked fieldmouse (Apodemus flavicollis)
and the Woodmouse (Apodemus sylvaticus) in the Western
Palearctic region. Heredity 94:52–63.
Mitchell-Jones AJ, Amori G, Bogdanowicz W, Krystûfek B,
Reijnders PJH, Spitzenberger F, Stubbe M, Thissen JB,
Vohralík V, Zima J. 1999. The Atlas of European mammals.
T & AD Poyser Ltd. London: Academic Press.
Mitsainas GP, Rovatsos MT, Giagia-Athanasopoulou EB.
2010. Heterochromatin study and geographical distribution
Downloaded by [University of Patras] at 01:52 12 September 2012
376
E. Thanou et al.
of Microtus species (Rodentia, Arvicolinae) from Greece.
Mammalian Biology 75:261–269.
Mitsainas GP, Tryfonopoulos GA, Thanou EG, Bisa R,
Fraguedakis-Tsolis SE, Chondropoulos BP. 2009. New data
on the distribution of Mus spicilegus Petenyi, 1882 (Rodentia,
Muridae) and a distinct mtDNA lineage in the southern
Balkans. Mammalian Biology 74:351–360.
Musser GG, Carleton MD. 2005. Superfamily Muroidea. In:
Wilson D, Reeder DM, editors. Mammal Species of the World:
A Taxonomic and Geographic Reference. Baltimore, MD:
Johns Hopkins University Press. pp. 510–756.
Niethammer J. 1982a. Microtus felteni (Malec und Storch, 1963).
In: Niethammer J, Krapp F, editors. Handbuch der Säugetiere
Europas. Wiesbaden: Akademische Verlagsgesellschaft. pp.
438–441.
Niethammer J. 1982b. Microtus subterraneus (de SélysLongchamps, 1836)-Kurzohrmaus. In: Niethammer J, Krapp
F, editors, Handbuch der Säugetiere Europas. Wiesbaden:
Akademische Verlagsgesellschaft. pp. 397–418.
Ondrias JC. 1966. The taxonomy and geographic distribution
of the rodents of Greece. Säugetierkundliche Mitteilungen
14:1–136.
Palyvos N. 2001. Geomorphological study of the broader Atalanti
area (Fthiotis, Central Greece). Ph.D. Thesis, University of
Athens, Department of Geology, 233 pp. [in Greek].
Perissoratis C, Conispoliatis N. 2003. The impacts of sea-level
changes during latest Pleistocene and Holocene times on
the morphology of the Ionian and Aegean seas (SE Alpine
Europe). Marine Geology 196:145–156.
Piertney SB, Stewart WA, Lambin X, Telfer S, Aars J, Dallas
JF. 2005. Phylogeographic structure and postglacial evolutionary history of water voles (Arvicola terrestris) in the United
Kingdom. Molecular Ecology 14:1435–1444.
Posada D, Crandall KA. 1998. MODELTEST: testing the model
of DNA substitution. Bioinformatics 14:817–818.
Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian
phylogenetic inference under mixed models. Bioinformatics
19:1572–1574
Rovatsos MTh, Giagia-Athanasopoulou EB. 2011. Taxonomical
status and phylogenetic relations between the “thomasi”
and “atticus” chromosomal races of the underground vole
Microtus thomasi (Rodentia, Arvicolinae). Mammalian Biology
(in press) doi:10.1016/j.mambio.2011.09.001.
Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL-W
- improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Research
22:4673–4680.
Tougard C, Renvoisé E, Petitjean A, Quéré J-P. 2008. New insight
into the colonization processes of common voles: inferences
from molecular and fossil evidence. PLoS ONE 3(10):e3532.
doi:10.1371/journal.pone.0003532.
Tryfonopoulos G, Thanou E, Chondropoulos B, FraguedakisTsolis S. 2008. MtDNA analysis reveals the ongoing
speciation on Greek populations of Microtus (Terricola) thomasi
(Arvicolidae, Rodentia). Biological Journal of the Linnean
Society 95:117–130.
Vega R, Amori G, Aloise G, Cellini S, Loy A, Searle JB.
2010. Genetic and morphological variation in a Mediterranean
glacial refugium: evidence from Italian pygmy shrews, Sorex
minutus (Mammalia: Soricomorpha). Biological Journal of the
Linnean Society 100:774–787.
Walsh HE, Kidd MG, Moum T, Friesen VL. 1999. Polytomies
and the power of phylogenetic inference. Evolution
53:932–937.
Wilson DE, Reeder DM. 2005. Mammal Species of the World.
Baltimore, MD: John Hopkins University Press.
Xia X. 2000. Data analysis in molecular biology and evolution.
Boston, MA: Kluwer Academic Publishers.
Xia X, Xie Z. 2001. DAMBE: Software package for data analysis in molecular biology and evolution. Journal of Heredity
92:371–373.
Zima J. 2004. Karyotypic variation in mammals of the Balkan
Peninsula. In: Griffiths HI, Kryštufek B, Reed JM, editors,
Balkan Biodiversity: Patterns and Process in the European
Hotspot. Dordrecht: Kluwer Academik. pp. 109–133.