Genetic variation in Sri Lankan leopards

Zoo Biology 10:139-146 (1991)

Genetic Variation in Sri Lankan Leopards S. Miththapala, J. Seidensticker, L.G. Phillips, K.L. Goodrowe, S.B.U. Fernando, L. Forman, and S.J. O’Brien

National Zoological Park, Smithsonian Institution, Washington, DC (S. M., J.S., L.G. P., L. F.); Deparfment of Wildlife and Range Sciences, University of Florida, Gainesville (S. M.); The National Zoological Gardens, Dehiwela, Sri Lanka (S. B. U. F.); Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland (S. J. 0.); Metropolitan Toronto Zoo, Toronto, Ontario, Canada (K. L. G.)

Electrophoretic variation of 50 gene-enzyme systems was typed in a population of 33 captive leopards (Panthera pardus) from the island of Sri Lanka. The captive leopard population was composed of several lineages: 1) wild-caught leopards of the island subspecies (P. p . kotiya), 2) captive-born animals of the same subspecies, 3) a melanistic lineage whose founders were obtained from Malaysia (P. p . delacouri), and 4) leopards of known mixed lineage and unknown status. Two loci, APRT and PGD, were polymorphic in all samples, whereas 48 loci were invariant. Percent polymorphism (P) and percent average heterozygosity (H) were calculated as 4% and 1.4%, respectively, for the wild-caught leopards; 4% and 1.2% for the captive-born kotiya leopards; and 4% and 2.0% for the melanistic lineage. The overall results revealed a detectable decrease in genetic variability compared with a previous study of captive leopards from mainland origins. The mainland leopards had three additional polymorphic loci, ADA, E S I , and HBB. Reexamination of the TF locus using a revised protocol resolved a new allele in the sample of mainland leopards but not in the Sri Lankan sample. With this new polymorphism, recalculated P and H values for the mainland sample are 10% and 3.1 %, respectively. No significant differences in polymorphic loci were observed between the leopard subspecies examined.

Key words: Panthera pardus kotiya, APRT, PGD, polymorphism

INTRODUCTION

How much genetically determined variation should be retained in the captive leopard (Pantheru pardus) population to maintain long-term viability and flexibility

Received for publication September 13, 1988; revision accepted October 3, 1990.

L.G. Phillips’ present address is Chicago Zoological Park, Brookfield, IL 60513.

L. Forman’s present address is Cellmark Diagnostics, Germantown, MD 20876.

Address reprint requests to John Seidensticker, National Zoological Park, Smithsonian Institution, Wash- ington, DC 20008.

0 1991 Wiley-Liss, Inc.

140 Miththapala et al.

to environmental changes? Should leopard subspecies be recognized in designing a long-term captive breeding program? Historically, the geographic distribution of the leopard (P. pardus) extended through most of Africa, the Middle East, Asia Minor, South and Southeast Asia, to the Amur Valley in the Asian Far East. Some 27 subspecies, 13 in Africa and 14 in Asia, have been described based on geographic distribution and phenotypic variation [Deraniyagala, 1949, 1956; Ellerman and Mor- rison-Scott, 1951; Smithers, 19711. Newman et al.’s [1985] allozyme survey of captive leopards placed them among the most polymorphic of the big cats. There are no genetic data available for any wild population of leopards, but, given the number of subspecies that have been described, it is reasonable to assume that geographically distinct wild populations will differ in the extent and character of genetic variation.

One entree into this question is to examine genetic variation in geographically isolated leopard population. In the present study, we examined allozyme variation of Sri Lankan leopards, which form one of only two extant island leopard populations; the other is on Java. (The status of leopards on Zanzibar and Kangean is uncertain [Pakenham, 1984; Helvoort et al., 19851). Leopards on Sri Lanka (P. p . katiya) [Deraniyagala, 1949, 19561 probably have been isolated from the mainland population at least since the Pleistocene-Holocene interface (10,000 years BP) when the island was last joined with the Indian subcontinent [Jacob, 19491.

Using leopards in the collection of the National Zoological Gardens of Sri Lanka (NZGSL), we conducted a survey of gene variation from electrophoretic data. In this report, we compare our findings with those of Newman et al. [1985] for the general population of captive leopards derived from mainland founders to determine if there are differences in biochemical genetic variation among captive-born mainland leopards, wild-caught island leopards, and captive-born island leopards. In addition, we wanted to determine whether there were genetic markers among the subspecies of leopards that we examined.

MATERIALS AND METHODS Leopards

The NZGSL had 41 leopards in its collection in March, 1987, when we collected our samples. From this collection we sampled 33, of which 1) seven were wild- caught leopards from Sri Lanka; 2) 15 were captive-born leopards from this subspecies; 3) five were related melanistic leopards, whose founders were P. p . delacouri that were obtained from peninsular Malaysia in the mid- 1960s; 4) two were leopards of known hybrid lineage of these two subspecies; and 5 ) four were leopards of unknown status (possibly hybrid).

Anesthesia and Tissue Collection

The leopards were initially immobilized with 4-6 mg/kg tiletimaine hydrochloride and zolazepam hydrochloride (Zoletil, Virbac) administered by blowdart. An- esthesia was maintained for a sample period of 3-4 hr with intramuscular supple- ments of Zoletil ranging from 50 to 100 mg per supplement. This period of anesthesia was required to collect serial blood samples for a concurrent hormonal study [Brown et al., 19891. Blood (30 ml) was collected from each leopard. Each leopard was weighed, measured, given a physical examination to assess health status, ear-tagged, and photographed for future identification [Miththapala et al., 19891.

Leopard Genetics 141

Electrophoretic Procedures

Using procedures described previously [Ford, 1978; O’Brien et al., 1980; New- man, 19831, freshly collected heparinized blood was separated into plasma, erythrocytes, and leukocytes and stored in liquid nitrogen containers for transport to the United States. In the laboratory, proteins were extracted from erythrocytes and leukocytes [Newman, 19831. These extracts and plasma were subjected to standard electrophoresis on starch and acrylamide gels and stained histochemically for 50 enzymes [O’Brien, 19801 using protocols described by Harris and Hopkinson [1976] and refined for felids by O’Brien [ 19801, O’Brien et al. [ 19801, and Newman [ 19831. Transferrin was reexamined in the leopards originally screened by Newman [ 19831 and Newman et al. [1985] concurrently with our sample, using a revised procedure described in O’Brien et al. [1987a].

Analysis of Electrophoretic Phenotypes

Allozyme variation was assessed by comparison with electrophoretic phenotypes that Newman et al. [1985] obtained for leopards. Initially, the frequency of polymorphic loci (P) and the percent average heterozygosity per individual (H) were calculated for the sample as a whole. Next, the computer program BIOSYS-1 [Ver- sion 1.7; Swofford, 19881 was used to calculate P and H in each lineage. Finally, percent average heterozygosities for each lineage were tested for differences using Nei’s [1987] test of significance. The standard x2 test for concordance with Hardy- Weinberg equilibrium was not done because these were zoo animals.

RESULTS

All samples were invariant at 48 loci but were polymorphic for two loci, which encode Adenosine phosphoribosyl transferase (APRT) and 6-Phosphogluconate de- hydrogenase (PGD) (Fig. 1). Allele frequencies for these two loci for separate lineages are presented in Table 1.

Newman [ 19831 and Newman et al. [ 19851 surveyed 18 leopards: some from the mainland subspecies P. p. japonensis, P. p . saxicolor, and P. p . panthera (whose founders were probably from Northern China, Iran, and North Africa, respectively), and some from unknown geographic origin housed in U.S. zoos. They reported five polymorphic loci APRT, PGD, ADA, E S I , and HBB (Table 1). Using a modified protocol for Transferrin (TF), we reexamined plasma samples from the same animals in Newman’s [1983] sample and discovered a new electrophoretic allele at this locus. Each of the 48 invariant loci from the Sri Lankan sample had mobilities identical to those resolved in the Newman [1983] and Newman et al. [1985] survey. Alleles at APRT and PGD loci specified allozymes identical in mobility to those described in other leopard subspecies [Newman, 19831. Thus the leopards in this study exhibited no fixed electrophoretic differences from African and other Asian leopards previously studied.

Percent polymorphism (P) and percent average heterozygosity (H) were 4% and 1.4% when all kotiya leopards in this study were treated as a single sample. When separate lineages were examined, P = 4% and H = 1.4% for wild-caught kotiya leopards, P = 4% and H = 1.2% for captive-born kotiya leopards, and P = 4 and H = 2.0% for the captive-born delacouri leopards. Results for leopards of mixed


+

bb ab ab ab ab aa

6-PGD Phenotype a

] Hemoglobin

+

aa ab ab ab bb

b APRT Phenotype

Fig. 1. Allozyme phenotypes of polymorphic loci in Sri Lankan leopards.

lineage (N = 2) and unknown status (N = 4) are not considered further because of extremely small sample sizes. Percent average heterozygosity was not significantly different between mainland captive and the kotiya samples nor between wild-caught/ captive-born and kotiyaldelacouri samples (Table 2).

DISCUSSION

This study revealed a detectable decrease in percent polymorphism of P. pardus kotiya compared with that found in the general population of captive leopards derived from mainland founders [Newman et al., 198.51 (Table 1). Loci polymorphic in captive mainland leopards-Adenosine deaminase (ADA), Esterase-a (ESU-a), He- moglobin (Hb), and Transferrin (TF)-were invariant in all 33 leopards screened at the NZGSL (Table 1).

The captive-born delacouri lineage had been inbred to maintain melanism. The level of polymorphism in this inbred lineage was remarkably similar to that found in both wild-caught and captive-born kotiya leopards. There are no data from wild- caught delacouri leopards for comparison.

The level of variation in the kotiya leopards ranks low in comparison with other big cats (Table 3). The revised values of percentage polymorphism (P) and average heterozygosity (H) for mainland captive leopards in Newman’s [ 19831 study place them with tigers (Panthera tigris), among the most polymorphic of big cats. The degree of polymorphism in the kotiya leopards was comparable to free-ranging lions


TABLE 1. Comparison of polymorphic loci in leopards

Allele frequency Newman et al. [I9851 sum of This study

japonensis, Wild- Captive- Captive- saxicolor., All caught born born punrheru kotiya kotiya kotiyu delucouri

Locus (N = 18) (N = 22) (N = 7) (N = 15) (N = 5 )

ADA a = 0.65 b = 0.32 c = 0.03

APRT a = 0.75 b = 0.25

ESI a = 0.83 b = 0.17

HBB a = 0.97 b = 0.03

PGD a = 0.81 b = 0.19

TF a = 0.85 b = 0.15

a = 1.0 b = 0.0 c = 0.0 a = 0.88 b = 0.12 a = 1.0 b = 0.0 a = 1.0 b = 0.0

a = 0.56 b = 0.44 a = 1.0 b = 0.0

a = 1.0 b = 0.0 c = 0.0 a = 0.93 b = 0.07 a = 1.0 b = 0.0 a = 1.0 b = 0.0

a = 0.5 b = 0.5 a = 1.0 b = 0.0

a = 1.0 b = 0.0 c = 0.0 a = 0.97 b = 0.03 a = 1.0 b = 0.0 a = 1.0 b = 0.0

a = 0.47 b = 0.53 a = 1.0 b = 0.0

a = 1.0 b = 0.0 c = 0.0 a = 0.40 b = 0.60 a = 1.0 b = 0.0

a = 1.0 b = 0.0 a = 0.70 b = 0.30 a = 1.0 b = 0.0

TABLE 2. Comparison of levels of polymorphism in captive mainland and Sri Lankan leopards (sample sizes in parentheses)

Percent Percent average Study polymorphism heterozygosity

Captive mainland”

Wild-caught kotiyub

Captive-born kotiyab

Captive-born delacourib

(18) 10 3.1‘

(7) 4 1.4‘

(15) 4 1.2‘

( 5 ) 4 2.0‘

“Newman et al. [ 19851, revised in this study. bThis study. ‘Values are not significantly different from each other (Nei’s [1987] test of significance).

(Panthera leo) from the Ngorongoro Crater. Located near the Serengeti, the Ngor- ongoro lion population is virtually isolated geographically, with only about 36% of the variation observed in Serengeti lions [O’Brien et al., 1987bl. The reduction observed in the Ngorongoro population was attributed to a demographic bottleneck caused by a Stomoxys fly epizootic. The decrease in polymorphism between the leopards in Newman’s [I9831 study and the Sri Lankan leopards is about 40% and parallels the reduction observed in the isolated lion population. The loci that we found polymorphic in this study were a subset of the variable loci in Newman’s study (Tables 1 and 2), again paralleling the profile observed in the Ngorongoro and Serengeti lions [O’Brien et al., 1987bl. Our study also presents further evidence that


TABLE 3. Genetic variation in large felids (based on approximately 50 allozyme loci run in the same laboratorv)

Locale No. of P Species (No. of animals) loci (%I African lion

Panthera leo leo

Tiger Panthera tigris

Leopard Panthera pardus

Atlas lion

African lion

African lion

Panthera leo leo

Panthera leo leo

Panthera leo leo

Clouded loepard

African lion Neofelis nebulosa

Panthera leo leo

Cheetah Acinonyx jubatus raineyi

Leopard Panthera pardus delacouri

Leopard Panthera pardus kotiyu

Leopard Panthera pardus kotiya

Cheetah Acinonyx jubatus jubatus

Asian lion Panthera leo persica

Serengeti, free-ranging (26)

Captive- mixed

Mainland, captive-mixed (18)

Captive (9)

Captive (9)

Kruger, free-ranging

(40)

(15) Captive

(20) Ngorongoro,

free-ranging (17)

East African, captive and wild-caught

Captive-born, Malaysian founders (5)

Sri Lankan, wild-caught (7)

captive-born (15)

South African, captive and wild-caught (98)

captive and wild-caught (28)

(30)

Sri Lankan,

Gir Forest,

46

50

50

50

50

50

49

46

49

50

50

50

52

46

1 1

10

10

7

1

7

6

4

4

4

4

2

2

0

H (%I

3.8"

3 . 9

3.1b

3.1"

2.9"

2.3"

2.3b

1.5"

1.4'

2.0d

1.4d

1.2d

0.04"

0.00"

aO'Brien et al. [1987b]. bNewman et al. [1985]. "O'Brien et al. [1987c]. dThis study.


island populations are usually less variable genetically than mainland conspecifics [Selander and Johnson, 1973; Nevo, 19781.

APRT was one of the loci polymorphic in both the Newman et al. [1985] study and our study (Table 1). In both the Newman et al. sample and the delacouri leopards, both alleles that are present are well represented. However, in the kotiya leopards (both wild-caught and captive-born), the frequency of the commoner allele is much higher (Table 1). Both the loss of allele variation and this shift in allele frequency suggest a founder event in the history of the Sri Lankan subspecies. We assume that this founder event occurred when Sri Lanka last separated from India by rising sea levels about 10,000 years BP [Jacob, 19491 and not by leopards swimming across the Palk Strait, which today is about 24 km wide and 10 m deep between Dhanuskodi, India, and Talaimannar, Sri Lanka. Tigers (Panthers tigris) do swim across wide tidal rivers ( 5 km) in the Sundarbans at the mouth of the Ganges [Hendrichs, 19751, but open-ocean swimming has not been observed in leopards. Other biogeographic anal- yses of Southeast Asian mammals have shown that 5-25 km-wide sea channels during the Pleistocene were substantial barriers to colonization by nonvolant mammals [Heaney, 19861.

Genetic markers distinguishing delacouri and kotiya subspecies were not observed in this study, although differences in polymorphic loci have been observed that distinguish other subspecies of big cats. Newman et al. [1985] reported allelic differences between tiger subspecies P. t. altaica and P. t. tigris, and O’Brien et al. [ 1987al demonstrated biochemical differences between Asian lions (P. 1. persica) and African lions (P. 1. Zeo).

Our results emphasize that the maintenance of genetic variation within the captive lineage of P. p . kotiya requires careful attention to founder contribution and avoidance of inbreeding [Shoemaker, 19821. The results of this survey do not provide sufficient information to make management decisions concerning the maintenance of the various subspecies in captive leopard breeding programs. This can be done only when samples from across the loepard’s geographic range become available for analysis.

ACKNOWLEDGMENTS

This project was undertaken with the financial support of the Smithsonian International Exchange Program; the National Zoological Park; the National Zoolog- ical Gardens of Sri Lanka; the Laboratory of Viral Carcinogenesis, National Cancer Institute; and the School of Forest Resources and Conservation, University of Florida. The Smithsonian Research Opportunities Fund, Smithsonian Special Foreign Cur- rency Program, and Friends of the National Zoo provided additional financial support. The Bombay Natural History Society provided essential logistic support. We are grateful to J. Ballou, B. Beck, F. Berkowitz, J. Block, S. Boyle, M. Bush, J.C. Daniel, J.F. Eisenberg, E. Gould, B. Kalashantha, S. Lumpkin, J.S. Martenson, A.M.V.R. Manatunga, J. McConville, M.M. Miyamoto, L.M.H. Molligoda and the animal keepers at the NZGSL, M. Robinson, R. Rudran, A, Shoemaker, M. Sun- quist, and C. Wemmer for all their assistance in this effort. Tissues were collected in full compliance with specific Federal Fish and Wildlife permits (CITES and Endan- gered and Threatened Species) issued to the National Zoological Park, Smithsonian Institution, principal officer Michael H. Robinson, Director.


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Genetic variation in Sri Lankan leopards