A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

The genetic structure of Australasian green turtles (Chelonia mydas): Exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long-term mark-recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Φ = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.

Genetic population structure of red snappers (Lutjanus malabaricus Bloch & Schneider, 1801 and Lutjanus erythropterus Bloch, 1790) in central and eastern Indonesia and northern Australia

The genetic population structure of red snapper Lutjanus malabaricus and Lutjanus erythropterus in eastern Indonesia and northern Australia was investigated by allozyme electrophoresis and sequence variation in the control region of mtDNA. Samples were collected from eight sites in Indonesia and four sites in northern Australia for both species. A total of 13 allozyme loci were scored. More variable loci were observed in L. malabaricus than in L. erythropterus. Sequence variation in the control region (left domain) of the mitochondrial genome was assessed by RFLP and direct sequencing. MtDNA haplotype diversity was high (L. erythropterus, 0.95 and L. malabaricus, 0.97), as was intraspecific sequence divergence, (L. erythropterus, 0.0-12.5% and L. malabaricus, 0.0-9.5%). The pattern of mtDNA haplotype frequencies grouped both species into two broad fisheries stocks with a genetic boundary either between Kupang and Sape (L. malabaricus) or between Kupang and Australian Timor Sea (L. erythropertus). The allozyme analyses revealed similar boundaries for L. erythropterus. Seven allozymes stocks compared to two mtDNA stocks of L. malabaricus including Ambon, which was not sampled with mtDNA, however, were reported. Possible reasons for differences in discrimination between the methods include: i) increased power of multiple allozyme loci over the single mtDNA locus, ii) insufficient gene sampling in the mtDNA control region and iii) relative evolutionary dynamics of nuclear (allozyme loci) and mitochondrial DNA in these taxa. Allozyme and haplotype data did not distinguish separate stocks among the four Australian locations nor the central Indonesian (Bali and Sape locations) for both L. malabaricus and L. erythropterus.

Concordant molecular and phenotypic data delineate new taxonomy and conservation priorities for the endangered mountain yellow-legged frog

The mountain yellow-legged frog Rana muscosa sensu lato, once abundant in the Sierra Nevada of California and Nevada, and the disjunct Transverse Ranges of southern California, has declined precipitously throughout its range, even though most of its habitat is protected. The species is now extinct in Nevada and reduced to tiny remnants in southern California, where as a distinct population segment, it is classified as Endangered. Introduced predators (trout), air pollution and an infectious disease (chytridiomycosis) threaten remaining populations. A Bayesian analysis of 1901 base pairs of mitochondrial DNA confirms the presence of two deeply divergent clades that come into near contact in the Sierra Nevada. Morphological studies of museum specimens and analysis of acoustic data show that the two major mtDNA clades are readily differentiated phenotypically. Accordingly, we recognize two species, Rana sierrae, in the northern and central Sierra Nevada, and R. muscosa, in the southern Sierra Nevada and southern California. Existing data indicate no range overlap. These results have important implications for the conservation of these two species as they illuminate a profound mismatch between the current delineation of the distinct population segments (southern California vs. Sierra Nevada) and actual species boundaries. For example, our study finds that remnant populations of R. muscosa exist in both the southern Sierra Nevada and the mountains of southern California, which may broaden options for management. In addition, despite the fact that only the southern California populations are listed as Endangered, surveys conducted since 1995 at 225 historic (1899-1994) localities from museum collections show that 93.3% (n=146) of R. sierrae populations and 95.2% (n=79) of R. muscosa populations are extinct. Evidence presented here underscores the need for revision of protected population status to include both species throughout their ranges.

Trapping to monitor tephritid movement: Results, best practice, and assessment of alternatives

Movement of tephritid flies underpins their survival, reproduction, and ability to establish in new areas and is thus of importance when designing effective management strategies. Much of the knowledge currently available on tephritid movement throughout landscapes comes from the use of direct or indirect methods that rely on the trapping of individuals. Here, we review published experimental designs and methods from mark-release-recapture (MRR) studies, as well as other methods, that have been used to estimate movement of the four major tephritid pest genera (Bactrocera, Ceratitis, Anastrepha, and Rhagoletis). In doing so, we aim to illustrate the theoretical and practical considerations needed to study tephritid movement. MRR studies make use of traps to directly estimate the distance that tephritid species can move within a generation and to evaluate the ecological and physiological factors that influence dispersal patterns. MRR studies, however, require careful planning to ensure that the results obtained are not biased by the methods employed, including marking methods, trap properties, trap spacing, and spatial extent of the trapping array. Despite these obstacles, MRR remains a powerful tool for determining tephritid movement, with data particularly required for understudied species that affect developing countries. To ensure that future MRR studies are successful, we suggest that site selection be carefully considered and sufficient resources be allocated to achieve optimal spacing and placement of traps in line with the stated aims of each study. An alternative to MRR is to make use of indirect methods for determining movement, or more correctly, gene flow, which have become widely available with the development of molecular tools. Key to these methods is the trapping and sequencing of a suitable number of individuals to represent the genetic diversity of the sampled population and investigate population structuring using nuclear genomic markers or non-recombinant mitochondrial DNA markers. Microsatellites are currently the preferred marker for detecting recent population displacement and provide genetic information that may be used in assignment tests for the direct determination of contemporary movement. Neither MRR nor molecular methods, however, are able to monitor fine-scale movements of individual flies. Recent developments in the miniaturization of electronics offer the tantalising possibility to track individual movements of insects using harmonic radar. Computer vision and radio frequency identification tags may also permit the tracking of fine-scale movements by tephritid flies by automated resampling, although these methods come with the same problems as traditional traps used in MRR studies. Although all methods described in this chapter have limitations, a better understanding of tephritid movement far outweighs the drawbacks of the individual methods because of the need for this information to manage tephritid populations.

Grey Mackerel (Scomberomorus semifasciatus) microsatellite genotypes (nine loci) representing twelve regions along Australia's northern coastline from the eastern coast of Queensland to the western coast of Western Australia.

Scomberomorus semifasciatus is an Australian endemic found in tropical, coastal waters from eastern to western Australia. Commercial and recreational exploitation is common and regulated by state-based authorities. This study used mitochondrial DNA sequence and microsatellite markers to elucidate the population structure of Scomberomorus semifasciatus collected from twelve, equidistant sampling locations. Samples (n=544) were genotyped with nine microsatellite loci, and 353 were sequenced for d-loop (384 bp) and ATP (800bp) mitochondrial DNA gene regions. Combined interpretation of microsatellite and mtDNA data identified four genetic stocks of S. semifasciatus: Western Australia, northwest coast of the Northern Territory, Gulf of Carpentaria and the east coast of Queensland. Connectivity among stocks across northern Australia from the Northern Territory to the east coast of Queensland was high, but in contrast, there was a clear genetic break between populations in Western Australia compared to the rest of northern Australia. This indicates a restriction to gene flow possibly associated with suboptimal habitat along the Kimberley coast (northwestern Australia). The appropriate scale of management for this species corresponds to the jurisdictions of the three Australian states, except that the Gulf of Carpentaria stock should be co-managed by authorities in Queensland and Northern Territory.

Migration of green turtles (Chelonia mydas) from Australasian feeding grounds inferred from genetic analyses

Coastal seagrass habitats in tropical and subtropical regions support aggregations of resident green turtles (Chelonia mydas) from several genetically distinct breeding populations. Migration of individuals to their respective dispersed breeding sites provides a complex pattern of migratory connectivity among nesting and feeding habitats of this species. An understanding of this pattern is important in regions where the persistence of populations is under threat from anthropogenic impacts. The present study uses mitochondrial DNA and mixed-stock analyses to assess the connectivity among seven feeding grounds across the north Australian coast and adjacent areas and 17 genetically distinct breeding populations from the Indo-Pacific region. It was hypothesised that large and geographically proximate breeding populations would dominate at nearby feeding grounds. As expected, each sampled feeding area appears to support multiple breeding populations, with two aggregations dominated by a local breeding population. Geographic distance between breeding and feeding habitat strongly influenced whether a breeding population contributed to a feeding ground (wi = 0.654); however, neither distance nor size of a breeding population was a good predictor of the extent of their contribution. The differential proportional contributions suggest the impact of anthropogenic mortality at feeding grounds should be assessed on a case-by-case basis.

Population genetic evidence for the east–west division of the narrow-barred Spanish mackerel (Scomberomorus commerson, Perciformes: Teleostei) along Wallace’s Line

Mitochondrial DNA D-loop (control) region (426-bp) was used to infer the genetic structure of Spanish mackerel (Scomberomorus commerson) from populations in Southeast Asia (Brunei, East and West Malaysia, Philippines, Thailand, Singapore, and China) and northern Australia (including western Timor). An east–west division along Wallace’s Line was strongly supported by a significant AMOVA, with 43% of the total sequence variation partitioned among groups of populations. Phylogenetic and network analyses supported two clades: clade A and clade B. Members of clade A were found in Southeast Asia and northern Australia, but not in locations to the west (Gulf of Thailand) or north (China). Clade B was found exclusively in Southeast Asia. Genetic division along Wallace’s Line suggests that co-management of S. commerson populations for future sustainability may not be necessary between Southeast Asian nations and Australia, however all countries should share the task of management of the species in Southeast Asia equally. More detailed genetic studies of S. commerson populations in the region are warranted.

The extent of population genetic subdivision differs among four co-distributed shark species in the Indo-Australian archipelago

Background: The territorial fishing zones of Australia and Indonesia are contiguous to the north of Australia in the Timor and Arafura Seas and in the Indian Ocean to the north of Christmas Island. The area surrounding the shared boundary consists of a variety of bio-diverse marine habitats including shallow continental shelf waters, oceanic trenches and numerous offshore islands. Both countries exploit a variety of fisheries species, including whaler (Carcharhinus spp.) and hammerhead sharks (Sphyrna spp.). Despite their differences in social and financial arrangements, the two countries are motivated to develop complementary co-management practices to achieve resource sustainability. An essential starting point is knowledge of the degree of population subdivision, and hence fisheries stock status, in exploited species. Results: Populations of four commercially harvested shark species (Carcharhinus obscurus, Carcharhinus sorrah, Prionace glauca, Sphyrna lewini) were sampled from northern Australia and central Indonesia. Neutral genetic markers (mitochondrial DNA control region sequence and allelic variation at co-dominant microsatellite loci) revealed genetic subdivision between Australian and Indonesian populations of C. sorrah. Further research is needed to address the possibility of genetic subdivision among C. obscurus populations. There was no evidence of genetic subdivision for P. glauca and S. lewini populations, but the sampling represented a relatively small part of their distributional range. For these species, more detailed analyses of population genetic structure is recommended in the future. Conclusion: Cooperative management between Australia and Indonesia is the best option at present for P. glauca and S. lewini, while C. sorrah and C. obscurus should be managed independently. On-going research on these and other exploited shark and ray species is strongly recommended. Biological and ecological similarity between species may not be a predictor of population genetic structure, so species-specific studies are recommended to provide new data to assist with sustainable fisheries management.

Determination of management units for grey mackerel fisheries in northern Australia.

The requirement for Queensland, Northern Territory and Western Australian jurisdictions to ensure sustainable harvest of fish resources and their optimal use relies on robust information on the resource status. For grey mackerel (Scomberomorus semifasciatus) fisheries, each of these jurisdictions has their own management regime in their corresponding waters. The lack of information on stock structure of grey mackerel, however, means that the appropriate spatial scale of management is not known. As well, fishers require assurance of future sustainability to encourage investment and long-term involvement in a fishery that supplies lucrative overseas markets. These management and fisher-unfriendly circumstances must be viewed in the context of recent 3-fold increases in catches of grey mackerel along the Queensland east coast, combined with significant and increasing catches in other parts of the species' northern Australian range. Establishing the stock structure of grey mackerel would also immensely improve the relevance of resource assessments for fishery management of grey mackerel across northern Australia. This highlighted the urgent need for stock structure information for this species. The impetus for this project came from the strategic recommendations of the FRDC review by Ward and Rogers (2003), "Northern mackerel (Scombridae: Scomberomorus): current and future research needs" (Project No. 2002/096), which promoted the urgency for information on the stock structure of grey mackerel. In following these recommendations this project adopted a multi-technique and phased sampling approach as carried out by Buckworth et al (2007), who examined the stock structure of Spanish mackerel, Scomberomorus commerson, across northern Australia. The project objectives were to determine the stock structure of grey mackerel across their northern Australian range, and use this information to define management units and their appropriate spatial scales. We used multiple techniques concurrently to determine the stock structure of grey mackerel. These techniques were: genetic analyses (mitochondrial DNA and microsatellite DNA), otolith (ear bones) isotope ratios, parasite abundances, and growth parameters. The advantage of using this type of multi-technique approach was that each of the different methods is informative about the fish’s life history at different spatial and temporal scales. Genetics can inform about the evolutionary patterns as well as rates of mixing of fish from adjacent areas, while parasites and otolith microchemistry are directly influenced by the environment and so will inform about the patterns of movement during the fishes lifetime. Growth patterns are influenced by both genetic and environmental factors. Due to these differences the use of these techniques concurrently increases the likelihood of detecting different stocks where they exist. We adopted a phased sampling approach whereby sampling was carried out at broad spatial scales in the first year: east coast, eastern Gulf of Carpentaria (GoC), western GoC, and the NW Northern Territory (NW NT). By comparing the fish samples from each of these locations, and using each of the techniques, we tested the null hypothesis that grey mackerel were comprised of a single homogeneous population across northern Australia. Having rejected the null hypothesis we re-sampled the 1st year locations to test for temporal stability in stock structure, and to assess stock structure at finer spatial scales. This included increased spatial coverage on the east coast, the GoC, and WA. From genetic approaches we determined that there at least four genetic stocks of grey mackerel across northern Australia: WA, NW NT (Timor/Arafura), the GoC and the east Grey mackerel management units in northern Australia ix coast. All markers revealed concordant patterns showing WA and NW NT to be clearly divergent stocks. The mtDNA D-loop fragment appeared to have more power to resolve stock boundaries because it was able to show that the GoC and east coast QLD stocks were genetically differentiated. Patterns of stock structure on a finer scale, or where stock boundaries are located, were less clear. From otolith stable isotope analyses four major groups of S. semifasciatus were identified: WA, NT/GoC, northern east coast and central east coast. Differences in the isotopic composition of whole otoliths indicate that these groups must have spent their life history in different locations. The magnitude of the difference between the groups suggests a prolonged separation period at least equal to the fish’s life span. The parasite abundance analyses, although did not include samples from WA, suggest the existence of at least four stocks of grey mackerel in northern Australia: NW NT, the GoC, northern east coast and central east coast. Grey mackerel parasite fauna on the east coast suggests a separation somewhere between Townsville and Mackay. The NW NT region also appears to comprise a separate stock while within the GoC there exists a high degree of variability in parasite faunas among the regions sampled. This may be due to 1. natural variation within the GoC and there is one grey mackerel stock, or 2. the existence of multiple localised adult sub-stocks (metapopulations) within the GoC. Growth parameter comparisons were only possible from four major locations and identified the NW NT, the GoC, and the east coast as having different population growth characteristics. Through the use of multiple techniques, and by integrating the results from each, we were able to determine that there exist at least five stocks of grey mackerel across northern Australia, with some likelihood of additional stock structuring within the GoC. The major management units determined from this study therefore were Western Australia, NW Northern Territory (Timor/Arafura), the Gulf of Carpentaria, northern east Queensland coast and central east Queensland coast. The management implications of these results indicate the possible need for management of grey mackerel fisheries in Australia to be carried out on regional scales finer than are currently in place. In some regions the spatial scales of management might continue as is currently (e.g. WA), while in other regions, such as the GoC and the east coast, managers should at least monitor fisheries on a more local scale dictated by fishing effort and assess accordingly. Stock assessments should also consider the stock divisions identified, particularly on the east coast and for the GoC, and use life history parameters particular to each stock. We also emphasise that where we have not identified different stocks does not preclude the possibility of the occurrence of further stock division. Further, this study did not, nor did it set out to, assess the status of each of the stocks identified. This we identify as a high priority action for research and development of grey mackerel fisheries, as well as a management strategy evaluation that incorporates the conclusions of this work. Until such time that these priorities are addressed, management of grey mackerel fisheries should be cognisant of these uncertainties, particularly for the GoC and the Queensland east coast.

Molecular Background of Common Dyslipidemias

The leading cause of death in the Western world continues to be coronary heart disease (CHD). At the root of the disease process is dyslipidemia an aberration in the relevant amounts of circulating blood lipids. Cholesterol builds up in the arterial wall and following rupture of these plaques, myocardial infarction or stroke can occur. Heart disease runs in families and a number of hereditary forms are known. The leading cause of adult dyslipidemia presently however is overweight and obesity. This thesis work presents an investigation of the molecular genetics of common, hereditary dyslipidemia and the tightly related condition of obesity. Familial combined hyperlipidemia (FCHL) is the most common hereditary dyslipidemia in man with an estimated population prevalence of 1-6%. This complex disease is characterized by elevated levels of serum total cholesterol, triglycerides or both and is observed in about 20% of individuals with premature CHD. Our group identified the disease to be associated with genetic variation in the USF1 transcription factor gene. USF1 has a key role in regulating other genes that control lipid and glucose metabolism as well as the inflammatory response all central processes in the progression of atherosclerosis and CHD. The first two works of this thesis aimed at understanding how these USF1 variants result in increased disease risk. Among the many, non-coding single-nucleotide polymorphisms (SNPs) that associated with the disease, one was found to have a functional effect. The risk-enhancing allele of this SNP seems to eradicate the ability of the important hormone insulin to induce the expression of USF1 in peripheral tissues. The resultant changes in the expression of numerous USF1 target genes over time probably enhance and accelerate the atherogenic processes. Dyslipidemias often represent an outcome of obesity and in the final work of this thesis we wanted to address the metabolic pathways related to acquired obesity. It is recognized that active processes in adipose tissue play an important role in the development of dyslipidemia, insulin resistance and other pathological conditions associated with obesity. To minimize the confounding effects of genetic differences present in most human studies, we investigated a rare collection of identical twins that differed significantly in the amount of body fat. In the obese, but otherwise healthy young adults, several notable changes were observed. In addition to chronic inflammation, the adipose tissue of the obese co-twins was characterized by a marked (47%) decrease in amount of mitochondrial DNA (mtDNA) a change associated with mitochondrial dysfunction. The catabolism of branched chain amino acids (BCAAs) was identified as the most down-regulated process in the obese co-twins. A concordant increase in the serum level of these insulin secretagogues was identified. This hyperaminoacidemia may provide the feed-back signal from insulin resistant adipose tissue to the pancreas to ensure an appropriately augmented secretory response. The down regulation of BCAA catabolism correlated closely with liver fat accumulation and insulin. The single most up-regulated gene (5.9 fold) in the obese co-twins was osteopontin (SPP1) a cytokine involved in macrophage recruitment to adipose tissue. SPP1 is here implicated as an important player in the development of insulin resistance. These studies of exceptional study samples provide better understanding of the underlying pathology in common dyslipidemias and other obesity associated diseases important for future improvement of intervention strategies and treatments to combat atherosclerosis and coronary heart disease.

Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction.

A multiplex real-time PCR was developed for the detection and differentiation of two closely related bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5). The multiplex real-time PCR combines a duplex real-time PCR that targets the DNA polymerase gene of BoHV-1 and BoHV-5 and a real-time PCR targeting mitochondrial DNA, as a house-keeping gene, described previously by Cawthraw et al. (2009). The assay correctly identified 22 BoHV-1 and six BoHV-5 isolates from the Biosecurity Sciences Laboratory virus collection. BoHV-1 and BoHV-5 were also correctly identified when incorporated in spiked semen and brain tissue samples. The detection limits of the duplex assay were 10 copies of BoHV-1 and 45 copies of BoHV-5. The multiplex real-time PCR had reaction efficiencies of 1.04 for BoHV-1 and 1.08 for BoHV-5. Standard curves relating Ct value to template copy number had correlation coefficients of 0.989 for BoHV-1 and 0.978 for BoHV-5. The assay specificity was demonstrated by testing bacterial and viral DNA from pathogens commonly isolated from bovine respiratory and reproductive tracts. The validated multiplex real-time PCR was used to detect and differentiate BoHV-1 and BoHV-5 in bovine clinical samples with known histories.

White shark (Carcharodon carcharias) microsatellite genotypes (six loci) from regions along Australia's southern and eastern coastline.

Despite international protection of white sharks (Carcharodon carcharias), important conservation parameters such as abundance, population structure and genetic diversity are largely unknown. The tissue of 97 predominately juvenile white sharks sampled from spatially distant eastern and southwestern Australian coastlines was sequenced for the mitochondrial DNA (mtDNA) control region and genotyped with six nuclear-encoded microsatellite loci. MtDNA population structure was found between the eastern and southwestern coasts (FST = 0.142, p < 0.001), implying female natal philopatry. This concords with recent satellite and acoustic tracking findings which suggest the sustained presence of discrete east coast nursery areas. Furthermore, population subdivision was found between the same regions with biparentally inherited microsatellite markers (FST = 0.009, p <0.05), suggesting that males may also exhibit some degree of reproductive philopatry. Five sharks captured along the east coast had mtDNA haplotypes that resembled western Indian Ocean sharks more closely than Australian/New Zealand sharks, suggesting that transoceanic dispersal or migration resulting in breeding may occur sporadically. Our most robust estimate of contemporary genetic effective population size was low and below the threshold at which adaptive potential may be lost. For a variety of reasons, these contemporary estimates were at least one, possibly two orders of magnitude below our historical effective size estimates. Further population decline could expose these genetically isolated populations to detrimental genetic effects. Regional Australian white shark conservation management units should be implemented until genetic population structure, size and diversity can be investigated in more detail. Reference: Blower, D. C., Pandolfi, J. M., Gomez-Cabrera, M. del C., Bruce, B. D. & Ovenden, J. R. (In press - April 2012). Population genetics of Australian white sharks reveals fine-scale spatial structure, transoceanic dispersal events and low effective population sizes. Marine Ecology Progress Series.

Pleistocene isolation, secondary introgression and restricted contemporary gene flow in the pig-eye shark, Carcharhinus amboinensis across northern Australia

We examine the structure and phylogeography of the pig-eye shark (Carcharhinus amboinensis) common in shallow coastal environments in northern Australia using two types of genetic markers, two mitochondrial (control region and NADH hydrogenase 4) and two nuclear (microsatellite and Rag 1) DNA. Two populations were defined within northern Australia on the basis of mitochondrial DNA evidence, but this result was not supported by nuclear microsatellite or Rag 1 markers. One possibility for this structure might be sex-specific behaviours such as female philopatry, although we argue it is doubtful that sufficient time has elapsed for any potential signatures from this behaviour to be expressed in nuclear markers. It is more likely that the observed pattern represents ancient populations repeatedly isolated and connected during episodic sea level changes during the Pleistocene epoch, until current day with restricted contemporary gene flow maintaining population genetic structure. Our results show the need for an understanding of both the history and ecology of a species in order to interpret patterns in genetic structure.

Population Expansion and Genetic Structure in Carcharhinus brevipinna in the Southern Indo-Pacific

Background:Quantifying genetic diversity and metapopulation structure provides insights into the evolutionary history of a species and helps develop appropriate management strategies. We provide the first assessment of genetic structure in spinner sharks (Carcharhinus brevipinna), a large cosmopolitan carcharhinid, sampled from eastern and northern Australia and South Africa. Methods and Findings:Sequencing of the mitochondrial DNA NADH dehydrogenase subunit 4 gene for 430 individuals revealed 37 haplotypes and moderately high haplotype diversity (h = 0.6770 ±0.025). While two metrics of genetic divergence (ΦST and FST) revealed somewhat different results, subdivision was detected between South Africa and all Australian locations (pairwise ΦST, range 0.02717–0.03508, p values ≤ 0.0013; pairwise FST South Africa vs New South Wales = 0.04056, p = 0.0008). Evidence for fine-scale genetic structuring was also detected along Australia’s east coast (pairwise ΦST = 0.01328, p < 0.015), and between south-eastern and northern locations (pairwise ΦST = 0.00669, p < 0.04).Conclusions: The Indian Ocean represents a robust barrier to contemporary gene flow in C. brevipinna between Australia and South Africa. Gene flow also appears restricted along a continuous continental margin in this species, with data tentatively suggesting the delineation of two management units within Australian waters. Further sampling, however, is required for a more robust evaluation of the latter finding. Evidence indicates that all sampled populations were shaped by a substantial demographic expansion event, with the resultant high genetic diversity being cause for optimism when considering conservation of this commercially-targeted species in the southern Indo-Pacific.