Genetic Diversity and Population History of the Red Panda (Ailurus fulgens) as Inferred from Mitochondrial DNA Sequence Variations
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Genetic Diversity and Population History of the Red Panda (Ailurus
fulgens) 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 interest
in 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 polymorphic
sites 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 consensus
phylogenetic tree of the 25 haplotypes was starlike. The pairwise mismatch distribution fitted into a pattern of
populations 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 in
the Sichuan population was both larger and more stable than that in the Yunnan population, implying a southward
expansion from Sichuan to Yunnan.
Introduction
The red panda (Ailurus fulgens) (also known as the received sufficient attention in population genetic stud-
lesser panda) is one of the earth’s living fossils. Its an- ies, partly due to the difficulty in obtaining large sam-
cestor can be traced back to tens of millions of years ago ples for such studies, a difficulty which is also common
with a wide distribution across Eurasia (Mayr 1986). Fos- for many other endangered species. Here, we report the
sils of the red panda have been unearthed from China in first study of mitochondrial DNA sequence variations in
the east to Britain in the west (Hu 1990a). However, due a large sample of red pandas.
to recent environmental destruction, the red panda is be-
coming an endangered species and has drawn a lot of Materials and Methods
attention in the conservation efforts, being rated as one DNA Samples
of the flagship species (Hu 1990a; Wei and Hu 1992;
IUCN red list of threatened animals, 1996: http:// A total of 74 samples were collected, including
www.wcmc.org.UK/species/animals/animalpredlist.html). blood samples (16), hair samples (16), and dried leather
The red panda lives in the bamboo forests of the Hima- samples (42). Due to degradation, DNA extractions were
layan and Heng-Duan Mountains. Its current habitat ex- successful for only 21 of the 42 dried leather samples
tends through Nepal, Bhutan, Myanmar, and Southwest- (table 1). Therefore, the total number of DNA samples
ern China (Tibet, Yunnan, and Sichuan provinces), over- was reduced to 53. Both of the two subspecies were
lapping with the distribution of the giant panda (Gao included, with five of them being Ailurus fulgens fulgens
1987). Molecular phylogenetic studies showed that as an and the others being Ailurus fulgens styani (table 1). The
ancient species in the order Carnivora, the red panda is blood and hair samples were obtained from the Chong-
relatively close to the American raccoon (family Pro- qing Zoo and Chengdu Zoos of China, and their wild
cyonidae) and may be either a monotypic family or a origins were known. Blood samples were anticoagulated
subfamily within the procynonid (Mayr 1986; Zhang and with heparin and stored at 2708C before DNA extrac-
Ryder 1993; Slattery and O’Brien 1995). tion. The hair samples were collected by plucking and
Genetic variation in a sample is informative in stored at 2708C. The dried leather samples were ob-
studying population DNA history. Patterns of mismatch tained from collections of the Kunming Institute of Zo-
distribution and phylogenetic analyses among genes ology, Chinese Academy of Sciences, and stored at
have been utilized to delineate population processes 2708C after sampling. The 53 red pandas were origi-
(Slatkin and Hudson 1991; Rogers and Harpending nally from 8 different geographic locations in the Sich-
1992; Nee et al. 1994; Moritz 1995; Glenn, Stephan, uan and Yunnan provinces of China (fig. 1). Although
and Braun 1999). In addition, several methods were also efforts were made to avoid sampling related individuals,
developed to estimate population parameters and to test the relationships among animals in the sample were gen-
biological hypotheses (Watterson 1975; Tajima 1983, erally unknown.
1989; Fu and Li 1993; Fu 1994, 1996, 1997). Compared
with its relative the giant panda, the red panda has not DNA Extraction, Polymerase Chain Reaction, and
Sequencing
DNA extractions from blood samples follow the
Key words: red panda, mitochondrial DNA, D-loop, sequence di-
versity, neutrality test, population expansion. standard phenol-chloroform method. The fresh hair and
dried leather samples were first treated with proteinase
Address for correspondence and reprints: Bing Su, Human Ge-
netics Center, University of Texas–Houston, 6901 Bertner Avenue, K at 568C for 2 h and then incubated with 10% Chelex
Houston, Texas 77030. E-mail: bsu@sph.uth.tmc.edu. 100 (Bio-Rad) at 988C for 30 min. After centrifugation
Mol. Biol. Evol. 18(6):1070–1076. 2001 at a high speed (10,000 rpm) for 10 min, the superna-
q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 tants were collected and directly used as DNA templates
1070Population Expansion in the Red Panda 1071
Table 1
Red Pandas Sampled in this Study
Population Location Sample ID Subspecies Total
Yunnan. . . . . . . . Lu-Shui R16–R31, R62 Ailurus fulgens styani 17
Gong-Shan R56, R58, R66, R68, Rn Ailurus fulgens fulgens 5
Sichuan . . . . . . . Lei-Bo R1, R5, R9 A. f. styani 3
Mian-Ning R2, R8 A. f. styani 2
Shi-Mian R6, R7, R12–R15 A. f. styani 7
Kang-Ding R49, R50 A. f. styani 2
Mu-Li R51 A. f. styani 1
E-Bian R32 A. f. styani 1
Unknown R11, R34–R37, R39–R48 A. f. styani 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 detailed
geographic information.
for PCR (Walsh 1990). The PCR was conducted by pre- bution was generated using Arlequin, version 2.000
denaturing at 948C for 2 min, cycling at 948C for 1 min, (Schneider, Roessli, and Excoffier 2000). The essential
568C for 1 min, and 728C for 1 min for 35–40 cycles, population parameter u was estimated using Watterson’s
and a final extension at 728C for 5 min. The primer (1975) estimate, Tajima’s (1983) estimate, and Fu’s
sequences are CAC CAT CAA CAC CCA AAG CTG (1994) UPBLUE estimate. Watterson’s estimate is based
(forward) and TTC ATG GGC CCG GAG CGA G (re- on the number of segregating sites among the sequences.
verse), which amplify a 276-bp fragment located up- Tajima’s estimate is based on the calculation of the mean
stream of the mtDNA D-loop region. The PCR products number of pairwise differences of the sequences, while
were purified through low-melting-point agarose gel Fu’s UPBLUE estimate is done by incorporating the ge-
electrophoresis. Sequencing was conducted on an nealogical information of the sequences. A statistical
ABI377 automatic sequencer with both forward and re- test of neutrality was carried out using Fu’s (1997) FS
verse primers. test. Strictly speaking, all three of these estimators of u
are based on the infinite-sites model (Watterson 1975;
Phylogenetic Analysis and Statistical Tests of Tajima 1983; Fu 1997). Since the sequences generated
Neutrality in this study are from the D-loop region that has mu-
tation hot spots, the infinite-sites model is violated to
For phylogenetic analysis, parsimony (PAUP, ver- some extent. To minimize the effect of violation of the
sion 3.1.1; Swofford 1993) and median-joining network model on the estimation of u, as well as statistical tests
analyses (Bandelt, Forster, and Röhl 1999) were used. of neutrality, we inferred all the required information for
The homologous sequence of the raccoon (Procyon lo- parameter estimation and neutrality testing from the par-
tor), the closest living relative of the red panda, was simony analysis. This was done by first reconstructing
included as an outgroup. The pairwise mismatch distri- a parsimony tree from the sequences and then inferring
the required information from the tree. For example, to
infer the total number of mutations in the sample, we
counted 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 parsimony
tree as well.
Fu’s FS test of neutrality was used to infer the pop-
ulation history of the red panda. The FS value tends to
be negative when there is an excess of recent mutations,
and therefore a large negative value of FS will be taken
as evidence against the neutrality of mutations, an in-
dication of deviation caused by population growth and/
or selection.
Results and Discussion
D-Loop Sequence Variations in the Red Panda
A total of 236 bp of the sequence of the D-loop
upstream region was generated from the 53 samples,
with 22 of them from the Yunnan population and 31
from the Sichuan population. The aligned sequences are
FIG. 1.—The geographic distribution of red pandas sampled in shown in figure 2, including the homologous segment
this study. (1) Lu-shui, (2) Gong-Shan, (3) Lei-bo, (4) Mian-ning, (5) of the raccoon. There are 17 variant sites; 16 of them
Shi-mian, (6) Kang-ding, (7) Mu-li, (8) E-bian. are transitions and 1 is a transversion (fig. 2). A total of1072 Su et al.
Table 2
Mitochondrial DNA Haplotype Distribution of Red
Pandas
Haplo-
type Sample No. Count
Hap01 . . R01, R09, R10, R12, R34, R39, R44–R46 9
Hap02 . . R02 1
Hap03 . . R05 1
Hap04 . . R06 1
Hap05 . . R07, R11, R13–R15, R35, R37, R43, R48 9
Hap06 . . R08 1
Hap07 . . R16 1
Hap08 . . R17, R19, R21, R27, R28 5
Hap09 . . R18, R20, R22 3
Hap10 . . R23–R25, R29, R31 5
Hap11 . . R26 1
Hap12 . . R30 1
Hap13 . . R40 1
Hap14 . . R49 1
Hap15 . . R50 1
Hap16 . . R56 1
Hap17 . . R58 1
Hap18 . . R62 1
Hap19 . . R66 1
Hap20 . . R68 1
Hap21 . . Rn 1
Hap22 . . R36 1
Hap23 . . R42, R47 2
Hap24 . . R41, R32 2
Hap25 . . R51 1
25 haplotypes were obtained from the 53 individual se-
quences, with 13 from the Sichuan population and 12
from the Yunnan population, respectively (table 2). Con-
sidering the nonrecombinant nature and high mutation
rate of mtDNA, multiple recurrent mutations were re-
sponsible for the excessive number of haplotypes ob-
served in the red panda. Among the 25 haplotypes, 18
of 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 based
on Nei’s (1987) method.
Mismatch Distribution and Phylogenetic Analysis
The pairwise sequence difference among the 53 red
panda sequences was calculated using Arlequin, version
2.000 (Schneider, Roessli, and Excoffier 2000), and the
mismatch distribution is shown in figure 3. The pairwise
differences range from 0 to 12 substitutions. Interesting-
ly, the mismatch distribution is a better fit to a bell-like
curve of a population undergoing exponential growth
than a typical L-shaped one at equilibrium (Slatkin and
Hudson 1991; Rogers and Harpending 1992). The pair-
wise sequence differences among the 25 haplotypes and
the raccoon sequence are shown in table 3.
Furthermore, phylogenetic analysis was performed
with PAUP, version 3.1.1 (Swofford 1993). Based on the
FIG. 2.—The mitochondrial DNA D-loop sequences of the 25
haplotypes in the 53 red pandas. parsimony rule, we obtained a total of 13 equal most-
parsimonious trees (tree length 5 74, tree length among
ingroups 5 37). The strict consensus tree is shown in
figure 4a. As revealed, the consensus tree demonstrated
a very shallow phylogenetic structure among haplo-
types. The starlike phylogeny in figure 4a again indi-Population Expansion in the Red Panda 1073
Sichuan population (31 individuals) and the Yunnan
population (22 individuals). Phylogenetic analyses using
parsimony generated 25 and 160 equal most-parsimo-
nious trees for the Sichuan and Yunnan populations, re-
spectively. As explained earlier, special care was made
to reduce bias in our analysis by inferring all of the
required 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 homoplasy
on our analyses should be minimal. In addition, Fu
(1994) showed that there is little difference in u esti-
FIG. 3.—The mismatch distribution of the 53 mtDNA D-loop se- mates from different most-parsimonious trees. The re-
quences of the red panda. The data points are connected to make a sults of the u estimations and the neutrality tests are
smooth curve, indicating the bell-shaped distribution.
summarized in table 4.
Fu’s FS test of neutrality, based on 5,000 simulated
samplings, was significant at the 5% level (FS 5
cates the signature of population expansion in the red 27.573) for the total population, a strong indication of
panda (Slatkin and Hudson 1991; Moritz 1995). We also population expansion, which was already implicated by
constructed a network using the median-joining method the mismatch and phylogenetic analyses. However,
(Bandelt, Forster, and Röhl 1999). Similarly, the hap- when the Sichuan and Yunnan populations were ana-
lotypes from the Sichuan and Yunnan populations were
lyzed separately, no significant FS values were obtained.
mixed together, and no phylogenetic inference could be
The FS value of the Yunnan population was still negative
made from the network in view of either geographic
(FS 5 22.283) while that for the Sichuan population
distribution or subspecies of the red panda (fig. 4b).
was positive. Hence, the Sichuan population seems to
be relatively stable, and the Yunnan population shows a
Tests for Population Expansion
tendency for population growth (Fu 1997). We also ap-
We conducted neutrality tests in two ways. First, plied several other statistical tests, including Tajima’s
all the 53 sequences were considered as one population, (1989) and Fu and Li’s (1993) tests (results not shown).
in which a total of 13 most-parsimonious trees existed. None of them were able to reject the null hypothesis.
Second, based on the geographic information, the 53 red This was likely due to a lack of power in these tests for
pandas were separated into two subpopulations, the population expansion (Fu 1997).
Table 3
Pairwise Sequence Differences Among the 25 Haplotypes of the Red Panda and the Homologous Sequence of the
Raccoon (outgroup)
Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Hap- Rac-
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 coon
Hap01 . . . . —
Hap02 . . . . 6 —
Hap03 . . . . 7 5 —
Hap04 . . . . 7 7 6 —
Hap05 . . . . 7 1 6 6 —
Hap06 . . . . 6 4 7 7 3 —
Hap07 . . . . 2 6 5 5 7 6 —
Hap08 . . . . 1 5 6 6 6 5 1 —
Hap09 . . . . 5 7 8 6 6 3 5 4 —
Hap10 . . . . 5 3 6 8 4 1 5 4 4 —
Hap11 . . . . 5 5 8 6 4 5 5 4 4 6 —
Hap12 . . . . 5 3 6 4 2 3 5 4 4 4 2 —
Hap13 . . . . 7 5 4 6 4 5 7 6 6 6 4 4 —
Hap14 . . . . 5 5 6 6 4 3 5 4 4 4 4 2 4 —
Hap15 . . . . 8 6 1 5 5 6 6 7 7 7 7 5 3 5 —
Hap16 . . . . 6 9 8 2 8 9 5 6 6 10 6 6 8 8 7 —
Hap17 . . . . 6 4 5 9 5 2 6 5 5 1 7 5 7 5 6 11 —
Hap18 . . . . 6 4 5 7 3 4 6 5 5 5 5 3 3 1 4 9 6 —
Hap19 . . . . 6 6 7 5 5 2 6 5 1 3 5 3 5 3 6 7 4 4 —
Hap20 . . . . 7 3 6 6 2 5 7 6 6 6 4 2 4 4 5 8 7 3 5 —
Hap21 . . . . 5 5 6 6 6 7 5 4 6 6 4 4 6 6 7 6 7 7 7 6 —
Hap22 . . . . 5 2 3 9 3 4 6 5 7 3 7 5 5 5 4 11 2 4 6 5 7 —
Hap23 . . . . 1 6 7 7 7 6 2 1 5 5 3 5 5 5 8 7 6 6 6 7 5 6 —
Hap24 . . . . 7 3 4 10 4 3 7 6 6 2 8 6 6 6 5 12 1 5 5 6 8 1 7 —
Hap25 . . . . 7 7 2 4 6 5 5 6 6 6 6 4 4 4 1 6 5 5 5 6 6 5 7 6 —
Raccoon . . 39 39 37 39 40 39 39 40 40 38 39 40 39 39 38 24 37 40 28 34 30 37 38 38 37 —1074 Su et al.
of the two estimates could give some clues as to how
population size has changed over time. Since u 5 2Nm
for the mitochondrial genome, the ratio of population
size change is positively correlated with the u values
given a constant mutation rate. Table 4 shows that for
the total population, the UPBLUE estimate is about two
times as large as that of the Tajima estimate, indicating
that the population size has been at least doubled re-
cently. A similar situation was also seen in the Yunnan
population (UPBLUE u/Tajima’s u 5 1.889), but not in
the Sichuan population (UPBLUE u/Tajima’s u 5
1.105).
According to the fossil record, the red panda di-
verged from its common ancestor with bears about 40
MYA (Mayr 1986). With this divergence, by comparing
the sequence difference between the red panda and the
raccoon, the observed mutation rate for the red panda
was calculated to be on the order of 1029 for the D-loop
region, which is apparently an underestimate compared
with 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 between
the red panda and the raccoon is extremely deep.
It should be noted that population expansion may
not be the only explanation for a significant FS test (Fu
1997). Other evolutionary forces, e.g., genetic hitchhik-
ing and background selection, can also lead to similar
patterns of variation. However, we did not observe any
obvious population subdivision in the phylogenetic anal-
ysis, and we have not seen any data showing selection
pressure on the mitochondrial DNA genome of the red
panda, especially considering the noncoding nature of
the D-loop region. Furthermore, selection would likely
produce similar polymorphism patterns in the Sichuan
and Yunnan populations, which is not the case in our
observations. Therefore, the data presented in this study
suggest that population expansion is the most likely
cause of the significant FS test for the red panda.
It should also be noted that no shared haplotypes
were observed between the Sichuan and Yunnan popu-
lations. This is probably due to either the sample size
FIG. 4.—a, The starlike phylogenetic tree of the 25 mtDNA D- in this study or an implication of limited genetic diver-
loop haplotypes in the red panda. This is the strict-consensus tree of gence between these two populations, even though it
the 13 most-parsimonious trees constructed (PAUP, version 3.1.1; was not observed in the phylogenetic analysis. The
Swofford 1993). b, The median-joining network of the red panda hap- Yangtze River, the second largest river in China, lining
lotypes. The solid circles represent the haplotypes from the Sichuan
population, while the empty circles represent those from the Yunnan between the Sichuan and Yunnan provinces could serve
population. Due to data missing in several samples at site 71 (see fig. as a natural barrier in recent history (fig. 1). However,
2), this site was not included in the network analysis, which resulted how complete the separation could be is unclear. Ac-
in the pooling of Hap01 and Hap08. The haplotypes are connected by cording to the FS tests shown above, the effective pop-
line segments proportional to the number of substitutions between hap-
lotypes. The sizes of the circles are proportional to the haplotype ulation size of the Sichuan population is larger and more
frequencies. stable than that of the Yunnan population. Therefore,
historically, Sichuan might be the homeland of the red
panda, and population growth might have led to a south-
ward expansion to Yunnan.
It is interesting to note that different estimators of It is well known that genetic diversity exists in nat-
u put different weights on mutations occurring in dif- ural populations and is considered the raw material of
ferent time periods. The UPBLUE puts heavy emphasis evolution. When a population grows rapidly, genetic
on recent mutations, thus revealing relatively recent variations will be accumulated and maintained and in
population process, while Tajima’s estimator put more the long run will be beneficial to the success of this
weights on ancient mutations, therefore reflecting an- species. It has been reported that rare and endangered
cient population events (Fu 1997). Hence, a comparison animal species usually show extremely low levels of ge-Population Expansion in the Red Panda 1075
Table 4
Summary of Estimtations of u and Neutrality Tests
Estimates and Tests Total Population Yunnan Population Sichuan Population
UPBLUE estimate (u) . . . . . . . . . . . 13.382 9.227 8.904
Variance 5 8.506 Variance 5 7.817 Variance 5 8.056
Tajima’s estimate (u) . . . . . . . . . . . . 6.212 4.883 8.056
Watterson’s estimate (u) . . . . . . . . . 8.685 6.803 7.474
Fu’s FS test. . . . . . . . . . . . . . . . . . . . 27.573 (26.92) 22.283 (24.63) 0.38 (26.20)
NOTE.—Values in parentheses indicate the 5% level of significance.
netic variation, which were interpreted as one of the FU, Y. X., and W. H. LI. 1993. Statistical tests of neutrality of
critical reasons leading to extinction (O’Brien et al. mutations. Genetics 133:693–709.
1985; Su et al. 1994; Wayne 1994). In this study, we GAO, Y. T. 1987. Mammals in China. Chinese Scientific Pub-
showed that the red panda harbors a considerable lishing, Beijing, China [in Chinese].
GLENN, T. C., W. STEPHAN, and M. J. BRAUN. 1999. Effects of
amount of genetic variation resulting from both a rela- a population bottleneck on whooping crane mitochondrial
tively large effective population size and a recent pop- DNA variation. Conserv. Biol. 13:1097–1107.
ulation expansion, although its population size has been HU, J. C. 1990a. Proceedings of studies of the red panda. Chi-
decreasing in the past several decades due to human nese Scientific Publishing, Beijing, China [in Chinese].
activity. For the conservation of this endangered species, ———. 1990b. Proceedings of biological research in the giant
our results are encouraging. With a high level of genetic panda. Sichuan Scientific Publishing, Chengdu, China [in
variation, the red panda would be more viable than its Chinese].
relative the giant panda, a well-known species with ex- LI, W. H. 1997. Molecular evolution. Sinauer, Sunderland,
tremely low genetic variation (Su et al. 1994). This com- Mass.
MAYR, E. 1986. Uncertainty in science: is the giant panda a
parison coincides with the field observation and the ex bear or a raccoon? Nature 323:769–771.
situ breeding of both endangered animals, for which the MORITZ, C. 1995. Uses of molecular phylogenies for conser-
newborn death rate is much higher for the giant panda vation. Philos. Trans. R. Soc. Lond. B Biol. Sci. 349:113–
than that for the red panda in the field, and the breeding 118.
of the red panda is much more successful than that of NEE, S., E. C. HOLMES, R. M. MAY, and P. H. HARVEY. 1994.
the giant panda (Hu 1990a, 1990b). Therefore, as long Extinction rates can be estimated from molecular phyloge-
as efforts are made to protect the natural habitats, the nies. Phil. Trans. R. Soc. Lond. B Biol. Sci. 344:77–82.
recovery of red panda populations should be expected. NEI, M. 1987. Molecular evolutionary genetics. Columbia Uni-
versity Press, New York.
O’BRIEN, S. J., M. E. ROELKE, L. MARKER, A. NEWMAN, C.
Supplementary Material A. WINKLER, D. MELTZER, L. COLLY, J. F. EVERMANN, M.
BUSH, and D. E. WILDT. 1985. A genetic basis for species
GenBank accession numbers are AF294229—
vulnerability in the cheetah. Science 227:1428–1434.
AF294253 (see fig. 2 for the sequence alignment). ROGERS, A. R., and H. HARPENDING. 1992. Population growth
makes waves in the distribution of pairwise genetic differ-
Acknowledgments ences. Mol. Biol. Evol. 9:552–569.
SCHNEIDER, S., D. ROESSLI, and L. EXCOFFIER. 2000. Arlequin:
We are grateful to Dr. David S. Woodruff for provid- a software for population genetics data analysis. Version
ing lab resources for part of the sequencing work. Dr. 2.000. Genetics and Biometry Lab, Department of Anthro-
Ya-ping Zhang provided the primer and the raccoon se- pology, University of Geneva, Geneva, Switzerland.
quences. We also thank Hongguang Hu, Menghu Wu, SLATKIN, M., and R. R. HUDSON. 1991. Pairwise comparisons
Guangxin He, Lisong Fei, and Fuwen Wei for providing of mitochondrial DNA sequences in stable and exponential
samples. This project was supported by the Yunnan Nat- growing populations. Genetics 129:555–562.
SLATTERY, J. P., and S. J. O’BRIEN. 1995. Molecular phylogeny
ural Science Foundation, the National Natural Science
of the red panda (Ailurus fulgens). J. Hered. 86:413–422.
Foundation of China, and the Chinese Academy of SU, B., L. M. SHI, G. X. HE, A. J. ZHANG, Y. F. SONG, S. L.
Sciences. ZHONG, and L. S. FEI. 1994. Genetic diversity in the giant
panda: evidence from protein electrophoresis. Chin. Sci.
LITERATURE CITED Bull. 39:1305–1309.
SWOFFORD, D. L. 1993. Phylogenetic analysis using parsimony
BANDELT, H. J., P. FORSTER, and A. RÖHL. 1999. Median-join- (PAUP). Version 3.1.1. Smithsonian Institution, Washing-
ing networks for inferring intraspecific phylogenies. Mol. ton, D.C.
Biol. Evol. 16:37–48. TAJIMA, F. 1983. Evolutionary relationship of DNA sequences
FU, Y. X. 1994. A phylogenetic estimator of effective popu- in finite populations. Genetics 105:437–460.
lation size or mutation rate. Genetics 136:685–692. ———. 1989. Statistical method for testing the neutral muta-
———. 1996. New statistical tests of neutrality of mutations. tion hypothesis by DNA polymorphism. Genetics 123:585–
Genetics 143:557–570. 595.
———. 1997. Statistical tests of neutrality of mutations against WALSH, P. S. 1990. Chelex 100 as a medium for simple ex-
population growth, hitchhiking and background selection. traction of DNA for PCR-based typing from forensic ma-
Genetics 147:915–925. terial. Biotechniques 10:506–513.1076 Su et al. WATTERSON, G. A. 1975. On the number of segregation sites. ZHANG, Y. P., and O. A. RYDER. 1993. Mitochondrial DNA Theor. Popul. Biol. 7:256–276. sequence evolution in the Arctoidea. Proc. Natl. Acad. Sci. WAYNE, R. K. 1994. Molecular genetics of endangered species. USA 90:9557–9561. Pp. 92–117 in P. J. S. OLNEY, ed. Creative conservation. Interactive management of wild and captive animals. Chap- man and Hall Press, London. WOLFGANG STEPHAN, reviewing editor WEI, F. W., and J. C. HU. 1992. Status and protection of the lesser panda in Sichuan. J. Sichuan Normal Coll. (Nat. Sci.) 13:156–160. Accepted January 31, 2001
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