Biological Journal of the Linnean Society (200l), 74: 141-156. With 5 figures
03
doi:10.1006/bij1.2001.0561, available online at httpj/lwww.idealibrary.com on I D f
Humid forest refugia, speciation and secondary
introgression between evolutionary lineages:
differentiation in a Near Eastern brown frog, Rana
maerocnem is
DAVID TARKHNISHVILI*, AXEL HILLE and WOLFGANG BOHME
Zoologisches Forschungsinstitut und Museum Alexander Koenig, Adenauerallee 160, 53113 Bonn,
Germany
Receiued 22 September 2000; accepted for publication 6 April 2001
Brown frogs of the complex Rana macrocnemis demonstrate various degrees of differentiation between the two
widespread forms, macrocnemis and camerani, in different parts of Anatolia, the Caucasus Isthmus and northern
Iran. In order to reveal whether or not the forms represent monophyletic evolutionary lineages, we analysed the
geographic distribution of characters of external morphology, alleles at three polymorphic allozyme loci and
mitochondrial DNA haplotypes. All three character sets showed a highly congruent pattern in a limited area of
humid forests in the south-west Caucasus. Frogs from this area (form macrocnemis) represent a monophyletic
lineage which forms a narrow hybrid zone with populations of camerani inhabiting the southern Caucasus and
north-east Turkey. However, in other parts of the group’s range, hybridization between the two lineages resulted
in the merger of forms and the formation of a clinal pattern of variations. The role of landscape-dependent
diversifying selection in the evolution of the group is discussed. Conceptual difficulties in delimiting the borders of
0 2001 The Linnean Society of London
subspecies are pointed out.
ADDITIONAL KEY WORDS: geographic variation - coloration pattern
zones - landscape-dependent selection - genealogical concordance.
INTRODUCTION
Mayr (1969) defined ‘true’ biological taxa, i.e. species
and subspecies, as groups of individuals that share
ancestry by descent. In contrast, ‘ecological races’ o r
ecological subspecies may include morphologically and
ecologically similar individuals, not necessarily the
closest relatives of each other (Mayr, 1969). Evidence
for - or against - the monophyletic origin of forms
within a taxonomic complex is crucial t o understanding
its evolutionary pattern, and separating between historical and on-going reasons of differentiation. However, if interbreeding is a n outcome of the interaction
between forms, such evidence is not easily come by.
Even the narrow transition zone between two races
may reflect either the primary outcome of diversifying
selection or hybridization in an area of secondary
* Corresponding author. E-mail: d.tarkhnishvili.zfmk&-unibonn.de
00244066/01/100141+ 16 $35.00/0
-
allozymes - mitochondrial DNA - hybrid
contact (Endler, 1973). If there is gene flow across a
transition zone, or if the zone expands, the question
is further complicated (Boecklen & Howard, 1997;
Riesenberg & Linder, 1999).
Avise & Ball (1990) proposed the use of congruence
in the distribution of multiple unlinked loci (‘principle
of genealogical concordance’) as a practical criterion
for delimiting species and subspecies. The authors’
original aim was to overtake difficulties met by traditional species concepts (Dobzhansky, 1937; Cracraft,
1987), which do not address the problem of incomplete
lineage sorting a t early stages of speciation. The authors claimed that the congruence between unlinked
loci reflects a period of isolation long enough t o attain
monophyly (Avise & Ball, 1990; Avise, 1994). The
probability of repetitive evolution of the congruence
between unlinked characters is low. Hence, the concordance principle is a useful tool to resolve questions
of monophyly for interbreeding forms. Significant areas
of congruence between unlinked diagnostic characters
indicate reciprocal monophyly attained by forms at
141
0 2001 The Linnean Society of London
22 2 3 , ~
,6 24
25,
-it
I
I
r -
BlackSca
L
7
)
A
10
\1
44
13
,-
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45
Figure. 1. Map of the Near Eastern region, with sample localities and numbers, and the transition zone between the two forms as given in Tarkhnishvili ct
al., 1999 and Baran & Atatur, 1986 (indicated by thick black lines). Uncircled numbers - sample localities of macrocnriiiis (regions where forest landscapes
dominate); circled nu nerals - sample localities of camerani (regions where treeless landscapes dominate). Darker stippled area: number of frogs with midi
dorsal stripe exceeds ciO?41 for individual populations; medium stippled area: number of striped frogs varies between 30-60'%1for individual populations; lighter
stippled area: number of striped frogs is below 30'X). White area: no Rana inacrocnrniis are found. The distribution of' frogs with stripe is reconstructed from
our data, along with data published in Baran & Atatur (1986) and Ishchenko (1978).
21
/
:I_
E:
ci
EVOLUTIONARY LINEAGES OF R. MACROCNEMIS
a certain stage of their evolution. In contrast, noncorrelated patterns in the distribution of individual
genes demonstrate that a form is composed of more
than one evolutionary lineage.
The advantage of the concordance principle in comparison with the use of individual genes for revealing
monophyly is obvious. For many hybridizing taxa,
introgression of individual genes across the hybrid
zone has been demonstrated (Gollmann, 1991; Prager,
Sage & Gyllenstein, 1993;Comes & Abott, 1999;Martin
& Crusan, 1999). Use of introgressive loci for reconstruction of phylogeny can lead to erroneous
conclusions. For instance, if the distribution of mitochondrial DNA haplotypes alone is taken into consideration, some populations of the common house
mouse Mus musculus should be unified in a single
monophyletic clade with the other species, M. domesticus. However, concordance of diagnostic loci away
from the contact zone provides evidence of reciprocal
monophyly for most populations of musculus and
domesticus (Prager et al., 1993).
The concordance principle can be applied in the
resolution of the evolutionary history of forms, irrespective of the degree of their differentiation. This
is especially important for taxonomic complexes that
include lineages with a differing scale of divergence,
such as, the leopard frog (Rana pipiens complex). This
group has some reproductively isolated taxa (Frost &
Platz, 1983),together with those forming narrow stable
hybrid zones (Kocher & Sage, 1986) or those in which
the expansion of hybrids is prevented by solely external
reasons (Parris, 1999). Another good (but not quite as
extensively studied) example of the various degrees of
differentiation between morpho-ecological forms involves brown frogs of the Near East and the Caucasus,
viz. Rana macmcnemis Boulenger, 1885 (see Tarkhnishvili, Arntzen & Thorpe, 1999 for recent review;
Fig. 1). This complex is represented by two widespread
races originally described as distinct species: Rana
macmcnemis Boulenger, 1885 and Rana camerani
Boulenger, 1896 [Three other forms, R. holtzi, R.
macmcnemis pseudodalmatina and R. m. tavasensis
(Werner, 1898; Baran, 1969; Eiselt & Schmidtler, 1973;
Baran & Atatur, 1986), have local ranges in southern
Turkey and Iran]. At least in the south-west Caucasus
(Fig. 1) these forms are geographically distinct, and
separated by a narrow transition zone. This transition
zone is marked by coinciding stepped clines in eight
morphological characters (Tarkhnishvili et al., 1999).
In addition, frogs from different sides of the transition
zone differ in growth rates and age of maturation
irrespective of the climatic conditions (Tarkhnishvili
& Gokhelashvili, 1996). However, it is not obvious
whether both forms are clearly delimited throughout
the entire group’s range. In Turkey and in the rest of
the Caucasus, morphological differentiation between
143
them is unclear (Ishchenko, 1978, 1987; Baran, 1969;
Baran & Atatur, 1986), and genetic differentiation is
negligible in comparison with ‘good’ species of Rana
(Mensi et al., 1992; Green & Borkin, 1993; Kosuch,
Vences & Veith, 1999). The distribution of the forms
is landscape-dependent (Papanyan, 1961; Ishchenko,
1978; Tarkhnishvili et al., 1999), and potentially characters separating ‘macmcnemis’ and ‘camerani’ could
independently evolve in different parts of the species’
range as a result of diversifying selection.
In the present study, we compared the distribution
of morphological characters, mitochondria1 DNA and
alleles a t polymorphic allozyme loci in populations of
Rana macmcnemis throughout the large part of the
group’s range. Our aim was to reveal (1)whether or not
macmcnemis and camerani represent monophyletic
evolutionary lineages; (2) whether both forms are
clearly separated throughout the entire group’s range;
and (3) if a n introgression across the contact zone
takes place.
MATERIAL AND METHODS
SAMPLING
The origin and size of samples from different localities
(Fig. 1)used for the morphological and genetic analyses
are given in Appendix 1.Frogs from Georgia that were
later processed biochemically and genetically were collected, measured and described in 1993-1999 (Tarkhnishvili et al., 1999); parts of individual tissue samples
were preserved in 96% ethanol for DNA extraction/
analysis, others in a 2% buffered solution of 2-phenoxyethanol PPS (Nakanishi et al., 1969) for allozyme
electrophoresis. Frogs from Turkey, Iran, Armenia and
the northern Caucasus were taken from ethanol collections of the Museum A. Koenig (ZFMK), the British
Museum of Natural History (BMNH), and the Natural
History Museum of Magdeburg (NHMM). Specimens
from Daghestan (north-eastGreat Caucasus) were provided courtesy of E. Roitberg.
In the analysis of data, all sampled localities were
pooled into seven groupdregions (Fig. 2A): (1) the
Great Caucasus mountain system and the mountains
of eastern Georgia; (2) the humid forests of the southwest Caucasus; (3) the treeless uplands of southern
Georgia; (4) Armenia, eastern and central Turkey;
(5) the Ponto mountains of northern Turkey; (6) the
mountains of western and southern Turkey, and (7)
northern Iran. In regions 1, 2 , 6 and 7 mountain forest
landscapes with limited areas of subalpine fields are
predominant. Frog populations from these regions
are assumed to represent the nominative form,
macmcnemis. In regions 3 and 4 treeless mountain
landscapes are predominant. Populations from these
regions represent camerani. There are no clear indications to ascribe frogs from region 5 to either of the
144
D. ‘I’ARKHNISHVILI ET AL.
two forms (Papanyan, 1961;Eiselt & Schmidtler, 1973;
Haran & Atatiir, 1986; Tarkhnishvili et al., 1999).
MORPHOLOGY
The results of the morphometric study of Georgian
populations are described elsewhere (Tarkhnishvili
P t al., 1999). For purposes of the present work, we reanalysed ’Georgian’samples along with new samples
from Turkey, Iran, Armenia and the northern Caucasus. The coloration pattern and skin structure were
described for 566 adult and sub-adult frogs, irrespective of sex and body size. Fifteen characters were
scored for each individual, and coded on a scale of
three to five rankings for the character concerned.
The description and states of coloration/skin structure
characters are given in Appendix 2.
By means of Principal Component Analysis (€‘CAI,
we analysed the degree of morphological differentiation
between frogs from the seven outlined regions. Multivariate analysis of body shape was based on ten
measurements, taken from each of 277 adult males.
Original measurements were In-transformed and
standardized residuals of individual measurements on
the snout-urostile length were applied to the analysis.
For the full description of characters and cietaildexplanations of the transformations see Tarkhnishvili et
al. (1999).
In addition, the proportion of frogs with a mid-dorsal
stripe found a t individual localities was counted both
on the basis of our data and additional data published
by Ishchenko (1978) and Baran & Atatur (1986). This
was done in order to reveal whether transitions between ‘striped and ‘non-striped’populations are abrupt
or gradual in the Turkish versus the Caucasian parts
of the group’s range. The genetic background of the
mid-dorsal stripe is a single dominant allele S (Schupak & Ishchenko, 1981; Tarkhnishvili, 1995).
ALLOZYMES
R
:i
~
I
2
1
In order to test whether the morphological transition
between the two forms in the south-west Caucasus
has a genetic background, we analysed the distribution
of allozyme alleles in populations from the Caucasus.
Muscle tissue of frogs from 13 Caucasian localities
(8-9, 15-19, 23-28, see Appendix 1, Fig. 1)was homogenized and processed by vertical starch gel electrophoresis. Four of 15 allozyme systems studied in toto
-1
2
->
2
1
_..
0
-2
-1
0
PC 1
1
2
3
Fig. 2. Morphological variations between Runa inacrocneinis throughout the Near East. (A) Parts of the region
corresponding to one or another nominal taxon of brown
frogs. (1) Great Caucasus mountain system and mountains of eastern Georgia (macrocnenris);(2) humid forests
of the south-west Caucasus (inacrocnemis);(3) treeless
uplands of southern Georgia (canierani): ( 4 ) Armenia.
eastern and central Turkey (camerani ); ( 5 ) P(into mountains in northern Turkey (no clear indications concerning
the frog form); (6) mountains of western and southwn
Turkey (macrocnernis);(7) northern Iran ( inat:~~(-nein
is).
White part of the map indicates the range of R. nzocrocnenzis complex. (B) Individual scores of frogs from different geographic areas (see A ) plotted against the first
and the second PCA axes based on coloration pattern and
skin texture characters. Ellipses delimit 75% of points
from individual regions. (C) Individual scores of males
frogs from different geographic areas (see A) plotted
against the second and the third PCA axes based o n body
shape characters. Ellipses delimit 75% of points from
individual regions.
EVOLUTIONARY LINEAGES OF R. MACROCNEMIS
were polymorphic: Lactate dehydmgenase (LDHb),
Mannose phosphoisomerase (MPI), Malate dehydmgenase (MDHa), and Phosphoglucose isomerase (PGI).
Only the three last-named enzymes were used in the
further analysis, because these were scored for large
numbers of individuals. Running buffers, staining systems and technical details of electrophoresis are given
in Hille & Meinig (1996). Prior t o analysis, in order to
increase sample size, small samples from neighbouring
localities (15 and 16, 17-19, 23 and 24) were pooled
in such a way that the number of samples was reduced
to nine. Four of these re-coded samples (1,2,8,9) were
from localities of macmcnemis in Great and Minor
Caucasus, five (3-7) from localities of camerani in
Southern Georgia (see Fig. 4).
We applied a PCA on multi-locus allele frequencies
in which each sample was described by its allelic counts
at the three variable loci, in order to demonstrate
whether genetic differentiation among populations reflects the geographic position of these populations.
Pair-wise F , , (Goudet, 1994; Weir, 1996) values were
calculated by means of the FSTAT software (Goudet,
1994), t o obtain an index of genetic differentiation
between localities. Genotype frequencies were tested
€or deviation from Hardy-Weinberg equilibrium and
linkage disequilibrium of allelic associations a t different loci (Louis & Dempster, 1987; Weir, 1996). For
the analysis we used the POPGENE package (1997).
MT-DNA HAPLOTYPES
In order to reveal whether local forms of R. macmcnemis harbour distinct mitochondrial haplotypes, we
analysed DNA sequences of 149 frogs from 22 localities
in Caucasus, Northeastern and central Turkey, representing both ‘macmcnemis’ and ‘camerani’.
DNA was extracted from alcohol-preserved muscle
tissue following the protocol of Sambrook, Fritsch &
Maniatis (1989). The modified primers L14841(5’-AAC
CCC ATC AAA CAT ?r‘C ATC ATT ATG AAA-3‘, Moritz,
Schneider & Wake, 1992) and cytb702 (5’-GGC AAA
TAG GAA GTA TCA TTC TG-3’, Moritz et al., 1992,
modified) were used for double-strand amplifications.
Polymerase chain reaction (PCR) was performed in a
50 p1 volume in 35 cycles using Taq DNA polymerase
(Sigma) under the following conditions: denaturation
at 92°C for 9Os, annealing a t 50°C for 6 0 s and extension a t 72°C for 90 s. This yielded a 712 bp fragment
of the cytochrome-b gene of mitochondrial DNA. The
amplicons were purified with the Wizard PCR Preps
DNA Purification system (Promega Corp.). Purified
PCR products were directly sequenced with the Thermosequenase labelled primer cycle sequencing kit with
7-deaza-dGTP (Amersham-Pharmacia, RPN 2438),
following a slightly modified cycle protocol provided
by the manufacturer (MWG-Biotech). 2 pmol of
145
the forward dye-labelled primer L14841mod-IR800
(IR800>5’-CCA TCC AAC ATC TCA GCA TGA TGA
AA-3’;MWG-Biotech) was used in separate single
strand sequencing reactions a t 55°C annealing temperature. Gel electrophoresis and visualization of the
chain termination sequencing products were accomplished using the LiCOR model 4200 IR2 automated DNA sequencer and the image analysis software
(IMAGIRB). The resolvable DNA stretches of mtDNA
partial cytochrome b framed 504 base pairs for 62
individuals representing 22 localities (prior to some
analyses, small samples from neighbouring populations were pooled in order to increase sample size,
reducing the total number of samples to 19), corresponding t o positions 16 399 (5’ end) and 16 902 (3’
end) of the Xenopus laevis mtDNA alignment (Roe et
al., 1985). 300 bp sequences obtained for 87 frogs from
the same localities were used for allocation of these
individuals to alignments of the 504 bp sequence haplotypes (Appendix 1).Sequences were aligned manually,
the open reading frame was identified, and both the
nucleotide and the amino acid sequences were compared to published sequences of Rana japonica
(Tanaka, Matsui & Takenaka, 1996).
Phylogenetic relationships between mtDNA haplotypes (ingroups only) were analysed with the MedianJoining algorithm (Bandelt, Forster & Rohl, 1999),
designed for comparisons a t the lower taxonomic level,
when the number of variable characters is too low to
obtain a single maximum parsimony tree with strong
bootstrap support for individual clades. The algorithm
provides a unique network, linking all individual ingroup haplotypes in a way that all possible evolutionary steps between them are reflected in a pathdiagram. The software applied was NETWORK, version 2.0b (Rohl, 1998).
TEST OF HYPOTHESES
In order to discriminate between the possible reasons
for divergence between frog populations, the partial
Mantel permutation tests were applied (Manly, 1986)
that are recommended for the separation of the direct
influence of environment from historical reasons, and
checking for the association between morphological
versus genetic distances (Thorpe et al., 1995, Thorpe,
Black & Malhotra, 1996; Thorpe, 1996; Malhotra &
Thorpe, 1997). Four alternative hypotheses were
tested: (1) differences between populations are associated with the geographic distance between populations measured from maps with the scale of u200 000
published by the Ministry of Defence of the USSR
(1978); (2) differences are associated with the distance
of a population from Tertiary forest refugia in the
south-west Caucasus as given in Gvozdetsky (1963),
Tuniyev (1990) and Tarkhnishvili, Thorpe & Arntzen
(2000); (3) differences are associated with geographic
146
D. TARKHNISHVILI ET AL.
positions that concern areas covered by forest during
the Holocene as given in Janelidze (1980), scored as
’within’versus ‘outside’forest landscapes, and (4) differences are associated with altitudes of individual
localities. To determine the significance of each of the
alternative hypotheses, the partial Mantel test with
I 0 000 randomizations was applied. The software used
was R. S. Thorpe’s version of the program originally
written by B. Manly (1986). As dependent variables,
we subsequently used: (1)the matrix of pair-wise Fsr
values estimated from composition of cyt-b haplotypes
at individual localities, calculated with AMOVA algorithm. (Excoffier, Smouse & Quattro, 1992; software
applied was Arlequin 1.1, Schneider et al., 1997); ( 2 )
the matrix of pair-wise FbT
values estimated over nine
alleles at the three variable allozyme loci; (3-7) the
differences in allele frequencies of the individual alleles
- hfDHa-1, PGI-1, MPI-1 - MPZ-3; (8, 9) Euclidean
distances between population arithmetic means, derived from 15 characters of coloration pattern and skin
structure and from ten characters of body shape; (10)
the frequency of individuals without the mid-dorsal
stripe, i.e. the proportion of homozygotes of the recessive allele S. Sequential Bonferroni correction (Rice,
1989) was applied t o correlation tables across rows.
In order to define an integrative morphological and
genetic similarity between the nine Caucasian populations. a nieta-analysis of principal components was
performed on seven ‘synthetic’ variables: (1, 2) population mean scores along the first two principal axes
based on the analysis of allozyme allele frequencies;
( 3 , 3 ) mean scores along the first two principal axes
based on analysis of coloration patterns; (5, 6) scores
along the second and third axes based on the analysis
of body shape, and (7) frequencies of the two main
haplotype clades. For all calculations the software
package SPSS 9.0 (1999) was used.
RESULTS
EXTERNAL MORPHOLOGY
The first t w o principal axes explained 30°/0 of the
observed variation in 15 coloration pattern characters
(Table 1A). The first axis separated individuals from
the humid forests of the south-west Caucasus (region
2) and individuals from treeless uplands of southern
Georgia (region 3) (Fig. 2A,B). The display along the
second axis made this separation even clearer. Individuals from distant parts of Georgia, from the northern Caucasus, Armenia, Turkey and Iran (regions 1,
4-7), regardless of the exact area they were collected
from (i.e. both ‘macmcnenzis‘ and ‘camerani’ populations), kept an intermediate position between these
two geographically adjacent groups.
The first three principal axes explained about 50Yo
of the total variation observed in male body shape.
The first axis showed comparable positive loadings for
almost all analysed characters, and could therefore
be assumed t o reflect body size differences between
individuals (Manly, 1994). The second axis demonstrated the contrast between leg measurements,
on the one hand, and head and metatarsal tubercle
measurements, on the other (Table lb). Along this axis,
frogs from humid forests of‘ the south-west Caucasus
(region 2), and those from the southern Georgian treeless uplands (region 3) were relatively well separated.
Frogs from other regions showed a wide scale of variation along this axis, irrespective of their geographic
origin (Fig. 2C).
The highest percentage of frogs with stripe (80-98‘%1)
was found in the mountain steppes of southern Georgia, western Armenia and eastern Turkey. and i n a
limited area of south-west Turkey (regions 3 and 4)
(Figs 1,3B, Appendix 1). In Turkey, stripe frequency
increases gradually from ‘peripheral’(forested) regions
(5 and 6) to ‘inner’ (treeless) parts of the country
(region 4). In the southern Caucasus (regions 2 and
3), the border between ‘striped and ‘non-striped’populations is pronounced, and coincides with the border
between the forest and mountain steppe belt of the
Minor Caucasus (Fig. 3B). In the exhaustively sampled
Borjomi area (central Georgia), the percentage of
striped frogs increases from 3 to 75 a t a distance of
15km, following the landscape transition from mixed
forest to subalpine belt.
ALLOZYME LOCI
The allozyme locus MPI exhibited four distinct alleles,
the locus MDHa had three alleles, and PGI had two
alleles. Allele frequencies of nine Georgian localities
are given in Figure 3C-E. The multivariate analysis
based on the frequency distribution of these nine alleles
showed a clear and separate plot for each of the sampling sites. The first two principal component axes
discriminate localities of the Great Caucasus from
those of the Caucasus Minor, and localities inhabited
by inacrocnemis from those inhabited by camerani.
Interestingly, the niacmcnemis populations inhabiting
humid forests in the Borjomi area of south-western
Georgia (locality 8) demonstrated the highest amount
of differentiation from canierani populations of neighbouring subalpine landscapes (localities 6 and 7 ) (Fig.
4A,B).
Genotypic distribution patterns across the ‘Borjomi’
transition zone are sufficiently noteworthy to merit
description in detail. The frequency of allele MDHa-2
strongly increased in treeless upland localities (Fig.
3E). In locality 6 (Fig. 4A), a significant deviation from
the Hardy-Weinberg equilibrium at the locus h1H
was observed (x2= 15.2, N=41,P=O.O19). Pairwise
FST
estimated from three polymorphic loci reached 0.17
EVOLUTIONARY LINEAGES OF R. MACROCNEMIS
147
Table 1. Percentage variance (O/o Var.) explained and scores of the first three axes of a principal component analysis
for coloration pattedskin structure (left panel) and body proportions of males (right panel) in Rana macrocnemis from
throughout the species’range. Abbreviations (left panel): BC - background coloration; SE - spot expression; ST - skin
texture; TC - throat coloration; VC - belly coloration; S - mid-dorsal stripe; P - speckles; V - V-shaped spot; NS number of spots; DS - dark dorso-lateral stripes; SS - spot shape; SP - spot position; LS - light dorso-lateral stripes;
RS - ring-shaped spots; (right panel): L - snout-urostile length; LC - head length; LTC - head width; DRO - distance
from eye to tip of snout; SPOC - distance between eyes; LO - diameter of eye; LTYM - diameter of tympanum; F femur length; T - tibia length; DP - length of first toe; CINT - length of inner metatarsal tubercle.
Axis
First
Second
Third
AXiS
Var.
BC
SE
19.6
0.42
0.57
0.55
-0.31
0.19
0.76
0.15
-0.11
0.30
0.36
0.63
-0.39
0.49
0.42
9.9
0.55
-0.09
0.29
0.08
-0.41
0.00
0.28
0.57
0.10
0.19
-0.03
0.56
0.19
-0.21
9.3
0.08
0.45
-0.08
-0.11
0.29
-0.01
-0.19
0.14
0.65
- 0.40
- 0.04
0.26
-0.52
0.12
Yo Var.
22.3
17.8
L
LC
LTC
DRO
SPOC
LO
LTYM
F
T
DP
CINT
- 0.02
- 0.02
Oh
ST
TC
vc
S
P
V
NS
DS
SS
SP
LS
RS
-
-
for populations of opposite forms, separated by distance
c. 20 km (localities 6 and 8). Pairwise FST
did not exceed
0.01 for populations of the same form separated by a
distance of 50 km (localities 3-6 and 8, 9) (Fig. 4A).
Second
First
0.63
0.51
0.41
0.60
0.67
0.45
0.40
0.48
0.36
0.32
- 0.29
- 0.55
-0.36
-0.22
0.23
-0.08
0.60
0.71
0.50
-0.47
Third
9.8
-0.01
- 0.264
0.201
-0.50
0.12
-0.11
- 0.18
-0.16
0.08
0.47
0.65
than M and is distributed from the north-east Caucasus (Daghestan) through the Taurus Mountains in
central Turkey. It is missing only from humid forest
areas in the south-west Caucasus (Fig. 3A).
MT-DNA HAPLOTYPES
TEST OF HYPOTHESES
Sequencing a 504 bpfragment ofthemitochondrial cytochrome-b gene in 62 individuals revealed the presence
of 11different haplotypes differing by 1-7 substitutions
(Table 2; GenBank Accession Nos AF373114AF373160). 12 variable site positions were found. No
transversions were observed, and T-C transitions were
4 times more frequent than A-G transitions. The median-joining network showed that all haplotypes are
clearly separated into two groups, M and C. Individual
haplotypes within each of these groups differ by 1-3
substitutions. M and C groups of haplotypes are connected by one single possible path and appear to be
monophyletic in respect to one another (Fig. 5).
Geographically, M is restricted to central and western Georgia and the adjacent area of north-east Turkey
(Table 2, Fig. 3A). In the humid forests of south-west
Georgia (region 2) haplotypes of the group M are found
exclusively, while in eastern and northern Georgia
(region 1) both M and C haplotypes coexist in the same
locality in different proportions, as well as in localities
of camerani from southern Georgia (region 3). The C
group of haplotypes has a much broader distribution
Results of the partial Mantel tests are given in Table
3. (1)The geographic distance between localities significantly affected differences of coloration pattern between frog populations. (2) The proximity of a locality
to the humid forests refugia in the south-west Caucasus as defined by Gvozdetsky (1963) and Tuniyev
(1990) significantly affected frequency of PGlf and
genetic differentiation estimated from mtDNA haplotype composition; even after removal of the influence
of interaction between factors, the influence on the
mtDNA haplotype composition remained significant.
(3) The position of a population, with respect to the
distribution of forest landscapes, was an important
factor in determining the proportion of allele MDHal.
(4) Finally, the differences in elevation between
localities significantly affected inter-population differences in body proportions.
THE CONGRUENCE BETWEEN MORPHOLOGICAL AND
GENETIC DATA
Figure 4C demonstrates the similarity between
nine Georgian populations, based on combined
148
13. TARKHNISHVILI ET AL.
.
~~
D
...
‘J
Figure 3. Frequencies of some alleles in Rana maemenemis populations in the western Caucasus. Bold line indicates
inountains separating humid forest landscape from treeleess uplands in the south-west Caucasus. (A) Mitochondria1
DNA haplotypes: white-C; grey-Ml, M3, M4; black-M2. Note: Frequencies of M2 and M1, M2, M3 decrease in
areas distant from refugial humid forests in the south-west Caucasus with different rates. (B) Frequencies of frogs
with a contrast mid-dorsal stripes (genotypes SS + Ss). Sharp transition coincides with the border bet.ween the
landscapes. (C) Frequencies of allele PGIZ. Significantly differs between populations from the Great and Minor
Caucasus, irrespectively to inacrocneinis-camerani transition. (D) Frequencies of allele MDHa2. Sharply decreases
lollowing t h e border between the landscapes. (E) Frequencies of four alleles a t locus MPI. Frequencies of alleles
significantly change in populations from the centre of the transition zone, in comparison both with macrriown~isand
cr17nt’rtrni.
EVOLUTIONARY LINEAGES OF R. MACROCNEMIS
morphological, allozyme and mtDNA variations. The
position of populations 8 and 9 (humid forests of the
south-west Caucasus, region 2) in a two-dimensional
space spanned by the first two PCA axes describing
66% of the total variance between localities (Table 4)
is noteworthy. The first axis separates populations
of macmcnemis and camerani, and along this axis,
populations from the transition zone (localities 8 and
6) form themselves into distinct orientations. This
vector has high loadings of variables ascribing body
shape, coloration pattern and allozyme characters.
Macrocnemis from the Greater Caucasus mountains
maintains a n intermediate position between southwest Caucasian populations and camerani.
DISCUSSION
There are clear morphological (see also Tarkhnishvili
et al., 1999) and genetic differences between 'macmcneinis' from humid forests in the south-west Caucasus
(region 2, Fig. 2 ) and 'camerani' from the treeless
uplands of southern Georgia, western Armenia and
north-east Turkey (region 3). In this area, the two
forms are separated by a narrow transition zone. R.
macmcnemis from region 2 is marked by a group of
closely related haplotypes, fixed on the population
level, which is absent from the greater part of the
group's range, from central Turkey to the north-east
Caucasus (Fig. 3A). Frogs from this region demonstrate
a highly congruent pattern in the distribution of genetic-based traits (morphology, allozymes and mitochondrial DNA), and represent a monophyletic
evolutionary lineage. In camerani from region 3,
results obtained from allozymes and morphology are
concordant, although populations have haplotypes,
marking both lineages. Frogs from other study areas
have character states intermediate between macrocnemis and camerani from regions 2 and 3, although
they also have mitochondria1 DNA marking camerani
populations. One can conclude that a sharp transition
between opposite forms occurs only in a limited area
of the south-west Caucasus, whereas over the greater
part of the range, the characters are dissolved in a
rather mosaic-like mode.
42"N
44"E
B
@
I
I 0,
4
PRIMARY OR SECONDARY DIFFERENTIATION?
The narrow transition zone between macmcnemis and
camerani in the south-west Caucasus coincides with
the clearly-defined border between humid mountain
~~
1
C
"
ri
j
()---
@ @
I
1
I
8
m
v
a
I
1
-0.5
PC 2
149
w
I
~
Figure 4. Differentiation between nine frog populations
from the western part of the Caucasus Isthmus. Populations of ( ) Rana macroenemis and (0)
R. camerani.
(A) Position of the localities on the geographic map (geographic coordinates indicated). Bold line indicates mountains separating humid forest from treeless landscapes
in the south-west Caucasus. (B) Scores along the first
and the second principal component axes based on the
frequencies of nine alleles at three variable loci for populations indicated in (A). Note the distinct orientation of
neighbouring populations of the opposite forms, 7 and 8.
(C) PCA ordination of the nine Georgian populations (A)
based on the mixed data set including coloration, body
shape, allele frequencies and mtDNA haplotype frequencies. Note the distinct orientation of populations 7
and 8 and intermediate position of macroenemis populations from the Great Caucasus, 1 and 2.
150
L). TL4RKHNISHVILIET AL.
~Table 2. Vdriahle sites of the aligned sequences (504 bp of the mitochondrlal cytochrome-b gene)
I,oc
alltl(5
('1
2 8 , 9 11 21.25, 26, 27 36
c2
('3
('1
1 2. 11
Pi
(26
\I1
\I2
\13
8
15. Xi, 27, 28. 31
27, 31 36
36
8 1 1 11-19. 21-28, 37
15-1 7 , 23. 24
16
511 28
V < i 13
T
T
T
T
T
T
T
T
T
C
T
T
C
C
C
C
C
C
G
G
G
G
G
G
A
A
A
A
G
G
T
T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
T
C
T
C
T
T
T
T
C
C
T
C
C
C
A
A
A
A
A
G
C
C
C
C
C
C
T
C
T
T
T
T
T
T
T
T
T
T
T
T
T
A
A
A
A
A
A
A
A
A
A
T
T
T
T
C
T
T
C
C
T
T
T
T
T
C
C
A
A
T
C
c
c
T
T
C
C
c
.i\
rr
C
C
A
C
A
1'
forests and treeless uplands with a n expressed continental climate, maintained by the mountain ridges
of the Caucasus Minor. Neighbouring populations at
opposite sides of this zone demonstrate significant
morphological and genetic differences, exceeding those
between geographically distant populations which becounted for three
long to t h e same form (pairwise FST
allozyme loci; Figs 2-4). Nonetheless, genes do flow
across t h e transition zone. The evidence includes: (a)
shifted distribution of alleles across the zone (Fig. 3);(b)
a remarkable number of non-identifiable individuals in
both macrocnemis and camerani populations (Fig. 2):
(c) free interbreeding between the forms (Ishchenko &
F'yastolova, 1973) and amplexus formation irrespective
of phenotype (Tarkhnishvili et al., 1999), and (d) the
distribution of alleles a t locus PGI and individual mtDNA haplotypes more similar between neighbouring
populations of opposite forms t h a n between distant
populations of the same form (Fig. 3).
Can the narrow transition zone reflect a primary
outcome of intense diversifying selection within a
single continuously distributed population? The sharp
clines in the distribution of characters alone could
not exclude such a possibility (Endler, 1973, 1986).
However, macrocnemis from the entire mountain forest
area in the south-west Caucasus (region 2) are homogenous concerning their mitochondria1 DNA (with here
and there a n M group of haplotypes) which indicates
their monophyletic origin. Moreover, although some of
the M haplotypes dominate in border populations of
camerani, haplotypes of another group (here and there
C) are also present in these populations, and they
appear to be related to morphologically similar cumerani from central Turkey. Consequently, stepped
clines between rnacrocnemis and camerani characters
in the south-west Caucasus reflect a zone of secondary
hybridization between the two monophyletic evolutionary lineages t h a t have undergone a certain period
of independent evolution.
rm5
"47
$3
iii;
'.cR
Figure 5. Median-joining network of Rana macrocnemis
haplotypes from central and eastern Turkey and the
Caucasus. See Table 3 and Figure 1 for localities of
individual haplotypes. C1-C6-kamerani'
haplotypes
found from central Turkey through Daghestan; M I M~--'rritrcrocnr~mi.s'haplotypesfound in western and central Georgia and the adjacent Artvin area in Turkey.
Haplotypes M2-M;S are found exclusively in humid forests
of the south-west Caucasus. Size of circles indicate numher of individuals with given haplotype.
EVOLUTIONARY LINEAGES OF R. MACROCNEMIS
151
Table 3. Mantel tests for associations of morphological and genetic distances between nine Georgian populations of
Rana macmcnemis with geographic distance (GEODIS), the position concerning refugia in the south-west Caucasus
(REFDIS), the position concerning forest borders in Holocene (HISTFDIS) and ecological transience reflected by
differences in elevation (ELDIS)
Dependant variable
GEODIS
REFDIS
HISTFDIS
ELDIS
FST(MtDNA)
FST(nine allels)
PGI-1
MDHa--1
MPI-1
MPI-2
MPI-3
Coloration (15 char.)
Body shape
Stripe
0.024/0.669
0.026/0.435
0.019/0.332
0.979/0.556
0.415/0.565
0.76q0.665
0.841/0.327
0.010*/0.002**
0.63q0.999
0.047/0.111
0.001**/O.OlO*
0.021/0.272
0.011*/0.233
0.719/0.448
0.406/0.927
0.764/0.741
0.730/0.155
0.297/0.048
0.266/0.568
0.351/0.452
0.95v0.946
0.021/0.072
0.299/0.287
0.009*/0.006*
0.21v0.484
0.45v0.362
0.697/0.896
0.015/0.035
0.83v0.448
0.017/0.016
0.299/0.125
0.976/0.761
0.476/0.376
0.048/0.193
0.290/0.288
0.624/0.465
0.970/0.738
0.103/0.091
0.008*/0.007*
0.09 l/O. 197
P-values given: left of the slash, for simple Mantel tests; right of the slash, for partial Mantel tests (Manly, 1986; Thorpe,
1996); for dependent variables see text. All significant or nearly significant (Pc0.05 before Bonferroni correction) regression
coefficients are positive. *P<0.05; **P<O.Ol, after Bonferroni correction applied.
Table 4. Percentage variance (O/u Var.) explained and
scores of the first three axes of a PCA for meta-analysis
based on combined morphological, allozyme and mtDNA
variations. ALL01 and ALL02 - scores on the first two
PCA axes based on allozyme allele frequencies; COLl and
COL2 -population mean scores on the two first PCA axes
based on the analysis of coloration pattern; SHAP2 and
SHAP3 - population mean scores on the second and third
PCA axes based on the analysis of body shape; mtDNA frequencies of haplotypes that belong to the group M
Axis
First
Second
Third
o/o Var.
36.81
0.541
0.791
0.829
-0.344
-0.838
0.242
0.304
28.10
0.590
-0.371
0.530
0.647
0.368
0.530
0.638
24.61
0.488
0.416
-0.133
0.627
0.122
-0.674
0.657
ALL0 1
ALL02
COLl
COL2
SHAF’2
SHAP3
MtDNA
~
~
POSSIBLE REASONS FOR THE SPLIT BETWEEN THE
TWO FORMS
The molecular differentiation between the two groups
of haplotypes, C and M , is minute when compared
with the level of geographic subspecies of other frogs
(Tanaka et al., 1996; Barber, 1999); it appears that the
split between the two lineages is of relatively recent
occurrence. The contemporary distribution of M is
significantly associated with Quaternary refugia of the
south-west Caucasus (Table 4).This fact, when taken
together with the high congruence between characters
marking macmcnemis in the same region, leads us to
conclude that the split followed the full or partial
isolation of macmcnemis ancestors in the Quaternary
refugia. These refugia maintained forest habitats
throughout the Ice Age (Gvozdetsky, 1963; Tuniyev,
1990).Morphological evolution of macmcnemis is likely
to have been triggered by adaptation t o mesophylic
forest landscapes, as opposed to camerani inhabiting
treeless regions with a continental temperate climate.
It is likely that the initial steps of differentiation
between the two lineages corresponded t o the peripatric speciation model as described by Nevo (1989).
According to this author, “. . . speciation in the isolates
may occur primarily because of their multiple genetic
and phenotypic adaptation to their new ecological . . .
frontier.. .”. Such a frontier has been provided by
humid forest refugia.
SELECTION AND SPATIAL STRUCTURE OF THE R A N A
MACROCNEMIS COMPLEX
The morphological and genetic characters of either
form are only consistently expressed in limited areas
of north-east Turkey and the south-west Caucasus.
Morphological and genetic character states of frogs
from the major part of the group’s range are intermediate between ‘central’ populations of macmcnemis
and camerani, and populations of opposite forms are
connected with gradual transitions rather than with
sharp clines. This pattern can reflect an extensive
introgression between lineages in early Holocene,
when forests spread over formerly treeless areas of
the Great Caucasus and Turkey (Janelidze, 1980;
Margalitadze, 1977; Hesselbarth, van Oorschot &
152
11. TAHKHNISHVILI E T A L .
Wagener, 1995). This process could trigger dispersal
of macroenemis genes from the south-west Caucasus
throughout the entire region. Remarkably, morphological characters spread far beyond mt-DNA
haplotypes.
Why prominent morphological and genetic differences between macrocnenais and camerani are
maintained at both sides of the ‘Caucasian’ narrow
transition zone, in spite of gene flow? There are several
explanatory models dealing with stable hybrid zones,
which can be subdivided into ‘environment-dependent’
and ‘environment-independent’ones (Barton & Hewitt,
1985,1989;Arnold, 1997).The association of diagnostic
characters of both forms with certain landscape types
support an important role of environment in maintaining morphological and genetic differences. The
possible mechanism includes landscape-dependent selection, providing an advantage to inacrocneinis in
forest areas and to camerani in treeless uplands. This
corresponds t o the ‘mosaic model’ described for a number of animals and plants (Arnold, 1997). Different t o
environment -independent models (‘tension zone’), the
concept of mosaic hybrid zone does not exclude differential rates of introgression for unlinked geneticbased traits. if their selective value differs.
Moreover, the ‘mosaicmodel’ fits well the mosaic-like
distribution of inacrocneinis and camerani characters
throughout the largest part of the group’s range. The
refugial forests of the south-west Caucasus (habitats
of ‘typical’macrocnernis) and volcanic uplands of region
3 (habitats of ‘typical’ camerani) represent extreme
cases of mild humid versus cold continental climate
throughout the range of Rana macrocnemis. At a distance of a 10-30 km, mean January temperature declines from 0 ( i 2 ) to - 10”C, and the level of annual
precipitation declines from 1000-3000 mm per annum
to 500-700 mm (Djavakhishvili, Aslanikashvili &
Dzotsenidze, 1964). Nowhere else in the Caucasus or
in Asia Minor is the level of humidity so high a t an
elevation below 1500m as in region 2, and nowhere
else is winter so cold and, simultaneously, relatively
dry as in region 3 (Djavakhishvili et al., 1964; Hesselbarth ei ai.. 1995). Transition between regions 2 and
3 represents a unique case for the group’s range, when
humidity decreases rather than increases with elevation. Mountains of the Great Caucasus and Turkey
have environmental conditions intermediate between
regions 2 and 3. Diversifying landscape-dependent selection in these regions may not be strong enough to
maintain a narrow hybrid zone between macroenemis
and cainerani; the introgression takes place resulted
in the occurrence of intermediate phenotypes.
The observed distribution of characters in the Rana
iriacrocnein is group provides an interesting case of
spatial organization. In the south-west Caucasus, adjacent populations of opposite forms maintain clear
differences. However, neither inacrocnenzis nor camerani is clearly delimited in geographic space. This
situation is not unique. Bohme (1978) described the
distribution of sand lizard subspecies in Central Europe, stressing that differences between these can be
clearly outlined only for central populations of either
subspecies; he suggested the use of the term ’polycentric species’ as more adequately describing the complex than the ‘polytypic species’ of Mayr (1969). The
problem with the unclear geographic borders of a taxon
is a general one. All dominating species concepts (Cracraft, 1987; Frost & Hillis, 1990; Arntzen & Bauer,
1996) address the question: What degree of differentiation between organisms is enough for them
t o be regarded as different taxa? However, even full
reproductive isolation between particular populations
(therefore representing different biological species)
cannot yet be taken to mean that each of these species
can be clearly delimited morphologically o r spatially.
Widely discussed examples of ‘ring species’ (Wake,
1997; Wake & Schneider, 1998; Gavrilets, Li & Vose,
1998) illustrate the problem: the effectivity of isolation
mechanisms can vary geographically. Two evolutionary
lineages of Rana inacrocneinis formed an extended
zone of secondary contact in Caucasus and Asia Minor.
Separation between the lineages is maintained by
extrinsic factors, which are less effective in the western
than in the northern part of the group’s range. ‘Environment-dependent’ hybrid zones appear to be quite
wide-spread (Arnold, 1997). If such a zone is geographically extended and covers wide spectra of ecological conditions, one can expect patterns similar to
that described here: taxa will remain distinct in some
parts of the zone and merge in other parts of it.
ACKNOWLEDGEMENTS
The work was financed and supported by the Alexander
von Humboldt Foundation (grant No. IV GEO 1064249)
and the Alexander Koenig Foundation. We used facilities of the ZFMK molecular genetic laboratory. A.
Kandaurov and R. Mamradze helped in collecting and
storing frog tissues. W. Bischoff provided samples from
the eastern Turkey and the Northern Caucasus. B.
Clarke assisted in work with the collection of London
Museum of Natural History. E. Roitberg and L. Mazanaeva provided frog sample from Daghestan. €2. S.
Thorpe kindly allowed to use his software for Mantel
test analysis. Two anonymous referees, Pim Arntzen,
M. Schafer; B. Misof and E. Roitberg made helpful
comments on the first draft. G. G. Wood corrected the
English of the manuscript.
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21
22
23
24
25
26
27
28
29
30
17
18
19
20
12
13
14
15
16
2
3
4
5
6
7
8
9
10
11
I
No.
Mamisoni
‘CrossPass’
Sioni
Duruju
Tbilisi-Tbkhneti
Satovle
Gostibe
Ateni
Nedzura
Baniskhevi
Abastumani
Goderzi Pass
Shavsheti
Batumi
Chitakhevi
Aspindza
Bakuriani
Gujareti
Tskhratskaro
Thbatskuri
‘Tsalka’
Paravani
Samsari
Khanchali
BZYP
3
3
3
3
3
3
3
3
3
2
2
3
2
2
2
I
2
1
1
I
I
I
1
1
pc
TU, PC
PC
TU
TU
TU
Pc
PC
Pc
TU
PC, TU
TU
TU
PC
TU, PC
PC
1900-2000
600-800
1350
1600
1000
900-1200
900-1100
1200-1700
1900
1000-1 500
50
1000-1 100
1100-1400
1500-1600
1600-1900
2200-2400
2000
1550
2100
2500-2600
1950
2550
2000-2500
?
?
?
?
?
?
Treeless mt.
Treeless mt.
Mountain forest
Mountain forest
Forest/steppe
Mountain forest
Alpine
Alpine
Carpinus forest
Subalp. meadow
Carpinus forest
Beach forest
Subalp. forest
Carpinus forest
Mixed forest
Mixed forest
Mixed forest
Subalp. meadow
Mountain forest
Deciduous forest
Carpinus forest
Forest/steppe
Subalpine forest
Subalp. meadow
Alpine
Mountain steppe
Mountain steppe
Mountain steppe
Alpine
Mountain steppe
PC
PC
Magd
Magd
Magd
Magd
TU
PC, ZFMK
1
1
‘S. Daghestan’
‘Centl: Daghestan’
Wadiha u kaz
Dombaj
Kislovodsk
I
I
I
I
Altitude
Habitat
Source
A
Locality
0
10
40
59
75
83
77
74
70
73
91
0
4.0
10
15
9.1
26
23
-
62
10
-
9
11
14
10
5
13
47
-
70
22
34
35
-
-
-
-
22
47
46
62
34
20
26
23
16
8
-
27
2
13
-
-
-
16
-
52
5
1
-
21
26
33
4
-
1
3
2
2
1
21
1
Nlc
1
70
10
9
11
11
0
0
2.9
2.9
30
0
11
-
Nlb
4
7
4
3
4
2
22
23
18
22
34
35
Nla
0
4
0
7
0
4
5.8
3
0
4
0
2
0
22
0
5
S
N2a
N2b
N3a
N3c
continued
N3b
Sample sizes are coded as follows: N l a : coloration pattern; Nlb: presencdabsence of the mid-dorsal stripe; Nlc: body proportions of adult males; N2a:
504 bp mtDNA fragment, N2b: 300 bp mtDNA fragment; N3a: genotypes scored for locus MPI; N3b: genotypes scored for the locus M D H a ; N3c: genotypes
scored for the locus PGI. S - percentage of frogs with the mid-dorsal stripe. Neighbour localities from Turkey, Iran and the northern Caucasus were pooled.
A - seven areas as used in morphometric analysis (see Material and Methods). Italicized numerals - ‘macrocnemis’ areas a s described in the literature;
remainder are ‘camerani’ areas. Bold italics indicates type and paratype localities. Data sources: Anonymous, 1996; Ministry of Defence of the USSR, 1978;
Djavakhishvili et al., 1964.
GEOGRAPHICAL AND ECOLOGICAL DESCRIPTION OF SAMPLING LOCALITIES
APPENDIX 1
‘Basum m t ‘
‘Sevan’
‘Van’
Kars
‘Rize-Chresun
Artuin
‘Taurus’
‘Kayseri’
‘Bolkar Dag1’
Tzmir’
Uludag
Cha2an
32
33
34
35
36
37
38
39
40
MM,HM
ZFMK
4
4
4
5
2
5
4
6
6
6
7
7
7
contrnued
APPENDIX 2
2200
500-1500
1500-2500
1500-2000
1500-2000
0-1000
1000-1500
?
?
?
?
?
?
?
?
Altltud?
20
27
0
0
-
-
22
13
1
5
-
11
-
-
1
5
1
5
22
13
3
-
6
-
-
2
-
-
2
-
-
-
-
1
I
-
3
2
-
-
-
2
-
6
1
7
1
6
8
I
Very light
Spots absent
Smooth
White
White
Absent
Absent
Absent
Absent
Spots absent
Spots absent
Spots absent
Absent
Spots absent
Background coloration
Spot expression
Skin texture
Throat coloration
Belly coloration
Mid-dorsal stripe
Speckles
V-shaped spot
Dark lateral stripes
Spot number
Spot shape
Spot position
l i g h t dorso-lateral stripes
King-sliuped spats
Fragmented
N<4
Small spots
Asymmetric
Poorly expressed
‘Pull‘spots
Intermediate
Spots with vague borders
Warts in the hind part
Light grey
Light grey
Light elongated spot
Hare points
Two elongated spots with angle
Gradation 2
Clearly expressed
4<N<12
Large round spots
Some pairs symmetric
Contrasting
King-shaped spots
Very dark
Clearly expressed
Small warts
Spotted
Spotted
Stripe with vague borders
Speckles and spots
V-shaped spot
Gradation 3
-
-
-
-
-
-
-
-
-
-
Mergcd in stripes
-
-
-
Large elongat.ed
Symmetric pattern
-
-
N> 12
-
-
-
-
N3c
Contrasting, reaches sn
-
Gradation 5
-
-
-
-
N3b
-
-
-
N k
Contrasting borders
Large warts
Dark
Dark
Contrasting, reaches head
Speckles only
-
Gradation 4
-
-
-
-
1
-
-
-
t5
-
2
3
2
N2h
11
-
N2a
5
Nlc
29
Nlb
29
Nla
7
43
61
6
81
1
43
7
0
1
4
3
6
70
8
1 1 1 1
60
5
97
60
6
CHARACTERS OF THE COLORATION PATTERN AND SKIN STRUCTURE SCORED FOR STUDIED INDIVIDUALS
ZFMK
ZFMK
ZFMK
ZFMK,BM
ZFMK, BM
BM
BM
ZFMK
ZFMK
ZFMK
Gradation 1
Be] Noorg
RM
4
BM
Mountain steppe
TU
3
Steppe, forest
Treeless upland
Treeless upland
Steppe. forest
Forest, subalps
Forest
Dry forest
Treeless areas
Forest
Forest
Forest, subalps
Forest
Forest
Forest
Hahitat
Sourcr
11
Character
43
44
45
Makzdz
Kurjanchaj
31
41
42
1A x d l It)
No
-
~ ~ € C O ~ ~ R A l ~ l i I AN11
C ‘ A l ECOi,W+ICAl,
,
I~ESC’KIPrlOiLOF hAMI’LIKG IJO(~AI~ITIES
APPENDIX I
Y
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