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Mol. Biol. Evol. 18(6):1070–1076. 2001q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

Genetic Diversity and Population History of the Red Panda (Ailurusfulgens) as Inferred from Mitochondrial DNA Sequence Variations

Bing Su,*† Yunxin Fu,† Yingxiang Wang,* Li Jin,† and Ranajit Chakraborty†*Laboratory of Comparative Genomics, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China; and†Human Genetics Center, University of Texas–Houston

The red panda (Ailurus fulgens) is one of the flagship species in worldwide conservation and is of special interestin evolutionary studies due to its taxonomic uniqueness. We sequenced a 236-bp fragment of the mitochondrial D-loop region in a sample of 53 red pandas from two populations in southwestern China. Seventeen polymorphicsites were found, together with a total of 25 haplotypes, indicating a high level of genetic diversity in the red panda.However, no obvious genetic divergence was detected between the Sichuan and Yunnan populations. The consensusphylogenetic tree of the 25 haplotypes was starlike. The pairwise mismatch distribution fitted into a pattern ofpopulations undergoing expansion. Furthermore, Fu’s FS test of neutrality was significant for the total population(FS 5 27.573), which also suggests a recent population expansion. Interestingly, the effective population size inthe Sichuan population was both larger and more stable than that in the Yunnan population, implying a southwardexpansion from Sichuan to Yunnan.

Introduction

The red panda (Ailurus fulgens) (also known as thelesser panda) is one of the earth’s living fossils. Its an-cestor can be traced back to tens of millions of years agowith a wide distribution across Eurasia (Mayr 1986). Fos-sils of the red panda have been unearthed from China inthe east to Britain in the west (Hu 1990a). However, dueto recent environmental destruction, the red panda is be-coming an endangered species and has drawn a lot ofattention in the conservation efforts, being rated as oneof the flagship species (Hu 1990a; Wei and Hu 1992;IUCN red list of threatened animals, 1996: http://www.wcmc.org.UK/species/animals/animalpredlist.html).The red panda lives in the bamboo forests of the Hima-layan and Heng-Duan Mountains. Its current habitat ex-tends through Nepal, Bhutan, Myanmar, and Southwest-ern China (Tibet, Yunnan, and Sichuan provinces), over-lapping with the distribution of the giant panda (Gao1987). Molecular phylogenetic studies showed that as anancient species in the order Carnivora, the red panda isrelatively close to the American raccoon (family Pro-cyonidae) and may be either a monotypic family or asubfamily within the procynonid (Mayr 1986; Zhang andRyder 1993; Slattery and O’Brien 1995).

Genetic variation in a sample is informative instudying population DNA history. Patterns of mismatchdistribution and phylogenetic analyses among geneshave been utilized to delineate population processes(Slatkin and Hudson 1991; Rogers and Harpending1992; Nee et al. 1994; Moritz 1995; Glenn, Stephan,and Braun 1999). In addition, several methods were alsodeveloped to estimate population parameters and to testbiological hypotheses (Watterson 1975; Tajima 1983,1989; Fu and Li 1993; Fu 1994, 1996, 1997). Comparedwith its relative the giant panda, the red panda has not

Key words: red panda, mitochondrial DNA, D-loop, sequence di-versity, neutrality test, population expansion.

Address for correspondence and reprints: Bing Su, Human Ge-netics Center, University of Texas–Houston, 6901 Bertner Avenue,Houston, Texas 77030. E-mail: [email protected].

received sufficient attention in population genetic stud-ies, partly due to the difficulty in obtaining large sam-ples for such studies, a difficulty which is also commonfor many other endangered species. Here, we report thefirst study of mitochondrial DNA sequence variations ina large sample of red pandas.

Materials and MethodsDNA Samples

A total of 74 samples were collected, includingblood samples (16), hair samples (16), and dried leathersamples (42). Due to degradation, DNA extractions weresuccessful for only 21 of the 42 dried leather samples(table 1). Therefore, the total number of DNA sampleswas reduced to 53. Both of the two subspecies wereincluded, with five of them being Ailurus fulgens fulgensand the others being Ailurus fulgens styani (table 1). Theblood and hair samples were obtained from the Chong-qing Zoo and Chengdu Zoos of China, and their wildorigins were known. Blood samples were anticoagulatedwith heparin and stored at 2708C before DNA extrac-tion. The hair samples were collected by plucking andstored at 2708C. The dried leather samples were ob-tained from collections of the Kunming Institute of Zo-ology, Chinese Academy of Sciences, and stored at2708C after sampling. The 53 red pandas were origi-nally from 8 different geographic locations in the Sich-uan and Yunnan provinces of China (fig. 1). Althoughefforts were made to avoid sampling related individuals,the relationships among animals in the sample were gen-erally unknown.

DNA Extraction, Polymerase Chain Reaction, andSequencing

DNA extractions from blood samples follow thestandard phenol-chloroform method. The fresh hair anddried leather samples were first treated with proteinaseK at 568C for 2 h and then incubated with 10% Chelex100 (Bio-Rad) at 988C for 30 min. After centrifugationat a high speed (10,000 rpm) for 10 min, the superna-tants were collected and directly used as DNA templates

Population Expansion in the Red Panda 1071

Table 1Red Pandas Sampled in this Study

Population Location Sample ID Subspecies Total

Yunnan. . . . . . . .

Sichuan . . . . . . .

Lu-ShuiGong-ShanLei-BoMian-NingShi-Mian

R16–R31, R62R56, R58, R66, R68, RnR1, R5, R9R2, R8R6, R7, R12–R15

Ailurus fulgens styaniAilurus fulgens fulgensA. f. styaniA. f. styaniA. f. styani

175327

Kang-DingMu-LiE-BianUnknown

R49, R50R51R32R11, R34–R37, R39–R48

A. f. styaniA. f. styaniA. f. styaniA. f. styani

211

15

NOTE.—Refer to the map in figure 1 for geographic locations. In the Sichuan population, 15 of the samples (R11, R34–37, and R39–R48) did not have detailedgeographic information.

FIG. 1.—The geographic distribution of red pandas sampled inthis study. (1) Lu-shui, (2) Gong-Shan, (3) Lei-bo, (4) Mian-ning, (5)Shi-mian, (6) Kang-ding, (7) Mu-li, (8) E-bian.

for PCR (Walsh 1990). The PCR was conducted by pre-denaturing at 948C for 2 min, cycling at 948C for 1 min,568C for 1 min, and 728C for 1 min for 35–40 cycles,and a final extension at 728C for 5 min. The primersequences are CAC CAT CAA CAC CCA AAG CTG(forward) and TTC ATG GGC CCG GAG CGA G (re-verse), which amplify a 276-bp fragment located up-stream of the mtDNA D-loop region. The PCR productswere purified through low-melting-point agarose gelelectrophoresis. Sequencing was conducted on anABI377 automatic sequencer with both forward and re-verse primers.

Phylogenetic Analysis and Statistical Tests ofNeutrality

For phylogenetic analysis, parsimony (PAUP, ver-sion 3.1.1; Swofford 1993) and median-joining networkanalyses (Bandelt, Forster, and Rohl 1999) were used.The homologous sequence of the raccoon (Procyon lo-tor), the closest living relative of the red panda, wasincluded as an outgroup. The pairwise mismatch distri-

bution was generated using Arlequin, version 2.000(Schneider, Roessli, and Excoffier 2000). The essentialpopulation parameter u was estimated using Watterson’s(1975) estimate, Tajima’s (1983) estimate, and Fu’s(1994) UPBLUE estimate. Watterson’s estimate is basedon the number of segregating sites among the sequences.Tajima’s estimate is based on the calculation of the meannumber of pairwise differences of the sequences, whileFu’s UPBLUE estimate is done by incorporating the ge-nealogical information of the sequences. A statisticaltest of neutrality was carried out using Fu’s (1997) FS

test. Strictly speaking, all three of these estimators of uare based on the infinite-sites model (Watterson 1975;Tajima 1983; Fu 1997). Since the sequences generatedin this study are from the D-loop region that has mu-tation hot spots, the infinite-sites model is violated tosome extent. To minimize the effect of violation of themodel on the estimation of u, as well as statistical testsof neutrality, we inferred all the required information forparameter estimation and neutrality testing from the par-simony analysis. This was done by first reconstructinga parsimony tree from the sequences and then inferringthe required information from the tree. For example, toinfer the total number of mutations in the sample, wecounted the total number of steps in the parsimony tree.For each pair of sequences, the distance needed for UP-BLUE could easily be computed from the parsimonytree as well.

Fu’s FS test of neutrality was used to infer the pop-ulation history of the red panda. The FS value tends tobe negative when there is an excess of recent mutations,and therefore a large negative value of FS will be takenas evidence against the neutrality of mutations, an in-dication of deviation caused by population growth and/or selection.

Results and DiscussionD-Loop Sequence Variations in the Red Panda

A total of 236 bp of the sequence of the D-loopupstream region was generated from the 53 samples,with 22 of them from the Yunnan population and 31from the Sichuan population. The aligned sequences areshown in figure 2, including the homologous segmentof the raccoon. There are 17 variant sites; 16 of themare transitions and 1 is a transversion (fig. 2). A total of

1072 Su et al.

FIG. 2.—The mitochondrial DNA D-loop sequences of the 25haplotypes in the 53 red pandas.

Table 2Mitochondrial DNA Haplotype Distribution of RedPandas

Haplo-type Sample No. Count

Hap01 . .Hap02 . .Hap03 . .Hap04 . .Hap05 . .

R01, R09, R10, R12, R34, R39, R44–R46R02R05R06R07, R11, R13–R15, R35, R37, R43, R48

91119


R08R16R17, R19, R21, R27, R28R18, R20, R22R23–R25, R29, R31

11535


R26R30R40R49R50

11111


R56R58R62R66R68

11111


RnR36R42, R47R41, R32R51

11221

25 haplotypes were obtained from the 53 individual se-quences, with 13 from the Sichuan population and 12from the Yunnan population, respectively (table 2). Con-sidering the nonrecombinant nature and high mutationrate of mtDNA, multiple recurrent mutations were re-sponsible for the excessive number of haplotypes ob-served in the red panda. Among the 25 haplotypes, 18of them were singletons (9 in Yunnan and 9 in Sichuan),indicating a high level of recent sequence diversity.Gene diversity was estimated to be 0.93 6 0.02 basedon Nei’s (1987) method.

Mismatch Distribution and Phylogenetic Analysis

The pairwise sequence difference among the 53 redpanda sequences was calculated using Arlequin, version2.000 (Schneider, Roessli, and Excoffier 2000), and themismatch distribution is shown in figure 3. The pairwisedifferences range from 0 to 12 substitutions. Interesting-ly, the mismatch distribution is a better fit to a bell-likecurve of a population undergoing exponential growththan a typical L-shaped one at equilibrium (Slatkin andHudson 1991; Rogers and Harpending 1992). The pair-wise sequence differences among the 25 haplotypes andthe raccoon sequence are shown in table 3.

Furthermore, phylogenetic analysis was performedwith PAUP, version 3.1.1 (Swofford 1993). Based on theparsimony rule, we obtained a total of 13 equal most-parsimonious trees (tree length 5 74, tree length amongingroups 5 37). The strict consensus tree is shown infigure 4a. As revealed, the consensus tree demonstrateda very shallow phylogenetic structure among haplo-types. The starlike phylogeny in figure 4a again indi-


FIG. 3.—The mismatch distribution of the 53 mtDNA D-loop se-quences of the red panda. The data points are connected to make asmooth curve, indicating the bell-shaped distribution.

Table 3Pairwise Sequence Differences Among the 25 Haplotypes of the Red Panda and the Homologous Sequence of theRaccoon (outgroup)

Hap-01

Hap-02

Hap-03

Hap-04

Hap-05

Hap-06

Hap-07

Hap-08

Hap-09

Hap-10

Hap-11

Hap-12

Hap-13

Hap-14

Hap-15

Hap-16

Hap-17

Hap-18

Hap-19

Hap-20

Hap-21

Hap-22

Hap-23

Hap-24

Hap-25

Rac-coon

Hap01 . . . .Hap02 . . . .Hap03 . . . .Hap04 . . . .Hap05 . . . .

—6777

—571

—66

—6 —


62155

46573

75686

75668

37664

—6531

—155

—44

—4 —


55758

53556

86461

64665

42445

53536

55756

44647

44647

64647

—2447

—425

—43

—5 —


66667

94463

85576

29756

85352

92425

56667

65556

65516

101536

67554

65332

87354

85134

76465

—11978

—647

—43

—5 —

Hap21 . . . .Hap22 . . . .Hap23 . . . .Hap24 . . . .Hap25 . . . .Raccoon . .

55177

39

52637

39

63742

37

697

104

39

63746

40

74635

39

56275

39

45166

40

67566

40

63526

38

47386

39

45564

40

65564

39

65564

39

74851

38

6117

126

24

72615

37

74655

40

76655

28

65766

34

—7586

30

—615

37

—77

38

—6

38—37 —

cates the signature of population expansion in the redpanda (Slatkin and Hudson 1991; Moritz 1995). We alsoconstructed a network using the median-joining method(Bandelt, Forster, and Rohl 1999). Similarly, the hap-lotypes from the Sichuan and Yunnan populations weremixed together, and no phylogenetic inference could bemade from the network in view of either geographicdistribution or subspecies of the red panda (fig. 4b).

Tests for Population Expansion

We conducted neutrality tests in two ways. First,all the 53 sequences were considered as one population,in which a total of 13 most-parsimonious trees existed.Second, based on the geographic information, the 53 redpandas were separated into two subpopulations, the

Sichuan population (31 individuals) and the Yunnanpopulation (22 individuals). Phylogenetic analyses usingparsimony generated 25 and 160 equal most-parsimo-nious trees for the Sichuan and Yunnan populations, re-spectively. As explained earlier, special care was madeto reduce bias in our analysis by inferring all of therequired information from the parsimony analyses.Since homoplasy in the data did not seem to be severe(fig. 4b), the parsimony trees should recover most mu-tations in the sample, and the influence of homoplasyon our analyses should be minimal. In addition, Fu(1994) showed that there is little difference in u esti-mates from different most-parsimonious trees. The re-sults of the u estimations and the neutrality tests aresummarized in table 4.

Fu’s FS test of neutrality, based on 5,000 simulatedsamplings, was significant at the 5% level (FS 527.573) for the total population, a strong indication ofpopulation expansion, which was already implicated bythe mismatch and phylogenetic analyses. However,when the Sichuan and Yunnan populations were ana-lyzed separately, no significant FS values were obtained.The FS value of the Yunnan population was still negative(FS 5 22.283) while that for the Sichuan populationwas positive. Hence, the Sichuan population seems tobe relatively stable, and the Yunnan population shows atendency for population growth (Fu 1997). We also ap-plied several other statistical tests, including Tajima’s(1989) and Fu and Li’s (1993) tests (results not shown).None of them were able to reject the null hypothesis.This was likely due to a lack of power in these tests forpopulation expansion (Fu 1997).

1074 Su et al.

FIG. 4.—a, The starlike phylogenetic tree of the 25 mtDNA D-loop haplotypes in the red panda. This is the strict-consensus tree ofthe 13 most-parsimonious trees constructed (PAUP, version 3.1.1;Swofford 1993). b, The median-joining network of the red panda hap-lotypes. The solid circles represent the haplotypes from the Sichuanpopulation, while the empty circles represent those from the Yunnanpopulation. Due to data missing in several samples at site 71 (see fig.2), this site was not included in the network analysis, which resultedin the pooling of Hap01 and Hap08. The haplotypes are connected byline segments proportional to the number of substitutions between hap-lotypes. The sizes of the circles are proportional to the haplotypefrequencies.

It is interesting to note that different estimators ofu put different weights on mutations occurring in dif-ferent time periods. The UPBLUE puts heavy emphasison recent mutations, thus revealing relatively recentpopulation process, while Tajima’s estimator put moreweights on ancient mutations, therefore reflecting an-cient population events (Fu 1997). Hence, a comparison

of the two estimates could give some clues as to howpopulation size has changed over time. Since u 5 2Nmfor the mitochondrial genome, the ratio of populationsize change is positively correlated with the u valuesgiven a constant mutation rate. Table 4 shows that forthe total population, the UPBLUE estimate is about twotimes as large as that of the Tajima estimate, indicatingthat the population size has been at least doubled re-cently. A similar situation was also seen in the Yunnanpopulation (UPBLUE u/Tajima’s u 5 1.889), but not inthe Sichuan population (UPBLUE u/Tajima’s u 51.105).

According to the fossil record, the red panda di-verged from its common ancestor with bears about 40MYA (Mayr 1986). With this divergence, by comparingthe sequence difference between the red panda and theraccoon, the observed mutation rate for the red pandawas calculated to be on the order of 1029 for the D-loopregion, which is apparently an underestimate comparedwith the average rate in mammals (Li 1997). This un-derestimation is probably due to multiple recurrent mu-tations in the D-loop region, as the divergence betweenthe red panda and the raccoon is extremely deep.

It should be noted that population expansion maynot be the only explanation for a significant FS test (Fu1997). Other evolutionary forces, e.g., genetic hitchhik-ing and background selection, can also lead to similarpatterns of variation. However, we did not observe anyobvious population subdivision in the phylogenetic anal-ysis, and we have not seen any data showing selectionpressure on the mitochondrial DNA genome of the redpanda, especially considering the noncoding nature ofthe D-loop region. Furthermore, selection would likelyproduce similar polymorphism patterns in the Sichuanand Yunnan populations, which is not the case in ourobservations. Therefore, the data presented in this studysuggest that population expansion is the most likelycause of the significant FS test for the red panda.

It should also be noted that no shared haplotypeswere observed between the Sichuan and Yunnan popu-lations. This is probably due to either the sample sizein this study or an implication of limited genetic diver-gence between these two populations, even though itwas not observed in the phylogenetic analysis. TheYangtze River, the second largest river in China, liningbetween the Sichuan and Yunnan provinces could serveas a natural barrier in recent history (fig. 1). However,how complete the separation could be is unclear. Ac-cording to the FS tests shown above, the effective pop-ulation size of the Sichuan population is larger and morestable than that of the Yunnan population. Therefore,historically, Sichuan might be the homeland of the redpanda, and population growth might have led to a south-ward expansion to Yunnan.

It is well known that genetic diversity exists in nat-ural populations and is considered the raw material ofevolution. When a population grows rapidly, geneticvariations will be accumulated and maintained and inthe long run will be beneficial to the success of thisspecies. It has been reported that rare and endangeredanimal species usually show extremely low levels of ge-


Table 4Summary of Estimtations of u and Neutrality Tests

Estimates and Tests Total Population Yunnan Population Sichuan Population

UPBLUE estimate (u) . . . . . . . . . . .

Tajima’s estimate (u) . . . . . . . . . . . .Watterson’s estimate (u) . . . . . . . . .Fu’s FS test. . . . . . . . . . . . . . . . . . . .

13.382Variance 5 8.5066.2128.68527.573 (26.92)

9.227Variance 5 7.8174.8836.80322.283 (24.63)

8.904Variance 5 8.0568.0567.4740.38 (26.20)

NOTE.—Values in parentheses indicate the 5% level of significance.

netic variation, which were interpreted as one of thecritical reasons leading to extinction (O’Brien et al.1985; Su et al. 1994; Wayne 1994). In this study, weshowed that the red panda harbors a considerableamount of genetic variation resulting from both a rela-tively large effective population size and a recent pop-ulation expansion, although its population size has beendecreasing in the past several decades due to humanactivity. For the conservation of this endangered species,our results are encouraging. With a high level of geneticvariation, the red panda would be more viable than itsrelative the giant panda, a well-known species with ex-tremely low genetic variation (Su et al. 1994). This com-parison coincides with the field observation and the exsitu breeding of both endangered animals, for which thenewborn death rate is much higher for the giant pandathan that for the red panda in the field, and the breedingof the red panda is much more successful than that ofthe giant panda (Hu 1990a, 1990b). Therefore, as longas efforts are made to protect the natural habitats, therecovery of red panda populations should be expected.

Supplementary Material

GenBank accession numbers are AF294229—AF294253 (see fig. 2 for the sequence alignment).

Acknowledgments

We are grateful to Dr. David S. Woodruff for provid-ing lab resources for part of the sequencing work. Dr.Ya-ping Zhang provided the primer and the raccoon se-quences. We also thank Hongguang Hu, Menghu Wu,Guangxin He, Lisong Fei, and Fuwen Wei for providingsamples. This project was supported by the Yunnan Nat-ural Science Foundation, the National Natural ScienceFoundation of China, and the Chinese Academy ofSciences.

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Accepted January 31, 2001

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