I02 ZOO CHALLENGES: PAST. PRESENT ANLI FLIT11Kt

Znt. Zoo Yb. (2003) 3 8 102-1 11 0 The Zoological Society of London

Genetic studies in zoological parks and their application to conservation: past, present and future 0. A. RYDER Center for Reproduction of Endangered Species, Zoological Society of San Diego, PO Box 120551, San Diego, California 92112-0551, USA

The development of new scientific techniques has led to significant advances in our understanding of bio- diversity and the threats facing animal populations. Zoos have been at the forefront of the application of these techniques, ranging from cytogenetics to the analysis of small-population biology, with the aim of improving animal management and facilitating in situ conservation. Many of the key applications of genetic analysis are discussed; for example, assessing species diversity, utilizing studbook data, under- standing genetic diseases and the related implications for captive-breeding and reintroduction, together with the latest technological developments. The increasing power of genetic analysis will offer fun- damental insights into aspects of biology that are of direct concern to zoos.

Key-words: cytogenetics, DNA, evolution, genetic management, genome. reintroduction, RNA, speci- ation. studbook

Although the number of organisms being studied using genetic techniques is increasing rapidly, a detailed under- standing, in terms of genomic sequence information, relies on data from only a few model systems. In an era when bio- logical diversity is disappearing rapidly and considerable effort is being invested in determining the number of extant species and their distribution and diver- sity, the importance of understanding the fundamental biology of threatened species in order to facilitate conservation efforts has never been clearer. Genetic analysis will provide valuable insights and as the discovery, interpretation and comparative evaluation of genomes takes place, the interests of zoological parks in applying science to animal management and con- tributing to species conservation in situ

will mandate the application of new technologies.

The traditional scientific and conserva- tion interests of zoological parks have broadened as scientific technologies have developed. Descriptions of new species now often include genetic information in support of the classification, for example, Pseudoryx (Dung et al., 1993; Gatesy & Arctander, 2000). In addition, taxa that were previously identified as subspecies are sometimes elevated to species level and, increasingly, genetic data are also included in support of the recognition as a separate species (e.g. Arctander et a/., 1996; Omland et al., 2000). It is clear that genetic studies have an important role to play in identifying and classifying bio- logical diversity, together with the more traditional descriptions of anatomy and behaviour.

The investigation of reproductive incompatibilities, mutation and morpho- logical variation, for example, is facili- tated by studies which realistically can only be undertaken in zoological parks. A comparatively recent increase in aware- ness of issues relating to small-population management, including the vulnerability of small populations to loss of genetic var- iation and the vagaries of chance events, has resulted in a profound change in animal management in zoological parks (e.g. de Boer, 1994; Foose et al., 1995; Ballou & Foose, 1996). The science of small-population management, which draws strongly on population genetic theory and, increasingly, on empirical

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studies, has created a framework for the coherent management of animal popu- lations in zoos, with defined goals for the amount of genetic variation to be retained over specific time periods (Mace & Ballou, 1990). The issues related to small-popu- lation management, as have been addressed in zoological parks, are increas- ingly being applied to small and frag- mented populations in nature (Lacy, 1997).

HISTORICAL DEVELOPMENT OF GENETIC STUDIES IN ZOOS The initial interest in genetic phenomena in zoos was largely focused on anomalies, including albinism, melanism and pelage variation. For example, an interest in the striping pattern of zebras in equid hybrids as a means to test the concept of telegony (the supposed carrying over of the influ- ence of a sire to the offspring of sub- sequent matings of the dam with other dd) led Cossar Ewart to conduct the Penicuik experiments (Ewart, 1899).

Zoological parks, with their interest in species diversity and speciation, were ideally placed to participate in the deve- loping field of genetics. Furthermore, as concepts of speciation that required a knowledge of reproductive isolation were proposed (e.g. Mayr, 1963), the informa- tion which could be collected in zoological parks became highly relevant (Gray, 1972). For example, cytogenetic studies and the identification of a chromosomal basis of sex determination led to the formulation of Haldane’s rule concerning infertility in interspecific crosses; that is, in first-generation hybrids, the heteroga- metic sex is usually sterile, rare or absent.

Many of these studies were only feasible in zoological parks where the appropriate animal-management staff, veterinarians and access to research laboratories were available (e.g. Loughman et al., 1970). For some species, for example, Hyaenas (Hyaenidae), the sex of individuals is ambiguous and a method was developed for sex determination (Wurster et al.,

1970). Monomorphic species, such as Cal- ifornia condor Gymnogyps calfornianus, could be sexed by chromosome analysis (Chemnick et al., 200Ci).

The development of methods for pre- paring well-spread nietaphase chromo- somes facilitated comparative cytogenetic studies and resulted in the emergence of a field of study that continues today (Benir- schke & Kumamoto, 1991; Robinson & Elder, 1993). Cytologisal investigations of zoo animals provided examples of mam- malian (e.g. Hsu & Benirschke, 1967-1977) and avian (e.g. Belterman & de Boer, 1990) chromosome diversity. The discovery of seven chromosomes in the 6 Indian muntjac Muntiacus muntjak yet only six in the represented a challenge to the understanding of the mechanisms of chromosomal evolution and how these might operate in different evolutionary lineages (Wurster & Benirschke, 1970). In some cases where conflicting hypothesis had previously existed, for example, for mules and other equine hybrids, chromo- somal studies provided clear evidence of the basis of the hybrid infertility (Benir- schke, 1967). Species of muntjac are still being discovered or rediscovered in native habitat (Schaller & Vrba, 1996; Rabi- nowitz etal., 1998; Amato etal., 1999) and the elucidation of’ their chromosomes represents an important step in under- standing mammalian diversification and speciation.

Notable early studies of genetic markers involved the variable immunological markers that are located on the surface of red blood cells and used to define blood groups (Socha & Moor-Jankowski, 1986). These studies were soon aided by the development of electrophoretic tech- niques. However, bo .h approaches were limited initially because invasive sampling andor chemical restraint were in the early stages of development and acquiring sam- ples was difficult.

The magnitude of the task of under- standing biological diversity of interest to zoological parks has resulted in a con-

104 ZOO CHALLENGES PAST PRESENT A N D t l ' T U R F

sistent hierarchy of questions which can be addressed by genetic analysis. Many of the questions that were asked in previous decades are still relevant today; a notable example is the question of subspeciation and the identification of appropriate units for conservation management (Ryder, 1986a,b; Ryder et al., 1988; Bowen, 1999; Crandall et al., 2000; Goldstein et at., 2000). Although the appropriate emphasis remains to be determined, there is no doubt that increasingly powerful tools of genetic analysis will be applied to under- stand and interpret the evolution of popu- lations and species in a manner that will benefit the conservation effort. None the less, genetic studies are providing new insights into the evolution and zoogeog- raphy of diverse taxa; for example, Komodo monitor lizard Varunus komo- doensis (Ciofi & Bruford, 1999; Ciofi et al., 1999). Many studies, such as systematic studies of Orang-utan Pongo pygmaeus, have been conducted using samples that would have been difficult, if not impossible, to obtain from sources other than zoological parks (Janczewski et al., 1990; Ryder & Chemnick, 1993; Zhi et al., 1996).

STUDBOOKS AND GENETIC MANAGEMENT A major initiative of zoos has been the expansion of record keeping, particularly for basic genetic and demographic data. Demographic data minimally comprise the dates of birth and death, and the sex of an individual. Genetic data can include the unique identification of an individual and its sire and dam. Some of the earliest collections of genetic data in zoological parks involved the establishment of stud- books, which in many ways set the stage for current concepts of ex situ manage- ment in zoological parks (see also Olney, this volume; Flesness, this volume). Without studbook data, conjecture about inbreeding effects in small populations would persist in a framework of unin- formed dialogue. The studies of Katherine

Ralls and others have relied on studbook data to demonstrate that the small popu- lations maintained in zoos are vulnerable to recruitment losses as a result of inbreeding effects (Ralls et al., 1979). These concerns quickly broaden to incor- porate the larger issue of the factors that contribute to the vulnerability of small populations to extinction: founder effect, genetic drift, inbreeding and selection (Foose & Ballou, 1988).

The concerns of zoos over the long- term management of their captive popu- lations opened an interface with theoretical studies of population biology and demography, resulting in the formulation and ongoing revision of cap- tive-management technology which could define and meet goals for the retention of genetic diversity over a specific period of time. The 200 European Endangered Species Programmes (EEPs) and 107 Species Survival Plans (SSP) for the co- ordinated captive management of small populations in zoos stand as remarkable accomplishments in zoo-based conserva- tion. As the collection of studbook data has expanded, the testing of hypotheses and the further elaboration of technology for small-population management (Hed- rick, 2000), such as assisted reproduction using appropriate individuals selected from collections worldwide, have become possible (Ballou, 1992; Lasley et at., 1994; Johnston & Lacy, 1995; Wildt & Wemmer, 1999; Loskutoff, in press; see also Wildt, this volume).

To population geneticists, all popu- lations in zoos, even those managed for retention of genetic diversity, are small populations. Thus, we can anticipate that founder effect and genetic drift, as agents of chance, will result in the appearance of genetic diseases in some populations of zoo animals (Ryder, 1988) and studbook data can be used to obtain information about potential carriers of, for example, the extension locus in Przewalski's horse Equus przewalskii (Marklund et at., 1996) or chondrodystrophy in California condor

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(Ralls et al., 2000). The inevitability of a genetic disease occurring in a significant number of intensively managed zoo popu- lations strongly suggests that accurate studbook data should be maintained and, furthermore, that reference samples for genetic analysis should be systematically collected and maintained as a research resource (see also Bailey et al., this volume). Emerging genetic technologies will make it feasible for future zoo genet- icists to understand the mechanisms of transmission of genetic diseases and develop tests for carriers. Such develop- ments will allow management of the incidence of genetically anomalous indi- viduals in the context of population persistence and retention of genetic variation.

REINTRODUCTION A greater knowledge of the California condor genome may play a crucial role in the recovery of the species in the wild. The recovery effort will require a specific design to establish the full extent of the genetic variation in reintroduced popu- lations. The source population of captive individuals comprises the entire extant gene pool of California condors and birds that are genetically valuable may also be potential carriers of the apparently reces- sive allele associated with the lethal chon- drodystrophic condition that has been observed in the remnant population (Ralls et a/., 2000). Without knowledge of whether an individual condor is a carrier of the chondrodystrophic allele, a random selection of birds into the cohort of can- didates for release could lead to a com- paratively high or low frequency of this allele in reintroduced populations. While potential carriers can be identified by ana- lysis of the studbook data, only progeny testing or the development of a test for a linked marker will identify obligate car- riers of the chondrodystrophic allele. With advancing technology in human and animal genetic mapping and genomics, the development of a test to identify carriers

of the chondrodystrophic allele that could be carried out at hatching is feasible and should be viewed as appropriate tech- nology for the California condor recovery effort.

The reintroduction of threatened species from zoological collections into formerly occupied habitats introduces a second bottleneck for populations that were extirpated and genetic management will continue to be of long-term concern. Integrated captive-population manage- ment and reintroduction efforts benefit from genetic monito-ing which also pro- vides useful informat Lon as reintroduction projects are refined and evaluated as, for example, with Black-footed ferret Mustela nigripes (Russell et al., 1994), Przewalski’s horse (van Dierendonck et al., 1996) and Arabian oryx Oryx leucoryx (Kumamoto et al., 1999; Marshall et al., 1999; see also Stanley Price & SooIae, this volume).

NEW METHODS AND APPLICATIONS The trend in genetic research to develop new methods for detecting genetic varia- tion at an even higher resolution has pro- vided, on the one hand, more detailed insights into phylogenetic systematics and, on the other, a previously unobtainable view of the biology of populations in an evolutionary context. Studies of mater- nally inherited niitochondrial DNA (mt DNA) variation, initially by restric- tion fragment length polymorphism (RFLP) analysis arid more recently by sequencing part or all of the mt DNA molecule, have become requisite items on the research agenda for species main- tained in zoos (e.g. Wayne et al., 1997; Caccone et al., 1999). In mammals, analysis of the variation in the Y chromosome is also being utilized in studies of Humans Homo sapiens (Semino et al., 2000) and other species, for example, Macaques Mucaca spp (Tosi et al., 2000), to infer aspects of population history, migration and sex bias in dispersal.

I06 ZOO CHALLENGES PAST. PRESENT A N D FL’TCIRF

An increased ‘resolving power’, derived from the analysis of greatly increased amounts of genotype information from geographically and temporally distributed populations, offers an opportunity to con- nect the studies of behavioural ecology, zoogeography, population biology and molecular systematics. At the expanding interface between behavioural biology and genetics, the availability of sufficient amounts of genetic data allows for the testing of hypotheses generated by behav- ioural studies (Altmann et al., 1996). In wild populations it may be difficult to determine the reproductive success of individuals and infer breeding patterns within populations but mate fidelity can now be investigated in species with varying social structures using genetic approaches (e.g. Piper etal., 1997). The ability to evaluate kinship, including maternity and paternity, in managed and wild populations has not only served to increase the accuracy of data for manage- ment purposes but also provided useful information for interpreting social struc- ture and mating strategies in wild popu- lations (Morin et al., 1994; Zhang et a/., 1994; Field et al., 1998; Gagneux et al., 1999).

Analysis of nuclear microsatellite vari- ation in canids (Goldstein et a/., 1999) and felids (Eizirik et al., 2001) has recently been used to infer and date demographic events, such as bottlenecks and periods of population expansion. An estimate of the effective population size N, may be critical for assessments of the level of endanger- ment and population viability (Mace & Lande, 1991). Evaluation of genetic vari- ation across nuclear and mitochondria1 loci may be used to infer effective popu- lation size (Kohn et af., 1999) and inbreeding within populations (Moehlman et al., 1996).

Enthusiasm for the application of such comparatively new approaches is high because the potential is enormous. How- ever, the discovery of artefacts associated with the polymerase chain reaction (PCR)

amplification of mt DNA (e.g. Arctander, 1995; Greenwood & Paabo, 1999) and nuclear microsatellite DNA sequences (e.g. Smith et al., 2000) suggests that the PCR method may occasionally lead to erroneous conclusions.

None the less, it is safe to say that the increasing power of genetic analysis will offer fundamental insights into aspects of biology that are of direct relevance to the concerns of zoos, including the study, appreciation and conservation of bio- logical diversity. Geneticists in zoos will participate in studies that will broaden our understanding of the natural history and evolution of species in order to help conserve them and the ecosystems that sustain them. Understanding components of fitness in model populations, such as those maintained in zoos, may contribute to a greater understanding of the evolu- tion of social systems within the context of individual reproductive strategies in wild populations (Wooninck et al., 2000). Dissecting the genetic components of mate choice will not only provide information that is fundamental to under- standing molecular mechanisms of selec- tion but also contribute to the technology of captive-breeding (e.g. Jordan & Bru- ford, 1998). The evaluation of allelic var- iation in populations may provide information about historic patterns of population movement and secondary con- tact. It may also link genealogical evalua- tion with evolutionary population genetics for many nuclear markers and sex chromosome markers (Semino et id., 2000), and also have relevance to the con- servation of threatened species (e.g. Thompson, 1995).

Single nucleotide polymorphisms (SNPs) are being recognized as valuable markers for studies of human genetic var- iation, evolution and medicine. It is likely that the same will eventually hold true for other species, particularly those that are closely related to model organisms, including humans. As the human genome project progresses to the analysis of vari-

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ation within, and selection on, the human genome, comparisons with Chimpanzees Pun troglodytes, Bonobos Pan paniscus, Gorillas Gorilla gorilla, Orang utans and other primates will become of increasing interest. Advanced technologies, such as DNA oligonucleotide arrays, may also be utilized in comparative studies of humans and apes (e.g. Hacia et al., 1999) and large-scale comparative genomic studies of apes will become more routine (Kaess- mann et al., 1999; Huttley et al,, 2000), furthering our understanding of medicine for humans and apes alike.

Closely related species differ little in their overall DNA sequence and the level of genetic differentiation between subspe- cies is even lower. Therefore it has been suggested that much of the adaptive evo- lutionary change between closely related taxa must take place at the level of gene regulation; through the timing, duration and intensity of gene expression. The methods available for the analysis of gene expression have undergone a major expansion over the past few years; from a time when the expression of only one or a few genes could be analysed at one time, to the ability to analyse the expression of many thousands of genes simultaneously, which is possible at time of writing. Such technologies will provide a greater knowl- edge of adaptive differences and will help us to understand the variety of genetic mechanisms that lead to reproductive isolation and, ultimately, the process of speciation itself. A more detailed understanding of gene expression will also be required to gain a deeper appreciation of animal intelligence and its role in adaptation.

Consequently, it is likely that genetic approaches to understanding animal development and physiology will benefit from the comparative analysis of samples collected for the study of gene expression. Zoos are already experiencing an increase in requests for a variety of samples that are collected and stored in ways that are appropriate for the extraction of DNA

and, particularly, messenger RNA (which identifies the genes that were being expressed at the time the sample was collected).

THE FUTURE The concern for conservation of biological diversity and the emerging interest in comparative genomic studies may now find common ground. The collection and documentation of samples and associated data including, for eKample, geographic and ecological information, and the demographic and parrmtage data on indi- viduals from the sou -ce population, will provide a crucial rssource for future studies (Ryder et al., 2000). Efforts to secure the long-term persistence of man- aged populations must involve geneticists to ensure that the necessary samples and data are available for future investiga- tions. As the genomic sequence and gene expression information from threatened primates, as ambassadors for biological diversity, becomes increasingly valuable for human medicine, the opportunity for zoological institutions to contribute to species conservation is enhanced.

The Human Genome Organization Ethics Committee has argued that the data obtained about the human genome are the collective property of humankind (Human Genome Organization Ethics Committee, 2000). A s the genomic sci- ences are applied to medical development and practice, there will be an increasing disparity between rich and poor. This rec- ognition was accompanied by a call to establish a means of sharing the wealth that is derived from advancement in the understanding of the human genome. Should not a similar call be made for sharing the wealth tfat accrues from our increased knowledge of biodiversity, that may be applied to the conservation of eco- systems and the species and processes they sustain? The call for some form of com- pensation for these ;pecies that contrib- utes directly to their in situ protection should come from zoological parks that,

10s ZOO CHALLENGES PAST, PRFSENT A N D FUTURF

because of their unparalleled roles in edu- cation (Andersen, this volume), research and conservation, are in a unique position to serve as advocates for threatened species.

The number of opportunities for util- izing the scientifically managed collections to benefit the conservation of species is increasing (e.g. Ryder, 1995). Zoological parks now link their efforts and expertise with the conservation of species in situ (Olney et al., 1994; Hutchins 8z Conway, 1995). As institutions whose popularity with the public has provided many oppor- tunities to educate about the diversity of animal life, zoological parks have an increasingly important role to play in an era when public perceptions and behav- iours will shape human conduct, influ- encing the survival of many species on Earth.

ACKNOWLEDGEMENT

Support of the Caesar Kleberg Wildlife Foundation is gratefully acknowledged.

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The role of reproductive technologies in zoos: past, present and future D. E. WILDT Conservation & Research Center, National Zoological Park, Smithsonian Institution, Front Royal, Virginia 22630, USA

Reproductive technologies have been playing a role in zoos for more than two decades. However, the value of these techniques has largely been misunder- stood. There has been an over-emphasis on hyper- bole and the ‘quick-fix’ (the attempted use of assisted-breeding techniques to produce offspring rapidly) and too little prominence on the prerequisite need to understand fundamental reproductive pro- cesses. The real value of these technologies is in delving into species-specific mechanisms that regu- late reproductive success. Thus, the priority should always be using the technologies as tools to generate new knowledge that can then have applied benefits to management, ex situ or in situ. Models of using this strategy to develop successful assisted-breeding programmes are discussed, as well as the importance of integrating science between researchers and animal managers.

Kqv-words: artificial insemination, assisted breeding, embryo transfer, genetic management, reproduction, reproductive technology

The reproductive sciences span a broad range of disciplines and venues, from behaviour to molecular biology, and from sophisticated laboratories to remote field sites. During the last 50 years there have been remarkable advances in assisted breeding to enhance livestock production and, more recently, to help overcome human infertility. During the late 1970s, interest grew in one particular area of the

reproductive sciencems: assisted breeding for propagating threatened species. In general, assisted breeding refers to any technique that circumvents natural pro- creation to produo: young. The most commonly used of these involves artificial insemination (AI) (Johnston et al., this volume) and embryo transfer (ET). Arti- ficial insemination relies on fresh or frozen-thawed sperm, whereas ET uses fresh or thawed embryos. In vitro fertiliza- tion (IVF) and intracellular sperm injec- tion (ICSI) are methods of producing embryos in the laboratory and, when used in conjunction with ET, are considered to be a component of assisted breeding. In recent years, a host of other methods, including gamete iritrafallopian transfer (GIFT), embryo splitting and nuclear transfer or cloning, have been developed to create embryos. F;or reasons to be dis- cussed, these techniques are likely to have little immediate relevance to the manage- ment of most threatened species. Of the procedures described here, only A1 with fresh or thawed spe -m holds promise for contributing to wildlife propagation in the near future.