Analysis of Genetic Diversity of the Desert Hamster (Phodopus roborovskii) in the Northern Part of Its Range

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1 ISSN , Biology Bulletin, 2011, Vol. 38, No. 1, pp Pleiades Publishing, Inc., Original Russian Text I.G. Meshcherskii, N.Yu. Feoktistova, 2011, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2011, No. 1, pp SHORT COMMUNICATION Analysis of Genetic Diversity of the Desert Hamster (Phodopus roborovskii) in the Northern Part of Its Range I. G. Mescherskii and N. Yu. Feoktistova Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii pr. 33, Moscow, Russia Received June 3, 2010 Abstract The of sequences of the cytochrome b gene and control region of mitochondrial DNA of 30 Desert hamsters (Phodopus roborovskii; Rodentia: Cricetinae) captured at ten geographical points from Zaisanskaya Depression (Kazakhstan) in the northwest to the Xilin Gol area (China) in the southeast were analysed. The lack of a correspondence between the closeness of the found haplotypes and their geographical distribution allows us to assume that the recent genetic diversity of the species on the investigated area formed as a result of several migrators streams from more southern territories. DOI: /S INTRODUCTION Two species of dwarfhamsters of the genus Phodopus the Campbell s hamster (P. campbelli, Thomas, 1905) and Djungarian hamster (P. sungorus, Pallas, 1773) have been an object of laboratory studies for about 50 years (Feoktistova, 2008). The hamsters are used for investigations in morphology, physiology, ethology, and chemocommunication. However, the phylogeography of these popular species has been studied only since 2008 (Feoktistova, 2008; Meschersky, Feoktistova, 2009). The Desert hamster (Phodopus roborovskii, Satunin, 1903) is the least studied of the three species of dwarfhamsters (Phodopus Rodentia: Cricetinae). In the last decade, it has been widely used as a model for a number of physiological and evolutionary studies (Feoktistova, 2008). The Desert hamster occurs north China, south, central, and northwestern Mongolia, the southern regions of the Tyva Republic (Russia), and the south of the Zaisanskaya Depression in east Kazakhstan (Fig. 1). Both in Kazakhstan and in Russia, the Desert hamster is included in lists of rare and protected species (Krasnaya kniga Red Data Book, 2001; Postanovlenie, 2006). The aim of this study was to evaluate the genetic diversity of the species in the northern part of its range, including the territories of Russia and Kazakhstan. MATERIAL AND METHODS The analyzed sample consisted of 30 Desert hamsters captured at ten geographical points (Fig. 1): in the south of the Zaisanskaya Depression (47 28 N, E, Kazakhstan Republic 7 specimens, Zai); in the area of Tere Khol and Shira Nuur lakes in the northeast of the Ubsunurskaya Depression (50 03 N, E and N, E, respectively, Tyva Republic 10 specimens, Tuv); in the area of Lake Khar Nuur in the southeastern part of Ubsunurskaya Depression (48 17 N, E, Mongolia 1 specimen, XaN); in southwestern Mongolia (four geographical points in the area between N and 95 36' 'E 9 specimens, Alb, Idz, BoN, and OrN), southern Mongolia (42 58 N, E 1 specimen, SGb), southeastern Mongolia (43 43 N, E 1 individual, EGb), and northeastern China (43 57 N, E, Xilin Gol area, Inner Mongolia province 1 specimen, InM). A genetic analysis was also done for six individuals from a laboratory colony of the A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, whose founders were captured at the area of Lake Tere Khol in DNA was extracted from muscle tissue (or ear tissues in the case of living animals) fixed in 96% ethanol. Sequences of the cytochrome b gene (1143 bp) and control region (862 bp) of mitochondrial DNA (mtdna), combined and analysed as a single locus, were used as a genetic marker. The methods of DNA exctraction, the two loci amplification, sequencing, and the sequences alignment were the same as used in the analysis of the phylogeographical structure of two other species of the genus Phodopus (Meschersky, Feoktistova, 2009). The unique nucleotide sequences (haplotypes) were deposited in the Genbank (accession numbers GU GU and GU GU812890). Sequences of the cytochrome b gene and control region of hamsters from the laboratory colony, identical in all studied individuals, are represented (as part of a larger mtdna fragment) in Genbank under no. EF Homologous sequences of both fragments 82

2 ANALYSIS OF GENETIC DIVERSITY OF THE DESERT HAMSTER N Kazakhstan Russia N China 85 E China 115 E Fig. 1. Supposed boundary of the range of the Desert hamster (1) and places where samples was collected (2). of mtdna of Campbell s hamster (P. campbelli: EF025538) were used as an outgroup. The treatment of the obtained data was done according to the approach and using software described earlier (Meschersky, Feoktistova, 2009). Relationships between haplotypes were stated by the Neighbor Joining method based on the HKY model (Hasegawa et al., 1985) chosen by the results of an analysis of sequences (2005 bp) using the Modeltest v.3.7 program (Posada, Crandall, 1998). For the Bayesian approach settings for the best-fit model were estimated using the MrModeltest v.2.3 program (Nylander, 2004). RESULTS AND DISCUSSION In 30 Desert hamsters captured in the wild, 26 haplotypes were described; the identical sequences were found for individuals in the samples from Kazakhstan and Tyva. The average genetic distance for 27 found haplotypes (including the one registered in the animals of the laboratory colony) was 0.80% when pairwise values varied from 0.05 to 1.53%. The analysis of relationships between the haplotypes (Fig. 2) showed an unambiguously high (0.9 1) support of only two clades formed by nucleotide sequences of mtdna of the animals caught in the Zaisanskaya Depression (clade I) and southeastern Tyva (clade II). The average genetic distances within each of these clades are relatively low: 0.18% with a range of values from 0.05% (the only substitution for the 2005 b.p. sequence) to 0.35% for the sample from Zaisan and 0.24% ( %) for the sample from south Tyva. Both Zaisanskaya Depression and south Tyva are extreme northwestern protuberances of the Desert hamsters' range. It can be assumed that the genetic diversity found here formed over the time of existence of local populations, which originated from a limited number of founders. Collection points in south Tyva are situated within the Ubsunurskaya Depression, which is a single orographical region where there are no barriers, which might separate populations of hamsters that live here. The colonization of the Ubsunurskaya Depression by Desert hamsters could only have occurred from the south since the depression is confined from three other sides by the mountain chains of Altai, Tannu Ola, and Khangai. Nonetheless, the haplotypes of the animal caught in another point of the Depression, near Khar Nuur Lake, in 200 km to the south from collection points in Tyva, differs from any of Tyva haplotypes no less than 0.96% (1.02% on average). The inclusion of this haplotype in the general sample of the Ubsunurskaya Depression increases its diversity 1.7-fold (table). The methods of analysis used do not allow us to determine the Khar Nuur haplotype as related to the Tyva ones but places it with a certain degree of probability among haplotypes of animals captured in the regions most remote from the Ubsunurskaya Depression: southeastern Mongolia and northeastern China (Fig. 2, clade IV). The difference between nucleotide sequences of animals from Khar Nuur Lake and Inner Mongolia (distance between the collection points is 1600 km in a straight

3 84 MESHCHERSKII, FEOKTISTOVA 55; ; ; ; ; ; OrN056 IDz002 III Bon271 Alb006 XaN003 InM305 IV EGb306 Zai020 Zai015 Zai002 I Zai014 Zai023 Tuv005 Tuv013 Tuv040 EF II Tuv119 Tuv789 Tuv834 Tuv836 Tuv835 OrN022 OrN021 OrN016 IDz004 OrN023 SGb307 P. campbelli Fig. 2. Polytomic tree of known mtdna haplotypes (cytochrome b gene and control region) of the Desert hamster. Nodes with support 50 (0.50) and higher revealed by the methods of Neighbor Joining (HKY model) (first value above the line), Maximum Parsimony (second value above the line), and Bayesian approach (under the line) are shown. The support values are given for nodes that unite three or more haplotypes. Bootstrap test replicates. I IV are haplogroups. line and about 1800 km bypassing forested areas of Khangai) is 0.55%, i.e., less than between the most distanced haplotypes of the Tyvinian clade. Thus, it can be assumed that the Ubsunurskaya Depression is inhabited by descendants of at least two separated genetic lines of the Desert hamster. It can be expected that more plentiful collections (if they are ever made) will discover in the southern part of the Ubsunurskaya Depression haplotypes closed to the Tyva ones, and in the north, those closed to the Khar Nuur type, though it would not negate the fact of the presence in the region of two different mitochondrial lines. Moving to the south (approximately 250 km from Lake Khar Nuur), the next collection point is the Mongolian region Al Bulak. This place is not part of the Ubsunurskaya Depression and was accepted here as another area southwestern Mongolia". Nevertheless, this region belongs to a lowland route between the Altai and Khangai mountains, which could be the only way for hamsters to occupy the more northern Ubsunurskaya Depression. However, the haplotype of the animal caught here (Alb) was not close to haplotypes of the Tyva line (as well as all other haplotypes found in Mongolia), and differs from the Khar Nuur one by a greater number of substitutions (0.65%) than that one differs from the Inner Mongolian haplotypes. Four haplotypes described in animals from four different points of southwestern Mongolia at a distance of km from each other (which does not allow to consider them as representatives of a common population) form clade III with a relatively low but stable support (Fig. 2) and mean level of intergroup distance 0.34%. It can be assumed that these animals are descendants of a single genetic line. However, within one true population in this region, in the area of Lake Orog Nuur (haplotypes OrN), representatives of other lines not related to the clade III, or to the Tyva, Khar Nuur, or Zaisanskaya lines were found. Their (as well as other haplotypes described in this region and in South Mongolia) positions on phylogenetic tree cannot be determined reliably(fig. 2). For all nine haplotypes described in southwestern Mongolia, the mean intergroup distance is 0.67% whereas for haplotypes of five animals captured in one point (Lake Orog Nuur)the averaged distance is 0.73%. The latter value is comparable with the diversity of the entire sample (0.80%) collected on the territory of about 2500 km from northwest to southeast. The presence of several different genetic lines of Desert hamster within one geographical region makes differences between geographic groups of haplotypes being lower than between the lines themselves i.e. between haplogroups, which form supported clades (table). The results allow us to suppose that the present diversity of P. roborovskii in the northern part of the range should be consider as mostly introduced. The colonization of northern and central parts of Mongolia, Tyva, and southeastern Kazakhstan by Desert hamsters took place, probably, in relatively recent (on an evolutionary scale) times from more southern regions the territory of modern China. The lack of a correspondence between the closeness of the found haplotypes and their geographical distribution also indicates several possible waves of colonization the investigated area by hamsters of different genetic lines. This assumption is illustrated by the median-joining network of known haplotypes (Network program Bandelt et al., 1999; engineering.com) given in Fig. 3. The described haplotypes of Desert hamster form a star-like structure with central (ancestor) haplotypes absent in the sample. At the same time sequences differ by a relatively small number of mutations can be found in different geographical regions. Earlier V.E. Flint (1977) suggested the probable high degree of isolation (and, therefore, genetic separation) of P. roborovskii populations divided even by not extended territories. This suggestion is based on

4 ANALYSIS OF GENETIC DIVERSITY OF THE DESERT HAMSTER 85 Net average distances (Da, %) between haplotype groups described for different geographical regions and supported phylogenetic haplogroups* Geographical region Da, % Haplogroup Da, % Zaisanskaya Depression Ubsunurskaya Depression n = 10, π = 0.4 Zaisanskaya Depression Southwestern Mongolia** n = 9, π = 0.67 Ubsunurskaya Depression n = 10, π = 0.4 Southwestern Mongolia** n = 9, π = I. Zaisanskaya Depression II. Tyva n = 9, π = I. Zaisanskaya Depression III. Southwestern Mongolia*** n = 4, π = II. Tyva n = 9, π = 0.24 III. Southwestern Mongolia*** n = 4, π = 0.34 Note: n is the number of haplotypes in the group and π is the averaged intergroup distance, %. * See Fig. 2. ** The southwestern Mongolia region is restricted to the areas where samples were collected. *** Haplogroup III unites only 4 of 9 haplotypes described for the Southwestern Mongolia region SGb307 Zai014 Zai023 Zai015 IDz004 OrN056 IDz002 Zai002 Zai020 OrN016 Bon271 Alb006 XaN003 InM305 Tuv834 Tuv119 Tuv040 Tuv789 Tuv836 Tuv005 EF02... OrN OrN021 Tuv835 EGb306 OrN022 Tuv013 Fig. 3. Median-joining network of known haplotypes of the Desert hamster. Origin of samples: (1) Zaisanskaya Depression, (2) Ubsunurskaya Depression, (3) southwestern Mongolia, (4) south Mongolia, (5) southeastern Mongolia and northeastern China. psammophily of the Desert hamster areas with a different soils theoretically can be a significant barrier for the distribution of this species. However, in our opinion, the high level of genetic differences found within one population along with haplotypes close in nucleotide composition found in distant regions does not support this point of view. ACKNOWLEDGMENTS The authors thank A.V. Surov, A.V. Abramov, V.S. Lebedev, O.G. Gorbunov, and R. Nazarov for supplying samples for analysis. This study was supported by the Biological Diversity Program of Basic Research of the Presidium of the Russian Academy of Sciences; Scientific and Scientific Pedagogical Personnel of Innovative Russia Federal Target Program for no. GK ; a grant of the Russian Federation NSH ; and the Russian Foundation for Basic Research, grant no REFERENCES Bandelt, H.-J., Forster, P., and Rohl, A., Median-Joining Networks for Inferring Intraspecific Phylogenies, Mol. Biol. Evol., 1999, vol. 16, pp Feoktistova, N.Yu., Khomyachki roda Rhodorus. Sistematika, filogeografiya, ekologiya, fiziologiya, povedenie, khimicheskaya kommunikatsiya (Hamsters of the Genus Phodopus: Systematics, Phylogeny, Ecology, Physiology, Behavior, and Chemical Communication), Moscow: Tovar. Nauch. Izd. KMK, Flint, V.E., Prostranstvennaya struktura populyatsii melkikh mlekopitayushchikh (Spatial Structure of Populations of Small Mammals), Moscow: Nauka, Hasegawa, M., Kishino, H., and Yano, T., Dating of the Human Ape Splitting by a Molecular Clock of Mitochondrial DNA, J. Mol. Evol., 1985, vol. 22(2), pp

5 86 MESHCHERSKII, FEOKTISTOVA Krasnaya kniga Rossiiskoi Federatsii. Zhivotnye (Red Data Book of the Russian Federation), Moscow: AST Astrel, Meschersky (Meshchersky), I.G. and Feoktistova, N.Yu., Intraspecific Organization of Dwarf Hamsters Phodopus campbelli and Phodopus sungorus (Rodentia: Cricetinae) Basing on mtdna Analysis Dokl. Akad. Nauk, 2009, vol. 424, no. 2, pp [Dokl. (Engl. Transl.), vol. 424, no. 2, pp ]. Nylander, J.A.A., MrModeltest 2.3. Program Distributed by the Author, Evol. Biol. Centre, Uppsala Univ., html. Posada, D. and Crandall, K.A., Modeltest: Testing the Model of DNA Substitution, Bioinformatics, 1998, vol. 14, no. 9, pp Postanovlenie Pravitel stva Respubliki Kazakhstan ot 31 oktyabrya 2006 goda ¹ 1034 Ob utverzhdenii Perechnei redkikh i nakhodyashchikhsya pod ugrozoi ischeznoveniya vidov zhivotnykh i rastenii (Governmental Regulation of the Republic of Kazakhstan no Approval of Checklists of Rare and Endangered Species of Animals and Plants Dated October 31, 2006,