ARTICLE IN PRESS www.elsevier.de/mambio Short communication Karyological study of Nasillus gracilis (Insectivora, Talpidae, Uropsilinae) By S. Kawada, S. Li, Y. Wang and S. Oda Laboratory of Animal Management and Resources, Graduate School of Bio-Agricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan; Division of Mammal, Kunming Animal Research Institute, Chinese Academy of Science, Kunming, Yunnan 650223, China Receipt of Ms. 24.5.2005 Acceptance of Ms. 21.11.2005 Key words: Nasillus gracilis, Uropsilinae, karyotype, G-band The Asiatic shrew moles, subfamily Uropsili- regarded them as a single species, U. soricipes. nae, are a group of Talpidae that are Recently, Wang (2002) classified Asiatic distributed exclusively in montane areas from shrew moles into two genera, including four southwestern China to Myanmar (Hutterer species: U. soricipes, N. gracilis, N. andersoni, 1993). Unlike other subterranean talpids, the and N. investigator. These taxonomies were Uropsilinae have terrestrial lifestyles, similar based on morphological and ecological data; to soricid shrews, and their external morpho- objective genetic information is now required. logical characteristics include apparent ear Although morphological and molecular phy- lids and a long tail. Their skulls and dental logenetic data are available for a species of characters are similar to those of other Uropsilinae, N. gracilis (Hoffman 1984; talpids, and the tympanic bone forms a Shinohara et al. 2003; Motokawa 2004), its complete auditory bulla. Based on these karyotype remains unknown. Molecular data distinct characters, the subfamily Uropsilinae have confirmed that the subfamily Uropsili- is generally accepted as belonging within the nae is basal in the family Talpidae (Shinohara Talpidae (Van Valen 1967; Corbet and Hill et al. 2003), but the phylogenetic relation- 1991; Hutterer 1993). According to Hoffman ships within this genus remain unclear. To re- (1984), three species belong to the Uropsili- evaluate the confusing specific treatments, nae:three species belong to the Uropsilinae: karyological data for these species are valu- Uropsilus andersoni, U. gracilis, and U. able. The aim of this study was to describe soricipes. Because these species have distinct the karyotype of one species of Uropsilinae, dental formulae, they were considered inde- N. gracilis. The karyotype should allow pendent genera in the original descriptions of comparison with published karyotypes of the species: Uropsilus, Nasillus, and Rhyncho- Talpidae, and to consider the systematic nax, respectively (Thomas 1911). This classi- implications. fication was followed by some systematic We followed the classification of Wang reviews (Allen 1938; Stroganov 1948). After (2002), who defined two distinct genera, and the generic descriptions by Thomas (1911), based species identification on dental formu- Allen (1912) and subsequent researchers lae (Hoffman 1984; Wang 2002). Karyologi- noted that the dentition of Uropsilus was cal data for N. gracilis were obtained from variable in some local populations and eight specimens collected on Mt. Laojun 1616-5047/$ - see front matter r 2005 Deutsche Gesellschaft fu¨r Sa¨ugetierkunde. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.mambio.2005.11.006 Mamm. biol. 71 (2006) 2 Á 115–119 ARTICLE IN PRESS 116 S. Kawada et al. (altitude: 3900 m, N: 2613709400,E:9914305200), of one submetacentric pair (Fig. 1a, arrow- Lijiang District, Yunnan Province, China. De- head). tails on the collection and some external The G-band karyotype of N. gracilis is shown measurements of the specimens are listed in in Fig. 1b. The chromosomes were named Table 1. The specimens were sacrificed, and based on the characteristics of the G-band bone marrow cells and epithelial tissue samples sequences. Again, a secondary constriction were cultured for chromosome preparation. appeared in one M pair, which we identified Chromosomes were prepared using standard as chromosome 3. The results of silver nitrate methods and stained with 4% Giemsa solution. staining demonstrated the localization of the G-banding and C-banding were performed nucleolar organizing region (NOR; data not using the ASG method of Sumner et al. shown). (1971) and the BSG method of Sumner Fig. 2 shows a C-banded metaphase of (1972), respectively. Silver nitrate staining was N. gracilis. In this species, very weak C-bands also performed according to the one-step method of Howell and Black (1980).The chromosome pairs were categorized as meta- submetacentric (M), subtelocentric (ST), or acrocentric (A) chromosomes according to Levan et al. (1964). The conventional karyotype of a male N. gracilis is shown in Fig. 1a. In the eight animals studied, no variation was seen in the diploid number or in the chromosomal morphology. The diploid chromosome num- ber (2n) and autosomal fundamental number (NFa) of N. gracilis were determined as 2n ¼ 34 and NFa=46, respectively. The karyotype consisted of four M, three ST, and nine A chromosome pairs and the sex pair (Fig. 1a). The X chromosome was a small M chromosome, and the Y chromo- some was minute, although it appeared to Fig. 1. Conventional (a) and G-band (b) karyotypes of be an A chromosome. A secondary constric- Nasillus gracilis. The arrowhead in (a) indicates a tion was obvious on the proximal long arm secondary constriction. Table 1. Collecting data and external measurements of the examined specimens. No. Sex Date Measurementsa Weight H.B. Tail F. Foot H. Foot Ear Tail ratio (%) SIK0604 ~ 2002 Oct. 29 7.4 68.0 64.5 8.0 12.0 6.5 94.9 SIK0605 # 2002 Oct. 29 7.0 71.0 57.5 7.0 12.5 8.5 81.0 SIK0607b # 2002 Oct. 30 7.5 74.0 32.0 7.5 13.0 5.5 43.2 SIK0608 ~ 2002 Oct. 31 6.6 71.0 64.0 7.5 14.5 6.5 90.1 SIK0616 # 2002 Nov. 1 7.2 71.0 63.5 6.5 14.0 9.0 89.4 SIK0619 # 2002 Nov. 2 6.9 68.5 61.0 7.5 13.0 7.0 89.1 SIK0621 # 2002 Nov. 2 5.9 72.0 65.0 8.0 13.5 7.5 90.3 SIK0624 ~ 2002 Nov. 3 7.5 72.0 63.5 7.5 13.0 7.5 88.2 aH.B., Head and body length (mm); Tail, tail length (mm); F. Foot, fore foot length (mm); H. Foot, hind foot length (mm); Ear, length of ear lid (mm). bThe tail of this specimen was cut off by trap. ARTICLE IN PRESS Karyological study of Nasillus gracilis 117 the short-faced mole is an exception. In the short-faced mole, the secondary constriction is localized on the proximal long arm of an A chromosome, although this is thought to have arisen secondarily via Robertsonian rearrangement from an M chromosome (Kawada et al. 2002b). Based on these studies, the NOR-bearing chromosome in the family Talpidae is assigned as karyologi- cally conservative. The NOR localization on the subtelocentric element in N. gracilis is considered a good indicator for the distinct- ness of the subfamily Uropsilinae within the Fig. 2. C-band metaphase plate of Nasillus gracilis. talpids. Moreover, the G-band karyotype of The scale bar indicates 10 mm. this species was very different from known talpid karyotypes. We attempted to compare were localized exclusively at the centromere them but we were unable to reconstruct the positions of all chromosomes. The Y chro- pattern, despite G-band homologies demon- mosome was stained positively throughout strated in some groups of Talpidae (Kawada the entire chromosome arm. and Obara 1999; Kawada et al. 2001, 2002a). In the Talpidae, the diploid number ranges in Again, the uniqueness of the Asiatic shrew 23 species from 2n ¼ 32248 (Tsuchiya 1985; mole is stressed in this context. Given this Reumer and Meylan 1986; Aniskyn and karyological information on the Uropsilinae, Romanov 1990; Yates and Moore 1990; subsequent discussion must center on the Kawada et al. 2002b). According to Kawada certification of this group as a distinct et al. (2002b), most of the species have 34 or subfamily, based on genetic information. 36 chromosomes in their karyotypes. Nasillus Recent molecular phylogenetic data (Shino- gracilis also has the 2n ¼ 34 karyotype, hara et al. 2003) and comparative skull which seems to invoke karyological kinship morphology (Motokawa 2004) also suggest with other talpid species. In known talpid that this group is very distinct and the most karyotypes, the Y chromosome is dot-shaped primitive of Talpidae, and myological and and morphologically undistinguishable, osteological cladistic analyses support this although it is somewhat larger in N. gracilis. view (Whidden 2000; Sanchez-Villagra and In general, the evolution of the mammalian Y Menke 2005; Sanchez-Villagra et al. 2005). chromosome tends toward miniaturization Grenyer and Purvis (2003) examined pub- with sex chromosome diversification (for a lished phylogenetic data for 47 species of review, see Delbridge and Graves 1999). If we Insectivora and clearly placed the Asiatic consider the external characters of this shrew mole as the root taxon of the Talpidae. species to indicate that it is a primitive talpid, In addition, the external morphology of the the Y chromosome feature of this species Uropsilinae, including the long tail, ear lids, may also reflect an ancient karyological and limbs for terrestrial locomotion, are condition. Furthermore, it is useful to discuss similar to the features of soricid shrews. the localization of the NOR because it is a Nevertheless, its cranial and dental morphol- good marker in talpid karyotypes. A second- ogy do not provide convincing support for ary constriction in this species was seen on its taxonomic treatment. Moreover, fossil the proximal long arm of chromosome 3. In evidence of Uropsilinae exists not only contrast, the secondary constriction is loca- from the Oligocene in Asia, but also from lized on the proximal short arm of a large M the early Miocene in North America chromosome pair in other talpid species (McKenna and Bell 1998), although highly (Yates et al.