Museum Genomics Reveals the Hybrid Origin of an Extinct Crater Lake Endemic.

Crater lake fishes are common evolutionary model systems, with recent studies suggesting a key role for gene flow in promoting rapid adaptation and speciation. However, the study of these young lakes can be complicated by human-mediated extinctions. Museum genomics approaches integrating genetic data from recently extinct species are, therefore, critical to understanding the complex evolutionary histories of these fragile systems. Here, we examine the evolutionary history of an extinct Southern Hemisphere crater lake endemic, the rainbowfish Melanotaenia eachamensis. We undertook a comprehensive sampling of extant rainbowfish populations of the Atherton Tablelands of Australia alongside historical museum material to understand the evolutionary origins of the extinct crater lake population and the dynamics of gene flow across the ecoregion. The extinct crater lake species is genetically distinct from all other nearby populations due to historic introgression between 2 proximate riverine lineages, similar to other prominent crater lake speciation systems, but this historic gene flow has not been sufficient to induce a species flock. Our results suggest that museum genomics approaches can be successfully combined with extant sampling to unravel complex speciation dynamics involving recently extinct species.

Genomic Vulnerability to Climate Change of an Australian Migratory Freshwater Fish, the Golden Perch (Macquaria ambigua).

Genomic vulnerability is a measure of how much evolutionary change is required for a population to maintain optimal genotype-environment associations under projected climates. Aquatic species, and in particular migratory ectotherms, are largely underrepresented in studies of genomic vulnerability. Such species might be well equipped for tracking suitable habitat and spreading diversity that could promote adaptation to future climates. We characterised range-wide genomic diversity and genomic vulnerability in the migratory and fisheries-important golden perch (Macquaria ambigua) from Australia's expansive Murray-Darling Basin (MDB). The MDB has a steep hydroclimatic gradient and is one of the world's most variable regions in terms of climate and streamflow. Golden perch are threatened by fragmentation and obstruction of waterways, alteration of flow regimes, and a progressively hotter and drying climate. We gathered a genomic dataset of 1049 individuals from 186 MDB localities. Despite high range-wide gene flow, golden perch in the warmer, northern catchments had higher predicted vulnerability than those in the cooler, southern catchments. A new cross-validation approach showed that these predictions were insensitive to the exclusion of individual catchments. The results raise concern for populations at warm range edges, which may already be close to their thermal limits. However, a population with functional variants beneficial for climate adaptation found in the most arid and hydrologically variable catchment was predicted to be less vulnerable. Native fish management plans, such as captive breeding and stocking, should consider spatial variation in genomic vulnerability to improve conservation outcomes under climate change, even for dispersive species with high connectivity.

Evolutionary relationships and fine-scale geographic structuring in the temperate percichthyid genus Gadopsis (blackfishes) to support fisheries and conservation management.

Gadopsis (Percichthyidae) is a freshwater genus distributed in south-eastern Australia, including Tasmania, and comprises two recognized species. Previous molecular phylogenetic investigations of the genus, mostly conducted in the pre-genomics era and reflecting a range of geographic and molecular sampling intensities, have supported the recognition of up to seven candidate species. Here we analyze a genome-wide SNP dataset that provides comprehensive geographic and genomic coverage of Gadopsis to produce a robust hypothesis of species boundaries and evolutionary relationships. We then leverage the SNP dataset to characterize relationships within candidate species that lack clear intraspecific phylogenetic relationships. We find further support for the seven previously identified candidate species of Gadopsis and evidence that the Bass Strait centered candidate species (SBA) originated from ancient hybridization. The SNP dataset permits a high degree of intraspecific resolution, providing improvements over previous studies, with numerous candidate species showing intraspecific divisions in phylogenetic analysis. Further population genetic analysis of the Murray-Darling candidate species (NMD) and SBA finds support for K = 6 and K = 7 genetic clusters, respectively. The SNP data generated for this study have diverse applications in natural resource management for these fishes of conservation concern.

Long-term climatic stability drives accumulation and maintenance of divergent freshwater fish lineages in a temperate biodiversity hotspot.

Anthropogenic climate change is forecast to drive regional climate disruption and instability across the globe. These impacts are likely to be exacerbated within biodiversity hotspots, both due to the greater potential for species loss but also to the possibility that endemic lineages might not have experienced significant climatic variation in the past, limiting their evolutionary potential to respond to rapid climate change. We assessed the role of climatic stability on the accumulation and persistence of lineages in an obligate freshwater fish group endemic to the southwest Western Australia (SWWA) biodiversity hotspot. Using 19,426 genomic (ddRAD-seq) markers and species distribution modelling, we explored the phylogeographic history of western (Nannoperca vittata) and little (Nannoperca pygmaea) pygmy perches, assessing population divergence and phylogenetic relationships, delimiting species and estimating changes in species distributions from the Pliocene to 2100. We identified two deep phylogroups comprising three divergent clusters, which showed no historical connectivity since the Pliocene. We conservatively suggest these represent three isolated species with additional intraspecific structure within one widespread species. All lineages showed long-term patterns of isolation and persistence owing to climatic stability but with significant range contractions likely under future climate change. Our results highlighted the role of climatic stability in allowing the persistence of isolated lineages in the SWWA. This biodiversity hotspot is under compounding threat from ongoing climate change and habitat modification, which may further threaten previously undetected cryptic diversity across the region.

Diversification of the sleepers (Gobiiformes: Gobioidei: Eleotridae) and evolution of the root gobioid families.

Eleotridae (sleepers) and five smaller families are the earliest diverging lineages within Gobioidei. Most inhabit freshwaters in and around the Indo-Pacific, but Eleotridae also includes species that have invaded the Neotropics as well as several inland radiations in the freshwaters of Australia, New Zealand, and New Guinea. Previous efforts to infer phylogeny of these families have been based on sets of mitochondrial or nuclear loci and have yielded uncertain resolution of clades within Eleotridae. We expand the taxon sampling of previous studies and use genomic data from nuclear ultraconserved elements (UCEs) to infer phylogeny, then calibrate the hypothesis with recently discovered fossils. Our hypothesis clarifies ambiguously resolved relationships, provides a timescale for divergences, and indicates the core crown Eleotridae diverged over a short period 24.3-26.3 Ma in the late Oligocene. Within Eleotridae, we evaluate diversification dynamics with BAMM and find evidence for an overall slowdown in diversification over the past 35 Ma, but with a sharp increase 3.5 Ma in the genus Mogurnda, a clade of brightly colored species found in the freshwaters of Australia and New Guinea.

Phylogenomics and biogeography of arid-adapted Chlamydogobius goby fishes.

The progressive aridification of the Australian continent from âˆ¼ 20 million years ago posed severe challenges for the persistence of its resident biota. A key question involves the role of refugial habitats - specifically, their ability to mediate the effects of habitat loss and fragmentation, and their potential to shape opportunities for allopatric speciation. With freshwater species, for example, the patchiness, or absence, of water will constrain distributions. However, aridity may not necessarily isolate populations if disjunct refugia experience frequent hydrological connections. To investigate this potential dichotomy, we explored the evolutionary history of the Chlamydogobius gobies (Gobiiformes: Gobiidae), an arid-adapted genus of six small, benthic fish species that exploit all types of waterbodies (i.e. desert springs, waterholes and bore-fed wetlands, coastal estuarine creeks and mangroves) across parts of central and northern Australia. We used Anchored Phylogenomics to generate a highly resolved phylogeny of the group from sequence data for 260 nuclear loci. Buttressed by companion allozyme and mtDNA datasets, our molecular findings infer the diversification of Chlamydogobius in arid Australia, and provide a phylogenetic structure that cannot be simply explained by invoking allopatric speciation events reflecting current geographic proximity. Our findings are generally consistent with the existing morphological delimitation of species, with one exception: at the shallowest nodes of phylogenetic reconstruction, the molecular data do not fully support the current dichotomous delineation of C. japalpa from C. eremius in Kati Thanda-Lake Eyre-associated waterbodies. Together these findings illustrate the ability of structural (hydrological) connections to generate patterns of connectivity and isolation for an ecologically moderate disperser in response to ongoing habitat aridification. Finally, we explore the implications of these results for the immediate management of threatened (C. gloveri) and critically endangered (C. micropterus, C. squamigenus) congeners.

Environmental selection, rather than neutral processes, best explain regional patterns of diversity in a tropical rainforest fish.

To conserve the high functional and genetic variation in hotspots such as tropical rainforests, it is essential to understand the forces driving and maintaining biodiversity. We asked to what extent environmental gradients and terrain structure affect morphological and genomic variation across the wet tropical distribution of an Australian rainbowfish, Melanotaenia splendida splendida. We used an integrative riverscape genomics and morphometrics framework to assess the influence of these factors on both putative adaptive and non-adaptive spatial divergence. We found that neutral genetic population structure was largely explainable by restricted gene flow among drainages. However, environmental associations revealed that ecological variables had a similar power to explain overall genetic variation, and greater power to explain body shape variation, than the included neutral covariables. Hydrological and thermal variables were the strongest environmental predictors and were correlated with traits previously linked to heritable habitat-associated dimorphism in rainbowfishes. In addition, climate-associated genetic variation was significantly associated with morphology, supporting heritability of shape variation. These results support the inference of evolved functional differences among localities, and the importance of hydroclimate in early stages of diversification. We expect that substantial evolutionary responses will be required in tropical rainforest endemics to mitigate local fitness losses due to changing climates.

Delayed Adaptive Radiation among New Zealand Stream Fishes: Joint Estimation of Divergence Time and Trait Evolution in a Newly Delineated Island Species Flock.

Adaptive radiations are generally thought to occur soon after a lineage invades a region offering high levels of ecological opportunity. However, few adaptive radiations beyond a handful of exceptional examples are known, so a comprehensive understanding of their dynamics is still lacking. Here, we present a novel case of an island species flock of freshwater fishes with a radically different tempo of adaptive history than that found in many popular evolutionary model systems. Using a phylogenomic data set combined with simultaneous Bayesian estimation of divergence times and trait-based speciation and extinction models, we show that the New Zealand Gobiomorphus gudgeons comprise a monophyletic assemblage, but surprisingly, the radiation did not fully occupy freshwater habitats and explosively speciate until more than 10 myr after the lineage invaded the islands. This shift in speciation rate was not accompanied by an acceleration in the rate of morphological evolution in the freshwater crown clade relative to the other species, but is correlated with a reduction in head pores and scales as well as an increase in egg size. Our results challenge the notion that clades always rapidly exploit ecological opportunities in the absence of competing lineages. Instead, we demonstrate that adaptive radiation can experience a slow start before undergoing accelerated diversification and that lineage and phenotypic diversification may be uncoupled in young radiations. [Adaptive radiation; Eleotridae; freshwater; Gobiomorphus; New Zealand.].

The roles of aridification and sea level changes in the diversification and persistence of freshwater fish lineages.

While the influence of Pleistocene climatic changes on divergence and speciation has been well-documented across the globe, complex spatial interactions between hydrology and eustatics over longer timeframes may also determine species evolutionary trajectories. Within the Australian continent, glacial cycles were not associated with changes in ice cover and instead largely resulted in fluctuations from moist to arid conditions across the landscape. We investigated the role of hydrological and coastal topographic changes brought about by Plio-Pleistocene climatic changes on the biogeographic history of a small Australian freshwater fish, the southern pygmy perch Nannoperca australis. Using 7958 ddRAD-seq (double digest restriction-site associated DNA) loci and 45,104 filtered SNPs, we combined phylogenetic, coalescent and species distribution analyses to assess the various roles of aridification, sea level and tectonics and associated biogeographic changes across southeast Australia. Sea-level changes since the Pliocene and reduction or disappearance of large waterbodies throughout the Pleistocene were determining factors in strong divergence across the clade, including the initial formation and maintenance of a cryptic species, N. 'flindersi'. Isolated climatic refugia and fragmentation due to lack of connected waterways maintained the identity and divergence of inter- and intraspecific lineages. Our historical findings suggest that predicted increases in aridification and sea level due to anthropogenic climate change might result in markedly different demographic impacts, both spatially and across different landscape types.

A fossil-calibrated time-tree of all Australian freshwater fishes.

Australian freshwater fishes are a relatively species-poor assemblage, mostly comprising groups derived from older repeated freshwater invasions by marine ancestors, plus a small number of Gondwanan lineages. These taxa are both highly endemic and highly threatened, but a comprehensive phylogeny for Australian freshwater fishes is lacking. This has hampered efforts to study their phylogenetic diversity, distribution of extinction risk, speciation rates, and rates of trait evolution. Here, we present a comprehensive dated phylogeny of 412 Australian fishes. We include all formally recognized freshwater species plus a number of genetically distinct subpopulations, species awaiting formal description, and predominantly brackish-water species that sometimes enter fresh water. The phylogeny was inferred using maximum-likelihood analysis of a multilocus data set comprising six mitochondrial and three nuclear genes from 326 taxa. We inferred the evolutionary timescale using penalized likelihood, then used a statistical approach to add 86 taxa for which no molecular data were available. The time-tree inferred in our study will provide a useful resource for macroecological studies of Australian freshwater fishes by enabling corrections for phylogenetic non-independence in evolutionary and ecological comparative analyses.

Surprising Pseudogobius: Molecular systematics of benthic gobies reveals new insights into estuarine biodiversity (Teleostei: Gobiiformes).

Snubnose gobies (genus Pseudogobius: Gobionellinae) are ubiquitous to, and important components of, estuarine ecosystems of the Indo-west Pacific. These small benthic fishes occur in freshwater, brackish and marine habitats such as mangroves, sheltered tide pools and lowland streams, and represent a model group for understanding the biodiversity and biogeography of estuarine fauna. To develop the species-level framework required for a concurrent morphological taxonomic appraisal, we undertook thorough sampling around the extensive Australian coastline, referenced to international locations, as part of a molecular systematic review using both nuclear and mitochondrial markers. The results indicate that while there are currently eight recognised species, the true diversity is close to double this, with a hotspot of endemism located in Australia. Complicated patterns were observed in southern Australia owing to two differing zones of introgression/admixture. Key drivers of diversity in the group appear to include plate tectonics, latitude, and historic barriers under glacial maxima, where an interplay between ready dispersal and habitat specialisation has led to regional panmixia but frequent geographic compartmentalisation within past and present landscapes. The findings have significant implications for biodiversity conservation, coastal and estuarine development, the basic foundations of field ecology, and for applied use such as in biomonitoring.

Unravelling the taxonomy and identification of a problematic group of benthic fishes from tropical rivers (Gobiidae: Glossogobius).

Flathead gobies (genus Glossogobius) include c. 40 small- to medium-sized benthic fishes found primarily in freshwater habitats across the Indo-Pacific, having biodiversity value as well as cultural and economic value as food fishes, especially in developing countries. To help resolve considerable confusion regarding the identification of some of the larger-growing Glossogobius species, a systematic framework was established using nuclear genetic markers, mitochondrial DNA barcoding and phenotypic evidence for a geographically widespread collection of individuals from the waterways of tropical northern Australia. Species boundaries and distribution patterns were discordant with those previously reported, most notably for the tank goby Glossogobius giuris, which included a cryptic species. Genetic divergence was matched with accompanying unique visual characters that aid field identification. Additional taxonomic complexity was also evident, by comparison with DNA barcodes from international locations, suggesting that the specific names applicable for two of the candidate species in Australia remain unresolved due to confusion surrounding type specimens. Although flathead gobies are assumed to be widespread and common, this study demonstrates that unrealised taxonomic and ecological complexity is evident, and this will influence assessments of tropical biodiversity and species conservation. This study supports the need for taxonomic studies of freshwater fishes to underpin management in areas subject to significant environmental change.

Latitudinal variation in climate-associated genes imperils range edge populations.

The ecological impacts of increasing global temperatures are evident in most ecosystems on Earth, but our understanding of how climatic variation influences natural selection and adaptive resilience across latitudes remains largely unknown. Latitudinal gradients allow testing general ecosystem-level theories relevant to climatic adaptation. We assessed differences in adaptive diversity of populations along a latitudinal region spanning highly variable temperate to subtropical climates. We generated and integrated information from environmental mapping, phenotypic variation and genome-wide data from across the geographical range of the rainbowfish Melanotaenia duboulayi, an emerging aquatic system for studies of climate change. We detected, after controlling for spatial population structure, strong interactions between genotypes and environment associated with variation in stream flow and temperature. Some of these hydroclimate-associated genes were found to interact within functional protein networks that contain genes of adaptive significance for projected future climates in rainbowfish. Hydroclimatic selection was also associated with variation in phenotypic traits, including traits known to affect fitness of rainbowfish exposed to different flow environments. Consistent with predictions from the "climatic variability hypothesis," populations exposed to extremes of important environmental variables showed stronger adaptive divergence and less variation in climate-associated genes compared to populations at the centre of the environmental gradient. Our findings suggest that populations that evolved at environmental range margins and at geographical range edges may be more vulnerable to changing climates, a finding with implications for predicting adaptive resilience and managing biodiversity under climate change.

Comparison of genetic structure in co-occurring freshwater eleotrids (Actinopterygii: Philypnodon) reveals cryptic species, likely translocation and regional conservation hotspots.

Freshwater systems are naturally fragmented and heterogeneous habitats that promote genetic sub-division and speciation for aquatic biota. Here we provide a novel nuclear genetic perspective (49 allozyme loci) complimented with updated mitochondrial data for the eleotrid genus Philypnodon to investigate broad genetic sub-structure across south-eastern Australia as a foundation for management and conservation. The genus is nominally comprised of two small benthic fishes with contrasting physical and ecological traits, namely the Flathead Gudgeon P. grandiceps and the Dwarf Flathead Gudgeon P. macrostomus. Extensive sample coverage included 99 sites across 5 major drainage divisions and 48 river basins. Nuclear markers revealed strong, geographically-based divergence and sub-structure, contrasting with shallower but largely congruent patterns for mtDNA. The results flag that each nominal species represents a hyper-cryptic species complex, including both broadly distributed and narrow-range taxa, with complicated biogeographic patterns. Predictions on dispersal and genetic structure based on ecological traits were only partially supported and varied by region, with the potential signature of human-assisted translocation evident in several catchments. Further intensive sampling in an important area of high genetic diversity, coastal south-east Queensland, is recommended to better resolve species boundaries and conservation units. The findings provide new insights on regional ecology and biogeography, demonstrating that even supposedly common species can, in reality, have complex conservation and management needs.

On the roles of landscape heterogeneity and environmental variation in determining population genomic structure in a dendritic system.

Dispersal and natural selection are key evolutionary processes shaping the distribution of phenotypic and genetic diversity. For species inhabiting complex spatial environments however, it is unclear how the balance between gene flow and selection may be influenced by landscape heterogeneity and environmental variation. Here, we evaluated the effects of dendritic landscape structure and the selective forces of hydroclimatic variation on population genomic parameters for the Murray River rainbowfish, Melanotaenia fluviatilis across the Murray-Darling Basin, Australia. We genotyped 249 rainbowfish at 17,503 high-quality SNP loci and integrated these with models of network connectivity and high-resolution environmental data within a riverscape genomics framework. We tested competing models of gene flow before using multivariate genotype-environment association (GEA) analysis to test for signals of adaptive divergence associated with hydroclimatic variation. Patterns of neutral genetic variation were consistent with expectations based on the stream hierarchy model and M. fluviatilis' moderate dispersal ability. Models incorporating dendritic network structure suggested that landscape heterogeneity is a more important factor determining connectivity and gene flow than waterway distance. Extending these results, we also introduce a novel approach to controlling for the unique effects of dendritic network structure in GEA analyses of populations of aquatic species. We identified 146 candidate loci potentially underlying a polygenic adaptive response to seasonal fluctuations in stream flow and variation in the relative timing of temperature and precipitation extremes. Our findings underscore an emerging predominant role for seasonal variation in hydroclimatic conditions driving local adaptation and are relevant for informing proactive conservation management.

Genomewide SNP markers breathe new life into phylogeography and species delimitation for the problematic short-necked turtles (Chelidae: Emydura) of eastern Australia.

Understanding the evolutionary history of diversifying lineages and the delineation of evolutionarily significant units and species remains major challenges for evolutionary biology. Low-cost representational sampling of the genome for single nucleotide polymorphisms shows great potential at the temporal scales that are typically the focus of species delimitation and phylogeography. We apply these markers to a case study of a freshwater turtle, Emydura macquarii, whose systematics has so far defied resolution, to bring to light a dynamic system of substantive allopatric lineages diverging on independent evolutionary trajectories, but held back in the process of speciation by low level and episodic exchange of alleles across drainage divides on various timescales. In the context of low-level episodic gene flow, speciation is often reticulate, rather than a bifurcating process. We argue that species delimitation needs to take into account the pattern of ancestry and descent of diverging lineages in allopatry together with the recent and contemporary processes of dispersal and gene flow that retard and obscure that divergence. Underpinned by a strong focus on lineage diagnosability, this combined approach provides a means for addressing the challenges of incompletely isolated populations with uncommon, but recurrent gene flow in studies of species delimitation, a situation likely to be frequently encountered. Taxonomic decisions in cases of allopatry often require subjective judgements. Our strategy, which adds an additional level of objectivity before that subjectivity is applied, reduces the risk of taxonomic inflation that can accompany lineage approaches to species delimitation.

Ecological disturbance influences adaptive divergence despite high gene flow in golden perch (Macquaria ambigua): Implications for management and resilience to climate change.

Populations that are adaptively divergent but maintain high gene flow may have greater resilience to environmental change as gene flow allows the spread of alleles that have already been tested elsewhere. In addition, populations naturally subjected to ecological disturbance may already hold resilience to future environmental change. Confirming this necessitates ecological genomic studies of high dispersal, generalist species. Here we perform one such study on golden perch (Macquaria ambigua) in the Murray-Darling Basin (MDB), Australia, using a genome-wide SNP data set. The MDB spans across arid to wet and temperate to subtropical environments, with low to high ecological disturbance in the form of low to high hydrological variability. We found high gene flow across the basin and three populations with low neutral differentiation. Genotype-environment association analyses detected adaptive divergence predominantly linked to an arid region with highly variable riverine flow, and candidate loci included functions related to fat storage, stress and molecular or tissue repair. The high connectivity of golden perch in the MDB will likely allow locally adaptive traits in its most arid and hydrologically variable environment to spread and be selected in localities that are predicted to become arid and hydrologically variable in future climates. High connectivity in golden perch is likely due to their generalist life history and efforts of fisheries management. Our study adds to growing evidence of adaptation in the face of gene flow and highlights the importance of considering ecological disturbance and adaptive divergence in biodiversity management.

Cryptic biodiversity in the freshwater fishes of the Kimberley endemism hotspot, northwestern Australia.

The prevalence of unrecognised cryptic species impairs biodiversity estimates, clouds biological research and hinders conservation planning. As the rate of cryptic species detection increases globally, research is needed to determine how frequent cryptic species are, whether they are more common in given management regions, and whether these patterns are consistent across taxonomic groups. The Kimberley region in remote northwestern Australia harbours some of the most speciose, and morphologically and functionally diverse, endemic animal and plant communities on the continent. The rugged and changeable landscape also appears to contain a large proportion of cryptic terrestrial species, raising the question of whether similar patterns are also found among aquatic taxa, which have yet to be studied using integrative systematic approaches. If true, then the actual levels of aquatic biodiversity are yet to be fully realised. Here we conducted a molecular assessment of where species boundaries may exist in the Kimberley regions' most speciose freshwater fish family, the Terapontidae (grunters), with a combined morphological assessment of the regions' most speciose terapontid genus, Syncomistes. Assessment of nuclear markers (54 allozyme loci), sequence data (mitochondrial cytochrome b (cytb); nuclear recombination activation gene one (RAG1)) and 31 meristic and 36 morphometric characters provides evidence for 13 new candidate species across three different genera. Many of these candidate species are narrow range endemics. Our findings raise several questions about the evolutionary origin of the Kimberley's endemic fish fauna and highlight the likelihood that freshwater fish species diversity in the Kimberley is severely under-represented by current systematic frameworks, with significant implications for ecological research, conservation and management.

Comparative ecological transcriptomics and the contribution of gene expression to the evolutionary potential of a threatened fish.

Understanding whether small populations with low genetic diversity can respond to rapid environmental change via phenotypic plasticity is an outstanding research question in biology. RNA sequencing (RNA-seq) has recently provided the opportunity to examine variation in gene expression, a surrogate for phenotypic variation, in nonmodel species. We used a comparative RNA-seq approach to assess expression variation within and among adaptively divergent populations of a threatened freshwater fish, Nannoperca australis, found across a steep hydroclimatic gradient in the Murray-Darling Basin, Australia. These populations evolved under contrasting selective environments (e.g., dry/hot lowland; wet/cold upland) and represent opposite ends of the species' spectrum of genetic diversity and population size. We tested the hypothesis that environmental variation among isolated populations has driven the evolution of divergent expression at ecologically important genes using differential expression (DE) analysis and an anova-based comparative phylogenetic expression variance and evolution model framework based on 27,425 de novo assembled transcripts. Additionally, we tested whether gene expression variance within populations was correlated with levels of standing genetic diversity. We identified 290 DE candidate transcripts, 33 transcripts with evidence for high expression plasticity, and 50 candidates for divergent selection on gene expression after accounting for phylogenetic structure. Variance in gene expression appeared unrelated to levels of genetic diversity. Functional annotation of the candidate transcripts revealed that variation in water quality is an important factor influencing expression variation for N. australis. Our findings suggest that gene expression variation can contribute to the evolutionary potential of small populations.

Resolution of the enigmatic phylogenetic relationship of the critically endangered Western Swamp Tortoise Pseudemydura umbrina (Pleurodira: Chelidae) using a complete mitochondrial genome.

Pseudemydura umbrina is one of the most endangered turtle species in the world, and the imperative for its conservation is its distinctive morphology and relict status among the Chelidae. We use Illumina sequencing to obtain the complete mitogenome for resolving its uncertain phylogenetic position. A novel nuclear paralogue confounded the assembly, and resolution of the authentic mitogenome required further Sanger sequencing. The P. umbrina mitogenome is 16,414bp comprising 37 genes organized in a conserved pattern for other vertebrates. The nuclear paralogue is 547bp, 97.8% identity to the corresponding mitochondrial sequence. Particular features of the mitogenome include an nd3 174+1A frameshift, loss of DHC loop in tRNASer (AGN), and a light-strand replication initiation site in Wancy region that extends into an adjacent tRNA gene. Phylogenetic analysis showed that P. umbrina is the monotypic sister lineage to the remaining Australasian Chelidae, a lineage probably dating back to the Cretaceous.