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 molecular genetic survey of all leopard subspecies was performed using 25 microsatellite feline-specific loci and sequence comparison of two mtDNA segments (NADH-5, 611 bp and CR, 116 bp). This analysis revealed subspecies level distinctiveness for the wild Far Eastern leopards [2]. The study has provided the basis for recognition of nine leopard subspecies distinguished at evolutionary and genetic levels, with the Far Eastern leopard, P. p. orientalis, being one of the nine. Further P. p. orientalis was shown to be close genetically to the North Chinese leopard, P. p. japonensis, likely evolving from it rather recently [2,7].

As the next step, we have undertaken genetic analysis 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].

We found that all seven leopards from the Primorsky Kray population possessed a single mitochondrial 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-founder with unknown origin – 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. japonensis leopards [2]. A pedigree analysis of the haplotype Jap2 transmission in the captive population indicated its origin from a single founder-female SB-89.

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 leopards comprise together a monophyletic 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 phylogenetic position of all captive leopards was intermediate between wild P. p. orientalis and P. p. japonensis. Additionally, leopards carrying a greater portion of leopard 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 unknown origin, SB2, most probably belonged to 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. japonensis or within both of them, confirming our hypothesis. Thus, we clarify another very important question for the conservation program – genetic status of the captive population. We confirmed that the population has a mixed origin, but this mixture is due to interbreeding 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 period of 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 severe fitness cost of genetic depletion caused 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 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 microsatellite allele frequencies [12], varied in wild P. p. orientalis from 60% to 90% confirming our worst expectation: 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 conclusions in our study: the last viable population remaining in Russia is dominated by very close relatives. Similarly, 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 genetically compromised populations [8,10] would raise the prospect that physiological and reproductive abnormalities 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 authorities, based on recent ecological and genetic studies, developed further recommendations for conservation 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 impoverishment with associated inbreeding depression and demographic threats of small populations. Primary conservation efforts should be concentrated around creation of additional population(s) in the wild, perhaps in areas 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 genetically 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. According to them, to preserve the integrity of a morphologically and genetically distinct leopard is a major goal of the conservation program.

The Far Eastern leopard, P. p. orientalis, is a genetically unique subspecies of leopard [2]. This leopard is particularly distinguished among the otherwise tropical species, since it is the only subspecies adapted to a cold snowy climate. From this perspective, conservation efforts should strive to save the integrity of the subspecies. However, the single remaining population is severely threatened by both genetic and demographic impoverishment, and a rescue strategy with genetic augmentation/restoration should be considered as a major priority. The captive population that derived 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 border in their natural habitats, and the accidental subspecies 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].