Applying molecular genetic tools to the conservation and action plan for the critically endangered Far Eastern leopard (Panthera pardus orientalis).

Abstract

A role for molecular genetic approaches in conservation of endangered taxa is now commonly recognized. Because conservation genetic analyses provide essential insights on taxonomic status, recent evolutionary history and current health of endangered taxa, they are considered in nearly all conservation programs. Genetic analyses of the critically endangered Far Eastern, or Amur leopard, Panthera pardus orientalis, have been done recently to address all of these questions and develop strategies for survival of the leopard in the wild. The genetic status and implication for conservation management of the Far Eastern leopard subspecies are discussed.

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C. R. Biologies 326 (2003) S93–S97
Applying molecular genetic tools to the conservation and action
plan for the critically endangered Far Eastern leopard
(Panthera pardus orientalis)
Olga Uphyrkinaa, Stephen J. O’Brienb,∗
aLaboratory of Evolutionary Zoology and Genetics, Institute of Biology and Soil Sciences, Russian Academy of Sciences,
Vladivostok, 690022, Russia
bLaboratory of Genomic Diversity, National Cancer Institute, Frederick, MD 21702-1201, USA
Abstract
A role for molecular genetic approaches in conservation of endangered taxa is now commonly recognized. Because
conservation genetic analyses provide essential insights on taxonomic status, recent evolutionary history and current health
of endangered taxa, they are considered in nearly all conservation programs. Genetic analyses of the critically endangered Far
Eastern, or Amur leopard, Panthera pardus orientalis, have been done recently to address all of these questions and develop
strategies for survival of the leopard in the wild. The genetic status and implication for conservation management of the Far
Eastern leopard subspecies are discussed. To cite this article: O. Uphyrkina, S.J. O’Brien, C. R. Biologies 326 (2003).
 2003 Académie des sciences. Published by Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Résumé
Application des outils de génétique moléculaire à la conservation et au plan de sauvegarde d’une espèce en grave
danger, le léopard d’Extrême-Orient (Panthera pardus orientalis). Le rôle des approches de la génétique moléculaire dans
la conservation des espèces menacées d’extinction est généralement reconnu. L’analyse génétique, qui assure la compréhension
nécessaire du statut taxinomique, détermine la viabilité actuelle et prédit le destin futur des taxons menacés, est dans l’agenda
de presque tous les programmes de la conservation et la restauration. Pour répondre à ces questions et élaborer la stratégie de
la survie dans l’état naturel du léopard d’Extrême-Orient ou d’Amur (Panthera pardus orientalis), qui représente une espèce
gravement menacée d’extinction, l’analyse génétique a été effectuée. L’état génétique et la gestion ensuite dans des programmes
de la conservation et la restauration concernant des sous-espèces sont discutés. Pour citer cet article:O. Uphyrkina, S.J.
O’Brien, C. R. Biologies 326 (2003).
 2003 Académie des sciences. Published by Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords: endangered species; conservation genetics; Panthera pardus; reintroduction
Mots-clés : espèce en danger; génétique de la conservation ; Panthera pardus; réintroduction
*Corresponding author.
E-mail address: obrien@ncifcrf.gov (S.J. O’Brien).
1631-0691/$ – see front matter  2003 Académie des sciences. Published by Éditions scientifiques et médicales Elsevier SAS. All rights
reserved.
doi:10.1016/S1631-0691(03)00044-1

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O. Uphyrkina, S.J. O’Brien / C. R. Biologies 326 (2003) S93–S97
1. Introduction
The Far Eastern leopard (Panthera pardus orien-
talis) is one of nine recently recognized subspecies of
leopard [1,2], yet is almost extinct in the wild. This
subspecies, that only a hundred years ago occupied
North-eastern China, the southern part of the Russian
Far East and the whole Korean peninsula today sur-
vives in the wild as only a single population of 25–
40 animals in the southwestern part of the Primorsky
Kray, Russia [3]. Habitat loss and alteration resulting
from direct humandevelopments,poachingand illegal
markets have led to dramatic consequences. The sub-
speciesis wellrepresentedincaptivity;howeverallbut
ten of the almost 200 captive leopards descend from a
founder mixture of P. p. orientalis that also included
at least one male of another subspecies [4]. The other
ten leopards were brought recently to European zoos
from the Zoo of Pyongyang and are thought to possi-
bly represent another population of the subspecies in
the wild in North Korea, although reliable information
concerning the origin of these animals is lacking.
To prevent the immediate extinction of this leopard
in the wild, the subspecies survival and conservation
program were developed by group of international cat
specialists during their first international meeting in
Vladivostok, Russia in November 1996 [5]. The Far
Eastern leopards from the wild had never been stud-
ied genetically; and its taxonomic status has remained
unsolved after a recent genetic survey that used the
captive individuals has remained uncertain [1]. The
size of the Primorsky Kray population has been sta-
ble for the 30 years since it became isolated from
another two populations that disappeared by end of
1970s [6]. Following the commonfate of all small iso-
lated populations, the leopards must have undergonea
close inbreeding that may have resulted in inbreeding
depression as a direct consequence of genetic deple-
tion. Detailed physiological and reproductive studies
of the wild leopards have not been undertaken, partly
due to enormous difficulties in catching these remain-
ing animals in the wild, partly due to disagreement
among ecologists concerning the conservation strat-
egy and necessity of such an analysis. Meanwhile, a
decrease in litter sizes over the years (from about 2
in 1973 to 1.0 in 1997) has been noted by field biol-
ogists [6]. Therefore, for conservation and restoration
efforts it was essential to resolve the taxonomic sta-
tus of the Far Eastern leopard and its relation to other
leopard subspecies and to estimate the level of ge-
netic depletion in the remaining population. A captive
population, established in zoos and private collections
throughoutthe world, represents a back-up population
of the subspecies, but is suspected to be of hybrid ori-
gin [4]. Thus the origin of the founders also had to be
determined by genetic analysis.
2. Material and methods
The complete genetic status of the Far Eastern
leopard in the wild and captivity has become clear
through a few steps of genetic assessment. First, a
moleculargeneticsurveyofall leopardsubspecieswas
performed using 25 microsatellite feline-specific loci
and sequence comparison of two mtDNA segments
(NADH-5, 611 bp and CR, 116 bp). This analysis re-
vealedsubspecies level distinctivenessforthe wild Far
Eastern leopards [2]. The study has provided the ba-
sis for recognition of nine leopard subspecies distin-
guishedat evolutionaryandgeneticlevels,withtheFar
Eastern leopard, P. p. orientalis, being one of the nine.
Further P. p. orientalis was shown to be close genet-
ically to the North Chinese leopard, P. p. japonensis,
likely evolving from it rather recently [2,7].
As the nextstep,we haveundertakengeneticanaly-
sis of seven leopards available from the Primorsky
Kray population, 5 leopards from North Korea and 22
leopards from the captive zoo population maintained
worldwide [9].
3. Results and discussion
We found that all seven leopards from the Pri-
morsky Kray population possessed a single mitochon-
drial haplotype, designated Ori2. The North Korean
leopards had two haplotypes, Ori1 and Ori2. Twenty-
two captive leopards (21 of which were descended
from the leopard-founderwith unknownorigin – SB2,
or studbook number 2) had three haplotypes: Ori1,
Ori2, and Jap2. The third captive haplotype, Jap2, was
identical to a common haplotype found in P. p. japo-
nensis leopards [2]. A pedigree analysis of the haplo-
type Jap2 transmission in the captive population indi-
cated its origin from a single founder-female SB-89.

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O. Uphyrkina, S.J. O’Brien / C. R. Biologies 326 (2003) S93–S97
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Table 1
Microsatellite diversity in leopard (Panthera pardus) and some other cat populations
SubspeciesPolymorphism Average
heterozygosity
Average
number
alleles/
locus
2.32/2.25∗
2.20
3.12
Average
range
repeat/
locus
2.72/1.66∗
2.36
3.64
Microsatellite
variance
Relatedness,
Rxy(%)
(1) P. p. orientalis (a)
(2) P. p. orientalis (b)
(3) P. p. orientalis (c)
0.80/0.88∗
0.76
1
0.341/0.340∗
0.322
0.521
1.59/1.4∗
1.70
2.38
77.5
75.8
57.5
(4) P. p. pardus
(5) P. p. fusca
(6) P. p. kotiya
(7) P. p. japonensis
1
1
0.96
1
0.803
0.696
0.485
0.549
8.52
5.52
3.52
3.76
9.72
6.2
4.58
4.44
7.28
5.38
4.25
2.70
–
40.7
66.2
–
(8) Puma concolor coryi
(9) Panthera leo persica
(10) Panthera leo leo∗∗
(a) Seven leopards from the Primorsky Kray population, the Russian Far East; (b) five leopards from North Korea; (c) 22 leopards from the
managed zoo (captive) population; see the text.
(1)–(7) Diversity summarized across the same 25 microsatellite loci [2].
(1)∗, (8)–(10) Diversity summarized across the same 16 microsatellite loci [7].
∗∗Ngorongoro population.
0.63
0.19
1
0.240
0.087
0.567
1.88
1.37
3.37
1.19
0.62
3.13
1.49
0.25
4.36
–
–
–
By analysing 25 microsatellite loci in all three
P. p. orientalis populations in a phylogenetic context
with leopards from other subspecies, we found that
wild leopards from the Primorsky Kray population
and North Korean leopardscomprise togethera mono-
phyletic group and genetically are not distinguished
between each other [7]. This suggests that the two
groups have become separated in the very recent past,
perhaps less than a hundred years ago. The phyloge-
netic position of all captive leopards was intermediate
between wild P. p. orientalis and P. p. japonensis. Ad-
ditionally, leopards carrying a greater portion of leop-
ard SB2’s genes clustered close to P. p. japonensis and
leopards carrying a lesser portion of leopard SB2’s
genes clustered close to wild P. p. orientalis. These
data have led us to the hypothesis that the leopard
with unknownorigin, SB2, most probablybelongedto
the P. p. japonensis subspecies. Analysis of STR allele
distribution in the captive leopards allocated all alleles
to within wild P. p. orientalis, or within P. p. japonen-
sis or within both of them, confirming our hypothesis.
Thus, we clarify another very important question for
the conservation program – genetic status of the cap-
tive population. We confirmed that the population has
a mixed origin, but this mixture is due to interbreed-
ing founders of two subspecies, P. p. orientalis and its
close neighbor P. p. japonensis.
To estimate the relative genetic health of all P. p.
orientalis populations we have compared indices of
genetic variation in P. p. orientalis populations (1)
with populations of other leopard subspecies, and (2)
with populations of other cat species known to have
low genetic variation and loss of fitness. Then, we
estimated relatedness values between individuals in
P. p. orientalis populations and compared to those in
some other leopard subspecies. The Primorsky Kray
population and the North Korea group both showed
lower genetic variation than any of the other leopards
subspecies examined: there was no mtDNA genetic
variation among living animals; and all STR diversity
parameters were considerably decreased (Table 1).
A considerable portion of microsatellite alleles had
frequencies less than 0.1 or more than 0.9, suggesting
that the populations can lose additional variation in a
short periodof time due to genetic drift. Microsatellite
variation in these two groups was comparable to
variation in the genetically depauperate populations
of Florida panther and Asiatic lions that show the
severefitness cost ofgenetic depletioncaused by close
inbreeding [8,9]. The captive population of the Far
Eastern leopards, despite its origination from only 9
founders [4], revealed an appreciable level of mtDNA
and STR diversity, comparable with populations of
some other leopard subspecies (Table 1). The high

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O. Uphyrkina, S.J. O’Brien / C. R. Biologies 326 (2003) S93–S97
fecundity of the captive population may have been
explained by subspecies hybridization, the positive
outcome of which has been already shown for big cats
[10,11].
Relatedness values, estimated based on microsatel-
lite allele frequencies [12], varied in wild P. p. ori-
entalis from 60% to 90% confirming our worst ex-
pectation: the leopards have been exposed to close
inbreeding for a number of generations. The results
of this analysis have led to one of the major conclu-
sions inourstudy:thelast viablepopulationremaining
in Russia is dominated by very close relatives. Simi-
larly, leopards in North Korea (if they still exist) have
been surviving at a very small number and also must
have experienced close inbreeding. This extraordinary
low level of genetic diversity by parallels to geneti-
cally compromised populations [8,10] would raise the
prospect that physiological and reproductive abnor-
malities might be occurring in the remaining leopards.
Indeed, three kittens recently born in captivity from
North Korean founders had either bone deformity or
reproductive abnormalities [13].
In May 2001, the Far Eastern leopard second
international workshop took place in Vladivostok,
where international cat specialists and local author-
ities, based on recent ecological and genetic stud-
ies, developed further recommendations for conser-
vation of the Far Eastern leopard in the wild. It was
agreed that the remaining Primorsky Kray population
is at considerable risk due to both genetic impoverish-
mentwithassociatedinbreedingdepressionanddemo-
graphic threats of small populations. Primary conser-
vation efforts should be concentrated around creation
of additional population(s) in the wild, perhaps in ar-
eas of the leopard’s former range. A suitable source
of potential leopards for reintroduction seemed to be
leopards from the managed zoo population that due
to accidental hybridization appeared to be more ge-
netically diverse and viable than the animals from the
wild. However, because they are hybrid, some argued
that they could not be released into the wild. Accord-
ing to them, to preserve the integrity of a morpholog-
ically and genetically distinct leopard is a major goal
of the conservation program.
The Far Eastern leopard, P. p. orientalis, is a ge-
netically unique subspecies of leopard [2]. This leop-
ard is particularly distinguished among the otherwise
tropical species, since it is the onlysubspecies adapted
to a cold snowy climate. From this perspective, con-
servation efforts should strive to save the integrity of
the subspecies. However, the single remaining popu-
lation is severely threatened by both genetic and de-
mographicimpoverishment,anda rescuestrategywith
geneticaugmentation/restorationshouldbeconsidered
as a major priority. The captive population that de-
rived from gene flow of two neighboring subspecies,
P. p. orientalis and P. p. japonensis, should be taken
as an acceptable and genetically beneficial source of
leopards for potential reintroduction. Perhaps only a
hundred yeas ago the two subspecies had a common
borderin their natural habitats, and the accidental sub-
species interbreeding in the captive population would
be equal to natural gene flow between subspecies. The
captive leopards would maximize genetic diversity of
homogenized wild leopards strengthening their health
and fitness and helping to withhold the environmental
changes. A similar conservation strategy was used for
restoration from near certain extinction of the Florida
panther relict population [8,14], and results of such
conservation efforts have exceeded many expectations
[11].
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