Abstract

Myotis petax is a common and widespread Asian bat species, whose intraspecific sequence variability remains poorly understood. In this work we analyzed the variability of the mitochondrial control region and craniometric measurements for an extensive sample set originating from the entire species range. This made it possible to identify the main genetic lineages and to compare their distribution with the morphological groups. From our investigations, we found that the prevalent genetic lineages, namely, “Siberia,” “Amur,” and “Okhotsk,” appear to be connected to large river systems. The cohabitation of various genetic lineages occurs only in territories where different river basins are connected, such as the Primorsky Territory, Khabarovsk Territory, Transbaikalia Territory, and Mongolia. Moreover, we discovered that the five morphological groups (Siberia, Okhotsk, Amur, Kunashir, and Korea) are partially correlated with previously identified genetic lineages and subspecies. However, M. p. petax and M. p. loukashkini were the only two out of the five subspecies that could be well-defined using specific mtDNA sequences and morphological descriptions. Nonetheless, the subspecies M. p. ussuriensis does not have a distinct genetic lineage to allow for their classification. Notably, a specific mix of morphological group and a genetic lineage characterize the “Amurian morphological form,” which may support its validity as a subspecies rank. That notwithstanding, more information is needed to fully unravel the intraspecific structure of M. petax in the southern Far East and potential contact zones of diverse forms.

Bat, control region, craniometric variability, Far East, genetic variability, Siberia

The Eastern water bat, Myotis petax Hollister, 1912, is a common and widespread Asian bat species. It prefers near-water habitats and often forages at a height of less than 10 cm above the water surface (Tiunov 1997). Myotis petax is considered a sedentary species that spends the winter in caves or mines. However, there is a noticeable discrepancy between the species’ summer and winter abundance in the Russian Far East. It has also been observed that some individuals seem to prefer different kinds of hibernation sites while others migrate outside of summer habitat (Tiunov 1985). The species’ range extends from Western Siberia to the Far East of Russia, Northeast China, Korea, and Japan, and in the south reaches Northern Mongolia (Botvinkin 2002; Smith et al. 2008; Bernikov et al. 2011; Zhigalin and Khritankov 2014; Kawai 2015; Scheffler et al. 2016; Jo et al. 2018). The high density, vast range, and presence of the insular populations make M. petax a convenient object for studying intraspecific geographic variability.

However, M. petax remains a poorly studied bat species, which is largely due to the fact that M. petax was until recently considered as part of the widespread polytypic species Myotis daubentonii Kuhl, 1819 (Ognev 1928; Kuzaykin 1950; Gromov et al. 1963; Tiunov 1984, 1997; Yoshiyuki 1989; Bogdanowicz 1994; Koopman 1994), and only in the early 2000s the species rank of M. petax was confirmed by molecular (Matveev et al. 2005; Kruskop et al. 2012), karyological (Gorobeyko et al. 2020), and morphological data (Kruskop 2004; Matveev et al. 2005). As a consequence, many details of the lifestyle and ecology of M. daubentonii have been extrapolated to M. petax, leaving our data on the latter species actually deficient. Furthermore, only a small number of M. petax individuals have been included in molecular analyses.

Previously, based on mtDNA COI sequences, it was shown that M. petax has low nucleotide variability with a prevalence of the central, most abundant haplotype (Gorobeyko et al. 2020), while the intraspecific p distances between specimens amount to 0.28% to 1.16% (Kruskop et al. 2012; Gorobeyko et al. 2020). The differences between mtDNA cyt b sequences of M. petax from the Russian Far East and Northeast China are amounted to 0.2% (Wang et al. 2010). At the same time, the coefficient of genetic distances (DL) within M. petax calculated on the basis of Inter-SINE(MIR)-PCR data according to Link et al. (1995) range from 0.13–0.55, which may indicate genetic heterogeneity (Matveev et al. 2005).

The Far Eastern specimens of M. petax have significant differences in the amount and locality of heterochromatic materials in chromosomes that are not common for the genus Myotis (Gorobeyko et al. 2020). Another unusual trait of M. petax is the presence of short, 30 bp R1 repeats in the control region of mtDNA found in individuals from the Amur Region and Primorsky Territory of Russia (Gorobeyko et al. 2023). Long, 81 bp R1 repeats in M. petax are presented in a variable copy number (4–7), exhibit inter-individual sequence diversity, and consist of parts of the ETAS1 and ETAS2 conservative blocks of mtDNA. The size heteroplasmy previously described for Myotis species was not detected in M. petax (Gorobeyko et al. 2023).

The following subspecies have been described for the species based on morphometric characters: nominotypical, common in Western Siberia; M. p. ussuriensis Ognev, 1927 inhabiting the Far East; M. p. loukashkini Shamel, 1942, and M. p. chasanensis Tiunov, 1997, whose ranges need to be clarified (Tiunov 1997; Kruskop 2004; Tiunov and Makarikova 2007; Wang et al. 2010; Gorobeyko et al. 2021). In addition, an analysis of the morphometric variability of M. petax revealed the presence in the Russian Far East of the “Amurian morphological form” which differs from the formerly known subspecies by a combination of craniometric parameters (Gorobeyko et al. 2021). It is important to take into account that only M. p. loukashkini was described as a subspecies of M. petax (Shamel 1942), whereas the identification of the subspecies M. p. ussuriensis and M. p. chasanensis was based on the differences of these morphological forms from M. daubentonii sensu lato. Therefore, it appears that the validity of subspecies and the intraspecific structure of M. petax are still unclear.

Given the above context, the goal of this study was to ascertain the intraspecific structure of M. petax by examining their genetic and morphological structures. Accordingly, we analyzed the control region of mtDNA and craniometric variability for an extensive sample from the entire range, which made it possible to define M. petax intraspecific structure for the first time. We then discuss the correspondence of M. petax subspecies to genetic lineages and their possible contact zones of different forms.

It was shown that natural habitats of M. petax and, in particular, the foraging sites are closely associated with different types of water bodies (lakes, ponds) and river systems (Tiunov 1997; Botvinkin 2002; Didorenko et al. 2022). The approximate borders of the mitochondrial genetic lineages’ distributions appear to coincide with the major watersheds of northeast Asia. So, the eastern border of the mitochondrial lineage “Siberia” and the western border of the lineage “Amur” are apparently the Yablonovy Range and the Khentei-Daurian Highlands, representing watersheds of the Arctic and Pacific oceans (Geniatulin 2009). The northern limit of the mitochondrial lineage “Amur” distribution is the Stanovoy Range, another watershed between the Arctic and Pacific oceans. Thus, the approximate range of the lineage “Amur” generally coincides with the Amur River basin, with the exception of the Lower Amur.

The mitochondrial lineage “Siberia” predominates in the Southern Sikhote-Alin and in the foothills of the Chernye Gory Range (and possibly in the Jilin province of China), while haplotypes of the lineage “Okhotsk” are found here in a mosaic manner. This suggests that the pattern of distribution of these two lineages probably represents a gradient, where in the southern part the haplotypes of the lineage “Siberia” predominate, but further north the ratio changes in the opposite direction, and starting from the central Sikhote-Alin, the haplotypes of the lineage “Okhotsk” are prevalent. Indicators of demographic and spatial expansion in the lineages “Siberia” and “Okhotsk” are higher compared to all other lineages. Combined with statistically significant negative Tajima’s D values, this may indicate past population growth with range expansion of these lineages, which, consequently, could lead to the expanding of the contact zone between lineages in Southern and Central Sikhote-Alin.

Due to the lack of genetic data from northeast China, the mitochondrial lineage “Siberia” is assumed to have a significant distribution gap (about 1800 km around). As a result, it is not possible to say unequivocally that the connection between the eastern and western portions of the range is completely interrupted. However, it appears that both parts of the range of the lineage “Siberia” were connected in the past, as has been shown for some Asian species with a ring range (Matyushkin 1976, 1982). Recent findings of M. petax in Jiangxi and Guangdong provinces, confirmed genetically (Wu et al. 2022), indicate that the species’ range in China may be much wider than previously thought.

The value of nucleotide diversity in the Primorsky Territory, Khabarovsk Territory, and Transbaikal Territory is significantly higher compared to neighboring territories (Table 2), which can be explained by the cohabitation of various genetic lineages at the confluence of different river basins. Thus, in Primorsky Territory, where the rivers of the Amur basin contact with the rivers of the basin of the Sea of Japan (East Sea), we discovered three genetic lineages: “Siberia,” “Okhotsk,” and “Amur.” Two genetic lineages, “Siberia” and “Okhotsk,” are found in Khabarovsk Territory, where Amur basin’s rivers connect with the rivers of the Sea of Okhotsk basin. The coexistence of “Siberia” and “Amur” lineages occurs in the Transbaikal Territory and Mongolia, where the rivers of the Amur basin relate with the rivers of the Selenga basin. A possible explanation for the presence of individuals of the lineage “Amur” in the Tayshet City could be incomplete sorting of mitochondrial lineages, but this issue requires further research. Although there are no reliable data on the migratory activity of M. petax, the ecologically similar Western Palearctic species M. daubentonii is considered a sedentary species or a local migrant, but is capable of covering distances of 280–300 km (Hutterer et al. 2005), which is sufficient to cross watersheds between river basins.

In full agreement with the previous studies of morphological variability of M. petax, this work also clearly confirms the similarity of the insular and mainland populations of the southern Far East (Maeda 1985; Yoshiyuki 1989; Tiunov 1997; Kruskop 2004; Gorobeyko et al. 2021), as well as the reliable differences between specimens from the Lake Khasan (Khasan group) and the rest of the Primorsky Territory sample (Tiunov 1997; Gorobeyko et al. 2021). The range of the Western Siberia morphological group coincides with the previously described distribution of the nominotypical subspecies (Kruskop 2004; Gorobeyko et al. 2021). The existence of a separate “Amurian morphological form” (Lower Amur group), similar to the Khasan group, is also supported (Gorobeyko et al. 2021). At the same time, we found that expanding the sample from the Middle Amur and Transbaikalia confirms the validity of the Baikal-Amur morphological group, which has not previously been proven in the studies (Kruskop 2004; Gorobeyko et al. 2021).

Unfortunately, it is not possible to genetically type all individuals whose morphological variability was analyzed, due to the fact that a large volume of museum material was used in the work. Furthermore, for many genotyped individuals, craniological material is not available. Nevertheless, the association between genetic lineages and morphological groups is partially present. We attempted to correlate the morphological groups identified during the morphometric analysis with previously described subspecies and genetic lineages discovered in this work (Fig. 6, Table 5).

Figure 6. 

Ranges of genetic lineages and morphological groups. For Roman numerals, see text. Asterisks indicate the type locality for each subspecies. Empty circles and filled circles represent localities from which only genetic or morphological data were obtained, respectively. The colors of the circles correspond to Figures 3, 5. The squares indicate localities where both genetic and morphological data were studied. The colors of the squares correspond to the colors of the morphological groups. Gray arrow heads show main watersheds within the species’ range.

1. (i) Specimens belonging to the Western Siberia morphological group and genetic lineage “Siberia” can be attributed to the nominotypical subspecies M. p. petax, described from the Chuya Steppe in the northwest of the Altai Mountains (Republic of Altai) (Hollister 1912).
2. (ii) Specimens with a combination of Baikal-Amur morphological group and genetic lineage “Amur” may be identified as M. p. loukashkini. The terra typica for the subspecies M. p. loukashkini is located in Wudalianchi City (Heilongjiang, China), and such a combination was found in the nearest localities, namely Lake Dolgoe (Amur Region) at a distance of 270 km and the Khalkhyn-Gol River (Mongolia) at a distance of 550 km. Moreover, the craniological measurements of the holotype and paratype of M. p. loukashkini (Shamel 1942) fall within the limits of variability for the Baikal-Amur group. All this allows us to assume that a combination of the Baikal-Amur morphological group and genetic lineage “Amur” (ii) may be attributed to the subspecies M. p. loukashkini. A similar combination (ii) was also found in one individual from Tayshet City (Irkutsk Region).
3. (iii) Individuals with a combination of genetic lineage “Siberia” and Baikal-Amur morphological group may probably represent the result of hybridization between the M. p. petax (i) and M. p. loukashkini (ii).
4. (iv) Most individuals of M. petax from Primorsky Territory (with the exception of Lake Khasan) are characterized by a combination of the Far East morphological group and the genetic lineage “Siberia.” It would be possible to assume that this combination may be inherent in the subspecies M. p. ussuriensis described from this territory. Nevertheless, in the putative range of M. p. ussuriensis, we also found combinations of the Far East morphological group with other genetic lineages.
5. (v) Specimens of M. petax belonging to the lineage “Okhotsk” and the Far East morphological group are distributed mainly on Sakhalin Island with isolated individuals in the south of Primorsky Territory. Presumably, this combination may be found in M. petax from adjacent areas of China (Jilin and Heilongjiang province).
6. (vi) Specimens of M. petax belonging to the lineage “Kunashir” and the Far East morphological group are found only on Kunashir Island. Previously, Maeda (1985) and Yoshiyuki (1989) did not find significant differences between M. petax from the Sakhalin Island, Kunashir Island, and Hokkaido Island, as well as from North Korea, and classified all of them as the subspecies M. p. ussuriensis.
7. (vii) The specimen from the Spasskaya Cave (Primorsky Territory) belongs to the lineage “Amur” and the Far East morphological group.

All this indicates the absence of a specific genetic lineage distinguishing the subspecies M. p. ussuriensis, in contrast to the condition observed in the nominotypical subspecies (i) and M. p. loukashkini (ii). Previously, a discrepancy in the distribution of two mitochondrial lineages and two morphological forms was revealed in Eptesicus serotinus Schreber, 1774 (Artyushin et al. 2009, 2012). The possible explanation for this may be incomplete lineage sorting, as well as introgression and fixation of alien mtDNA haplotypes.

1. (viii) Most of M. petax from Lower Amur (Khabarovsk Territory) are identified as the Lower Amur morphological group in combination with the genetic lineage “Okhotsk”. Previously we designated these specimens as the “Amurian morphological form” (Gorobeyko et al. 2021).
2. (ix) The exception is one specimen with a combination of the Lower Amur group and the lineage “Siberia”.

“The Amurian morphological form” has essential differences from the other morphological groups and, moreover, cannot be related to any described subspecies. Greater similarity is observed with the Khasan group; however, the Lower Amur group is distinguished by larger average values of condylobasal and condylocanine length and width between the maxillary teeth. It can be assumed that the genetic lineage “Okhotsk” is inherent in the Lower Amur group, which, in turn, can serve as another argument confirming the validity of the “Amurian morphological form” as a form of subspecific rank. In this case, specimens with a combination of the lineage “Okhotsk” and the Far East group (v) or the lineage “Siberia” with the Lower Amur group (ix) may be hybrids of the “Amurian morphological form” (viii) and individuals combining the Far East group and the lineage “Siberia” (iv).

1. (x) Part of the Middle Amur sample (Amur Region) belongs to the Lower Amur morphological group and genetic lineage “Amur,” while the other part of this sample is identified as M. p. loukashkini (ii).
2. (xi) Most specimens of M. petax from the Proschalnaya Cave (Khabarovsk Territory) belong to the genetic lineage “Okhotsk” and the Baikal-Amur morphological group.

These facts may indicate a possible contact zone between the “Amurian morphological form” (viii) and M. p. loukashkini (ii) at the border of their ranges.

1. (xii) Specimens of M. petax belonging to the Khasan group are found only near Lake Khasan, the southernmost part of the Primorsky Territory. These individuals are, on average, larger than the rest of the sample and were previously described as the subspecies M. p. chasanensis from Khasan District of the Primorsky Territory (Tiunov 1997). In certain publications, subspecies M. p. chasanensis was reduced to a synonym of M. p. loukashkini (Kruskop 2004). Nevertheless, the data obtained in the present work do not support such consolidation.

Unfortunately, due to the lack of material for genetic analysis, it was not possible to establish correspondence of the Khasan group to any genetic lineage. It is worth noting, however, that a distinct genetic lineage, “Korea,” has been identified in the relatively nearby Gangwon Province of South Korea. According to morphometric data (Yoon et al. 2010), specimens from Gangwon, North Gyeongsang, Jeonbuk and Jeonnam provinces (South Korea), presumably belonging to this genetic lineage, are on average larger in craniometric parameters than other M. petax (Gorobeyko et al. 2021). On the contrary, M. petax from South Pyongan Province (North Korea) shows a high degree of similarity with the Japanese specimens in craniometric measurements (Maeda 1985). The relationship between M. p. chasanensis and genetic lineage “Korea” appears to still require further study.

For the first time, a detailed description of the intraspecific structure of M. petax has been presented using both morphological and molecular information. From our investigations, we discovered three common genetic lineages: “Okhotsk,” “Amur,” and “Siberia,” and uncovered that the range of these genetic lineages seems to be connected to large river systems. Notably, “Korea” and “Kunashir,” the two local and most genetically distinct lineages, are exclusive to the Korean Peninsula and Kunashir Island, respectively. The cohabitation of various genetic lineages has been established only for territories where different river basins are connected, such as the Primorsky Territory, Khabarovsk Territory, Transbaikalia Territory, and Mongolia.

We revealed the five morphological groups, which only partially correlated with genetic lineages and morphological subspecies. The two subspecies previously described for M. petax sensu stricto can apparently be defined as a specific combination of a morphological group with a genetic lineage. Thus, M. p. petax can be characterized as specimens belonging to the Western Siberia morphological group and genetic lineage “Siberia,” while M. p. loukashkini represents a combination of the Baikal-Amur morphological group with the genetic lineage “Amur.” Accordingly, a distinctive feature of M. p. loukashkini is the presence of additional R1 repeats in the control region of mtDNA, found only in the lineage “Amur.”

The subspecies structure of M. petax in the southern Far East remains unclear and still requires further study. We found the absence of a specific genetic lineage distinguishing the individuals in the putative range of M. p. ussuriensis, in contrast to the condition observed in the nominotypical subspecies and M. p. loukashkini. On the contrary, the lack of genetic data from the type locality does not allow establishing the relation of M. p. chasanensis with any genetic lineage.

Most specimens of the “Amurian morphological form” are characterized by a specific combination of the Lower Amur morphological group and the genetic lineage “Okhotsk,” which may possibly serve as confirmation of the validity of “Amurian morphological form” as a form of subspecific rank.

We are especially grateful to the members of Vladivostok Caving Club for the organization of field works in the caves of Primorsky Territory. We would like to thank Dr. Oleg N. Morozov (Center of Children’s Complementary Education and Evenkis’ Folk Crafts, Bagdarin, Russia), Yulia A. Mel’nikova and Denis N. Kochetkov (Khingan Nature Reserve), Sergey Yu. Ignatenko and Elena V. Ignatenko (Zeya Nature Reserve), Dr. Alexander D. Botvinkin (Irkutsk State Medical University, Irkutsk, Russia), Dr. Maxim A. Khasnatinov (Federal State Public Science Institution “Scientific Centre for Family Health and Human Reproduction Problems,” Irkutsk, Russia), Vladimir S. Lebedev and Yaroslav A. Red’kin (Zoological Museum of the Lomonosov Moscow State University, as well as part of Joint Russian-Mongolian Complex Biological Expedition of the Russian Academy of Sciences and Mongolian Academy of Sciences), Alexandra P. Shumkina, Elena Yu. Shumkina, Alexander B. Alekseev and Evgeny Raspopov (“Mechta,” Irkutsk, Russia), Nikolai V. Yakovchic (Irkutsk, Russia), Vadim V. Bobrovsky and Polina S. Van (Petrenko) (Komsomolsk-on-Amur, Russia), Evgeny E. Kozlovsky (Yuzhno-Kurilsk, Russia), Vasily V. Gorobeyko (Vladivostok, Russia) for their help in mounting the expeditions. We express our sincere gratitude to the reviewers for their adequate assessment of the manuscript and valuable comments that allowed us to improve the manuscript. We sincerely thank Haneef Ahmed Amissah, a native English speaker, who corrected language errors in the article.

The research was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation theme No. 124012200182-1 (Federal Scientific Center of the East Asia Terrestrial Biodiversity of FEB RAS), theme No. 121030900138-8 (Institute of General and Experimental Biology of SB RAS), and state theme of scientific work of the ZMMU No. 121032300105-0.