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Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage. This work was completed in part with resources provided by the University of Massachusetts' Green High Performance Computing Cluster (GHPCC). Funding was provided by the University of Massachusetts Amherst, NSF-IOS (grant #1904439 to L.M.K.), NOAA-Fisheries, National Research Council postdoctoral fellowship program to L.M.K., VGP, Rockefeller University, to E.D.J., HHMI to E.D.J., the Sanger Institute, Max-Planck-Gesellschaft, and grant contributions from Tom Gilbert, Paul Flicek, R.W.M., Karen A. Bjorndal, Alan B. Bolten, Ed Braun, N.J.G., T.M.-B., and A.F.S. We acknowledge CONICYT-DAAD for scholarship support to T.C.-V., the São Paulo Research Foundation to E.K.S.R.–FAPESP (grant #2020/10372-6). BeGenDiv is partially funded by the German Federal Ministry of Education and Research (BMbF, Förderkennzeichen 033W034A). The work of F.T.-N. and P.M. was supported by the Intramural Research Program of the National Library of Medicine, NIH. The work of M.P. was partially funded through the Federal Ministry of Education and Research (grant 01IS18026C). H.P. was supported by a Formació de Personal Investigador fellowship from Generalitat de Catalunya (FI_B100131). M.K. was supported by “la Caixa” Foundation (ID 100010434; code LCF/BQ/PR19/11700002), the Vienna Science and Technology Fund (WWTF), and the City of Vienna (VRG20-001). Funding for green turtle resequencing was provided by a Welsh Government Sêr Cymru II and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 663830-BU115 and the Sea Turtle Conservancy, Florida Sea Turtle Grants Program (17-033R).

Sex determination and differentiation varies widely across vertebrates, but is most dramatically diverse in fishes. Among fishes sex reversal and sex change are observed in 41 teleost families spanning 7 orders. These sex-changing fish perhaps highlight better than any other system that sex determination is not the narrow and fixed construct we once thought, but a plastic trait that is better viewed as a reaction norm. However, while this stunning transformation is increasingly understood, a fundamental question arises, which is why some fish species have retained this inherent plasticity in sexual fate, while others have not? Here, we explore our current understanding of sex change in fish, some of the factors that permit and constrain sex reversal, and posit that gene duplication and neofunctionalization contribute to the sexual lability observed in fish.

Fish show extraordinary sexual plasticity, changing sex naturally as part of their life cycle or reversing sex because of environmental stressors. This plasticity shows that sexual fate is not an irreversible process but the result of an ongoing tug-of-war for supremacy between male and female signaling networks. The behavioral, gonadal, and morphological changes involved in this process are well described, yet the molecular events that underpin those changes remain poorly understood. Epigenetic modifications emerge as a critical link between environmental stimuli, the onset of sex change, and subsequent maintenance of sexual phenotype. Here we synthesize current knowledge of sex change, focusing on the genetic and epigenetic processes that are likely involved in the initiation and regulation of sex change. We anticipate that better understanding of sex change in fish will shed new light on sex determination and development in vertebrates and on how environmental perturbations affect sexual fate.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings. Here we have established the protogynous New Zealand spotty wrasse, Notolabruscelidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using aromatase inhibition or manipulation of social groups. Complete female-to-male transition occurred over 60 days in both cases and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy. Early-stage decreases in plasma 17β-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.

Sex change occurs as a usual part of the life cycle for many teleost fish and the modifications involved (behavioural, gonadal, morphological) are well studied. However, the mechanism that transduces environmental cues into the molecular cascade that underlies this transformation remains unknown. Cortisol, the main stress hormone in fish, is hypothesised to be a key factor linking environmental stimuli with sex change by initiating gene expression changes that shift steroidogenesis from oestrogens to androgens but this notion remains to be rigorously tested. Therefore, this study aimed to experimentally test the role of cortisol as an initiator of sex change in a protogynous (female-to-male) hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). We also sought to identify potential key regulatory factors within the head kidney that may contribute to the initiation and progression of gonadal sex change. Cortisol pellets were implanted into female spotty wrasses under inhibitory conditions (presence of a male), and outside of the optimal season for natural sex change. Histological analysis of the gonads and sex hormone analyses found no evidence of sex change after 71 days of cortisol treatment. However, expression analyses of sex and stress-associated genes in gonad and head kidney suggested that cortisol administration did have a physiological effect. In the gonad, this included upregulation of amh, a potent masculinising factor, and nr3c1, a glucocorticoid receptor. In the head kidney, hsd11b2, which converts cortisol to inactive cortisone to maintain cortisol balance, was upregulated. Overall, our results suggest cortisol administration outside of the optimal sex change window is unable to initiate gonadal restructuring. However, our expression data imply key sex and stress genes are sensitive to cortisol. This includes genes expressed in both gonad and head kidney that have been previously implicated in early sex change in several sex-changing species.

Many teleost fishes undergo natural sex change, and elucidating the physiological and molecular controls of this process offers unique opportunities not only to develop methods of controlling sex in aquaculture settings, but to better understand vertebrate sexual development more broadly. Induction of sex change in some sequentially hermaphroditic or gonochoristic fish can be achievedin vivothrough social manipulation, inhibition of aromatase activity, and steroid treatment. However, the induction of sex changein vitrohas been largely unexplored. In this study, we established anin vitroculture system for ovarian explants in serum-free medium for a model sequential hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). This culture technique enabled evaluating the effect of various treatments with 17β-estradiol (E2), 11-ketotestosterone (11KT) or cortisol (CORT) on spotty wrasse ovarian architecture for 21 days. A quantitative approach to measuring the degree of ovarian atresia within histological images was also developed, using pixel-based machine learning software. Ovarian atresia likely due to culture was observed across all treatments including no-hormone controls, but was minimised with treatment of at least 10 ng/mL E2. Neither 11KT nor CORT administration induced proliferation of spermatogonia (i.e. sex change) in the cultured ovaries indicating culture beyond 21 days may be needed to induce sex changein vitro. Thein vitrogonadal culture and analysis systems established here enable future studies investigating the paracrine role of sex steroids, glucocorticoids and a variety of other factors during gonadal sex change in fish.

The stunning sexual transformation commonly triggered by age, size or social context in some fishes is one of the best examples of phenotypic plasticity thus far described. To date our understanding of this process is dominated by studies on a handful of subtropical and tropical teleosts, often in wild settings because sex change has been challenging to achieve in captivity. Here we have established the protogynous New Zealand spotty wrasse, Notolabrus celidotus, as a temperate model for the experimental investigation of sex change. Captive fish were induced to change sex using either aromatase inhibition or manipulation of social groups. Complete transition from female to male occurred over 60 days and time-series sampling was used to quantify changes in hormone production, gene expression and gonadal cellular anatomy using radioimmunoassay, nanoString nCounter mRNA and histological analyses, respectively. Early-stage decreases in plasma 17β-estradiol (E2) concentrations or gonadal aromatase (cyp19a1a) expression were not detected in spotty wrasse, despite these being commonly associated with the onset of sex change in subtropical and tropical protogynous (female-to-male) hermaphrodites. In contrast, expression of the masculinising factor amh (anti-Müllerian hormone) increased during early sex change, implying a potential role as a proximate trigger for masculinisation. Expression of male-related genes responsible for androgen production cyp11c1 and hsd11b2 increased from mid sex change. Gonadal expression of the glucocorticoid and mineralocorticoid receptors nr3c1 and nr3c2, putative mediators of the stress hormone cortisol, increased in late stages of sex change. Collectively, these data provide a foundation for the spotty wrasse as a temperate teleost model to study sex change and cell fate in vertebrates.Summary statementThe spotty wrasse, Notolabrus celidotus, is a new temperate model for the study of vertebrate sex change, this work characterises endocrine and genetic markers based on laboratory induced sex change.

Mollusc shells are an abundant resource that have been long used to predict the structures of ancient ecological communities, examine evolutionary processes, reconstruct paleoenvironmental conditions, track and predict responses to climatic change, and explore the movement of hominids across the globe. Despite the ubiquity of mollusc shell in many environments, it remains relatively unexplored as a substrate for molecular genetic analysis. Here we undertook a series of experiments using the New Zealand endemic greenshell mussel, Perna canaliculus, to explore the utility of fresh, aged, beach-cast and cooked mollusc shell for molecular genetic analyses. We find that reasonable quantities of DNA (0.002–21.48 ng/mg shell) can be derived from aged, beach-cast and cooked mussel shell and that this can routinely provide enough material to undertake PCR analyses of mitochondrial and nuclear gene fragments. Mitochondrial PCR amplification had an average success rate of 96.5% from shell tissue extracted thirteen months after the animal’s death. A success rate of 93.75% was obtained for cooked shells. Amplification of nuclear DNA (chitin synthase gene) was less successful (80% success from fresh shells, decreasing to 10% with time, and 75% from cooked shells). Our results demonstrate the promise of mollusc shell as a substrate for genetic analyses targeting both mitochondrial and nuclear genes.

Phenotypic plasticity represents an elegant adaptive response of individuals to a change in their environment. Bluehead wrasses (Thalassoma bifasciatum) exhibit astonishing sexual plasticity, including female-to-male sex change and discrete male morphs that differ strikingly in behavior, morphology, and gonadal investment. Using RNA-seq transcriptome profiling, we examined the genes and physiological pathways underlying flexible behavioral and gonadal differences among female, dominant (bourgeois) male, and female-mimic (sneaker) male blueheads. For the first time in any organism, we find that female mimicry by sneaker males has a transcriptional signature in both the brain and the gonad. Sneaker males shared striking similarity in neural gene expression with females, supporting the idea that males with alternative reproductive phenotypes have "female-like brains." Sneaker males also overexpressed neuroplasticity genes, suggesting that their opportunistic reproductive strategy requires a heightened capacity for neuroplasticity. Bourgeois males overexpressed genes associated with socio-sexual behaviors (e.g., isotocin), but also neuroprotective genes and biomarkers of oxidative stress and aging, indicating a hitherto unexplored cost to these males of attaining the reproductively privileged position at the top of the social hierarchy. Our novel comparison of testicular transcriptomes in a fish with male sexual polymorphism associates greater gonadal investment by sneaker males with overexpression of genes involved in cell proliferation and sperm quality control. We propose that morphological female-mimicry by sneaker male teleosts entails pervasive downregulation of androgenesis genes, consistent with low androgen production in males lacking well-developed secondary sexual characters.

Ovary-to-testis transformation in a sex-changing fish involves transcriptomic and epigenomic reprogramming., Bluehead wrasses undergo dramatic, socially cued female-to-male sex change. We apply transcriptomic and methylome approaches in this wild coral reef fish to identify the primary trigger and subsequent molecular cascade of gonadal metamorphosis. Our data suggest that the environmental stimulus is exerted via the stress axis and that repression of the aromatase gene (encoding the enzyme converting androgens to estrogens) triggers a cascaded collapse of feminizing gene expression and identifies notable sex-specific gene neofunctionalization. Furthermore, sex change involves distinct epigenetic reprogramming and an intermediate state with altered epigenetic machinery expression akin to the early developmental cells of mammals. These findings reveal at a molecular level how a normally committed developmental process remains plastic and is reversed to completely alter organ structures.

Fishes exhibit remarkably diverse, and plastic, patterns of sexual development, most striking of which is sequential hermaphroditism, where individuals readily reverse sex in adulthood. How this stunning example of phenotypic plasticity is controlled at a genetic level remains poorly understood. Several genes have been implicated in regulating sex change, yet the degree to which a conserved genetic machinery orchestrates this process has not yet been addressed. Using captive and in-the-field social manipulations to initiate sex change, combined with a comparative qPCR approach, we compared expression patterns of four candidate regulatory genes among three species of wrasses (Labridae) - a large and diverse teleost family where female-to-male sex change is pervasive, socially-cued, and likely ancestral. Expression in brain and gonadal tissues were compared among the iconic tropical bluehead wrasse (Thalassoma bifasciatum) and the temperate spotty (Notolabrus celidotus) and kyusen (Parajulus poecilepterus) wrasses. In all three species, cyp19a1a (encoding gonadal aromatase that converts androgens to oestrogens) and amh (encoding anti-müllerian hormone that primarily regulates male germ cell development) were downregulated and upregulated, respectively, at the initiation of gonadal sex change, and may act concurrently to orchestrate ovary-testis transformation. In the brain, our data argue against a role for brain aromatase (cyp19a1b) in initiating behavioural sex change, as its expression trailed behavioural changes. However, we find that isotocin (it, that regulates teleost socio-sexual behaviours) expression correlated with dominant male-specific behaviours in the bluehead wrasse, suggesting it upregulation mediates the rapid behavioural sex change characteristic of blueheads and other tropical wrasses. However, it expression was not sex-biased in temperate spotty and kyusen wrasses, where sex change is more protracted and social groups may be less tightly-structured. Together, these findings suggest that while key components of the molecular machinery controlling gonadal sex change are phylogenetically conserved among wrasses, neural pathways governing behavioural sex change may be more variable.

Sexual fate can no longer be considered an irreversible deterministic process that once established during early embryonic development, plays out unchanged across an organism's life. Rather, it appears to be a dynamic process, with sexual phenotype determined through an ongoing battle for supremacy between antagonistic male and female developmental pathways. That sexual fate is not final and is actively regulated via the suppression or activation of opposing genetic networks creates the potential for flexibility in sexual phenotype in adulthood. Such flexibility is seen in many fish, where sex change is a usual and adaptive part of the life cycle. Many fish are sequential hermaphrodites, beginning life as one sex and changing sometime later to the other. Sequential hermaphrodites include species capable of female-to-male (protogynous), male-to-female (protandrous), or bidirectional (serial) sex change. These natural forms of sex change involve coordinated transformations across multiple biological systems, including behavioral, anatomical, neuroendocrine and molecular axes. Here we review the biological processes underlying this amazing transformation, focusing particularly on the molecular aspects, where new genomic technologies are beginning to help us understand how sex change is initiated and regulated at the molecular level.

Teleost fish exhibit remarkably diverse and plastic patterns of sexual development. One of the most fascinating modes of plasticity is functional sex change, which is widespread in marine fish including species of commercial importance; however, the regulatory mechanisms remain elusive. In this review, we explore such sexual plasticity in fish, using the bluehead wrasse (Thalassoma bifasciatum) as the primary model. Synthesizing current knowledge, we propose that cortisol and key neurochemicals modulate gonadotropin releasing hormone and luteinizing hormone signaling to promote socially controlled sex change in protogynous fish. Future large-scale genomic analyses and systematic comparisons among species, combined with manipulation studies, will likely uncover the common and unique pathways governing this astonishing transformation. Revealing the molecular and neuroendocrine mechanisms underlying sex change in fish will greatly enhance our understanding of vertebrate sex determination and differentiation as well as phenotypic plasticity in response to environmental influences. Mol. Reprod. Dev. 84: 171-194, 2017. © 2016 Wiley Periodicals, Inc.

Reference is regularly made to the power of new genomic sequencing approaches. Using powerful technology, however, is not the same as having the necessary power to address a research question with statistical robustness. In the rush to adopt new and improved genomic research methods, limitations of technology and experimental design may be initially neglected. Here, we review these issues with regard to RNA sequencing (RNA-seq). RNA-seq adds large-scale transcriptomics to the toolkit of ecological and evolutionary biologists, enabling differential gene expression (DE) studies in nonmodel species without the need for prior genomic resources. High biological variance is typical of field-based gene expression studies and means that larger sample sizes are often needed to achieve the same degree of statistical power as clinical studies based on data from cell lines or inbred animal models. Sequencing costs have plummeted, yet RNA-seq studies still underutilize biological replication. Finite research budgets force a trade-off between sequencing effort and replication in RNA-seq experimental design. However, clear guidelines for negotiating this trade-off, while taking into account study-specific factors affecting power, are currently lacking. Study designs that prioritize sequencing depth over replication fail to capitalize on the power of RNA-seq technology for DE inference. Significant recent research effort has gone into developing statistical frameworks and software tools for power analysis and sample size calculation in the context of RNA-seq DE analysis. We synthesize progress in this area and derive an accessible rule-of-thumb guide for designing powerful RNA-seq experiments relevant in eco-evolutionary and clinical settings alike.

Sexual fate can no longer be considered an irreversible deterministic process that once established during early embryonic development, plays out unchanged across an organism's life. Rather, it appears to be a dynamic process, with sexual phenotype determined through an ongoing battle for supremacy between antagonistic male and female developmental pathways. That sexual fate is not final and is actively regulated via the suppression or activation of opposing genetic networks creates the potential for flexibility in sexual phenotype in adulthood. Such flexibility is seen in many fish, where sex change is a usual and adaptive part of the life cycle. Many fish are sequential hermaphrodites, beginning life as one sex and changing sometime later to the other. Sequential hermaphrodites include species capable of female-to-male (protogynous), male-to-female (protandrous), or bidirectional (serial) sex change. These natural forms of sex change involve coordinated transformations across multiple biological systems, including behavioral, anatomical, neuroendocrine and molecular axes. Here we review the biological processes underlying this amazing transformation, focusing particularly on the molecular aspects, where new genomic technologies are beginning to help us understand how sex change is initiated and regulated at the molecular level.

Bluehead wrasses undergo dramatic, socially-cued female to male sex change. We apply transcriptomic and methylome approaches in this wild coral reef fish to identify the primary trigger and subsequent molecular cascade of gonadal metamorphosis. Our data suggest that the environmental stimulus is exerted via the stress axis, that repression of the aromatase gene (encoding the enzyme converting androgens to estrogens) triggers a cascaded collapse of feminizing gene expression, and identifies notable sex-specific gene neofunctionalization. Furthermore, sex change involves distinct epigenetic reprogramming and an intermediate state with altered epigenetic machinery expression akin to the early developmental cells of mammals. These findings reveal at a molecular level how a normally committed developmental process remains plastic and is reversed to completely alter organ structures.One Sentence SummaryOvary to testis transformation in a sex-changing fish involves transcriptomic and epigenomic reprogramming.

Sexual fate is no longer seen as an irreversible deterministic switch set during early embryonic development but as an ongoing battle for primacy between male and female developmental trajectories. That sexual fate is not final and must be actively maintained via continuous suppression of the opposing sexual network creates the potential for flexibility into adulthood. In many fishes, sexuality is not only extremely plastic, but sex change is a usual and adaptive part of the life cycle. Sequential hermaphrodites begin life as one sex, changing sometime later to the other, and include species capable of protandrous (male-to-female), protogynous (female-to-male), or serial (bidirectional) sex change. Natural sex change involves coordinated transformations across multiple biological systems, including behavioural, anatomical, neuroendocrine, and molecular axes. We here review the biological processes underlying this amazing transformation, focussing particularly on its molecular basis, which remains poorly understood, but where new genomic technologies are significantly advancing our understanding of how sex change is initiated and progressed at the molecular level. Knowledge of how a usually committed developmental process remains plastic in sequentially hermaphroditic fishes is relevant to understanding the evolution and functioning of sexual developmental systems in vertebrates generally, as well as pathologies of sexual development in humans.

Teleost fish are the only vertebrate lineage that includes sequentially hermaphroditic species, which begin life as one sex and change sometime later to the opposite sex as a natural part of their life cycle. Sequential hermaphrodites may be protandrous (male to female), protogynous (female to male) or bidirectional (serial sex change in both directions) sex changers. Sex change in fish is a remarkable example of sexual plasticity and involves cross-talk between multiple biological systems, including neuroendocrine, hormonal and molecular systems, to coordinate dramatic changes at a whole-body level.

Cortisol is the main glucocorticoid (GC) in fish and the hormone most directly associated with stress. Recent research suggests that this hormone may act as a key factor linking social environmental stimuli and the onset of sex change by initiating a shift in steroidogenesis from estrogens to androgens. For many teleost fish, sex change occurs as a usual part of the life cycle. Changing sex is known to enhance the lifetime reproductive success of these fish and the modifications involved (behavioral, gonadal and morphological) are well studied. However, the exact mechanism behind the transduction of the environmental signals into the molecular cascade that underlies this singular process remains largely unknown. We here synthesize current knowledge regarding the role of cortisol in teleost sex change with a focus on two well-described transformations: temperature-induced masculinization and socially regulated sex change. Three non-mutually exclusive pathways are considered when describing the potential role of cortisol in mediating teleost sex change: cross-talk between GC and androgen pathways, inhibition of aromatase expression and upregulation ofamh(the gene encoding anti-Müllerian hormone). We anticipate that understanding the role of cortisol in the initial stages of sex change will further improve our understanding of sex determination and differentiation across vertebrates, and may lead to new tools to control fish sex ratios in aquaculture.

The influence of Pleistocene climatic cycles on Southern Hemisphere biotas is not yet well understood. Australia's eastern coastal margin provides an ideal setting for examining the relative influence of landscape development, sea level fluctuation, and cyclic climatic aridity on the evolution of freshwater biodiversity. We examined the impact of climatic oscillations and physical biogeographic barriers on the evolutionary history of the wide-ranging Krefft's river turtle (Emydura macquarii krefftii), using range-wide sampling (649 individuals representing 18 locations across 11 drainages) and analysis of mitochondrial sequences (∼1.3-kb control region and ND4) and nuclear microsatellites (12 polymorphic loci). A range of phylogeographic (haplotype networks, molecular dating), demographic (neutrality tests, mismatch distributions), and population genetic analyses (pairwise F ST, analysis of molecular variance, Bayesian clustering analysis) were implemented to differentiate between competing demographic (local persistence vs. range expansion) and biogeographic (arid corridor vs. drainage divide) scenarios. Genetic data reveal population genetic structure in Krefft's river turtles primarily reflects isolation across drainage divides. Striking north-south regional divergence (2.2% ND4 p-distance; c. 4.73 Ma, 95% higher posterior density (HPD) 2.08-8.16 Ma) was consistent with long-term isolation across a major drainage divide, not an adjacent arid corridor. Ancient divergence among regional lineages implies persistence of northern Krefft's populations despite the recurrent phases of severe local aridity, but with very low contemporary genetic diversity. Stable demography and high levels of genetic diversity are inferred for southern populations, where aridity was less extreme. Range-wide genetic structure in Krefft's river turtles reflects contemporary and historical drainage architecture, although regional differences in the extent of Plio-Pleistocene climatic aridity may be reflected in current levels of genetic diversity.

Aim: Our aim was to investigate the evolutionary response of a freshwater Gondwanan relictual lineage to ongoing aridification of the Australian landmass. Australian snapping turtles (Elseya spp.) are riverine specialist species and were used to examine biogeographical hypotheses associated with changes to riverine connectivity through eustatic sea-level change, landform development and aridity across this understudied region. Location: Northern and Eastern Australia and New Guinea. Methods: Phylogenetic relationships were inferred for all extant species of Elseya plus two putative species not yet described, from molecular data comprising mitochondrial (control region, ND4 and 16S) and nuclear (R35 intron) loci, using maximum likelihood and Bayesian inference methods. A calibrated relaxed molecular clock was used to estimate divergence times. Intraspecific lineage structure and diversity were investigated using control region sequences analysed via haplotype networks and AMOVA. Results: Elseya species exhibited a striking degree of local endemism across their range. Four divergent clades corresponded geographically to New Guinea, southern New Guinea plus northern Australia, north-eastern Australia, and south-eastern Australia. These arose in the late Miocene (c. 5.82-9.7Ma), diversifying further in the early Pleistocene (c. 2.2-2.43Ma and 1.36-1.66Ma), coincident with major phases of aridity and climatic upheaval. Main conclusions: The genus Elseya has a long vicariant history in Australia, closely tied to disconnection of fluvial habitat through landform evolution, sea-level rise and ongoing aridification. Our analysis paints a more complete picture of Australian freshwater biogeography, including evidence for periodic connectivity with New Guinea, important regional biogeographical barriers, and the location of potential freshwater refugia. Congruence with patterns described for terrestrial groups implies a collective response of the Australian fauna to aridification.

Effective spatial classification of freshwater biodiversity remains a worldwide conservation challenge. The isolating nature of catchment boundaries over evolutionary timescales makes them potentially important in defining natural units for biodiversity management. We sought to clarify biogeographical relationships amongst drainages within Australia's biodiverse mid-eastern coastal region (Fitzroy, Burnett, and Mary Catchments) where freshwater communities face considerable urban pressure, using a locally endemic riverine specialist, the white-throated snapping turtle, Elseya albagula. Mitochondrial and nuclear microsatellite data sets were employed to investigate past and present influences on population connectivity and to identify units for management. Populations within catchments were largely well connected genetically. However, the Fitzroy Catchment contained a distinct genetic lineage, deeply divergent from a second lineage present across the Burnett and Mary Catchments. The two lineages can be considered evolutionarily significant units that reflect historical isolation of the Fitzroy and recent coalescence of the Burnett-Mary Catchments during lowered Pleistocene sea levels. Congruence with geological evidence and patterns reported for fish and macroinvertebrates supports a shared biogeographical history of a diverse regional biota. This work highlights the need for better spatial classification of freshwater biodiversity at local as well as regional scales, including recognition of potentially cryptic diversity amongst individual river drainages.

The ornate spiny lobster Panulirus ornatus is a commercially important fishery and aquaculture species throughout south-east Asia and Australia. Thirty microsatellite markers were developed for P. ornatus of which 15 loci were consistently amplified and exhibited adequate levels of polymorphism for parentage and population genetic applications. Number of alleles observed per locus ranged from 2 to 23 (mean NA = 9.1 ± 1.4). Observed and expected heterozygosities ranged from 0.03–0.98 (HO mean = 0.61 ± 0.06) to 0.03–0.94 (HE mean = 0.66 ± 0.06), respectively. Microsatellite markers were also tested in three related Panulirus species, P. homarus, P. versicolor, and P. argus, with varying levels of amplification success and polymorphism exhibited.

454 shotgun sequencing was used to identify thousands of in silico microsatellite loci from two Australian freshwater turtle species (Pleurodira, Chelidae), Elseya albagula and Emydura macquarii krefftii. Twenty-nine polymorphic microsatellites were subsequently developed and tested as markers, 15 for E. m. krefftii and 14 for E. albagula. The former marker set showed greater allelic richness (mean N A = 7.7, range 3–15) than the latter (mean N A = 4.4, range 2–8). Levels of expected heterozygosity were high for both marker suites (mean H E = 0.718, range 0.177–0.875; and mean H E = 0.567, range 0.317–0.827, respectively), indicating both are suitable for studies of population genetic structure, connectivity and mating systems in these taxa. Cross-species amplification in 12 other short-necked freshwater turtle taxa highlighted the potential utility of these markers in other species, notably Emydura spp., Rheodytes leukops and Elusor macrurus.

Litoria nannotis is an endangered waterfall frog from the wet tropics region in north Queensland that has suffered significant population declines due to the emerging fungal disease known as chytridiomycosis. The species has two deeply divergent lineages, and we used 454 shotgun sequencing of DNA extracted from one individual of the northern lineage to identify and design PCR primers for 576 microsatellite loci. Thirty markers were tested for amplification success and variability in a population sample from each lineage. Of these, 17 were found to be polymorphic in the northern lineage and 10 loci were polymorphic in the southern lineage. Numbers of alleles per locus ranged from 2 to 14 (mean = 6.47, s.d. = 4.02) for the northern lineage (17 polymorphic loci), and from 2 to 8 (mean = 5.40, s.d. = 2.55) in the southern lineage (10 polymorphic loci). Levels of heterozygosity were high in both lineages (northern mean HE = 0.63, s.d. = 0.21, range = 0.27–0.89; southern mean HE = 0.57, s.d. = 0.25, range = 0.18–0.81). These loci will be useful in understanding the genetic variation and connectivity amongst populations of this species recovering from mass population declines due to disease.

Genetic parentage studies can provide detailed insights into the mating system dynamics of wild populations, including the prevalence and patterns of multiple paternity. Multiple paternity is assumed to be common among turtles, though its prevalence varies widely between species and populations. Several important groups remain to be investigated, including the family Chelidae, which dominate the freshwater turtle fauna of the Southern Hemisphere. We used seven polymorphic microsatellite markers to investigate the presence of multiple fathers within clutches from the white-throated snapping turtle (Elseya albagula), an Australian species of conservation concern. We uncovered a high incidence of multiple paternity, with 83% of clutches showing evidence of multiple fathers and up to three males contributing to single clutches. We confirm a largely promiscuous mating system for this species in the Burnett River, Queensland, although a lone incidence of single paternity indicates it is not the only strategy employed. These data provide the first example of multiple paternity in the Chelidae and extend our knowledge of the taxonomic breadth of multiple paternity in turtles of the Southern Hemisphere.