Chromosome Research 9: 107^120, 2001. # 2001 Kluwer Academic Publishers. Printed in the Netherlands
107
Comparative chromosome and mitochondrial DNA analyses and phylogenetic relationships within common voles (Microtus, Arvicolidae) Nina A. Mazurok1, Nadezhda V. Rubtsova1, Albina A. Isaenko1, Marina E. Pavlova1, Sergey Ya. Slobodyanyuk1, Tatyana B. Nesterova1,2* & Suren M. Zakian1 1 Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Department, Novosibirsk 630090, Russia; 2 MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK; Tel: 44 208 383 8286/8297; Fax: 44 208 383 8303; E-mail:
[email protected] *Correspondence Received 22 September 2000; received in revised form and accepted for publication by M. Schmid 29 November 2000
Key words: common vole, chromosome, GTG-banding, NORs, mitochondrial DNA, phylogeny
Abstract The four species of common voles within the genus Microtus ^ M. kirgisorum, M. transcaspicus, M. arvalis, and M. rossiaemeridionalis ^ are so closely related that neither morphological features nor paleontological evidence allow clari¢cation of their phylogeny. Analysis of vole karyotypes and mitochondrial DNA sequences, therefore, is essential for determining their phylogenetic relationships. A comparison of high resolution GTG-banding patterns allows us to ascertain the similarity between the karyotypes of these species, revealing that they are composed of rearrangements of the same chromosomal elements. Based on this analysis, we propose possible routes of chromosomal divergence involved in speciation within this group of voles and construct a phylogenetic tree of their karyotypes. We suggest that two different karyotypic variants existed during the course of vole evolution ^ one resulting in M. rossiaemeridionalis and M. transcaspicus, the other, M. kirgisorum and M. arvalis. As an alternative approach FITCH and KITSCH computer programs were used to construct a phylogenetic tree of vole molecular evolution based on a pairwise comparison of mitochondrial cytochrome b sequences and the divergence time of the species was determined. The correlation between the trees constructed using karyologic and molecular approaches is discussed in the context of other available data.
Introduction The group of common voles (arvalis) belongs to the genus Microtus (grey voles) and comprises four closely related species: M. kirgisorum, M. transcaspicus, M. arvalis, and M. rossiaemeridionalis. Two of the species, M. kirgisorum (previously known as M. ilaeus) and M.
transcaspicus, are morphologically distinct allopatric species found in Middle Asia. The other two species, M. arvalis and M. rossiaemeridionalis (previously knows as M. subarvalis or M. epiroticus), are sympatric sibling species and are widespread in Eurasia. Members of the M. arvalis species display chromosome forms ``arvalis'' and ``obscurus'' differing in centromere positions
108 on several pairs of small autosomes and also the Y chromosome. The karyoforms substitute for one another geographically and never occur together (Orlov & Malygin 1969, Meyer et al. 1972). It is therefore possible that these karyoforms represent individual species or that they re£ect speciation in progress (Zagorodnyuk 1991). Intensive divergence of the grey voles began relatively recently (1.2^0.7 million years ago) and the chromosome sets of many phenotypically similar Microtus species have undergone rapid rearrangement during a short span of geological time (Gromov & Polyakov 1977, Meulen 1978). Since neither morphological features nor paleontological evidence allow clari¢cation of the phylogeny within the arvalis group, one approach to tracing the evolutionary relationships between common voles has been to study the patterns of their chromosome sets (Malygin 1983, Meyer et al. 1996). By employing a method for differential banding of metaphase chromosomes, earlier cytogenetic studies have established the taxonomic status of four common vole species (see Nesterova et al. 1998b for review). It was shown that M. transcaspicus and M. rossiaemeridionalis are the most karyotypically close compared to M. kirgisorum and M. arvalis, which are more divergent (Meyer et al. 1985, 1996). These studies suggested a split from the ancestor karyotype into three branches giving rise to the future M. transcaspicus, M. rossiaemeridionalis and M. kirgisorum ( M. ilaeus) species, with subsequent evolution of M. arvalis from the M. kirgisorum branch. However, a problem with such analysis is that common vole karyotypes contain numerous small chromosomes with obscure individual patterns. Thus GTG- banding of metaphase chromosomes fails to distinguish between all the chromosome pairs and reveal more details of vole phylogeny. Previously we developed a reliable nomenclature for the differential chromosomal banding patterns of these four common vole species using a technique for obtaining extended chromosomes at the prometaphase stage (Mazurok et al. 1994, 1995, 1996a, 1996b). High resolution GTG-banding of these chromosomes allowed a more precise determination of homology between certain regions and entire chromosomes than pre-
N. A. Mazurok et al. viously (Meyer et al. 1985, Malygin & Sablina 1994). This paper describes further homologies in GTG-banding of several chromosomes and proposes signi¢cant changes to the interspecies chromosome homologies reported earlier (Meyer et al. 1985, Malygin & Sablina 1994). This has enabled us to ¢nd homologies for all the elements of the karyotypes under study and to propose routes of chromosome divergence during speciation within the group of common voles. In addition, a pairwise comparison of mitochondrial cytochrome b gene fragments was performed allowing us to determine the divergence time of these species and, in combination with cytogenetic data, to construct a phylogenetic tree for the arvalis group. Materials and methods Animal stocks Animals belonging to M. kirgisorum, M. transcaspicus, M. rossiaemeridionalis and two karyoforms of M. arvalis, ``arvalis'' and ``obscurus'', (subgenus Microtus) were used for cytogenetic analysis. ``Arvalis'' and ``obscurus'' karyoforms used in this study display four and ten acrocentric autosomes, respectively. The main characteristics of their karyotypes are listed in Table 1. The animals were trapped from their natural populations, transferred to the vivarium at the Institute of Cytology and Genetics (Novosibirsk, Russia), and bred in captivity. Animals of species Microtus oeconomus (subgenus Pallasiinus), Microtus agrestis (subgenus Microtus), and Microtus gregalis (subgenus Stenocranius) were trapped in the Novosibirsk region and Arvicola terrestris (genus Arvicola) were obtained from the vivarium, Institute of Cytology and Genetics. M. agrestis, the closest relative of the arvalis group, belongs to another group of the same subgenus; M. oeconomus and M. gregalis, members of the other two subgenera with Microtus; A. terrestris belongs to the genus Arvicola, closest to genus Microtus and was used as outgroup species. These species were used in the analysis of mitochondrial DNA.
Common vole phylogeny
109
Table 1. Main karyotype characteristics of voles of ``arvalis'' group. Species
M. M. M. M.
rossiaemeridionalis transcaspicus kirgisorum arvalis ``arvalis'' ``obscurus''
n
27 26 27 23 23
Type of chromosome, n acrocentric
metacentric
25 X/Y 24 X/Y 14 X/Y 4Y 10
1 1 12 18 X 12 X/Y
Cytogenetic preparations and differential staining Metaphase chromosomes were obtained from spleen and primary ¢broblast cell cultures. Spleen cell cultures were made by routine technique (Graphodatsky & Radjabli 1988). The primary lung ¢broblast cultures were generated by trypsinization and cultivated as described (Nesterova et al. 1994). Chromosome preparations from both types of cell cultures were made using ethidium bromide, added to the culture 2^2.5 h prior to ¢xation (at a ¢nal concentration of 6 mg/ml). Colchicine was added 40 min before ¢xation (at a ¢nal concentration of 0.3 mg/ml). Cell hypotony and ¢xation were performed as described elsewhere (Mazurok et al. 1994). GTG-staining was performed according to Seabright (1971) with minor modi¢cations. NORs were visualized by Ag-staining as described by Howell & Black (1980). To identify NOR-bearing chromosomes, Ag-staining was followed by GTG-banding of the same metaphase spreads (Graphodatsky 1981). Cytochrome b Total DNA was isolated conventionally from the liver of adult animals (Sambrook et al. 1989). A 400-bp fragment of the mitochondrial cytochrome b gene was ampli¢ed using primers L14724 (50 -CGAAGCTTGATATGAAAAACCATCGTTG) and H15149 (50 -AAACTGCAGCCCCTCAGAATTGATATTTGTCCTCA) (Kocher et al. 1989, Meyer et al. 1990). PCR products were sequenced directly by linear polymerase chain reaction (Murray 1989). Phylogenetic trees were constructed with the PHYLIP package (Felsenstein 1993) using FITCH and KITSCH programs, which employ a matrix of genetic dis-
Number of arms, NF
Chromosomes with NORs, n
28 27 39 41/42 36
16 11 13 X 5 10
tances. Genetic distances between species were determined using the DNADIST program by the Kimura method (Kimura 1980). Results and discussion Homologies in GTG-banding patterns of common vole chromosomes Previously we obtained high resolution G-banding of chromosomes of four vole species from the arvalis group (Mazurok et al. 1994, 1995, 1996a, 1996b). In this paper, we compare the chromosomes of these species to determine the similarity between the different karyotypic elements. Figure 1 shows haploid sets of G-stained chromosomes for M. rossiaemeridionalis, M. transcaspicus, M. kirgisorum, and two chromosome forms of M. arvalis. The chromosomes are combined in 22 groups according to homology of their G-banding patterns (Figure 1a). G-banding of X chromosomes is similar only within the euchromatic region and differs in the size and location of the heterochromatic block. Y chromosomes are composed exclusively of C heterochromatin (Zakian et al. 1991) and, despite the lack of GTG-banding homology, are combined in one group (Figure 1b). All chromosomes, excluding the Y, display homologies with either entire chromosomes of other species or their large fragments. Our analysis shows that the karyotypes of the species studied are composed of the same elements in different combinations. A pairwise species comparison detected the highest number of chromosomes with similar G-banding pattern between M. rossiaemeridionalis and M. transcaspicus (22) and between the ``arvalis'' and ``obscurus''
110
N. A. Mazurok et al.
Common vole phylogeny
111
Figure 1. Comparison of G-banded chromosomes of common voles. (a) chromosomes with detected homology and (b) Y chromosomes lacking observable homology in their G-banding. (R) M. rossiaemeridionalis; (T) M. transcaspicus, (K) M. kirgisorum, ) region of inversion. (O) ``obscurus'', (A) ``arvalis'', ( ÿ ) centromeric region, (+) NOR, and (
karyoforms of M. arvalis (21; Table 2). However, many acrocentric chromosomes displaying similar G-banding patterns differ in the locations of their centromeres so that the number of truly identical chromosomes between species pairs is lower than expected. For example, there are only ¢ve identical chromosomes in the pair ``arvalis'' and M. rossiaemeridionalis and three in the pair ``arvalis'' and M. transcaspicus.
Comparison of NOR chromosomal localizations Subchromosomal NOR localizations were established for all the species studied using successive Ag- and GTG-banding of vole chromosomes (Mazurok et al. 1996b, Nesterova et al. 1998b). The total numbers of NOR-bearing chromosome pairs detected between common vole species are indicated in Table 1. Chromosomes of different
112
N. A. Mazurok et al.
Table 2. Number of autosomes homologous in their G-banding pattern and morphology within the arvalis group of voles. Species
M. M. M. M.
rossiaemeridionalis transcaspicus kirgisorum arvalis, ``arvalis''
n
27 26 27 23
M. transcaspicus
22(17)
M. kirgisorum
15(8) 13(8)
M. arvalis ``arvalis''
``obscurus''
11(5) 10(3) 13(9)
13(10) 14(10) 13(10) 21(17)
Number of chromosomes similar in G-banding pattern. The number of chromosomes similar in both G-banding pattern and centromere position is shown in parentheses.
species with similar G-banding may differ in both NOR occurrence and their subchromosomal localizations (Figure 1, Table 1). All the NORs detected in M. rossiaemeridionalis and M. transcaspicus are localized to pericentromeric regions of long arms of acrocentric chromosomes (secondary constriction regions under routine staining). In M. kirgisorum and ``arvalis'' chromosomes, NORs are localized to short arms of acrocentric chromosomes, while in ``obscurus'' they are additionally found at the telomeres of large metacentrics (Figure 1). Certain homeologous chromosomes harbour NORs in all, or the majority, of the species studied. Other chromosomes have NORs only in one species. In general, all but three chromosomal elements of common voles carry NORs in at least one species. It is possible that the differences observed in NOR localization relative to the centromere might have appeared during species divergence as a result of inversions involving pericentromeric regions. Structural rearrangements and evolution of common vole chromosomes The choice of the correct ancestral karyotype is very important in phylogenetic studies since this will affect the estimation of number and types of chromosome rearrangements that have occurred during the course of evolution. According to earlier studies, the primitive Arvicolidae karyotype, which includes genus Microtus, comprises acrocentrics and one small metacentric pair with a modal number of 2n 56 (Matthey 1973, Modi 1987). The putative primitive X chromosome of Arvicolidae is acrocentric and entirely euchromatic. It has been postulated that vole karyotypes evolve predominantly towards a
decrease in chromosome number due to tandem rearrangements, namely Robertsonian, centromeric^telomeric, and telomeric fusions (Meyer et al. 1985, Modi 1987). The M. rossiaemeridionalis (2n 54) and M. transcaspicus (2n 52) karyotypes, containing all acrocentrics and one metacentric pair, closely match the description of the Arvicolidae ancestor. Therefore a karyotype consisting of M. rossiaemeridionalis and/or M. transcaspicus acrocentric chromosomes and small metacentric speci¢c for M. rossiaemeridionalis was hypothesized to be the primitive form ^ the ancestor of common voles. Where the homeologous M. rossiaemeridionalis and M. transcaspicus chromosomes differed in centromere position, the chromosome version either occurring in other common vole species or ancestral to the entire Arvicolidae family (Modi 1987) was selected as a primitive. A determination of the type of chromosome alterations during the common vole karyotypic evolution relative to the hypothetical ancestral form is presented in Table 3. All the rearrangements detected in common vole chromosomes through interspecies comparison of high resolution G-banding patterns fall into four types: centric transpositions, peri- and paracentric inversions, and tandem fusions (Robertsonian and centromeric^telomeric). Frequencies of these rearrangement types in the species studied are shown in Table 4. Centric transpositions (displaying the same G-banding but differing in the centromere position) are typical of common voles. They may result from both pericentric and paracentric inversions involving the entire acrocentric long arm. They can also arise through telomeric and telomeric^centromeric fusions of original excess acrocentric chromosomes of the ancestral
Common vole phylogeny
113
Table 3. Homology between autosomes in common voles. M. rossiaemeridionalis
M. transcaspicus
Chr.
Rearrangement type
Chr.
1 2 3 4 5 6 7 8 9 10* 11* 12* 13* 14* 15* 16* 17* 18* 19* 20* 21* 22* 23* 24* 25* 26
p.inv c.transa c.transa
1 2 3 4 5 6 7 8* 9* 10* 11* 12 13 14 15* 16* 17* 25q 18* 19* 20* 21 22 23* 25p 24
c.transa c.transa
Rearrangement type
c.transa
rob
rob inv
M. kirgisorum
M. arvalis ``arvalis''
``obscurus''
Chr.
Rearrangement type
Chr.
Rearrangement type
Chr.
Rearrangement type
1 13 2 15* 14* 16* 17* 3 4 18* 5 8 6 7 19* 10 11 21* 9 20* 23* 24* 22* 25* 26* 12
c.trans
5 1q 1p 2q 3q 4p 2p 4q 6 3p 8 7 13 9 12 14 19* 15 11 10* 21* 17 16 20* 22* 18
c.trans rob rob rob rob tan rob tan inv rob c.trans inv inv c.transb c.trans inv
5 1q 1p* 2q 3q 4p* 2p* 4q 6 3p* 13 7 15 14* 16* 10 18* 17 9 8 21* 19 11 20* 22* 12
c.trans rob rob rob rob tan rob tan inv rob
c.trans p.inv
c.transb inv inv inv inv c.transb p.inv inv c.transb p.inv inv p.inv c.transa
inv inv c.transb c.trans c.transb
inv c.transa inv inv c.transb c.transb
Chromosome numbering corresponds to the species nomenclature. The type of rearrangement speci¢c of particular chromosome compared to its ancestor variant is indicated: inv ^ pericentric inversion; p.inv ^ paracentric inversion; tan ^ centromere-telomere tandem translocation; rob ^ Roberobsonian translocation; c.trans ^ centric transposition; a ^ G-banding pattern does not exlude the possibility of paracentric inversion; b ^ G-banding pattern does not exlude the possibility of pericentric inversion; * ^ chromosomes with localized NORs. Table 4. Frequencies of different type rearrangements of common vole autosomes. Type of rearrangement
R
Tandem translocation Centric transposition Inversion periparaTotal
^ 4 ^ 1 5
T
1 1 1 ^ 3
K
^ 6 (2) 6 4 16
A ``arvalis''
``obscurus''
4 7 (3) 6 ^ 17
4 4 (1) 4 ^ 12
Total
%
9 22 (6) 17 5 53
17 41.5(11.3) 32.1 41:5 9.4 100
Number of centric transpositions that are unexplainable with inversions is shown in parenthesis. (R) M. rossiaemeridionalis; (T) M. transcaspicus, (K) M. kirgisorum, (A) M. arvalis.
karyotype followed by inactivation of one of the two centromeres (Meyer et al. 1985). Previously inactivated centromeres might be subsequently reactivated or alternatively centric transpositions
may result from an interchromosome translocation of a centromere-containing region. Paracentric inversions are mainly typical of the M. kirgisorum karyotype while pericentric
114 inversions are more typical of M. kirgisorum and M. arvalis. Tandem fusions were detected only in the M. arvalis karyotype. Consequently, its chromosomes are divided into clear-cut groups of large and small chromosomes, whereas the karyotypes of other species are represented by chromosome series with continuously decreasing sizes. Frequencies of different types of chromosome rearrangements in common vole karyotypes display certain differences from those of the Arvicolidae family (Modi 1987). Interspecies euchromatin chromosome rearrangements of nine types are described in Arvicolidae (Modi 1987) whereas we found only four types in common voles. The tandem fusion rate is 41.2% in Arvicolidae but 17% in common voles. The rates of pericentric inversions are 12.8% and 32.1%, respectively; of paracentric inversions, 8.5% and 9.4% and of centric transpositions, 12.1% and 41.5%, respectively. Note that centric transpositions may have resulted from inversions during chromosome divergence; therefore the percentage of inversions in the rearrangements within the group of common voles may be considerably higher. The data on comparative analysis of GTG-banded chromosomes and NOR localization suggest that inversions of various types including both small regions and entire chromosome arms have played an important role in the evolution of common voles. This conclusion is supported by the data on subchromosomal mapping of ¢ve X-linked genes (Nesterova et al. 1998a). Evolution of the common vole X chromosome comprises at least two inversions ^ one in M. rossiaemeridionalis, the other in M. arvalis and M. kirgisorum. These inversions are undetectable by G-banding, as they cover the region of 3^4 bands with a central symmetry. G-banding also fails to detect small inversions occurring within a band. For example, the MS3 repeat was detected in homologous M. kirgisorum and M. arvalis autosomes as a discrete signal located on different sides of the positive band, indicating that this inversion escaped detection by differential staining (Elisaphenko et al. 1998). Thus the actual number of inversions having occurred during chromosome divergence may be higher than detected by analysis of differentially stained chromosomes due to small inversions
N. A. Mazurok et al. and inversions covering the regions with symmetrical G-banding patterns. Karyology-based phylogenetic tree We used the karyotypic analysis presented above to construct a hypothetical phylogenetic tree for the four common vole species (Figure 2), re£ecting the number and types of putative changes during chromosome divergence of the species in question. M. rossiaemeridionalis and M. transcaspicus karyotypes are the most closely related of the four species studied, while the M. arvalis and M. kirgisorum karyotypes display numerous differences. However, this latter pair contains chromosomes absent in the other species, suggesting their assignment to a common branch of the tree (Meyer et al. 1996). We have detected four common pericentric inversions in M. kirgisorum and M. arvalis karyotypes, absent in the other two species. NOR patterns suggest that there were two karyotypic variants in common vole evolution ^ one gave origin to M. rossiaemeridionalis and M. transcaspicus; the other to M. kirgisorum and M. arvalis. The data obtained also indicate different rates of chromosome divergence in the two evolutionary branches. M. kirgisorum differs from M. arvalis at least as much as from M. rossiaemeridionalis and M. transcaspicus. The karyotype of M. kirgisorum exhibits one unique pericentric and four unique paracentric inversions. The pattern of the M. arvalis karyotype suggests a new stage of fusions when large two-arm chromosomes are formed from the already composite chromosomes. It is likely that the M. arvalis karyotype was formed later than those of the other three species. In addition, numerous chromosome rearrangements and differences in NOR localization were detected while comparing the two karyotype patterns of M. arvalis. The karyotype divergence between ``arvalis'' and ``obscurus'' is comparable with interspecies divergence of common voles. Mitochondrial DNA analysis Sequences of various mtDNA regions are now widely used for assessing genetic distances between related species and reconstructing evolutionary
Common vole phylogeny
115
Figure 2. Karyology-based phylogenetic tree of common voles: The rearrangement type speci¢c to a particular chromosome of given species (number in bold) relative to the ancestral variant (number in parenthesis, M. rossiaemeridionalis or M. transcaspicus (t)): & tandem fusion, * paracentric inversion, * pericentric inversion, centric transposition, putative centric transposition resulting from paracentric inversion, and putative centric transposition as a result of pericentric inversion. Two numbers in bold, separated by a comma, correspond to M. arvalis and M. obscurus chromosome numbers respectively; three comma-separated numbers in bold correspond to M. arvalis, M. obscurus and M. kirgisorum chromosome numbers, respectively.
relationships. The mitochondrial gene encoding cytochrome b is most frequently used for constructing phylogenetic trees of mammals (Kocher et al. 1989). To gain independent data on
phylogenetic relationships within the group of common voles, comparative analysis of a 400-bp sequence of the mitochondrial cytochrome b gene was performed. mtDNA sequences of several
116 other vole species were also included in the analysis: M. agrestis, M. oeconomus, M. gregalis and A. terrestris. A pairwise comparison of mtDNA sequences has demonstrated that the majority of substitutions occur at the third codon position. Intraspeci¢c mtDNA polymorphisms were estimated to occur at a frequency of less than 1%, which is similar to the rate found for the other species studied (Wilcox et al. 1997). KITSCH and FITCH programs from the software package PHYLIP (Felsenstein 1993) were
N. A. Mazurok et al. used to construct phylogenetic trees. The former implies the existence of a molecular clock, that is, a constant molecular evolutionary rate within different branches of the tree. KITSCH trees allow the divergence times to be estimated. The latter program permits different evolutionary rates within individual branches and, as a consequence, FITCH trees more accurately re£ect the species branching order. A KITSCH tree is shown in Figure 3. The time scale implies that the molecular evolutionary rate
Figure 3. Rooted mitochondrial cytochrome b gene tree inferred by the KITSCH program. Numbers at the nodes are the bootstrap values obtained in 100 replicates. Digits at the tips mean the number of specimens with certain hyplotype.
Common vole phylogeny
117
Figure 4. Unrooted mitochondrial cytochrome b gene tree inferred by the FITCH program. Numbers at the nodes are the bootstrap values obtained in 100 replicates.
of rodent mtDNA equals 7^8% substitutions per 1 million years (Catze£is et al. 1992); the time depth of the tree approximating to 1.17 million years. This upper age estimate of the species studied dates their evolutionary radiation from the Pleistocene age. Typical of this era in geological history were numerous cyclic glaciations favouring origination of geographical isolates and consequently species £ocks. An unrooted FITCH tree (Figure 4) with a pronounced ``explosive'' evolution (from one point) elucidates the evolution of the species studied. The four common vole species in question are located on one branch of the FITCH tree, matching their systematic position as forming a separate group. High bootstrap values for both FITCH (94%) and KITSCH (89%) trees support a monophyletic origin of this group. A bootstrap value is a quantitative estimate of con¢dence limits on the branching order within a phylogenetic tree and is usually used as a criterion of monophyletic origin of a group of species (Felsenstein 1985). The analysis of mtDNA performed suggests the following evolution of common vole species: the common ancestor of the arvalis group appears
to have separated from the other grey vole species about 0.5^0.6 million years ago; ancestral M. kirgisorum was the ¢rst to diverge within this group; it is likely that ancestral M. arvalis, M. rossiaemeridionalis, and M. transcaspicus appeared through geographical isolation from the ancestral population followed by evolution to individual species rather than successive evolution. Phylogenetic relationships of common voles Phylogenetic trees of common voles within the genus Microtus were constructed based on both chromosome and mtDNA analyses of the species in question. Both trees demonstrate a considerable divergence of M. kirgisorum compared with the other common voles. However, the molecular and cytogenetic trees present different branching order for the other three species; the former implies radiating evolution of all three species, the latter demonstrates a splitting event from the ancestor into two evolutionary branches,
118 one comprising M. rossiaemeridionalis and M. transcaspicus, the other, M. kirgisorum and M. arvalis. To understand the causes of this discrepancy and to establish the genuine phylogenetic relationships within the common vole group, we analysed the differences underlying both molecular and karyotypic evolution assays. Analysis of mtDNA detects gradually accumulating substitutions in nucleotide sequences which are random and reversible as far as the gene product remains functional. The majority of phylogenetic models, including KITSCH program, imply that these substitutions are occurring at the constant rate, allowing an estimation of the time of separation and phylogenetic distances between different lineages. Our mtDNA analysis dated the age of grey voles and the time of common vole divergence to Pleistocene era and these estimates are in agreement with paleontological evidence on Pleistocene arvalis-like voles (Malygin 1983, Meyer et al. 1996). However, it has been demonstrated in many instances that molecular evolutionary rates vary signi¢cantly among orders and between species of the same order leading to distortion of phylogenetic trees (Janke et al. 1994, Gissi et al. 2000, Yoder & Yang 2000, Bromham et al. 2000). We tried to avoid this problem by using the FITCH algorithm, which allows unequal evolutionary rates in different species in order to determine more accurate branching order. Despite this method being effective in many phylogenetic studies, some signs of inaccuracy and bias were found when the method was tested on simulated data for rates of evolution which varied between different sites (Kuhner & Felsenstein 1994). Therefore, the distortion of the FITCH tree branching might occur as a result of unequal rates of nucleotide substitutions in different vole species. In contrast to gradual accumulation of nucleotide substitutions occurring in the mitochondrial genome, the cytogenetic analysis reveals major (revolutionary) changes, re£ecting a higher level of genomic organization. Considerable and fast reorganization of karyotype often accompany speciation and was suggested to occur in the order Carnivora during Canidae formation and in evolution of the cotton rats (Rubtsov et
N. A. Mazurok et al. al. 1988, Elder 1980). The most spectacular and gross chromosome evolution was found in Muntiacus family (Lee et al. 1993, Yang et al. 1997). The main difference between karyotypic evolution and molecular evolution is that once chromosome rearrangement has occurred the mutation is irreversible and detected in all following generations of the lineage. It is very unlikely that exactly the same chromosome rearrangement would occur in two independent lineages. This allows to use chromosome rearrangements as reliable markers for phylogenetic studies. Comparing these two methods we can conclude that molecular analysis is preferential for establishing the divergence time on a large scale, but karyotypic analysis appears to be more reliable for determining the phylogenetic relationships between closely related species. To verify our conclusions and clarify the common vole phylogeny further we combined all data obtained by various methods. Experimental mating data has demonstrated that M. transcaspicus and M. rossiaemeridionalis are the most closely related, while M. kirgisorum and M. arvalis have diverged as much as M. rossiaemeridionalis and M. kirgisorum (Meyer et al. 1985). In spite of this zoological evidence, previous cytogenetic studies suggested independent karyotypic divergence of M. transcaspicus and M. rossiaemeridionalis and the existence of a common ancestor for M. arvalis and M. kirgisorum (Meyer et al. 1996). The same order of ¢ve X chromosome genes we found in M. arvalis and M. kirgisorum but not in M. rossiaemeridionalis and M. transcaspicus supports the hypothesis that M. arvalis and M. kirgisorum belong to the same evolutionary branch (Nesterova et al. 1998a). The additional evidence for the common ancestor for these two species was provided from the localization of the repeat, typical of common vole sex chromosomes, on homologous autosomes of M. arvalis and M. kirgisorum only (Elisaphenko et al. 1998). In spite of high homology of M. rossiaemeridionalis and M. transcaspicus karyotypes and mating data, these two species were considered to arise from different evolutionary branches. Our comparative karyotypic analysis and data on NOR chromosome localization
Common vole phylogeny suggested the existence of two distinct karyotypic variants ^ one giving rise to M. rossiaemeridionalis and M. transcaspicus; the other to M. kirgisorum and M. arvalis. Comparison of taxonoprint patterns of common vole repetitive DNA also supports the existence of two evolutionary branches, since the repeat distribution patterns of M. rossiaemeridionalis and M. transcaspicus are as similar as those of M. arvalis and M. kirgisorum (Shevchenko et al. 1999). The taxonoprint method allows the species whose genomes have undergone drastic changes in different repeat numbers (ampli¢cation of certain sequences and chromosome rearrangements) to be distinguished; however, it fails to trace the point mutation accumulation. Therefore, the taxonoprint data agree more closely with the cytogenetic evidence than with the mtDNA analysis. Each method we used in this study is able to assess only part of the vole evolutionary history, but integrated analysis of all available data obtained by various approaches allows us to get a broader perspective of vole phylogenetic relationships. Overall, our data suggest the following evolution of common voles. The common ancestor of the arvalis group separated from the rest of the grey vole species in the Pleistocene era, about 0.5^0.6 million years ago, resulting in two evolutionary branches. It is likely that the ancestor of M. kirgisorum and M. arvalis separated earlier than the ancestor of M. rossiaemeridionalis and M. transcaspicus, and that speciation occurred through partition of the common ancestral population and formation of geographical isolates rather than through successive evolution. The degree of divergence between species within individual evolutionary branches is different since M. kirgisorum and M. arvalis appear to have diverged to a greater extent than the other two species. M. arvalis is considered to be the youngest species among common voles and is still undergoing active evolution, as indicated by considerable intraspecies polymorphism and chromosome forms. Acknowledgements We would like to thank Dr. Neil Brockdorff for the advice and support of this work and Drs. Sarah
119 Duthie, Alistair Newall, Maria Gomez and Colette Johnston for the discussion and valuable comments during preparation of the paper. This work was supported by a grant from the Russian Foundation for Basic Research (98-04-49561).
References Bromham L, Penny D, Rambaut A, Hendy MD (2000) The power of relative rates tests depends on the data. J Mol Evol 50: 296^301. Catze£is FM, Aguilar J-P, Jaeger J-J (1992) Muroid rodents: phylogeny and evolution. Tree 7: 122^126. Elder FF (1980) Tandem fusion, centric fusion, and chromosomal evolution in the cotton rats, genus Sigmodon. Cytogenet Cell Genet 26:199^210. Elisaphenko EA, Nesterova TB, Duthie SM et al. (1998) Repetitive DNA sequences in the common vole: cloning, characterisation and chromosome localisation of two novel complex repeats MS3 and MS4 from the genome of the East European vole Microtus rossiaemeridionalis. Chrom Res 6: 351^360. Felsenstein J (1985) Con¢dence limits on phylogenesis: an approach using the bootstrap. Evolution 39: 783^791. Felsenstein J (1993) PHYLIP (Phylogenetic Inference Package), version 3.5C, Dept. of Genetics, Univ. of Washington, Seattle. Gissi C, Reyes A, Pesole G, Saccone C (2000) Lineage-speci¢c evolutionary rate in mammalian mtDNA. Mol Biol Evol 17: 1022^31. Graphodatsky AS (1981) Nucleolus organiser regions of the chromosomes of domestic pig. Tsitologiya i Genetica 15: 29^31. Graphodatsky AS, Radjabli SI (1988) Chromosomes of farm and laboratory mammals. Novosibirsk: Nauka. Gromov IM, Polyakov IY (1977) The fauna of Russia. Mammals. Voles (Microtinae). Leningrad: Nauka. Howell WM, Black DA (1980) Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36: 1014^1015. Janke A, Feldmaier-Fuchs G, Thomas WK, von Haeseler A, Paabo S (1994) The marsupial mitochondrial genome and the evolution of placental mammals. Genetics 137: 243^56. Kimura M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 16: 111^120. Kocher TB, Thomas WK, Meyer A et al. (1989) Dynamics of mitochondrial DNA evolution in animals: ampli¢cation and sequencing with conserved primers. Proc Nat. Acad Sci USA 86: 6196^6200. Kuhner MK, Felsenstein J (1994) A simulation comparison of phylogeny algorithms under equal and unequal evolutionary rates. Mol Biol Evol 11: 459^68. Lee C, Sasi R, Lin CC (1993) Interstitial localization of telomeric DNA sequences in the Indian muntjac chromosomes: further evidence for tandem chromosome
120 fusions in the karyotypic evolution of the Asian muntjacs. Cytogenet Cell Genet 63: 156^9. Malygin VM (1983) Systematics of common voles. Moscow: Nauka. Malygin VM, Sablina OV (1994) Karyotypes. In: Sokolov VE & Bashenina NV, eds. Common vole: the sibling species. Moscow: Nauka, pp. 7^25. Matthey R (1973) The chromosome formulae of eutherian mammals. In: Chiarelli AB & Capanna F, eds. Cytotaxonomy and vertebrate evolution. Academic Press: London and New York, pp. 531^616. Mazurok NA, Rubtsov NB, Nesterova TB, Zakian SM (1994) High-resolution G-banding of chromosomes in Microtus kirgisorum (Muridae, Rodentia). Cytogenet Cell Genet 67: 208^210. Mazurok NA, Nesterova TB, Zakian SM (1995) High-resolution G-banding of chromosomes in Microtus subarvalis (Rodentia, Arvicolidae). Hereditas 123: 47^52. Mazurok NA, Isaenko AA, Nesterova TB, Zakian SM (1996a) High-resolution G-banding of chromosomes in the common vole Microtus arvalis (Rodentia, Arvicolidae). Hereditas 124: 229^232. Mazurok NA, Rubtsova NV, Isaenko AA, Nesterova TB, Zakian SM (1996b) Comparative analysis of chromosomes in Microtus transcaspicus and Microtus subarvalis (Arvicolidae, Rodentia): high-resolution G-banding and localisation of NORs. Hereditas 124: 243^250. Meulen AJ (1978) Microtus and Pitymys (Arvicolidae) from Cumberland Cave, Maryland, with a comparison of some new and old world species. Ann Carnegie Mus 47: 101^145. Meyer A, Kocher TB, Basasiwaki P, Wilson AC (1990) Monophyletic origin of Lake Victoria cichlid ¢shes suggested by mitochondrial DNA sequences. Nature 347: 550^553. Meyer MN, Orlov VN, Sholl ED (1972) Sibling species in Microtus arvalis group (Rodentia, Cricetidae). Zool J 51: 724-738. Meyer MN, Radjabli SI, Bulatova NS, Golenishchev FN (1985) Karyological pecularities and probable relations of common voles of the group ``arvalis'' (Rodentia, Cricetidae, Microtus). Zool J 64: 417^428. Meyer MN, Golenishchev FN, Radjabli SI, Sablina OV (1996) Grey voles (Subgenus Microtus) of the fauna of Russia and adjacent territories. Trans Zool Inst Ross Akad Nauk (St. Petersburg), 232: 90^112. Modi WS (1987) Phylogenetic analyses of chromosomal banding patterns among the nearctic Arvicolidae (Mammalia, Rodentia). Syst Zool 36: 109^136.
N. A. Mazurok et al. Murray V (1989) Improved double-stranded DNA sequencing using linear polymerase chain reaction. Nucl Acid Res 17: 88^89. Nesterova TB, Mazurok NA, Matveeva NM et al. (1994) Demonstration of the X-linkage and order of the genes GLA, G6PD, HPRT and PGK in two vole species of the genus Microtus. Cytogenet Cell Genet 64: 250^255. Nesterova TB, Duthie SM, Mazurok NA et al. (1998a) Comparative mapping of X chromosomes in rat and ¢ve vole species of the genus Microtus. Chrom Res 6: 41^48. Nesterova TB, Mazurok NA, Rubtsova NV, Isaenko AA, Zakian SM (1998b) The vole gene map. ILAR J 39: 138^ 144. Orlov VN, Malygin VM (1969) Two forms of 46 chromosome Microtus arvalis Pallas. In: Vorontsov NN, ed. The Mammals (evolution, karyology, systematics, faunistics). Materials of the 2nd All-Union Symposium on Mammals. Novosibirsk: Nauka, pp. 143^144. Rubtsov N, Graphodatsky A, Matveeva VG et al. (1988) Silver fox gene mapping: conserved chromosome regions in the order Carnivora. Cytogenet Cell Genet 48: 95^98. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Second edition, Vol. I^III, Cold Spring Harbor: New York. Seabright M (1971) A rapid banding technique for human chromosomes. Lancet 2: 971^972. Shevchenko AI, Slobodyanyuk SY, Zakiyan SM (1999) Variability of DNA repeats in four species of common voles. Moleculyarnaya Biologia 33: 622^627. Wilcox TP, Hugg L, Zeh JA, Zeh DW (1997) Mitochondrial DNA sequencing reveals extreme genetic differentiation in a cryptic species complex of neotropical pseudoscorpions. Mol Phylogenet Evol 7: 208^216. Yang F, O'Brien PCM, Wienberg J, Neitzel H, Lin CC, Ferguson-Smith MA (1997) Chromosomal evolution of the Chinese muntjac (Muntiacus reevesi). Chromosoma 106: 37^43. Yoder AD, Yang Z (2000) Estimation of primate speciation dates using local molecular clocks. Mol Biol Evol 17: 1081^90. Zagorodnyuk IV (1991) Karyotypic variability of 46-chromosome forms of Microtus arvalis (Rodentia) voles: taxinomic estimate. Vestnik Zoologii 1: 36^45. Zakian SM, Nesterova TB, Cheryaukene OV, Bochkarev MN (1991) Heterochromatin as a factor affecting the inactivation of X-chromosome in interspeci¢c hybrid voles (Microtidae, Rodentia). Genet Res 58: 105^110.
