Karyology of the Bats from the Russian Far East Karyology of the Bats from the Russian Far East

Recent studies based on morphologic and molecular genetic data have revealed quite a seri- ous variety in the trans-Palearctic species, which brought about taxonomic status changes in 14 of 18 Russian Far Eastern bat species. Far Eastern bat status revisions resulted in species growth whose chromosome characteristics have been described either under other names or have not been studied at all. This paper has inventoried bat chromosome research in the Russian Far East and neighboring regions and has improved the accuracy of chromosome characteristics for 17 of 18 valid species today. For the first time, the karyotypes and their variation type for the valid bat species in the Russian Far East have been described.


Introduction
Till the middle of the twentieth century, most of the Russian bats were considered to belong to widespread Palearctic species. Since the mid-1960s, a gradual transition from the "wide" polytypic species concept appears to be replaced by the "narrow" monotypic one [1]. This is largely due to the improved morphological data processing methods [2][3][4] and the use of the molecular genetic [5,6] and the karyological [7][8][9] methods in bat systematics. Many of the Far Eastern bat taxa were treated formerly as eastern subspecies within polytypical trans-Palearctic species. Recently, most of the Far Eastern subspecies have been elevated to a species rank, which resulted in taxonomic status changes of 14 Far Eastern bat species [5,6,[10][11][12][13][14][15][16][17][18][19][20][21]. However, the taxonomic status of certain forms needs to be clarified [22]. Most of these species are restricted to Northeast Asia, with the western species distribution bordering the Trans-Baikal and the Altai regions [22,23].
The so-called Myotis-type karyotype with 2n = 44 and fundamental number (NFa) being 50 is accepted to be the ancestral karyotype of family Vespertilionidae Gray, 1821 [37]. The chromosomal arms are usually numbered using Bickham's scheme, in which ordinal numbers have been assigned to all the autosomal arms based on GTG-banding patterns [40].
The position and number of the nucleolus organizer regions (NORs) and the amount and location of heterochromatic material (C-band) on chromosomes of many vespertilionid species have been shown to represent species-specific characteristics. The sequential staining methods (G-band; NOR; С-band) revealed karyological differences in species of the same karyotype [7,8,39,[41][42][43][44][45].
Species composition revision of the Far Eastern bats caused an increase in the number of species, whose chromosomal characteristics were reported either under the wrong species names or were not studied at all.
The paper presents an inventory of available karyological data on bats from the Russian Far East and neighboring regions. It provides revision of specified chromosomal characteristics of 18 valid bat species from the Russian Far East. The karyotype descriptions of valid Far Eastern bat species and their chromosomal variability are given for the first time. Table 1 shows valid Russian Far Eastern bat species. The columns represent species belonging to geographically various regions. The last one gives the species names describing the karyotypes. The table demonstrates the level of karyological knowledge available of certain bat species in every region studied. European and Northeastern Asian karyotype species have been studied to the fullest extent possible. Less data have been obtained regarding karyotype species in Siberia and the Russian Far East.

Karyotypes of Far Eastern bat
To illustrate the intrageneric and intraspecific variability of the Russian Far Eastern bat karyotypes based on data available,

Notes:
The geographical regions with the names abbreviated karyotypes investigated: E-Europe, S-Siberia, FE-Far East, J-Japan, C-China, K-Korea, T-Thailand, M-Malaysia.
Sources for species of Europe: [7,39,42,44,50], of Siberia: [47,51], of the Far East-see Table 2. "no"-unknown.     M. fuliginosus  regions for the first time and to reveal availability or lack of this variability. For simplicity sake, three size groups have been introduced to analyze size variability of two-arm (M-SM) chromosomes: large, medium-sized, and small ones, with their respective karyotype numbers assigned. This allowed us to show the karyotype variability based on this feature. Besides, Table 2 also shows the previous study of the species by using different sequential staining methods for the chromosomes, thus making it possible to differentiate species with a similar chromosome formula.
Integrated data on the karyotypes, extent of their studies, and chromosome variability of the Russian Far Eastern bats are provided below.
The amount and location of C-band in Eurasian Myotis chromosomes varies intra-and interspecifically [39,41,43,54,55]. Eurasian Myotis species proved to have small heterochromatic segments close to the centromere on most of the chromosomal arms. Certain Myotis species show a distinct intercalary heterochromatic segments found in the proximal part of chromosome 15, in the vicinity of the centromere on chromosomal arm 16, and in the short arm of the X-chromosome adjacent to the centromere [39]. The size and morphology of Y-chromosome were species-specific and depended on amount of heterochromatic material in chromosome [39]. Asian bat species karyotypes have a distinctly pronounced totally heterochromatic short arm on one of the dot-like chromosomes 24 and 25. There might be a tiny second arm in several species or a large heterochromatic secondary arm of the same size as the euchromatic arm [39,41,43].
The genus Myotis is the most frequently found bats genus in the Russian Far East, with seven recorded species. Of these, six species are also spread in Northeast Asia and five species are common in Siberia. Karyotype of one species was reported found in Siberia. The karyotypes of four Myotis species studied are common for the Russian Far East. The karyotypes of five Myotis species were described from Northeast Asia. Intraspecific variability is likely to exist here regarding the large heterochromatic short arms on the 25 autosomal pairs [41].

M. longicaudatus
Ognev, 1927. The karyotype was studied using the Japan species. The cmc-NORs were located on 13 autosomal pairs: from 8 to 11, from 13 to 15, and from 18 to 23. The morphology of Y-chromosome seems to vary from acrocentric [41] and subtelocentric [43] to submetacentric [55]. The morphology of chromosome 25 appears to vary from acrocentric to submetacentric due to the presence or absence of heterochromatic short arms [41,43]. (Temminck, 1840). The karyotype was described using Northeast Asia and the Russian Far East specimens (Figure 1). The cmcNORs were located on 18-23 autosomal pairs. The morphology of chromosome 25 seems to vary from acrocentric chromosome in M. macrodactylus from the Russian Far East [46], Korea [58], and Japan [54] to metacentric chromosome in other Japanese M. macrodactylus [41,43,53,55,56]. The presence of one B-chromosome for M. macrodactylus from Japan has been showed [56].  most important differentiating characteristics for the Myotis species. Various levels of the data studied for differently staining Myotis chromosomes from various Northeastern regions make it impossible to do species comparative analysis based on the above features.
There are two species of Plecotus in the Russian Far East: P. ognevi and P. sacrimontis. Kishida, 1927. The karyotype of P. ognevi was described from the Russian Far East (Figure 2). Four NORs were found belong to acrocentric chromosomes of P. ognevi; but it was impossible to determine the numbering of these chromosomal arms according to Myotis-type karyotype because of G-banding failure [47]. The distributional patterns of heterochromatic material in karyotype were shown: large heterochromatic segments were found in all biarmed autosomal pairs, while small C-band emerged in the most acrocentric chromosomes except the first pair [47].

P. ognevi
G-staining, Q-banding, and Zoo-FISH of Siberian P. ognevi karyotypes were studied. A pericentric inversion or centromere shift on the smallest metacentric P. ognevi chromosome 16/17 using the HSA 16 probe was revealed, which accounted for the differences between G-banding patterns and the homologous Myotis species chromosome [51].
So, all Plecotus species have similar karyotypes with 2n = 32, NFa = 50. P. auritus and P. sacrimontis had different NORs distribution on chromosomes. For P. ognevi, it was impossible to determine the numbering and NOR location on chromosomal arms. Heterochromatic distribution pattern in karyotype was studied only for P. ognevi from the Russian Far East, thus making it impossible to compare data from various species and regions.
There is only one species of genus Barbastella in the Russian Far East-B. darjelingensis Hodgson, 1855. It can be found exclusively on the island of Kunashir [23,78]. The chromosomal set was reported only from B. darjelingensis from Northeast Asia. Five NORs were found on 21-25 autosomal pairs of standard Myotis-type karyotype [43].
There is one pipistrelles species inhabiting the Russian Far East, i.e., P. abramus Temminck, 1840. Karyotype of P. abramus was described from Northeast Asia. Unlike other pipistrelles, P. abramus has low number 2n and NFa (2n = 26, NFa = 44) due to centric fusions. Chromosome rearrangements complexity makes it impossible to identify the chromosomal arms by G-banding that were involved in composition of 5 out of 10 biarmed pairs of P. abramus karyotype. Therefore, the numbering of P. abramus chromosomes differs from Myotis-type karyotype [43,54,63,65].

The distributional pattern of NORs is interstitial (intNORs). The large NOR was located in secondary constriction (SC) of five metacentric pairs consisting of 14 and 7 autosomal pairs of
Myotis-type karyotype [43].
The intraspecific variations of sex chromosomes in karyotype of especially P. abramus were likely to be found. Many researchers identified X chromosome morphology as a mediumsized acrocentric, while the X chromosome of the P. abramus from Fukuoka prefecture (Japan) was described as subtelocentric [54]. The Y chromosome of P. abramus was usually characterized as the smallest acrocentric, while the Y chromosome of the same species from Gunma prefecture (Japan) was described as a small metacentric [52].
High intraspecific variability of heterochromatic material seems to be specific of the P. abramus karyotype. This variability for P. abramus from Northeast Asia is presented in Table 3.
The P. abramus karyotype is described only from Northeastern Asia specimens, which can be possibly accounted for by existing intraspecific variability based on morphology of sex chromosomes, number and localization of structural heterochromatin in karyotype.
All Vespertilio species showed location of two large intNORs in the SC of 15 and 23 autosomal pairs [42,43]. There are two Vespertilio species in the Russian Far East-V. murinus and V. sinensis.

V. murinus Linnaeus, 1758
is the trans-Palearctic bat species, whose karyotype was described from Europe, Siberia, and the Russian Far East. The NOR distributional pattern was reported from Europe [42]. The chromosome characteristics show stability across the entire area of its distribution (Figure 3).
V. sinensis Peters, 1880 belongs to the East Asian bat species. The karyotype was described from Northeast Asia and the Russian Far East. NFa = 54 was shown to characterize some specimens from Japan, probably due to the fact that certain researchers included small heterochromatic secondary arms on the two smallest acrocentric in NFa [68]. The distributional pattern of NORs was reported from Northeast Asia [43]. The significant intraspecific polymorphism seems to exist in regard to amount and location of heterochromatic material in karyotype of Japanese V. sinensis ( Table 4).
Geographical regions abbreviations: J-Japan, C-China.  The localization of telomeric sequences (TTAGGG) n was described by FISH for V. sinensis from Japan. Hybridization signals were observed at both ends of all V. sinensis chromosomes along with very faint and small-sized interstitial signals that were also present at centromeric sites of all seven biarmed chromosomes. Large and intense hybridization signals revealed themselves at the centromeric regions in eight pairs of acrocentric autosomes (18)(19)(20)(21)(22)(23)(24)(25) and the Y chromosome of V. sinensis. It is interesting to note that C-band of the smallest acrocentric pair 25 and of the Y chromosome displayed a complete hybridization, while interstitial C-band in 5/6, 7/13, and 15 autosomal pairs in V. sinensis exhibited no hybridization. Internal telomeric sequences were observed in the heterochromatic regions or satellite DNA on chromosomes that may indicate recent chromosomal rearrangements occurred in the evolution process [69].
While the chromosome characteristics of V. murinus show stability across the entire area of its distribution, the karyotype of V. sinensis seems to have a significant intraspecific polymorphism regarding the content of structural heterochromatin in the karyotype.

Genus Hypsugo Kolenati, 1856: high pipistrelles
The diploid number and fundamental number of genus Hypsugo chromosomes noticeably vary due to the centric fusions as well as inversions and centromere shift [44].
Note: ○-totally euchromatic chromosomes; +-heterochromatic band in vicinity of the centromere; •-totally heterochromatic chromosomes; *-secondary construction on the chromosome.  (Figure 4). 2n and NFa are the same for most of the studied E. nilssonii excepting this one from Hokkaido with one biarmed autosomal pair in karyotype [69]. The large intNORs is located on secondary constriction in chromosome 15 [44,47].
The amount and location of heterochromatic material in karyotype was described for E. nilssonii from the Russian Far East. There were small C-bands on all chromosomes pairs, and the fourth largest pair showed a large interstitial heterochromatic segment. The SC on chromosome 15 showed C-band [47].
The chromosome characteristics of E. nilssonii including distributional pattern of NORs show stability across the entire area of its distribution. Structural heterochromatin distribution pattern was studied only for the Far Eastern E. nilssonii, which prevented us from evaluating variability of this feature.

M. ussuriensis Ognev, 1914.
Karyotype of M. ussuriensis was described from Japan. With the known localization type, the localization of multiple cmcNORs on chromosomes has not been determined yet because G-banding has not been done [43].
The amount and location of heterochromatic material in M. ussuriensis karyotype were described from Japan. The autosomal pairs 5/6, 16/17, 20, 24 and X chromosome showed small centromeric C-bands, while the Y chromosome was totally heterochromatic. The interstitial faintly stained C-band was revealed in the distal part of X chromosome [72].
M. hilgendorfi Gray, 1842. Karyotype of M. hilgendorfi was described from Siberia, Northeast Asia, and the Russian Far East region ( Table 2).
Karyotype of one specimen from Primorsky Velican cave (the Russian Far East) was clearly different from other M. hilgendorfi ones by the number of large biarmed pairs: there were only two large metacentric pairs, one medium-sized submetacentric pair being approximately equal to a long arm of large metacentric pair and one small metacentric pair [47]. The same karyotype was previously described for a tube-nosed bat from Thailand [70]. It was originally reported as M. leucogaster Milne-Edwards, 1872, though later the bat was redefined as M. harrisoni Csorba & Bates, 2005 [82]. However, karyotypes of other specimens of M. harrisoni [81,83] and M. leucogaster [84] exhibited karyotype with three large biarmed chromosomal pairs.
The amount and location of heterochromatic material in karyotype were shown for M. hilgendorfi from Japan. There were small C-band close to centromere on chromosomes 5/6, 16/17, 20, 24 and X chromosome with totally heterochromatic Y chromosome [72].
The location of structural heterochromatin of M. ussuriensis and M. hilgendorfi from Japan scarcely differs from each other. M. hilgendorfi karyotype with two large metacentric pairs, one medium-sized submetacentric pair and 1 small metacentric pair described from the Russian Far East, seemed to be either in error or an isolated case that requires verification.

Genus Miniopterus Bonaparte, 1837: bent-winged bats
Karyotypes of bent-winged bats are clearly different from standard Myotis-type karyotype due to chromosomal rearrangements. By using GTG-staining and FISH methods, the biarmed chromosome 3/4 of Myotis-type karyotype was shown to be similar to two acrocentric pairs of Miniopterus, due to centric fissions the metacentric pair 16/17 assumed the shape of an acrocentric, and the acrocentric pair 12 became biarmed due to pericentric inversions, with the G-banding pattern of 7 and 10 autosomal arms being different from standard Myotis-type karyotype [76].
There is one species of the monotypic family Miniopteridae found in the Russian Far East that is M. fuliginosus Hodgson, 1835. Karyotype (2n = 46, NFa = 50-52) was described from Northeast Asia.
The M. fuliginosus seems to exhibit intraspecific polymorphism by the number of biarmed autosomal pairs. Karyotype with two large and one small biarmed pairs is most common. M. fuliginosus, with its mostly encountered karyotype, was found in Malaysia, Thailand, China, and Japan [43,[74][75][76]. Karyotype with two large and one medium biarmed chromosomal pair was described from China [65,77]. Karyotype of M. fuliginosus from Thailand was similar to the previous one with one exception: it had one subtelocentric pair [71]. Karyotype with two large, one medium, and one small biarmed pairs was described from Japan [53,73].
One cmcNORs was shown to be located on 20 autosomal pair and one intNOR is located on chromosome 23 in the M. fuliginosus karyotype from Japan [43]. The small C-band close to centromere was described to be located on all chromosomal pairs of Chinese M. fuliginosus [77].
So, M. fuliginosus from Northeastern Asia seems to be characterized by intraspecific chromosome polymorphism based on the number of autosomal pairs.

Conclusion
For the first time, the references' analysis undertaken enabled us to demonstrate the extent of chromosome characteristics studied for bats from the Russian Far East. It also illustrated the nature of the intrageneric and intraspecific chromosome variability of the bats from the Russian Far East.
The data available enable us to suggest Miniopterus fuliginosus, Murina hilgendorfi, and some Myotis species to show intraspecies chromosome polymorphism regarding biarmed autosomal pairs. Intraspecies variability could be fairly assumed to exist as regards X,Y chromosomes in P. abramus, M. longicaudatus and M. macrodactylus karyotypes from Northeastern Asia. A significant intraspecies polymorphism regarding structural heterochromatin in a karyotype seems to be available in V. sinensis, P. abramus, and Myotis species. Such important characteristic as the amount and localization of cmcNORs on chromosomes has been very irregularly studied for the Far Eastern bat species, which restricts our ability to compare data from different regions. There is not enough data to compare Barbastella and Hypsugo species in terms of their karyotype chromosome characteristics.
Thus, one might make a conclusion that karyotypes of the majority bats from the Russian Far East and Siberia still remain to be studied. The bats from Northeastern Asia and Europe have their bats' chromosome characteristics somewhat more fully explored, though we still have considerable gaps in our knowledge of karyotypes for certain bats' species.