International Journal of Primatology, Vol. 18, No. 3, 1997

A Phylogeny of Chinese Leaf Monkeys UsingMitochondria! ND3-ND4 Gene Sequences

Wen Wang,1 Michael R. J. Forstner,2 Ya-ping Zhang,1 Zi-min Liu,3 YuWei,3 Hua-qiang Huang,4 Hong-guang Hu,5 You-xin Xie,5 Deng-hu Wu,5and Don J. Melnick2,6,7

Received July 1, 1996; accepted December 16, 1996

The phylogeny of Chinese leaf monkeys, especially the snub-nosed monkeys(Rhinopithecus), has not been thoroughly investigated using molecularsequence data, perhaps due to their rarity in the wild and their poorrepresentation in institutional collections. Despite several proposedclassifications, systematic relationships of these species remain poorly definedand this has hindered their conservation. To clarify the phylogeneticrelationships of the leaf monkey clade in China, we sequenced themitochondrial ND3, ND4L, ND4, tRNA^, tRNAHh, (tRNASer, and tRNA1*11

genes for Rhinopithecus bieti, R. roxellana, Trachypithecus francoisi, T. f.leucocephalus, and T. phayrei as well as Pygathrix nemaeus and Colobusguereza We included a total of 2252 characters for each individual, excludinggaps in primary sequences. Our interpretation of the results from character-and distance-based phylogenetic analyses suggest that (1) Pygathrix nemaeusis sister to Rhinopithecus rather than to Trachypithecus though it is quite

'Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, Kunming,Yunnan Province 650223, P.R. China.

2Center for Environmental Research and Conservation, Columbia University, New York, NewYork 10027.

3The Wildlife and Nature Reserves Managing Station of Guangxi Department of Forestry,Nanning, Guangxi Province 530022, P.R. China.

4Rare and Endangered Animal Protection Station of Fusui County, Fusui, Guangxi Province532100, P.R. China.

5Chongqing Zoo, Chongqing, Sichuan Province 630223, P.R. China.Department of Anthropology and Department of Biological Sciences, Columbia University,New York, New York 10027.

7To whom correspondence should be addressed at center for Environmental Research andConservation, Columbia University, New York, New York 10027.

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0164-0291/97/0600-0305$12.50/0 © 1997 Plenum Publishing Corporation

306 Wang el al.

divergent from the former; (2) the Yunnan snub-nosed monkey, Rhinopithecusbieti, represents a valid species; (3) the white-headed leaf monkey is not adistinct species, but instead is a subspecies of Trachypithecus francoisi (T. f.leucocephalus), though it should still be considered a separate evolutionarysignificant unit (ESU); and (4) because two individuals of the Phayrei's leafmonkey, T. phayrei, are genetically distinct from one another, a more extensiverevision of the taxonomy of this putative species in China is needed. Theseresults, plus ongoing work on the molecular systematics of the entire Asianleaf monkey radiation, can provide a sound basis for identifying the appropriateunits of conservation for this endangered group of primates.

KEY WORDS: Chinese leaf monkeys; phylogeny; mtDNA sequences.

INTRODUCTION

Chinese leaf monkeys, especially the snub-nosed monkeys (Rhinopi-thecus), are not well represented in the literature, possibly due to a combi-nation of isolated populations, restricted ranges, general rarity, and poorrepresentation in institutional collections. Therefore, it is not surprising thatthe taxonomy of Chinese leaf monkeys has been uncertain during most ofthe twentieth century. According to recent taxonomy (Li, 1993), there areeight species of leaf monkey (Colobinae, Cercopithecidae, Primates) occur-ring in China: Rhinopithecus roxellana, R. bieti, R. brelichi, Presbytis francoisi,P. phayrei, P. leucocephalus, P. pileatus, and P. entellus. However, not allauthors agree with this taxonomic arrangement. For example, Groves (1970)recognized Rhinopithecus as a subgenus of Pygathrix because of similaritiesin the skull and limb proportions, and this scheme has been followed by mostWestern authorities (Brandon-Jones, 1984; Napier, 1985; Groves, 1989; Oateset al., 1994). However, most Chinese primatologists have insisted upon thegeneric status of Rhinopithecus (Li and Lin, 1983; Ye, 1993; Peng et al., 1988;1993). This view has been accepted by several researchers outside China sincethe late 1980s (Eudey, 1987; Nowak, 1991; Jablonski and Peng, 1993). Trachypi-thecus is often considered a subgenus or synonym of Presbytis (Groves, 1970;Wolfheim, 1983), and Chinese primatologists agree with this assignment (Penget al., 1988; Ye, 1993; Li, 1993). Nevertheless, Groves (1989) designated it aseparate genus and also removed entellus from Presbytis, erecting the new ge-nus, Semnopithicus, to contain it. This arrangement has been followed by re-cent authorities (Eudey, 1987; Nowak, 1991; Oates et al., 1994). We treatfrancoisi, leucocephalus, and phayrei as members of the genus Trachypithecus.

At the specific level, Ellerman and Morrison-Scott (1951, 1966) rec-ognized bieti and brelichi as subspecies of Rhinopithecus roxellana; Groves(1970) placed brelichi as an independent species but retained bieti as a sub-

A Phylogeny of Chinese Leaf Monkeys 307

species of R. roxellana; and Peng et al. (1988) recognized all three snub-nosed monkeys as separate, valid species. Recent evidence fromcytochrome b sequences (Zhang and Ryder, 1996) and ribosomal DNA re-striction mapping (Wang et al., 1996) supported maintaining bieti as a fullspecies. Another notable debate is whether the white-capped langur (T.leucocephalus) is a valid species or a subspecies of the Francois's langur,T. francoisi. Li and Ma (1980) recognized it as a subspecies of T. francoisi,but Brandon-Jones (1984) and Tan (1985) separated it as a new species.Subsequently, Brandon-Jones (1995) tentatively concluded that leuco-cephalus may represent an albinistic morph of T. francoisi.

In spite of increasing attention to the study of the evolutionary relation-ships and conservation status of Chinese leaf monkeys by national and inter-national researchers, a paucity of data, especially genetic data, for them hasremained a barrier to a fuller understanding of their phylogeny. New dataof this type are thus indispensable in determining evolutionary relationshipsand developing sound conservation plans for this group of primates. We pre-sent a preliminary phylogeny of Chinese leaf monkeys based on mtDNA se-quence data from Rhinopithecus bieti, R. roxellana, Trachypithecus francoisi,T. phayrei, T. leucocephalus, Pygathrix nemaeus, and Colobus guereza.

The use of mitochondrial DNA in primate studies is summarized byMelnick et al., (1992; Melnick and Hoelzer, 1993). Briefly, mammalian mi-tochondrial DNA (mtDNA) is a haploid, nonrecombining, maternally inher-ited molecule. Additionally, it possesses a highly conservative genetic orderand, on average, a rapid substitution rate relative to the nuclear genome(Brown, 1983). All of these characteristics make it an ideal molecular systemfor generating phylogenetically informative data. Different regions of themtDNA genome evolve at different rates (Brown, 1983; Miyamoto andBoyle, 1989). Because ND3, ND4L, and ND4 evolve at an appropriate ratefor specific- and generic-level systematics in vertebrates (Cracraft and Helm-Bychowski, 1991; Arevalo et al., 1994; Forstner et al., 1995), and the terminalend of this region is known to vary even at the population level in leafmonkeys from restriction site analyses (Rosenblum and Melnick, 1994), weselected these three protein-coding genes and the four tRNA genes flankingor separating them to serve as the genetic data for our phylogenetic study.

MATERIALS AND METHODS

Sample Sources

Table I lists the species and individuals that we studied. We extractedtotal DNA from a variety of sources, including tissue, whole blood, and

308 Wang et al.

Table I. List of Species, Specific Localities, and Accession Numbers for All Individualsfrom Which the mtDNA Region Including the ND3, ND4L, ND4, tRNA^*, tRNAHis,

tRNASer, and tRNAUu Genes Was Amplified and Sequenced

Species

Rhinopithecus bietiRhinopithecus bietiRhinopithecus bietiRhinopithecus bietiRhinopithecus bietiRhinopithecus bietiRhinopithecus roxellanaRhinopithecus roxellanaRhinopithecus roxellanaTrachypithecus francoisiTrachypithecus francoisiTrachypithecus francoisiTrachypithecus francoisiTrachypithecus phayreiTrachypithecus phayreiTrachypithecus leucocephalusTrachypithecus leucocephalusTrachypithecus leucocephalusPygathrix nemaeusPygathrix nemaeusColobus guereza

Individualcode

DJ1DJ3DJ4DJ5DJ6DJ7CJ1CJ2CJ3HY1HY2HY3HY4FY1FY2BTY1BTY2BTY3BTUY1BTUY2Cg

Collection locality

Weixi, Yunnan, ChinaWeixi, Yunnan, ChinaWeixi, Yunnan, ChinaWeixi, Yunnan, ChinaWeixi, Yunnan, ChinaWeixi, Yunnan, ChinaShengnongjia, Hubei, ChinaAnxian, Sichuang, ChinaAnxian, Sichuang, ChinaGuangxi, ChinaGuangxi, ChinaGuangxi, ChinaGuangxi, ChinaXishhuangbanna, Yunnan, ChinaHekou, Yunnan, ChinaGuangxi, ChinaGuangxi, ChinaGuangxi, ChinaVietnamVietnamKenya, Africa

GENBANKaccession

No.

U92951U92956U92957U92958U92959U92960U92961U92962U92963U92952U92953U92954U92955U92969U92970U92966U92967U92968U92964U92965U92950

lymphocyte cell lines transformed by EB virus, following a protocol modi-fied by W. Wang et al. (1995) from Sambrook et al. (1989). All biologicalmaterials except those from Colobus guereza are from the repository of theWild Animal Cell Bank in the Kunming Institute of Zoology, China.

PCR and Sequencing

We amplified the mitochondrial DNA fragment between tRNAG'y andtRNA1-6", which encompasses the ND3, ND4L, and ND4 genes as well asthe tRNA^s, tRNAHis, tRNASer, and tRNAUu genes, for the individualslisted in Table I (except Colobus guereza) at the Laboratory of Cellular andMolecular Evolution, Kunming Institute of Zoology, China. The amplifi-cation primers are GLYLF-5' ACT TCC AAT TAG CTA GTT T 3' andMLEU-5' TGG TGC AAC TCC AAA TAA AAG TA 3'-for all samplesof Chinese leaf monkeys. We used the primer PIB—5' TAC TGA CAC

A Phylogeny of Chinese Leaf Monkeys 309

TTT GTA GAY GTT GTC TG 3' and Mleu to amplify the target fragmentin Pygathrix nemaeus and Colobus guereza (Fig. 1). We successfully obtainedamplifications via the following protocol: 35 cycles of denaturing at 94°Cfor 1 min, annealing at 50°C for 1 min, and extension for 3 min and 30sec at 72°C on a Robocycler Gradient 40 Temperature Cycler (Stratagene).We purified PCR products amplified in China by a low-melting pointagarose gel method (Sambrook et al., 1989) and then brought them to theCenter for Environmental Research and Conservation (CERC), ColumbiaUniversity, USA, to be sequenced. We cleaned the fragment amplified fromColobus guereza by a Qiaquick PCR purification kit (Qiagen, No. 28106)using the protocol provided by the manufacturer, modified by final elutionwith sterile distilled water after incubation at 55°C. We designed eight in-ternal sequencing primers (Table II, Fig. 1) and conducted cycle sequencingon a Perkin Elmer Model 480 thermal cycler with FS-DNA sequencing kit(Perkin Elmer, No. 402079) following the supplied protocol modified byhalving all reactions (total reaction volume = 10 \il). We cleaned sequenc-ing products of excess dyes with CentriSep Spin Columns (PrincetonSeparations, No. CS-901) and then electrophoresed them on a 4.25%polyacrylamide gel (19:1 Acryl/Bis gel stock, AMRESCO, No. 0496-500)and scored them on a 377 PRISM automated sequencer (Perkin Elmer).We aligned sequences by eye and via Sequence Navigator and AutoAs-

Fig. 1. The segment of the mitochondrial genome that we sequenced for the taxa listed inTable I. The region corresponds to the positions 10,061-12315 on the human mtDNA map(Anderson et al., 1981). Relative primer positions (Table II) and orientations are indicatedby arrows. The map is approximately to scale.

310 Wang el al.

Table II. Names and Sequences of the Eight Internal Sequencing Primers Utilized in thisStudy.

Primer name

ARGREV2ND4LMND4SREV2ND4#1NEWND4MNAP2MLEAF1FORMREV

Primer sequence

5' TAG ATT ART ATG CCT AGO AGT G3'5' CTA ATA TGC YTA GAA GGA ATA AT 3'5' AAG AAT TAT TTT TAG CAT TG 3'5' CTT CTA ACA CTR ACC GCC TGA CT 3'5' A ATA CCC CTA TAT GGY CTA CAC CTA TG 3'5' GG AGC TTC AAC GTG GGC TTT 3'5' CCC TGA AGC TTY ACT GGC GCT AT 3'5' CT TCA RAA GGC TAT TAG TGG 3'

Positionon human

mtDNA map

H 10536L10559H 10755L10954L11379H11429L11677H11837

sembler software (Applied Biosystem Inc., Perkin Elmer, 1994) against thehomologous region of a human mtDNA sequence (Anderson et al., 1981).We proofed each segment of sequence through each nucleotide positionat least three times.

Data Analysis

In order to examine the phylogenetic information content of the finalsequence data set, we generated tree distributional skewness (Hillis andHuelsenbeck, 1992) of 10,000 random trees via PAUP 3.1.1 (Swofford,1991). We determined substitution events using MacClade (Maddison andMaddison, 1992). We obtained phylogenetic trees by branch and bound orheuristic searches of 3000 bootstrap replications under maximum-parsi-mony criteria via the PAUP 3.1.1 package (Swofford, 1991) based on thewhole data set and for ND3, ND4L, and ND4 individually and the tRNAgenes as a group. We also constructed neighbor-joining (NJ) trees with thePHYLIP 3.57c package (Felsenstein, 1995), based on a distance matrix gen-erated from the entire sequence data under the Kimura 2-parameter model(Kimura, 1981). Homo sapiens was the outgroup in all tree searches, andall analyses included the more proximal outgroup, Colobus guereza.

RESULTS AND DISCUSSION

Sequence Variation

We deposited the sequence data for the sample taxa in GENBANK;the GENBANK accession numbers for these taxa are in Table I. Because

A Phytogeny of Chinese Leaf Monkeys 311

only a small part of the tRNA01? gene sequence was obtained, we excludedit from our analyses. We compiled transition-to-transversion ratios andsummary characteristics for individual genes and the combined data set viaMacClade software (Maddison and Maddison, 1992) (Table III). The ND3and ND4L genes possess the highest (7.6:1) and lowest (3:1) transition-to-transversion ratios, respectively, implying that ND3 is a more conservativegene, while ND4L has evolved at a relatively more rapid rate in colobines.The ratio along the whole segment sequenced across all taxa is 5:1, whichthe ND4 and tRNA genes very closely approximate. The proportions ofinformative substitutions are also summarized in Table III. The lowest pro-portion of informative characters is in the genes of tRNA, a result of boththeir highly conserved nature and the constraints on secondary structurein these sequences (Brown, 1983; Kumazawa and Nishida, 1993).

Phylogenetic Resolution at the Generic Level

The distribution of 10,000 random trees is left skewed (gl = -0.4511,P < 0.01), showing that the data set is significantly structured and contains

Table III. Summary of Variations Along the Sequences Across Taxa"

Genes

Total charactersConstant charactersUninformative charactersInformative characters% informative No. charactersNo. of transitions (TS)No. of transversions (TV)Ratio of TS/TVA->GG-»AC-»TT->CA-»TA->CC->AC->GT-»AT-»GG-»TG->C

ND3

35919944

10830.8106

147.6:1

253

563231602210

ND4L

2911723683

28.57525

3.0:1156

322344565100

tRNAs

2371623738

16.04911

4.5:1162

111312311100

ND4

137778921437427.2394

775.1:1

11314

1671202317132011242

Total

2252131833160326.8624127

4.9:116925

2661883124272719652

"All gaps have been excluded from analyses. The genes of tRNA*18, tRNAHis, tRNASer,and tRNA^" are combined. The numbers of unambiguous changes between basesare generated using MacClade 3.0 (Maddison and Maddison, 1992) based on theweighted parsimony tree (Fig. 2B).

312 Wang et al.

a strong phylogenetic signal (Hillis and Huelsenbeck, 1992). We con-structed parsimonious trees (not shown) by branch and bound searches thatequally weighted characters based on the entire data set as well as theindividual genes (the four tRNA genes were combined together in a singleanalysis). The trees from the total data and the ND4 gene are topologicallyidentical and quite similar to that shown in Fig. 2B, except that the mo-nophyly of the two individual T. phayrei collapse. However, the trees basedon the ND3 and ND4L genes fail to resolve intergeneric relationships re-sulting in a four-way polychotomy among the genera. This failure may bea result of both the short length of the ND3 and ND4L genes and theirrelatively extreme evolutionary rates: rapid or slow. Although the tRNAgene tree resolved most of the taxa, bootstrap support for most of thebranches is very low.

In addition to the unweighted analyses, we performed a heuristic searchon the entire data set that weighted mutations resulting in transversions fivetimes more than mutations resulting in transitions. This analysis results in12 most parsimonious trees, each with a length of 2912. The strict consensusfor these 12 trees is presented in Fig. 2B, including bootstrap support per-centages resulting from 3000 replications. This consensus tree is similar tothe unweighted tree (not shown), differing only in resolving the monophylyof the two individual T. phayrei. We also constructed a NJ tree (Fig. 2A)based on a Kimura two-parameter distance matrix (Table III) with thePHYLIP 3.57c package (Felsenstein, 1995). The NJ tree (Fig. 2A) displaysthe same topology as the weighted parsimony tree (Fig. 2B).

Both of the trees in Fig. 2 reveal that Colobus is the sister lineage tothe Asian colobines and that Rhinopithecus is closer to Pygathrix than toTrachypithecus. This support for a close relationship between Rhinopithecusand Pygathrix, also suggested by most morphological studies (Groves, 1970;Brandon-Jones, 1984; Napier, 1985; Peng, et al., 1988, 1993; Oates et al.,1994), is in conflict with the results of ribosomal DNA (rDNA) restrictionmapping (Wang et al., 1996), which imply that Rhinopithecus is closer toTrachypithecus than to Pygathrix. The disparity between our finding and therDNA mapping results may be ascribed either to homoplasy or a lack ofinformative sites in the rDNA sequence (Wang et al., 1996).

Although it is difficult to interpret the generic status of Rhinopithecusbased solely on the trees in Fig. 2, the long branch length on the NJ phylo-gram and the large genetic distance between Pygathrix and Rhinopithecus(about 15% on average) suggest at least that these two taxa have been in-dependent evolutionary entities for a considerable period of time. However,it is not always appropriate to assume that genetic distances and time ofdivergence have a linear relationship (Melnick and Hoelzer, 1993). While welean toward classifying Pygathrix and Rhinopithecus as separate genera, the

A Phylogeny of Chinese Leaf Monkeys 313

Fig. 2. The neighbor-joining tree (A) constructed from Kimura two-parameter distancematrix (Table IV) and the strict consensus parsimony tree (B) obtained by 3000 heu-ristic bootstrap searches of the raw sequence data weighting transversions over transi-tions 5:1 for the taxa listed in Table I. The bootstrap supporting percentages are shownabove the branches of the parsimony tree.

final resolution of this issue will require further systematic examination, in-cluding molecular, morphological, and possibly paleontological data. A morecomplete phylogeny, which will eventually contain at least one representativeof each Asian leaf monkey (species and genus) is now being constructed atthe CERC laboratory at Columbia University. This ongoing revision of thegroup as a whole will provide a more complete answer to questions regardingthe relationships among these higher level taxonomic groups.

Phylogenetic Resolution at the Specific Level

At the specific level our data provide additional information on thesystematics of Chinese leaf monkeys. Ellerman and Morrison-Scott (1951,1966) recognized Rhinopithecus bieti and R. brelichi as subspecies of R.roxellana. Groves (1970) placed R. brelichi as a independent species butretained R. bieti as a subspecies of R. roxellana. Peng et al. (1988) recog-nized all three Chinese snub-nosed monkeys as valid species. Recentevidence from cytochrome b sequencing (Zhang et al., 1996) and ribosomalDNA restriction mapping (Wang et al., 1996) also supports the designationof R. bieti and R. roxellana as full species. In the phylogenetic trees pre-sented here, R. bieti and R. roxellana are monophyletic sister groups. The

314 Wang et al.

branch length between them is comparable to that between Trachypithecusfrancoisi and T. phayrei (Fig. 2A), which have been widely accepted as twodistinct species (Nowak, 1991). The average sequence divergence betweenR. bieti and R. roxellana is about 6.4%, while that between T. francoisi andT. phayrei is about 8.4% (Table IV). Therefore, we interpret these resultsto support R. bieti as a full species.

Y.-X. Wang et al. (1995) classified the extant populations of R. roxellanainto three subspecies, R, r. roxellana, R. r. hubeiensis, and R. r. qinlingensis.In our sample, CJ2 and CJ3 geographically belong to R. r. roxellana andCJ1 belongs to R, r. hubeiensis. Ribosomal DNA restriction mapping re-vealed one site difference between R. r. roxellana and R. r. hubeiensis (Wanget al., 1996). CJ2 (R. r. roxellana) is the genetically distinctive individual.Sequence differences between CJ2 and the other two individuals are both2.2%, or three times that between CJ1 (R. r. hubeiensis) and CJ3 (R. r. roxel-lana) (Table IV). In other words, the intrapopulational variation exceedsthat found between populations (Hoelzer et al. 1994). This result emphasizesthe need to investigate additional individuals of this endangered primatespecies to elucidate its true geographic population genetic structure.

Li and Ma (1980) designated the white-capped langur as a subspeciesof Francois's langur (T. francoisi), while Tan (1985) recognized it as a fullspecies (T. leucocephalus). Brandon-Jones (1995) concludes that, basedupon the available evidence, leucocephalus is merely an albinistic morph ofT. francoisi. Beyond simple taxonomic interest, this question has additionalurgency as the population size of the white-capped langur is estimated tobe only 400-600 individuals (Wang and Jiang, 1995), and the debate overits status and importance in the animal protection plan of the GuangxiDepartment of Forestry is increasing. Our sequence data show that all threewhite-capped langurs share a number of synapomorphic characters that dis-tinguish them from other Trachypithecus. On the other hand, the averagegenetic distance between the white-capped langur and Francois's langur ofChina is only 1.6%, which is much less than that between T. francoisi andT, phayrei (8.4%) (Table IV). Thus, it is probably appropriate to retainleucocephalus as a subspecies of T. francoisi, although it should still be con-sidered a separate evolutionarily significant unit (ESU) (Ryder, 1986;Moritz, 1994) worthy of conservation.

The Yunnan snub-nosed monkey (R. bieti) is one of the most endan-gered species of mammals. The IUCN classifies it as endangered, andEudey (1987) ranked it second among the four primate species in Asiawith the highest priority for conservation action. There are only about1000-1500 individuals left in the wild (Long, personal communication).There are only 18 captive Yunnan snub-nosed monkeys in the world, 10of which are at the Kunming Institute of Zoology (KIZ). Protein electro-

A Phytogeny of Chinese Leaf Monkeys 315

316 Wang et al.

phoresis revealed extremely low genetic variation in all six founders of theKIZ population, which were captured in two field expeditions to the samelocality (Su and Shi, 1995). Five of the Yunnan snub-nosed monkeys in-volved in this study were sampled from the founders of the KIZ population;the sixth DJ7 was sampled from the wild at the same locality as the foun-ders. Our results reveal relatively low sequence variation (0.39% onaverage) among these individuals, but not strikingly different from someof the other Chinese leaf monkeys: the average interindividual distancewithin T. leucocephalus and T. francoisi is about 0.54 and 0.64%, respec-tively. Therefore, the low level of mtDNA sequence variation in R. bietimay not be the product of severe population reduction, but simply char-acteristic of most leaf monkey populations in China. Alternatively, allChinese leaf monkey populations and species may have been subjected tothe same anthropogenic processes, which in each case have diminishedpopulation size and eroded genetic variation.

A puzzling result is that the two individuals of T. phayrei representvery distinct entities. The sequence divergence between them (7.76%) isas high as those between T. francoisi and either of them: (8.62 and 8.22%,respectively (Table IV). In fact, the unweighted parsimony analyses fail toresolve the two individuals of T. phayrei beyond a basal polychotomy forTrachypithecus. The total DNA sample of each was extracted from livertissue, and we repeated PCR amplification and sequencing, which providedidentical sequences to those resolved initially. Thus, these differences arereal, and not a result of PCR contamination or sequencing artifacts. Thetwo individuals were collected from two localities (Table I) about 10 yearsago. Their skeletons are in the Kunming Institute of Zoology, China. De-spite the potential for highly divergent haplotypes within a population, e.g.,Macaca sinica (Hoelzer et al., 1994), we consider this unlikely to explainour results in the present context. Thus, the sequencing results require firsta reexamination of the identification of the two individuals. It will also benecessary to collect more leaf monkey samples from the two original lo-calities in order to examine their phylogenetic position within the clade ofTrachypithecus. Our ongoing study into the systematic relationships amongthe Asian leaf monkeys should also shed further light on the phylogeneticstatus of these two specimens ascribed to T. phayrei.

Conservation Implications

The leaf monkeys of China are in a precarious position as their habi-tats shrink and they are hunted for food and traditional medicines (Eudey,1987; Wang and Quan, 1986). To develop management plans to promote

A Phylogeny of Chinese Leaf Monkeys 317

their conservation, it is important to gain a firm understanding of the re-lationships among species and the variability within each species.Historically these data have been provided by a combination of morpho-logical and paleontological data, which ultimately form the hypotheses thatmolecular data are used to test. The use of genetic data provides a powerfultool for this purpose (Avise, 1989). In particular, the areas of populationgenetics and molecular systematics assist in the delineation of conservationunits and management units (Moritz, 1994), which along with behavioral,morphological, ecological, and biogeographic information, form the foun-dation of any successful conservation management strategy.

Our results provide preliminary information for use in the conservationof China's leaf monkeys. First, the larger taxonomic units—genera—areclearly defined and their cladistic and phenetic relationships are apparent.Second, within each genus the taxonomic status of certain species and thedegree of variation within each of them have been preliminarily deter-mined. Third, areas of taxonomic or population genetic uncertainty or bothhave been identified.

Clearly, the two taxa of Rhinopithecus represent separate conservationunits. Moreover, within Trachypithecus francoisi and T. phayrel there areseparate management units. In T. francoisi, these units correspond to pre-vious subspecific designations, representing both morphological and geneticdistinctiveness, while in T. phayrei more data are needed to determine thegeographic distribution of the distinct genetic types. Hence, from a rela-tively limited study, we defined six units worthy of separate conservationefforts. Each unit has little variation within it but consistent differencesbetween it and its closest taxonomic relatives. It is likely that as we extendthis study, other conservation or management units will emerge, providingus with a comprehensive picture of Chinese leaf monkey diversity, whichcan, in turn, be used to develop a blueprint for their future preservation.

ACKNOWLEDGMENTS

We are grateful to Profs. Y.-X. Wang, R.-Q. Liu, R.-J. Zou, W.-Z Ji,and A.-H. Liu; S. K. Davis, Drs. B. Su, J. C. Morales, and C. Lehn; Mr.F.-H. Yu, and Y.-J. Cheng; and Ms. S.-Y. Lin, C.-H. Wu, L. Rosenblum,R. Aziz, and J. Pastorini for their assistance with many aspects of the com-pleted research. We also thank L. A. Brooks, J. Marks, J. McKnight, andan anonymous reviewer for their comments on the manuscript. This workhas been supported by the Premier Fund of China for Young Scientists,the Chairman Fund of the Chinese Academy of Sciences, and grants fromthe John D. and Catherine T. MacArthur Foundation and the U.S. Na-

318 Wang et al.

tional Science Foundation to D.J.M. The MacArthur Foundation grant hasas its specific purpose the training of Southeast Asian scientists in the meth-ods of molecular conservation genetics. Wen Wang was trained under thisgrant.

REFERENCES

Anderson, S., Bankier, A. T., Barrell. B. G., de Bruijn, M. H. L., Coulson, A. R., Drouin, J.,Eperon, I. C, Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H.,Staden, R., and Young, I. G. (1981). Sequence and organization of the humanmitochondrial genome. Nature 290: 457-465.

Applied Biosystems, Inc., A Division of Perkin-Elmer Corporation (1994a). SequenceNavigator, DNA and protein sequence comparison software.

Applied Biosystems, Inc., A Division of Perkin-Elmer Corporation (1994b). AutoAssembler,DNA sequence assembly software.

Arevalo, E., Davis, S. K.., and Sites, J. W. (1994). Mitochondria! DNA sequence divergenceand phylogenetic relationships among eight chromosome races of the Sceloporusgrammicus complex (Phrynosomatidae) in central Mexico. Syst. Biol. 43: 387-418.

Avise, J. C. (1989). A role for molecular genetics in the recognition and conservation ofendangered species. Trends Ecol. Evol. 4: 279-281.

Brandon-Jones, D. (1984). Colobus and leaf monkeys. In Macdonald, D. (ed.), TheEncyclopedia of Mammals. Vol. 1, Allen and Unwin, London, pp. 398-410.

Brandon-Jones, D. (1995). A revision of the Asian pied leaf monkeys (Mammalia:Cercopithecidae: superspecies Semnopithecus auratus), with a description of a newsubspecies. Raffles Bull. Zool. 43(1): 3-43.

Brown, W. M. (1983). Evolution of mitochondrial DNA. In Nei, M., and Koehn, R. K. (eds.),Evolution of Genes and Proteins, Sinauer, Sunderland, MA, pp. 62-68.

Cracraft, J., and Helm-Bychowski, K. (1991). Parsimony and phylogenetic inference usingDNA sequences: Some methodological strategies. In Miyamoto, M. M. and Cracraft, J.(eds.), Phylogenetic Analysis of DNA Sequences, Oxford University Press, New York, pp.184-220.

Ellerman, J. R., and Morrison-Scott, T. C. S. (1951). Checklist of Palearctic and Indianmammals. British Museum (Nat. Hist.), London.

Ellmeran, J. R. and Morrison-Scott, T. C. S. (1966). Checklist of Palearctic and IndianMammals, British Museum (Nat. Hist.), London.

Eudey, A. A. (1987). Action Plan for Asian Primates Conservation: 1987-1991, Int. UnionConserv. Nat., Gland, Switzerland.

Felsenstein, J. (1995). PHYLIP (phylogeny inference package), Version 3.57c (distributed by theauthor), Department of Genetics, University of Washington, Seattle.

Forstner, M. R. J., Davis, S. K., and Arevalo, E. (1995). Support for the hypothesis ofAnguimorph ancestry for the suborder serpents from phylogenetic analysis ofmitochondrial DNA sequences. Mol. Phylogenet. Evol. 4: 93-102.

Groves, C. P. (1970). The forgotten leaf-eaters and the phylogeny of the Colobinae. In Napier,J. R., and Napier, P. H. (eds.), Old World Monkeys: Evolution, Systematics and Behavior,Academic Press, New York, pp. 555-588.

Groves, C. P. (1989). A Theory of Human and Primate Evolution, Clarendon Press, Oxford.Hillis, D. M., and Huelsenbeck, J. P. (1992). Signal, noise, and reliability in molecular

phylogenetic analyses. J. Hered. 83: 189-195.Hoelzer, G. A., Dittus, W. P. J., Ashley, M. V., and Melnick, D. J. (1994). The local

distribution of highly divergent mitochondrial DNA haplotypes in toque macaques(Macaca sinica) at Polonnaruwa, Sri Lanka. Mol. Ecol. 3: 451-458.

A Phylogeny of Chinese Leaf Monkeys 319

Jablonski, N. G., and Peng, Y. Z. (1993). The phylogenetic relationships and classification ofthe doucs and snub-nosed langurs of China and Vietnam. Folia Primatol. 60: 36-55.

Kimura, M. (1981). Estimation of evolutionary distances between homologous nucleotidesequences. Proc. Natl. Acad. Sci. USA 68: 454-458.

Kumazawa, Y,, and Nishida, M. (1993). Sequence evolution of mitochondria! tRNA genesand deep-branch animal phylogeny. J. Mol. Evol. 37: 380-398.

Li, Z.-X., and Lin, Z.-Y. (1983). Taxonomy and distribution of the Yunnan primates. Zoo/.Res. 4: 111-120.

Li, Z.-X., and Ma, S.-L. (1980). A taxonomic revision of the white-headed langur. ActaZootaxon Sinica 5: 440-442.

Li, Z.-Y. (1993). Taxonomy and geography of leaf monkeys in China. In Ye, Z.-Z. (ed.),Biology of Leaf Monkeys, Yunnan Scientific Press, Kunming, pp. 19-26.

Maddison, W. P., and Maddison, D. R. (1992). MacClade: Analysis of Phylogeny and CharacterEvolution, Version 3.0, Sinauer Associates, Sunderland, MA.

Melnick, D. J., and Hoelzer, G. A. (1993). What is mtDNA good for in the study of primateevolution? Evol. Anthropol. 2: 191-199.

Melnick, D. J., Hoelzer, G. A., and Honeycutt, R. L. (1992). Mitochondrial DNA: Its usesin Anthropological research. In Devor, E. J. (ed.), Molecular Applications in BiologicalAnthropology, Cambridge University Press, Cambridge, pp. 179-233.

Miyamoto, M, M., and Boyle, S. M. (1989). The potential importance of mitochondrial DNAsequence data to eutherian mammal phylogeny. In Fernholm, B., Brewewr, K., andJoenvall, H. (eds.), The Hierarchy of Life, Elsevier, Amsterdam, pp. 437-450.

Moritz, C. (1994). Defining " evolutionary significant units " for conservation. Trends Ecol.Evol. 9: 353-411.

Napier, P. H. (1985). Catalogue of Primates in the British Museum (Natural History) andElsewhere in the British Isles. HI. Family Cercopithecidae, Subfamily Colobinae, BritishMuseum (Natural History), London.

Nowak, R.M. (1991). Walker's Mammals of the World, 5th ed., The Johns Hopkins UniversityPress, Baltimore and London.

Oates, J. F., Davies, A. G., and Delson, E. (1994). The diversity of living colobines. In Davis,A. G. and Oates, J. F. (eds.), Colobine Monkeys: Their Ecology, Behavior and Evolution,Cambridge University Press, Cambridge, pp. 45-73.

Peng, Y.-Z., Ye, Z.-Z., and Pan, R.-L. (1988). The classification and phylogeny of snub-nosedmonkeys based on gross morphological characters. Zool Res. 9: 239-247.

Peng, Y. Z., Pan, R. L., and Jablonski, N. G. (1993). Classification and evolution of Asiancolobines. Folia Primalol. 60: 106-117.

Rosenblum, L. L., and Melnick, D. J. (1994). A comparison of mitochondrial DNA variationin Macaca nemestrina and Presbytis cristata. Am. J. Phys. Anthropol. S18: 173-174.

Ryder, O. A. (1986). Species conservation and systematics: The dilemma of subspecies. TrendsEcol. Evol. 1: 9-10.

Sambrook, J., Fritsch, E., and Maniatis, T. (1989). Molecular Cloning—A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring, Harbor, NY.

Su, B., and Shi, L.-M. (1995). Genetic diversity in the snub-nosed monkey (Rhinopithecusbieti) as estimated by protein electrophoresis. Conserv. Biol. 9: 947-951.

Swofford, D. L. (1991). PAUP: Phylogenetic Analysis Using Parsimony, Version 3.1.1, IllinoisNatural History Survey, Champaign.

Tan, B.-J. (1985). The status of primates in China. Primates Conserv. 5: 63-81.Wang, S., and Quan, G.-Q. (1986). Primates status and conservation in China. In Benirschkle,

K. (ed.), Primates. The Road to Self-Sustaining Populations, Springer-Verlag, New York,pp. 213-220.

Wang W., Lan, H., Su B., and Shi, L.-M. (1995). Analysis of random-amplified polymorphicDNA for four minorities in Yunnan province. Chinese Sci. Bull. 40: 158-163.

Wang, W., Su, B., Lan, H., Zhang Y.-P., Lin, S.-Y., Liu, A.-H., Liu, R.-Q., Ji, W.-Z., Hu,H.-G., Xie, Y.-X., and Wu, D.-H. (1996). Phylogenetic relationships among two speciesof golden monkeys and three species of leaf monkeys inferred from ribosomal DNAvariations. Folia Primatol. (in press).

320 Wang et al.

Wang, Y.-X., and Jiang, X.-L. (1995). Primate research in China: Today and the future. InXia, W.-P., and Zhang, Y.-Z. (eds.), Primate Research and Conservation, China ForestryPublishing House, Beijing, pp. 1-15.

Wang, Y.-X., Jiang, X.-L., and Li, D.-W. (1995). Classification of existing subspecies on goldensnub-nosed monkey, Rhinopithicus roxellana (Colobinae, Primates). Handbook andabstracts of XVth Congress of the International Primatological Society, 3-8 August 1994,Kuta-Bali, Indonesia, p. 277.

Wolfheim, J. H. (1983). Primates of the World, University of Washington Press, Seattle.Ye, Z.-Z. (1993). Taxonomy and distribution of leaf monkeys. In Ye, Z.-Z. (ed.), Biology of

Leaf Monkeys, Yunnan Scientific Press, Kunming, pp. 3-18.Zhang, Y.-P., and Ryder, O. A. (1996). Mitochondria! cytochrome b gene sequences of

snub-nosed langurs: Evolutionary inference and conservation relevance (submitted forpublication).