Mary V Ashley. American Scientist. Volume 87, Issue 1. Jan/Feb 1999.
Although the black rhinoceros (Diceros bicornis) was once distributed throughout most of sub-Saharan Africa, fewer than 5,000 individuals remain today, scattered in small, isolated groups. Like the other four species of rhinoceros, black rhinos are threatened not so much by loss of habitat as by poaching. Because their horns are valued in many parts of Asia for medicinal purposes, the taking of all species, ages, genders and sizes is a lucrative business.
Protecting the remaining black rhinos became a priority in the late 1980s.Unfortunately, the isolation and small size of the groups presented significant problems for wildlife managers. The rhinos were vulnerable not only to poaching but also to demographic and environmental fluctuations as well as inbreeding. Consolidating groups would have eased such problems, but the black rhino posed one major complication. The species had been divided into several named subspecies based on small variations in external morphology, such as horn shape and body size.
Did the different subspecies represent genetically or ecologically distinct units? If so, conservation strategies should probably have included separate management of each subspecies. Alternatively, were there only very small genetic differences among the named subspecies? If so, it was likely that the remaining populations were recently connected by gene flow and might be considered a single population for breeding purposes.
Such are the sorts of questions that molecular conservation genetics attempts to answer through an increasing variety of techniques. Such methods support in several ways the attempts of conservation biologists to maintain the distinctiveness of unique evolutionary lineages and genetic diversity within these lineages. Toward the first goal, molecular genetics can provide information for reconstructing evolutionary trees (phylogenies) that identify and relate evolutionary lineages. The levels of genetic divergence among such lineages, including populations, subspecies or species, aid in determining the units that should be the focus of conservation, as in the case of the black rhino. Molecular-genetic analyses can also directly measure genetic variation in species of interest and characterize how that variability is partitioned within and among populations of that species, providing clues to the likelihood of long-term survival and to the evolutionary flexibility of that species.
I shall return shortly to the plight of the black rhino and other endangered species. For the moment, however, let us look more closely at the sorts of tools used to help determine the most effective means of ensuring their survival.
It seems that every year or so a “hot” new method for studying or manipulating DNA comes along, leaving biologists scrambling to keep up. One technique that has been widely used for almost two decades in one form or another is the analysis of a genome located outside the nucleus of the cell, in the cellular organelles called mitochondria. Mitochondria generate adenosine triphosphate (ATP), an energy-rich molecule used to fuel cellular activities. For reasons that are not well understood, mitochondria have retained a handful of genetic information needed for the functioning of the organelle. The mitochondrial genome of vertebrates is a circular molecule of DNA about 16,000 nucleotide base pairs in length. The nucleotides-adenosine, guanine, cytosine and tyrosine (A, G, C and T)-pair in specific patterns, which can be altered by mutation. The genome is inherited only from the mother and codes for a total of 37 genes, probably only 0.1-0.2 percent of the genes present in the average vertebrate; the rest are found on the chromosomes in the nucleus of the cell and are inherited in two copies, one from the mother and one from the father.
The DNA found in the mitochondria (mtDNA) has several features that make it especially useful for evolutionary biologists and conservation geneticists. One of the most useful is its rapid rate of evolution; it accumulates mutations at a rate roughly five to ten times that of a typical nuclear gene. This high rate of mutation, generally in the form of single base-pair changes (or small insertions and deletions), allows differences to be easily detected between the mtDNAs of closely related species or even individuals of the same species. Such high resolution is crucial for conservation genetics, since an important goal is to identify evolutionary units or management units within species. Further, the small genome of the mitochondria does not undergo recombination, a phenomenon that complicates studies of nuclear genes. Thus the maternal inheritance of mtDNA provides a relatively unambiguous picture of the evolutionary history of female lineages.
One of the first mtDNA studies of an endangered species was my work on the black rhinoceros described in the introduction to this article. I chose to analyze mtDNA because its rapid mutation rate ensured that if the rhinos had separate evolutionary histories for a considerable period of time, the mtDNA should be one of the first DNA regions to accumulate mutational differences. Representatives of three of the original seven subspecies remain-Diceros bicornis, found in Namibia, D.b. minor, ranging from Kenya to South Africa, and D.b. michaeli, found in Kenya and Tanzania. (Most black rhinos in North American and European zoos belong to D.b. michaeli, so this subspecies is the principal focus of captive-breeding programs.)
I was able to examine the mtDNA of 23 black rhinos from D.b. minor and D.b. michaeli using a technique called restriction-fragment-length polymorphism (RFLP) analysis. Restriction enzymes occur naturally in bacteria and have the unusual property of recognizing specific short DNA sequences, usually four to six base pairs in length, and then cutting the DNA double helix wherever these sequences occur. One can survey the mtDNA of each individual in a study with a suite of restriction enzymes, each recognizing and cutting the DNA at different places where their recognition sequences occur. If there have been mutations in the mtDNA of an individual, restriction fragments may appear that are not present in other individuals. The proportion of shared restriction fragments can then be used as an estimate of DNA similarity between any two mtDNA types.
Using 18 restriction enzymes, I found that black rhinoceros ranging from Kenya to South Africa showed very few mtDNA differences, regardless of subspecies designation or geographic origin. In fact, only three distinct mtDNA types were found, and these differed from each other by less than 0.4 percent of their base-pair sequences. This suggested a very close genetic relationship among populations of black rhinos and provided no evidence of multiple evolutionary units. The genetic risks of mixing and interbreeding animals from different populations-if such mixing proved beneficial for management and protection-were probably small.
Along Came PCR
Although RFLP analysis still sees common use, the development of the polymerase chain reaction (PCR) in the late 1980s changed the way molecular biologists studied DNA, and it was not long before this new technology became standard practice in conservation genetics as well. PCR allows an investigator to obtain many, many copies of a particular gene or DNA region from only the tiniest amount of starting material. Thus the DNA sequence of the PCR-amplified target can be obtained directly, without the need for laborious cloning procedures. Still focusing on mtDNA, many conservation genetics researchers switched from RFLP analysis to PCR amplification and DNA sequencing of the mitochondrial genes themselves. This provided even greater resolution of the genetic structure and evolutionary history of species.
One such study conducted in my lab yielded results that in many ways were the opposite those in the black rhino work. The focus was the small, nocturnal owl monkey (genus Aotus), which is widespread in the neotropics from Panama to northern Argentina. Traditionally, all owl monkeys were thought to be a single species, Aotus trivirgatus. More recently, cytogenetic studies done by Nancy Shui-Fong Ma while at the New England Primate Research Center examined the chromosomes of owl monkeys and revealed that animals from different parts of their range had remarkably different sets of chromosomes. Individual monkeys were found to contain between 23 and 29 chromosome pairs arranged in at least 12 different sets, or karyotypes. It was unknown whether each karyotype belonged to a distinct species or subspecies. As a result, taxonomists argued that Aotus could be divided into as many as nine distinct species. Aotus taxonomy had become a mess, and from a conservation standpoint it was important to determine whether management practices should reflect multiple reproductively isolated species or a single wide-ranging species connected by gene flow.
I sequenced a PCR-amplified mitochondrial gene, cytochrome oxidase subunit II (COII), from owl monkeys originating in different geographic regions that represented several different karyotypes. For comparison, I also sequenced this gene from two other neotropical monkeys-the squirrel monkey (Saimiri sciureus) and Goeldi’s monkey (Callimino goeldii). The results were striking. Unlike the black rhinoceros situation, the mtDNA sequences from some of the different owl monkeys were quite distinct, differing by up to 6 percent in this gene.
Figure 1 shows a phylogenetic tree constructed from the sequences. The most important finding is that the length of the branches connecting different owl monkey lineages are approximately one-third as long as those connecting Aotus with the unrelated species of monkeys. This suggests that the different owl monkey lineages have been diverging for about one-third the time since the common ancestor of all neotropical monkeys lived. The date for this common ancestor is not well established, but both paleontological and molecular studies place it in the range of 18-30 million years before present. Thus owl monkey lineages must have separated some 6-10 million years ago. Such a long period of evolutionary isolation is likely accompanied by many unique ecological adaptations and reproductive isolation. The mtDNA evidence suggests that, although owl monkeys may look very similar, there are probably distinct species, which should be reflected in assessments of their conservation status in the wild and in future management plans.
PCR technology not only eliminated laborious cloning procedures, but it also opened the door for genetic analysis of new sample sources. Rather than the fresh blood or frozen organ tissue previously needed, dried museum skins, shed or plucked hairs from living animals, or even fresh feces could provide the starting material for genetic analysis. One outcome was the emergence of a new field in conservation biology: molecular wildlife forensics.
As is the case with the rhinoceros, many endangered species are threatened by exploitation for commercial products such as ivory, meat, feathers, fins and shells. Although many such species are protected by international regulations, compliance to these regulations is difficult to monitor. Wildlife forensics employs molecular-genetic techniques to help in the identification of commercial products from protected species. C. Scott Baker from the University of Auckland in New Zealand and his colleagues have focused their work on illegal whaling. Despite a worldwide moratorium on commercial whaling in 1986, whale meat is still sold in foreign fish markets. Is this whale meat from whales hunted legally (for example, Southern Hemisphere minke whales taken for scientific purposes by Japan), from bycatch of other species or from the illegal harvest of whales? The answer lies in identifying the species showing up in markets.
Using PCR and mtDNA sequencing, Baker examined the only portion of the mitochondrial genome that does not code for functional gene products, a rapidly evolving segment called the control region. Rather than containing genes, the control region houses the sites for regulating replication of mtDNA. They first established a data base of control-region sequences from known whale species and then purchased whale meat (including dried, marinated meat as well as raw slices) from retail markets in Japan and Korea. By comparing the control-region sequences of the commercial products with their whale sequence data base, they were able to identify Northern Hemisphere minke, fin and humpback products that had either been illegally hunted or had been in storage for seven or eight years (since before the moratorium). The latter explanation being unlikely, they concluded that illegal whaling continued.
In another study, Rob DeSalle and Vadim Birstein, with the American Museum of Natural History, analyzed mtDNA sequences amplified from black caviar purchased in gourmet shops in New York City. The found that many of the products were actually derived from sturgeon species other than the one claimed on the label. Consequently, accurate monitoring of the production and trade of black caviar will likely be best accomplished by DNA analysis.
Microsatellites-The Newest Tool
Although PCR amplification of mtDNA followed by direct DNA sequencing has become the workhorse of conservation genetics, mtDNA does have several limitations. Because mtDNA is maternally inherited, it does not provide a direct record either of the transmission of genes through male lineages or of gene flow that occurs through male dispersal.
Further, conservation biologists are concerned with problems of loss of genetic variability in endangered species, but mtDNA variation may be a poor indicator of nuclear genetic variation. Even though there are many copies of mtDNA in every individual, these copies are usually identical, so the mitochondrial genome acts as an effectively haploid (having a single set of chromosomes) genome. When a few individuals move into a new region founding a new population with only a small subset of the total genetic variations found in the initial population (called founder events), or during periods when conditions allow only a few individuals to survive (called population bottlenecks), there will generally be a greater impact on mtDNA diversity than on nuclear-DNA diversity. And there is no evidence suggesting that reduced levels of mtDNA alone are detrimental to a population. Geneticists must turn to the nuclear genome for additional insight.
The bulk of nuclear DNA has long been known to be noncoding-that is, it neither gets translated into polypeptide chains nor codes for RNAs that are used in cell function. The purpose, if any, of this so-called “junk DNA” is unknown, but much of it is highly repetitive, present in many copies scattered around or clustered on the chromosomes. One class of junk called microsatellite DNA was discovered about the same time that PCR technology was developed. Microsatellites are extremely short, repeated sequences, with the repeated unit being 1-6 base pairs in length. For example, a stretch of DNA often contains sequences such as CACACACACACA or ATTATTATTATTATTATTATTATT. The number of repeat units tends to change quite often as a result of mutation, so that for many microsatellite loci most individuals may be heterozygous (have different versions of the same genes), and there may be a dozen or more different alleles (forms of a gene) in a population at that locus. The different alleles for microsatellite loci reflect the number of repeat units, for example CA^sub 6^ or CA^sub 8^.
Microsatellites have proved useful for applications ranging from genome mapping to paternity testing. Primers for PCR amplification are made complementary to sequences that flank the microsatellite repeat, and these are used to amplify the microsatellite locus for all the individuals in a study. The length of the PCR product will be determined by the number of repeat units. Microsatellite alleles are transmitted as standard Mendelian traits, with one allele coming from each parent. Their high levels of variability make them extremely useful in determining relatedness among individuals and in characterizing the genetic structure of a species.
One application of microsatellite analysis was to help design management strategies for the Mexican gray wolf (Canis lupis bayileyi). Now extinct in the wild, a few wolves, known as “certified lineage,” were captured from Mexico in the late 1970s and brought to the U.S. to initiate a captive-breeding program. The certified lineage, which now numbers more than 100 animals, is descended from one adult male, one young male and a pregnant female whose wild mate was not captured. Two other lineages, Ghost Ranch and Aragon, were thought to be Mexican gray wolves as well, but the three lineages were never interbred because of concerns about ancestry and hybridization with domestic dogs or coyotes in one or both of these lineages.
If the purity of these lineages as Mexican gray wolves could be established by molecular analysis, combining lineages would increase levels of genetic variation and reduce the likelihood of inbreeding depression. This might be crucially important for the success of the reintroduction program, since all three lineages were founded by very few individuals and have experienced moderate to high levels of inbreeding.
Microsatellite results from work done by Jaime Garcia-Moreno and Robert Wayne (University of California, San Diego) and colleagues in 1996 and by Phillip W. Hedrick, from the University of Arizona, and colleagues in 1997 that the three putative Mexican wolf lineages were genetically similar to each other and distinct from other gray wolves, coyotes and dogs. These genetic data also showed that it was unlikely that any of the lineages had hybridized with coyotes or dogs. The investigators therefore propose that animals from Ghost Ranch and Aragon be crossed into the certified lineages. Furthermore, even though the founders of the certified lineage are long dead, the microsatellite data could be used to infer that the young male brought in from the wild was probably the pup of the pregnant female that was also captured. The number of genetic founders of the certified lineage was therefore lower than previously believed, and introducing new genes from the Ghost Ranch and Aragon lineages may be even more important. (Postscript: Eleven individuals in three Mexican gray wolf family groups were reintroduced to southeastern Arizona in late March 1998.)
In Situ Management
Although captive breeding and reintroduction may successfully reestablish some highly endangered species in the wild, many conservation biologists have come to question the effectiveness of this approach as a general conservation strategy. Many argue, instead, for in situ management of species in their native habitat, occupying all or part of their former range. This approach may demonstrate the greatest strength of molecular genetics in conservation, since DNA studies can often provide information on basic demographic properties of organisms that are difficult to study by traditional observational methods. The mating system of the species, the relative reproductive success of different individuals and the degree of genetic isolation of different populations are often crucial for developing appropriate management plans, and these parameters can all readily be investigated using molecular genetics.
An extensive study of wild chimpanzees (Pan troglodytes) was conducted by Phillip Morin, now with Sequana Therapeutics Inc., and his colleagues. They performed both microsatellite genotyping and mtDNA sequencing to examine genetic variation, social behavior and phylogenetic relationships among chimpanzees from 20 sites in Africa. A detailed look at the genetics of the Gombe National Park population (the group made famous by Jane Goodall’s long-term behavioral study) demonstrated that males were more related to each other than were females, supporting observational data that groups of related males cooperate to defend territories and thereby increase their access to resources and females.
Sequence data from mtDNA samples collected from a larger sample of African chimpanzees supported the division of P. troglodytes into three distinct and genetically differentiated subspecies, but populations within each subspecies were genetically connected, probably through females who occasionally disperse long distance and mate. Morin and his colleagues propose that the West African subspecies, P. t. verus, is genetically so different from the other two subspecies that it should be elevated to a full species. A remarkable feature about the chimpanzee study is that it was conducted without disturbing the free-ranging animals, using samples collected noninvasively. Chimpanzees make fresh sleeping nests in trees each night, and hairs used for genotyping were collected from the nests after the chimpanzees had left in the morning.
Most conservation biologists would agree that stemming the assaults on biodiversity by habitat destruction and fragmentation, human population growth and over-exploitation is of much greater concern than the genetics of individual species. Nevertheless, as with the black rhinoceros, management often works species by species. The new tools of molecular biology can lead to a much clearer understanding of the biology of such threatened species and aid in the development of more effective strategies for protection and management.