http://www.paper.edu.cn Rapid and Parallel Chromosomal Number Reductions in Muntjac Deer Inferred from Mitochondrial DNA Phylogeny Wen Wang1 and Hong Lan2 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, People's Republic of China Muntjac deer (Muntiacinae, Cervidae) are of great interest in evolutionary studies because of their dramatic chro- mosome variations and recent discoveries of several new species. In this paper, we analyze the evolution of kar- yotypes of muntjac deer in the context of a phylogeny which is based on 1,844-bp mitochondrial DNA sequences of seven generally recognized species in the muntjac subfamily. The phylogenetic results support the hypothesis that karyotypic evolution in muntjac deer has proceeded via reduction in diploid number. However, the reduction in number is not always linear, i.e., not strictly following the order: 46→14/13→8/9→6/7. For example, Muntiacus muntjak (2n 5 6/7) shares a common ancestor with Muntiacus feae (2n 5 13/14), which indicates that its karyotype was derived in parallel with M. feae's from an ancestral karyotype of 2n $ 13/14. The newly discovered giant muntjac (Muntiacus vuquangensis) may represent another parallel reduction lineage from the ancestral 2n 5 46 karyotype. Our phylogenetic results indicate that the giant muntjac is relatively closer to Muntiacus reevesi than to other muntjacs and may be placed in the genus Muntiacus. Analyses of sequence divergence reveal that the rate of change in chromosome number in muntjac deer is one of the fastest in vertebrates. Within the muntjac subfamily, the fastest evolutionary rate is found in the Fea's lineage, in which two species with different karyotypes diverged in around 0.5 Myr. Introduction Muntjac deer (Muntiacinae, Cervidae) are distrib- numbers, 46, 47, and 48, observed in natural populations uted throughout Southeast Asia, South China, and India. (Shi 1981; Shi, Yang, and Kumamoto 1991). This con- They are of great interest to evolutionary biologists and siderable karyotypic diversity makes muntjac deer spe- cytogeneticists because of the considerable diversity of cies excellent models for the study of chromosomal evo- their karyotypes, despite their morphological similarity lution and speciation. (Fontana and Rubini 1990). The Indian muntjac, Mun- Hsu, Pathak, and Chen (1975) hypothesized that tiacus muntjak, possesses the lowest diploid chromo- the large chromosomes in M. muntjak result from mul- somal number in mammals (2n 5 6 for females [F] and tiple tandem and centromeric fusions of small ancestral 7 for males [M]) (Wurster and Benirschke 1970; Wurster acrocentric chromosomes similar to those retained in M. and Atken 1972; Shi 1976), whereas the Chinese munt- reevesi. Comparative cytogenetic studies, especially jac (Muntiacus reevesi) has a 2n number of 46 in both those recent works using chromosome painting, strongly sexes (Wurster and Benirschke 1967). These two spe- support this hypothesis and ®nd tandem fusion to be the cies, however, can produce viable F1 hybrids (2n 5 27) major process in chromosome evolution of muntjacs in captivity, and partial spermatogenesis was observed (Shi, Ye, and Duan 1980; Brinkley et al. 1984; Lin et in hybrids (Shi, Ye, and Duan 1980; Shi and Pathak al. 1991; Yang et al. 1995, 1997a, 1997c). However, the 1981; Neiztel 1987). Other karyotyped species have in- scenario of chromosome evolution in muntjacs, such as termediate numbers of chromosomes; for example, 2n the chronology of the reduction events, is still unclear. 5 8F,9MinMuntiacus crinifrons (Shi 1983), 2n 5 8 A comprehensive phylogenetic analysis for muntjac deer F, 9 M i n Muntiacus gongshanensis (Shi and Ma 1988), would be helpful in addressing this question. and 2n 5 13 F (Soma et al. 1983, 1987), 14 M (L. M. Meanwhile, muntjac deer are also a fascinating Shi, personal communication) in Muntiacus feae. The subject to mammalogists. Although the discovery of tufted deer (Elaphodus cephalophus), which is the sole new large mammal species is very rare nowadays, a species in the other genus of the Muntiacinae subfamily, number of new muntjac species have been discovered has polymorphic karyotypes with three different diploid since the late 1980s. In 1988, the Gongshan muntjac (M. gongshanensis) was discovered on Gongshan Mountain Key words: muntjac deer (Muntiacinae), mitochondrial ND4L, in Southwest China (Shi and Ma 1988; Ma, Wang, and ND4 genes, phylogeny, chromosomal evolution. Shi 1990). In 1994, the giant muntjac (Megamuntiacus Abbreviations: MYA, million years ago; Myr, million years; vuquangensis or Muntiacus vuquangensis) was found in mtDNA, mitochondrial DNA; ND4, subunit 4 of nicotinamide adenine the Annamite mountains along the border of Laos and dinucleotide dehydrogenase; ND4L, subunit 4L of nicotinamide ade- nine dinucleotide dehydrogenase; NJ, neighbor joining. Vietnam (Evans and Timmins 1994; Touc et al. 1994) and was later con®rmed (Schaller and Vrba 1996; Tim- Address for correspondence and reprints: Wen Wang, Department of Ecology and Evolution, University of Chicago, Chicago, Illinois mins et al. 1998). In 1998, Muntiacus truongsonensis 60637. E-mail: [email protected]. was reported from the Annamite mountains of Laos (Giao et al. 1998). In 1999, Muntiacus putaoensis was 1 Present address: Department of Ecology and Evolution, Univer- sity of Chicago. discovered in northern Myanmar (Amato, Egan, and Ra- binowitz 1999). Some preliminary observations even in- 2 Present address: Department of Oncology, University of Wis- consin±Madison. dicate that there may exist more new muntjac species in Mol. Biol. Evol. 17(9):1326±1333. 2000 the Annamite mountains (Giao et al. 1998; R. J. Tim- q 2000 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 mins, personal communication). 1326 http://www.paper.edu.cn Phylogeny and Chromosomal Evolution of Muntjac Deer 1327 Table 1 Information of Diploid Number and Range for All Extant Muntjac Species Species Diploid No. Range Sample Collection Locality Elaphodus cephalophus* .............. 46, 47, 48 Most of South China Yunnan, China Muntiacus reevesi* ................... 46 Throughout South China Individual 1 ....................... Yunnan, China Individual 2 ....................... Yunnan, China Muntiacus vuquangensis*.............. ? Annamite Mountains, Laos and Vietnam Ban Lak, Laos Muntiacus feae* ..................... 14 F, 13 M Myanmar and Thailand Thailand Muntiacus crinifrons* ................. 8F,9M Zhejiang and Anhui, China Zhejiang, China Muntiacus gongshanenis* ............. 8F,9M Western Yunnan, China Gongshan, Yunnan, China Muntiacus muntjak* .................. 6F,7M Indo-China Muntiacus muntjak yunnanensis* ...... South Sichuan and West Yunnan, China South Sichuan, China Muntiacus muntjak menglasis*........ South Yunnan, China South Yunnan, China Muntiacus muntjak nigripes* ......... Hainan Hainan, China Muntiacus muntjak annamensis* ...... South Vietnam and Laos, Thai/Cambodia border Individual 1 ...................... Ban Lak, Laos Individual 2 ...................... Ban Lak, Laos Muntiacus atherodesa ................. ? Borneo Ð Muntiacus rooseveltorumb ............. ? Annamite Mountain, Laos and Vietnam Ð Muntiacus truongsonensis ............. ? Annamite Mountain, Laos and Vietnam Ð Muntiacus putaoensiss ................ ? North Myanmar Ð NOTE.ÐAsterisks indicate the taxa used in this study. F and M indicate female and male, respectively. a The muntjac in Borneo was ®rst considered as a distinct species (M. atherodes) in 1982 (Groves 1982). b The Roosevelt's muntjac, M. rooseveltorum, which was ®rst described by Osgood in 1932 but for which little evidence was accumulated afterward, had been controversial (Ma, Wang, and Xu 1986; Groves and Grubb 1990; Sheng and Ohtaishi 1993) until it was rediscovered recently by Amato et al. (1999) based on mitochondrial 16S DNA sequences. Although much attention has been paid to muntjac PCR and Sequencing deer, the taxonomy of this family is controversial and the phylogeny is still an open question. It has been ob- DNA was extracted from blood or tissue following served that sequences of mitochondrial ND4L and ND4 the protocol of Sambrook, Fritsch, and Maniatis (1989). Arg Leu genes can supply good phylogenetic information for re- The mtDNA fragment between the tRNA and tRNA Ser solving relationships from subspecies to genus level in genes, which contains the ND4L, ND4, tRNA , and His Arg Leu vertebrates (Forstner, Davis, and Arevalo 1995; Wang et tRNA genes and part of the tRNA and tRNA al. 1997). In this paper, we present a phylogenetic anal- genes, was PCR ampli®ed for each individual marked ysis based on mitochondrial ND4L and ND4 gene se- with an asterisk in table 1 by two pairs of primers: ND4 quences for the seven generally recognized Muntiacinae against Leu (Forstner, Davis, and Arevalo 1995), and species: E. cephalophus, M. reevesi, M. muntjak, M. Arg2 (59-ACT CAA AAA GGA CTA GAA TGA-39) feae, M. crinifrons, M. gongshanensis, and M. vuquan- against MuntND4R3 (59-GCT CGC TAA GAG TCA gensis. We then analyze the evolution of karyotype TCA GGT GGC-39), respectively. Additional oligonu- based on the mtDNA phylogeny obtained. cleotides were designed to be used as sequencing prim- ers to proof each nucleotide at least twice. The PCR Materials and Methods reaction was performed on a Robocycler Gradient 40 Sample Sources temperature cycler (Stratagene). Cycle sequencing was carried out on a thermal cycler (Amplitron I, Barnstead/ Table 1 lists all of the