Mammal Study 37: 1–00 (2012) © The Society of Japan

Karyotype of the Gansu (Scapanulus oweni): Further evidence for karyotypic stability in talpid

Kai He1,2, Jin-Huan Wang1, Wei-Ting Su1, Quan Li1,3, Wen-Hui Nie1 and Xue-Long Jiang1,* 1 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China 2 Institute of Eastern-Himalaya Biodiversity Research, Dali University, Dali 671003, China 3 University of Chinese Academy of Sciences, Beijing 100049, China

Abstract. Little is known about the ecology and evolution of the Gansu mole (Scapanulus oweni). The morphology of this monotypic genus (, , Mammalia) indicates that it should fall into the tribe Scalopini. Although all the other scalopines are distributed in North America, S. oweni is endemic to Central and Southwest China. Previous studies have indicated that the chromosomes of talpid moles exhibit remarkable stability. However, the karyotype of S. oweni has not been determined. In this study, we report the karyotypes including G-banding and C-banding patterns of S. oweni. The diploid and fundamental autosomal numbers are 34 and 64, respectively, identical with six other talpid species and thus providing another line of evidence for chromosomal uniformity in this family. The models of karyotype stability are discussed, none of which adequately explains the chromosomal conservatism. We suggest that comprehensive approaches are needed to test in which degree that the chromosomal rearrangement, phylogeny, phylogeography and ecological adaptation have shaped the chromosomal evolution in this family.

Key words: Gansu (Kansu) mole, G-band, karyotypic stability, Scapanulus oweni.

The talpid moles are subterranean within the Family Talpidae (Eulipotyphla, Mammalia). Because these fully fossorial rarely come to the surface of the Earth, specimens are often difficult to obtain. As such, the biology of many species, such as the Gansu mole (Scapanulus oweni), remains understudied. The Gansu (Kansu) mole is within the monotypic genus Scapanulus. Its morphology clearly indicates that it should fall into the tribe Scalopini, which includes three other genera (Parascalops, , and Scalopus) (Hutterer 2005), and its external appearance is incredibly similar to the hairy-tailed mole, P. breweri (Fig. 1). While S. oweni is endemic to Central and Southwest China, all other scalopines (5 species and 33 subspecies) are distributed in North America (Hutterer 2005). Fig. 1. External morphology of Scapanulus oweni (upper) from The first specimen of S. oweni was caught by G. Ningshan, Shaanxi, China (male, catalog number: KIZ027013) and Fenwick Owen in 1911 in Gansu, China (Thomas 1912). Parascalops breweri (lower) from North America (Photo by D. C. Since then, reports of its occurrence have been limited, Gordon, American Society of Mammalogists photo catalog number: all of which have been from Central and Southwest 852). China (Gansu, Qinghai, Shaanxi, and Sichuan Provinces)

*To whom correspondence should be addressed. E-mail: [email protected] 2 Mammal Study 37 (2012)

Fig. 2. Distribution and sample locality in this study. Grey areas are the known distribution of Scapanulus oweni based on Wang and Zhang (1997), Zhang (1997) and our collection. These include 22 counties in China: Chongqing (Wanzhou and Wushan), Gansu (Huanxian, Lanzhou, Lingtai, Lintan, Lintao, Pingliang, Tianshui, Zhengning. and Zhuoni), Qinghai (Tongren), Shaanxi (Liuba, Longxian, Luonan, Ningshan. and Taibai,) and Sichuan (Baoxing, Heishui, Maerkang, Pingwu, and Songpan). The arrow points to Ningshan County in Shaanxi, China where the two specimens were collected.

(Fig. 2) (Wang and Zhang 1997; Zhang 1997). To date, Sciences, China. Pitfall traps were set at about 1,500 m only eight specimens are known in museum collections above sea level at the Huoditang tree farm in Ningshan, in Germany, the UK, and the USA (GBIF data portal; Shaanxi, China (33.4°N, 108.5°E). The specimens were www.gbif.org). Given that the distribution, population, identified according to Thomas (1912) and deposited in and ecology of this species are barely known (Smith and the Kunming Institute of Zoology, Chinese Academy of Johnston 2008), it is not surprising that its karyotype Sciences. remains unknown. The chromosomal information for all other talpid Cell culture and cytological preparation genera is available (at least one species per genus) and Fibroblast cell cultures derived from the lung and exhibits remarkable stability (Table 1; Nevo 1979). In kidney of the specimen were stored in Kunming Cell 2011, we collected two specimens of S. oweni in Bank of the Chinese Academy of Sciences, Kunming, Ningshan, Shaanxi, one of which was caught alive and Yunnan 650223, P.R. China. Cell culture and metaphase sacrificed for karyotypic study. Here, we examined the preparations were performed following the conventional pattern of karyotype variation among the talpids and procedures (Hungerford 1965). G-banding, C-banding test multiple models of chromosomal evolution within and silver-stained were performed following Seabright the family. (1971), Summer (1972) and Goodpasture and Bloom (1975), respctively. Images were captured using the Materials and methods CytoVision system (Applied Imaging) with a CCD camera mounted on a Zeiss microscope. The chromo- Specimen collection somes of S. oweni were arranged based on their relative All samples were obtained following the regu- lengths, from the longest to the shortest. lations for the implementation of China on the protection of terrestrial wild animals (State Council Decree [1992] Reconstruction of karyotype evolution No. 13) and approved by the Ethics Committee of The phylogeny of the family from the most recent Kunming Institute of Zoology, Chinese Academy of study (Sanchez-Villagra et al. 2006) was considered as He et al., Karyotype of the Gansu mole 3

Table 1. Chromosomal numbers of talpids

Subfamily Tribe species 2N FNa Citation Lifestyles Uropsilinae andersoni 34 52 (Motokawa et al. 2009) Ambulatory Uropsilus gracilis 34 46 (Kawada et al. 2006a) Uropsilus investigator – Uropsilus soricipes – Scalopinae Condylurini Condylura cristata 34 64 (Reumer and Meylan 1986) Semi-aquatic/Semi-fossorial Scalopini Parascalops breweri 34 56 (Reumer and Meylan 1986) Fully fossorial Scalopus aquaticus 34 64 (Reumer and Meylan 1986) Scapanulus oweni 34 64 Present study Scapanus latimanus 34 60 (Reumer and Meylan 1986) Scapanus orarius 34 ? (Yates and Moore 1990) Scapanus townsendii 34 ? (Yates and Moore 1990) Desmanini Desmana moschata 32 60 (Aniskyn and Romanov 1990) Aquatic Galemys pyrenaicus 42 64 (Reumer and Meylan 1986) Neurotrichini gibbsii 38 72 (Kawada et al. 2008b) Semi-fossorial Scaptonychini Scaptonyx fusicaudus 34 64 (Kawada et al. 2008b) Talpini longirostris 34 52 (Kawada et al. 2008c) Fully fossorial Euroscaptor parvidens 36 60 (Kawada et al. 2008c) Euroscaptor malayana 36 52 (Kawada et al. 2005) Euroscaptor micrura – Euroscaptor grandis – Euroscaptor klossi 36 54 (Kawada et al. 2006b) Euroscaptor mizura 36 52 (Kawada et al. 2001) Euroscaptor subanura 36 56 (Kawada et al. 2012) imaizumii 36 54 (Kawada et al. 2001) Mogera insularis 32 58 (Lin et al. 2002) Mogera latouchei 30 52 (Kawada et al. 2010) Mogera kanoana 32 58 (Kawada et al. 2007) Mogera tokudae 36 54/60 (Kawada et al. 2001) Mogera uchidai – Mogera wogura 36 52/58 (Kawada et al. 2001) Parascaptor leucura 34 64 (Ye 2007) Scaptochirus moschatus 48 54? (Kawada et al. 2002) altaica 34 64 (Reumer and Meylan 1986) Talpa caeca 36 64 (Reumer and Meylan 1986) Talpa caucasica 38 62 (Reumer and Meylan 1986) Talpa europaea 34 64 (Reumer and Meylan 1986) Talpa davidiana – Talpa levantis 34 62 (Reumer and Meylan 1986) Talpa occidentalis 34 64 (Reumer and Meylan 1986) Talpa romana 34 64 (Reumer and Meylan 1986) Talpa stankovici 34 62 (Reumer and Meylan 1986) Urotrichini Dymecodon pilirostris 34 62 (Kawada and Obara 1999) Semi-fossorial Urotrichus talpoides 34 64 (Kawada and Obara 1999) The follows Hutterer (2005), but includes four new species: Euroscaptor malayana (Kawada et al. 2008a), Euroscaptor subanura (Kawada et al. 2012), Mogera kanoana (Kawada et al. 2007) and Mogera insularis (Kawada et al. 2010). 4 Mammal Study 37 (2012) the “true” phylogeny. We considered genera Euroscaptor, Mogera, and Talpa as monophyletic genera so that the diploid number (2N) and autosomal fundamental number (FNa) of each taxon could be marked on the tree. The ancestral states of 2N and FNa numbers and were recon- structed based on the most parsimonious (MP) assump- tion using Mesquite v2.75 (Maddison and Maddison 2008).

Result and discussion

We collected two specimens of Gansu mole. Among Fig. 3. Tail and hind foot of the S. oweni from Ningshan, Shaanxi, them, one male was caught alive in a pitfall trap (catalog ‘swimming’ in the moist soil. number: KIZ027013), and the other was found as a dead body and its sex could not be determined (KIZ027113). Specimens of S. oweni were identified by their long and thickly haired tails (Figs. 1 and 3), and their dental for- mula is identical to the type specimen (i 2/2, c 1/1, p 3/3, m 3/3, total 36) (Fig. 4). The external measurements of KIZ027013 in mm are: head-body length (HB) 91, tail length (TL) 49, hind foot (HF) 19; of KIZ027113 are: HB 80, TL 35 and HF 14. The skull measurements of KIZ027013 are as follow: greatest length 28.45, basal length 24.46, palatal length 12.90, interorbital breadth 6.24, zygomatic breadth 10.53, breadth of brain case Fig. 4. Dorsal view of the mandible and dorsal, ventral, lateral views of the cranium of the specimen of S. oweni from Ningshan, 13.34, breadth across molars 8.39, upper tooth row Shaanxi. 12.42, and lower tooth row 11.25. The external meas- urements of specimens from Ningshan are smaller than those from Gansu, but the skull is slightly larger (Allen (NORs) located at chromosome 8 indicating secondary 1938). More specimens and molecular data are needed constriction. The G-banding pattern and the position of to test whether the differences are due to geographical the NOR of this chromosome are identical with the variations. The halluces of the live animal are curved chromosome 8 of Talpa europaea (Volleth and Mueller and twisted away from the other digits (Fig. 3) as in the 2006) and chromosome 5 of Scaptonyx fusicaudus dead body as described by Thomas (1912), thus we (Kawada et al. 2008b). confirmed this twist natural, rather than resulting from Compared with the other scalopines in North America, distortion during the desiccation (Thomas 1912). S. oweni has the same diploid number (2N = 34), and The diploid number (2N) and autosomal fundamental its fundamental number (FNa = 64) is the same with number (FNa) are 34 and 64, respectively (Fig. 5a and Scalopus aquaticus (Table 1) even though the chromo- 5d). All autosomal chromosomes are biarmed. In total, somal morphology are not identical (Yates and Schmidly there are 24 metacentric + submetacentric, 8 subtelocen- 1975). The G-banding and C-banding patterns and the tric autosomes in the karyotype. The X chromosome is positions of NORs of the scalopines in North America metacentric; the dot-like Y chromosome is the smallest are warranted for karyotype comparison (Motokawa chromosome. The G-banding and C-banding karyotypes et al. 2009) and evolutionary analyses (Kawada et al. are shown in Figs. 5b and 5c. The G-banded karyotype 2008b) in this tribe. identified homologous chromosomes. The C-bands were Upon including S. oweni, karyotypes of 37 talpid distributed at the centromeric regions of all chromo- species (out of the 43 recognized species) representing somes as well as at the long arm of chromosome 12 and all 17 genera have been determined (Table 1). The 2N the terminal of the short arm of chromosome 13. Silver ranges from 30–48 (Standard deviation [SD] = 2.9) and staining demonstrates the nucleolar organizer regions the FNa ranges from 46–72 (SD = 5.5). Twenty species He et al., Karyotype of the Gansu mole 5

more specifically, the variability of diploid numbers is relatively low among mammals (Zima 2000). On the other hand, 14 different karyotypes were observed in Talpini, indicating relatively complicated chromosomal rearrangements happened in the tribe (Table 1). Different degrees of chromosome variation have been observed in vertebrates. This is likely the consequence of different rates of chromosomal evolution, which are strongly correlated with speciation rates (Bush et al. 1977). Thus, the karyotypic stability of talpids could also be related to the small number of species (n = 41; see Table 1), given that their sibling family, Soricidae (n = 376; Hutterer 2005), has an extraordinarily high degree of karyotype variation (SD of 2N = 10.2, SD of FNa = 15.7) (Zima 2000). Based on the most recent phylogenetic hypothesis (Sanchez-Villagra et al. 2006) and a simple MP assump- tion, we predicted the ancestral karyotype of the family to be 2N = 34 and FNa = 64. The ancestral diploid number is identical to that of paradoxus (Reumer and Meylan 1986), which is in the basal genus in Eulipotyphla (Roca et al. 2004). Note that this pre- diction relied on simple assumptions without taking intra-chromosomal rearrangements into consideration and should be tested, for example, using fluorescence in situ hybridization (FISH) in further study (Pinkel et al. 1988). Nonetheless, taking into account our working hypothesis in Fig. 6, the diploid number changed in Desmanini, Neurotrichini, and Talpini; the autosomal fundamental number also changed in these taxa as well as in Parascalops breweri and Dymecodon pilirostris (Fig. 6). The striking degree of chromosomal uniformity among the talpids has long been recognized, especially when compared with the other fossorial mammals, in which great amounts of chromosomal variation has been observed (e.g. Nevo 1979). Striking chromosomal rearrangement was only observed in the -mole, Neurotrichus gibbsii. Several models have been pro- posed to explain the karyotypic stability of animals. Arnason (1972) hypothesized that large deme size, inbreeding, high mobility, and a continuous range of Fig. 5. Karyotype of S. oweni collected from Ningshan, Shaanxi illustrating a) conventional, b) G-banding, c) C-banding, d) conven- distribution were the primary reasons for karyotypic tional metaphase plate and e) silver-stained metaphase plate. Arrows stability (deme size model), which is not necessarily indicate the position of active Ag-NORs. suitable for the whole talpid family, since the popula- tion densities are variable (0.42–12.4 moles/hectare; (54.1%) have the same diploid number (2N = 34) and Funmilayo 1977; Carraway et al. 1993; Hartman and twelve (34.3%) have the same autosomal fundamental Krenz 1993; Nores et al. 1998). number (FNa = 64). Thus, the karyotype variability, Bickham and Baker (1979) presumed that after a long 6 Mammal Study 37 (2012)

Fig. 6. Working hypothesis of karyotypic evolution in Talpidae. The phylogenetic tree was modified from Sanchez-Villagra et al. (Sanchez- Villagra et al. 2006). We assumed that Euroscaptor, Mogera, and Talpa are monophyletic genera, which may be not true (Shinohara et al. 2005; He 2011). Branches/species names in grey indicate changes of diploid number/fundamental autosomal number of the chromosome arms. period of karyotypic rearrangement, an “optimum”, or tions of a unique karyotype of N. gibbsii and karyotypic nearly optimum karyotype will have been achieved. variations in the tribe Talpini were observed. While a Accordingly, relatively ancient groups are more likely to clear relationship between the rates of chromosomal have evolved optimum karyotypes (canalization model). evolution and speciation exists, the reason for karyotype Alternatively, King’s (1985) “dead end model” hy- stability remains unclear. None of the previously pro- pothesized that when a karyotype has been saturated by posed models adequately explains the low level of chromosomal rearrangement, an evolutionary dead end chromosomal variation within this family. Therefore, will be reached. Despite the critiques of the canalization we suggest that intra- and inter chromosomal rearrange- model (King 1985, 1987), the most recent common an- ments should be taken into account for comparison cestor (MRCA) of the extant talpids was at about 52 ± 5 and evolutionary analyses. Further, because different Ma (Douady and Douzery 2003), which is not much phylogeny, phylogeographic histories (i.e. dispersal- older than the MRCA of the African mole rat family, vicariance) and ecological adaptation may have affected Bathyergidae at about 40–48 Ma (Faulkes et al. 2004), in the patterns of chromosomal evolution (Struwe et al. which a much higher degree of chromosomal variation 2011), a robust phylogenetic hypothesis is warranted with was observed (Faulkes et al. 2004; Deuve et al. 2008 and comparisons of ecology and habitat use (Elith et al. 2011). references therein). Further, both the canalization and dead end model cannot explain the relative higher level Acknowledgments: We thank Dr. Kawada and another of karyotype variability in one fully fossorial tribe anonymous reviewer for constructive suggestions and (Talpini) but not in the other (Scalopini). More karyo- comments. The project was supported by the Fund of typic and FISH studies are warranted at both inter- State Key Laboratory of Genetics Resources and Evolu- specific and intra-specific levels for North American tion, Kunming Institute of Zoology, Chinese Academy of scalopines to test these models. Sciences (1101090359) and the National Basic Research We determined the karyotype of an enigmatic talpid Program of China (973 Program) (2007CB411600 and species, S. oweni and provided further evidence for 2011CB302102). We thank Joseph D. Orkin for im- karyotype uniformity in the family despite the observa- proving the English. He et al., Karyotype of the Gansu mole 7

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