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

Karyotype of the Gansu mole (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 (Talpidae, Eulipotyphla, 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 mammals within the Family Talpidae (Eulipotyphla, Mammalia). Because these fully fossorial animals 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, Scapanus, 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). The first specimen of S. oweni was caught by G. Fenwick Owen in 1911 in Gansu, China (Thomas 1912). Since then, reports of its occurrence have been limited, all of which have been from Central and Southwest China (Gansu, Qinghai, Shaanxi, and Sichuan Provinces)

Fig. 1. External morphology of Scapanulus oweni (upper) from Ningshan, Shaanxi, China (male, catalog number: KIZ027013) and Parascalops breweri (lower) from North America (Photo by D. C. Gordon, American Society of Mammalogists photo catalog number: 852).

*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, only eight specimens are known in museum collections in Germany, the UK, and the USA (GBIF data portal; www.gbif.org). Given that the distribution, population, and ecology of this species are barely known (Smith and Johnston 2008), it is not surprising that its karyotype remains unknown. The chromosomal information for all other talpid genera is available (at least one species per genus) and exhibits remarkable stability (Table 1; Nevo 1979). In 2011, we collected two specimens of S. oweni in Ningshan, Shaanxi, one of which was caught alive and sacrificed for karyotypic study. Here, we examined the pattern of karyotype variation among the talpids and test multiple models of chromosomal evolution within the family.

Materials and methods Specimen collection All animal samples were obtained following the regulations for the implementation of China on the protection of terrestrial wild animals (State Council Decree [1992] No. 13) and approved by the Ethics Committee of Kunming Institute of Zoology, Chinese Academy of

Sciences, China. Pitfall traps were set at about 1,500 m above sea level at the Huoditang tree farm in Ningshan, Shaanxi, China (33.4°N, 108.5°E). The specimens were identified according to Thomas (1912) and deposited in the Kunming Institute of Zoology, Chinese Academy of Sciences. Cell culture and cytological preparation Fibroblast cell cultures derived from the lung and kidney of the specimen were stored in Kunming Cell Bank of the Chinese Academy of Sciences, Kunming, Yunnan 650223, P.R. China. Cell culture and metaphase preparations were performed following the conventional procedures (Hungerford 1965). G-banding, C-banding and silver-stained were performed following Seabright (1971), Summer (1972) and Goodpasture and Bloom (1975), respctively. Images were captured using the CytoVision system (Applied Imaging) with a CCD camera mounted on a Zeiss microscope. The chromosomes of S. oweni were arranged based on their relative lengths, from the longest to the shortest. Reconstruction of karyotype evolution The phylogeny of the family from the most recent study (Sanchez-Villagra et al. 2006) was considered as

He et al., Karyotype of the Gansu mole

3 Table 1.

Subfamily

Tribe

Uropsilinae

species

Chromosomal numbers of talpids 2N

FNa

Uropsilus andersoni

34

52

(Motokawa et al. 2009)

Uropsilus gracilis

34

46

(Kawada et al. 2006a)

Uropsilus investigator

Talpinae

Lifestyles Ambulatory

–

Uropsilus soricipes Scalopinae

Citation

–

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)

Desmana moschata

32

60

(Aniskyn and Romanov 1990) Aquatic

Galemys pyrenaicus

42

64

(Reumer and Meylan 1986)

Neurotrichus gibbsii

38

72

(Kawada et al. 2008b)

Scaptonychini Scaptonyx fusicaudus

34

64

(Kawada et al. 2008b)

Talpini

Euroscaptor longirostris

34

52

(Kawada et al. 2008c)

Euroscaptor parvidens

36

60

(Kawada et al. 2008c)

Euroscaptor malayana

36

52

(Kawada et al. 2005)

Desmanini Neurotrichini

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)

Mogera 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

Fully fossorial

–

Mogera wogura

36

52/58

Parascaptor leucura

34

64

(Ye 2007)

Scaptochirus moschatus

48

54?

(Kawada et al. 2002)

Talpa 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

Urotrichini

Semi-fossorial

(Kawada et al. 2001)

–

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)

Dymecodon pilirostris

34

62

(Kawada and Obara 1999)

Urotrichus talpoides

34

64

(Kawada and Obara 1999)

Semi-fossorial

The taxonomy 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 reconstructed based on the most parsimonious (MP) assumption using Mesquite v2.75 (Maddison and Maddison 2008).

Result and discussion We collected two specimens of Gansu mole. Among them, one male was caught alive in a pitfall trap (catalog 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 formula 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 13.34, breadth across molars 8.39, upper tooth row 12.42, and lower tooth row 11.25. The external measurements of specimens from Ningshan are smaller than those from Gansu, but the skull is slightly larger (Allen 1938). More specimens and molecular data are needed to test whether the differences are due to geographical variations. The halluces of the live animal are curved and twisted away from the other digits (Fig. 3) as in the dead body as described by Thomas (1912), thus we confirmed this twist natural, rather than resulting from distortion during the desiccation (Thomas 1912). The diploid number (2N) and autosomal fundamental number (FNa) are 34 and 64, respectively (Fig. 5a and 5d). All autosomal chromosomes are biarmed. In total, there are 24 metacentric + submetacentric, 8 subtelocentric autosomes in the karyotype. The X chromosome is metacentric; the dot-like Y chromosome is the smallest chromosome. The G-banding and C-banding karyotypes are shown in Figs. 5b and 5c. The G-banded karyotype identified homologous chromosomes. The C-bands were distributed at the centromeric regions of all chromosomes as well as at the long arm of chromosome 12 and the terminal of the short arm of chromosome 13. Silver staining demonstrates the nucleolar organizer regions

Fig. 3. Tail and hind foot of the S. oweni from Ningshan, Shaanxi, ‘swimming’ in the moist soil.

Fig. 4. Dorsal view of the mandible and dorsal, ventral, lateral views of the cranium of the specimen of S. oweni from Ningshan, Shaanxi.

(NORs) located at chromosome 8 indicating secondary constriction. The G-banding pattern and the position of the NOR of this chromosome are identical with the chromosome 8 of Talpa europaea (Volleth and Mueller 2006) and chromosome 5 of Scaptonyx fusicaudus (Kawada et al. 2008b). Compared with the other scalopines in North America, S. oweni has the same diploid number (2N = 34), and its fundamental number (FNa = 64) is the same with Scalopus aquaticus (Table 1) even though the chromosomal morphology are not identical (Yates and Schmidly 1975). The G-banding and C-banding patterns and the positions of NORs of the scalopines in North America are warranted for karyotype comparison (Motokawa et al. 2009) and evolutionary analyses (Kawada et al. 2008b) in this tribe. Upon including S. oweni, karyotypes of 37 talpid species (out of the 43 recognized species) representing all 17 genera have been determined (Table 1). The 2N ranges from 30–48 (Standard deviation [SD] = 2.9) and the FNa ranges from 46–72 (SD = 5.5). Twenty species

He et al., Karyotype of the Gansu mole

Fig. 5. Karyotype of S. oweni collected from Ningshan, Shaanxi illustrating a) conventional, b) G-banding, c) C-banding, d) conventional metaphase plate and e) silver-stained metaphase plate. Arrows indicate the position of active Ag-NORs.

(54.1%) have the same diploid number (2N = 34) and twelve (34.3%) have the same autosomal fundamental number (FNa = 64). Thus, the karyotype variability,

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 assumption, we predicted the ancestral karyotype of the family to be 2N = 34 and FNa = 64. The ancestral diploid number is identical to that of Solenodon paradoxus (Reumer and Meylan 1986), which is in the basal genus in Eulipotyphla (Roca et al. 2004). Note that this prediction 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 shrew-mole, Neurotrichus gibbsii. Several models have been proposed to explain the karyotypic stability of animals. Arnason (1972) hypothesized that large deme size, inbreeding, high mobility, and a continuous range of distribution were the primary reasons for karyotypic stability (deme size model), which is not necessarily suitable for the whole talpid family, since the population densities are variable (0.42–12.4 moles/hectare; Funmilayo 1977; Carraway et al. 1993; Hartman and Krenz 1993; Nores et al. 1998). 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. (SanchezVillagra 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 nearly optimum karyotype will have been achieved. Accordingly, relatively ancient groups are more likely to have evolved optimum karyotypes (canalization model). Alternatively, King’s (1985) “dead end model” hypothesized that when a karyotype has been saturated by chromosomal rearrangement, an evolutionary dead end will be reached. Despite the critiques of the canalization model (King 1985, 1987), the most recent common ancestor (MRCA) of the extant talpids was at about 52 ± 5 Ma (Douady and Douzery 2003), which is not much older than the MRCA of the African mole rat family, Bathyergidae at about 40–48 Ma (Faulkes et al. 2004), in which a much higher degree of chromosomal variation was observed (Faulkes et al. 2004; Deuve et al. 2008 and references therein). Further, both the canalization and dead end model cannot explain the relative higher level of karyotype variability in one fully fossorial tribe (Talpini) but not in the other (Scalopini). More karyotypic and FISH studies are warranted at both interspecific and intra-specific levels for North American scalopines to test these models. We determined the karyotype of an enigmatic talpid species, S. oweni and provided further evidence for karyotype uniformity in the family despite the observa-

tions of a unique karyotype of N. gibbsii and karyotypic variations in the tribe Talpini were observed. While a clear relationship between the rates of chromosomal evolution and speciation exists, the reason for karyotype stability remains unclear. None of the previously proposed models adequately explains the low level of chromosomal variation within this family. Therefore, we suggest that intra- and inter chromosomal rearrangements should be taken into account for comparison and evolutionary analyses. Further, because different phylogeny, phylogeographic histories (i.e. dispersalvicariance) and ecological adaptation may have affected the patterns of chromosomal evolution (Struwe et al. 2011), a robust phylogenetic hypothesis is warranted with comparisons of ecology and habitat use (Elith et al. 2011). Acknowledgments: We thank Dr. Kawada and another anonymous reviewer for constructive suggestions and comments. The project was supported by the Fund of State Key Laboratory of Genetics Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences (1101090359) and the National Basic Research Program of China (973 Program) (2007CB411600 and 2011CB302102). We thank Joseph D. Orkin for improving the English.

He et al., Karyotype of the Gansu mole

References Allen, G. M. 1938. The Mammals of China and Mongolia. Part I. American Museum of Natural History, New York, 620 pp. Aniskyn, V. and Romanov, P. 1990. Kariologicheskaya kharakteristika Desmana moschata. L. 5th Vsesoyuznogo Teriologicheskogo Obsshestva AN SSSR, No. I: 39 (in Russian). Arnason, U. 1972. The role of chromosomal rearrangement in mammalian speciation with special reference to Cetacea and Pinnipedia. Hereditas 70: 113–118. Bickham, J. W. and Baker, R. J. 1979. Canalization model of chromosomal evolution. Bulletin Carnegie Museum of Natural History 13: 70–84. Bush, G. L., Case, S. M., Wilson, A. C. and Patton, J. L. 1977. Rapid speciation and chromosomal evolution in mammals. Proceedings of the National Academy of Sciences of the United States of America 74: 3942–3946. Carraway, L. N., Alexander, L. F. and Verts, B. 1993. Scapanus townsendii. Mammalian Species 434: 1–7. Deuve, J. L., Bennett, N. C., Britton-Davidian, J. and Robinson, T. J. 2008. Chromosomal phylogeny and evolution of the African mole-rats (Bathyergidae). Chromosome Research 16: 57–74. Douady, C. J. and Douzery, E. J. P. 2003. Molecular estimation of eulipotyphlan divergence times and the evolution of “Insectivora”. Molecular Phylogenetics and Evolution 28: 285–296. Elith, J., Phillips, S. J., Hastie, T., Dudík, M., Chee, Y. E. and Yates, C. J. 2011. A statistical explanation of MaxEnt for ecologists. Diversity and Distributions 17: 43–57. Faulkes, C. G., Verheyen, E., Verheyen, W., Jarvis, J. U. and Bennett, N. C. 2004. Phylogeographical patterns of genetic divergence and speciation in African mole-rats (Family: Bathyergidae). Molecular Ecology 13: 613–629. Funmilayo, O. 1977. Distribution and abundance of moles (Talpa europaea L.) in relation to physical habitat and food supply. Oecologia 30: 277–283. Goodpasture, C. and Bloom, S. E. 1975. Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53: 37–50. Hartman, G. and Krenz, J. 1993. Estimating population density of moles Scalopus aquaticus using assessment lines. Acta Theriologica 38: 305–314. He, K. 2011. Phylogeny and phylogeography of some taxa in Soricomorpha in Southwest China. Ph.D. Thesis. Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming (in Chinese with English abstract). Hungerford, D. A. 1965. Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCl. Biotechnic and Histochemistry 40: 333–338. Hutterer, R. 2005. Order Soricomorpha. In (D. E. Wilson and D. A. Reeder, eds.) Mammal Species of the World: A Taxonomic and Geographic Reference, 3rd ed, pp. 220–311. John Hopkins University Press, Baltimore. Kawada, S., Harada, M., Koyasu, K. and Oda, S. 2002. Karyological note on the short-faced mole, Scaptochirus moschatus (Insectivora, Talpidae). Mammal Study 27: 91–94. Kawada, S., Harada, M., Obara, Y., Kobayashi, S., Koyasu, K. and Oda, S. 2001. Karyosystematic analysis of Japanese talpine moles in the genera Euroscaptor and Mogera (Insectivora, Talpidae). Zoological Science 18: 1003–1010. Kawada, S., Kobayashi, S., Endo, H., Rerkamnuaychoke, W. and Oda, S. 2006b. Karyological study on Kloss’s mole Euroscaptor klossi (Insectivora, Talpidae) collected in Chiang Rai Province,

7 Thailand. Mammal Study 31: 105–109. Kawada, S., Li, S., Wang, Y. and Oda, S. 2006a. Karyological study of Nasillus gracilis (Insectivora, Talpidae, Uropsilinae). Mammalian Biology 71: 115–119. Kawada, S., Li, S., Wang, Y., Mock, O. B., Oda, S. and Campbell, K.L. 2008b. Karyotype evolution of shrew moles (Soricomorpha: Talpidae). Journal of Mammalogy 89: 1428–1434. Kawada, S. and Obara, Y. 1999. Reconsideration of the karyological relationship between two Japanese species of shrew-moles, Dymecodon pilirostris and Urotrichus talpoides. Zoological Science 16: 167–174. Kawada, S., Oda, S. and Hideki, E. 2010. A comparative karyological study of Taiwanese and Vietnamese Mogera (Insectivora, Talpidae) and classification (Biodiversity inventory in the Western Pacific Region (3) Indochina and southeastern China). Memoirs of the National Museum of Nature and Science 46: 47–56. Kawada, S., Shinohara, A., Kobayashi, S., Harada, M., Oda, S. and Lin, L. 2007. Revision of the mole genus Mogera (Mammalia: Lipotyphla: Talpidae) from Taiwan. Systematics and Biodiversity 5: 223–240. Kawada, S., Shinohara, A., Yasuda, M., Oda, S. and Liat, L. B. 2005. Karyological study of the Malaysian mole, Euroscaptor micrura malayana (Insectivora, Talpidae) from Cameron Highlands, Peninsular Malaysia. Mammal Study 30: 109–115. Kawada, S., Son, N. T. and Can, D. N. 2008c. Karyological diversity of talpids from Vietnam (Insectivora, Talpidae). In (D. N. Can, H. Endo, N. T. Son, T. Oshida, L. X. Canh, D. H. Phuong, D. P. Lunde, S. Kawada, A. Hayashida and M. Sasaki, eds.) Checklist of Wild Mammal Species of Vietnam, pp. 384–389. Shoukadoh, Kyoto. Kawada, S., Son, N. T. and Can, D. N. 2012. A new species of mole of the genus Euroscaptor (Soricomorpha, Talpidae) from northern Vietnam. Journal of Mammalogy 93: 839–850. Kawada, S., Yasuda, M., Shinohara, A. and Lim, B. L. 2008a. Redescription of the Malaysian mole as to be a true species, Euroscaptor malayana (Insectivora, Talpidae). Memoirs of the National Museum of Nature and Science 45: 65–74. King, M. 1985. The canalization model of chromosomal evolution: A critique. Systematic Biology 34: 69–75. King, M. 1987. Chromosomal rearrangements, speciation and the theoretical approach. Heredity 59: 1–6. Lin, L., Motokawa, M. and Harada, M. 2002. Karyotype of Mogera insularis (Insectivora, Talpidae). Mammalian Biology 67: 176– 178. Maddison, W. P. and Maddison, D. R. 2008. Mesquite: a modular system for evolutionary analysis. Evolution 62: 1103–1118. Motokawa, M., Wu, Y. and Harada, M. 2009. Karyotypes of six soricomorph species from Emei Shan, Sichuan Province, China. Zoological Science 26: 791–797. Nevo, E. 1979. Adaptive convergence and divergence of subterranean mammals. Annual Review of Ecology and Systematics 10: 269– 308. Nores, C., Ojeda, F., Ruano, A., Villate, I., González, J., Cano, J. and García, E. 1998. Estimating the population density of Galemys pyrenaicus in four Spanish rivers. Journal of Zoology 246: 454– 457. Nowak, R. M. 1999. Walker’s Mammals of the World. Johns Hopkins University Press, Baltimore, 1936 pp. Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J. and Gray, J. 1988. Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proceedings of the National

8 Academy of Sciences 85: 9138–9142. Reumer, J. W. F. and Meylan, A. 1986. New developments in vertebrate cytotaxonomy IX Chromosome numbers in the order Insectivora (Mammalia). Genetica 70: 119–151. Roca, A. L., Bar-Gal, G. K., Eizirik, E., Helgen, K. M., Maria, R., Springer, M. S., O’Brien, S. J. and Murphy, W. J. 2004. Mesozoic origin for West Indian insectivores. Nature 429: 649–651. Sanchez-Villagra, M. R., Horovitz, I. and Motokawa, M. 2006. A comprehensive morphological analysis of talpid moles (Mammalia) phylogenetic relationships. Cladistics 22: 59–88. Seabright, M. 1971. A rapid banding technique for human chromosomes. Lancet 2: 971–972. Shinohara, A., Campbell, K. L. and Suzuki, H. 2005. An evolutionary view on the Japanese talpids based on nucleotide sequences. Mammal Study 30: S19–S24. Smith, A. T. and Johnston, C. H. 2008. Scapanulus oweni. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.1. Downloaded on 08 September 2012. Struwe, L., Smouse, P. E., Heiberg, E., Haag, S. and Lathrop, R. G. 2011. Spatial evolutionary and ecological vicariance analysis (SEEVA), a novel approach to biogeography and speciation research, with an example from Brazilian Gentianaceae. Journal of Biogeography 38: 1841–1854. Summer, A. 1972. A simple technique for demonstrating centromeric heterochromatin. Experimental Cell Research 75: 304–306. Thomas, O. 1912. On a collection of small Mammals from the Tsinling Mountains, Central China, presented by Mr. G. Fenwick

Mammal Study

37 (2012)

Owen to the National Museum. Annals and Magazine of Natural History 10: 395–403. Volleth, M. and Mueller, S. 2006. Zoo-FISH in the European mole (Talpa europaea) detects all ancestral Boreo-Eutherian human homologous chromosome associations. Cytogenetic and Genome Research 115: 154–157. Wang, Y. and Zhang, Z. 1997. Insectivores from Sichuan I: Erinaceidae and Talpidae. Sichuan Journal of Zoology 16: 78–82 (in Chinese with English abstract). Yates, T. L. and Moore, D. W. 1990. Speciation and evolution in the family Talpidae (Mammalia: Insectivora). Progress in Clinical and Biological Research 335: 1–22. Yates, T. L. and Schmidly, D. J. 1975. Karyotype of the eastern mole (Scalopus aquaticus), with comments on the karyology of the family Talpidae. Journal of Mammalogy 56: 902–905. Ye, J. 2007. Comparative Molecular Cytogenetics of the Core Insectivores. Ph.D. Thesis. Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 108 pp. (in Chinese with English abstract) Zhang, R. 1997. Distribution of Mammalian Species in China. China Forestry Publishing House, Beijing, 228 pp. Zima, J. 2000. Chromosomal evolution in small mammals (Insectivora, Chiroptera, Rodentia). Hystrix: the Italian Journal of Mammalogy 11: 5–15. Received 25 June 2012. Accepted 14 September 2012.