Chromosome Analysis and Morphometric of Croslet Horseshoe Bat, Rhinolophus Coelophyllus and Least Horseshoe Bat, Rhinolophus Pusillus (Chiroptera, Rhinolophidae)

Chromosome Analysis and Morphometric of Croslet Horseshoe Bat, Rhinolophus coelophyllus and Least Horseshoe Bat, Rhinolophus pusillus (Chiroptera, Rhinolophidae)

Isara Patawang1,7, Bundit Tengjaroenkul2,6, Praween Supanuam3, Wanpen Kakampuy4, Sumpars Khunsook5 and Alongklod Tanomtong5,6*

1 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Muang 50200, Thailand 2 Department of Veterinary Clinical Medicine, Faculty of Veterinary Medicine, Khon Kaen, Muang 40002, Thailand 3 Biology Program, Faculty of Science, Udonratchathani Rajabhat University, Udon Ratchathani, Muang 34000, Thailand 4 Faculty of Agriculture and Technology, Nakhon Phanom University, Nakhon Phanom, Muang 48000, Thailand 5 Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 6 Toxic Substances in Livestock and Aquatic Animals Research Group, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 7 Center of Excellence in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University, Chiang Mai, Muang 50200, Thailand

Received April 25, 2016; accepted June 25, 2017

Summary Chromosome and morphometric analyses of the croslet horseshoe bat (Rhinolophus coelophyllus) and least horseshoe bat (R. pusillus) from Northeast of Thailand were studied. Bat chromosome preparations were conducted by squash technique from bone marrow and testis. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of R. coelophyllus and R. pusillus were 2n=62, and the fundamental number (NF) were 64 in both species. The types of autosomes observed were 26 large telocentric, 12 medium telocentric, and 22 small telocentric chromosomes in R. coelophyllus and R. pusillus. The X-chromosome was a largest acrocentric chromosome and the Y-chromosome was a small acrocentric chromosome in both species. NORs are located at the secondary constriction near the centromere on the long arm of the large acrocentric chromosome pair 13 in R. coelophyllus and R. pusillus. We found that during metaphase I the homologous chromosomes showed synapsis, which can be deﬁned as 31 bivalents. Six external morphological characters were measured as well as 13 cranial and dental measurements. The karyotype formulae were as follows: t tt Rhinolophus coelophyllus; 2 n (62) = L26 + M 12 + S 22 + sex chromosomes t tt Rhinolophuspusillus; 2 n (62) = L26 + M 12 + S 22 + sex chromosomes

Key words Chromosome, Morphometric, Rhinolophus coelophyllus, Rhinolophus pusillus.

The croslet horseshoe bat (Rhinolophus coelophyl- hawking insects in flight or gleaning them from surfaces lus) and least horseshoe bat (R. pusillus) belongs to the (Newak 1991, Vaughan et al. 2000, Csorba et al. 2003). order Chiroptera, Suborder Microchiroptera, and fam- Studies of chromosomes have led to a better under- ily Rhinolophidae (Corbet and Hill 1992, Francis 2008) standing of systematic and phylogenetic relationships of (Figs. 1 and 2). Rhinolopidae includes approximately 77 bats and other groups but only a few studies have made species in a single genus, Rhinolophus. The closely re- use of banding techniques. In addition to traditional tax- lated Hipposideridae are sometimes included within the onomical studies of morphological characteristics, kary- horseshoe bats as subfamily, Hipposiderinae. Both fami- ological comparisons can also provide very useful data lies are classified in the suborder Yinpterochiroptera and on phylogenetic and taxonomic problems (Volleth et al. were previously included in Microchiroptera. Rhinolo- 2001). Up to the present, there is one genus and 18 spe- phids inhabit temperate and tropical regions of southern cies of Rhinolophid bats recorded in Thailand (Francis Europe, Africa, Asia, parts of Australasia, and many 2008). Most cytogenetic studies on Rhinolophid bats in parts of the Pacific Island. All species are insectivorous, Thailand have been conducted. Eleven species have been studied cytogenetically, each having a different/similar * Corresponding author, e-mail: [email protected] diploid number: R. acuminatus, 2n=62; R. malayanus, DOI: 10.1508/cytologia.82.471 2n=62; R. coelophyllus, 2n=62; R. pusillus, 2n=62; R. 472 L. Neeratanaphan et al. Cytologia 82(5)

Fig. 1. Morphological characters of the croslet horseshoe bat (Rhinolophus coelophyllus) from Northeast of Thailand, including external morphology (A), dorsal view of skull (B), lateral view of skull (C), ventral view of skull and mandible (D), scale bars indicate 0.5 cm.

affinis, 2n=62; R. stheno, 2n=62; R. marshalli, 2n=62; from bone marrow and testis (mitotic and meiotic cell R. yunnanensis, 2n=60; R. luctus, 2n=32; R. luctus per- division). Conventional staining and Ag-NOR band- niger, 2n=32 and R. thailandensis, 2n=60 (Harada et al. ing techniques were applied to stain the chromosome 1982, 1985, Hood et al. 1988, Wu et al. 2009). (Howell and Black 1980). The length of short arm (Ls) In this article, we report karyotype and other chromo- and long arm (Ll) chromosomes were measured and somal markers such as Ag-stained nucleolar organizer calculated for the length of total arm chromosomes (LT, region (NOR) in the R. coelophyllus and R. pusillus LT=Ls+Ll). Relative length (RL) and centromeric index populations from Northeast of Thailand. This study is (CI) were estimated. CI was also computed to clas- the first report on Ag-NOR chromosome of R. coelophyl- sify the types of chromosomes according to Chaiyasut lus and R. pusillus. In the future, the knowledge on basic (1989). All parameters were used in karyotyping. Exter- cytogenetics could be applied to numerous breeding nal and cranial morphologies were measured for species studies and also have advantages in species conservation identification (Duengkae 2006, Francis 2008, Lekagul and chromosome evolution studies. and McNeely 1988).

Materials and methods Results and discussion

The R. coelophyllus and R. pusillus samples were No signiﬁcant intra-individual variation in chromo- obtained from Nongbualamphu Province, Northeast of some constitution, banding pattern, and chromosome Thailand. The bats, 10 males and 10 females, were trans- number was observed in any of the specimens exam- ferred to the laboratory and were kept under standard ined. The present investigation revealed that the somatic conditions for one day prior to the experiments. Chro- chromosome numbers of R. coelophyllus and R. pusillus mosome preparation was conducted by squash technique were 2n=62, the fundamental numbers (NF, number of 2017 Morphometric of Croslet Horseshoe Bat, Rhinolophus coelophyllus and Least Horseshoe Bat, R. pusillus 473

Fig. 2. Morphological characters of the least horseshoe bat (Rhinolophus pusillus) from Northeast of Thailand, including external morphology (A), dorsal view of skull (B), lateral view of skull (C), ventral view of skull and mandible (D), scale bars indicate 0.5 cm.

chromosome arms) were 64 in both sexes of these spe- nanensis and R. thailandensis (Harada et al. 1985, Wu cies. The X-chromosome was a largest acrocentric chro- et al. 2009); 2n=32 for R. luctus and R. luctus perniger mosome and the Y-chromosome was a small acrocentric (Harada et al. 1985, Hood et al. 1988). The karyotype chromosome in both species. of R. coelophyllus and R. pusillus were composed of 26 large telocentric, 12 medium telocentric, and 22 small Conventional staining karyotype telocentric autosomes (mono-arm telocentric chromo- The cytogenetic study of R. coelophyllus and R. pu- somes). In the genus Rhinolophus, most species previ- sillus using squash technique from bone marrow and ously studied have a common karyotype consisting of 30 the conventional staining procedures revealed that the pairs of telocentric autosomes (Harada et al. 1982, 1985, chromosome number was 2n (diploid)=62, which con- Hood et al. 1988) (Table 1). sists of 60 (30 pairs) autosomes and two (1 pair) sex chromosomes. The NF of R. coelophyllus and R. pusil- Ag-NOR banded karyotype lus were 64 in both males and females (Figs. 3 and 5). Our cytogenetic study of R. coelophyllus and R. pusil- According to Harada et al. (1982, 1985) and Hood et al. lus performed by Ag-NOR staining technique is the ﬁrst (1988), who reported the chromosome number and NF of its kind. The objective of this technique is to detect of R. coelophyllus and R. pusillus to be 62 and 64, re- the nucleolar organizer regions/NORs, which represent spectively. Cytogenetic studies on the Rhinolophid bats the location of genes that function in 18S and 28S ribo- are few. Eleven species in Thailand have been studied somal RNA synthesis. NORs produce numerous gene cytogenetically, each having the same diploid number expressions, and they are composed of more non-histone (2n=62) (Harada et al. 1982, 1985, Hood et al. 1988). protein than other chromosome regions. These proteins In some species, a different diploid number from these induce speciﬁc dark bands (NOR positive) by the reduc- studies has been reported, such as 2n=60 for R. yun- tion of organic silver that changes silver to be dark (Gold 474 L. Neeratanaphan et al. Cytologia 82(5)

Fig. 3. Metaphase chromosome plates and karyotypes of male (A) and female (B) croslet horseshoe bats (Rhinolophus coelophyllus), 2n=62 by conventional straining technique (scale bars=10 µm).

et al. 1990). In R. coelophyllus and R. pusillus, Ag-NOR be caused by the centric fusion or pericentric inversion bands were observed on the subcentromeric regions of between two telocentric chromosomes. the long arm of the chromosome pairs 13 (two positions) (Figs. 4 and 6). Meiotic cell division The amount and location of NORs can explain the The present study on the meiotic cell division from evolution of each chromosome. Normally, most bats testis of R. coelophyllus and R. pusillus found that dur- have one pair of NORs in a chromosome complement. ing metaphase I (meiosis I) the homologous chromo- By comparing two bats in Thailand including the lesser somes showed synapsis, which can be deﬁned as the 31 Asiatic house bat (Scotophilus kuhlii) and intermediate bivalent, and 31 haploid chromosomes at metaphase II as roundleaf bat (Hipposideros larvatus), we found that S. diploid species. The largest telocentric chromosome pair kuhlii (2n=36) had two NORs positions on the long arm 1 is the largest bivalent. In prophase I (meiosis I), we subcentromeric region of small telocentric chromosome found that R. coelophyllus and R. pusillus had the dis- pair 17 (Supanuam et al. 2012), and H. larvatus (2n=32) tinct character of the observable leptotene (initiation of had two NORs positions on the long arm near the cen- chromosome shrinking), pachytene (completion of chro- tromere of large metacentric chromosome pair 8 (Janta- mosome synapsis), and diplotene (chiasma and crossing rat et al. 2014). Furthermore, NORs are usually located over) (Figs. 7 and 8). close to a subcentromeric region of a chromosome arm. If a NOR appears between an interstitial region, it may 2017 Morphometric of Croslet Horseshoe Bat, Rhinolophus coelophyllus and Least Horseshoe Bat, R. pusillus 475

Table 1. Review cytogenetic reports of the Rhinolophid bats in Thailand (Chiroptera, Rhinolophidae).

Species 2n NF m sm a t X Y NORs Reference

R. acuminatus 62 64 0 0 0 60 a a ̶ Harada et al. 1982 62 64 ̶ ̶ ̶ ̶ a a ̶ Hood et al. 1988 R. malayanus 62 64 0 0 0 60 a a ̶ Harada et al. 1982 62 64 ̶ ̶ ̶ ̶ a a ̶ Hood et al. 1988 R. coelophyllus 62 64 0 0 0 60 a a ̶ Harada et al. 1982 62 64 0 0 0 60 a a ̶ Hood et al. 1988 62 64 0 0 0 60 a a 13 (SCR) Present study R. pusillus 62 64 0 0 0 60 a bi-armed ̶ Harada et al. 1985 62 63 0 0 0 60 a t ̶ Wu et al. 2009 62 64 0 0 0 60 a a 13 (SCR) Present study R. afﬁnis 62 64 0 0 0 60 a bi-armed ̶ Harada et al. 1985 62 64 ̶ ̶ ̶ ̶ a a ̶ Hood et al. 1988 R. stheno 62 64 0 0 0 60 a bi-armed ̶ Harada et al. 1985 R. marshalli 62 64 0 0 0 60 a bi-armed ̶ Harada et al. 1985 R. yunnanensis 60 67/68 4 2 0 52 a t ̶ Harada et al. 1985 R. luctus 32 60 ̶ ̶ ̶ ̶ a S ̶ Hood et al. 1988 R. luctus perniger 32 33/34 30 0 0 0 sm a ̶ Harada et al. 1985 R. thailandensis 60 64 4 2 0 52 a t ̶ Wu et al. 2009

Notes: 2n=diploid chromosome, NF=fundamental number (number of chromosome arm), m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric chromosome, NORs=nucleolar organizer regions, S=small chromosome, SCR=subcentromeric region and ̶=not available.

Fig. 4. Metaphase chromosome plates and karyotypes of male (A) and female (B) croslet horseshoe bats (Rhinolophus coelophyllus), 2n=62 by Ag-NOR banding technique. Arrows indicate nucleolar organizer regions/NORs (scale bars=10 µm). 476 L. Neeratanaphan et al. Cytologia 82(5)

Fig. 5. Metaphase chromosome plates and karyotypes of male (A) and female (B) least horseshoe bats (Rhinolophus pusillus), 2n=62 by conventional straining technique (scale bars=10 µm).

Fig. 6. Metaphase chromosome plate and karyotype of the male (A) least horseshoe bat (Rhinolophus pusillus), 2n=62 by Ag- NOR banding technique. Arrows indicate nucleolar organizer regions/NORs (scale bar=10 µm). 2017 Morphometric of Croslet Horseshoe Bat, Rhinolophus coelophyllus and Least Horseshoe Bat, R. pusillus 477

Fig. 7. Meiotic cell division of the croslet horseshoe bat (Rhinolophus coelophyllus), 2n=62 on interphase (A), leptotene (B), pachytene (C), early diplotene (D), and metaphase I (E), scale bars indicate 5 µm.

Fig. 8. Meiotic cell division of the least horseshoe bat (Rhinolophus pusillus), 2n=62 on interphase (A), leptotene (B), pachytene (C), diplotene (D), and metaphase I (E), scale bars indicate 5 µm.

Chromosome markers in centimeters of 20 cells (males and females) in mitotic For R. coelophyllus and R. pusillus, chromosome metaphase was measured. The mean length of short arm markers are the autosome pairs 1 and 30, which is the chromosome (Ls), length of long arm chromosome (Ll), largest telocentric chromosome and the smallest telocen- total length of arm chromosome (LT), relative length tric chromosome, respectively. The important karyotype (RL), centromeric index (CI), standard deviation of RL feature of R. coelophyllus and R. pusillus are the asym- and CI, and the size and type of chromosomes are shown metrical karyotypes, which were found in two types in Tables 2 and 3. The karyotype formula for R. coelo- of chromosomes (acrocentric and telocentric chromo- phyllus and R. pusillus could be deduced as: somes). The largest chromosome is three times larger 2n (62) = Lt + M tt + S + sex chromosomes. than the smallest chromosomes. The chromosome length 26 12 22 478 L. Neeratanaphan et al. Cytologia 82(5)

Table 2. Mean of length short arm chromosomes (Ls), length long arm chromosomes (Ll), length total arm chromosomes (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from metaphase chromosomes in 20 cells of male and female croslet horseshoe bat (Rhinolophus coelophyllus), 2n (diploid)=62. Chro. pair Ls Ll LT RL±SD CI±SD Chro. size Chro. type 1 0.000 3.480 3.480 0.049±0.004 1.000±0.000 Large Telocentric 2 0.000 3.290 3.290 0.047±0.006 1.000±0.000 Large Telocentric 3 0.000 3.220 3.220 0.046±0.005 1.000±0.000 Large Telocentric 4 0.000 3.180 3.180 0.045±0.004 1.000±0.000 Large Telocentric 5 0.000 3.055 3.055 0.043±0.004 1.000±0.000 Large Telocentric 6 0.000 3.510 3.510 0.050±0.003 1.000±0.000 Large Telocentric 7 0.000 2.880 2.880 0.041±0.006 1.000±0.000 Large Telocentric 8 0.000 2.840 2.840 0.040±0.003 1.000±0.000 Large Telocentric 9 0.000 2.810 2.810 0.040±0.003 1.000±0.000 Large Telocentric 10 0.000 2.720 2.720 0.039±0.003 1.000±0.000 Large Telocentric 11 0.000 2.600 2.600 0.037±0.002 1.000±0.000 Large Telocentric 12 0.000 2.460 2.460 0.035±0.004 1.000±0.000 Large Telocentric 13* 0.000 2.400 2.400 0.034±0.002 1.000±0.000 Large Telocentric 14 0.000 2.200 2.200 0.031±0.003 1.000±0.000 Medium Telocentric 15 0.000 2.135 2.135 0.030±0.002 1.000±0.000 Medium Telocentric 16 0.000 2.080 2.080 0.030±0.002 1.000±0.000 Medium Telocentric 17 0.000 1.920 1.920 0.027±0.003 1.000±0.000 Medium Telocentric 18 0.000 1.840 1.840 0.026±0.002 1.000±0.000 Medium Telocentric 19 0.000 1.810 1.810 0.026±0.001 1.000±0.000 Medium Telocentric 20 0.000 1.730 1.730 0.025±0.001 1.000±0.000 Small Telocentric 21 0.000 1.680 1.680 0.024±0.000 1.000±0.000 Small Telocentric 22 0.000 1.660 1.660 0.024±0.001 1.000±0.000 Small Telocentric 23 0.000 1.625 1.625 0.023±0.001 1.000±0.000 Small Telocentric 24 0.000 1.570 1.570 0.022±0.001 1.000±0.000 Small Telocentric 25 0.000 1.420 1.420 0.020±0.000 1.000±0.000 Small Telocentric 26 0.000 1.200 1.200 0.017±0.000 1.000±0.000 Small Telocentric 27 0.000 1.100 1.100 0.016±0.000 1.000±0.000 Small Telocentric 28 0.000 1.090 1.090 0.016±0.000 1.000±0.000 Small Telocentric 29 0.000 1.060 1.060 0.015±0.000 1.000±0.000 Small Telocentric 30 0.000 0.980 0.980 0.014±0.000 1.000±0.000 Small Telocentric X 0.790 2.900 3.690 0.052±0.004 0.786±0.022 Large Acrocentric Y 0.280 0.800 1.080 0.015±0.001 0.741±0.017 Small Acrocentric Remarks: *=Nucleolar organizer region/NOR and Chro.=chromosome

Table 3. Mean of length short arm chromosomes (Ls), length long arm chromosomes (Ll), length total arm chromosomes (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from metaphase chromosomes in 20 cells of male and female least horseshoe bat (Rhinolophus pusillus), 2n (diploid)=62. Chro. pair Ls Ll LT RL±SD CI±SD Chro. size Chro. type 1 0.000 3.738 3.738 0.050±0.004 1.000±0.000 Large Telocentric 2 0.000 3.625 3.625 0.048±0.003 1.000±0.000 Large Telocentric 3 0.000 3.563 3.563 0.047±0.004 1.000±0.000 Large Telocentric 4 0.000 3.513 3.513 0.047±0.002 1.000±0.000 Large Telocentric 5 0.000 3.350 3.350 0.045±0.003 1.000±0.000 Large Telocentric 6 0.000 3.325 3.325 0.044±0.002 1.000±0.000 Large Telocentric 7 0.000 3.163 3.163 0.042±0.002 1.000±0.000 Large Telocentric 8 0.000 3.150 3.150 0.042±0.003 1.000±0.000 Large Telocentric 9 0.000 3.125 3.125 0.042±0.002 1.000±0.000 Large Telocentric 10 0.000 3.038 3.038 0.040±0.002 1.000±0.000 Large Telocentric 11 0.000 2.925 2.925 0.039±0.001 1.000±0.000 Large Telocentric 12 0.000 2.780 2.780 0.037±0.002 1.000±0.000 Large Telocentric 13* 0.000 2.700 2.700 0.036±0.001 1.000±0.000 Large Telocentric 14 0.000 2.350 2.350 0.031±0.004 1.000±0.000 Medium Telocentric 15 0.000 2.338 2.338 0.031±0.003 1.000±0.000 Medium Telocentric 16 0.000 2.313 2.313 0.031±0.002 1.000±0.000 Medium Telocentric 17 0.000 2.188 2.188 0.029±0.002 1.000±0.000 Medium Telocentric 18 0.000 2.100 2.100 0.028±0.001 1.000±0.000 Medium Telocentric 19 0.000 1.977 1.977 0.026±0.002 1.000±0.000 Medium Telocentric 20 0.000 1.850 1.850 0.025±0.001 1.000±0.000 Small Telocentric 21 0.000 1.840 1.840 0.024±0.001 1.000±0.000 Small Telocentric 22 0.000 1.825 1.825 0.024±0.001 1.000±0.000 Small Telocentric 23 0.000 1.788 1.788 0.023±0.001 1.000±0.000 Small Telocentric 24 0.000 1.763 1.763 0.023±0.001 1.000±0.000 Small Telocentric 25 0.000 1.638 1.638 0.021±0.000 1.000±0.000 Small Telocentric 26 0.000 1.350 1.350 0.018±0.000 1.000±0.000 Small Telocentric 27 0.000 1.200 1.200 0.016±0.000 1.000±0.000 Small Telocentric 28 0.000 1.188 1.188 0.015±0.000 1.000±0.000 Small Telocentric 29 0.000 1.130 1.130 0.015±0.000 1.000±0.000 Small Telocentric 30 0.000 1.050 1.050 0.014±0.000 1.000±0.000 Small Telocentric X 0.870 3.100 3.970 0.052±0.004 0.781±0.012 Large Acrocentric Y 0.263 0.900 1.163 0.015±0.000 0.774±0.025 Small Acrocentric Remarks: *=Nucleolar organizer region/NOR and Chro.=chromosome 2017 Morphometric of Croslet Horseshoe Bat, Rhinolophus coelophyllus and Least Horseshoe Bat, R. pusillus 479

Table 4. External, cranial and dental measurement of croslet horse- Table 5. External, cranial and dental measurement of least horseshoe bat (Rhinolophus coelophyllus) from Thailand (n=10). shoe bat (Rhinolophus pusillus) from Thailand (n=10).

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

FA 36.68 34.50 41.13 1.78 FA 36.68 34.50 41.13 1.78 HB 39.73 36.00 43.62 2.27 HB 39.73 36.00 43.62 2.27 T 19.21 16.20 22.18 1.67 T 19.21 16.20 22.18 1.67 E 14.15 8.20 19.80 2.65 E 14.15 8.20 19.80 2.65 Ti 13.78 9.20 19.50 2.95 Ti 13.78 9.20 19.50 2.95 HF 7.86 5.52 10.50 1.57 HF 7.86 5.52 10.50 1.57 GTL 15.85 14.80 17.30 0.75 GTL 15.85 14.80 17.30 0.75 CBL 13.83 13.40 14.50 0.31 CBL 13.83 13.40 14.50 0.31 CCL 13.49 13.00 14.10 0.39 CCL 13.49 13.00 14.10 0.39 ZB 7.55 7.20 8.00 0.21 ZB 7.55 7.20 8.00 0.21 BB 7.32 6.70 7.90 0.38 BB 7.32 6.70 7.90 0.38 IC 1.87 1.70 2.10 0.14 IC 1.87 1.70 2.10 0.14 PC 1.95 1.70 2.20 0.15 PC 1.95 1.70 2.20 0.15 RW 5.28 5.20 5.50 0.10 RW 5.28 5.20 5.50 0.10 ML 9.85 9.20 10.50 0.41 ML 9.85 9.20 10.50 0.41 C–M3 5.45 5.00 6.10 0.34 C–M3 5.45 5.00 6.10 0.34

C–M3 6.05 5.70 6.40 0.18 C–M3 6.05 5.70 6.40 0.18 M3–M3 5.36 5.10 5.70 0.17 M3–M3 5.36 5.10 5.70 0.17 C1–C1 3.81 3.50 4.00 0.15 C1–C1 3.81 3.50 4.00 0.15

Notes: Forearm length (FA), head-body length (HB), tail length Notes: Forearm length (FA), head-body length (HB), tail length (T), ear length (E), tibia length (TIB) and hind foot length (HF). (T), ear length (E), tibia length (TIB) and hind foot length (HF). The following cranial and dental measurements were taken: The following cranial and dental measurements were taken: greatest length of skull (GTL), condylo-basal length (CBL), greatest length of skull (GTL), condylo-basal length (CBL), condylo-canine length (CCL), zygomatic breadth (ZB), braincase condylo-canine length (CCL), zygomatic breadth (ZB), braincase breadth (BB), interorbital constriction (IC), post orbital constric- breadth (BB), interorbital constriction (IC), post orbital constriction (PC), mandible length (ML), rostral width (RW), maxillary tion (PC), mandible length (ML), rostral width (RW), maxillary 3 3 toothrow (C–M ), mandibular toothrow (C–M3), posterior palatal toothrow (C–M ), mandibular toothrow (C–M3), posterior palatal width (M3–M3), anterior palatal width (C1–C1), and standard de- width (M3–M3), anterior palatal width (C1–C1) and standard deviation (SD). viation (SD).

Morphological descriptions The braincase was globular elevate. The rostrum was The small size bat, R. coelophyllus from caves of slightly upwards and elongated. The upper tooth row Nong Bua Lam Phu province, Thailand with average (C–M3) was an average length of 5.45 mm (5.00– forearm length of 46.07 mm (43.50–49.10 mm) was ex- 6.10 mm). The mandibular length was an average length amined (Table 4). The pelage on the head and back var- of 9.85 mm (9.20–10.50 mm). The dental formula was ies from greyish brown to darker brown, with paler hair 1/2, 1/1, 2/3, 3/3=32. roots. The ventral surface is pale buff-brown, almost cream white in some individuals. R. coelophyllus has a Acknowledgements broadly arched connecting process. The bat also has a round lancet, densely covered with hair, connecting pro- The authors are grateful to thank Toxic Substances cess rounded. The skull had an average condylo-basal in Livestock and Aquatic Animals Research Group, length of 18.93 mm (18.00–19.40 mm). The braincase Khon Kaen University for the financial support. The was globular and elevated. The rostrum was slightly project was approved by the Institute of Animals for upwards and elongated. The upper tooth row (C–M3) Scientific Purpose Development of National Research was an average length of 7.71 mm (6.20–8.10 mm). The Council of Thailand (Resolution U1-04491-2559). mandibular length was an average length of 13.84 mm (13.30–14.20 mm). The dental formula was 1/2, 1/1, 2/3, References 3/3=32. R. pusillus is very small size bat, from caves of Nong Chaiyasut, K. 1989. Cytogenetics and Cytotaxonomy of the Family Bua Lam Phu province, Thailand. The average forearm Zephyranthes. Department of Botany, Faculty of Science, Chul- alongkorn University, Bangkok. length of 36.68 mm (34.50–41.13 mm) was examined Corbet, G. B. and Hill, J. E. 1992. The Mammals of the Indomalayan (Table 5). Length of ear was average of 14.15 mm Region: A Systematic Review. Oxford University Press, Oxford. (8.20–19.80 mm). The pelage is rather furry, with upper Csorba, G., Ujhelyi, P. and Thomas, N. 2003. Horseshoe Bats of the part brown to pale brown and the under part lighter. The World: (Chiroptera: Rhinolophidae). Alana Books, Shropshire. noseleaf of R. pusillus, lancet sharply point, connecting Duengkae, P. 2006. Bats of Thailand the Identification Field Guide. Department of Forest Biology, Faculty of Forestry, Kasetsart process point, sella parallel size. The skull had an aver- University, Bangkok. age condylo-basal length of 13.83 mm (13.40–14.50 mm). 480 L. Neeratanaphan et al. Cytologia 82(5)

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