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Rapid and Parallel Chromosomal Number Reductions in 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 ϭ 6/7) shares a common ancestor with Muntiacus feae (2n ϭ 13/14), which indicates that its karyotype was derived in parallel with M. feae's from an ancestral karyotype of 2n Ն 13/14. The newly discovered giant muntjac (Muntiacus vuquangensis) may represent another parallel reduction lineage from the ancestral 2n ϭ 46 karyotype. Our phylogenetic results indicate that the giant muntjac is relatively closer to Muntiacus reevesi than to other 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 , 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 (2n ϭ 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 ϭ 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. ϭ 8F,9MinMuntiacus crinifrons (Shi 1983), 2n ϭ 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 ϭ 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 (Elaphodus cephalophus), which is the sole new large 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. (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- ᭧ 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 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 (5Ј-ACT CAA AAA GGA CTA GAA TGA-3Ј) feae, M. crinifrons, M. gongshanensis, and M. vuquan- against MuntND4R3 (5Ј-GCT CGC TAA GAG TCA gensis. We then analyze the evolution of karyotype TCA GGT GGC-3Ј), 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 species or putative species Thermolyne) using an FS-DNA sequencing kit (ABi, in Muntiacinae. Asterisks indicate those taxa used in this number 402079) following the supplied protocol. Se- study. The Chinese (Hydropotes inermis)is quences were scored on a 377 PRISM automated se- used as an outgroup because it retains the hypothetical quencer (ABi). ancestral Cervidae karyotype (2n ϭ 70) and is believed to be closely related to the ancestral lineage (Groves and Data Analysis Grubb 1987; Neiztel 1987; Fontana and Rubini 1990; Yang et al. 1997c). The Chinese water deer used to be Sequences were aligned manually in the interface widely distributed in East Asia but is now found only of PAUP, version 3.1.1 (Swofford 1993), using the bo- in east China and Korea (Sheng and Ohtaishi 1993). The vine mtDNA sequence (Anderson et al. 1982) as a ref- DNA sample of H. inermis was a gift from Dr. F. Yang erence. Sequence divergence between mtDNA types and at Cambridge University. The blood samples of M. vu- neighbor-joining (NJ) trees (Saitou and Nei 1987) were quangensis and M. muntjak annamensis were kindly obtained using MEGA (Kumar, Tamura, and Nei 1994) provided by Dr. R. J. Timmins (Wildlife Conservation assuming the Kimura (1980) two-parameter model. The Society) with the transportation help of Dr. W. Bleisch base composition of each DNA sequence was also an- in 1994. All of the other samples were from the collec- alyzed using MEGA. Maximum-parsimony trees were tions of the Kunming Cell Bank of Type Culture Col- constructed using the branch-and-bound option in PAUP, lection of the Chinese Academy of Sciences. version 3.1.1 (Swofford 1993). The sequences of http://www.paper.edu.cn

1328 Wang and Lan

Table 2 Summary of Sequence Variations %of %of Mean Variable Informative No. of A ϩ T Ts/Tv Gene Sites Sites Sites (%)a Ratio ND4L ..... 32.0 18.2 291 63.2 6.9 ND4 ...... 31.2 17.6 1,378 61.9 8.5 tRNA ..... 25.0 15.6 180b 69.9 4.0 Total ...... 30.7 17.6 1,848 62.9 7.6

a No signi®cant difference among taxa. b Four gaps included. FIG. 1.Ð(a) Parsimony tree and (b) neighbor-joining tree. The supporting percentages of 1,000 bootstrap replicates are shown for taurus and H. inermis were de®ned as outgroups in all each internal branch. Tree length is shown in the neighbor-joining tree. tree searches. One thousand bootstrap replications were Diploid numbers are listed in parentheses. performed for both phylogenetic analyses in order to test the robustness of branches. The ratio of transitions (Ts) over transversions (Tv) was estimated based on the con- ble 3). This NJ tree has the same topology as the par- sensus tree using MacClade, version 3.0 (Maddison and simony tree (®g. 1). Maddison 1992). Uniform distribution analysis of sub- Some phylogenetic information has previously stitutions along the DNA segment was also conducted been provided for the genus Muntiacus based on mor- using MacClade, version 3.0. To examine the phyloge- phological data (Ma, Wang, and Xu 1986; Groves and netic information content of the ®nal data set, we used Grubb 1990). Ma, Wang, and Xu (1986) proposed that the method of Hillis and Huelsenbeck (1992) to study crinifrons and rooseveltorum were derived from the re- the skewness in the distribution of trees, using 10,000 evesi lineage, whereas feae and muntjak were derived random trees generated with PAUP, version 3.1.1. In ad- from a different lineage. Groves and Grubb (1990) dition, a relative-rate test (Wu and Li 1985) was con- thought that rooseveltorum was a synonym of feae and ducted using K2WuLi, version 1.0 (Jermiin 1996). that feae was a sister group to muntjak and crinifrons. Molecular markers have also been used to study the Results and Discussion phylogeny of genus Muntiacus (Lan, Shi, and Suzuki Phylogeny of Muntiacinae 1993; Lan, Wang, and Shi 1995; Giao et al. 1998). The results of these previous studies, however, could not We obtained 1,844 bp of unambiguous sequences supply a sound phylogeny for muntjac deer due to either for muntjac species but 1,846 bp for H. inermis, which poor representation of species or lack of informative contains ND4L, ND4, tRNASer, and tRNAHis genes and characters. part of the tRNALeu gene. Muntiacus muntjak yunnanensis In this study, a more inclusive phylogenetic tree is and Muntiacus muntjak nigripes share the same sequence. generated for the muntjac subfamily. The proposed tree GenBank accession numbers for these sequences are topology appears quite robust; the same consensus tree AF190673±AF190685. Base compositions are rather ho- is always obtained using different phylogenetic infer- mogeneous across these taxa, with the AϩT content rang- ence methods (parsimony or NJ) or using different gene ing from 62.2% (M. feae) to 63.2% (M. vuquangensis). segments (single gene or combined data set; ®g. 1). All Uniform distribution analysis shows that substitutions are branches in the trees are well supported by bootstrap quite homogeneously distributed along the fragment (data analyses (Ͼ70%), with one exception for the vuquan- not shown). Including the bovine sequence, we summa- gensis-reevesi clade (53%) in the parsimony tree. How- rize the sequence variations of the three gene fragments ever, this branch is well supported by the NJ tree with (tRNA genes were pooled) in table 2. a probability of 0.82. In our phylogenetic trees (®g. 1), The distribution of 10,000 random trees is strongly the basal split separates Elaphodus and Muntiacus. left-skewed (g1 ϭϪ0.8293, P Ͻ 0.01), indicating that There are two major clades within the genus Muntiacus; the data set is signi®cantly structured and contains a one includes vuquangensis and reevesi, and the other strong phylogenetic signal. A single shortest tree with a includes feae, gongshanensis, crinifrons, and muntjak. length of 937 steps was obtained using the unweighted In the latter clade, muntjak is positioned in a distinct maximum-parsimony method (®g. 1a). When a single branch as sister to the Fea's muntjac lineage. Within the gene or gene cluster (all tRNA genes) was used under Fea's muntjac lineage, gongshanensis is most closely an unweighted scheme, the topologies of resulting trees related to crinifrons, with feae as their sister species. were more or less different from the tree in ®gure 1a. The four subspecies of muntjak are monophyletic. The Nevertheless, if we assign more weight to transversions newly discovered giant muntjac (M. vuquangensis)is than to transitions (e.g., 7:1, as shown in table 2), the relatively closer to M. reevesi. When it was initially de- trees obtained from a single gene become quite consis- scribed, the giant muntjac was granted generic status, tent with the tree in ®gure 1a, and the tree obtained from Megamuntiacus (Touc et al. 1994). This classi®cation, the combined data set remains the same as that in ®gure however, was subsequently questioned by Schaller and 1a. The NJ tree, shown in ®gure 1b, was constructed Vrba (1996) and Timmins et al. (1998). Our result sup- based on the Kimura two-parameter distance matrix (ta- ports it as a member of Muntiacus. The sequence di- http://www.paper.edu.cn

Phylogeny and Chromosomal Evolution of Muntjac Deer 1329

vergences between M. vuquangensis and other muntjacs

9/3 are at the same level as those among other distinct Mun- 25/4 29/3 102/8 103/3 106/5 217/79 225/53 187/29 114/8 122/7 119/7 tiacus species (table 3). Given the constant evolutionary rate of this mtDNA segment in muntjacs as shown be- low by the molecular-clock test, this result indicates that the giant muntjac has not diverged much from other 24/5 28/4 101/9 102/4 103/6 219/80 223/54 187/30 111/9 120/8 117/8

0.0065 Muntiacus species.

Molecular-Clock Test 12/5 103/9 106/4 111/6 220/80 229/54 194/30 119/9 121/8 120/8

0.0176 0.0176 The concept of a constant substitution rate along each muntjac lineage was tested with Wu and Li's (1985) relative-rate test. Using H. inermis as the out- group, all pairwise comparisons between ingroup taxa 0.0093 0.0159 0.0159 103/10 106/5 105/7 219/81 228/55 195/31 118/10 121/9 120/9 were performed. None of the comparisons could reject the molecular-clock hypothesis (data not shown, avail- able on request). Therefore, the molecular clock has

65/5 37/2 been ticking well in muntjac mtDNAs. Assuming this 0.0645 0.0676 0.0627 0.0640 239/76 240/52 198/30 121/7 128/6 127/6 rate constancy, by referring to the fossil records for some extant muntjac species, we tentatively estimated the evolutionary rate of this mtDNA fragment (mainly 60/5 0.0216 0.0640 0.0634 0.0609 0.0610 242/76 239/50 193/28 111/5 124/4 121/4 ND4L and ND4). Fossils of M. reevesi ®rst appeared in the early Pleistocene (ϳ1±2 MYA), while M. muntjak and M. feae appeared in the middle Pleistocene (ϳ0.5± 1 MYA) (Ma, Wang, and Xu 1986; Dong 1993). Based 0.0365 0.0394 0.0650 0.0644 0.0632 0.0632 229/81 232/55 195/33 112/10 122/9 121/9 on the genetic distance in table 3, we reach two esti- mations for the mtDNA evolution: 3.8%±7.5%/Myr and 3.8%±7.6%/Myr, which are quite compatible with each

13/0 other. According to these rates, the following chronol- 0.0756 0.0727 0.0776 0.0750 0.0744 0.0725 0.0732 229/78 234/52 198/30 113/7 ogy of muntjac speciation is inferred: the two muntjac genera, Elaphodus and Muntiacus, diverged about 1.9± 3.7 MYA; vuquangensis separated from reevesi about 0.0762 0.0746 0.0783 0.0756 0.0750 0.0744 0.0751 0.0071 228/78 238/52 199/30 108/7 0.9±1.8 MYA; feae and muntjak shared a common an- cestor until 0.8±1.5 MYA; and the divergence between crinifrons and gongshanensis was a very recent event (0.3±0.5 MYA). Of course, because one species could 0.0706 0.0670 0.0744 0.0743 0.0744 0.0694 0.0707 0.0664 0.0694 221/79 234/53 198/31 have existed for a while before the earliest known fossil was formed, these datings could turn out to be under- estimates, as all fossil-based molecular calibrations tend to be. Attempts (data not shown) to use rates calibrated 0.1394 0.1348 0.1396 0.1381 0.1368 0.1319 0.1313 0.1402 0.1403 0.1396 226/78 240/44 using the well-known artiodactyl-cetacean divergence (Arnason and Gullberg 1996) led to divergence times that were clearly contradicted by the fossil data, indi- 0.1806 0.1826 0.1847 0.1776 0.1777 0.1732 0.1741 0.1794 0.1808 0.1832 0.1802 219/88 cating that either the muntjac clock might tick more rap- idly than that of mammals in general or artiodactyls and cetaceans have diverged from each other so much that 12345678910111213 underestimation of multiple hits become inevitable for 0.1959 0.2025 0.2002 0.1883 0.1883 0.1876 0.1854 0.1930 0.1915 0.1884 0.1931 0.1938 these mtDNA genes. .... 1 ...... Rapid Reduction of Chromosomal Numbers ......

...... Considerable diploid number variations among re-

...... lated species have been reported from grasshoppers and ...... 2 ...... 1 ...... some other insects (reviewed in White 1978), gibbons ...... (2n ϭ 38, 44, 50, and 52) (Jauch et al. 1992), and mice

...... (Nachman and Searle 1995). In particular, there was a 2 ...... recent report of a group of island mice in which chro- mosome number has nearly halved in less than 500 years (Britton-Davidian et al. 2000). However, tandem Muntiacus crinifrons Muntiacus gongshanensis Muntiacus muntjak annamensis Muntiacus muntjak menglasis Muntiacus muntjak yunnanensis Muntiacus feae M. m. annamensis Bos taurus Hydropotes inermis Elaphodus cephalophus Muntiacus vuquangensis Muntiacus reevesi M. reevesi fusions are rarely involved in the chromosome number 8. 9. 7. 1. 2. 3. 4. 5. 6. 10. 12. 13. 11. Table 3 Pairwise Kimura Two-Parameter Distances (below the diagonal) and Transition/Transversion Numbers (above the diagonal) changes in either gibbons or mice. http://www.paper.edu.cn

1330 Wang and Lan

As mentioned above, tandem fusion is the major and 9 M, were independently derived within a short time cytological change during the karyotypic evolution of (probably Ͻ0.5 Myr). muntjacs. However, the rate of reduction is unknown. In However, the reduction in number is not always this study, with the help of molecular phylogenetic data, linear, i.e., not strictly following the order 46→14/ we ®nd a remarkably rapid chromosomal reduction rate 13→8/9→6/7. The 2n ϭ 6/7 karyotype in the Indian in muntjac deer. Bush et al. (1977) calculated chromo- muntjac (M. muntjak) was not sequentially derived from some evolution rates in vertebrates. They found rapid the 2n ϭ 8/9 karyotype existing in crinifrons or gong- chromosomal evolution and speciation in mammals, and shanensis. Rather, M. muntjak may share a common an- the fastest rate they observed was in horses, in which cestor with feae (2n ϭ 14/13), suggesting that it results the rates of chromosome number and fundamental num- from a reduction pathway parallel to the Fea's lineage. ber (FN) changes were 0.61 and 0.79 per Myr, respec- Since the karyotype of 2n ϭ 8 has been found in one tively. Based on the diploid numbers and FNs observed individual of Muntiacus muntjak muntjak (Wurster and in muntjacs (Fontana and Rubini 1990), using the for- Atken 1972), it is possible that the 2n ϭ 6/7 karyotype mula of Bush et al. (1977), we obtained the result that, held by other M. muntjak subspecies was derived from in the genus Muntiacus, the chromosome number chang- an intermediate 2n ϭ 8/9 ancestor represented by M. m. es by 1.08±2.11 per Myr and the FN changes by 0.97± muntjak. Nevertheless, this 2n ϭ 8 karyotype is still a 1.91 per Myr. Even the conservative estimates of these descendant of the reduction pathway parallel to the Fea's two rates are faster than those for horses. Therefore, the lineage. It is even possible that the 2n ϭ 6/7 karyotype evolutionary rate of chromosomal number in muntjac was independently derived from the common 2n Ն 14 deer is among the fastest in vertebrates. ancestor of M. muntjak and the Fea's lineage. Within muntjacs, the most drastic reduction process Evidence below supports the above interpretations. occurred in the Fea's lineage. According to our diver- Chromosome painting data have suggested that the 2n gence time estimates, the karyotypes of M. gongshanen- ϭ 6/7 karyotype of M. muntjak cannot be derived sim- sis and M. crinifrons, which have been found apparently ply from the 2n ϭ 8/9 karyotypes of crinifrons or gong- different (Yang et al. 1995, 1997b), were derived from shanensis. In fact, an unpublished chromosome tree the ancestral karyotype less than 0.5 MYA. In fact, the based on chromosomal painting for reevesi, gongsha- genetic distances among the three species of the Fea's nensis, crinifrons, muntjak, and another 2n ϭ 70 deer lineage are very small compared with the divergences (Mazama gouazoubira) reveals phylogenetic relation- among other deer. Cronin (1991) has shown that the ships for these species similar to those of the mtDNA mtDNA divergence within deer species and between tree (F. Yang, personal communication). In addition, the species of the same deer subfamily are usually Ͻ3% and large population size, wide range, and well-de®ned sub- 4%±12%, respectively. In a phylogenetic study on musk species differentiation of M. muntjak all suggest that M. deer (Moschus) based on the sequences of mitochondrial muntjak is a species with a relatively long history, un- cytochrome b genes, Su et al. (1999) also reported that likely to be derived from the young feae lineage. around 3% sequence divergences were found only be- More reduction episodes and parallel events may tween very close species or subspecies. In table 3, the be identi®ed after the determination of karyotypes of sequence divergences between crinifrons, gongshanen- those newly reported species. For example, M. vuquan- sis, and feae are less than 4%, and the divergence be- gensis may represent another chromosomal reduction tween crinifrons and gongshanensis is as small as 2.2%. pathway from the ancestral 2n ϭ 46 karyotype, as this These data show that the Fea's lineage has apparently species is a member of Muntiacus and holds more de- experienced extremely rapid chromosome evolution. rived characters than M. reevesi does (Schaller and Vrba 1996; Timmins et al. 1998). The most recently reported Parallel Chromosomal Number Reduction two species might be closely related to the giant muntjac based on the overlapping geographic distribution. If this Our phylogenetic results reveal that karyotypic is the case, then those two species could be the progeny evolution in muntjac deer has proceeded via reduction of the reduction descent represented by the giant in diploid number, supporting the fusion hypothesis of muntjac. muntjac chromosome evolution. Based on the phyloge- ny (®g. 1), together with all available chromosome data, Chromosomal Number Reduction and Speciation six chromosomal reduction episodes can be deduced in these species. From the hypothetical ancestral karyotype The inferred process of chromosome evolution in of deer (2n ϭ 70), the ®rst two reductions result in the muntjac deer implies that there is a general, constant karyotypes of the tufted deer (E. cephalophus, 2n ϭ 46, tendency toward chromosome reduction, with tandem 47, 48) and the Chinese muntjac (M. reevesi, 2n ϭ 46), fusion as a major factor, in the speciation process of this respectively. The reevesi-like muntjac ancestor subse- group of species. The correlation between chromosomal quently experienced further chromosomal reduction, and rearrangement and speciation has been discussed exten- thereby the karyotypic array of Muntiacus species was sively (White 1978; Patton and Sherwood 1983; Sites derived. One clade of the progeny leads to the lineage and Moritz 1987), although tandem fusion has seldom of Fea's muntjac (M. feae, 2n ϭ 13 F, 14 M). From this been taken into account, probably due to its rarity in lineage, the black muntjac (M. crinifrons) and the Gong- nature. Compared with other types of chromosome re- shan muntjac (M. gongshanensis), both with 2n ϭ 8F arrangements, multiple tandem fusions would lead to http://www.paper.edu.cn

Phylogeny and Chromosomal Evolution of Muntjac Deer 1331 high frequency of unbalanced gametes in heterozygotes The molecular mechanism whereby the muntjac and hence can serve effectively in erecting a reproduc- telomere and centromere repetitive sequences induce tive isolation barrier once new multiply fused chromo- frequent tandem fusions is unknown. Further study on somes are ®xed. However, how to overcome the nega- muntjac repetitive DNAs, as well as on the mutation tive heterotic effects prior to the ®xation is a problem mechanism of these repeats, will bring us new insight (Patton and Sherwood 1983). As suggested by other au- into the essential nature of the evolution of muntjac thors, this kind of stasipatric speciation (sympatric spe- chromosomes. By elucidating the driving force behind ciation caused by chromosome rearrangements) most the tandem fusions, we may one day be able to recon- likely occurs in taxa with small demes (White 1978; struct the reduction process in laboratories. Patton and Sherwood 1983; Neiztel 1987; Sites and Mo- ritz 1987). Patton and Sherwood (1983) further pointed Acknowledgments out that in the case of multiple rearrangements, in order for the new karyotype to be ®xed, the ®xation has to be We dedicate this paper to our mentor, the late Pro- rapid. Also, the chance to overcome the negative het- fessor Liming Shi, who was one of the pioneers in erosis will increase if the speci®c chromosomal rear- studying muntjac chromosome evolution. Special thanks rangement is not unique. Alternatively, this problem can to Dr. Chung-I Wu at the University of Chicago for his also be overcome by successive accumulation of slightly great help and encouragement during the preparation of deleterious rearrangements. In other words, individual the manuscript, and to Dr. Fengtang Yang at Cambridge tandem fusions in muntjacs may not be so deleterious University for his helpful discussion and generosity in as to cause a rapid elimination of fused karyotype. After showing us his unpublished chromosomal painting data. more fusions accumulate, postmating isolation may have We also thank Drs. James Patton, Manyuan Long, Janice been established between individuals with severely dif- Spofford, A. Hon-Tsen Yu, and Esther Betran and Ms. ferent karyotypes. Haijing Yu for their critical review of the manuscript. Interestingly, several pieces of evidence from We appreciate Mr. Shikang Gou's technical assistance. muntjac deer suggest that these mammals may have suc- We further thank Drs. Rob Timmins and William ceeded in stasipatric speciation, with chromosomal tan- Bleisch from the Wildlife Conservation Society (New dem fusion as the major genetic mechanism. First, munt- York) for providing the blood samples of M. vuquan- jacs have been found to have the smallest breeding gensis and Muntiacus muntjak annamensis, Dr. Feng- group in Cervidae (Clutton-Brock and Albon 1980), tang Yang for the DNA sample of H. inermis, and the while many Muntiacus species are sympatric with one Kunming Cell Bank of Type Culture Collections of the or more other species (see table 1). Second, the muntjac Chinese Academy of Sciences for samples of the other chromosomes appear to have ``sticky ends'' that in- species used in this study. This study was supported by crease fusion ability. A number of satellite DNAs have various fundings from the Chinese Academy of Science been found at or near the centromeric and telomeric re- and the Chinese Science Foundation granted to Liming gions of muntjac chromosomes (Bogenberger, Neu- Shi, W.W., and H.L. Both authors contributed equally to maier, and Fittler 1985; Benedum et al. 1986; Lin et al. this work. 1991; Scherthan 1991). Some of these repetitive ele- ments, such as C5 and the mammalian telomere se- LITERATURE CITED quence, have been found at the putative fusion points (Lee, Sasi, and Lin 1993; Fronicke and Scherthan 1997; AMATO, G., M. G. EGAN, and A. RABINOWITZ. 1999. A new Yang et al. 1997a). It has been proposed that homolo- species of muntjac, Muntiacus putaoensis (Artiodactyla: gous repetitive elements at the centromeres and telo- Cervidae) from northern Myanmar. Anim. Conserv. 2:1±7. meres are able to cause illegitimate recombination be- AMATO, G., M. G. EGAN,G.B.SCHALLER,R.H.BAKER,H. tween nonhomologous chromosomes and thus result in C. ROSENBAUM, and W. G. ROBICHAUD. 1999. Rediscovery tandem fusion (Ferguson-Smith 1973; Yang et al. of Roosevelt's barking deer (Muntiacus rooseveltorum). J. 1997a). The stickiness of the muntjac chromosome ends Mammal. 80:639±643. ANDERSON, S., M. H. L. DEBRUDN,A.R.COULSON,I.C.EPE- might have caused chromosome fusion at a higher rate RON,F.SNAGER, and I. G. YOUNG. 1982. Complete se- than that of other mammals, as discussed above. In fact, quence of bovine mitochondrial DNA. J. Mol. Biol. 156: the occurrence of tandem fusion has been observed in 683±717. an established cell line of M. muntjak (Neiztel 1987). ARNASON, U., and A. GULLBERG. 1996. Cytochrome b nucle- We can imagine that while the karyotypic lability con- otide sequences and the identi®cation of ®ve primary line- tinuously creates variations, some of the new karyotypes ages of extant cetaceans. Mol. Biol. Evol. 13:407±417. can be ®xed in the small breeding populations by virtue BENEDUM, U. M., H. NEITZEL,K.SPERLING,J.BOGENBERGER, of either successive accumulations of slightly deleteri- and F. FITTLER. 1986. Organization and chromosomal dis- ous fusions or coexistence of similar fusions. Each of tribution of a novel repetitive DNA component from Mun- the surviving karyotypes would represent a new Mun- tiacus muntjak vaginalis with a repeat length of more than 40 kb. Chromosoma 94:267±272. tiacus species. Species with a recently survived karyo- BOGENBERGER, J. M., P. S. NEUMAIER, and F. FITTLER. 1985. type would not have had enough time to disperse to a The muntjak satellite IA sequence is composed of 31-base- large area, which is compatible with the distribution pat- pair internal repeats that are highly homologous to the 31- tern of Muntiacus species: most are limited to small ar- base-pair subrepeats of the bovine satellite 1.715. Eur. J. eas, except for M. reevesi and M. muntjak (table 1). Biochem. 148:55±59. http://www.paper.edu.cn

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