ISSN 1062-3590, Biology Bulletin, 2017, Vol. 44, No. 9, pp. 1049–1055. © Pleiades Publishing, Inc., 2017. Original Russian Text © I.N. Sheremetyeva, I.V. Kartavtseva, T.V. Vasiljeva, 2017, published in Zoologicheskii Zhurnal, 2017, Vol. 96, No. 4, pp. 477–484.

Does Alexandromys evoronensis Inhabit the Northeastern Part of Verkhnezeiskaya Plain? I. N. Sheremetyeva*, I. V. Kartavtseva, and T. V. Vasiljeva Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences, Vladivostok, 690022 *e-mail: [email protected] Received October 16, 2015

Abstract⎯Gray of the “maximowiczii” group of the genus Alexandromys were found in the Argi River of the region (northeastern part of Verkhnezeiskaya Plain). Analysis of mtDNA showed its similarity to A. evoronensis. The karyotype was 2n = 36, and NFa = 51–52. The karyotypes of these individuals did not contain the marker chromosome typical for Alexandromys maximowiczii.

Keywords: Alexandromys, Verkhnezeiskaya Plain, inhabit, karyotypes, control region of mtDNA DOI: 10.1134/S1062359017090126

Alexandromys maximowiczii (Schrenck 1858) is (Verkhnebureinskaya Depression), which, according a wide-ranging species of voles of eastern Asia, which to the results of molecular genetic analysis, were close is characterized by a complex chromosomal polymor- to A. evoronensis (Sheremetyeva et al., 2016). It should phism (2n = 36–44, NFa = 50–60). Five chromo- be noted that the karyotype of the from the vicin- somal forms with geographical confinement have been ity of Chegdomyn (2n = 37, NF = 55) did not fit the distinguished. In the northern peripheral area in range of variability of the karyotype known for A. evo- opposite parts of the species range, two forms were ronensis (2n = 38–40, NF = 53–56) (Koval’skaya and 1 described in intermountain isolates. Later, as a result Sokolov, 1980; Golenishchev and Radzhabli, 1981; of the study of their karyotypes and hybridization Meyer et al., 1996; Kartavtseva et al., 2007) but was experiments, these forms acquired the species status: similar in the number and morphology of chromo- A. evoronensis (Kowalskaia et Sokolov 1980) (2n = 38– somes to the karyotype of A. maximowiczii from Khen- 40, NFa = 51–54) and A. mujanensis (Orlov et Kowal- tei in Mongolia (Orlov et al., 1978). The similarity in skaia 1978) (2n = 38, NFa = 46–50). A. evoronensis is the number and morphology of chromosomes (with- one of the sibling species of gray voles of the “maxi- out differential staining) for these species cannot be mowiczii” group, which was described 35 years ago regarded as evidence of their identity. However, near the source of the Devyatka River on the shore of Koval’skaya and Sokolov (1980) assumed that, Evoron Lake, Solnechnyi district, krai according to the types of mutations known, the diploid only on the basis of the results of karyological analysis number in A. evoronensis theoretically may vary from (Koval’skaya and Sokolov, 1980). Until recently, this 36 to 42. However, extreme variants of chromosome species was reliably known only from the Evoron- numbers have not yet been found. These authors also Chukchagirskaya Lowland, (Fig. 1, assumed that the distribution of A. evoronensis is not points 1–3): 1, in the vicinities of Evoron Lake (Gole- limited to the Evoron-Chukchagirskaya Lowland and nishchev and Radzhabli, 1981; Meyer et al., 1996; that this species may be found in some lacustrine basins Shenbrot and Krasnov, 2005); 2, in the environs of the in the eastern part of the Baikal–Amur Railroad. village of Kharpichan (51°19′ N, 136°35′ E) (Kartavt- Within the framework of the study of the variability seva et al., 2007; Sheremetyeva et al., 2010); and 3, in of marginal and isolated populations of species of gray the environs of the village named after Polina voles of the “maximowiczii” group, in July 2015 we Osipenko, Khabarovsk krai, near the confluence of made a second attempt to catch a representative of this Amgun and Nimelen rivers (Sheremetyeva et al., group (A. maximowiczii) in the northeastern part of the 2010). It was believed that the penetration of A. evo- Verkhnezeiskaya Plain. The first attempt was made ronensis to the west is hampered by the , Dusse- one year ago (Kartavtseva et al., 2015), when we Alin, and Yam-Alin ranges (Meyer et al., 1996). How- caught only a pair of root voles in the vicinity of the vil- ever, in July 2014, a pair of gray voles was caught in the lage of Verkhnezeysk. At this time, attempts to catch the Urgal River valley near the village of Chegdomyn (Fig. 1, were made in the Argi River valley (Fig. 1), point 4), Verkhnebureinskii district, Khabarovsk krai Amur region, near the place where, according to

1049 1050 SHEREMETYEVA et al.

(a)(a) – A. evoronensis

Yam-Alin Range Stanovoi Range – New find Tugur

Уда Dusse-Alin Range Argi 3

Dzhagdy Range Nimelen Ezol Range 2 Amur Nora 1 Soktakhan RangeDep Amgun 4 Zeya

Turana Range

Bureinskii Range

Bureya

Amur China

((b)b) a ey ZeyaZ

ЗеяЗ ея

l i ne ArgiArg han a c 4 ZeyaZey channel 3

1 2

Fig. 1. (a) Sites of finds and (b) scheme of location of the biotopes in which the catches of A. evoronensis were performed.

Sapaev (Sapaev, 1973; Sapaev and Voronov, 1976), completely covered with water. In total, 15 gray voles A. maximowiczii was previously caught. To set traps, were caught in 80 traps. Also we caught two gray red- we selected four biotopes: (1) the left bank of the Zeya backed voles, five wood mice, one pika, and one shrew Channel with a broadleaf forest site (54°40′17″ N, of the genus Sorex. The biological diversity in the catches 129°6′36″ E); (2) the left bank of the Zeya channel was maximum in biotope 1, and the number of gray voles overgrown with willows, at the confluence into the was maximum (eight individuals) in biotope 3. Argi River (54°40′6″ N, 129°6′39″ E); (3) an island on The species diagnostics of gray voles was performed the Argi River, overgrown willow and horsetail using karyological analysis and sequencing of the (54°40′31″ N, 129°5′49″ E); and (4) a high bank of the mtDNA control region. For ten individuals, species Argi River with a larch forest, overgrown with rosehip identification was performed in vivo, and the individ- (54°40′40″ N, 129°6′1″ E). It should be noted that, uals themselves were left for further breeding. Chro- during a heavy flood in 2013, biotopes 2 and 3 were mosome preparations were obtained in the field from

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(a)(a)

XY

((b)b)

XY

Fig. 2. (a) Karyotype and (b) C-banding of chromosomes of the vole (male no. 3859) from the Argi River basin (Amur region). the femoral bone marrow cells (Grafodatskii and and the live specimens are kept in the vivarium of the Radzhabli, 1988). Staining for structural heterochro- Institute of Biology and Soil Science, Far East matin was performed according to Sumner (1972) Branch, Russian Academy of Sciences (Vladivostok). without pretreatment of preparations with hydrochlo- ric acid. G-bands on chromosomes were identified The analysis of the chromosomal preparations pre- using trypsin (Seabright, 1971). Chromosome images pared for one male (no. 3950) from the natural popu- were obtained with an AxioImager M1 microscope. lation and one female (no. 4017), which was obtained as a result of vivarium breeding, showed that the DNA was isolated by salt extraction (Aljanabi and karyotypes of these individuals contained 36 chromo- Martinez, 1997) from tissues fixed with alcohol. The somes (Fig. 2a). The number of autosomal arms of the procedure of the reaction mixture preparation, the male (no. 3950) and the female (no. 4017) was 51 and scheme of PCR reaction for obtaining the fragment, 52, respectively. Thus, this population is characterized and the preparation of samples for sequencing were by polymorphism, which requires a more detailed described previously (Sheremetyeva et al., 2015). The analysis. The Y chromosome is acrocentric and con- nucleotide sequence was determined with an ABI sists of heterochromatin only. The Y chromosome was Prizm 3130 automatic sequencer (Applied Biosystems, determined by the structural heterochromatin staining United States) on the basis of the Institute of Biology method (Fig. 2b). The X chromosome is a medium- and Soil Science, Far East Branch, Russian Academy size submetacentric (it was determined by G-banding) of Sciences (Vladivostok). The editing and alignment and has a slight C-block in the pericentromeric region. of the sequences obtained was performed using the Pericentromeric C-blocks were identified in three BioEdit 7.0.9.0 software (Hall, 1999). Constructing pairs of meta-submetacentric chromosomes (one pair phylogenetic trees and the calculation of genetic dis- of medium-size chromosomes and two pairs of tances performed using MEGA 6.0 program (Tamura smaller chromosomes) as well as in the first pair and et al., 2011). In constructing phylogenetic trees, the the two last pairs of acrocentric chromosomes. Other mtDNA control region sequences of seven species of acrocentric chromosome pairs may contain small eastern Asian voles (A. fortis, A. sachalinensis, A. gro- amounts of heterochromatin. In addition, the short movi, A. mujanensis, A. middendorffii, A. evoronensis, arms of one of the subtelocentric chromosomes con- and A. maximowiczii), obtained previously (Haring sist solely of heterochromatin. When comparing the et al., 2011; Sheremetyeva et al., 2015), as well as the newly described karyotype of the individuals from the mtDNA control region sequences of the voles that Argi River basin (2n = 36, NF = 54) (Verkhnezeiskaya were caught in July 2015 in the vicinity of Chegdomyn Plain) with the karyotype of the male (no. 3859) from (Sheremetyeva et al., 2016), were taken as the control. the vicinity of Chegdomyn (2n = 37, NF = 55) The collected material (chromosomal preparations, (Sheremetyeva et al., 2016) and A. maximowiczii from skulls, and DNA collection) is stored in the collections Khentei (2n = 36, NF = 54) showed similarities in the of the laboratory of evolutionary zoology and genetics, morphology of the chromosomes; however, this does

BIOLOGY BULLETIN Vol. 44 No. 9 2017 1052 SHEREMETYEVA et al.

(a)(a)

XX

(b)(b)

XY

(c)(c)

XY

Fig. 3. Differential staining of (a) female no. 4017, which was obtained as a result of vivarium breeding of the voles from the Argi River basin (Amur region), (b) A. maximowiczii from the (male no. 2063), and (c) A. evoronensis from the vicinities of the village of Polina Osipenko (male no. 2050).

Table 1 . Genetic p-distances (below the diagonal) and the error (above the diagonal) between the isolated populations of A. evoronensis (marked with an asterisk) and two species of the genus Alexandromys Sample 1 2 3 4 5 1 A. maximowiczii 0.0047 0.0039 0.0039 0.0068 2 A. evoronensis (Verkhnebureinskaya Depression)* 0.0331 0.0041 0.0045 0.0074 3 A. evoronensis (Verkhnezeiskaya Plain)* 0.0296 0.0215 0.0034 0.0073 4 A. evoronensis (Evoron–Chukchagirskaya Lowland) * 0.0290 0.0228 0.0212 0.0073 5 A. sachalinensis 0.0589 0.0536 0.0573 0.0577

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96 HM135889 HM135890 КМ403506 63 КМ403497 A. maximowiczii КМ403535 99 КМ403525 НМ135883 99 НМ135873 НМ135853 100 100 НМ135856 A. mujanensis 99 НМ135855 НМ135854 50 НМ135859 61 НМ135858 97 НМ135861 56 НМ135860 A. evoronensis 82 НМ 135862

НМ135857

146-14 78 100 Verkhnebureinskaya Depression 99 139-14(3859) Urgal River basin 98 25argi 56 100 20argi 28argi Verkhnezeiskaya Plain 88 75 29argi Argi River basin 15argi 76 27argi 71 92 3argi 66 HM135904 79 HM135905 HM135902 A. sachalinensis 100 HM135901 92 HM 135906 40 HM135903 HM 135892 HM135893 100 A. gromovi 61 HM135891 HM135894 HM135899 A. middendorffii HM135821 100 HM135815 A. fortis HM135816 0.01

Fig. 4. Minimum evolution tree for seven Alexandromys species. The haplotypes of the voles caught in the basins of the Urgal (Verkhnebureinskaya Depression) and Argi (northeastern part of Verkhnezeiskaya Plain) rivers are marked by rectangles.

BIOLOGY BULLETIN Vol. 44 No. 9 2017 1054 SHEREMETYEVA et al. not testify to their identity. Differential staining of However, to determine uniquely the species status chromosomes showed that the karyotypes of the indi- of the voles from the Argi River valley and the Verkh- viduals from the Argi River basin (2n = 36) signifi- nebureinskaya Depression (Sheremetyeva et al., 2016), cantly differ from the karyotype of A. maximowiczii further research is required. Only a detailed karyolog- (Fig. 3). First of all, the karyotypes of the individuals ical analysis with the use of differential staining meth- from the Argi River basin lack a large metacentric ods and fluorescence in situ hybridization (FISH) of chromosome pair, which in A. maximowiczii is typical chromosomes, a detailed study of the intrapopulation (marker) and is formed as a result of fusion of meta- chromosomal polymorphism of voles from different centric chromosome pairs (nos. 3 and 4) (Kartavtseva isolates, and the hybridization of voles from the popu- et al., 2013). This type of fusion stably occurs in all lations of the Argi River valley and the Evoron-Chuk- A. maximowiczii, except for two animals from the chagirskaya and Verkhnebureinskaya depressions will vicinitty of the village of Romanovka (Buryatiya), in make it possible to determine the taxonomic position which the diploid number is 44. In addition, the indi- of the voles with the new chromosomal form. viduals from the Argi River basin, similarly to A. evo- ronensis and A. mujanensis, have a submetacentric X chromosome, whereas in A. maximowiczii this chro- ACKNOWLEDGMENTS mosome is always acrocentric. Thus, the results of This study was supported by the Russian Founda- karyological analysis showed that the gray voles caught tion for Basic Research, project no. 15-04-03871. in the Argi River basin do not belong to A. maximowic- zii and are, apparently, a new karyomorph of A. evo- ronensis. REFERENCES For 14 samples of gray voles from the Argi River Aljanabi, S.M. and Martinez, I., Universal and rapid salt extraction of high quality genomic DNA for PCR-based valley, the nucleotide sequences of the full-length techniques, Nucleic Acids Res., 1997, vol. 25, no. 22, mtDNA control region 940 bp long were obtained. 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SPELL: 1. intermountain

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