Karyology of eight species of bats (Mammalia: Chiroptera) from Hainan Island, China.

Karyotypes and chromosomal data are presented for eight bat species representing two families (Rhinolophidae and Vespertilionidae) from Hainan Island, China. The species investigated were Rhinolophus lepidus (2n = 62, FN = 60), R. pusillus (2n = 62, FN = 60), R. affinis (2n = 62, FN = 60), R. sinicus (2n = 36, FN = 60), Myotis horsfieldi (2n = 44, FN = 52), Pipistrellus abramus (2n = 26, FN = 44), Miniopterus australis (2n = 46, FN = 50) and M. schreibersii (2n = 46, FN = 50). The karyotype of Rhinolophus lepidus is reported for the first time.

1. Introduction

Hainan Island is separated from the mainland of southern China by the narrow Strait of Hainan, which 17 to 20 km wide. It is the second largest island in China, with a land area of 34,000 km2, and the most southern province in China. The surface of Hainan Island is diversified geomorphologically, rising in the interior to mountains with altitudes exceeding 1,400 m near Wuzhishan and Jianfengling, which protect the southern part of the island from the monsoons. Hence, this region is quite different from the adjacent mainland of southern China climatically. The island's vegetation types include lowland shrubs, lowland evergreen forest, semi-evergreen forest, tropical rain forest, and montane evergreen forest. It has the best protected and largest area of primary tropical rain forest in China.

The fauna of Hainan Island comprises about 112 species of mammal, including 27 species of bat belonging to seven families. In a survey of bats, three species were only recently recorded from Hainan Island 1-4. Although all 27 bat species are considered to be conspecific with populations in southern China, subspecific status has been proposed for the Hainan populations of many taxa. Comparative studies of the populations in southern China and Hainan Island are needed to evaluate their degree of differentiation and to clarify their taxonomic status based on morphological and genetic perspectives. Conventional karyotyping is an important taxonomic tool for assessing the diversification between species, subspecies, and populations. Although 52 bat species in China have been karyotyped so far 5-17, no information is available for the bats of Hainan Island. This study reports the conventional karyotypes of eight species of bat representing two families (Rhinolophidae and Vespertilionidae) collected from Hainan Island as well as G- and C-banding karyotypes for selected species, and discusses their karyological relationships with conspecific populations and congeneric species in southern China and adjoining regions.

2. Materials and Methods

Twenty-eight specimens of eight species of bats were captured on Hainan Island, China in February 2005 and September 2008 (Table 1). The identification of these bats was based on standard taxonomic papers 18-21. Voucher specimens are deposited in the College of Life Science, Guangzhou University, Guangzhou. Chromosome preparations were made from primary culture cells of lung and tail tissue using the methods of Harada and Yosida 22. G- and C-banding differential stainings were applied for selected species following the methods of Seabright 23 and Sumner 24, respectively. The nomenclature of chromosomes follows Levan et al. 25. The diploid number (2n) was determined by observing 30 metaphase cells in each specimen, and the fundamental number (FN) was defined as the total number of autosomal arms (Table 2).

Table 1

Species, numbers, and localities of bat specimens collected from Hainan Island, China examined in this study.

Species		Male	Female	Locality
Rhinolophidae
	Rhinolophus lepidus	1	0	Mt. Diao Luo (caves)
	Rhinolophus pusillus	1	0	Mt. Diao Luo (caves)
		4	2	Qiongzhong, Caocan
	Rhinolophus affinis	0	1	Jianfengling, Tianchi
		4	1	Qiongzhong, Caocan
	Rhinolophus sinicus	2	1	Mt. Diao Luo (caves)
Vespertilionidae
	Myotis horsfieldi	1	0	Wuzhishan, Diyicun
	Pipistrellus abramus	1	7	Jianfengling, Tianchi
	Miniopterus australis	1	0	Wuzhishan, Diyicun
	Miniopterus schreibersii	1	0	Mt. Diao Luo (caves)

Table 2

Karyotype data for the eight bat species collected from Hainan Island, China.

Species	2n	FN	Autosome pair			Sex chromosomes
			M-SM	ST	A	X	Y
Rhinolophus lepidus	62	60	0	0	30	ST	A
Rhinolophus pusillus	62	60	0	0	30	ST	A
Rhinolophus affinis	62	60	0	0	30	ST	A
Rhinolophus sinicus	36	60	13	0	4	ST	ST
Myotis horsfieldi	44	50	4	0	17	SM	A
Pipistrellus abramus	26	44	10	0	2	A	A
Miniopterus australis	46	50	3	0	19	SM	A
Miniopterus schreibersii	46	50	3	0	19	SM	A

3. Results and Discussion

Karyotypes of four species in the genus Rhinolophus

This is the first karyotype description for Rhinolophus lepidus, which is widely distributed in Asia from Afghanistan to China, as well as in Sumatra 26. The karyotype of R. lepidus from Hainan Island was 2n = 62 and FN = 60 (Fig. 1), consisting of 30 medium-sized to small acrocentric autosomal pairs, a large subtelocentric X chromosome, and a small acrocentric Y chromosome. Secondary constrictions were detected adjacent to one pair of medium-sized acrocentric chromosomes.

Figure 1

Conventional (A), G-banding (B), and C-banding karyotypes of Rhinolophus lepidus from Hainan Island, China. Secondary constrictions are indicated by arrows.

The karyotypes of R. pusillus and R. affinis from Hainan Island were also 2n = 62, FN = 60 (Fig. 2A, B), consisting of 30 medium-sized to small acrocentric autosomal pairs, a large subtelocentric X chromosome, and a small acrocentric Y chromosome. A secondary constriction was detected in one pair of medium-sized acrocentric chromosomes in both species. Rhinolophus pusillus and R. affinis are widely distributed in the Indomalayan region and China and each contains nine subspecies 26. The two subspecies from Hainan (R. pusillus parcus and R. affinis hainanus) examined in this study showed no karyological differences from previous reports for the following taxa: R. p. szechwanus from the Chinese provinces of Sichuan 14 and Guizhou 13 (reported as R. cornutus); R. p. calidus from the Chinese provinces of Anhui 6 (as R. cornutus pumilus) and Guangdong 15 (as R. cornutus); R. p. lakkhanae from Thailand 27; R. a. himalayanus from the Chinese provinces of Guizhou 12 and Henan 28; R. a. macrurus from the Chinese provinces of Anhui 6 and Guangdong 15 (as R. affinis); and R. a. macrurus from Thailand 27.

Figure 2

Conventional karyotypes of R. pusillus (A) and R. affinis (B) from Hainan Island, China. Secondary constrictions are indicated by arrows.

The reported comparative karyological data for 35 Rhinolophus species in the world 6, 15, 27-31 suggest that the karyotype 2n = 62, FN = 60 is likely the conserved among the species and subspecies of the genus Rhinolophus and is shared by R. lepidus, R. pusillus, R. affinis, R. macrotis, R. rex, and R. huananus (reported as R. macrotis 15). Karyotype of R. lepidus is also considered a member of this 2n = 62, FN = 60 karyotype without recognized difference in G-banding pattern compared with Mao et al. 17. This 2n = 62, FN = 60 karyotype was thought to be ancestral state of the genus Rhinolophus, but recent karyotype study suggested that 2n=58 karyotype is more closely related to ancestral karyotype of the genus 17.

The karyotype of R. sinicus was 2n = 36, FN = 60 (Fig. 3), consisting of 13 large-to-small metacentric or submetacentric pairs and four medium-sized to small acrocentric pairs of autosomes, a medium-sized subtelocentric X chromosome, and a small subtelocentric Y chromosome. This species had a prominent secondary constriction involving one pair of medium-sized acrocentric chromosomes. The karyotype of R. sinicus from Hainan Island (2n = 36, FN = 60) does not differ from conspecific populations in the Chinese provinces of Anhui 6 (reported as R. rouxii sinicus), Sichuan 14, and Guangdong 15 in conventional karyotype and that from "south-western China" also in G-banding pattern 17, but does differ from the 2n = 56, FN = 60 karyotype of R. rouxii reported from India and Sri Lanka 31. These karyological differences between R. sinicus and R. rouxii support the view that these two taxonomically confused taxa both represent distinct species, as previously proposed 14, 15, 26.

Figure 3

Conventional (A), G-banding (B), and C-banding karyotypes of Rhinolophus sinicus from Hainan Island, China. Secondary constrictions are indicated by arrows.

The karyotype of Myotis horsfieldi

The karyotype of Myotis horsfieldi was 2n = 44, FN = 50, consisting of three large and one small metacentric or submetacentric pairs and 17 medium-sized to small acrocentric pairs in autosomes, a medium-sized submetacentric X chromosome, and a small acrocentric Y chromosome (Fig. 4A). The karyotype of Myotis horsfieldi from Hainan Island does not differ from conspecific population in Borneo 32. The vespertilionid genus Myotis is one of the most diverse mammalian genera and includes 103 species 26, but it is also one of the most karyologically conservative genera. A diploid number 2n = 44 characterizes all 47 species for which karyotypes have been reported 22, 33. Chromosomal variation is essentially restricted to the presence of heterochromatic short arms on the smaller autosomes, variation in the location of active nucleolus organizer regions (NORs), as well as differences in the size of the Y chromosome 22, 33, 34. In addition, an inverted autosome and a duplicated translocation involving the X chromosome have been reported in M. pruinosus 35. An extra pair of acrocentric autosomes (2n = 46) has also been reported in M. annectans from Thailand 36 and in M. davidii from Guangdong 16. The karyotype of M. horsfieldi in this study is the conservative one and showed no additional translocation.

Figure 4

Karyotypes of Myotis horsfieldi (A), Miniopterus australis (B), and M. schreibersii (C). Secondary constrictions are indicated by arrows.

The karyotype of Pipistrellus abramus

The karyotype of Pipistrellus abramus from Hainan Island was 2n = 26, FN = 44 (Fig. 5), consisting of ten large to medium-sized metacentric or submetacentric pairs and two small acrocentric pairs in autosomes, a medium-sized acrocentric X chromosome, and the smallest acrocentric Y chromosome. The conventionally stained karyotype of P. abramus from Hainan Island does not differ from conspecific populations from the Chinese provinces of Zhejiang, Henan, and Sichuan, Taiwan, Japan, or Korea 8, 14, 37-43. Large negative G-band and positive C-band areas were found in the pericentromeric regions of chromosome 1 through 4, 10, 11 and X; and these are considered centromeric constitutive heterochromatin. This characteristic was also reported from Japan and Taiwan 38, 41, 42, and any difference in G- and C-banding patterns among populations from Hainan Island, Japan and Taiwan was detected.

Figure 5

Conventional (A), G-banding (B), and C-banding karyotypes of Pipistrellus abramus from Hainan Island, China.

The karyotype of two species in the genus Miniopterus

The karyotypes of Miniopterus australis and M. schreibersii from Hainan Island were both 2n = 46, FN = 50 (Fig. 4B, C), both consisting of two large and one small metacentric or submetacentric pairs and 19 medium-sized to small acrocentric pairs in autosomes, a submetacentric X chromosome, and an acrocentric Y chromosome. One small acrocentric pair (No. 20) had a secondary constriction adjacent to the centromere. The karyotype of M. australis from Hainan Island is very similar to a previous report for the same species from Borneo 32. The karyotype of M. schreibersii from Hainan Island is similar to those for M. schreibersii from Borneo and Taiwan 9, 32, M. s. chinensis from Anhui Province, China 44, M. s. pallidus from Russia 45, and M. s. fuliginosus from Japan 40, 46 and Guizhou Province, China 47 (as M. fuliginosus). The members of this genus have likely retained the karyologically conservative state.

Diversification of bats from Hainan Island

Eight species of bats from Hainan Island examined in this study did not show any karyological differences from the conspecific populations or congeneric species from southern China and adjoining regions. Because karyological analyses suggest no evidences for differentiation, we follow the current taxonomic status to consider the Hainan bats as conspecific with the populations in southern China and adjoining regions. The karyological characteristics, however, are very conservative in some genera as discussed above. Morphological and genetic studies are also desired for more precise evaluation of the diversification and the taxonomic status of bats in Hainan Island.