AUSTRALASIAN FAUNA 103

Board of Victoria and the collective ‘mission’ of our visitors.

Pessimists (and economic rationalists) would predict further losses of staff. It would be tragic to lose those teachers because they provide a critical mass that achieves much more than a high rate of service. However, to take full advantage of the potential we must do two things: we must find out much more about our audience, particularly the non-school visitors; we must evaluate our pro- grammes and services using more than visitor numbers or revenue as our yard- stick for both the public and the schools programme. We must look to meeting objectives that relate directly to our mission.

In 1991 we can articulate more clearly

our aims and objectives and we have a greater range of strategies for meeting these objectives. This does not mean that we are more clever, more efficient or more creative than the 1970 zoo educator; our achievements through the 1990s will continue to be based on a strong founda- tion of commitment, creativity and enthu- siasm. The 1990s ‘wheel’ is not a new one, just more refined than the 1970s model.

REFERENCES HOTCHKISS, N. A. (1991): The pros and cons of live animal contact. J . Mus. Educ. q2). SHERWOOD, K. P., JR, RALLIS, S. F. & STONE, J. (1989): Effects of live animals vs preserved specimens on student learning. Zoo Biol. 8: 99-104.

Manuscript submitted 3 December 1991

Int. Zoo Yb. (1992) 31: 103-108 0 The Zoological Society of London

Application of DNA profiling to the management of enda ng ered species GRAHAM HALL, DAVID GROTH & JOHN WETHERALL School of Biomedical Sciences, Curtin University, GPO Box U1987, Perth, Western Australia 6001

It is well known that individual members of a species manifest numerous differences many of which are genetically determined. The extent of these differences reflects the degree of genetic diversity characteristic of that species or particular subpopula- tion of animals. In the final analysis all inherited differences will be accounted for in the detailed structure of the deoxyribose nucleic acid (DNA) which comprises the genetic endowment of the individual.

DNA PROFILES Over the past decade a new technique has become available which permits an objec-

tive assessment of genetic diversity within species. The biochemical analysis of DNA from hypervariable genetic loci character- ized by short repeating segments of DNA results in a distinctive pattern (Jeffreys et al., 1985a). This DNA profile consists of a series of DNA fragments of decreasing size which is usually visualized as bands on an autoradiograph (Plates 1 and 2). DNA profiles from individual animals may be compared and the extent to which they differ quantified by measuring the number of shared bands. Several para- meters related to genetic diversity of the group (such as, heterozygosity) may then be derived from this statistic. Further-

104 AUSTRALASIAN F A U N A

more, DNA profiles may be used to predict close family relationship with a high degree of certainty since the indivi- dual bands represent small fragments of genetic material which are inherited in a predictable manner.

Complex DNA profiles covering numerous genetic loci and sometimes containing up to 30 bands may be gener- ated for many different species. The pro- cedures for generating these follow from the pioneering work of Alec Jeffreys on repetitive segments of DNA, ‘minisatellite DNA’, which, because they are so highly discriminatory for identification of indivi- duals have become widely known as ‘DNA fingerprints’ (Jeffreys et al., 1985a, 1985b).

For some species it is possible to generate hypervariable DNA profiles from a single genetic locus containing only two bands. These profiles are very much easier to interpret and compare but their application is at present restricted to the relatively few species for which appro- priate DNA probes have been developed (Hanotte et al., 1991). The use of DNA profiles has revolutionized many aspects of forensic science and human genetic analysis and is now being applied with considerable success to many animal species within the broad field of conserva- tion genetics. Despite the difficulties inherent in analysing the complex multi- banded DNA profiles these remain the most effective way of objectively measuring genetic heterogeneity in animal populations and of predicting close family relationships.

Management of the genetic resources of an endangered species requires knowledge of its life-history characteristics, extent of genetic diversity within and among popu- lations, relationships between individuals and defined management goals for future populations. Individuals can then be managed so that genetic diversity is retained (Foose & Ballou, 1988) and the risk of inbreeding minimized (Ralls et al., 1988). The use of behavioural observa- tions as indicators of blood relationships

is often unreliable because of the poss- ibility of extra-pair copulations. In species lacking pair-bonding the problem is exacerbated and it is necessary to rely on genetic markers to deduce such relation- ships (Lynch, 1988). Until recently most attempts to ascertain relationships have relied on polymorphic enzyme encoding loci (Wilkinson & McCracken, 1986). However, the analysis of each locus requires a separate protocol and many loci, since they are monomorphic, provide no information. The process is therefore time-consuming. In addition is has been shown by Chakraborty et al. (1988) that individual relatedness estimates based on isozyme surveys are statistically unreliable.

The application of DNA profiling based on hypervariable genetic loci promises to revolutionize our under- standing of animal social systems and to become a useful addition to the approach currently used in behavioural ecology. The technique estimates relatedness with an unprecedented degree of accuracy and has two major advantages over conven- tional isozyme analysis. First, the DNA profile permits direct identification of a genotype rather than a phenotype and multiple loci may be examined simul- taneously. Secondly, the average number of variants, that is alleles, per genetic locus examined is much greater than for the isozyme genes. Although DNA profiling technology has generated much enthusiasm, there have been relatively few studies to date involving wild populations. However, the following examples may help to illustrate the expanding usefulness of the technique over a wide range of species and situations.

BIRDS There have been a number of applications of the technique to bird populations. Wetton et al. (1987) were among the first to use DNA profiling to study the demo- graphics of a bird species in the wild (the House sparrow Passer domesticus) in an investigation which has become a model

AUSTRALASIAN FAUNA 105

for subsequent work. Other early studies were on House sparrows (Burke & Bruford, 1987) and Dunnocks Prunella modularis (Burke et al., 1989). There have also been genetic studies on Cygnus sp by Bacon & Andersen-Harild (1987) and by Meng et al. (1990), who recognized that knowledge of genetic variability among swans may be important for their protec- tion and breeding. Meng and his co- workers hybridized DNA from blood cells of three species, Cygnus olor, C.cygnus and C . columbianus, with a human DNA probe, obtaining both individual-specific and species-specific profiles and concluded that, since the three did not share any bands within the size range 1-30 kb, that there had been rapid divergent evolution of DNA profiles among these species.

MAMMALS Determining paternity may be difficult in social species, such as the African lion Panthera leo which lives in complex social prides consisting of adult 99 and their dependent young and a coalition of dd which have entered from elsewhere. Incoming 88 kill dependent young of prior 88 but sire only one cohort per pride before being evicted (Bygott et al., 1979). Packer et al. (1991) described a novel application of DNA profiling to demonstrate the kinship structure of lion prides. Since most 99 are recruited into their mothers’ pride all are closely related, but almost all 88 gain residence in new prides; resident 38 are therefore either closely related or unrelated, but mating partners are unrelated. Paternity analysis revealed that 8 reproductive success increased as coalition size increased, as the 88 acted as non-reproductive helpers in coalitions composed of close relatives. DNA profiling allowed the authors to assess whether companions and mating partners were related. Their data revealed that the degree of band sharing could be used as an estimate of kinship demon- strating that the technique can be used to go beyond simple paternity analysis and

address other fundamental questions in

Many of the early studies of DNA profiling were limited to larger species and the technique has been difficult to apply to small species because of the inherent problem of obtaining sufficient DNA from small living animals. Hoagland et al. (199 1) successfully applied the technique to Prairie voles Microtus ochrogaster by obtaining DNA from only 150pl of blood, which yielded 3-5pg of DNA. It was concluded that a combination of DNA profiling and behavioural and ecological considerations d l provide information for the precise determination of kinship relationships in natural popula- tions of small species.

ecology.

INVERTEBRATES Although DNA profiling procedures for some species of vertebrates have been well documented (for example, for dogs and cats (Jeffreys & Morton, 1987) and domestic animals (Georges et al., 1988)) much less is known regarding such profiles in invertebrates. Blanchetot (1989) has obtained profiles in several species of insect. Carvalho et al. (1991) reported on the feasibility of using such methods to examine genetic differentia- tion in aphid clones. These workers succeeded in obtaining DNA profiles from individual aphids and used the method to measure reproductive success. The genetic variability detectable by DNA profiling was also contrasted with the paucity of genetic variation previously detected by isoenzyme analysis (May & Holbrook, 1978). Carvalho and col- leagues concluded that DNA profiling provided a means of studying gene flow in an invertebrate species and analysing the genetic basis of the clonal diversity exhibited by the study species.

APPLICATION IN CAPTIVITY Captive stocks of animals are, by neces- sity, maintained at relatively small popu- lation sizes. Hence the risk of inbreeding and the concomitant loss of genetic diver-

106 AUSTRALASIAN FAUNA

sity as measured by allelic variation are considerably heightened. DNA profiling has been increasingly applied as a management tool to small populations of endangered species held in captivity for preservation programmes, among them Galapagos tortoises Geochelone elephan- topus (Ryder et al., I989), the Chimpanzee Pan troglodytes (Ely & Ferrell, 1990), the Isle Royale gray wolf Cunis lupus (Wayne et ul., 1991), the Lion-tailed macaque Mucaca silenus (Morin & Ryder, 1991) and the Arabian oryx Oryx leucoryx (Tudge & Flint, 1991). In Western Australia the technique has been applied to several species kept for breeding both in zoos and private collections. Further- more, it is being used to identify indivi- duals of rare species to help to prevent illegal trading in specimens which have been removed from the wild under the guise of ‘captive breeding success’.

WESTERN SWAMP TORTOISE The Western swamp tortoise Pseud- emydura umbrinu is Australia’s most endangered reptile. A captive-breeding programme has been undertaken by Perth Zoo and the Western Australian Depart- ment of Conservation and Land Manage- ment for some years (Kuchling & DeJose, 1989; Kuchling et al., this volume). DNA profiling has been applied to assess the genetic diversity of the captive stocks. Two DNA probes generated complex profiles for 16 adult tortoises, which contained numerous bands varying both in size and intensity. Under the conditions employed an average of 26 scorable bands per animal were detected (?late 1). The mean probability of one band being in another individual randomly selected in the captive population was calculated to be approximately 0.8. This value is much greater than that obtained with most other species studied to date and indicates that the degree of genetic variation in the population under study is relatively limited. Only one animal had a DNA profile quite different from the rest sharing, on average, only 28% of its

Plate 1. DNA profiles for the adult Western swamp tortoise Pseudemydura umbrina obtained using a multilocus hypervariable DNA probe derived from the bacteriophage MI3 (Vassart et at., 1987). With one exception the profiles show a high degree of similarity.

bands with any other animal and unfor- tunately this proved to be an elderly 6 contributing little genetic input into the breeding colony.

The small size of tortoise hatchlings and their slow growth rate makes collec- tion of suitable tissue specimen for DNA analysis difficult. However, after hatching DNA can be successfully removed from the allantoic membranes. At present it is not known whether this membrane is of maternal or embryonic origin but this question is currently being resolved by comparison of maternal and allantoic membrane D-NA. If the membranes are embryonic in origin then a simple non- invasive method of obtaining DNA will be available.

NARETHA BL1JE BONNET PARROTS In 1990 40 adult Naretha blue bonnet parrots Psephotus (Northiella) haemato- gaster narethae were legally collected from the wild to establish a private breeding programme by selected aviculturists in Western Australia. Prior to the distribu- tion of breeding pairs all birds were DNA profiled. The profiles (Plate 2) showed

AUSIKALASIAN F 4 l l h A I 0 7

Plate 2. DNA profiles for the Naretha blue bonnet parrot Psephotus (Northiellu) huemutogaster narrthue obtained using a multilocus hypervanahle DNA probe derived from D16S85, the human a-globin locus. The profiles, which contain many bands per animal, are all quite distinct indicating a high degree of genetic diversity within the group tested.

that there was a high degree of variability in the population. Randomly selected progeny from these birds will be subjected to parentage testing in order to ensure their legitimacy of breeding. In addition, maintaining a database of DNA profiles for this group and their progeny will permit any changes in the degree of genetic diversity in the population to be monitored. The experience gained with this group of parrots is being used in the management of other species with captive- breeding programmes.

Several endangered species of parrots and cockatoos in Australia are subject to

severe poaching of wild stocks for commercial gain. In the past proof of illegal activities has been difficult to obtain since it has not been possible to determine unequivocally the parentage of confiscated birds. The advent of DNA profiling now permits definitive confirma- tion of parentage for several species of parrots and cockatoos. Wolfes et al. (1991) also used DNA profiles on birds of prey to establish whether they were obtained by poaching and has reported that the use of this technique resulted both in the confiscation of illegally collected birds and a reduction in the claims of ‘breeding success’ by dealers. Similar observations have been made from experience in Western Australia.

CONCLUSION The technology for generating DNA profiles for an ever-increasing variety of species of plants and animals continues to progress at an impressive rate. The application of the ‘polymerase chain reac- tion’ technique (Mullis & Faloona, 1987; Saiki ef al., 1988) to analysis of the hyper- variable loci which generate highly informative DNA profiles promises to revolutionize this field of genetics. The technique utilizes a thermostable form of DNA polymerase to amplify selectively the concentration of a particular segment of DNA; thus it may be used to analyse very small amounts of DNA quickly and at less cost. Furthermore, the fact that it is now known that there are many genetic loci characterized by very short repeating segments of DNA (microsatellites) offers great promise for generating DNA profiles for exotic species. We confidently predict that DNA profiling will have an increasingly important impact on the management of endangered species for many years to come.

ACKNOWLEDGEMENTS

We are grateful to our colleagues, especially those in the WA Department of Conservation and Land Management and Perth Zoo, for the opportunity to pursue these studies for the benefit of both the animals and mankind.

108 AUSTRALASIAN FAUNA

REFERENCES BACON, P. J. & ANDERSEN-HARILD, P. (1987): Colonial breeding in mute swans (Cygnus olor) associated with an allozyme of lactate dehydrogenase. Biol. J. Linn. Soc. 30: 193-228. BLANCHETOT, A. (1989): Detection of highly polymorphic regions in insect genomes. Nucl. Acids Res. 17: 3313. BURKE, T. & BRUFORD, M. W. (1987): DNA fingerprinting in birds. Nature, Lond. 327: 149-152. BURKE, T., DAVIS, N. B., BRUFORD, M. W. 8r HATCHWELL, B. J. (1989): Parental care and mating behaviour of polyandrous dunnocks Prunella modularis related to paternity by DNA fingerprinting. Nature. Lond. 338: 249-25 1. BYGOIT, J. D., BERTRAM, B. C. R. & HANBY, J. P. (1979): Male lions in large coalitions gain reproductive advantages. Nature, Lond. 282

CARVALHO, G. R., MACLEAN, N., WRATTEN, S. D., CARTER, R. E. & THURSTON, J. P. (1991): Differentiation of aphid clones using DNA fingerprints from individual aphids. Proc. R. Soc., Lond. B 243 109-1 14. CHAKRABORTY, R., MEAGHER, T. R. & SYORSE, P. E. (1988): Parentage analysis with genetic markers in natural population. I. The expected proportion of offspring with unambiguous paternity. Genetics 118:

ELY, J. dr FERRELL, R. E. (1990): DNA ‘fingerprints’ and paternity ascertainment in chimpanzees (Pan troglodytes). Zoo Biol. 9 91-98. FOOSE, T. J. & BALLOU, J. D. (1988): Population management: theory and practice. Int. Zoo Yb. 27: 2 H 1 . GEORGES, M., LEQUARRB, A.-S., CASTELLI, M., HANSET, R. & VASSART, G. (1988): DNA fingerprinting in domestic animals using four different minisatellite probes. Cytogenet. Cell Genet. 47: 127-131. HANOTTE, O., BURKE, T., ARMOUR, J. A. L. & JEFFREYS, A. J. (1991): Cloning, characterization and evolution of Indian peafowl Pavo cristatus minisatellite loci. In DNA fingerprinting: approaches and applications. Burke, T., Dolf, G., Jeffreys, A. J. & Wolff, R. (Eds). Basel: Birkhauser Verlag. HOAGLAND, D. B., TILAKARATNE, N., WEAVER, R. F. & GAINES, M. S. (1991): ‘DNA fingerprinting’ of prairie voles (Microtus ochrogaster). J. Mamm. 7 2 422426. JEFFREYS, A. J. & MORTON, D. B. (1987): DNA fingerprinting of dogs and cats. Anim. Genet. 1 8 1-15. JEFFREYS, A. J., WILSON, V. & THEIN, S. L. (1985a): Hypervariable ‘minisatellite’ regions in human DNA. Nature. Lond. 314: 67-73. JEFFREYS, A. J., WILSON, V. & THEIN, S. L. (1985b): Individual-specific ‘fingerprints’ of human DNA. Nature, Lond. 316 76-79. KUCHLING, G. & DEJOSE, J. P. (1989): A captive breeding operation to rescue the critically endangered Western swamp turtle Pseudemydura

839-841.

527-536.

umbrina from extinction. Int. Zoo Yb. 28: 103-109. LYNCH, M. (1988): Estimation of relatedness by DNA fingerprinting. Molec. Biol. Evol. 5: 584-599. MAY, B. & HOLBROOK, F. R. (1978): Absence of genetic variability in the green peach aphid Myzus persicae (Hemiptera: Aphididae). Ann. ent. Soc. Am.

MENG, A,, CARTER, R. E. & PARKIN, D. T. (1990): The variability of DNA fingerprints in three species of swan. Heredity 64: 73-80. MORIN, P. A. & RYDER, 0. A. (1991): Founder contribution and pedigree inference in a captive breeding colony of lion-tailed macaques, using mitochondria1 DNA & DNA fingerprint analyses. Zoo Biol. 1 0 341-352. MULLIS, K. B. & FALOONA, F. A. (1987): Specific synthesis of DNA in vitro via a polymerasecatalysed chain reaction. Method- Enzymol. 155: 335-350. PACKER, C., GILBERT, D. A., PUSEY, A. E. &

O’BRIEN, S. J. (1991): A molecular genetic analysis of kinship and cooperation in African lions. Nature, Lond. 351: 562-565. RALLS, K., BALLOLJ, J. D. & TEMPLETON, A. (1988): Estimates of lethal equivalents and the cost of inbreeding in mammals. Conserv. Biol. 2 185-193. RYDER, 0. A., CHEMNICK, L. G., SCHAFER, S. F. & SHIMA, A. L. (1989): Individual DNA fingerprints from Galapagos tortoises Geochelone elephantopus. Int. Zoo Yb. 28: 84-87. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, S. J., HIGUCHI, R., HORN, G. T., MULLIS, K. B. a ERLICH, H. A. (1988): Primer-directed amplification of DNA with a thermostable DNA polymerase. Science, Wash. 239 487-491. TUDGE, C. & FLINT, A. P. F. (1991): Science for conservation: research of the Zoological Society of London. London: Zoological Society of London. VASSART, G., GEORGES, M., MONSIEUR, R., BROCAS, H., LEQUARRE, A. S. & CHRISTOPHE, D. (1987): A sequence in M 13 phage detects hypervariable minisatellites in human and animal DNA. Science, Wash. 235 683-684. WAYNE, R. K., LEHMAN, N., GIRMAN, D., MAN, P. J. P., GILBERT, D. A., HANSEN, K., PETERSON, R. O., SEAL, U. S., EISENHAWER, A., MECH, L. D. B KRUMENAKER, R. J . (1991): Conservation genetics of the endangered Isle Royale gray wolf. Conserv. Biol.

WETTON, J. H., CARTER, R. E., PARKIN, D. T. 8r WALTERS, D. (1987): Demographic study of a wild house sparrow population by DNA fingerprinting. Nature. Lond. 327: 147-149. WILKINSON, G. S. & MCCRACKEN, G. F. (1986): On estimating genetic relatedness using genetic markers. Evolution 3 9 1169-1 174. WOLFS, R., MATHE, J. & SEITZ, A. (1991): Forensics of birds of prey by DNA fingerprinting with I2P labelled oligonucleotide probes. Electrophoresis 12:

71: 809-812.

5: 41-51.

175-180.

Manuscript submitted 18 December 1991