Tibetan macaques (Macaca thibetana) are a threatened primate species endemic to China. The current taxonomy of the species is based on external morphological and anatomical variations. To further understand the intraspecific variation and relationships among populations, we analyzed 44 mitochondrial DNA control region sequences (475 bp fragment) from individuals across the species range. Results revealed 11 major haplotypes with a high nucleotide diversity (0.792), but nucleotide diversity within haplotype lineages was only 0.042. Neighbor-joining phylogenetic analyses indicated support for four distinct haplotype clades corresponding to regional groups consistent with the recognized subspecies M .t. thibetana, M. t. guizhonensis, M. t. huangshanensis and M. t. pullus. As a result of regional geographic variation and genetic differences, we recommend the four subspecies should be considered different management units for conservation efforts.
Tibetan macaque (Macaca thibetana), also known as the Chinese stump-tailed macaque or Milne-Edwards' macaque, occurs in east-central China. It is found in China from 25–33° N, 102°30′-119°30′ E; the range extends south into Guangxi at 23°48′ N, about 110° E, and as far west as the Yangtze Gorge in western and northwestern Sichuan (Groves 2001). It is commonly considered as an endemic monkey species in China and occupies subtropical, deciduous and broadleaf evergreen forests, between 800 and 2500 m above sea level (Jiang et al. 1996, Wang 1998).
Its historic range has retreated in response farmland development and associated pressures resulting in drastically decreased population abundance and local extirpation (Wang 2000). The most serious threat to the Tibetan macaque comes from humans, including habitat destruction, herbicide and pesticide poisoning, human-transmitted diseases and illegal poaching. Tibetan macaques are protected by Chinese wildlife law, and listed on Appendix II of the Conventionon International Trade in Endangered Species.
The genus Macaca includes 19 different species, which have been divided into four species groups according to morphological data (Fooden 1976, 1983). Tibetan macaques are classified within the sinica group, and closely related to the Assamese macaque (M. assamensis) (Eudey 1980, Deinard & Smith 2001). M. thibetana is divided into four subspecies (M. t. thibetana, M. t. pullus, M. t. huangshanensis, and M. t. guizhouensis) based on morphological trait analysis (Jiang et al. 1996, Groves 2005) (Table 1). In order to provide effective management for this endangered species, it is necessary to understand its genetic diversity. Previous studies reported on genetic variation of M. t. huangshanensis (Liu et al. 2006) and M. t. thibetana (Li et al. 2008), however, there are no molecular data to test the genetic variation and the specific phylogenetic relationships among all the four subspecies.
To get further understanding of genetic variation and phylogeny of M. thibetana, we analysed 44 mitochondrial DNA control region sequences which represent all taxa of the macaques. Objectives in this study will be: (1) to test the genetic variation and phylogeny relations of M. thibetana, (2) to define which groups should be protected as independent management units.
The subspecies and distribution of Macaca thibetana taxonomy after Jiang et al. (1996).
Subspecies, samples origin, samples type, GenBank accession numbers (NO.).
The sampling sites and the sites information were presented in Table 2 and Fig. 1. Mt. Emei is located in the west of Sichuan province (103.3° E, 29.5° N), Tangjiahe National Nature Reserve is located in the north of Sichuan province (104.7° E, 32.5° N), Mt. Wuyi is located in the north of Fujian province (117.6° E, 27.8° N), Mt. Huangshan is located in the south of Anhui province (118.2° E, 30.5° N), and the county named Jiangkou is located in the north of Guizhou province (108.7° E, 27.6° N).
This species is almost never seen in captivity outside of Asia, and it is quite rare in zoos even there. It is also difficult to collect fecal samples in the wild. We collected feces from 39 individuals, representing individuals across their range, and 5 sequences were retrieved from GenBank. Two of the five GenBank samples belong to an M. t. thibetana population (Li et al. 2008), and the other three belong to M. t. huangshanensis population (Liu et al. 2006). The fresh fecal samples were gathered in the wild and preserved in 80% or absolute ethanol, then stored at -20°C.
Total genomic DNA was extracted from feces using the standard phenol/chloroform protocols (Zhao & Li 2005). The products were inspected on 1.2% agarose gel.
PCR reactions and sequencing
The mtDNA control region was amplified using the PCR primers LqqF (TCCTAGGGCAATCA GAAAGAAAG) and TDKD (CCTGAAGTAGGA ACCAGATG) (Li & Zhang 2004). Each mixture included at least 25ng DNA template, 5ul 10X reaction buffer, 1 pmol/L forward and reverse primer, 2 mmol/L mixed dNTPs, 3 mmol/L MgCl2, 0.25 unit of Taq polymerase, adding sterile distilled water to 50ul. Amplification was performed on a RoboCycler Gradient 40 (Stratagene) thermal cycler. The PCR procedure began with an initial 2 min denaturation at 95°C; followed by 35 cycles of 1 min denaturation at 94°C, 1 min reannealing at 60°C and 1 min extension at 72°C; a 5 min final extension at 72°C. The PCR products were tested using agarose gel electrophoresis and purified with the Gel Extraction Mini Kit (Warson Co, Shanghai, China) following the manufacturer's instructions. Purified PCR products were labeled using the BigDyeTM Terminator cycle sequencing kits (PE Biosystems, Foster City, CA) with an ABI377 DNA automatic sequencer using the same primers as used in the PCR reactions. Cycle sequencing reactions were performed following the instructions provided by the manufacturer.
44 mitochondrial DNA control region sequences were evaluated. GenBank accession numbers are reported in Table 2. Sequences were aligned using the program Clustal X (Thompson et al. 1997). Genetic diversity was estimated using haplotype diversity (Hd) and nucleotide diversity (Pi), genetic diversity and the genotype were calculated using DnaSP Version 4.10 (Rozas et al. 2003). Genetic distances were calculated via Kimura's 2-parameter method (Kimura 1980) in MEGA3.1. Phylogenetic tree was conducted via Neighbor-Joining (NJ) according to mitochondrial DNA sequences in MEGA3.1. The NJ trees were reconstructed using the Kimura's 2-parameter evolutionary models and tested with 1000 bootstrap replicates (Kumar et al. 2004). M. assamensis was the outgroup in the phylogenetic analysis. Analysis of molecular variance (AMOVA) was carried out to assess differentiation between geographical units using Arlequin 3.1.
For all individuals, 475-bp of the mitochondrial DNA control region sequences were examined. The overall base composition of the sequences were A, 30.9%; G, 14.0%; T, 26.6% and C, 28.5%. On the basis of genetic diversity analysis, the haplotype diversity within the four subspecies is lower than the total haplotype diversity (Hd = 0.792) of M. thibetana, and the nucleotide diversity (Pi = 0.042) of the Tibetan macaque is low. Both the haplotype diversity and the nucleotide diversity of M. t. thibetana (Hd = 0.576, Pi = 0.024) is higher the other three subspecies (M. t. pullus: Hd = 0.222, Pi = 0.002; M. t. guizhouensis: Hd = 0.478, Pi = 0.006; M. t. huangshanensis: Hd = 0.341, Pi = 0.004).
The calculation of genetic distances shows: (1) Mean genetic distances between four populations (0.0115–0.1367) of M. thibetana are greater than the mean genetic distances within each population (0.0053–0.0019). (2) It must be noted that the mean genetic distances within M. t. thibetana population are greater than that between any two of other three subspecies. The genetic differentiation degree (Fst) among the four subspecies is significant (P < 0.05) (Table 3). Among the 44 sequences of the M. thibetana examined, 11 haplotypes were identified. Haplotype distribution is indicated in Table 4, and the four subspecies do not share haplotypes. In these haplotypes, four were present in M. t. thibetana population (Mt. Emei: Hap1, Hap2; Tangjiahe: Hap1; Mabian: Hap3, Hap4) and three in M. t. huangshanensis population (Mt. Hangshan: Hap5, Hap6, Hap7), two haplotypes were identified in M. t. pullus population (Mt. Wuyi: Hap8, Hap9) and two in M. t. guizhouensis (Jiangkou: Hap10, Hap11). The phylogenetic trees which were constructed with the haplotypes revealed two deep branching lineages (Fig. 2). M. t. thibetana population, is a sister group to all other subspecies, which compose a separate monophyletic group.
Mitochondrial DNA variation in Tibetan macaques
Compared with other primates, the observed Hd is similar to golden monkeys (Rhinopithecus roxellana, Hd = 0.845; Li et al. 2007), and barbary macaques (M. sylvanus, Hd = 0.872; Modolo et al. 2005), whereas Pi is higher than the reported values for those species (Pi = 0.0331, 0.026, respectively). These data suggest that the overall genetic diversity in M. thibetana is not low, which is congruent with Li's (2008) result. Haplotype diversity of M. t. thibetana population is higher than each of other three subspecies. These data imply that the populations belonging to the nominate subspecies have been separated from the other three subspecies for a relatively long time. The genetic structure of the other subspecies is most likely shaped by inbreeding, genetic drift, or rapid range expansion resulting in lower genetic diversity. However, a lack of variability in the mitochondrial control region does not necessarily reflect low levels of genetic diversity in the nuclear genome (Rosel & Rojas-Bracho 1999).
Mean genetic distances between four subspecies (above the diagonal) and mean genetic distances within each subspecies (below the diagonal). Mean genetic differentiation degree between the four subspecies (Fst).
Phylogenetic relationships among Tibetan macaques
The Old World genus Macaca is one of the most successful groups of monkeys. Its representatives are distributed in both Asia and Africa, and the genus has the widest geographical range among primates after Homo (Fooden 1980). As a species of genus Macaca, Tibetan macaques (M. thibetana) are a threatened primate species endemic to China (Jiang et al. 1996). Our results presented here enable us to resolve the phylogenetic relationships within the M. thibetana. There are two clear branches of the NJ tree, one includes M. t. huangshanensis, M. t. pullus, M. t. guizhouensis, and the other is M. t. thibetana. This branching pattern may indicate geographical separation of ancestral populations along the River Yangtze. This riverine geographical barrier might contribute to the divergence between the nominate subspecies and other populations.
Implication for conservation
Tibetan macaques live in small isolated populations in east-central China (Li 1999). The IUCN Red list classifies them as near threatened/conservation dependent (IUCN 2009). Their habitat area has retreated with farmland development and their populations have decreased drastically (Wang 2000). In the light of the genetic variation among those four subspecies, we suggest defining the individual subspecies as four management units (MUs). According to the model proposed by Moritz (1994), evolutionary significant units (ESU) are designated on the basis of reciprocal monophyly at mitochondrial markers, whereas MUs are identified by significant differences in allele frequency distributions and significant divergence in mitochondrial or nuclear loci. Considering these criteria, populations with genotypes that are closely related to but not shared with other populations would be described as MUs. Additionally, molecular variance analysis consistently indicate that the four subspecies populations are considerably divergent. Furthermore, studies based on morphologic characters of the four subspecies have described significant differences (Jiang et al. 1996). Thus, we believe that these four subspecies should be conserved as different MUs.
The haplotype distribution and abbreviation.
This research was funded by the grants from National Natural Science Foundation of China (No.30270209, 30770288), Key Teacher Program of the Ministry of Education of China (No.18557), Excellent Youth Foundation of Anhui Province (No.04043070), Natural Science Foundation of Anhui Province (No.99042409), and China Scholarship Council (No.99834011). We specially thank the Tangjiahe National Nature Reserve, Sichuan province, Wuyi Moutian National Nature Reserve, Fujian province, Huangshan Monkey Center, Anhui province, Garden Forest Bureau of Emei mountain, Sichuan province, Garden Forest Bureau of Jiangkou county, Guizhou province. We also specially thank Dr. Megan Matheson and Dr. Steven Wagner in Central Washington University for comments and English revision, which greatly improved the draft.