Using Molecular Techniques to Answer Ecological Questions

As far back as the late 1800s, researchers realized that answers to some ecological questions could be obtained by examining the molecular composition of organisms. One of the earliest attempts at using molecules to address an ecological question was by Church in the late 1860s. Church studied relationships among birds and found that the pigment turacin was present only in birds of the Musophagidae family (Figure 1). He and others went on to determine that evolutionary relationships could be inferred according to whether species shared particular molecules. Early studies were limited to organic molecules obtained through diet and so may at times have confused relationships among organisms. However, the idea that the study of molecules could be a useful technique for understanding animals, their relationships, and their evolution had been firmly planted within the mind of the scientific community. And it is from this idea that the discipline of molecular ecology eventually emerged.

What exactly is molecular ecology? As we will see, it is an interdisciplinary approach to some of the most fundamental questions in organismal biology. Some scientists today do not consider it to be its own discipline but rather an "approach" taken in certain cases to answer certain questions. Most scientists, however, agree that it is distinct from other studies of organismal biology. Molecular ecology is defined as "the application of molecular techniques to answer ecological questions" (Beebee & Rowe 2004). In this article we explore the tools used in molecular ecology and how these tools enhance traditional ecological studies. We examine a number of seminal studies that have used molecular ecology tools. We also discuss the limitations of molecular ecology.

Molecular ecology's development as a field of study can be seen to run parallel to advances in what would become the tool of the trade: the molecular marker. Molecular markers are sections of an organismal genome. These sections of DNA can be more readily obtained through a procedure known as the polymerase chain reaction (PCR) (Figure 2). There are many different types of DNA markers used in molecular ecology, including: microsatellites (highly repetitive sequences of DNA that mutate rapidly and are often used to identify individuals), minisatellites (similar to microsatellites but with longer repetitive sequences), restriction fragment length polymorphisms (RFLPs, specific sites of DNA that can be cut by enzymes yielding different-sized fragments of DNA in different species, populations, and — rarely — individuals), and DNA sequence data (the bases of DNA are determined and similarities and differences are compared to identify species, populations, and individuals) (Figure 3). These markers are by no means a comprehensive list, and the marker (or markers) one chooses to use depends greatly on the type of question being addressed in the study.

One of the reasons why molecular ecology has advanced rapidly as a field of study is the advent of PCR. PCR makes it possible to amplify billions of copies of a specific piece of DNA from the genome with very few starting copies. In other words, it is possible to take a small sample of tissue to obtain enough DNA for study. This contrasts with earlier approaches that often required large amounts of DNA or protein, which often meant killing the organism of study. Obviously, killing one's study organism can be counter-productive, particularly if the intent of the study is to advance conservation efforts or protect endangered species. The fact that only a small starting amount of DNA is needed now for molecular ecology studies has opened the door for non-invasive sampling methods. It is now possible to isolate DNA from hair, urine, shed skin, and feces, thus preventing harm to endangered and non-endangered species. PCR also amplifies old and/or degraded DNA, such as that found in fossils (Figure 2).

The development of molecular markers has led to an explosion of studies that have used them to answer questions ranging from relatedness among species, to the evolutionary history of populations, the amount of genetic variation within a species, patterns of behavior, how patterns of gene expression can vary among closely related populations, and many other aspects of organismal variation. For example, in one of the earliest molecular ecological studies, O'Brien and his colleagues (1983) found that the genetic diversity among cheetahs in South Africa was extremely low (Figure 4). In fact, O'Brien et al. (1985) transplanted skin grafts between different cheetahs and found that the cheetahs were so genetically similar, their immune systems did not reject the tissue grafts. This and other early studies triggered the debate, which continues today, over the importance of genetic diversity to the persistence of a population and how genetic diversity is related to environmental change. If every organism in a population has the same genetic make up, it is likely each will respond the same way to any environmental change. If this environmental change hinders an organism's ability to survive or reproduce, it is probable that all genetically similar individuals in the population will be affected in the same way, thus increasing the chance of extinction. However, this is not always the case since some genetically similar populations appear to thrive. As such, the importance of genetic diversity for population survival continues to be a subject of debate.

Molecular techniques can be useful to, and sometimes necessary for, the field of ecology. Traditional approaches to ecology have limitations that can sometimes be addressed with molecular techniques. For one, traditional ecological approaches have relatively narrow timeframes of observation. Unless a long history of data has been directly collected on a particular organism through the years, traditional ecological approaches are limited to the period over which a study is conducted. Extrapolations can be made, but support for these extrapolations can be tenuous. On the other hand, historical events leave distinct signatures in the molecules of organisms that can be accurately interpreted.

The divergence time between species, for example, can be readily calculated provided the divergence can be associated with significant historical and environmental events. For instance, if one can associate the divergence between two species with a known geologic event, one can calibrate a molecular clock. In turn, a molecular clock can be used to determine the time since other species diverged from each other based on the amount of genetic differences observed at a specific molecular marker. Beerli et al. (1996) were able to calibrate a protein clock for populations of the Rana esculenta frog group separated by saltwater barriers in the Aegean Sea using known geological dates of island isolation. Since frogs can't survive in salt water, they were most likely separated when these salt water barriers appeared. As a result, the researchers were able to date the separation of frogs on these islands. They then measured how many genetic differences exist between these groups and used these data to estimate how many genetic changes occurred over time.

A second important limitation of traditional ecological approaches is dependence on direct observation. One of the most commonly used methods of tracking animal movements and immigration into new populations is telemetry (Figure 5). Simply understanding an animal's physical movement from one place to another, however, often gives an incomplete picture of that animal's behavior and how it relates to its environment. One fundamental limitation of telemetry is that it cannot detect whether a dispersing animal successfully mates in its new territory or upon joining a new population. Reproductive success is an indication of fitness and, therefore, of the long-term survival of a population — a fundamental issue in ecology. But a DNA-based approach can provide much more insight into the mating behaviors of dispersing animals. By looking at molecular markers in a particular group of animals, researchers can establish the familial relationships among the members of that group; therefore, they can get an accurate picture of who is mating with whom. Recent immigrants will have slight differences in their molecular markers and they can be identified. If those immigrants are mating successfully in their new group, those differences will be passed onto their offspring and will appear more frequently. If immigrants do not mate, those differences will disappear.

Finally, since traditional ecological approaches are based on direct observations of organisms, they frequently do not detect underlying variation in organisms that does not influence physical appearances. It is possible for organisms to appear the same physically while exhibiting as much genetic divergence as found between distinct species. Cryptic variation can sometimes only be detected by comparing DNA.

These examples are intended to illustrate how molecular techniques can supplement and enhance information gained through traditional methodologies. We can get an even better idea of how molecular techniques have refined traditional ecology by looking at some of the fundamental questions answered by molecular ecology.

Some of the earliest molecular ecology studies involved examining the mating systems of birds. It was long thought that birds were monogamous. DNA samples of parents and their offspring challenged this idea. Indeed, it was found that extra-pair copulations were very frequent among bird species thought to be monogamous. As a result of the extensive use of DNA sampling of parents and their offspring, it is now understood that only a very small number of bird species adhere to a strictly monogamous mating system. Mating behaviors have also been described using molecular techniques in many other organisms including (among others) pipefish, frogs, beetles, and turtles.

Our understanding of habitat use has changed as the result of molecular techniques. Habitat use can be relatively simple to assess when animals can be tracked and directly observed using their habitat. But what happens when specific patterns of habitat use cannot be directly observed? There have been a number of studies on genetic structuring of populations that appeared to be uniformly distributed across a particular landscape. These studies found that populations were highly structured, indicating that organisms preferred to settle and mate in certain habitats over others. These findings contradicted observed distribution patterns at the landscape level because observational approaches were unable to track mating patterns.

Early studies involving use of molecular techniques also found evidence of extreme variation among apparently physically-identical individuals within a species. Indeed, several studies among salamanders of the genus Plethodon demonstrated extreme genetic divergence among animals that appeared identical. These early studies also demonstrated a mixture of different Plethodontid genomes within individuals, providing evidence of hybridization between different species, information that was not yet available by traditional ecological methods. Identifying cryptic species has become extremely important in the field of conservation genetics, especially with regard to the protection of endangered species. For example, the spotted frog Rana pretiosa, which occurs in the Pacific Northwest of the United States, displays almost no physical variation among populations (Figure 6). However, genetic analysis has revealed that populations in Oregon are as genetically distinct from other populations in the Pacific Northwest as separate species. The discovery of genetic divergence within the species led to the classification of two separate species, both of which are now protected. The existence of cryptic species is not surprising given that these organisms, and many others, often rely on non-visual cues, such as chemicals and/or sound, to identify a mate. Molecular ecology has allowed researchers to explore why there are genetic differences in the absence of morphological differences. Not only does molecular ecology allow the detection of genetic differences, but it also allows for the interpretation of how the differences came to be since certain phenomena (e.g., natural selection, mate choice, differential habitat use) may leave different genetic signatures.

Molecular approaches have also played a significant role in endangered species conservation besides addressing the question of cryptic variation. For example, molecular studies have have been used to identify migration corridors between populations that can prevent isolation of endangered populations. Molecular approaches have also been used to identify ideal populations for transfer to extinct or declining populations, methods for maximizing genetic variation in captive-bred animals, and species identification of contraband goods. Identifying species has been especially important for prosecuting poachers for harvesting endangered species when the only remains of the harvested animal is a piece of meat, bone, or fur. In fact, molecular techniques have been developed that can identify species based on the DNA from tissue even if the tissue has been cooked and mixed with other ingredients.

Molecular ecology has important limitations to consider. First, marker development can be time-consuming and expensive. Second, while it can be beneficial that molecular ecology is not dependent on direct observation of behaviors, this benefit can often be a limitation. Since the behavior is not directly observed, scientists must deduce the behavior that led to a specific molecular pattern, and there can often be multiple explanations for the same observed pattern. Third, it is not practical to look at the entire genome of all organisms, so one must look at a small subset of markers. Different markers may show discordant patterns or may not be representative of the entire genome. Finally, there are some questions that molecular ecology simply cannot answer and must be addressed with direct observation. For example, some behaviors important to the natural history of an organism, such as parental care and courtship behavior, can only be documented through direct observation.