Karyological Analysis and Morphometrics of the Lesser Asiatic House Bat, Scotophilus Kuhlii (Chiroptera, Vespertilionidae)

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Karyological Analysis and Morphometrics of the Lesser Asiatic House Bat, Scotophilus kuhlii (Chiroptera, Vespertilionidae)

Praween Supanuam1, Alongklod Tanomtong1*, Sumpars Khunsook1, Krit Pinthong2 and La-orsri Sanoamuang1

1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 2 Biology Program, Faculty of Science and Technology, Surindra Rajabhat University, Muang, Surin 32000, Thailand

Received January 19, 2012; accepted September 27, 2012

Summary Karyological analysis and morphometrics of the lesser Asiatic house bat (Scotophilus kuhlii) from Thailand were studied. Blood samples were taken from 5 male and 5 female bats, lymphocytes were cultured at 37°C for 72 h in presence of colchicine, and metaphase spreads were performed on microscopic slides and air-dried. Conventional staining and GTG-banding techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of S. kuhlii was 2n=36, and the fundamental number (NF) was 52 in both male and female. The types of autosomes were 6 large metacentric, 4 large submetacentric, 6 large telocentric, 2 medium submetacentric, 10 medium telocentric, 2 small metacentric, and 4 small telocentric chromosomes. The X chromosome was a large submetacentric chromosome and the Y chromosome was the smallest acrocentric chromosome. The GTG-banding technique, indicated that the number of bands and locations in S. kuhlii was 167, and that each chromosome pair could be clearly differentiated. In addition, a pair of long arms near the centromere of chromosome pair 17 showed clearly observable nucleolar organizer regions (NORs). In terms of morphological measurement, the following measurements were taken 6 external morphological characters as well as 13 cranial and dental characters. The karyotype formula of S. kuhlii was as follows:

m sm t sm t m t 2n (36)=L 6 + L 4 + L6 + M 2 + M10 + S 2 + S4 + XY

Key words Lesser Asiatic house bat, Scotophilus kuhlii, Karyotype, Morphometrics, GTG- banding.

The lesser Asiatic house bat (Scotophilus kuhlii Leach, 1822) is a member of the order Chiroptera (bats), the family Vespertilionidae (common bats) and the subfamily Vespertilioninae. The family Vespertilionidae contains about 48 genera and 407 species (Corbet and Hill 1992, Nowak 1999, Simmons 2006) of which 13 genera and 34 species occur in Thailand. Vespertilionids are the largest, most diverse and most widespread family of bats, occurring on every continent except Antarctica (Lekagul and McNeely 1988, Francis 2008). The characteristics of S. kuhlii are that the fur is soft and short, brownish on top, with light brown parts underneath. The hairs are light brown at the base, gradually darkening; however, pelage color is widely variable. The tragus is long, narrow, and pointed. The occipital helmet is less developed, and the dentition is less robust (Fig. 1). S. kuhlii distribution ranges from Pakistan to Taiwan, south Sri Lanka to western Malaysia, southeast Philippines to Indonesia (Lekagul and McNeely 1988, Bates 1997, Francis 2008).

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.77.401 402 P. Supanuam et al. Cytologia 77(3)

Fig. 1. Morphological characters of the Asiatic house bat (Scotophilus kuhlii) from northeast Thailand, including external morphology (A), ventral view of skull (B), lateral view of skull (C), and mandible (D), scale bars 0.5 cm.

A series of intensive bat surveys have been conducted in Thailand in recent years (Francis 2008). In addition to traditional taxonomic comparisons by morphological characters, karyological studies can not only provide information on phylogenetic relationships but also have great value in solving systematic problems (Yoshiyuki 1989, Yoo and Yoon 1992, Sreepada et al. 1996, Volleth et al. 2001). Although standard karyotypes are of limited value in assessing chromosomal evolution and phylogenetic relationships at lower taxonomic levels, such information can be useful for clari- fying systematic relationships, either for detecting cryptic species or for confirming species status. Standard karyotypes also suffice for determining general karyotypic trends within taxa (Yi and Harada 2006). At the present time, there are 15 genera and 52 species of Vespertilionid bats in Thailand (Francis 2008). Few cytogenetic studies on Vespertilionid bats in Thailand have been conducted (Table 1). Nine species have been studied cytogenetically, each having a different/similar diploid number: S. kuhlii, 2n=36 (Harada et al. 1982), Myotis sirigorensis, 2n=44; M. mystacinus, 2n=44; Pipistrellus puveratus, 2n=44; Tylonyteris robustula, 2n=32; Miniopterus schreibersiv, 2n=46 (Harada et al. 1985a), M. ater, 2n=44; M. muricola, 2n=44 and M. annactens, 2n=46 (Bickham et al. 1986) (Table 1). There are 6 reports on cytogenetic studies of S. kuhlii, namely Pathak and Sharma (1969), Harada and Kobayashi (1980), Harada et al. (1982), Sreepada and Gururaj (1994), Lin et al. (2002) and Volleth et al. (2006). In addition, we confirm the results by comparing them with previous reports. This study is the first report on the chromosome size, standardized idiogram, and karyotype formula of S. kuhlii. This knowledge represents significant cytogenetic information that will aid further study on taxonomy and evolutionary relationships. Moreover, it is useful basic information for conservation and for studying chromosomal evolution in this bat. 2012 Karyological Analysis and Morphometrics of the Lesser Asiatic House Bat, Scotophilus kuhlii 403

Materials and methods

Chromosome preparation Blood samples were collected from 5 males and 5 females of S. kuhlii and kept using aseptic techniques in Mahasarakham Province, northeast Thailand. The samples were kept in 10 ml vac- uum tubes containing heparin to prevent blood clotting and cooled on ice until arriving at the laboratory. The lymphocytes were cultured using the whole blood microculture technique adapted from Rooney (2001) and Campiranont (2003).

Cell culture Five milliliters of RPMI 1640 medium was prepared with 2% PHA (Phytohemagglutinin) as a mitogen and kept in blood culture ﬂasks. A blood sample of 0.5 ml was dropped into a medium bot- tle, mixed, loosely capped, incubated at 37°C under 5% of carbon dioxide environment and regu- larly shaken in the morning and evening. When reaching harvest time at 72nd hour of incubation, colchicine was added and well mixed, followed by further incubation for 30 min.

Cell harvest The blood sample mixture was centrifuged at 3,000 rpm for 5 min and the supernatant was discarded. Ten milliliters of hypotonic solution (0.075 M KCl) was applied to the pellet and the mixture incubated for 30 min. KCl was discarded from the supernatant after centrifugation at 3,000 rpm for 5 min. Cells were fixed in fresh cool fixative (3 : 1, methanol: glacial acetic acid) which was gradually added up to 8 ml before centrifuging again at 3,000 rpm for 5 min, after which the supernatant was discarded. The fixation was repeated until the supernatant was clear and the pellet was mixed with 1 ml fixative. The mixture was dropped onto a clean and cold slide by micropipette followed by the air-dry technique. The slide was conventionally stained with 20% Giemsa’s solution for 30 min.

GTG-banding method GTG-banding technique was adapted from Campiranont (2003). The slide was well dried and then soaked in working trypsin (0.025% trypsin EDTA) at 37°C before terminating trypsin activity by washing the slide with Sorensen buffer. The slide was stained with 20% Giemsa’s solution for 30 min.

Chromosomal checks, karyotyping and idiogramming Chromosome counting was performed on mitotic metaphase cells under light microscope. Twenty clearly observable and well-cells spread chromosomes of each male and female were se- lected and photographed. The length of short arm chromosome (Ls) and the length of long arm chromosome (Ll) were measured and calculated to the length of total arm chromosome (LT, LT=Ls+Ll). The relative length (RL) and the centromeric index (CI) were estimated. CI was also computed to classify the types of chromosomes according to Chaiyasut (1989). All parameters were used in karyotyping and idiogramming.

Morphological measurement The following external measurements were taken: forearm length (FA), head-body length (HB), tail length (T), ear length (E), tibia length (TIB), and hind foot length (HF). The following cranial and dental measurements were taken: greatest length of skull (GTL), condylo-basal length (CBL), condylo-canine length (CCL), zygomatic breadth (ZB), braincase breadth (BB), interorbital constriction (IC), post orbital constriction (PC), mandible length (M), rostral width (RW), maxillary toothrow (C–M3), mandibular toothrow (C–M3), posterior palatal width (M3–M3), and anterior pal- 404 P. Supanuam et al. Cytologia 77(3) atal width (C1–C1) (Bates 1997).

Results and discussions

Chromosome number, fundamental number and karyotype of S. kuhlii Karyological analysis using lymphocyte culture revealed that the chromosome number was 2n (diploid)=36. This is the same chromosome number reported for S. kuhlii in previous studies. The karyotype of S. kuhlii from Malaysia (Harada and Kobayashi 1980, Volleth et al. 2006), Thailand (Harada et al. 1982), India (Pathak and Sharma 1969, Sreepada and Gururaj 1994) and Taiwan (Lin et al. 2002) share the same 2n=36. This examination also revealed that the fundamental number (NF, number of chromosome arms) of S. kuhlii was 52 in both male and female. This is not consis- tent with the reports of Lin et al. (2002) and Volleth et al. (2006) which revealed that S. kuhlii has an NF of 48. The difference between the outcomes of the studies may be due to the different crite- ria used for the classiﬁcation of chromosome types.

Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) Asiatic house bat (Scotophilus kuhlii) 2n (diploid)=36 by conventional staining technique, arrows indicate sex chromosomes and nucleolar organizer regions (NORs), scale bars 10 μm. 2012 Karyological Analysis and Morphometrics of the Lesser Asiatic House Bat, Scotophilus kuhlii 405

S. kuhlii has 3 types of autosomes, 8 metacentric, 6 submetacentric and 20 telocentric chromosomes. The 8 metacentric autosomes were classiﬁed by size into 6 large and 2 small chromosomes; the 6 submetacentric autosomes were classiﬁed by size into 4 large and 2 medium chromosomes; while the 20 telocentric autosomes were distinguished to be 6 large, 10 medium, and 4 small chromosomes (Fig. 2). Similar chromosomal features were reported by Sreepada and Gururaj (1994), Lin et al. (2002) and Volleth et al. (2006) which indicated that the autosomes of S. kuhlii consists of 14 metacentric to submetacentric and 20 acrocentric autosomes. The X chromosome of S. kuhlii is a large submetacentric chromosome and the Y chromosome is the smallest acrocentric chromosome. These features are similar to the reports of Harada and Kobayashi (1980), Harada et al., (1982) and Lin et al. (2002) that revealed S. kuhlii to have submetacentric X and acrocentric Y chromosomes. The GTG-banding technique indicates that each chromosome pair is distinctively differenti-

Fig. 3. Metaphase chromosome plates and karyotypes of male (A) and female (B) Asiatic house bat (Scotophilus kuhlii) 2n (diploid)=36 by GTG-banding technique, arrows indicate sex chromosomes and nucleolar organizer regions (NORs), scale bars 10 µm. 406 P. Supanuam et al. Cytologia 77(3) ated. The GTG-banding revealed that the number of bands on 1 set of haploid chromosomes, which includes autosomes, X and Y chromosomes, is 167 for S. kuhlii (Fig. 3). The G-banding provides clearly defined chromosome bands which are represented in the black (heterochromatin) and white regions (euchromatin) on chromosomes. The level of band numbers is defined by a visible and in a haploid set which is composed of autosomes, X and Y chromosomes. Thus, the haploid set of S. kuhlii consists of 34 autosomes including X and Y chromosomes. The genus Scotophilus is included in the Eptesicus-like group. This group includes chromo- somally derived taxa and is composed of Eptesicus, Rhogeessa, Nycticeius, Antrozous, and Scotophilus. The primitive chromosomal condition of Vespertilionidae is represented by the Myotis-like group in which chromosome arms 1/2, 3/4, and 5/6 form 3 large metacentric autosomes. In the Eptesicus-like group, these are present either as acrocentric autosomes or have been rearranged. Therefore, all genera in this group are thought to have evolved from an ancestor with an all-acrocentric karyotype like that of Eptesicus. In Scotophilus, arms 1–4 and 6 are present as acrocentric autosomes, and 5 is fused with 12 to form a metacentric autosome. Thus, the 3 chromosomal evolutionary trends described by McBee et al. (1986) are found independently in each of the chromosomal groups described by Bickham (1979). This suggests that chromosomal evolution in vespertilionids may be more complex than previously thought. McBee et al. (1986) suggested that karyotypic data of the family Vespertilionidae show 3 patterns of chromosomal variability among genera. The first pattern includes conservative genera in which all species have the same or nearly the same standard karyotypes, such as Myotis, Eptesicus, Plecotus, Scotophilus, Miniopterus, and Murina. The second pattern includes the genera that ex- hibit interspecific variability such as Pipistrellus and Nyctalus. The third pattern includes species that possess certain distinct karyotypes such as the genus Rhogeessa. The idiogram of S. kuhlii shows the gradually decreasing length of the autosomes. The karyotype formula of S. kuhlii is as follows:

m sm t sm t m t 2n (36)=L 6+L4 +L6+M2 +M10+S 2+S4+XY

Chromosome markers of S. kuhlii In the present investigation, 2 nucleolar organizer regions, NORs (satellite chromosomes), which represent the chromosome marker, are located on the long arm near centromere of chromosome pair 17. This result is in agreement with previous reports of genus Scotophilus (S. kuhii, S. dinganii, and S. nux) which has 1 pair of satellite chromosomes (Ruedas et al. 1990, Lin et al. 2002). Volleth and Yong (1987) suggested that NORs may be used to distinguish among chromo- somally conservative genera. Variation in the morphology of chromosomes bearing NORs was ob- served in each of the Scotophilus species. NORs have been localized in the karyotypes of numerous vertebrate species (Howell 1982). During the course of evolution, NORs have often been translocated. This has also been proven to be true for vespertilionids (Volleth and Yong 1987). Therefore, it is almost impossible to determine from which chromosomes in the ancestral karyotype the NORs have been borne. A comparison with the related families Natalidae and Molossidae, where 1–3 NOR-bearing pairs were found (Ono and Obara 1989), was not helpful in this respect since the banding patterns of the NOR-bearing chromosomes differ from those of the vespertilionids. In addition, the NORs are located both in the short arms and the secondary constrictions in these families. The members of the vespertilionid genera close to the root of the tree show NORs on 2 or more chromosomal pairs. It is therefore un- likely that the NOR-bearing secondary constriction on chromosome pair 15, present in the tribes Pipistrellini, Vespertilionini and Eptesicini, represents the ancestral position. Assuming that this feature is derived, 2 possibilities for the relationships between the vespertilionine tribes mentioned can be considered: 1) the NOR on chromosome pair 15 is a synapomorphic feature of these 3 2012 Karyological Analysis and Morphometrics of the Lesser Asiatic House Bat, Scotophilus kuhlii 407

Table 1. Review of cytogenetic reports on bats from Thailand.

Species 2n NF m sm st a X Y References

Pteropodidae Pteropus lylei 40 72 32 6 0 sm a Hood et al. (1988) Rousettus amplexicaudatus 36 68 ̶ ̶ ̶ ̶ sm a Hood et al. (1988) Rousettus leschenaulti 36 68 24 10 0 sm a Harada et al. (1982) Cynopterus sphinx 34 58 22 4 6 st a Harada et al. (1982) 34 58 ̶ ̶ ̶ ̶ sm a Hood et al. (1988) Megaerops ecaudatus 26 ̶ 10 10 6 ̶ ̶ Harada et al. (1982) Megaerops niphanae 26 42 18 0 6 sm a Hood et al. (1988) Eonycteris spelaea 36 68 24 10 0 sm sm Harada et al. (1982) 36 66 ̶ ̶ ̶ ̶ sm sm Hood et al. (1988) Macroglossus sobrinus 34 60 ̶ ̶ ̶ ̶ m a Hood et al. (1988) Emballonuridae Thaphozous theobaldi 42 64 24 0 16 sm a Harada et al. (1982) T. melanopogon 42 64 ̶ ̶ ̶ ̶ sm a Hood and Baker (1986) T. melanopogon 42 64 ̶ ̶ ̶ ̶ sm s Hood et al. (1988) Emballonura monticola 24 44 22 0 0 ̶ ̶ Hood et al. (1988) Megadermatidae Megaderma lyra 54 104 52 0 sm a Hood et al. (1988) M. spasma 38 64 28 0 8 st a Hood et al. (1988) Rhinolophidae Rhinolophus acuminatus 62 60 0 0 60 st st Harada et al. (1982) 62 60 ̶ ̶ ̶ st st Hood et al. (1988) R. malayanus 62 60 0 0 60 st st Harada et al. (1982) 62 60 ̶ ̶ ̶ st st Hood et al. (1988) R. coelophyllus 62 60 0 0 60 st st Harada et al. (1982) 62 60 ̶ ̶ ̶ st st Hood et al. (1988) R. pusillus 62 64 0 0 60 st biarm Harada et al. (1985b) R. affinis 62 64 0 0 60 st biarm Harada et al. (1985b) 62 60 ̶ ̶ ̶ st st Hood et al. (1988) R. stheno 62 64 0 0 60 st biarm Harada et al. (1985b) R. marshalli 62 64 0 0 60 st biarm Harada et al. (1985b) R. yunnanensis 60 68/67 4 2 0 52 st a Harada et al. (1985b) R. luctus 32 60 ̶ ̶ ̶ st s Hood et al. (1988) R. luctus perniger 32 34/33 30 0 0 sm a Harada et al. (1985b) R. thailandensis 60 64 4 2 0 52 st a Wu et al. (2009) Hipposideridae Hipposideros larvatus 32 60 30 0 0 sm st Harada et al. (1982) 32 60 ̶ ̶ ̶ sm st Hood et al. (1988) H. lekaguli 32 60 26 4 0 sm st Harada et al. (1982) 32 60 ̶ ̶ ̶ sm st Hood et al. (1988) H. fulvus 32 64/63 24 0 0 sm a Harada et al. (1985a) 32 60 ̶ ̶ ̶ ̶ sm a Hood et al. (1988) H. armiger 32 60 ̶ ̶ ̶ ̶ sm st Hood et al. (1988) Aselliscus stoliczkanus 30 60/59 28 0 0 sm a Harada et al. (1985a) Vespertilionidae Scotophilus kuhlii 36 48 14 0 20 m a Harada et al. (1982) 36 52 8 6 0 20 sm a Present study Myotis sirigorensis 44 56/55 6 4 0 32 sm a Harada et al. (1985a) M. mystacinus 44 56/55 6 4 0 32 sm a Harada et al. (1985a) M. ater 44 50 8 0 32 sm ̶ Bickham et al. (1986) M. muricola 44 50 8 0 32 sm ̶ Bickham et al. (1986) M. annactens 46 54 10 bi-arms 32 sm a Bickham et al. (1986) Pipistrellus puveratus 44 56/55 8 2 0 32 sm a Harada et al. (1985a) Tylonycteris robustula 32 52 16 4 10 a a Harada et al. (1985a) Miniopterus schreibersi 46 56 4 2 2 36 sm ̶ Harada et al. (1985a) Mollossidae Tadarida plicata 48 54 8 0 38 sm a Harada et al. (1982)

Notes 1. Harada et al. (1982): NF=NFa 2. Harada et al. (1985a): Used subacrocentric not subtelocentric 3. Harada et al. (1985b): Did not report NF 4. Bickham et al. (1986): NF=NFa 5. Hood and Baker (1986): Did not show Taphozous melanopogon karyotype 6. Hood et al. (1988): NF=NFa; Studied 19 species of bats but showed karyotypes of only 5 species Pteropus lylei, Megaerops niphanae, Emballonura monticola, Megaderma lyra and M. spasma. Detailes of karyotype not given for some species not detail of karyotype. 7. Present study: acrocentric=telocentric chromosome 408 P. Supanuam et al. Cytologia 77(3) tribes; an inversion in the distal part of the same chromosome, however, took place only in the Vespertilionini and Pipistrellini; or 2) the NORs on chromosome pair 15 appeared by 2 independent events. Then, by means of Robertsonian ﬁssions of the 3 large metacentrics, the Eptesicini could be grouped together with the Nycticeiini, e.g. Scotophilus. In our opinion, considering the morphological similarities of the tribes in question, the former possibility seems to be more likely (Volleth and Heller 1992). The mean length of short arm chromosome (Ls), length of long arm chromosome (Ll), total length of arm chromosome (LT), relative length (RL), centromeric index (CI), standard deviation of RL, CI, size and type of chromosome are presented in Table 2. For S. kuhii the chromosome marker is chromosome pair 1 which is the largest metacentric chromosome. The important karyotype feature is the asymmetrical karyotype, which is includes metacentric, submetacentric, acrocentric and telocentric chromosomes. The largest and smallest chromosomes show a 3-fold difference in size. Figures 4 and 5 show the idiograms for S. kuhii from conventional staining and the GTG-banding technique respectively.

Chromosome evolution of S. kuhlii The previous report by Volleth et al. (2001) demonstrated that there was some chromosome change in Eptesticus bottae (2n=50), but that it has the most similar karyotype to the Vespertilionid ancestor (2n=52). Changes in the 16 chromosome of E. bottae were induced by tandem fusion of

Fig. 4. Idiogram of the Asiatic house bat (Scotophilus Fig. 5. Idiogram of the Asiatic house bat (Scotophilus kuhlii) 2n (diploid)=36 by conventional stain- kuhlii) 2n (diploid)=36 by GTG-banding tech- ing technique. A pair of the short arms of nique. A pair of the short arms of chromosome chromosome pair 17 showed clearly observ- pair 17 showed clearly observable satellite able satellite chromosome. chromosome. 2012 Karyological Analysis and Morphometrics of the Lesser Asiatic House Bat, Scotophilus kuhlii 409

Table 2. Mean of length short arm chromosome (Ls), length long arm chromosome (Ll), length total arm chromosome (LT), relative length (RL), centromeric index (CI), and standard deviation (SD) of RL, CI from metaphase chromosomes in 20 cells of male and female lesser Asiatic house bat (Scotophilus kuhlii) 2n=36.

Chromosome Chromosome Chromosome Ls Ll LT RL±SD CI±SD pair sizes types

1 1.211 1.508 2.718 0.0870±0.0050 0.5547±0.0014 Large Metacentric 2 1.127 1.353 2.479 0.0794±0.0060 0.5456±0.0015 Large Metacentric 3 0.788 1.357 2.145 0.0687±0.0050 0.6327±0.0008 Large Submetacentric 4 0.844 1.020 1.864 0.0597±0.0040 0.5472±0.0067 Large Metacentric 5 0.689 1.163 1.852 0.0593±0.0010 0.6280±0.0040 Large Submetacentric 6 0.000 1.828 1.828 0.0585±0.0020 1.0000±0.0000 Large Telocentric 7 0.000 1.781 1.781 0.0570±0.0017 1.0000±0.0000 Large Telocentric 8 0.000 1.704 1.704 0.0545±0.0018 1.0000±0.0000 Large Telocentric 9 0.000 1.644 1.644 0.0526±0.0020 1.0000±0.0000 Medium Telocentric 10 0.614 1.004 1.618 0.0518±0.0022 0.6205±0.0081 Medium Submetacentric 11 0.000 1.598 1.598 0.0511±0.0027 1.0000±0.0000 Medium Telocentric 12 0.000 1.540 1.540 0.0493±0.0019 1.0000±0.0000 Medium Telocentric 13 0.000 1.466 1.466 0.0469±0.0028 1.0000±0.0000 Medium Telocentric 14 0.000 1.354 1.354 0.0433±0.0024 1.0000±0.0000 Medium Telocentric 15 0.530 0.649 1.179 0.0378±0.0023 0.5506±0.0009 Small Metacentric 16 0.000 0.973 0.973 0.0312±0.0032 1.0000±0.0000 Small Telocentric 17* 0.000 0.628 0.628 0.0201±0.0029 1.0000±0.0000 Small Telocentric X 0.716 1.411 2.127 0.0681±0.0057 0.6635±0.0030 Large Submetacentric Y 0.202 0.656 0.858 0.0275±0.0015 0.7642±0.0085 Small Acrocentric

Remark: *=satellite chromosome (nucleolar organizer region, NOR).

Table 3. External, cranial and dental measurement of lesser Asiatic house bat (Scotophilus kuhlii) from Thailand (n=12).

Morphological Mean (mm) Minimum (mm) Maximum (mm) SD measurement

FA 45.13 39.61 49.63 3.65 HB 62.85 52.76 69.61 6.30 T 43.42 39.58 47.06 3.12 E 11.43 7.52 15.18 3.17 TIB 14.77 8.76 19.51 3.94 HF 8.73 6.77 12.13 2.01 GTL 18.23 17.40 19.20 0.54 CBL 17.03 16.10 18.00 0.45 CCL 16.51 15.90 16.90 0.34 ZB 13.14 12.80 13.70 0.25 BB 9.25 8.90 9.60 0.27 IC 4.71 4.30 5.00 0.20 PC 2.73 2.60 2.90 0.12 RW 7.58 7.00 8.00 0.32 ML 13.52 13.00 13.90 0.31 C–M3 6.13 6.00 6.40 0.14 C–M3 6.97 6.20 7.30 0.27 M3–M3 8.15 7.90 8.60 0.24 C1–C1 6.15 5.90 6.60 0.19

Remarks: Forearm length (FA), head-body length (HB), tail length (T), ear length (E), tibia length (TIB), and hind foot length (HF). The following cranial and dental measurements were taken: greatest length of skull (GTL), condylo-basal length (CBL), condylo-canine length (CCL), zygomatic breadth (ZB), braincase breadth (BB), interorbital constriction (IC), post orbital constriction (PC), mandible length (ML), rostral width (RW), maxillary toothrow (C–M3), mandibular toothrow (C–M3), posterior palatal width (M3–M3), and anterior palatal width (C1–C1). 410 P. Supanuam et al. Cytologia 77(3)

Table 4. Karyotype comparison of Vespertilionid ances- the 16 and 17 chromosomes of its ancestor. For tors, Eptesticus bottae and Scotophilus kuhlii the karyotype of S. kuhlii (2n=36) from from Thailand. Thailand, there are also several changes in Vespertilionid Eptesticus bottae Scotophilus kuhlii chromosomes. Chromosomes 1 to 5 were de- Ancestor (2n=52) (2n=50) (2n=36) rived from the centric fusion of chromosomes Volleth et al. Volleth et al. Present study (2001) (2001) 8 and 14, 9 and 13, 10 and 20, 15 and 21, 12 and 23, respectively. Tandem fusion between 1 1 6 chromosomes 16 and 17 caused chromosome 2 2 7 3 3 8 16 of S. kuhlii. In addition, we assume that the 4 4 9 complex changing on chromosomes 10 and 15 5 5 11 may have resulted from the centric fusion and 6 6 12 pericentric fusion of Vespertilionid ancestral 7 7 13 8 8 1q karyotypes, respectively (Eick et al. 2007) 9 9 2q (Table 4). 10 10 3q 11 11 14 Morphological descriptions 12 12 5q 13 13 2p A medium-to-small size Scotophilus with 14 14 1p an average forearm length of 45.13 mm 15 15 4q (39.61–49.63 mm) is for the specimen from 16 16qprx 16qprx Mahasarakham province, Northeast Thailand. 17 16qdis 16qdis 18 17 10 The pelage is brownish above and light brown 19 18 15 underneath. The muzzle is relatively short and 20 19 3p round. The ears have a long narrow tragus and 21 20 4p blunt forward (Fig. 1A). The skull has an aver- 22 21 10 23 22 5p age condylo-basal length of 17.03 mm (16.10– 24 23 17 18.00 mm). The skull is broad with shortened 25 24 15 rostrum and point lambda (Figs. 1B–D). The upper tooth row (C–M3) length is average Remarks: prx=proximal, dis=distal, p=short arm chromosome, and q=long arm chromosome. 6.13 mm (6.00–6.40 mm). The mandible length is between 13.00 and 13.90 mm (average 13.52 mm). The dental formula is 1/3, 1/1, 3/2, 3/3. Table 3 shows external, cranial and dental measurements.

Acknowledgments

This work was supported by the Applied Taxonomic Research Center (ATRC), Khon Kaen University grant ATRC-R5304 and Development and Promotion of Science and Technology talents project (DPST). We wish to acknowledge the support of the Khon Kaen University Publication Clinic, Research and Technology Transfer Affairs, Khon Kaen University, for their assistance.

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