ISSN 1062-3590, Biology Bulletin, 2017, Vol. 44, No. 2, pp. 159–171. © Pleiades Publishing, Inc., 2017. Original Russian Text © I.N. Sheremetyeva, I.V. Kartavtseva, M.V. Pavlenko, V.A. Kostenko, I.S. Sheremetyev, I.O. Katin, M.E. Kosoy, 2017, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2017, No. 2, pp. 129–141. ZOOLOGY Morphological and Genetic Variability in Small Island Populations of the Striped Field Mouse Apodemus agrarius Pallas, 1771 I. N. Sheremetyevaa, *, I. V. Kartavtsevaa, M. V. Pavlenkoa, V. A. Kostenkoa, I. S. Sheremetyeva, I. O. Katinb, and M. E. Kosoyc a Institute of Biology and Soil Science, Far East Branch, Russian Academy of Sciences, pr. 100 let Vladivostoku 159, Vladivostok, 690022 Russia b Research and Educational Center Primorskii Aquarium, Far East Branch, Russian Academy of Sciences, ul. Pal’chevskogo 17, Vladivostok, 690059 Russia c Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, 80521 United States *e-mail: sheremet76@yandex.ru Received April 15, 2015 AbstractMorphological (craniometrical characteristics and variations of cusp t3 on the second upper molar (M2)) and genetic (polymorphism of chromosomes and blood proteins) variability was analyzed in small island populations of the striped field mouse Apodemus agrarius Pallas, 1771 from the Peter the Great Bay (Sea of Japan). It was found that the absence of t3 on M2 is not a specific trait for A. agrarius. It was demonstrated that the population of field mice on the Bolshoy Pelis Island significantly differs from the populations from other islands and from the mainland according to the craniometrical parameters, teeth phenes, and variants of blood transferrin. The possible age of establishment of the island populations of the striped field mouse is discussed. DOI: 10.1134/S1062359016050113 INTRODUCTION Microevolution is an early stage of evolutionary processes proceeding in populations and resulting in generation of new populations, subspecies, or species. The discovery of the mechanisms of the microevolutionary process (Timofeev-Resovskii et al., 1973; Shvarts, 1980) is one of the fundamental problems of biology. In this connection, isolated and island populations of wide-distributed species present the significant theoretical interest. The islands located in the Peter the Great Bay of the Sea of Japan (number >15) have different sizes and periods of the isolation. The time of their separation dates back to 8000—11000 years ago (Velizhanin, 1976). All islands of the Peter the Great Bay formed a single whole with the territory of the present-day mainland during the period of the last glacial maximum in the Pleistocene that resulted in the common flora and fauna. Most of the land submerged as a consequence of the climate warming. Only tops of some ridges remained as islands above the ocean surface The islands are inhabited by separate populations of some plant and animal species that were able to adapt to the new conditions. These islands present the convenient model for a study of features specific for initial stages of microevolutionary processes in small isolated populations of mammals, including rodents. Each island population has evolved independently, and therefore each population of mammals is apparently unique. The striped field mouse Apodemus agrarius Pallas, 1771 is a widespread and ecologically plastic species within the Palearctic realm. The extensive area of distribution of this species, separated by a disjunction in Transbaikalia and Mongolia, extends from Central Europe to the edges of Asian continent, including Eastern and Western China, and Korea (Corbet, 1978; Xia, 1984; Karaseva et al., 1992; Gromov and Erbaeva, 1995; Musser et al., 1996; Zhang et al., 1997). A number of isolated island populations, which were given a status of subspecies (Wilson and Reeder, 2005) distributed along the eastern periphery of the region including both relatively large islands of Taiwan and Jeju and many small islands close to the coast of the southern part of the Korean Peninsula (Jones and Johnson, 1965; Koh, 1991) and of the southern part of the Far East of Russia (Nazemnye…, 1984; Kostenko, 2000; Sheremetyev, 2001). In Primorye, the striped field mouse is the most numerous rodent species inhabiting diverse habitats in treeless spaces and areas occupied by the secondary forest (Volkov et al., 1979; Nazemnye…, 1984; Karaseva et al., 1992). On the islands of the Peter the Great Bay, the striped field 159 160 SHEREMETYEVA et al. Table 1. Methods and size of studied striped field mouse Apodemus agrarius samples from island and mainland populations Code* POP PUT AS RUS FUR PEL '' '' '' US '' '' '' SPA '' LAZ KHA Place of collection S, km2 P, km Cra Phen Kar All Popov Island Putyatin Island Askold Island Russky Island Furugelm Island Bolshoy Pelis Island, 1965 '', 1982 '', 1999 '', 2009 Ussuri Reserve, 1982 '', 1986 '', 1990 '', 1982, 1987, 1988, 1995 Spasskii district, vicinity of the village of Bussevka, 1986 '', 1990 Lazovskii district Khasanskii district 12 22.7 14.1 93 2.4 3.3 3.3 3.3 3.3 – – – – – 0.6 1.8 7.2 1.6 5.2 17 17 17 17 – – – – – 16 17 4 6 8 0 0 39 15 0 0 26 0 0 5 25 0 12 0 6 31 15 31 47 42 16 0 30 7 10 0 3 2 0 0 3 8 0 0 33 0 0 3 13 2 10 4 0 16 24 9 0 26 0 58 0 – – – – – – 103 25 55 61 41 24 0 0 16 30 0 25 S, island area; P, distance from supposed source of colonization (Sheremetyev, 2001). Methods of study: Cra, craniometrical; Phen, phenetics of second upper molar (M2); Kar, karyological; All, blood protein electrophoresis. * For Tables 1, 4, 5–7 and Fig. 1. mouse along with the reed vole is a dominated rodent species and it was reported on eight islands, including the Russky, Popov, Putyatin, Bolshoy Pelis, Furugelm, Askold, Reyneke, and Vera islands (Nazemnye…, 1984; Chugunov and Katin, 1984; Sheremetyev, 2001). These islands differ in size, distance from the continent (Table 1), separation time, and habitat conditions (Chugunov and Katin, 1984; Sheremetyev, 2001). The island populations of the striped field mouse in the Far East of Russia have not been studied sufficiently. The most complete data on genetic and morphological characterization are available mice from Jeju Island and from a number of small islands close to the coast of the Korean Peninsula (Koh, 1991; Koh and Yoo, 1992; Han et al., 1996; Koh et al., 2000, 2014; Oh et al., 2003, 2013; Yoon et al., 2004). For islands in the Peter the Great Bay, only preliminary data on genetic uniqueness of the mice from the Bolshoi Pelis Island are available. In this population, a variant of the blood transferrin (Tf) rarely reported in mainland samples (Pavlenko, 1994) and a presence of the cusp t3 on the second upper molar (M2) in most studied individuals was found. These reports raised a question whether mice from this island indeed belong to the species of A. agrarius (Kostenko et al., 2003). The absence of t3 on M2 is one of the diagnostic traits for identification of mice belonging the species A. agrarius (Ruprecht, 1978). Although limited material from islands of the Peter the Great Bay (4 from Putyatin Island; 2, from Askold; 5, from Reyneke; 5, from Russky) was included in the phylogeographic study of Apodemus species in Asia based on the mtDNA cyt b gene (Sakka et al., 2010), differentiation of island populations of the striped field mouse was not specially considered in this work. In other studies, variability of nuclear genes (Atopkin et al., 2007; Zasypkin et al., 2007; Dokuchaev et al., 2008) and mitochondrial (Pereverzeva and Pavlenko, 2014) genes was analyzed in mice from mainland populations of the Far East of Russia. The objectives of this study were: an analysis of microevolutionary processes in the isolated island populations of striped field mouse based on morphological and genetic (chromosomal and allozyme) variability of field mice from the Bolshoy Pelis Island compared with both small island populations from the Peter the Great Bay and from three mainland populations; study of the variability and differentiation patterns according to craniological traits; estimationn of the significance of t3 on M2 as a differentiating and diagnostic trait; description of the blood Tf variability; and analysis of karyotypes. BIOLOGY BULLETIN Vol. 44 No. 2 2017 MORPHOLOGICAL AND GENETIC VARIABILITY SPA 161 US LAZ Vladivostok POP RUS KHA AS PUT PEL FUR Peter the Great Bay Fig. 1. Places of Apodemus agrarius material collection. MATERIALS AND METHODS Striped field mice were caught on six islands of the Peter the Great Bay (Sea of Japan) and on the mainland territory of southern part of Primorskii krai (Russia) (Fig. 1). The number of studied samples is presented in Table 1. The skulls of the studied animals, as well as chromosomal slidess and blood serum samples, are stored in the collection of the Institute of Biology and Soil Science, the Far East Branch, Russian Academy of Sciences. Craniometrical analysis. The following cranial parameters were measured in 314 individuals: the condylobasal length (CBL), upper diastema length (DL), zygomatic breadth (ZB), interorbital distance (ID) breadth, cranium width near hearing capsules (NCW), cranium height near hearing capsules (NCH), and length of top row molars (LM) (Fig. 2), (Table 1). All measurements were performed by use of the electronic caliper. Only adult individuals were included in the analysis. The animal age was estimated based on reproductive status of the animals and by the degree of teeth effacement. The sexual dimorphism was estimated using the Kruskal–Wallis coefficient for each parameter (differences are significant at the significance level of p < 0.01). Randomly selected (not specifically assigned) mice were the object for the analysis. The analysis has followed the scheme proposed by Puzachenko (Boeskorov and Puzachenko, 2001; Puzachenko, 2001) that was previously used for the study of craniometrical variability of gray voles (Sheremetyeva, 2007; Sheremetyeva et al., 2009) and Siberian roe deer (Sheremetyeva, Sheremetyev, 2008). Eighty-five individuals were randomly selected out of the 314 mice (a “working” sample set). The Euclidean distance was used for estimation of the distance BIOLOGY BULLETIN Vol. 44 No. 2 2017 between each pair of individuals from this sample set. All measurements were standardized beforehand. After that a minimal number of hypothetically mutually independent (not correlated) variables for multidimensional scaling axes (MSA) was found on the basis of the distance matrix. The minimal number of MSA was preliminarily estimated based on the compliance method to measure stress (S). The second criterion was based on interpretation of the MSA axes by analysis of the correlations between measured parameters. For this purpose, the rank Spearman correlation was calculated. Then the hierarchical classification of individuals was conducted using the MSA as variables, and the unweighted pair group method with the arithmetic mean (UPGMA) and standard Euclidean distance. The maximal number of groups at the lower level of classification was selected. Quality was estimated by means of the “median” test for each parameter (nonparametric analogue of the rank dispersion Kruskal– Wallis analysis). The most significant parameters to separate individuals into clusters were selected from a totality of parameters by the stepwise discriminant analysis method. By means of the selected parameters and the “working” sample set, it was determined to which a particular group of other specimens was belonged. The results were compared with classes of individuals from the geographical locations by using the cross-tabulation method. The χ2 Pearson’s agreement criterion was used for testing the classification independence. All calculations were conducted by means of the Statistica 8.0 program package for Windows (StatSoft, MLTsa, OK, United States). SHEREMETYEVA et al. ID CBL LM DL 162 A B C D Fig. 3. Morphotypes of cusp t3 on M2 (Kawamura, 1989). A, absence of cinture and cusp t3; B, presence of cincture; C, presence of small cusp t3; D, presence of well-expressed cusp t3. ZB NCH NCW Fig. 2. Cranium parameters of Apodemus agrarius: CBL, condylobasal length; DL, upper diastema length; ZB, zygomatic breadth; ID, interorbital distance; NCW, cranium width near hearing capsules; NCH, cranium height near hearing capsules; LM, length of top row molars. Analysis of М2 variability. An additional cusp t3 on was studied in 380 individuals of island and mainland populations, including 119 individuals from populations of six islands and 261 individuals from four mainland populations (Table 1). We used the morphology study method M2 developed of cusp t3 for the A. speciosus Temminck, 1844 (Kawamura, 1989), and four morphotypes of the cusp t3 were differentiated (Fig. 3). Chromosomal analysis. The slides were prepared according to the standard method from bone marrow cells (Ford and Hamertom, 1956). Forty minutes before slaughter, 0.04% colchicine solution (calculated 1 mL solution per 100 g of individual weight) was introduced into the animals. The bone marrow cells were washed out from the femoral bone with a 0.56% hypotonic potassium chloride solution and kept at room temperature for 20 min. The fixation was conducted in ethanol/acetic solution (3 : 1) with triple change of the fixator. Nucleolus organizer regions (NOR) were stained according to the Münke and Schmiady method (Münke and Schmiady, 1979). Analysis of blood proteins as biochemical gene markers. The blood serum samples obtained from the animals caught by animal traps were used for this. In total, 220 animals were analyzed. The places of material collection and the sizes of the studied samples are presented in Table 1. The method of electrophoresis in starch gel was used in this study. Tf was detected using the buffer system (BS) of Christianson in the modification of FomiM2 cheva (1973) and using BS-1 (Glazko, 1985). The gel sections were stained by the Amido Black Solution 10B. We previously described the methodological details for the interpretation of the Tf spectrum for analysis of mice of the genus Apodemus (Pavlenko et al., 1984; Pavlenko, 1989). RESULTS Craniometrical analysis. No significant differences in any of the craniometrical parameters were found between males and females of striped field mice. It allowed to ignore gender differences in the cranium sizes and to unite the individuals of both sexes for further analysis of variability. As a result of multidimensional scaling of the matrix reflecting distances between individuals by sizes, the minimal dimension was 6. Six values of MSA coordinates were compared for each individual. The ratio between measurements was calculated based the analysis of their correlation for each MSA (Table 2). All parameters (with the exception of LM) correlate statistically significantly with the first axis; CBL, DL, ID, NCW, and NCH, with the second axis. Significant correlations with the third axis were registered for the length of the top row molars. The fourth axis mainly describes the interorbital width; the fifth axis, cranium width and height near the ear drums; and the sixth axis, the zygomatic breadth. The variability of the cranium is not random, since there is a bend on the graph of intercluster distances, which gives grounds to assume the possibility of separation of some groups. Three main clusters and two hierarchical levels were discriminated as a result of the classification of the specimens using MSA (Fig. 4a). Differences in the cluster I specimens from the others were the most significant for CBL, DL, ZB, NCW, and NCH. The biggest differences between clusters II and III were detected for ID and NCH. Other specimens (not included in the “working” sample set) were classified by the method of discriminant analysis using the set of differentiating cranium parameters as independent variables. The efficiency of the specimen classification was 99.9% (Fig. 4b). Two parameters (CBL and ZB with standardized coeffiBIOLOGY BULLETIN Vol. 44 No. 2 2017 MORPHOLOGICAL AND GENETIC VARIABILITY 163 Table 2. Spearman correlation values of Apodemus agrarius cranium parameters with multidimensional scaling axes (D1– D6) obtained based on the variability similarity matrix Multidimensional scaling axes Parameter CBL DL ZB ID NCW NCH LM D1 D2 D3 D4 D5 D6 –0.789273* –0.735324* –0.757654* –0.542605* –0.621485* –0.693676* –0.056859 0.511452* 0.568758* 0.08145 –0.481103* –0.459981* –0.364747* –0.16179 0.198904 0.093501 –0.147978 –0.217943 0.057868 –0.114559 0.938395* –0.121511 –0.149287 0.304867 –0.594693* 0.221727 0.219413 0.005073 0.001114 –0.099189 0.208418 0.089867 –0.466861* 0.3999* 0.064083 –0.014413 0.064807 –0.46076* –0.241744 0.010839 0.299053 –0.106241 CBL, condylobasal length; DL, upper diastema length; ZB, zygomatic breadth; ID, interorbital distance; NCW, cranium width near hearing capsules; NCH, cranium height near hearing capsules; LM, length of top row molars; for Tables 2 and 3. * Values for which р < 0.01. Table 3. Cranium measurements (mm) of Apodemus agrarius individuals generating three morphological clusters Cluster I II III CBL DL 25.19–29.69 6.8–8.65 26.57 ± 0.06 7.6 ± 0.03 24.1–26.8 6.36–7.6 25.16 ± 0.12 7.02 ± 0.07 23.49–27.57 6.02–7.86 25.07 ± 0.07 7.02 ± 0.03 ZB 12–14.5 12.88 ± 0.03 11.5–12.8 12.27 ± 0.05 10.58–13.58 12.14 ± 0.04 ID NCW 3.99–4.83 10.06–12.3 4.41 ± 0.02 11.44 ± 0.03 4.2–4.8 10.2–12.09 4.57 ± 0.03 11.33 ± 0.07 3.9–4.85 10.1–11.98 4.27 ± 0.02 11.01 ± 0.03 NCH LM 8.65–10.3 9.39 ± 0.02 9–10.1 9.47 ± 0.05 8.14–9.54 8.84 ± 0.03 3.52–4.7 4.07 ± 0.02 3.73–4.5 4.12 ± 0.04 3.33–4.7 4.06 ± 0.02 Upper line, limits of the parameter variability; lower line, average value and standard error. cients –0.697 and –0.611, respectively) entered the discriminant function in the case of two clusters (I and II + III). CBL (standardized coefficients for the first function –0.823; for the second, –0.572), ZB (0.004, 0.787), and NCH (–0.499, 0.568) entered the discriminant functions at the level of three clusters. Differences between individuals of clusters I and III are manifested in all cranium measurements with the exception for the length of the top tooth row (Table 3). Differences of individuals of cluster II from individuals of other groups are manifested in the measured craniometrical variable. Relatively big dimensions for height and width of the skulls near the ear drums and interorbital distance were typical for individuals belonging to this cluster. The distribution of individuals from different clusters for each of the eight sample sets was significantly different from random distribution (χ2 = 79.867, p < 0). In the mainland populations (MAT), most individuals belonged to the clusters I and III (51.2 and 42.6%, respectively), while individuals belonging to the cluster II had 6.2% (Table 4). In the island populations, individuals belonging to the clusters I and III also prevailed, while the portion of the cluster II individuals did not exceed 25%. The portion of cluster I individuals in most island samples exceeds 62.5%. Exceptions BIOLOGY BULLETIN Vol. 44 No. 2 2017 are samples collected from the Bolshoi Pelis Island in 1999 and 2009 with most individuals (64.1–73.3% of) belonging to the cluster II (Fig. 5a). Individuals of clusters I and III were also found in these samples however, their portion did not exceed 20.5%. Thus, three groups of striped field mouse individuals can be differentiated by craniometrical measurements. The first group included individuals from the mainland part of Primorskii krai and from the Askold Island. Both mice with small and large values of the cranium parameters prevailed equally there. The second group (where individuals with a larger cranium prevailed) included mice from other islands of the Peter the Great Bay (the Popov, Putyatin, Russky, and Furugelm islands), except for the Bolshoy Pelis Island. The third group included individuals from the Bolshoy Pelis Island, where individuals had a small cranium, but a relatively large cranium height and width near the ear drums and interorbital distance, prevail. Analysis of М2 variability. It was demonstrated that all four morphotypes determined by appearance of the cusp t3 on the second upper molar (typical for the members of the Apodemus genus) are present in field mice from Primorskii krai (Fig. 5b), but their distribution varied between different populations (Table 5). 164 SHEREMETYEVA et al. of both a well-expressed (D morphotype) and a small cusp t3 (C morphotype) prevailed. The study of samples of different years (1965, 1982, 1999, and 2009) of the collected mice demonstrated the decrease in the number of animals with the cusp t3 and the increase in the number of animals with cincture on the upper molar. It is interesting that the number of animals with a small cusp t3 increased over 44 years (from 1965 to 2009). The portion of mice with the cusp t3 on the Popov and Russky islands was also high (60 and 75.7%, respectively). It was registered that the animals with a small cusp t3 prevailed in these populations. The ratio of animals with the cincture per M2 in all three island populations was approximately the same (Table 5), while the portion of animals with the A phenotype (without cusp t3 and cincture) varied from 8.3 (Russky Island) to 44% (Putyatin Island). (а) ED 1.6 1st level 2nd level 1.2 0.8 0.4 III II All four variants of anteroexternal cusp t3 manifestation were registered in four mainland populations; their frequency varied widely, the A phenotype (without cusp t3 and cincture) prevailed (Table 5). The study of nonsimultaneous striped field mouse samples from two mainland populations (located approximately 100 km from each other) demonstrated a relative stability in this trait (Table 5). Thus, the portions of animals with the cusp t3 were 30, 23.8, and 25% in the samples of 1982, 1986, and 1990, respectively (taken from the population of Ussuri Reserve). The portions of animals with a cusp t3 in two samples (1986 and 1990) in animals from Spasskii district were lower than in the Ussuri population, but also changed insignificantly in different years and were 16.7 and 18%, respectively. The cusp t3 in both populations was always very small (C phenotype). I (b) F2 4 0 −4 −4 0 4 F1 Fig. 4. UPGMA classification of individuals of “working” sample (a), in which multidimensional scaling axes were used as variables. Differences between individuals were estimated by the Euclidean distance (ED). (b) Position of individuals in the space of discriminant functions (F1, F2) united in three clusters (I—III) corresponding to the second level of classification. In the population on the Bolshoy Pelis Island, the A morphotype (without cusp t3 and cincture) was completely absent, while individuals with the presence In addition, we also studied two mainland populations of striped field mice, each represented with one sample set. One population, located in Khasanskii district of Primorskii krai, is located approximately 200 km southwest of the Ussuri population. Another population is located in Lazovskii district approximately 200 km southeast of the Ussuri population. The portions of animals with the cusp t3 (C + D phenotype) in these populations did not differ significantly (33.3 and 39%, respectively). The animals with a large cusp t3 (D phenotype) appear in populations of Lazovskii district (12.2%), while such a phenotype was not registered in other mainland populations (Table 5). Thus, the mainland populations (MAT) are usually characterized by animals without an additional cusp t3 (A phenotype), and the portion of such animals is, on average, 63.5%. The portions of animals without the cusp t3 vary from 37.5 to 77%. The minimal number of animals with such a trait was registered in the Lazovskii population (37.5%). The portion of animals with the cusp t3 (C + D phenotypes) on the mainland was smaller (on average, by 24.6%) and varied from 16.7 to 39%. BIOLOGY BULLETIN Vol. 44 No. 2 2017 MORPHOLOGICAL AND GENETIC VARIABILITY The island populations differ from the mainland populations by the A phenotype. If the animals with such phenotype were absent on the Bolshoy Pelis island, a portion of such animals in populations of the Popov, Putyatin, and Russky islands was different (20, 44, and 8%, respectively), that brings together the first two populations closer to the mainland populations. The portion of animals with the cusp t3 (75.7%), C and D phenotypes, was high in the Russky island population. The presence of a well-pronounced additional cusp t3 (the D phenotype) is typical for all island populations. This trait is extremely rare for the mainland populations (12.2%) and was registered only in one population. Chromosomal analysis. All studied specimens of striped field mice from island and mainland populations demonstrated stable number of chromosomes (2n = 48); no B-chromosomes were detected. The karyotype of mice is characterized by 19 pairs of acrocentric–subtelocentric (A–St) chromosomes gradually decreasing in size and usually by four pairs of small metacentric autosomes (M). Variability in the number of the chromosome arms (NF = 54–56) caused by the morphology of small autosomes and previously described was detected in certain populations (Kartavtseva and Pavlenko, 2000; Kartavtseva, 2002). The number of small metacentric chromosomes in the karyotype of mice from the Bolshoy Pelis Island varied from 6 to 8, while there were 8 chromosomes of this type in other island populations. Previously, a variability in distribution of heterochromatin material was detected in the mainland and island populations of the south of the Far East of Russia (Kartavtseva and Pavlenko, 2000; Kartavtseva, 2002). Due to incomplete data for the island population, no analysis of the variability of heterochromatin material in the chromosomes was conducted in the present work. The staining on NOR in the striped field mouse karyotype demonstrated the presence of NOR in telomeric regions of two middle size pairs of telocentric chromosomes and in the short arms of the small size acrocentric chromosome. No differences in the character of NOR-staining of the chromosomes were detected among the populations studied. Analysis of blood proteins as biochemical gene markers. In total, we detected eight electrophoretic Tf variants. Frequencies of Tf variants in striped field mice are presented in Table 6. The AA variant was the most prevalent variant with the frequency varied from 0.5 to 1.0. The presence of four Tf alleles is supposed. According to other researchers, at least two Tf alleles were also detected in the striped field mouse (Dobrowolska et al., 1983; Tsuchiya, 1984; Dobrowolska and Wolanska, 1985). The allele a, found both in the homozygous state (in most individuals) and in the correspondent heterozygotes, was the most common allele among investiBIOLOGY BULLETIN Vol. 44 No. 2 2017 165 Table 4. Portion (%) of morphological clusters I–III in geographical samples of Apodemus agrarius Code n, specimens I II III POP PUT AS RUS FUR PEL, 1999 '' , 2009 US, 1990 SPA, 1990 LAZ KHA MAT 16 17 4 6 8 39 15 26 103 25 55 209 68.8 70.6 50 66.7 62.5 20.5 13.35 80.8 75.7 36.4 14.5 51.2 25 5.9 0 0 12.5 64.1 73.3 3.8 5.8 5.5 6.5 6.2 6.2 23.5 50 33.3 25 15.4 13.35 15.4 18.5 58.1 79 42.6 n, sample size; for Tables 4, 5. Table 5. Portion of Apodemus agrarius individuals (%) in geographical samples of Primorskii krai with different phenes of the second upper molar Code n, specimens A B POP PUT RUS PEL, 1965 '' , 1982 '' , 1999 '' , 2009 US, 1982 '' , 1986 '' , 1990 SPA, 1986 '' , 1990 LAZ KHA MAT 5 25 12 6 31 15 31 47 42 16 30 61 41 24 261 20 44 8.3 0 0 0 0 60 69 75 70 77 46.3 37.5 63.5 20 20 16.7 0 3.2 20 32.2 10 7.2 0 13.3 5 14.7 29.2 11.9 C D C+D 40 20 60 32 4 36 59 16.7 75.7 0 100 100 12.9 83.9 96.8 26.7 53.3 80 48.4 19.4 67.8 30 0 30 23.8 0 23.8 25 0 25 16.7 0 16.7 18 0 18 26.8 12.2 39 33.3 0 33.3 22.3 2.3 24.6 gated field mice. Its frequency varied from 0.7 to 0.75 between different sample sets from the mainland part of Primorskii krai. Additional b and c alleles were registered in almost all sample sets with frequencies varied from 0.15 to 0.24 for the allele b and from 0.028 to 0.077 for the allele c. They were presented in both heterozygous and homozygous variants. The rarest allele f was mainly found in the heterozygous state in single animals from several sample sets from the southern mainland part of Primorye. The mosaic distribution of 166 SHEREMETYEVA et al. (а) SPA MAT US Vladivostok LAZ RUS KHA POP PUT ASK PEL FUR I II III (b) SPA US MAT Vladivostok LAZ RUS KHA POP PUT PEL A B C D (c) SPA US Vladivostok MAT RUS KHA PUT PEL b c f a Fig. 5. Occurrence of individuals belonging to different morphological clusters (a), with different phenes of the second upper molar (b), and frequency of transferrin alleles (c) in geographical samples. Tf variants and alleles was observed in the part of the studied area (Fig. 5c). Two groups can be distinguished among the island populations from the Peter the Great Bay. The main Tf variant (AA) was mainly detected in the first group, and, respectively, the allele a dominated on the Big Putyatin, Popov, and Russky islands close to the coast (Table 7). The second group included separated populations on the Bolshoy Pelis, Furugelm and Askold islands, where allele b dominated, while the common BIOLOGY BULLETIN Vol. 44 No. 2 2017 MORPHOLOGICAL AND GENETIC VARIABILITY 167 Table 6. Occurrence of variants and allele frequencies of transferrin in samples from island and mainland field striped mouse populations in Primorskii krai Code POP PUT AS RUS FUR PEL, 1982 '', 1999 '', 2009 US SPA KHA MAT n specimens 3 13 2 10 4 16 24 9 84 30 25 139 Variants AA BB CC AB AC AF BC 3 13 0 8 1 0 1 3 49 17 11 77 0 0 2 0 1 14 23 6 2 1 1 4 0 0 0 0 0 0 0 0 2 0 0 2 0 0 0 1 2 2 0 0 21 6 10 37 0 0 0 1 0 0 0 0 6 4 1 11 0 0 0 0 0 0 0 0 1 1 2 4 0 0 0 0 0 0 0 0 3 1 0 4 allele a was found only in heterozygous state and only in few individuals. In the population from the Bolshoy Pelis Island, represented by the samples of 1982, 1999, and 2009 the Tf variant BB and, respectively, allele b, constantly dominated. This allele was also frequently registered among samples from the Furugelm and Askold islands, and both studied individuals from the Askold Island were Tf BB homozygotes. DISCUSSION Craniometrical variability. Multidimensional analysis of the cranium variability within populations of the striped field mouse from the mainland territory of Primorskii krai and from islands in the Peter the Great Bay demonstrated a high degree of variability of the craniometrical parameters. The main differences between individuals and their groups are manifested in the size of skulls and in the ratio between height of cranium to the condylobasal length. The formal classification allowed us differentiate three morphologically isolated groups of individuals based on the difference in measurements of the skulls. The variability of craniometrical parameters of striped field mice was not found accidentally. Significant differences between striped field mice from all island populations (except those from the Askold Island) and the mainland, as well as between the island populations themselves, were detected (Table 7). Thus, each island population of the striped field mouse demonstrated specific craniometrical feature that was formed as a result of unique history of each population. The largest level of differences by craniometrical traits was found for the population of mice from the Bolshoy Pelis Island. Variability of the cusp t3 on the second upper molar. The presence or absence of t3 on the second molar M2 BIOLOGY BULLETIN Allele frequencies Vol. 44 No. 2 2017 a b 1 0 1 0 0 10 0.9 0.05 0.5 0.5 0.062 0.938 0.042 0.958 0.333 0.667 0.75 0.167 0.75 0.15 0.7 0.24 0.741 0.176 c f 0 0 0 0.05 0 0 0 0 0.077 0.083 0.028 0.068 0 0 0 0 0 0 0 0 0.006 0.017 0.002 0.014 is one of the main diagnostic traits for identification of species of the genus Apodemus (Musser et al., 1996). For A. agrarius (the cusp t3 was present in 7.2% out of 131 mice studied in collections from China) and Apodemus chevieri (Milne-Edwards, 1868), the absence of cusp t3 in most individuals was typical; for A. speciosus (endemic of Japanese islands), a low frequency of animals with this trait was registered (Kawamura, 1989; Musser et al., 1996); for A. gurkha (Thomas 1924), a decrease in the cusp t3 sizes was typical (Martens and Niethammer, 1972). For the A. specious, a variability in presence/absence of cusp t3 was described (for the first time for the genus), and variability of the cusp t3 sizes from well-pronounced to small (which is always located at an overflowing dentinal area similar to the cincture) was analyzed. In addition, the cincture Table 7. Degree of differences estimated according to Pearson’s χ2 criterion of geographical Apodemus agrarius samples by composition of morphological groups (clusters) included in them POP +++ PUT + +++ AS – – – PUS ++ +++ – – FUR +++ +++ +++ – – PEL +++ +++ +++ +++ +++ +++ MAT POP PUT AS RUS FUR “–”, no significant differences; “+”, weak differences (p < 0.05); “++”, considerable differences (p < 0.001); “+++”, maximal differences (p < 0.00001). 168 SHEREMETYEVA et al. sometimes can be presented on the tooth without the cusp t3 (Kawamura, 1989). Thus, four morphotypes of the studied trait (the presence of a cincture with a well-pronounced cusp t3, with a poorly pronounced cusp t3, without a cusp t3, and the absence of the cincture and cusp t3) were described for M2. Previously, two traits (the presence and absence of cusp t3) were studied for European populations of the striped field mouse. The detection of cusp t3 frequency variability (by the presence of an additional cusp t3) demonstrated that, on average, 3.9% out of 3228 analyzed teeth of striped field mice from Poland had this cusp t3 on M2. The portion of individuals with the additional cusp t3 varied from 0.6 to 33 and even 40% between different sample sets. Thus, the diagnostic significance of this trait for identification of the species (first of all, for the striped field mouse) was questioned (Ruprecht, 1978). The analysis of the top molar morphotypes demonstrated a sharp difference of mice from the Bolshoy Pelis Island, where mice without an additional cusp t3 on M2 of the Apodemus genus were not found at all. This supports our previous results (Kostenko et al., 2003). The presence of the cincture (B phenotype) was registered in both all island and mainland populations. The portions of individuals with this phenotype can reach 32.2% on islands and 29.2% in mainland populations. Individuals from all studied islands were different from the mainland populations by the presence of a well-pronounced cusp t3 (C + D phenotypes). In turn, the portion of individuals with the A phenotype was always higher in the mainland populations of field mice. Consequently, the presence/absence of t3 on M2 cannot be considered as a differentiating feature for the Apodemus genus. At the same time, parameters such as the well-pronounced cusp t3 (C + D phenotype) and the absence of a cusp t3 (A phenotype) can differentiate populations of A. agrarius. Chromosomal variability. The study of the chromosomes of striped field mice demonstrated a similarity of karyotypes between populations on the islands of the Peter the Great Bay and all previously studied mainland populations by number of the 2n. The variability by the number of arms (NF = 54–56) was in the range of variability reported for this species (Kartavtseva, 2002; Chassovnikarova et al., 2009). It was found that NF in the island mouse karyotype was stable (56) except for individuals from the Bolshoy Pelis Island (54–56). Such variants were also registered for a number of mainland populations from the Far East. The mice had evident diversity in the number of NOR (Boeskorov et al., 1995). The observed number and localization of NOR-blocks on the chromosomes was similar to those for A. agrarius from the southern part of the Far East (Kartavtseva, 2002). No differentiation by this trait was detected between the island and mainland populations in the southern Far East of Russia. It was reported a single case that an isolated population of the Saaremaa Island (Baltic Sea) in Estonia was different from all other studied populations by the number of NOR in chromosomes (NOR blocks are located in two, but not three pairs of autosomes) in the western part of the striped field mouse area (Boeskorov et al., 1995). No additional or B chromosomes that we previously described for this species from the mainland populations of Primorskii krai (Kartavtseva, 1994) were found in the studied island populations. Thus, the results of our study demonstrate that the lasting separation of the populations of the striped field mouse island in the Peter the Great Bay has resulted neither in a variability of the number of chromosomes or morphology and differentiation of their karyotype. Blood protein variability. It was demonstrated the replacement of the main Tf allele for an alternative variant, which occurs in a small frequency (0.176, on average) on the mainland. The tendency towards homozygotization of populations by the allele b was observed in the population of striped field mice from the Bolshoy Pelis Island comparing to those on the mainland and to a number of big islands close to the coast. The field mice from the Russky, Putyatin, and Reyneke islands were characterized by presence of the main Tf variant, which is also prevalent on the mainland. Ecological features and possible history of population of striped field mice on the bolshoy pelis island. The populations of striped field mice on the islands of the Peter the Great Bay live under pessimal conditions comparing to the mainland populations. Significant fluctuations in density of the mouse populations are common on the Bolshoy Pelis Island (Chugunov and Katin, 1983, 1984). According to the long-term observations, the trapping success measured by a number of individuals per 100 trap-nights varied as follows: 1978, 0.7; 1979, 9.7; 1981, 6.2; 1982, 51.3; 1983, 1.8; and 1984, 8.2%. The first observation that we were made in 1982 during a rapid increase in the number followed by the population depression that lasted for four years (Chugunov and Katin, 1984). Of 34 animals, more than a half could not survive a short-term stay in the vivarium under standard conditions. The autopsy of dead animals revealed presence of helminthes in the internal organs and increased size of spleen. Though special studies are absent, these observed details can indirectly indicate a weak status of the mouse population. In 1999, the high density was observed (59% of trapping success), but investigations made in 2009 have also demonstrated the low number of mice (7.3%). We can assume that significant fluctuations in the small population along with isolation and so-called “founder effect” could play an important role in developing the morphological and genetic features BIOLOGY BULLETIN Vol. 44 No. 2 2017 MORPHOLOGICAL AND GENETIC VARIABILITY observed in the population of striped field mice on the Bolshoy Pelis Island. It remains problematic to estimate the age of small isolated populations of striped field mice, including the populations on the Bolshoy Pelis Island. The striped field mouse is known to be ecologically limited synanthropic species (Karaseva et al., 1992; Kucheruk and Karaseva, 1992), and distribution of this species at present is closely associated with human economic activity (Volkov et al., 1979; Karaseva et al., 1992; Tikhonova et al., 1992). In this regard, a possibility of invasion of striped field mice to the islands of the Peter the Great Bay cannot be completely excluded; however, we have no data directly demonstrated an introduction of striped field mice to the Bolshoy Pelis Island. It is probably necessary to consider the history of the modern distribution of striped field mice in the southern part of Primorskii krai in the light of paleontological and paleogeographical data. Fossilized remains of the striped field mouse are known from the Holocene layers in the cave sediments found the southern Primorye (Panasenko and Tiunov, 2010). Intensive warming was registered in the beginning of the Holocene (10500–10200 years ago). The climate change led to increase in the portion of deciduous trees in the composition of vegetation in the southern Far East (Korotky et al., 1988) and the development of steppe and meadow vegetation at low relief levels in southern Primorskii krai (Korotky et al., 1996). The expanding distribution of striped field mice to the north from Eastern Asia probably happened during this period. The time of the last connection between the islands of the Peter the Great Bay and the mainland dates back to the same period (Korotky et al., 1988). Therefore, the time of their separation was estimated around 7000—11000 years ago, and the Bolshoy Pelis Island together with the neighboring Matveev Island was first to be separated from the mainland (approximately 10000 years ago). We cannot exclude that striped field mice have already occupied coastal regions that were still not completely separated by the Late Glacial (Korotky et al., 1988, 1996). At that time, the low-lying lands framed by separate elevated places wereI common in the area of the modern Peter the Great Bay. It is possibe that local land bridges, which connected islands with the mainland and provided habitats appropriate for striped field mice existed in the Boreal and the beginning of the Atlantic. The latter assumption is consistent with data on fluctuations in the level of the Sea of Japan in the Late Glacial (Korotky et al., 1980, 1988), according to which the sea level still was at the level 43–47 m at the beginning of the Holocene, while the modern shape of the coastline was developed in the Holocene optimum (approximately 6000 years ago) (Korotky et al., 1996). BIOLOGY BULLETIN Vol. 44 No. 2 2017 169 ACKNOWLEDGMENTS The authors are grateful to the workers of the Far East Marine Biosphere Reserve for the assistance in trapping the animals. This work was supported in part by the Russian Foundation for Basic Research, project no. 15-0403871. REFERENCES Atopkin, D.M., Bogdanov, A.S., and Chelomina, G.N., Genetic variation and differentiation in striped field mouse Apodemus agrarius inferred from RAPD–PCR analysis, Russ. J. Genet., 2007, vol. 43, no. 6, pp. 665–676. 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