Freeland - Molecular Ecology (Wiley, 2005) - Chapter 1 pps

1 Molecular Genetics in Ecology What is Molecular Ecology? Over the past 20 years, molecular biology has revolutionized ecological research. During that time, methods for genetically characterizing individuals, populations and species have become almost routine, and have provided us with a wealth of novel data and fascinating new insights into the ecology and evolution of plants, animals, fungi, algae and bacteria. Molecular markers allow us, among other things, to quantify genetic diversity, track the movements of individuals, measure inbreeding, identify the remains of individuals, characterize new species and retrace historical patterns of dispersal. These applications are of great academic interest and are used frequently to address practical ecological questions such as which endangered populations are most at risk, from inbreeding, and how much hybridization has occurred between genetically modified crops and their wild relatives. Every year it becomes easier and more cost-effective to acquire molecular genetic data and, as a result, laboratories around the world can now regularly accomplish previously unthinkable tasks such as identifying the geographic source of invasive species from only a few samples, or monitoring populations of elusive species such as jaguar or bears based on little more than hair or scat samples. In later chapters we will take a detailed look at many of the applications of molecular ecology, but before reaching that stage we must first understand just why molecular markers are such a tremendous source of information. The simplest answer to this is that they generate data from the infinitely variable deoxyribo- nucleic acid (DNA) molecules that can be found in almost all living things. The extraordinarily high levels of genetic variation that can be found in most species, together with some of the methods that allow us to tap into the goldmine of information that is stored within DNA, will therefore provide the focus of this chapter. We will start, however, with a retrospective look at how Molecular Ecology Joanna Freeland # 2005 John Wiley & Sons, Ltd. the characterization of proteins from fruitfly populations changed forever our understanding of ecology and evolution. The Emergence of Molecular Ecology Ecology is a branch of biology that is primarily interested in how organisms in the wild interact with one another and with their physical environment. Historically, these interactions were studied through field observations and experimental manipulations. These provided phenoty pic data, which are based on one or more aspects of an organism’s morphology, physiology, biochemistry or behaviour. What we may think of as traditional ecological studies have greatly enhanced our knowledge of many different species, and have made invaluable contributions to our understanding of the processes that maintain ecosystems. At the same time, when used on their own, phenotypic data have some limitations. We may suspect that a dwindling butterfly population, for example, is suffering from low genetic diversity, which in turn may leave it particularly susceptible to pests and pathogens. If we have only phenotypic data then we may try to infer genetic diversity from a variable morphological character such as wing pattern, the idea being that morphologically diverse populations will also be genetically diverse. We may also use what appear to be population-specific wing patterns to track the movements of individuals, which can be important because immigrants will bring in new genes and therefore could increase the genetic diversity of a population. There is, however, a potential problem with using phenoty pic data to infer the genetic variation of populations and the origins of individuals: although some physical characteristics are under strict genetic control, the influence of environmental conditions means that there is usually no overall one-to-one relationship between an organism’s genotype (set of genes) and its phenotype. The wing patterns of African butterflies in the genus Bicyclus, for example, will vary depending on the amount of rainfall during their larval development period; as a result, the same genotype can give rise to either a wet season form or a dry season form (Roskam and Brakefield, 1999). The potential for a single genotype to develop into multiple alternative phenoty pes under different environmental conditions is known as phenotypic plasticity. A spectacular example of phenotypic plasticity is found in the oak caterpillar Nemoria arizonaria that lives in the southwest USA and feeds on a few species of oaks in the genus Quercus. The morphology of the caterpillars varies, depending on which part of the tree it feeds on. Caterpillars that eat catkins (inflorescences) camouflage themselves by developing into catkin-mimics, whereas those feeding on leaves will develop into twig mimics. Experiments have shown that it is diet alone that triggers this developmental response (Greene, 1996). The difference in morphology between twig-mimics and catkin-mimics is so pro- nounced that for many years they were believed to be two different species. There 2 MOLECULAR GENETICS IN ECOLOGY is also a behavioural component to these phenotypes, because if either is placed on a part of the tree that it does not normally frequent, the catkin-mimics will seek out catkins against which to disguise themselves, and the twig-mimics will seek out leaves or twigs. Some other examples of phenotypic plasticity are given in Table 1.1. Phenotypic plasticity can lead to overestimates of genetic variation when these are based on morphological variation. In addition, phenotypic plasticity may obscure the movements of individuals and their genes between populations if it causes the offspring of immigrants to bear a closer resemblance to individuals in their natal population than to their parents. Complex interactions between genotype, phenoty pe and environment provided an important reason why biologists sought long and hard to find a reliable way to genotype wild organisms; genetic data would, at the ver y least, allow them to directly quantify genetic variation, and to track the movements of genes and therefore individuals or gametes between populations. The first milestone in this quest occurred around 40 years ago, when researchers discovered how to quantify individual genetic Table 1.1 Some examples of how environmental factors can influence phenotypic traits, leading to phenotypic plasticity Environmental Characteristic influence Example Gender Temperature during embryonic development Eggs of the American snapping turtle Chelydra serpentina develop primarily into females at cool temperatures, primarily into males at moderate temperatures, and exclusively into females at warm temperatures (Ewert, Lang and Nelson, 2005) Growth patterns in plants Soil nutrients and water availability Southern coastal violet (Viola septemloba) allocated a greater proportion of biomass to roots and rhizomes in poor-quality environments (Moriuchi and Winn, 2005) Leaf size Light intensity Dandelions (Taraxacum officinale) produce larger leaves under conditions of relatively strong light intensity (Brock, Weinig and Galen, 2005) Migration between host plants Age and nutritional quality of host plants Diamond-back moths (Plutella xylostella) are most likely to migrate as adults if the juvenile stage feed on mature plants (Campos, Schoereder and Sperber, 2004). Feeding-related morphology Food availability Sea-urchin larvae (Strongylocentrotus purpuratus and S. franciscanus) produce longer food-gathering arms and smaller stomachs when food is scarce (Miner, 2005) Plumage colouration Carotenoids in diet The plumage of male house finches (Carpodacus mexicanus) shows varying degrees of red, orange and yellow depending on the carotenoids in each bird’s diet (Hill, Inouye and Montgomerie, 2002) THE EMERGENCE OF MOLECULAR ECOLOGY 3 variation by identifying structural differences in proteins (Harris, 1966; Lewontin and Hubby, 1966). This discovery is considered by many to mark the birth of molecular ecology. Protein allozymes In the 1960s a method known as starch gel electrophoresis of allozymic proteins was an extremely important breakthrough that allowed biologists to obtain direct information on some of the genetic properties of individuals, populations, species and higher taxa. Note that we are not yet talking about DNA markers but about proteins that are encoded by DNA. This distinction is extremely important, and to eliminate any confusion we will take a minute to review the relationship between DNA, genes and proteins. Prokaryotes, which lack cell nuclei, have their DNA arranged in a closed double-stranded loop that lies free within the cell’s cytoplasm. Most of the DNA within the cells of eukaryotes, on the other hand, is organized into chromosomes that can be found within the nucleus of each cell; these constitute the nuclear genome (also referred to as nuclear DNA or nrDNA). Each chromosome is made up of a single DNA molecule that is functionally divided into units called genes. The site that each gene occupies on a particular chromosome is referred to as its locus (plural loci). At each locus, different forms of the same gene may occur, and these are known as alleles. Each allele is made up of a specific sequence of DNA. The DNA sequences are determined by the arrangement of four nucleotides, each of which has a different chemical constituent known as a base. The four DNA bases are adenine (A), thymine (T), guanine (G) and cytosine (C), and these are linked together by a sugar phosphate backbone to form a strand of DNA. In its native state, DNA is arranged as two strands of complementary sequences that are held together by hydrogen bonds in a double-helix formation (Figure 1.1). No two alleles have exactly the same DNA sequence, although the similarity between two alleles from the same locus can be very high. The function of many genes is to encode a particular protein, and the process in which genetic information is transferred from DNA into protein is known as gene expression. The sequence of a protein-coding gene will determine the structure of the protein that is synthesized. The first step of protein synthesis occurs when the coding region of DNA is transcribed into ribonucleic acid (RNA) through a process known as transcription. The RNA sequences, which are single stranded, are complementar y to DNA sequences and have the same bases with the exception of uracil (U), which replaces thymine (T). After transcription, the introns (non- coding segments of DNA) are excised and the RNA sequences are translated into protein sequences following a process known as translation. Translation is possible because each RNA molecule can be divided into triplets of bases (known as codons), most of which encode one of 20 different amino acids, which are the constituents of proteins (Table 1.2). Transcription and 4 MOLECULAR GENETICS IN ECOLOGY translation involve three types of RNA: ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA). Ribosomal RNA is a major component of ribosomes, which are the organelles on which mRNA codons are translated into proteins, i.e. it is here that protein synthesis takes place. Messenger RNA molecules act as templates for protein synthesis by carrying the protein-coding information that was encoded in the relevant DNA sequence, and tRNA molecules incorporate particular amino acids into a growing protein by matching amino acids to mRNA codons (Figure 1.2). Specific combinations of amino acids give rise to polypeptides, which may form either part or all of a particular protein or, in combination with other molecules, a protein complex. If the DNA sequences from two or more alleles at the same locus are sufficiently divergent, the corresponding RNA triplets will encode different amino acids and this will lead to multiple variants of the same protein. These variants are known as allozymes. However, not all changes in DNA sequences will result in different proteins. Table 1.2 shows that there is some redundancy in the genetic code, e.g. leucine is specified by six different codons. This redundancy means that it is possible for two different DNA sequences to produce the same polypetide product. GC AT A T C G TA C G G C A T 5´ 3´ 5´ 3´ CG AT 5´ 3´ 3´ 5´ A C C T T G A G C T A G G A A C T C G A T C T G B)A) AT GC Sugar− phosphate backbone Hydrogen bond Figure 1.1 (A) A DNA double helix. Each sequence is linked together by a sugar–phosphate backbone, and complementary sequences are held together by hydrogen bonds; 3 0 and 5 0 refer to the orientation of the DNA: one end of a sequence has an unreacted 5 0 phosphate group and the other end has an unreacted 3 0 hydroxyl group. (B) Denatured (single-stranded) DNA showing the two complementary sequences. The DNA becomes denatured following the application of heat or certain chemicals THE EMERGENCE OF MOLECULAR ECOLOGY 5 Allozymes as genetic markers The first step in allozyme genotyping is to collect tissue samples or, in the case of smaller species, entire organisms. These samples are then ground up with appropriate buffer solutions to release the proteins into solution, and the allozymes then can be visualized following a two-step process of gel electrophoresis Table 1.2 The eukaryotic nuclear genetic code (RNA sequences): a total of 61 codons specify 20 amino acids, and an additional three stop-codons (UAA, UAG, UGA) signal the end of translation. This genetic code is almost universal, although minor variations exist in some microbes and also in the mitochondrial DNA (mtDNA) of animals and fungi Amino acid Codon Amino acid Codon Leucine (Leu) UUA Arginine (Arg) CGU UUG CGC CUU CGA CUC CGG CUA AGA CUG AGG Serine (Ser) UCU Alanine (Ala) GCU UCC GCC UCA GCA UCG GCG AGU AGC Valine (Val) GUU Threonine (Thr) ACU GUC ACC GUA ACA GUG ACG Proline (Pro) CCU Glycine (Gly) GGU CCC GGC CCA GGA CCG GGG Glutamine (Gln) CAA Aspartic acid (Asp) GAU CAG GAC Asparagine (Asn) AAU Glutamic acid (Glu) GAA AAC GAG Lysine (Lys) AAA Cysteine (Cys) UGU AAG UGC Ty rosine (Tyr) UAU Histidine (His) CAU UAC CAC Isoleucine (Ile) AUU Phenylalanine (Phe) UUU AUC UUC AUA Methionine (Met) AUG a Tryptophan (Trp) UGG a Codes for Met when within the gene and signals the start of translation when at the beginning of the gene. 6 MOLECULAR GENETICS IN ECOLOGY and staining . Electrophoresis refers here to the process in which allozymes are separated in a solid medium such as starch, using an electric field. Once an electric charge is applied, molecules will migrate through the medium at different rates depending on the size, shape and, most importantly, electrical charge of the molecules, characteristics that are determined by the amino acid composition of the allozymes in question. Allozymes then can be visualized by staining the gel with a reagent that will acquire colour in the presence of a particular, active enzyme. A coloured band will then appear on the gel wherever the enzyme is located. In this way, allozymes can be differentiated on the basis of their structures, which affect the rate at which they migrate through the gel during electrophoresis. Genotypes that are inferred from allozyme data provide some information about the amount of genetic variation within individuals; if an individual has only one allele at a particular locus then it is homozygous, but if it has more than one allele at the same locus then it is heterozygous (Figure 1.3). Furthermore, if enough individuals are characterized then the genetic variation of populations can be quantified and the genetic profiles of different populations can be compared. This distinction between individuals and populations will be made repeatedly throughout this book because it is fundamental to many applications of molecular ecology. Keep in mind that data are usually collected from individuals, but if the sample size from any given population is big enough then we often assume that the rRNA (ribosomal) tRNA (transfer) mRNA (messenger) Ribosome Translation Protein DNA Transcription Figure 1.2 DNA codes for RNA via transcription, and RNA codes for proteins via translation THE EMERGENCE OF MOLECULAR ECOLOGY 7 individuals collectively provide a good representation of the genetic properties of that population. We will return to allozymes in subsequent chapters, but at this point it is enoug h to realize that the identification within populations of multiple allozymes (alleles) at individual loci was a seminal event because it provided the first snapshot of genetic variation in the wild. In 1966, one of the first studies based on allozyme data was conducted on five populations of the fruitfly Drosophila pseudoobscura. This revealed substantially higher levels of genetic variation within populations than were previously believed (Lewontin and Hubby, 1966). In this study 18 loci were characterized from multiple individuals, and in each population up to six of these loci were found to be polymorphic (having multiple alleles). There was also evidence of genetic variation within individuals, as revealed by the observed heterozygosity (H o ) values, which are calculated by averaging the heterozygosity values across all characterized loci (Table 1.3). Although unarguably a major breakthrough in population genetics, and still an important source of information in molecular ecology, allozyme markers do have some drawbacks. One limitation is that, as we saw in Table 1.2, not all variation in Allele A Allele A Allele A Allele A Allele B Allele A Allele B Allele B Allele A Allele B Allele C Allele B Locus 1 Locus 2 Locus 3 Locus 1 Locus 2 Locus 3 Individual 1 Individual 2 Figure 1.3 Diagrammatic representation of part of a chromosome, showing which alleles are present at three loci. Individual 1 is homozygous at loci 1 and 3 ( AA in both cases) and heterozygous at locus 2 ( AB ). Individual 2 is homozygous at locus 1 ( BB ) and heterozygous at locus 2 ( BC ) and locus 3 ( AB ) Table 1.3 Levels of polymorphism and observed heterozygosity ( H o ) at 18 enzyme loci calculated for five populations of Drosophila pseudoobscura (data from Lewontin and Hubby, 1966). This was one of the first studies to show that genetic variation in the wild is much higher than was previously believed Number of Proportion of Observed Population polymorphic loci polymorphic loci heterozygosity Strawberry Canyon 6 6/18 ¼ 0.33 0.148 Wildrose 5 5/18 ¼ 0.28 0.106 Cimarron 5 5/18 ¼ 0.28 0.099 Mather 6 6/18 ¼ 0.33 0.143 Flagstaff 5 5/18 ¼ 0.28 0.081 8 MOLECULAR GENETICS IN ECOLOGY DNA sequences will translate into variable protein products, because some DNA base changes w ill produce the same amino acid following translation. A wealth of information is contained within every organism’s genome, and allozyme studies capture only a small portion of this. Less than 2 per cent of the human genome, for example, codes for proteins (Li, 1997). The acquisition of allozyme data is also a cumbersome technique because organisms often have to be killed before adequate tissue can be collected, and this tissue then must be stored at very cold temperatures (up to À70  C), which is a logistical challenge in most field studies. These drawbacks can be overcome by using appropriate DNA markers, which are now the most common source of data in molecular ecology because they can potentially provide an endless source of information, and they also allow a more humane approach to sampling study organisms. In the following sections, there- fore, we shall switch our focus from proteins to DNA. An Unlimited Source of Data Even very small organisms have extremely complex genomes. The unicellular yeast Saccharomyces cerevisiae, despite being so small that around four billion of them can fit in a teaspoon, has a genome size of around 12 megabases (Mb; 1 Mb ¼ 1 million base pairs) (Goffeau et al., 1996). The genome of the considerably larger nematode worm Caenorhabditis elegans, which is 1 mm long, is approximately 97 Mb (Caenorhabditis elegans Sequencing Consortium, 1998), and that of the flowering plant Arabidopsis thaliana is around 157 Mb (Arabidopsis Genome Initiative, 2000). The relatively enormous mouse Mus musculus contains some- where in the region of 2600 Mb (Waterston et al., 2002), which is not too far off the human genome size of around 3200 Mb (International Human Genome Mapping Consortium, 2001). Within each genome there is a tremendous diversity of DNA. This diversity is partly attributable to the incredible range of functional products that are encoded by different genes. Furthermore, not all DNA codes for a functional product; in fact, the International Human Genome Sequencing Con- sortium has suggested that the human genome contains only around 20 000 25 000 genes, which is not much more than the $19 500 found in the substantially smaller C. elegans genome (International Human Genome Sequencing Consor- tium, 2004). Non-coding DNA includes introns (intervening sequences) and pseudogenes (derived from functional genes but having undergone mutations that prevent transcription). Many stretches of nucleotide sequences are repeated anywhere from several times to several million times throughout the genome. Short, highly repetitive sequences include minisatellites (motifs of 10 100 bp repeated many times in succession) and microsatellites (repeated motifs of 1 6 bp). Another class of repetitive gene regions that has been used sometimes in molecular ecology is middle-repetitive DNA. These are sequences of hundreds or thousands of base AN UNLIMITED SOURCE OF DATA 9 pairs that occur anywhere from dozens to hundreds of times in the genome. Examples of these include the composite region that codes for nuclear ribosomal DNA (Figure 1.4). In contrast, single-copy nuclear DNA (scnDNA) occurs only once in a genome, and it is within scnDNA that most transcribed genes are located. The proportion of scnDNA varies greatly between species, e.g. it comprises approximately 95 per cent of the genome in the midge Chironomus tentans but only 12 per cent of the genome in the mudpuppy salamander Necturus maculosus (John and Miklos, 1988). Although the structure and function of genes vary between species, they are typically conserved among members of the same species. This does not, however, mean that all members of the same species are genetically alike. Variations in both coding and non-coding DNA sequences mean that, with the possible exception of clones, no two individuals have exactly the same genome. This is because DNA is altered by events during replication that include recombination, duplication and mutation. It is worth examining in some detail how these occur, because if we remain ignorant about the mechanisms that generate DNA variation then our understanding of genetic diversity will be incomplete. Mutation and recombination Genetic variation is created by two processes: mutation and recombination. Most mutations occur during DNA replication, when the sequence of a DNA molecule is used as a template to create new DNA or RNA sequences. Neither reproduction nor gene expression could occur without replication, and therefore its importance cannot be overstated. During replication, the hydrogen bonds that join the two strands in the parent DNA duplex are broken, thereby creating two separate strands that act as templates along which new DNA strands can be synthesized. The mechanics of replication are complicated by the fact that the synthesis of new strands can occur only in the 5 0 3 0 direction (Figure 1.5). Synthesis requires an enzyme known as DNA polymerase, which adds single nucleotides along the template strand in the order necessary to create a complementary sequence in which G is paired with C, and A is paired with T (or U in RNA). Successive nucleotides are added until the process is complete, by which time a single parent NTS ETS 18S 5.8S 28S ITS1 ITS2 Figure 1.4 Diagram showing the arrangement of the nuclear ribosomal DNA gene family as it occurs in animals. The regions coding for the 5.8S, 18S and 28S subunits of rRNA are shown by bars; NTS ¼ non-transcribed spacer, ETS ¼ external transcribed spacer and ITS ¼ internal transcribed spacer. The entire array is repeated many times 10 MOLECULAR GENETICS IN ECOLOGY [...]... rule (Figure 1. 7) Part of the challenge to finding suitable genetic markers for ecological research involves Comparison between harbour seal and grey seal Comparison between fin whale and blue whale Comparison between mouse and rat Percentage sequence divergence 0.3 0.2 0 .1 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Pairwise comparisons of 18 different mitochondrial regions Figure 1. 7 Sequence... contains fragments that are 1, 5, and 10 bp long, then the first, fifth and tenth bases in the sequence must be adenine (A) The fragments from each of the four reactions can be pieced together to recreate the entire sequence (Figure 1. 11) G 10 A T C AGGCATCGTA AGGCATCGT 8 AGGCATCG 7 AGGCATC 6 AGGCAT 5 AGGCA 4 AGGC 3 AGG 2 AG 1 Increase in fragment size (bases) 9 A Figure 1. 11 Representation of a sequencing... Hartl, D.L and Jones, E.W 19 98 Genetics: Principles and Analysis, (4th edn) Jones and Bartlett Publishers, Boston, MA Li, W.-H 19 97 Molecular Evolution Sinauer Associates, Sunderland, Massachusetts Review articles Baker, G.C., Smith, J.J and Cowan, D.A 2003 Review and re-analysis of domain-specific 16 S primers Journal of Microbial Methods 55: 5 41 555 Benard, M.F 2004 Predator-induced phenotypic plasticity... Annual Review of Ecology, Evolution, and Systematics 35: 6 51 673 Gachon, C., Mingam, A and Charrier, B 2004 Real-time PCR: what relevance to plant studies? Journal of Experimental Botany 55: 14 45 14 54 REVIEW QUESTIONS 29 Gugerli, F., Parducci, L and Petit, R.J 2005 Ancient plant DNA: review and prospects New Phytologist 16 6: 409 418 ´ ¨¨ Paabo, S., Poinar, H., Serre, D., Jaenicke-Despres, V., Hebler,... sequencing reaction gives target DNA sequence fragments of the following sizes: Reaction Reaction Reaction Reaction with with with with ddG: 2, 4, 5 and 8 bases ddA: 6, 7, 9, 13 and 14 bases ddT: 1, 3, 12 and 15 bases ddC: 10 and 11 bases What is the sequence of this region of DNA? ... with short-wavelength ultraviolet light The sizes of DNA bands then can be extrapolated from ladders that consist of DNA fragments of known sizes (Figure 1. 10) If the amplified products are of variable AN UNLIMITED SOURCE OF DATA 23 Negative electrode Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Wells into which samples are loaded Size markers (bases) 500 400 300 200 10 0 Positive electrode Figure 1. 10 Representation... and Bunnell, 19 94), 22 MOLECULAR GENETICS IN ECOLOGY but further investigation showed that the most likely source of this DNA was human contamination (Zischler et al., 19 95) Nevertheless, characterization of ancient DNA has been successful on numerous occasions, for example DNA sequences from 3000-year-old moas provided novel insight into the evolution of flightless birds (Cooper et al., 19 92), and ancient... used in molecular ecology CHAPTER SUMMARY 27 Chapter Summary  Before the emergence of molecular ecology it was very difficult to obtain genetic data from wild populations, and biologists often had to rely on visible polymorphisms Phenotypic data are useful for many things, although phenotypic plasticity often obscures the relationships between phenotypes and genotypes  The first studies to link molecular. .. Step 1 Denaturation Reverse primer Step 1 Denaturation Original DNA sequence Figure 1. 8 The first two cycles in a PCR reaction Solid black lines represent the original DNA template, short grey lines represent the primers and hatched lines represent DNA fragments that have been synthesized in the PCR reaction Forward primer Original template DNA Primer Newly synthesized DNA 18 MOLECULAR GENETICS IN ECOLOGY. .. as EMBL-Bank)  DNA Data Bank of Japan (DDBJ): www.ddbj.nig.ac.jp  The Web Guide of Polymerase Chain Reaction: www.pcrlinks.com  List of primer-design software provided by the UK Human Genome Mapping Project Resource Centre: http://www.hgmp.mrc.ac.uk/GenomeWeb/ nuc-primer.html  ClustalX (Thompson et al., 19 97), software for aligning sequences: ftp://ftpigbmc.u-strasbg.fr/pub/ClustalX/  Molecular . heterozygosity Strawberry Canyon 6 6 /18 ¼ 0.33 0 .14 8 Wildrose 5 5 /18 ¼ 0.28 0 .10 6 Cimarron 5 5 /18 ¼ 0.28 0.099 Mather 6 6 /18 ¼ 0.33 0 .14 3 Flagstaff 5 5 /18 ¼ 0.28 0.0 81 8 MOLECULAR GENETICS IN ECOLOGY DNA sequences. (Figure 1. 7). Part of the challenge to finding suitable genetic markers for ecological research involves Pairwise comparisons of 18 different mitochondrial regions 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 . 1 Molecular Genetics in Ecology What is Molecular Ecology? Over the past 20 years, molecular biology has revolutionized ecological research. During