Mitochondrial DNA control region polymorphisms: genetic markers for ecological studies of marine turtles

We describe a rapid and sensitive method for the detection of population-specific genetic markers in mitochondrial DNA (mtDNA) and the use of such markers to analyse population structure of marine turtles. A series of oligonucleotide primers specific for the amplification of the mtDNA control region in Cheloniid turtles were designed from preliminary sequence data. Using two of these primers, a 384–385-bp sequence was amplified from the 5′ portion of the mtDNA control region of 15 green turtles Chelonia mydas from 12 different Indo-Pacific rookeries. Fourteen of the 15 individuals, including some with identical whole-genome restriction fragment patterns, had sequences that differed by one or more base substitutions. Analysis of sequence variation among individuals identified a total of 41 nucleotide substitutions and a 1-bp insertion/deletion. Comparison with evidence from whole-genome restriction enzyme analysis of the same individuals indicated that this portion of the control region is evolving approximately eight times faster than the average rate and that the sequence analysis detected approximately one fifth of the total variation present in the genome. Restriction enzyme analysis of amplified products from an additional 256 individuals revealed significant geographic structuring in the distribution of mtDNA genotypes among five of the 10 rookeries surveyed extensively. Additional geographic structuring of genotypes was identified through denaturing gradient gel electrophoresis (DGGE) of amplified products. Only two of the 10 rookeries surveyed could not be differentiated, indicating that the Indo-Pacific C. mydas include a number of genetically differentiated populations, with minimal female-mediated gene flow among them. Important applications for genetic markers in the conservation and management of marine turtles include the identification of appropriate demographic units for research and management (i.e. genetically discrete populations) and assessment of the composition of feeding and harvested populations.     

Rapid assessment of single-copy nuclear DNA variation in diverse species

We investigated the use of PCR primers designed to conserved exons within nuclear DNA to amplify potentially variable regions such as introns or hypervariable exons from a wide range of species. We then explored various approaches to assay population-level variation in these PCR products. Primers designed to amplify regions within the histone H2AF, myoglobin , MHC DQA , and aldolase (ALD) genes gave clean amplifications in diverse mammals (DQA) , and in birds, reptiles and mammals ( aldolase, H2AF, myoglobin ). The sequenced PCR products generally, but not always, confirmed that the correct locus had been amplified. Several primer sets produced smaller size fragments consistent with preferential amplification of intronless pseudogenes; this was confirmed by sequencing seal and reptile H2AF PCR products. Digestion with randomly selected four-base recognizing enzymes detected variation in some cases but not in others. In species/gene combinations with either low (e.g. seal H2AF, ALD-A ) or high (e.g. skink ALD-1 ) nucleotide diversity it was more efficient to sequence a small number of distantly related individuals (e.g. one per geographic population) and from these data to identify informative or potentially informative restriction enzymes for 'targeted' digestion. We conclude that for studies of population-level variation, the optimal approach is to use a battery of primers for initial PCR of both mtDNA and scnDNA loci, select those that give clean amplifications, and sequence one sample from each population to (i) confirm gene identity, (ii) estimate the amount of variation and, (iii) search for diagnostic restriction sites. This will allow determination of the most efficient approach for a large-scale study.     

Isolation and characterization of nine polymorphic microsatellite loci from the desert locust,Schistocerca gregaria

Because of the scarcity of polymorphic genetic markers, only a few genetic studies on the population structure of the desert locust, Schistocerca gregaria, have been carried out. We isolated and characterized nine polymorphic dinucleotide microsatellite loci. These markers were evaluated using individuals from Niger and Senegal. Seven of these microsatellite markers are also applicable to the nongregarious subspecies Schistocerca gregaria flaviventris. Cross‐species applicability was limited to one of the loci in the sister species S. americana and in the locust Locusta migratoria.     

Comparative phylogeography and the identification of genetically divergent areas for conservation

Genetic diversity is recognized as a fundamental component of biodiversity and its protection is incorporated in several conventions and policies. However, neither the concepts nor the methods for assessing conservation value of the spatial distribution of genetic diversity have been resolved. Comparative phylogeography can identify suites of species that have a common history of vicariance. In this study we explore the strengths and limitations of Faith's measure of 'Phylogenetic Diversity' (PD) as a method for predicting from multiple intraspecific phylogeographies the underlying feature diversity represented by combinations of areas. An advantage of the PD approach is that information on the spatial distribution of genetic diversity can be combined across species and expressed in a form that allows direct comparison with patterns of species distributions. It also seeks to estimate the same parameter, feature diversity, regardless of the level of biological organization. We extend the PD approach by using Venn diagrams to identify the components of PD, including those unique to or shared among areas and those which represent homoplasy on an area tree or which are shared across all areas. PD estimation should be complemented by analysis of these components and inspection of the contributing phylogeographies. We illustrate the application of the approach using mtDNA phylogeographies from vertebrates resident in the wet tropical rainforests of north-east Queensland and compare the results to biodiversity assessments based on the distribution of endemic vertebrate species. The genetic vs. species approaches produce different assessments of conservation value, perhaps reflecting differences in the temporal and spatial scale of the determining processes. The two approaches should be seen as complementary and, in this case, conservation planning should incorporate information on both dimensions of biodiversity.     

Comparative phylogeography of two open forest frogs from eastern Australia

We investigated the phylogeography of two closely related Australian frog species from open forest habitats, Limnodynastes tasmaniensis and L. peronii , using mitochondrial ND4 sequence data. Comparison of our results with previous work on Litoria fallax allowed us to test the generality of phylogeographic patterns among non-rainforest anurans along the east coast of Australia. In general, there was no strong evidence for congruence between overall patterns of genetic structure in the three species. However, phylogenetic breaks congruent with the position of the Burdekin Gap were detected at some level in all species. As previously noted for closed forest taxa, this area of dry habitat appears to have been an important influence on the evolution of several open forest taxa. There were broad geographic similarities in the phylogenetic structuring of southern populations of L. peronii and L. tasmaniensis. Contrarily, although the McPherson Range has previously been noted to coincide geographically with a major mtDNA phylogenetic break in Litoria fallax this pattern is not apparent in L. peronii or L. tasmaniensis. It appears that major phylogeographic splits within L. peronii and L. tasmaniensis may predate the Quaternary. We conclude that phylogeographies of open forest frogs are complex and more difficult to predict than for rainforest taxa, mainly due to an absence of palæomodels for historical distributions of non-rainforest habitats.     

Applications of mitochondrial DNA analysis in conservation: a critical review

Patterns of variation in mitochondrial DNA (mtDNA) increasingly are being investigated in threatened or managed species, but not always with clearly defined goals for conservation. In this review I identify uses of mtDNA analysis which fall into two different areas: (i) 'gene conservation' - the identification and management of genetic diversity, and (ii) 'molecular ecology' - the use of mtDNA variation to guide and assist demographic studies of populations. These two classes of application have different conceptual bases, conservation goals and time-frames. Gene conservation makes extensive use of phylogenetic information and is, in general, most relevant to long-term planning. Appropriate uses here include identification of Evolutionarily Significant Units and assessment of conservation priority of taxa or areas from an evolutionary perspective. Less appropriate are inferences about fitness from within-population diversity and about species boundaries. Molecular ecology makes more use of allele frequencies and provides information useful for short-term management of populations. Powerful applications are to identify Management Units and to define and use naturally occurring genetic tags. Estimating demographic parameters, e.g migration rate and population size, from patterns of mtDNA diversity is fraught with difficulty, particularly where populations are fluctuating, and is unlikely to produce quantitative estimates sufficiently accurate to be useful for practical management of contemporary populations. However, through comparative studies, mtDNA analysis can provide qualitative signals of population changes, allowing efficient targeting of resource-intensive ecological studies. Thus, there are some relatively straightforward uses of mtDNA, preferably in conjunction with assays of nuclear variation, that can make a significant contribution to the long-term planning and short-term execution of species recovery plans.     

Molecular population genetics of the red kangaroo (Macropus rufus): mtDNA variation

The genetic population structure of a large, wide-ranging marsupial, the red kangaroo ( Macropus rufus ) was assessed using sequence and haplotype frequency data of mitochondrial DNA (mtDNA) from locations across the species range in Australia. Results from sequence data revealed extensive haplotype diversity within the red kangaroo (32/34 sequences were unique). Sequence diversity was distributed within rather than between geographical regions across the species range. Genetic connectivity across the range of the species has therefore been maintained over the long term. On a smaller within-region scale, significant genetic structuring was evident from heterogeneity of haplotype frequencies amongst sampling sites. The geographical scale of panmictic populations differed across the continent with more restricted genetic populations occurring in areas with greater topographic and habitat complexity. We propose that these differences in area of genetic populations are the result of population responses to limiting ecological factors during drought.     

Mitochondrial DNA diversity and historical biogeography of a wet forest-restricted frog (Litoria pearsoniana) from mid-east Australia

MtDNA sequencing was used to investigate the genetic population structure of Litoria pearsoniana, a wet forest-restricted hylid frog, endemic to southeast Queensland and northeast New South Wales, Australia. L. pearsoniana is regarded as endangered under Queensland legislation. Significant genetic divergence among populations of frogs from different rainforest isolates was identified, but the lack of reciprocal monophyly among adjacent isolates suggests this is the result of a relatively recent disruption to gene flow. A paired catchment study within a single rainforest isolate, the Conondale Range, revealed no substantial genetic structuring, indicating the occurrence of terrestrial dispersal among nearby streams either in the recent past or currently. Two major reciprocally monophyletic clades of mtDNA alleles were identified. These corresponded to two geographical regions separated by the Brisbane River valley; one consisting of the Conondale and D’Aguilar Ranges, and the other of the southern isolates in the Main, Border and Gibraltar Ranges. Sequence divergence between the two regions was more consistent with a late Miocene or Pliocene rather than late Pleistocene separation, and is similar to that found among phylogeographic divisions of rainforest reptiles and amphibians in north Queensland rainforests. The molecular evidence for long-term separation of these two regions is corroborated by the pattern of species turnover in the distributions of species of rainforest-restricted amphibians and reptiles. Bioclimatic modelling suggests that appropriate conditions for L. pearsoniana would have been restricted to isolated refuges in each phylogeographic division under cooler and drier climates, such as predicted for the last glacial maximum. Currently isolated montane areas may have been connected transiently during the past 2000 years. Identification of long-term zoogeographic divisions among southeast Queensland rainforest herpetofauna has important implications for conservation and management. Conservation management of L. pearsoniana should be applied at the scale of major rainforest isolates and the conservation status of the species should be assessed independently north and south of the historical division.