Nature and Human Society: The Quest for a Sustainable World (1997)
Chapter: Conservation Genetics: Applying Molecular Methods to Maximize the Conservation of Taxonomic and Genetic Diversity
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Conservation Genetics:
Applying Molecular Methods to Maximize the Conservation of Taxonomic and Genetic Diversity.
Conservation biology is an applied science that involves direct human intervention into the management of diminishing natural resources. However, unlike traditional resource management, the focus of conservation biology is not necessarily driven by direct economic incentive or the desire to manage a resource for the sake of harvesting it. Instead, the primary goal of this field is to stop the downward spiral of loss of biological diversity by mitigating factors that erode the biological integrity of intact ecosystems and the long-term evolutionary viability of populations, species, and communities of organisms. In this sense, conservation biologists attempt to manage biodiversity on two time scales, the ecological (present) and the geological (future), but management itself is inevitable.
By definition, conservation biology must be multidisciplinary, requiring an integration of many areas of biology, including biogeography, systematics, plant and animal ecology, reproductive biology and physiology, range and wildlife management, environmental toxicology, population biology, genetics, and molecular biology. Moreover, the coordination or planning of any conservation effort also involves issues outside the realm of biology, because most environmental-conservation solutions are compromises between the biological requirements of a natural system and the socioeconomic and political realities of the human populations that are associated with that system. Therefore, the conservation of biodiversity must strike a balance between the needs of a growing human population and the viability of biological systems in the face of a rapidly changing environment.
The act of preserving our natural or biological resources, as in so many action-oriented fields, can be distilled into five simple questions: why, what, where, how,
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and who? These questions are simple, but their answers have proved vexing and indeed have generated some intense debates. The answer to the first and perhaps most important societal, issue, why, has been framed in ways that range from the economics of ecosystem services to the psychological and cultural value of intact ecosystems and species to our moral and ethical obligations to pass on to our children the natural world in roughly the same condition in which we found it (Costanza and others 1997; Kellert and Wilson 1993; Pimm 1997). A detailed discussion of these issues is outside the scope of this paper; we will assume that the reader will find justification elsewhere for why we should conserve our natural resources.
Assuming we should develop a rational means to describe and conserve the world's biological diversity, we must rely on some scientific systems of measurement and theory to address the four remaining critical questions. Over the last 10 years, conservation genetics has emerged as a subfield of biological conservation that offers an objective approach to three of these questions: what, where, and how. Conservation genetics is more a focus than a field of study, but it has at its root the application of molecular and quantitative genetic methods to the preservation of genetic, species, and ecosystem diversity. Genetics can be applied to these issues in numerous ways, but the term conservation genetics usually refers specifically to molecular genetic techniques that help to
• identify evolutionarily distinct groups of organisms (for example, populations or species) that are worthy of separate conservation efforts (that is, conservation units) (Avise 1996; Moritz 1994, 1995; O'Brien 1996);
• define specific geographic regions that harbor genetically distinct populations, and/or species (that is, regions of genetic endemism) (Avise 1996; Riseberg and Swensen 1996; Templeton and Georgladis 1996; Vane-Wright and others 1994; Williams and Humphries 1994; Witting and others 1994); and
• estimate the distribution of genetic diversity within and among conservation and management units to develop plans that will conserve the greatest amount of that diversity and the evolutionary potential it offers (Burgman and others 1993; Caughley 1994; Frankham 1995; O'Brien 1994).
Researchers have used genetic approaches to address a variety of conservation problems in plants and animals found on many continents. This genetic research has involved diverse laboratory procedures and approaches to data analysis; the results have provided critical information for wildlife managers and environmental policy-makers. In the following sections, we present a brief overview of both the methods used in conservation genetics and some empirical studies that underscore the value of genetic analysis in conservation management.
Molecular Systematics
what are the Conservation Units?
A critical first step in designing appropriate conservation measures is properly defining and identifying the group one wishes to conserve, the so-called conser-
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vation unit (CU). In other words, we need to define what we wish to conserve before we can take measures to conserve it. Although this may seem trivial, the evolutionary process is often murky enough to lead to great difficulty in defining CUs, particularly when we are dealing with closely related species that have been thrown together recently by human-induced changes in the environment or those which were never isolated fully from one another reproductively (that is, hybridization has occurred). In these cases, careful examination of the biological characteristics of an organism that are most likely to carry evolutionary historical information (a field known as biological systematics)such as features of anatomy, behavior, and geneticsoften yields the clues necessary to place a series of populations and species on a synthetic family tree (also known as a phylogenetic tree). These clues even can be used to determine whether a group of organisms can be defined as a single evolutionarily significant unit (ESU), which may be but is not necessarily synonymous with what we usually refer to as a species.
Molecular-generic approaches to biological systematics have emerged as one of the most exciting new areas of biological research (Hillis and others 1996). A wide array of technical and analytical methods has been used to address issues of evolution and conservation at all levels of organization, ranging from genes within populations through the process of speciation to the reconstruction of the tree of life itself (Avise 1994). Of particular importance here are the contributions of molecular techniques to the identification and phylogenetic placement of rare and endangered species. Knowledge about diversity at the molecular level can be used to reconstruct the evolutionary history of an endangered organism (Avise and Hamrick 1996) and to identify the ESUs on which to focus our attention for conservation (Moritz 1994, 1995). Because much of conservation planning depends on taxonomic or species assignments (Taberlet 1996), identifying systematically based CUs aids considerably in developing management plans and in evaluating priorities for conservation (Smith and Wayne 1996).
Case Studies
Molecular-systematic studies can help clarify taxonomic issues at three different levels. First, we can identify cases of “oversplitting”, that is, when distinct morphological forms are considered different evolutionary entities but are in fact genetically indistinguishable. This implies that gene flow still may be occurring between the different forms and that they therefore should not be considered evolutionarily distinct. For example, the now-extinct dusky seaside sparrow (Ammodramus maritimus nigrescens) of Florida, originally described as a distinct species, was redefined later as a subspecies when it was shown to be genetically indistinguishable from other populations of seaside sparrows (Avise and Nelson 1989). Moreover, it was shown that all populations of seaside sparrows on the Atlantic coast (including the dusky seaside sparrow) are genetically more similar to each other than they are to populations of seaside sparrows that are found along the Gulf of Mexico. Clearly, molecular data supported the inclusion of the dusky seaside sparrow in the seaside-sparrow species and suggested that its loss, although regrettable, had little or no effect on the long-term evolutionary course of the entire species.
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Second, molecular systematics can help us distinguish between forms that are morphologically similar but are in fact ancient, unrelated lineages with little or no gene flow between them. One example is Darwin's fox (Dusicyon fulvipes) on Chiloé Island in Chile. Some scientists had considered Darwin's fox a small race of the common South American grey-fox species Dusicyon griseus, on the basis of morphological similarities. Chiloé island is only 5 km off the coast and likely was connected to the mainland during the last glaciation (about 13,000 years ago), which would have created opportunities for gene flow between Darwin's and grey foxes. However, genetic analyses of Darwin's fox and other South American fox species suggest that Darwin's fox is at least as divergent from the grey fox as the grey fox is from another well-recognized fox species, the culpeo fox (Dusicyon culpaeus), and that Darwin's fox probably evolved from the first immigrant foxes into South America 2–3 million years ago. Recently, a small population of Darwin's foxes was found on mainland Chile, and they were shown to be quite genetically divergent from the grey fox but closely related to the population on Chiloé island. This suggests little or no present or historical gene flow between Darwin's and grey foxes, and it supports the distinctiveness of Darwin's fox as a separate species (Wayne 1996).
Third, systematic analyses of genetic characters can provide an objective means of identifying evolutionarily distinct lineages among closely related groups. The Iberian lynx, Lynx pardinus, is considered to be the most vulnerable cat in the world. Its remaining populations are highly fragmented and of limited size. The species status of the Iberian lynx is complicated: Some consider it to be a geographic variant of the Eurasian lynx, Lynx lynx, and others consider it to be a distinct species. Because the taxonomic status of the Iberian lynx is important to the establishment of an effective management plan for lynxes in general, a molecular-systematic study was conducted recently (Beltran and others 1996). The results of this study revealed a close relationship between the Canadian lynx (Lynx canadensis) and the Eurasian lynx, but the Iberian lynx is evolutionarily more distinct. Thus, these molecular data give validity to the concept that the Iberian lynx is a phylogetically distinct species that deserves separate consideration for conservation.
Molecular Phylogeography
Where do those Conservation Units Reside?
Once we decide what groupings of organisms are distinct and worthy of separate efforts at conservation (that is, we identify our CUs), it becomes critical to determine the geographic location of important subsets of individuals within each CU. In other words, where will we focus our conservation efforts to preserve a CU or species?
The use of molecular systematics in a geographic context can contribute to answers to this question in two ways. First, detailed studies of intraspecific (within-species) variation can identify the geographic limits of either a CU or what Moritz (1994) calls a management unit (MU). Second, patterns of intraspecific phylogenies of unrelated groups of organisms may assist in identifying geographic regions
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whose populations and species have had a shared, unique evolutionary history, thus allowing for the conservation of communities of organisms that have high levels of genetic endemism or uniqueness.
Intraspecific Phylogeography
The term intraspecific phylogeography denotes the connection between biological systematics, population genetics, and biogeography, the study of the distribution of organisms in geographic space and the factors that led to that distribution (Avise and others 1987). In principle, any biological characteristic can be used for this purpose, but intraspecific phylogeography now mostly is associated with the study of molecular markers, especially mitochondrial DNA (in animals) or chloroplast DNA (in plants). By determining the detailed genetic and evolutionary relationships of populations within a species (or CU), and superimposing that intraspecific molecular phylogeny on a geographic map, one can infer the processes that historically determined the current distribution of organisms. One also can use this approach to identify the geographic location of genetically distinct populations (that is, populations that substantially differ from one another by the frequency of genetic traits rather than by the presence or absence of those traits) or MUs, which might deserve special attention if specific conservation measures become necessary to preserve a given species. The identification of specific MUs and their geographic location currently has one of the highest priorities in most efforts that use molecular markers for conservation purposes, and intraspecific phylogeography provides a theoretical framework to accomplish this.
Case Study. Among the animal species currently listed by the International Union for the Conservation of Nature (IUCN), the Convention on International Trade in Endangered Species (CITES), and the US Department of the Interior as endangered, the Sumatran rhinoceros (Dicerorhinus sumatrensis) is one in greatest need of special attention and immediate wild-population (or in situ) management. Historically, this species inhabited most of the Indochinese peninsula, from Burma (Myanmar) to Vietnam, and south to Malaysia and the islands of Sumatra and Borneo. Destruction of habitat and hunting have led to a rapid decline of this species over the last 2 decades. Only a few confirmed populations remain on peninsular Malaysia, Borneo, and Sumatra. Because of the dire situation of this species, translocation programs have been proposed that would move individuals that are scattered among fragments of unsustainable forest and concentrate them in protected zones of natural habitat (Foose and van Strien 1995). However, it is important to remember that the objectives of any conservation effort should be not only to maintain a collection of organisms, but also to preserve the maximal amount of existing genetic variability within a species and to maintain the evolutionary historical integrity of its wild populations.
Geographic mapping of the distribution of mitochondrial-DNA (mtDNA) variants among Sumatran rhinoceros populations (Morales and others 1997), using both molecular-systematic and population-genetic methods, reveals two phylo-geographic features that are important to the conservation of the Sumatran rhinoceros. First, a phylogenetic tree of mtDNA haplotypes, overlaid on the distri-
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bution of the Sumatran rhinoceros, suggests that the population in Borneo possesses a unique mtDNA variant that is not shared with other Sumatran rhinoceroses, indicating a long history of isolation from the remaining Sumatran and peninsular Malaysian populations. Therefore, the population in Borneo should be considered a separate ESU. Second, a geographically referenced population-genetic analysis suggests that the populations outside Borneo can be divided into two groupings or MUswest Sumatra, and east Sumatra and Malayaon the basis of significant differences in the frequency of mtDNA variants and the restriction of gene flow that they imply. Thus, translocation and other conservation efforts should take these three distinct units (ESUs or MUs) into consideration and try to maintain the evolutionary and genetic integrity of each unit.
Regional Phylogeography
More recently, and closely related to the common use of intraspecific phylogeography, efforts have been made to map intraspecific phylogeographic patterns simultaneously among a wide variety of species that occupy overlapping geographic ranges. This has been done to identify regions that harbor populations or species that are consistently genetically distinct from other populations within their species (so-called conspecifics) or other closely related (sister) species within their genus (so-called congenerics). These regions of genetic uniqueness or genetic endemism can be used to design reserves and other mechanisms of conservation (Avise and Hamrick 1996; Templeton and Georgladis 1996; Williams and Humphries 1994; Witting and others 1994). They also provide an effective shortcut to making decisions about conservation at the levels of species and community because it would be impossible to conduct individual genetic surveys of the hundreds of thousands of species in a particular region. Thus, a consistent pattern of regional genetic uniqueness across a diverse but logistically feasible number of species (including fungi, plants, invertebrates, and vertebrates) would allow one to assume reasonably that most populations or species within that particular region, the large majority of which will not have been analyzed, are genetically unique.
Case Studies. One compilation of several studies of invertebrate and vertebrate animals of the southeastern United States found major patterns of molecular phylogeographic congruence among populations of coastally distributed species (Avise 1996). These patterns were shared among varied groups of organisms, including horseshoe crabs, American oysters, diamondback terrapins, ribbed mussels, seaside sparrows, toadfish, black sea bass, and tiger beetles. Even species that have a greater ability to disperse, like white-tailed deer, showed a similar pattern, suggesting population differentiation in this region in response to a persistent set of historical biogeographic processes (Ellsworth and others 1994). The pattern revealed by most species indicates major molecular phylogenetic discontinuities between the Gulf of Mexico and the Atlantic coastline of the southeastern United States, whereas some patterns, like that seen for deer, indicate the uniqueness of the populations in southern Florida. Together, these findings suggest that maritime and other species in this region may have been subjected to the same bio-
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geographic influences and thus share a common biogeographic history. Although some exceptions to the common molecular-phylogeographic pattern exist, the evidence is strong that at least among animal species, populations on either side of southern Florida are likely to be genetically distinct from one another. The conservation of any one of those species must incorporate efforts on both sides of this important biogeographic divide.
Population Genetics
How will We Manage Genetic Diversity within each Conservation Unit?
Once we have determined what groupings of organisms (CUs) are distinct and worthy of separate efforts at conservation, and where the genetically unique populations of those units (MUs) or larger regions of general genetic uniqueness (regions of genetic endemism) are, it is critical for us to devise the means to conserve those species individually or regionally. In other words, how will we conserve each species's populations and their underlying genetic diversity well into the future?
Population genetics offers a key perspective on this issue because most critical evolutionary events occur at the level of the population. The potential rate of evolution depends on the amount of genetic diversity in a population; processes that erode levels of genetic diversity or increase the occurrence of deleterious combinations of genes (such as inbreeding) within populations limit the rate and scope of potential evolutionary changes in those populations to meet environmental challenges (Templeton and others 1990). Furthermore, biologists agree that levels of genetic diversity within individuals may confer important advantages of fitness on those individuals (Allendorf and Leary 1986). Thus, a fundamental concern of conservation biologists is to preserve genetic diversity in populations and species and the resulting evolutionary potential. The field of population genetics plays a critical role in determining how that diversity is distributed and how best to preserve it.
Population Genetic Structure
A species's genetic diversity can be distributed in various ways, depending on historical ecological, geological, and human-induced events, as well as on the current patterns of geographic distribution, individual dispersal, social organization, ecological adaptation, demographic transition (births, deaths, and generation length and overlap), and genetic migration (the flow of genes across a landscape). How one configures a conservation-management strategy to encompass the individuals and populations that are necessary to capture the greatest amount of a species's genetic diversity will be derived largely from knowledge of the existing distribution of that diversity across the species's range, otherwise known as the population genetic structure of a species. Information of this sort is essential for conservation planning but is often difficult to obtain.
Case Study. Among Asian macaque monkeys are species that have extensive geographic distributions, either contiguous through the mainland, like the rhesus
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monkey (Macaca mulatto), or fragmented, like the long-tailed macaque (Macaca fascicularis), whose distribution includes part of mainland Asia and many islands in the Malay Archipelago and the Philippines. Melnick (1988) and Melnick and Hoelzer (1992) have shown that 91% of the nuclear genetic variation in the rhesus monkey can be attributed to variation within a geographic region; in the more fragmented long-tailed macaque, this figure is reduced to 67%. In other words, nearly 4 times as much of a species's genetic variation can be attributed to differences between regions in the more fragmented long-tailed macaque as in the more contiguously distributed rhesus monkey. This pattern also holds true for species that have a much more restricted distribution, like the toque macaque (Macaca sinica) of Sri Lanka and the Japanese macaque (Macaca fuscata). In the toque macaque, which exists on only one island, only 3% of the species variation can be attributed to differences between regions, whereas in the Japanese macaque, which exists on a number of islands in Japan, that figure increases to 24%.
What does all this mean in terms of conservation? Very simply, the greater the percentage of overall genetic variation in a species that can be attributed to differences between populations, the greater the number of populations that must be included in a conservation-management plan that seeks to preserve some maximal level (for example, 90%) of existing genetic diversity. The Japanese macaque is considered an endangered species by the IUCN; thus, given its current population genetic structure, efforts to conserve this species must include a broad geographic representation of different island populations to maximize the genetic diversity to preserve. If the toque macaque ever shares a similar fate in Sri Lanka, reserves that harbor only a small number of sufficiently large populations likely will capture most of the species's existing variation.
Metapopulation Management
As human populations continue to grow, landscapes that are fragmented by human activities are becoming the predominant arena within which demographic and evolutionary processes in terrestrial plants and animals occur. Nevertheless, the effects of human-induced changes in the landscape on the distribution of genetic variation in wild populations remain largely unknown. It is important to examine the long-term genetic consequences of fragmentation of habitat so we can develop appropriate strategies for maintaining viable populations in remnants of habitat over hundreds to thousands of years. Such studies are only beginning to be conducted, but the new area of metapopulation analysis and management has emerged as a result of these issues (Hanski and Gilpin 1997). A metapopulation is characterized as a network of populations that have limited gene flow between them and have population extinction and recolonization in specific localities (Levins 1969). In the context of management, we define a situation as extinction if a population either has died out or has been removed for the purpose of translocation.
Metapopulation management brings together the fields of demography, population genetics, and resource management. The primary goal is to “fool” the evolutionary process into “believing” it is acting on one large contiguous population, with all the attendant complexities of births, deaths, dispersal, and local group ex-
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tinctions, when in fact the members of the species are distributed in many small patches that effectively are isolated from one another by intervening, unsuitable habitat through which they cannot cross. In general, a-metapopulation-management plan involves in-depth study of the behavior, demography, and genetics of a species to determine when, how many, and where individuals should be moved among the existing patches of suitable habitat so as to mimic one large panmictic (free-mixing) population or species. The critical long-term goal of such a strategy is to provide the largest number of breeding individuals, or effective population size, to maintain most of the population's or species's genetic diversity over the course of centuries (Wade and McCauley 1988). One can demonstrate both mathematically and experimentally that the larger the effective population size, the less likely that genetic variation will be lost to random processes that generally remove genetic variants from a population (so-called genetic drift). Hence, the general goal is to maintain as large an effective population as possible, thus buffering the forces that otherwise would inevitably erode genetic diversity.
Case Studies. Populations of the ocelot (Leopardus pardalis) in southern Texas provide an example of a habitat specialist that has been fragmented into many small populations after 50 years of converting land to agricultural uses. A recent examination of genetic variation has revealed a lack of gene flow between the populations in southern Texas and the historical source population in northern Mexico (Walker 1997). Furthermore, genetic variation within populations in southern Texas has eroded. Assuming that a generation in the ocelot is about 2 years, this means that the fragmentation of the ocelots' range into small, relatively isolated populations has resulted in a major loss of genetic variation in only 25 generations. If genetic diversity within populations of ocelots in southern Texas is to be restored and maintained, any future conservation plan must involve exchanging cats between these isolated populations and those in northern Mexico.
Black lion tamarins (Leontopithecus chrysopygus), endemic to the state of São Paulo, Brazil, exist in only seven forest fragments (Coimbra-Filho 1976; Valladares-Padua 1993). Researchers from the Instituto Pesquisas Ecolõgicas (IPÊ) in São Paulo and from the Center for Environmental Research and Conservation (CERC) at Columbia University in New York are undertaking a project to devise a program of metapopulation (translocation) and management for these animals. The immediate goal of this effort is to translocate individuals from one forest fragment to another. The ultimate goals are to ensure proper assimilation of introduced individuals into other populations or into unoccupied but suitable patches of habitat and to conserve a “natural” amount of genetic diversity in the combined forest fragments, including the empty ones that will be colonized by translocated lion tamarins. One way to ensure proper assimilation of introduced lion tamarins is to mimic their natural dispersal patterns and their current population genetic structure. The genetic data from this study will contribute immeasurably to what is known about the social organization, dispersal patterns, and population genetics of the black lion tamarin and thus will enhance the chances of successful translocation, demographic stability, genetic management, and long-term survival of this highly endangered species.
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Conservation Genetics Training
Who will Perform Genetic Analysis for Conservation?
Conservation-management recommendations that come from outside the nation in which they are to be implemented rarely are followed. Indeed, innumerable counterexamples teach us that conservation is done best when it is done at home. For this reason, scientists from each country in which the work is to be done should be trained as conservation geneticists. Thus, when asking who should be performing genetic analysis for conservation, the logical answer would be the countries' scientists who would use the resulting genetic information to establish and revise their conservation programs and policies.
This means that, in addition to training our own students and future conservation geneticists, the universities and other research institutions in developed countries should be providing opportunities for in-depth technical and analytical training to young scientists who have the best chance of establishing this type of research in their own countries. One program that is doing just that is the Conservation Genetics Training Program for Southeast-Asian Scientists, which is based at CERC (see Melnick and Pearl 2000) and is funded by the MacArthur Foundation. This year-long program provides training in research design, laboratory techniques, and data analysis. As a followup to this training, CERC staff help the trainees establish research programs in their home countries. This assistance ranges from technical guidance to the actual purchase and outfitting of small laboratories to do the work. This program has trained researchers from Indonesia, Malaysia, Thailand, Vietnam, and China, and the CERC training staff now includes a postdoctoral scientist from Sri Lanka. Out of this program is a rapidly growing regional cadre of researchers who publish in peer-reviewed international journals (Wang and others 1997). This group is likely to have a major effect on future decisions about conservation within Southeast Asia.
Conclusion.
In this chapter, we have highlighted the important uses of genetic analyses to define the units of conservation and the units of management, the geographic locations of those units, and the ways in which genetic variation is distributed within and among the populations that make up each unit. This discussion and the examples we have offered are meant to provide a brief introduction to the nonspecialist reader and to highlight the value of these approaches for wildlife managers, other conservation practitioners, and environmental policy-makers. It is important, however, to point out that other biological disciplines (for example, morphological systematics and behavioral ecology) also contribute significantly to the definition of evolutionary distinctiveness and that many other considerationssuch as overall evolutionary uniqueness, current vulnerability, and socio-cultural valuemust be considered when we are developing protective measures for a particular population or species. Ultimately, we must apply as much information as possible to decisions about designing and launching conservation efforts. We hope it is clear that genetic analysis is a powerful and timely mechanism for generating a great deal of valuable information for the purpose of conservation.
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