150. Woodruff, D.S. Biodiversity: conservation and genetics. In: Environment, Science and Technology: The Challenge of the 21st Century. Vol. 1, Proceedings of the 2nd Princess Chulabhorn Science Congress. Bangkok, Thailand, November 2-6, 1992. Chulabhorn Research Institute, Bangkok, pp. 589-598. (1999d).

REVIEW ARTICLE

A 149

BIODIVERSITY: CONSERVATION AND GENETICS

DAVID S. WOODRUFF

Department of BiologyUniversity of California, San DiegoLa Jolla, California 92093-0116USA

1. INTRODUCTION

The biodiversity of Thailand is the country's most undervalued and neglectedresource, both biologically and economically.l Yet the nation's plants and animals,the communities they form, and the ecological services they provide are major assetsthat have direct bearing on the quality of life today and in the future. Much of theTwentieth Century was devoted to building a global consensus on the value ofnature conservation, and even in Thailand where environmental damage has beenenormous, the century closes with increased recognition that nature conservation isin the national interest. Government condoned environmental destruction anddegradation is increasingly unacceptable to the population at large and major firststeps have been taken to begin to protect what remains of the national heritage.Although this increased love and appreciation of nature are critical prerequisites forenvironmental conservation they are unfortunately insufficient to insure futuregenerations the wealth and services of nature. Attention in Thailand and elsewheremust now shift to developing and implementing the science and technology ofbiodiversity conservation and community restoration. Environmental managementfor human welfare requires more than just a change in societal and political attitudes:nature, now disturbed, needs our active interventive stewardship. The purpose ofthis paper is to draw attention to the hitherto widely ignored genetic aspects ofbiodiversity conservation that will require more attention in the next century.Integrating genetics into biodiversity conservation efforts constitutes one of the greatchallenges of the Twenty-first Century -- a fitting focus for the Chulabhorn ResearchInstitute and this Congress on environment, science and technology.

2. BIODIVERSITY, GENETIC DIVERSITY AND FUTURE LIFE ON EARTH

The biosphere is that portion of the planet where physical conditions permitlife. These conditions, of temperature, oxygen concentrations, etc., occur andpersist only because of the activities of living organisms over the last 3.5 billionyears. This fact, that life sustains itself on an otherwise inhospitable planet,

provides the most important argument for the conservation of nature. The futurehabitability of our only home depends on our living in harmony with the planet'sother organisms.

The biological diversity (biodiversity for short) of the planet embraces thevariety and assemblages of organisms at all levels of taxonomic and ecologicalcomplexity. It involves the genetic variants (mutants, strains, races, subspecies)belonging to the same species as well as the arrays of species and their highertaxonomic groupings as monerans, protists, plants, fungi and animals. As very fewspecies can exist alone in nature, biodiversity also embraces the co-evolvedmultispecies associations and communities responsible for ecosystem functioning.Biodiversity thus embraces far more than just the species which are so often thefocus of conservation efforts. Excellent reviews of the status of biodiversity areprovided elsewhere.2-5

I will not belabor the nature and magnitude of the global biodiversity crisis.Its significance is underscored by the 1992 UN Conference on Environment andDevelopment - the first true Earth Summit Locally, the situation in Thailand isworse than in many countries: the largest mammals, birds, reptiles and fish are goneor endangereds and the country's stocks of ecological capital have been depleted tothe point where human lives are lost and rural communities are becomingdysfunctional. Natural resource based industries like forestry and tourism have beenseriously mis-managed for private profit rather than sustainability for the publicgood. Suffice here to note that species extinctions and community degradation arenot inevitable results of development. InThailand and elsewhere trends are beingreversed and biodiversity conservation is beginning to receive national politicalattention.

AIL'ioughmost discussions of the biodiversity crisis focus on habitats andspecies, conservationists are ultimately concerned with the genetic resourcesunderlying nature. Genetic diversity of individuals, populations and speciesprovides the raw materials from which our domesticated crops, animals andmicrobes can be artificially selected. The clearest arguments for the valuation ofgenetic resources come from specific agricultural, pharmaceutical andbiotechnological examples.Zf Less thought has been given, until recently, to therole of this same genetic diversity in enabling species to persist, to continue toevolve in nature in response to environmental change. If natural communities withhundreds of interacting species provide valuable ecological services then the geneticunderpinnings of wild plants and animals are also of concern to biologists anddevelopers. In the present century biodiversity conservation has been left primarilyto ecologists, in the next century nature will also require genetic management.Without such intervention species will continue to be lost and communities collapse.Genetic engineering will have to add population and species-level conservation to itstraditional concerns.? The urgent challenge is now to develop the science ofconservation genetics together with the appropriate technology to prevent theirreplaceable loss of more species, communities and ecological services. Failure toaccept stewardship of the genetic resources underpinning biodiversity wi11leavehumans on a duller and more dangerous planet

3. CONSERVATION OF GENETIC DIVERSITY

Extinction, as Lande!" has noted, is fundamentally a demographic processinfluenced by genetic and environmental factors. Before discussing these geneticfactors it is important to reiterate his opinion that for wild populations in natural orseminatural environments, demography is likely to be of more immediate importancethan genetics in determining population viability. Genetic factors do not figureamong the four major courses of extinction (the Evil. Quartet): overkill, habitatdestruction and fragmentation, impact of introduced species, and secondary orcascade effects. I I Thus, although genetic factors are major determinants of apopulation's long-term viability, conservationists can do more for a threatenedpopulation in the short-term by managing its ecology. Ecological management isthe cheapest and most effective way of conserving genetic diversity.

Having noted the interdependence of ecology and genetics in determining apopulation's viability let us now examine two aspects of the conservation of geneticdiversity that require scientific research and technology development. The firstinvolves the mitigation of the effects of genetic erosion in small populations. Thesecond concerns the recognition and genetic management of evolutionarilysignificant units (species, races, etc.).

Genetic Erosion.

I follow OuborgI2 in defining genetic erosion as the decrease inheterozygosity and the loss of alleles from a population due to random genetic driftand inbreeding. These inexorable processes that rob small isolated populations oftheir viability are insignificant in larger populations and in populations connectedwith one another as a metapopulation by frequent gene flow. Genetic drift (chanceloss of alleles during meiosis) leads to decreased heterozygosity and reduced geneticdiversity without regard for whether the alleles lost are beneficial or deleterious.Inbreeding (mating of related individuals) also leads to a decrease in the frequency ofheterozygotes. It may also lead to an increase of genetic disease as recessivedeleterious alleles are exposed in homozygous form. Such alleles. the genetic loadof all species, are routinely purged by natural selection in large outbred populationsand are rarely noticed. However, when historically outbred populations crashfollowing range destruction and fragmentation, the frequencies of deleterious allelescan increase by drift and inbreeding faster than they can be culled. Genetic erosionthus leads to reduced individual and population viability.

In theory, the rate of genetic erosion is simply related to effective populationsize (Ne) such that the change in frequency of heterozygotes is 1/(2NeJ. An effectivepopulation of 10 will therefore lose heterozygotes five times faster than an effectivepopulation of 100. Note that in this formulation we are using the effectivepopulation size (NeJ not the CCii5US population size (N). Ne is almost always lessthan N as a variety of life history factors will reduce the number of geneticallyeffective or different individuals in a given population. The predictions ofpopulation genetic theory are that small populations lose genetic variations faster

than large populations and have lower levels of variation (heterozygote frequencyand allelic diversity). Genetic erosion in small recently fragmented populations maythus contribute to their extinction.

The potentially deleterious consequences of genetic erosion are predicted bythe widely accepted axiom that genetic variability is positively correlated withindividual and population fitness. A central premise of evolutionary theory holdsthat genetic variability (especially additive genetic variation) is a prerequisite foradaptation by evolution. Without the variability to work on natural selection can dolittle to improve a population's chances of surviving significant environmentalchange. It follows that small, isolated populations have higher probabilities ofextinction than large widespread populations. Although this argument is supportedby theory and many observations it is not without its critics and conservationistsshould be aware of these controversies.U Some species with long histories ofinbreeding show little sign of genetic erosion and, in other species, individual fitnessdoes not appear to be related to genetic variability. In still other cases demographicbottlenecks have actually increased the additive genetic variation in a population andthus countered the predicted effects of genetic erosion.l+ It is important toremember that there have been very few studies of genetic erosion in nature. Untilmore research is undertaken and the apparent paradoxes resolved, genetic erosionmust be regarded as a potential but still unproven general enhancing factor in theextinction process.

Such scientific uncertainties are typical in conservation biology and noexcuse for inaction. The possibility that genetic erosion is already threateningfragmented populations of Thailand's endangered species is too serious to wait forthe resolution of the general issues.6,I5-I6 As noted at the outset, managers can takeimmediate action to relieve the pressures on a population's genetic diversity byconfronting the Evil Quartet, by securing the population ecologically. With time,they can begin to monitor population's genetic viability and counter loss of variationwith natural or artificial gene flow. Thais should not lose the opportunity to savewhat is left of their genetic resources by waiting for the development of a bettertheoretical understanding of the role of genetic erosion in human inducedextinctions. Although I have here followed convention in arguing that geneticerosion works primarily by reducing individual fitness it is possible that it could actin other ways. For example, if loss of genetic variation makes individuals moresusceptible to environmental variance then genetic erosion will be far more importantduring this time of major global climatic change than it has been in the past

Evolutionarily Significant Units.

If populations and metapopulations are the units of ecology, subspecies andspecies the units of systematists, and races and varieties the units of agriculturalists,what units should conservation biologists focus their attention on. The answer, Ibelieve, does not matter, as long as the units selected involve individuals with acommon recent genetic history. Our goal should be to conserve naturalevolutionarily significant units (ESUs) regardless of how they originated or theircurrent taxonomic status. Our goal should be to conserve evolutionary potential, a

process requiring genetic diversity, rather than very narrowly defmed types or kindsof organisms.8,17

Notwithstanding the need to focus on processes (ecosystem functioning andevolutionarily significant units) it is clear that for the foreseeable future mostbiodiversity conservation efforts will concentrate on species - the fundamental unitsof nature. The power of the species-level focus is well illustrated by regionalSpecies Survival Plans, national Endangered Species Acts and the internationalConvention on International Trade in Endangered Species. Species inventories areused to identify biodiversity "hot-spots" and species distributions are mapped toidentify "gaps" in the protected area systems.

Species level conservation plans have both strengths and weaknesses. AsThailand is beginning to place more emphasis on species management it is importantto review the weaknesses of this approach. First, the theoretical underpinnings ofthe species concept are still under active development Experts still disagree on justhow species originate and how they should be defined.If Numerous speciesconcepts are debated among evolutionary biologists and even the currently mostpopular biological, cohesion and phylogenetic species concepts may be hard to applyto real world situations. Conservation biologists cannot wait for the resolution ofthese issues; their energies and the enabling legislation behind their efforts must beunfettered by such scientific debates. Second, the use of the species level approachrequires' conservationists to make choices among the many species requiringattention. For pragmatic reasons we must allow that all species are not created equaland carefully select those whose management might conserve whole ecosystems.Such choices are very difficult and different groups around the world areexperimenting with programs targeting different types of species includingecologically pivotal keystone species, umbrella species with Iarge arearequirements, charismatic megavertebrate flagship andfocal species, and speciesthat are especially vulnerable to human activitles.t? Other groups are trying toidentify and conserve ecologically sensitive management indicator species. Almosteverywhere evolutionary relics (living fossils) and local endemics are affordedspecial consideration. Rarity per se may not be a sufflcient criterion to meritinterventive management.w With limited resources conservationists in Thailandmust now try to objectively establish their priorities for effective biodiversityconservation in the next century. Genetic data, as discussed below, comes into playonce the hard choices have been made.

Evolutionarily significant units, once selected for conservation management,require prompt genetic assessment. Whatever their current taxonomic designation(species, subspecies, unnamed but isolated geographic race) it is important to verifytheir genetic integrity and innate variability. The consequences of failing to carry outa full genetic study at this stage can be expensive, embarrassing and lead to loss ofgenetic resources and extinction. Consider the following examples:

• outbreeding depression led to the failure of a reintroduction program involvingibex, Capra ibex, in Czechoslovakia. The program involved genetically distantTurkish and Nubian subspecies; their hybrids were so poorly adapted that theentire population went extinct 8

• outbreeding depression led to reproductive waste (unnecessary deaths) in aspider monkey, Ateles sp., breeding colony when different chromosomal typeswere inadvertently mixed} 7

• failure to recognize the natural pattern of genetic variation in an endangeredSonoran topminnow, Poeciliopsis occidentalis, in Arizona and Mexico led to arestocking plan with a l,righprobability of failure.l?

The prime purpose of genetic screening early in any taxon-based conservation effortis therefore to preclude the inadvertent mixing of well-differentiated groups within asingle management program. This is especially important when the programinvolves both in situ and ex situ efforts and reintroductions and translocations arecontemplated. Comprehensive genetic screening will also provide baseline data oninnate genetic variation and its partitioning within and between populations(population structure).

Traditional taxonomy is often misleading when it comes to definingevolutionarily significant units for conservation management. Most vertebrates andflowering plants received their taxonomic names long before the development of thebiological species concept. Even conspicuous, well-known species may require re-assessment The magnitude of the genetic difference between the two subspecies oforangutan, Pongo pygmaeus+ and between the West African and the othersubspecies of chimpanzee, Pan troglodytest) suggests that they require separatemanagement. Large genetic distances (proportional to evolutionary divergence froma common ancestor) must be taken into account for effective conservationmanagement

Genetic relationships between individuals, populations, subspecies andspecies can be established by a number of laboratory methods. Karyotypes,allozymes, mitochondrial DNA RFLP (restriction fragment length polymorphism)patterns, mitochondrial and nuclear gene sequences have all been used effectively.With sufficient tissue, time, expertise and money the genetic parameters of interest toconservationists can all be estimated. That this was not done routinely in the pastreflects a failure of managers to appreciate the risks involved in ignoring geneticsand a real shortage of sufficiently motivated geneticists. Another problem is moreinsidious and reflects the inadequacies of the infant science of conservation genetics.Often laboratory studies provided managers with uninterpretable data or addedunacceptable complexities to on-going programs. In this regard it is worth notingthat genetic distance data alone may not answer the key question about an ESU'shomogeneity. The event of speciation is not simply related to genetic distance:although large distances may indicate significant genetic differentiation accompaniedby speciation, small distances do not necessarily indicate that populations arecon specific, Interpretation of genetic data for management purposes typicallyrequires a simultaneous assessment of other patterns of variation (morphology,distribution, ecology and behavior). Morphology, the criterion used traditionally todefine species is often unreliable, but coupled with genetic patterns permits therecognition of ESUs in some of the taxonomically most challenging syngameons.t

I have argued that species generally provide the best focus for taxon-orientedconservation but subspecific variants and unnamed isolated populations (restricted toislands, mountain tops, river basins, etc.) may be equally meritorious. Again,taxonomic status of the target population is less important than the ecosystem and thenumber of other species that will benefit indirectly from the conservation program.What is important, is that target taxon be genetically defined and representative ofsome natural ESU. Subsequent management of the ESU would then seek tomaintain or restore the genetic variability of the original population(s). Care wouldalso be taken to avoid "enlarging" the ESU by hybridization with individuals fromanother species or subspecies. Failure to manage genetic integrity along these linesresulted in the contamination of one line of endangered Przwalski's horse withdomestic horse genes and may have contributed to the extinction of the duskyseaside sparrow. 22 In a last ditch attempt to save the latter species from the eastcoast of Florida, the last birds were crossed with a subspecies from the west coast ofFlorida rather than the more closely related Atlantic coast subspecies from furthernorth. Had genetic relationships among the taxa been established earlier this mistakecould have been avoided.

In managing threatened ESUs the creation of "generic" populations _mixtures of previously isolated and well-differentiated subspecies, for example _should be avoided. It is justified only when there are no alternatives as hybrid orgeneric taxa are clearly preferable to the groups extinction. Such artificial hybridtaxa and their natural equivalents can not be excluded from conservationconsideration simply because they lack racial "purity". Such a notion is obsolete andruns counter to our present appreciation of the importance of conserving as muchgenetic diversity as possible.

It is not the purpose of this paper to discuss the theory and methods ofmanaging genetic diversity once it is identified and characterized. For recentdiscussions of population viability analysis and metapopulation analysis and thetechnologies of captive propagation the reader is referred elsewhere.23-29 Suffice itto note that the conservation of genetic diversity requires a multidisciplinarysynthetic approach combining the best basic science with the pragmatism of thetraditional nature manager.

4. RESEARCH INVOLVING THAI ANIMALS

I now offer a few examples from my own research to illustrate theimportance of considering genetic factors in biodiversity conservation. Theexamples were chosen to illustrate three common problems that managers will haveto deal with in the next century: proper identification of ESUs, selection ofappropriate individuals for reintroduction programs, and assessing levels ofvariation remaining in remnant populations. In some cases the research was basedon proven methods, in others it is frankly experimental. In all cases it involvedcollaboration between Thai scientists and managers and members of my laboratorygroup in the U.S. In each case the expertise and technology has or will betransferred to laboratories in Thailand.

Proper Identification of EvolutionarilySignificant Units.

Three studies of freshwater molluscs and their parasites illustrate theimportance of proper identificationof evolutionarily significant units even though theanimals themselves are not the subject of conservation efforts. First, allozymevariation was used to show that 21 nominal species of Thai clams, Corbicula, areactually all members of the same conchologically variable species, C.fluminea.30This means that an effort to save the clams previously referred to C. erosa and foundonly in Glaeng district, Rayong province, would betaxonomically unjustified,More generally, the taxonomic revision of the clams indicates that attempts toconserve the traditional "species" for their own sake would have been misguided.Second, a similar allozyme study of a small snail in the Mekong and Mun riversrevealed that "Neotricula aperta" is actually comprised of at least four separatesibling species.t! Misidentification of ESUs in this case has important biomedicalimplications as one or more members of the species group is the intermediate host ofthe human blood fluke, Schistosoma mekongi, The third example involves thissnail-transmitted parasite which had been confused with the species S.japonicumfrom Japan, China and the Philippines. Allozyme variation shows clearly that theThai schistosome is a different species and that it is more closely related to S.malayensis than the widespread S. Iaponicum.n These three examples show howboth taxonomic lumping and splitting can affect the definition of ESUs forconservation purposes. Each case involved an animal that was relatively well-known in Thailand (as a food source, disease vector, or pathogen) but the use oftraditional non-genetic taxonomy would have resulted in the adoption ofinappropriate units for conservationmanagement.

Selection of AppropriatelyMatched Individuals for Reintroduction.

Translocation and reintroduction are increasingly important conservationmanagement tools. Two on-going projects illustrate the need to consider geneticfactors in planning such actions. The first case involves the threatened Eld' s brow-antlered deer, Cervus eldi, and the Royal Forest Department's propagation project atPhu Khieo Wildlife Sanctuary in northeast Thailand. This is within the traditionalrange of the Southeast Asian subspecies, C. e. siamensis, which has been extirpatedin Thailand. Deer moved to Phu Khieo include Laotian C. e. siamensis, andindividuals of C. e. thamin, a subspecies centered in Myanmar that may survive inthe ranges of western Thailand. Two questions arise: are the Burmese and Siamesesubspecies genetically compatible and is it alright to release the western subspeciesinto the former range of the eastern subspecies? A very preliminary geneticcomparison in my laboratory suggests that the two subspecies are more differentthan expected: 13 differences including 2 transversions were detected among 167base-pairs of the mitochondrial cytochrome b gene sequence studied (Carlos Garza,pers. comm.). If this result were confirmed and extended then it would seemgenetically inadvisable to mix the subspecies. Similarly, it may be preferable toreintroduce Laotian deer to Phu Khieo rather than Burmese as long as the former areavailable.

The second case involves the genetic management of captive gibbons,Hylobates. The Thai Zoological Organization has long had a serious problem withabandoned pet gibbons; in the mid-1980's up to 20 per month would be abandonedat or turned over to the Dusit Zoo, Bangkok. CITES restrictions prevented theseanimals from being placed in captive breeding programs outside Thailand and theThai zoos were ill-equipped to house, let alone breed, these threatened apes. Onelong-term solution under discussion by officials of the Zoological Organization, theRoyal Forestry Department, concerned NGO's and primatologists would involverehabilitating and reintroducing selected individuals to sanctuaries within theirformer range. One problem with this laudable idea is that the animals themselves areof unknown geographic origin and there is some concern about inadvertently mixinggibbons of diverse origin. White-handed gibbons, H. lar, for example, range over1500 km from north to south in Thailand and several geographically defined butmorphologically indistinguishable subspecies have been named - the effects ofreleasing northern gibbons into south Thailand and vice versa, and the viability ofintersubspecific hybrids are unknown. The solution to this problem is to learn thegeographic patterns of genetic variation in this species and use these data to establishthe probable region of origin of all candidates for reintroduction. Our first attempt todo this failed; the 252 base-pair segment of the cytochrome b gene used was simplynot informative enough.V A second attempt with a more variable gene sequence isnow underway and promises to permit the sorting of captive far gibbons intoappropriate genetic groups.

Assessing Levels of Genetic Variation in Isolated Populations.

Managers need to be able to assess the extent and rate of genetic erosion inrecently fragmented and isolated populations. This has not been attempted inThailand or elsewhere to any significant extent because of technical obstacles.Animals had to be darted or captured and bled, and the tissue sample had to befrozen immediately and transported to the laboratory on dry ice or liquid nitrogen.For most free-ranging mammals and birds in remote areas this is too difficult tocontemplate. We have overcome these problems by developing a non-invasivegenotyping method which uses hair, feathers and even feces as the DNA source.34We are now demonstrating the method's utility for assessing levels of geneticvariation in recently fragmented populations of small mammals in Khlong SaengWildlife Sanctuary. At the same time we are testing theoretical predictions aboutrates of genetic erosion by monitoring these populations for their first 10 years post-isolation. The particular populations under investigation became isolated on smallislands in Chiew Lam Reservoir wben the River was dammed in 1987. The on-going extinction processes in these forest fragments are described elsewhere. 15.35-6It is premature to describe our genetic results and their significance.

5. CONCLUSION

The above research projects are suggestive of the urgent and enormouschallenge confronting scientists and managers in Thailand. To an outsider like

myself it is clear that in the last decade, despite horrific setbacks, there is a netimprovement in national efforts to conserve biodiversity. Increased interest amongstudents and academics, increased public perception of the importance ofbiodiversity, increased interagency cooperation on conservation projects, improvedlegal framework supporting conservation efforts, improved implementation ofgovernment policies (a narrowing of the gap between words and deeds), increasedpublic intolerance for government condoned destruction of natural resourcesincluding biodiversity are-among the highly encouraging signs that Thailand willtake the necessary steps to conserve her natural heritage, The research priorities andinfrastructure needs to meet this challenge are clearly defined in recent reports of theScience Society of Thailand and the Royal Forest Departrnenut-t? what remains tobe seen is how fast these recommendations will be addressed and implemented.

Thailand is very fortunate in having a Royal Family whose members playmajor roles in national conservation efforts. Their leadership has enabled numerousforeign scientists like myself to make small contributions to such important nationalefforts. Meeting the environmental challenges of the Twenty-first Century, asProfessor Dr. HRH Princess Chulabhorn correctly noted requires the cooperativeefforts of numerous specialists; it has been a privilege to collaborate with my Thaicolleagues in their critical endeavor. Environmental conservation and natureprotection are global problems and together we must seek both local and globalsolutions.

ACKNOWLEDGEMENTS

My collaborative research in Thailand would not have been possible withoutpermission of the National Research Council and the collaboration and support ofnumerous colleagues including: Warren Brockelman, Alongkorn Mahannop, ChiraMeckvichai, Jarujin Nabhitabhata, the late Seub Nakhasathien, Schwarm Tunhikorn,Suchart Upatham and Sawai Wanghongsa.This work was supported by grants fromU.S.A.I.D., U.S.N.S.F. and the University of California.

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