Voles of the Genus Alexandromys from the Verkhnebureinskaya Depression I

Home , Alexandromys, Amgun, Bureya (river), Chegdomyn, Microtus

ISSN 1062-3590, Biology Bulletin, 2017, Vol. 44, No. 7, pp. 813–819. © Pleiades Publishing, Inc., 2017. Original Russian Text © I.N. Sheremetyeva, I.V. Kartavtseva, T.V. Vasiljeva, L.V. Frisman, 2016, published in Zoologicheskii Zhurnal, 2016, Vol. 95, No. 5, pp. 597–603.

Voles of the Genus Alexandromys from the Verkhnebureinskaya Depression I. N. Sheremetyevaa, *, I. V. Kartavtsevaa, T. V. Vasiljevaa, and L. V. Frismana, b aInstitute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences, Vladivostok, 690022 Russia bInstitute of Complex Analysis of Regional Problems, Far East Branch, Russian Academy of Sciences, Birobidzhan, 679016 Russia *e-mail: [email protected] Received June 15, 2015

Abstract⎯Voles of an unclear taxonomic status have been found in the Bureya River depression (Khabarovsk krai) for the first time. An analysis of the nucleotide sequence of the control region in two individuals revealed their greatest similarity with species of the “maximowiczii” group. The minimum p-distance (0.0217 ± 0.0044) was observed between individuals from the Bureya River depression and Alexandromys evoronensis studied earlier. The karyotype of one individual studied had a chromosomal aberration distinguished at the heterozygous state (2n = 37; NF = 55). A similar karyotype was described in the polymorphic A. maximowiczii population from the Khentii Mountains in Mongolia.

Keywords: East Asian voles, Alexandromys, Verkhnebureinskaya Depression, distribution, chromosomes, mtDNA control region DOI: 10.1134/S1062359017070159

The taxonomy of the East Asian meadow voles of yaeva, 1965; Kuzikov et al., 1979). Later, the speci- the genus Alexandromys (=Microtus) has been widely mens caught in the valley of the Urgal River were discussed in scientific papers. The maximowiczii determined as Far Eastern voles: Alexandromys fortis group of species including sibling species (A. maximo- Buchner 1889 (Khamaganov, 1973; Kostenko, 1984, wiczii (Schrenk 1859), A. mujanensis (Orlov et Kowal- 2000). Despite the contradictory data, in the last atlas skaia 1978) and A. evoronensis (Kowalskaia et Sokolov of the distribution of rodents (Shenbrot and Krasnov, 1980)) is systematically one of the complex groups in 2005), the voles of the Urgal River valley are referred the genus (Pozdnyakov, 1996; Abramson and to as Maximowicz’s voles. Thus, the species status of Lisovskii, 2012). The species of this group were distin- the voles of this isolated population is still in question. guished mainly on the basis of chromosomal differ- This paper is dedicated to specification of the taxo- ences and the data of a hybridological analysis (Meyer nomic status of two vole individuals caught in July et al., 1996). The range of Maximowicz’s vole has a 2014 in the Verkhnebureinskaya Depression in the mosaic structure and extends from Baikal to Sikhote- vicinity of the village of Chegdomyn of the Verkhne- Alin’ in Russia, entering eastern Mongolia and north- bureinskii district of Khabarovsk krai (Fig. 1). eastern China, whereas two other species have been Karyological analysis and sequencing of the found in intermountain hollows: the Muya Valley vole mtDNA control region were used to diagnose voles. in northern Buryatia and the Evoron vole in southern Preparations of chromosomes were prepared using the Khabarovsk krai. technique of short-term culturing of bone marrow Despite the consistent interest of researchers in cells (Grafodatsky and Rajabli, 1988). Photographs of species of this group, the taxonomic status of a number chromosomes were obtained using an AxioImager M1 of isolated geographic populations of voles located in microscope. DNA extraction was performed using the the intermountain basins of Northeast Asia has not yet method of salt extraction (Aljanabi and Martinez, been fully established (Kartavtseva et al., 2015). One of 1997) from tissues fixed in 95% alcohol (liver and these isolates is a population of large meadow voles in muscle). The control region fragment was amplified the Verkhnebureinskaya Depression in Khabarovsk by polymerase chain reaction (PCR) using direct krai. According to the literature data, individuals from Pro+ and reverse Phe– primers. A detailed procedure the vicinity of the village of Chegdomyn situated in the for preparing the reaction mixture, the PCR reaction basin of the Urgal River, which flows into Bureya, schemes for obtaining a fragment, and preparation of were originally identified as Maximowicz’s voles (Bel- the samples for sequencing have been described previ-

813 814 SHEREMETYEVA et al.

50°

45°

40°

35°

110110° 115° 120° 125° 130° 135° 145° E

A. evoronensis A. maximowiczii Stanovoi Ridge New ﬁnd A. mujanensis

Dzhgdy Ridge Nimelen Ezop Ridge

Selemdzha

Amgun’

Turana Ridge

eya Bureinskii Ridge

Bur

Sea of Japan

Fig. 1. Ranges of the “maximowiczii” group species (by Shenbrot and Krasnov, 2005) and location of the new find. ously (Sheremetyeva et al., 2015). The sequence of ivostok). Editing and alignment of the sequences nucleotides was determined on an ABI Prizm 3130 obtained was carried out using the BioEdit 7.0.9.0 pro- automatic sequencer (Applied Biosystems, United gram (Hall, 1999). The selection of the model for the States) at the Institute of Biology and Soil Science, construction of the ME phylogenetic trees and the cal- Far East Branch, Russian Academy of Sciences (Vlad- culation of genetic distances were performed using the

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Table 1. Genetic p-distances (under the diagonal) and error (above the diagonal) between the species of the genus Alexan- dromys A. maximowiczii evoronensis A. A. mujanensis findNew A. sachalinensis gromovi A. A. middendorffii fortis A. A. maximowiczii 0.0048 0.0044 0.0041 0.0067 0.0089 0.0083 0.0093 A. evoronensis 0.0320 0.0051 0.0044 0.0071 0.0091 0.0092 0.0097 A. mujanensis 0.0310 0.0311 0.0047 0.0073 0.0091 0.0094 0.0096 New find 0.0289 0.0217 0.0281 0.0071 0.0089 0.0088 0.0094 A. sachalinensis 0.0564 0.0507 0.0559 0.0556 0.0088 0.0088 0.0094 A. gromovi 0.0830 0.0791 0.0857 0.0793 0.0817 0.0083 0.0095 A. middendorffii 0.0828 0.0921 0.0941 0.0870 0.0814 0.0740 0.0083 A. fortis 0.0940 0.0928 0.0953 0.0931 0.0855 0.0887 0.0802

MEGA 5.1 program (Tamura et al., 2011). Stability of individuals of the Verkhnebureinskaya depression and the order of clustering was estimated using bootstrap the previously studied specimens of Evoron voles analysis (1000 replicates). In the construction of the 0.0217 ± 0.0044 (Table 1). phylogenetic trees, as controls, sequences of the mtDNA control region of seven species of East Asian In the karyotype of the male (no. 3859) vole from voles obtained earlier (Haring et al., 2011, Sheremet- the vicinity of Chegdomyn (Fig. 3a), 37 chromosomes yeva et al., 2015) and placed in GenBank/NCBI were were found (NF = 55). The karyotype contains taken: A. fortis (nos. HM135815, HM135816, (1) three unpaired chromosomes represented by two HM135828), A. sachalinensis (nos. HM135901– large and medium-sized submetacentrics and one HM135906), A. gromovi (nos. HM135891– acrocentric; (2) eight pairs of meta-submetacentric HM135894), A. mujanensis (nos. HM135853– chromosomes smoothly decreasing in size from large HM135856), A. middendorffii (no. HM135899), to medium; and (3) nine pairs of acrocentric chromo- A. evoronensis (nos. HM135857–HM135862), and somes smoothly diminishing in size. Apparently, A. maximowiczii (nos. HM135871–HM135873, unpaired chromosomes represent a chromosomal HM135883, HM135887, HM135889, KM403506, rearrangement in the heterozygous state. In this work, KM403535). The collected material (chromosome owing to the strong spiralization of the metaphase preparations, carcasses in alcohol, skulls, and the col- chromosomes, we are hesitant to determine accurately lection of DNA) is stored in the collections of the Lab- either the chromosomal rearrangement or the sex oratory of Evolutionary Zoology and Genetics at the chromosomes, since only routine chromosome stain- Institute of Biology and Soil Science, Far East ing of one individual has been performed. The forma- Branch, Russian Academy of Sciences (Vladivostok). tion of a large submetacentric could occur due to the tandem fusion of a medium-sized submetacentric and For the samples of meadow voles from the vicinity acrocentric. To show the nature of the chromosomal of Chegdomyn, nucleotide sequences of the total transformation, it is necessary to investigate the karyo- mtDNA control region of 940 bp in length were types of a larger number of voles using chromosome obtained. A phylogenetic analysis showed that the differential staining methods. For Evoron voles, chro- individuals investigated (no. 139-14 (3859) and mosomal polymorphism (Kovalskaya and Sokolov, no. 146-14) on the ME tree clustered with Evoron 1980) was noted both in terms of the number of chro- voles (Fig. 2). An analysis of the sequences of the con- mosomes accompanied by the telomere fusion of two trol region showed that the haplotypes of the voles two-arm chromosome pairs with the formation of a captured earlier in the vicinity of the village of Polina large metacentric (2n = 38–40), and in different cen- Osipenko near the junction of the Nemelen and tromere positions in the three pairs of autosomes, Amgun’ rivers (nos. HM135858–HM135861) differ which leads to variability of the morphology of chro- from the Evoron vole specimen from a typical habitat mosomes (Meyer et al., 1996). (no. HM135857) in ten sites, whereas the voles caught in the vicinity of Chegdomyn differ in 22 sites. As a According to the literature, a karyotype with simi- result of the analysis of the genetic (p) distances, the lar characteristics (2n = 37, NF = 55) was described closest resemblance of the individuals studied was earlier for Maximowicz’s vole from Khentii (environs found with the species of the group “maximowiczii,” of Mungen Morot) in Mongolia (Orlov et al., 1978). with the minimum distance registered between the Unfortunately, the authors do not give a karyotype

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91 HM135887 HM135889 KM403506 79 99 HM135871 A. maximowiczii KM403535 96 HM135872 HM135883 99 HM135873 100 HM135853 100 HM135856 A. mujanensis 100 HM135855 HM135854

100 146-14 139-14(3859) 83 HM135857 100 HM135862 82 HM135860 A. evoronensis HM135861 99 HM135859 HM135858 HM135904 81 HM135905 HM135902 A. sachalinensis 100 HM135901 HM135906 97 HM135903 HM135892 100 HM135891 A. gromovi 86 HM135893 HM135894 HM135899 A. middendorf ﬁi HM135815 100 HM135828 A. fortis HM135816 0.01

Fig. 2. МЕ phylogenetic tree for seven species of the genus Alexandromys. The frame contains the haplotypes of the voles captured in the Verkhnebureinskaya Depression. The numbers correspond to the samples in GenBank/NCBI. layout with 2n = 37, but in the description, two skaya and Sokolov, 1980). When comparing the karyo- unpaired submetacentric chromosomes and an acro- type of Maximowicz’s voles from Khentii with 2n = centric were noted, without discussing the nature of 36, NF = 54 (Orlov et al., 1978) and the karyotype of the chromosome rearrangement. In a subsequent, the male (no. 3859) from the vicinity of Chegdomyn, more detailed study of voles from Mongolia, it was a similarity in the morphology of chromosomes was shown that the voles from Khentii have chromosomal found (Fig. 3b), which, however, does not indicate the polymorphism in terms of the number of chromo- identity of the karyotypes. Thus, due to the lack of full somes (2n = 36–38, NF = 52–55) and that the forma- information about the interpopulation chromosomal tion of large metacentrics occurs due to the Robertson variability of A. evoronensis and A. maximowiczii and fusion of acrocentric pairs of chromosomes (Koval- due to the absence of differential staining of the chro-

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(a) 1

(b)

Fig. 3. Comparison of the karyotypes: (a) voles from the vicinity of Chegdomyn of Khabarovsk krai (male no. 3859), (b) Maxi- mowicz’s voles from the environs of Mungen Morot, Mongolia (Orlov et al., 1978). (1) Unpaired chromosomes, (2) meta-submetacentric chromosomes, and (3) acrocentric chromosomes. mosomes, we cannot reliably state the species status of electric power plant was constructed and part of the the voles caught in the Verkhnebureinskaya Depres- Verkhnebureinskaya Depression was flooded. sion. The formation of the mountain systems surround- ing the Verkhnebureinskaya Depression occurred at Currently, there are two known habitats of Evoron the end of the Mesozoic and in the Cenozoic. Distinct voles located within the Evoron-Chukchagir Depres- traces of two glaciations corresponding to the Early sion in Khabarovsk krai: (1) a typical place is the shore (the maximum is 110000–100000 years) and Late (the of Evoron Lake at the source of the Devyatka River of maximum is 25000–18000 years) Wurm have been the Solnechnyi district, (2) near the confluence of the revealed for Yam-Alin’ and Bureinskii ridges. The gla- Nemelen and Amgun’ rivers (environs of Polina ciations of Yam-Alin’ were of a mountain–valley, and Osipenko, Polina Osipenko district) (Sheremetyeva in some places, of a glacial nature (Osipov et al., et al., 2010). According to the molecular genetic anal- 2000). At the end of the Late Wurm, a gradual climate ysis, the investigated voles of the Verkhnebureinskaya change took place in the Holocene. First, 10000– Depression from the vicinity of Chegdomyn are most 7000 years ago, a warming of the climate with moder- similar to the individuals of the previously studied ate humidification occurred. Later on, 7000–5000 Evoron voles from the Evoron-Chukchagir Depres- years ago, the climate warmed significantly (climatic sion. Both populations are isolated from each other by optimum of the Holocene), which led to the emer- the Bureinskii Range. gence of birch and pine and then spruce and fir forests, The Verkhnebureinskaya Depression is located while the upper boundary of the forest in the climatic between Bureinskii Ridge, which is the watershed of optimum of the Holocene exceeded the modern one the Bureya and Amgun’ rivers to the east and Turan and reached a mark of 1800–1900 m above sea level Ridge, which serves as the watershed of the Selemdzha (Korotkii et al., 1987; Korotkii and Kovalyukh, 1987). and Bureya rivers to the west. To the north, the Later, 5000–1000 years ago, an increase in continen- Depression is bounded by the Dusse-Alin’, Yam- tality with a general decrease in the average annual Alin’, and Ezop ridges, and to the south by a granite temperatures down to their present values took place. massif, which cuts through the Bureya River, forming The area of forest formations sharply decreased, and a narrow corridor called the Talakan point. At this broad-leaved elements completely disappeared from place the width of the river along the bottom is 500 m, the vegetation on the slopes (Osipov et al., 2000). the banks are formed by steep rocks with vertical According to paleogeographic data, in the period slopes. Recently, the dam of the Bureiskaya hydro- of the last great cooling, up to 8000–10000 years ago,

BIOLOGY BULLETIN Vol. 44 No. 7 2017 818 SHEREMETYEVA et al. reorganization of the lake–river systems of the Belyaeva, N.A., Murine rodents in the south of Khabarovsk Okhotsk Far East took place. Thus, for the basin of the krai, Izv. Irkutsk, Protivochumn. Inst., 1965, vol. 25, Amur, Amgun’, Tugur, and Uda rivers, traces of pp. 304–308. recent extensive lakes are characteristic, and on the Grafodatskii, A.A. and Radzhabli, S.I., Khromosomy slopes that border the watershed, there are through sel’skokhozyaistvennykh i laboratornykh mlekopitayushchikh: canyons. During this period, the Amgun River, as a Atlas (Chromosomes of Farm and Laboratory Mammals: result of ponding, flowed into Bureya in the middle An Atlas), Novosibirsk: Nauka, 1988. reaches and became part of the system of the middle Grosval’d, M.G., Traces of ice sheet expansion from the Amur (Grosval’d, 1996, 2009). In this paper, we con- shelf to the coast of Northeastern Siberia, Dokl. Ross. 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