Chromosome Polymorphism in Microtus (Alexandromys) Mujanensis (Arvicolinae, Rodentia)

Short Report

Cytogenet Genome Res Accepted: July 1, 2015 DOI: 10.1159/000439096 by M. Schmid Published online: August 29, 2015

Chromosome Polymorphism in Microtus (Alexandromys) mujanensis (Arvicolinae, Rodentia)

a e b Natalya A. Lemskaya Irina V. Kartavtseva Nadezhda V. Rubtsova d e a, c Fedor N. Golenishchev Irina N. Sheremetyeva Alexander S. Graphodatsky a b c Institute of Molecular and Cellular Biology SB RAS, Institute of Cytology and Genetics SB RAS, and Novosibirsk d e State University, Novosibirsk , Zoological Institute RAS, Saint Petersburg , and Biology and Soil Science Institute Far East Branch of RAS, Vladivostok , Russia

Key Words food specialization, resulting in a lack of significant dif- Chromosome polymorphism · Genus Microtus · Muya Valley ferences in the structure of molars [Musser and Carleton, vole · Pericentric inversion · Species group ‘maximowiczii’ 2005]. Despite the broad geographic distribution, many Microtus species live in similar ecological conditions, ad- aptation to which led to formation of analogous morpho- Abstract logical traits. For example, the sibling species Microtus The Muya Valley vole (Microtus mujanensis) has a constant arvalis (2n = 46) and M. rossiaemeridionalis (2n = 54) diploid chromosome number of 2n = 38, but an unstable have overlapping habitats but are reproductively isolated karyotype with polymorphic chromosome pairs. Here, we and are distinguished by chromosome number. So, often describe 4 karyotypic variants involving 2 polymorphic chro- the morphological similarity between different species is mosome pairs, MMUJ8 and MMUJ14, in 6 animals from Bur- determined less by relatedness but rather by convergence yatia using a combination of GTG-banding and chromosome or parallel development [Meyer et al., 1996]. On the oth- painting with M. agrestis probes. We suggest that the poly- er hand, karyological differences of Microtus voles are morphic pairs MMUJ8 and MMUJ14 were formed through much more pronounced. M. arvalis Pallas, 1778 has 2 pericentric inversions that played a major role during karyo- chromosomal forms ‘arvalis’ and ‘obscurus’ which have type evolution of the species. We also propose that the sta- the same diploid number (2n = 46) but differ in the posi- ble diploid number with some ongoing polymorphism in the tions of centromeres in some of the smaller autosomes number of chromosome arms indicates that this evolution- [Orlov and Malygin, 1969; Meyer et al., 1996]. Both spe- arily young endemic species of Russian Far East is on the way cies with stable and with polymorphic karyotypes were to karyotype and likely species stabilization. described in the genus. One of the main features of Mi- © 2015 S. Karger AG, Basel crotus evolution is significant inter- and intraspecific karyotype diversity that is not always reflected at the morphological level and often does not conform to the data Currently, the genus Microtus Schrank, 1798 compris- of experimental hybridization. For this reason the num- es more than 60 species that are characterized by weak ber of species within the genus as well as subspecies re- morphological isolation and pronounced uniformity of mains controversial.

© 2015 S. Karger AG, Basel N.A. Lemskaya 1424–8581/15/0000–0000$39.50/0 Institute of Molecular and Cellular Biology SB RAS 8, Ac. Lavrentieva ave. E-Mail [email protected] RU–630090 Novosibirsk (Russia) www.karger.com/cgr E-Mail lemnat @ mcb.nsc.ru Downloaded by: Rutgers University Alexander Library 198.143.38.97 - 9/4/2015 4:41:19 PM The ‘Asian’ lineage of Microtus voles includes 2 sub- Materials and Methods genera: Alexandromys Ognev, 1914 and Pallasiinus Kret- zoi, 1964 [Gromov and Erbaeva, 1995], which are merged Here, we analyzed the karyotypes of 6 M. mujanensis individuals (4 males and 2 females) from the Muya Valley in Buryatia into a single subgenus Alexandromys in the taxonomy of (N 56°21.877′ E 114°50.115′ ) on the west side of the Vitim River. Musser and Carleton [2005] based on morphological, cy- Fibroblast cell lines were established from lung and intercostal bi- togenetic and molecular data. This subgenus is represent- opsies. Chromosome preparations were obtained from early (be- ed by 9 species on the territory of the Russian Far East low 5) passages of the fibroblast cultures and also from bone mar- row without culturing. Images of GTG-banded metaphase chro- (RFE): M. oeconomus , M. sachalinensis , M. mongolicus , mosomes were captured prior to FISH [Sitnikova et al., 2007]. M. middendorffi , M. fortis , M. gromovi , M. maximowiczii , Chromosomes were numbered according to the karyotype pre- M. evoronensis , and M. mujanensis . The ‘maximowiczii’ sented in the Atlas of Mammalian Karyotypes [Sablina et al., 2006]. species group [Golenishchev, 1982] includes Maximo- We used the set of M. agrestis painting probes derived from flow- wizch’s vole (M. maximowiczii), Evoron vole (M. evoro- sorted chromosomes described in Sitnikova et al. [2007]. nensis) and the species of interest, the Muya Valley vole (M. mujanensis). These 3 vole species are included in the checklist of endemic fauna of the RFE. M. mujanensis was Results and Discussion described from a population found in the Muya hollow in a meadow steppe of northern Transbaikalia [Orlov and The ‘maximowiczii’ is a young species group that dis- Kovalskaya, 1978]. plays laboratory-proven reproductive isolation, morpho- The available fossil remains provide limited insights logical divergence and, importantly, remarkable chromo- into the origins of the Asian Microtus species group. The somal polymorphism as possible signs of the ongoing earliest remains of those voles are known from the cave speciation. ‘Bliznets’, RFE, late Pleistocene [Alexeeva and Goleni- The diploid number of M. mujanensis (2n = 38, NFa = shchev, 1986]. The remains with more precise dating 46–49) is constant in contrast to M. evoronensis (2n = (30,000–24,000 years before present, BP) were found in 38–40, NFa = 51–54) and M. maximowiczii (2n = 36–44, the cave ‘Medvezhyi Klyk’, RFE [Panasenko and Tiunov, NFa = 50–60). We hybridized all M. agrestis chromo- 2010]. According to phylogenetic data based on Cyt b some-specific DNA-probes onto GTG-banded chromo- gene sequences [Bannikova et al., 2010], the divergence somes of M. mujanensis ( figs. 1 , 2 ) and identified poly- time between M. maximowiczii and the closest species morphisms for 2 chromosome pairs of M. mujanensis , from the ‘maximowiczii’ group (M. sachalinensis) is MMUJ8 and MMUJ14, based on karyotype analyses of 270,000 BP. 6 individuals. In total, there were 4 karyotypic variants Species of the ‘maximowiczii’ group are characterized (fig. 3 ). Either both chromosomes MMUJ8 were acrocen- by weak morphological differentiation and wide intraspe- tric, or one was acrocentric and the other metacentric. cific variation of morphological and chromosomal fea- Chromosome pair MMUJ14 consisted of 2 acrocentric, 1 tures. Comparative analyses of conventionally stained acrocentric and 1 metacentric, or 2 metacentric chromo- and GTG-banded chromosomes of the group were per- somes. formed for the first time by Meyer et al. [1996]. The au- Previously, we hypothesized that the karyotype of the thors demonstrated that Evoron and Maximowizch’s ancestor of the genus Microtus had only acrocentric au- voles have karyotypes that are polymorphic both in dip- tosomes [Lemskaya et al., 2010]. Based on G-banding loid number and morphology of chromosomes [Meyer et comparative analyses, Meyer et al. [1996] suggested that al., 1996; Sablina et al., 2006]. However, based on karyo- the karyological polymorphism in M. mujanensis might type analyses of 7 individuals, it was shown that the M. be explained by several types of rearrangements such as mujanensis diploid number was invariable, 2n = 38. There centric or centromere-telomere fusions of acrocentric were 3 karyotypic variants with regard to the number of chromosomes or by inversions. The karyotypic variants metacentric autosomes (10, 11 and 12), and it was con- identified here clearly indicate a possible mechanism for cluded that heteromorphic variants of one chromosome the formation of heteromorphic chromosome pairs in M. pair were causing this variability. Chromosomes in the mujanensis (fig. 3 ). We presume that pericentric inver- study of Meyer et al. [1996] were not numbered, and it is sions are the main type of karyotype rearrangements ac- not possible to reliably identify the polymorphic chromo- companying M. mujanensis speciation. some pairs based on GTG-banding alone or to adopt the The Muya valley voles described by Meyer et al. [1996] current classification. (Dogopchan village, east side of the Vitim River) were

2 Cytogenet Genome Res Lemskaya et al. DOI: 10.1159/000439096 Downloaded by: Rutgers University Alexander Library 198.143.38.97 - 9/4/2015 4:41:19 PM b

a d

Fig. 1. GTG-banded karyotype of M. mujanensis male 1 (a ) and chromosome pairs 8 and 14 (b–d ) from the other 5 animals examined, showing the different observed chromosome morphologies (heteromorphisms) in MMUJ8 and MMUJ14. The lines and numbers on the right indicate localization of M. agrestis painting probes. The black dots indicate the positions of centromeres.

a c

b d

Fig. 2. a–d FISH with M. agrestis (MAG) whole-chromosome janensis (green signals). Precise identification of the chromosome painting probes onto GTG-banded chromosomes of M. mujanen- morphology on differentially stained chromosomes is problematic sis demonstrated 4 karyological variants. The painting probe for in M. mujanensis. The combination of GTG-banding with DAPI MAG4 highlighted segments on MMUJ8 and 13 (red signals). The staining reliably identifies the centromere (DAPI-positive, ar- MAG6 probe hybridized to the entire chromosomes 14 of M. mu- rows). A = Acrocentric; M = metacentric.

Chromosome Polymorphism in Microtus Cytogenet Genome Res 3 mujanensis DOI: 10.1159/000439096 Downloaded by: Rutgers University Alexander Library 198.143.38.97 - 9/4/2015 4:41:19 PM of more specimens from wider Muya Valley and Vitim Valley areas and subsequent chromosome analysis by GTG-banding and FISH could further illuminate ongoing speciation processes in M. mujanensis. We assume that the constant diploid number and the limited (2 pairs) chromosomal heteromorphism indicate that the Muya ac Valley vole is on the way to karyological and perhaps species stabilization. The origin of M. mujanensis can be indirectly deduced from paleoglaciological data [Enikeev and Staryshko, 2009]. During the Sartanskoe Glaciation (28,000–12,500 BP), the recent range of this species was covered by a gi- ant glacier-dammed lake for 14,000 years. So, we suppose b d that M. mujanensis could not originate earlier than 12,000 years ago. Presumably due to this short period of time, 2 chromosome pairs maintained their heterozygous status and remained in unfixed condition in populations. Such polymorphic karyological variants in young species like M. mujanensis are possibly representative of the shared ancestral polymorphism in the M. mujanensis -M. evoro- e nensis species complex. The polymorphic chromosome pairs could have possibly originated even earlier in the Fig. 3. Diagrams depicting the 4 karyological variants in M. muja- ancestor of the whole ‘maximowiczii’ group, the rapid nensis . a Both pairs 8 and 14 are composed of acrocentric chromo- formation of which is confirmed by paleontological and somes. b Chromosomes 8 are heteromorphic, both chromosomes 14 are acrocentric. c Both chromosomes 8 are acrocentric, while molecular data [Alexeeva and Golenishchev, 1986; Pana- chromosomes 14 are heteromorphic. d Both chromosomes 8 are senko and Tiunov, 2010; Haring et al., 2011]. acrocentric, both chromosomes 14 are metacentric. e Inversions Such revolving chromosomal polymorphisms in rap- leading to the heteromorphic pairs 8 and 14. A = Acrocentric; idly evolving species significantly complicate the use of M = metacentric; cen = centromere position; inv = inversion, red chromosomal rearrangements for phylogenetic recon- horizontal line indicates border of the inverted segment. struction due to the appearance of hemiplasic characters [Avise and Robinson, 2008; Robinson et al., 2008]. Kary- ological heteromorphism is found in many mammalian branches and can play a valid role in ‘phylogenetic discor- captured at a straight line distance of ∼ 60 km from our dance’ between species tree and the tree built using chro- collection site (N 56°21.877 ′ E 114°50.115′ , west side of mosomal rearrangements [e.g. Kovalskaya et al., 2011; the Vitim River, in the Muya River valley). The Muya Riv- Robinson and Ropiquet, 2011]. It is important to identify er flows into the river Vitim forming a single Vitim water and put less weight to such evolutionary chromosome re- area which also includes many river branches that reg- arrangements in the phylogenetic analysis to avoid or ularly flood surrounding silt marshes and grassy lakes minimize discrepancies, and to allow reconstruction of [Kropotkin and Polyakov, 1973; Snytko et al., 2014]. This the most plausible scheme of evolution. kind of swampy landscape surrounding the river does not represent a typical geographical barrier. Perhaps the 2 studied populations (Muya Valley and Vitim River) are Acknowledgements indeed distinct and differ by the presence of 2 and 1 heteromorphic pairs, respectively. However, we cannot ex- This study was funded in part by research grants of the RFBR, MCB and Biodiversity of RAS. The authors would like to thank Dr. clude that the limited number of individuals sampled F. Yang, P.C.M. O’Brien and Prof. M. Ferguson-Smith for kindly from both populations at a single occasion does not re- providing painting probes of M. agrestis. flect the whole range of the heteromorphism. Thus, it is possible that the karyotypes with 2 polymorphic chromosome pairs also occur in the Vitim population. Sampling

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Chromosome Polymorphism in Microtus Cytogenet Genome Res 5 mujanensis DOI: 10.1159/000439096 Downloaded by: Rutgers University Alexander Library 198.143.38.97 - 9/4/2015 4:41:19 PM