Museum skins enable identification of introgression associated with cytonuclear discordance.

Increased sampling of genomes and populations across closely related species has revealed that levels of genetic exchange during and after speciation are higher than previously thought. One obvious manifestation of such exchange is strong cytonuclear discordance, where the divergence in mitochondrial DNA (mtDNA) differs from that for nuclear genes more (or less) than expected from differences between mtDNA and nuclear DNA (nDNA) in population size and mutation rate. Given genome-scale datasets and coalescent modelling, we can now confidently identify cases of strong discordance and test specifically for historical or recent introgression as the cause. Using population sampling, combining exon capture data from historical museum specimens and recently collected tissues we showcase how genomic tools can resolve complex evolutionary histories in the brachyotis group of rock-wallabies (Petrogale). In particular, applying population and phylogenomic approaches we can assess the role of demographic processes in driving complex evolutionary patterns and assess a role of ancient introgression and hybridisation. We find that described species are well supported as monophyletic taxa for nDNA genes, but not for mtDNA, with cytonuclear discordance involving at least four operational taxonomic units (OTUs) across four species which diverged 183-278 kya. ABC modelling of nDNA gene trees supports introgression during or after speciation for some taxon pairs with cytonuclear discordance. Given substantial differences in body size between the species involved, this evidence for gene flow is surprising. Heterogenous patterns of introgression were identified but do not appear to be associated with chromosome differences between species. These and previous results suggest that dynamic past climates across the monsoonal tropics could have promoted reticulation among related species.

Functionally mediated cranial allometry evidenced in a genus of rock-wallabies.

In assessments of skeletal variation, allometry (disproportionate change of shape with size) is often corrected to examine size-independent variation for hypotheses relating to function. However, size-related trade-offs in functional demands may themselves be an underestimated driver of mammalian cranial diversity. Here, we use geometric morphometrics alongside dental measurements to assess craniodental allometry in the rock-wallaby genus Petrogale (all 17 species, 370 individuals). We identified functional aspects of evolutionary allometry that can be both extensions of, and correlated negatively with, static or ontogenetic allometric patterns. Regarding constraints, larger species tended to have relatively smaller braincases and more posterior orbits, the former of which might represent a constraint on jaw muscle anatomy. However, they also tended to have more anterior dentition and smaller posterior zygomatic arches, both of which support the hypothesis of relaxed bite force demands and accommodation of different selective pressures that favour facial elongation. By contrast, two dwarf species had stouter crania with divergent dental adaptations that together suggest increased relative bite force capacity. This likely allows them to feed on forage that is mechanically similar to that consumed by larger relatives. Our results highlight a need for nuanced considerations of allometric patterns in future research of mammalian cranial diversity.

Limited Introgression between Rock-Wallabies with Extensive Chromosomal Rearrangements.

Chromosome rearrangements can result in the rapid evolution of hybrid incompatibilities. Robertsonian fusions, particularly those with monobrachial homology, can drive reproductive isolation amongst recently diverged taxa. The recent radiation of rock-wallabies (genus Petrogale) is an important model to explore the role of Robertsonian fusions in speciation. Here, we pursue that goal using an extensive sampling of populations and genomes of Petrogale from north-eastern Australia. In contrast to previous assessments using mitochondrial DNA or nuclear microsatellite loci, genomic data are able to separate the most closely related species and to resolve their divergence histories. Both phylogenetic and population genetic analyses indicate introgression between two species that differ by a single Robertsonian fusion. Based on the available data, there is also evidence for introgression between two species which share complex chromosomal rearrangements. However, the remaining results show no consistent signature of introgression amongst species pairs and where evident, indicate generally low introgression overall. X-linked loci have elevated divergence compared with autosomal loci indicating a potential role for genic evolution to produce reproductive isolation in concert with chromosome change. Our results highlight the value of genome scale data in evaluating the role of Robertsonian fusions and structural variation in divergence, speciation, and patterns of molecular evolution.

Genetic mixing in conservation translocations increases diversity of a keystone threatened species, Bettongia lesueur.

Translocation programmes are increasingly being informed by genetic data to monitor and enhance conservation outcomes for both natural and established populations. These data provide a window into contemporary patterns of genetic diversity, structure and relatedness that can guide managers in how to best source animals for their translocation programmes. The inclusion of historical samples, where possible, strengthens monitoring by allowing assessment of changes in genetic diversity over time and by providing a benchmark for future improvements in diversity via management practices. Here, we used reduced representation sequencing (ddRADseq) data to report on the current genetic health of three remnant and seven translocated boodie (Bettongia lesueur) populations, now extinct on the Australian mainland. In addition, we used exon capture data from seven historical mainland specimens and a subset of contemporary samples to compare pre-decline and current diversity. Both data sets showed the significant impact of population founder source (whether multiple or single) on the genetic diversity of translocated populations. Populations founded by animals from multiple sources showed significantly higher genetic diversity than the natural remnant and single-source translocation populations, and we show that by mixing the most divergent populations, exon capture heterozygosity was restored to levels close to that observed in pre-decline mainland samples. Relatedness estimates were surprisingly low across all contemporary populations and there was limited evidence of inbreeding. Our results show that a strategy of genetic mixing has led to successful conservation outcomes for the species in terms of increasing genetic diversity and provides strong rationale for mixing as a management strategy.

Future-proofing the koala: Synergising genomic and environmental data for effective species management.

Climatic and evolutionary processes are inextricably linked to conservation. Avoiding extinction in rapidly changing environments often depends upon a species' capacity to adapt in the face of extreme selective pressures. Here, we employed exon capture and high-throughput next-generation sequencing to investigate the mechanisms underlying population structure and adaptive genetic variation in the koala (Phascolarctos cinereus), an iconic Australian marsupial that represents a unique conservation challenge because it is not uniformly threatened across its range. An examination of 250 specimens representing 91 wild source locations revealed that five major genetic clusters currently exist on a continental scale. The initial divergence of these clusters appears to have been concordant with the Mid-Brunhes Transition (~430 to 300 kya), a major climatic reorganisation that increased the amplitude of Pleistocene glacial-interglacial cycles. While signatures of polygenic selection and environmental adaptation were detected, strong evidence for repeated, climate-associated range contractions and demographic bottleneck events suggests that geographically isolated refugia may have played a more significant role in the survival of the koala through the Pleistocene glaciation than in situ adaptation. Consequently, the conservation of genome-wide genetic variation must be aligned with the protection of core koala habitat to increase the resilience of vulnerable populations to accelerating anthropogenic threats. Finally, we propose that the five major genetic clusters identified in this study should be accounted for in future koala conservation efforts (e.g., guiding translocations), as existing management divisions in the states of Queensland and New South Wales do not reflect historic or contemporary population structure.

Population genomics of a predatory mammal reveals patterns of decline and impacts of exposure to toxic toads.

Mammal declines across northern Australia are one of the major biodiversity loss events occurring globally. There has been no regional assessment of the implications of these species declines for genomic diversity. To address this, we conducted a species-wide assessment of genomic diversity in the northern quoll (Dasyurus hallucatus), an Endangered marsupial carnivore. We used next generation sequencing methods to genotype 10,191 single nucleotide polymorphisms (SNPs) in 352 individuals from across a 3220-km length of the continent, investigating patterns of population genomic structure and diversity, and identifying loci showing signals of putative selection. We found strong heterogeneity in the distribution of genomic diversity across the continent, characterized by (i) biogeographical barriers driving hierarchical population structure through long-term isolation, and (ii) severe reductions in diversity resulting from population declines, exacerbated by the spread of introduced toxic cane toads (Rhinella marina). These results warn of a large ongoing loss of genomic diversity and associated adaptive capacity as mammals decline across northern Australia. Encouragingly, populations of the northern quoll established on toad-free islands by translocations appear to have maintained most of the initial genomic diversity after 16 years. By mapping patterns of genomic diversity within and among populations, and investigating these patterns in the context of population declines, we can provide conservation managers with data critical to informed decision-making. This includes the identification of populations that are candidates for genetic management, the importance of remnant island and insurance/translocated populations for the conservation of genetic diversity, and the characterization of putative evolutionarily significant units.

Correction: Phylogeography of the iconic Australian red-tailed black-cockatoo (Calyptorhynchus banksii) and implications for its conservation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phylogeography of the iconic Australian red-tailed black-cockatoo (Calyptorhynchus banksii) and implications for its conservation.

Advances in sequencing technologies have revolutionized wildlife conservation genetics. Analysis of genomic data sets can provide high-resolution estimates of genetic structure, genetic diversity, gene flow, and evolutionary history. These data can be used to characterize conservation units and to effectively manage the genetic health of species in a broad evolutionary context. Here we utilize thousands of genome-wide single-nucleotide polymorphisms (SNPs) and mitochondrial DNA to provide the first genetic assessment of the Australian red-tailed black-cockatoo (Calyptorhynchus banksii), a widespread bird species comprising populations of varying conservation concern. We identified five evolutionarily significant units, which are estimated to have diverged during the Pleistocene. These units are only partially congruent with the existing morphology-based subspecies taxonomy. Genetic clusters inferred from mitochondrial DNA differed from those based on SNPs and were less resolved. Our study has a range of conservation and taxonomic implications for this species. In particular, we provide advice on the potential genetic rescue of the Endangered and restricted-range subspecies C. b. graptogyne, and propose that the western C. b. samueli population is diagnosable as a separate subspecies. The results of our study highlight the utility of considering the phylogeographic relationships inferred from genome-wide SNPs when characterizing conservation units and management priorities, which is particularly relevant as genomic data sets become increasingly accessible.

Analysis of Phylogenomic Tree Space Resolves Relationships Among Marsupial Families.

A fundamental challenge in resolving evolutionary relationships across the tree of life is to account for heterogeneity in the evolutionary signal across loci. Studies of marsupial mammals have demonstrated that this heterogeneity can be substantial, leaving considerable uncertainty in the evolutionary timescale and relationships within the group. Using simulations and a new phylogenomic data set comprising nucleotide sequences of 1550 loci from 18 of the 22 extant marsupial families, we demonstrate the power of a method for identifying clusters of loci that support different phylogenetic trees. We find two distinct clusters of loci, each providing an estimate of the species tree that matches previously proposed resolutions of the marsupial phylogeny. We also identify a well-supported placement for the enigmatic marsupial moles (Notoryctes) that contradicts previous molecular estimates but is consistent with morphological evidence. The pattern of gene-tree variation across tree-space is characterized by changes in information content, GC content, substitution-model adequacy, and signatures of purifying selection in the data. In a simulation study, we show that incomplete lineage sorting can explain the division of loci into the two tree-topology clusters, as found in our phylogenomic analysis of marsupials. We also demonstrate the potential benefits of minimizing uncertainty from phylogenetic conflict for molecular dating. Our analyses reveal that Australasian marsupials appeared in the early Paleocene, whereas the diversification of present-day families occurred primarily during the late Eocene and early Oligocene. Our methods provide an intuitive framework for improving the accuracy and precision of phylogenetic inference and molecular dating using genome-scale data.

Phylogenetic analysis of the tree-kangaroos (Dendrolagus) reveals multiple divergent lineages within New Guinea.

Amongst the Australasian kangaroos and wallabies (Macropodidae) one anomalous genus, the tree-kangaroos, Dendrolagus, has secondarily returned to arboreality. Modern tree-kangaroos are confined to the wet tropical forests of north Queensland, Australia (2 species) and New Guinea (8 species). Due to their behavior, distribution and habitat most species are poorly known and our understanding of the evolutionary history and systematics of the genus is limited and controversial. We obtained tissue samples from 36 individual Dendrolagus including representatives from 14 of the 17 currently recognised or proposed subspecies and generated DNA sequence data from three mitochondrial (3116 bp) and five nuclear (4097 bp) loci. Phylogenetic analysis of these multi-locus data resolved long-standing questions regarding inter-relationships within Dendrolagus. The presence of a paraphyletic ancestral long-footed and derived monophyletic short-footed group was confirmed. Six major lineages were identified: one in Australia (D. lumholtzi, D. bennettianus) and five in New Guinea (D. inustus, D. ursinus, a Goodfellow's group, D. mbaiso and a Doria's group). Two major episodes of diversification within Dendrolagus were identified: the first during the late Miocene/early Pliocene associated with orogenic processes in New Guinea and the second mostly during the early Pleistocene associated with the intensification of climatic cycling. All sampled subspecies showed high levels of genetic divergence and currently recognized species within both the Doria's and Goodfellow's groups were paraphyletic indicating that adjustments to current taxonomy are warranted.

Conservation and Genetics.

Humans are responsible for a cataclysm of species extinction that will change the world as we see it, and will adversely affect human health and wellbeing. We need to understand at individual and societal levels why species conservation is important. Accepting the premise that species have value, we need to next consider the mechanisms underlying species extinction and what we can do to reverse the process. One of the last stages of species extinction is the reduction of a species to a few populations of relatively few individuals, a scenario that leads invariably to inbreeding and its adverse consequences, inbreeding depression. Inbreeding depression can be so severe that populations become at risk of extinction not only because of the expression of harmful recessive alleles (alleles having no phenotypic effect when in the heterozygous condition, e.g., Aa, where a is the recessive allele), but also because of their inability to respond genetically with sufficient speed to adapt to changing environmental conditions. However, new conservation approaches based on foundational quantitative and population genetic theory advocate for active genetic management of fragmented populations by facilitating gene movements between populations, i.e., admixture, or genetic rescue. Why species conservation is critical, the genetic consequences of small population size that often lead to extinction, and possible solutions to the problem of small population size are discussed and presented.

Gene flow despite complex Robertsonian fusions among rock-wallaby (Petrogale) species.

Complex Robertsonian rearrangements, with shared arms in different fusions, are expected to prevent gene flow between hybrids through missegregation during meiosis. Here, we estimate gene flow between recently diverged and chromosomally diverse rock-wallabies (Petrogale) to test for this form of chromosomal speciation. Contrary to expectations, we observe relatively high admixture among species with complex fusions. Our results reinforce the need to consider alternative roles of chromosome change, together with genic divergence, in driving speciation.

A transcriptome resource for the koala (Phascolarctos cinereus): insights into koala retrovirus transcription and sequence diversity.

BACKGROUND: The koala, Phascolarctos cinereus, is a biologically unique and evolutionarily distinct Australian arboreal marsupial. The goal of this study was to sequence the transcriptome from several tissues of two geographically separate koalas, and to create the first comprehensive catalog of annotated transcripts for this species, enabling detailed analysis of the unique attributes of this threatened native marsupial, including infection by the koala retrovirus. RESULTS: RNA-Seq data was generated from a range of tissues from one male and one female koala and assembled de novo into transcripts using Velvet-Oases. Transcript abundance in each tissue was estimated. Transcripts were searched for likely protein-coding regions and a non-redundant set of 117,563 putative protein sequences was produced. In similarity searches there were 84,907 (72%) sequences that aligned to at least one sequence in the NCBI nr protein database. The best alignments were to sequences from other marsupials. After applying a reciprocal best hit requirement of koala sequences to those from tammar wallaby, Tasmanian devil and the gray short-tailed opossum, we estimate that our transcriptome dataset represents approximately 15,000 koala genes. The marsupial alignment information was used to look for potential gene duplications and we report evidence for copy number expansion of the alpha amylase gene, and of an aldehyde reductase gene.Koala retrovirus (KoRV) transcripts were detected in the transcriptomes. These were analysed in detail and the structure of the spliced envelope gene transcript was determined. There was appreciable sequence diversity within KoRV, with 233 sites in the KoRV genome showing small insertions/deletions or single nucleotide polymorphisms. Both koalas had sequences from the KoRV-A subtype, but the male koala transcriptome has, in addition, sequences more closely related to the KoRV-B subtype. This is the first report of a KoRV-B-like sequence in a wild population. CONCLUSIONS: This transcriptomic dataset is a useful resource for molecular genetic studies of the koala, for evolutionary genetic studies of marsupials, for validation and annotation of the koala genome sequence, and for investigation of koala retrovirus. Annotated transcripts can be browsed and queried at http://koalagenome.org.

Taxonomic uncertainty and the loss of biodiversity on Christmas Island, Indian Ocean.

The taxonomic uniqueness of island populations is often uncertain which hinders effective prioritization for conservation. The Christmas Island shrew (Crocidura attenuata trichura) is the only member of the highly speciose eutherian family Soricidae recorded from Australia. It is currently classified as a subspecies of the Asian gray or long-tailed shrew (C. attenuata), although it was originally described as a subspecies of the southeast Asian white-toothed shrew (C. fuliginosa). The Christmas Island shrew is currently listed as endangered and has not been recorded in the wild since 1984-1985, when 2 specimens were collected after an 80-year absence. We aimed to obtain DNA sequence data for cytochrome b (cytb) from Christmas Island shrew museum specimens to determine their taxonomic affinities and to confirm the identity of the 1980s specimens. The Cytb sequences from 5, 1898 specimens and a 1985 specimen were identical. In addition, the Christmas Island shrew cytb sequence was divergent at the species level from all available Crocidura cytb sequences. Rather than a population of a widespread species, current evidence suggests the Christmas Island shrew is a critically endangered endemic species, C. trichura, and a high priority for conservation. As the decisions typically required to save declining species can be delayed or deferred if the taxonomic status of the population in question is uncertain, it is hoped that the history of the Christmas Island shrew will encourage the clarification of taxonomy to be seen as an important first step in initiating informed and effective conservation action.

Multiple biogeographical barriers identified across the monsoon tropics of northern Australia: phylogeographic analysis of the brachyotis group of rock-wallabies.

The monsoon tropics of northern Australia are a globally significant biodiversity hotspot, but its phylogeography is poorly known. A major challenge for this region is to understand the biogeographical processes that have shaped the distribution and diversity of taxa, without detailed knowledge of past climatic and environmental fluctuations. Although molecular data have great potential to address these questions, only a few species have been examined phylogeographically. Here, we use the widely distributed and abundant short-eared rock-wallaby (Petrogale brachyotis; n = 101), together with the sympatric monjon (P. burbidgei; n = 11) and nabarlek (P. concinna; n = 1), to assess historical evolutionary and biogeographical processes in northern Australia. We sequenced ∼1000 bp of mitochondrial DNA (control region, ND2) and ∼3000 bp of nDNA (BRCA1, ω-globin and two anonymous loci) to investigate phylogeographic structuring and delineate the time-scale of diversification within the region. Our results indicate multiple barriers between the Top End (Northern Territory) and Kimberley (Western Australia), which have caused divergence throughout the Plio-Pleistocene. Eight geographically discrete and genetically distinct lineages within the brachyotis group were identified, five of which are separated by major river valleys (Ord, Victoria, Daly), arid lowlands and discontinuous sandstone ranges. It is likely that these barriers have similarly influenced genetic structure in other monsoonal biota.

Novel insights into the phylogenetic relationships of the endangered marsupial genus Potorous.

The three extant potoroo species of the marsupial genus Potorous -Potorous tridactylus, P. longipes and P. gilbertii - are all of conservation concern due to introduced predators and habitat loss associated with the European settlement of Australia. Robust phylogenies can be useful to inform conservation management, but past phylogenetic studies on potoroos have been unable to fully resolve relationships within the genus. Here, a multi-locus approach was employed, using three mitochondrial DNA (mtDNA): NADH dehydrogenase subunit 2, cytochrome c oxidase subunit 1 and 12S rRNA and four nuclear DNA (nuDNA) gene regions: breast and ovarian cancer susceptibility gene, recombination activating gene-1, apolipoprotein B and omega globin. This was coupled with widespread geographic sampling of the broadly distributed P. tridactylus, to investigate the phylogenetic relationships within this genus. Analyses of the mtDNA identified five distinct and highly divergent lineages including, P. longipes, P. gilbertii and three distinct lineages within P. tridactylus (northern mainland, southern mainland and Tasmanian). P. tridactylus was paraphyletic with the P. gilbertii lineage, suggesting that cryptic taxa may exist within P. tridactylus. NuDNA sequences lacked the resolution of mtDNA. Although they resolved the three currently recognised species, they were unable to differentiate lineages within P. tridactylus. Current management of P. tridactylus as two sub-species (mainland and Tasmania) does not recognise the full scope of genetic diversity within this species, especially that of the mainland populations. Until data from more informative nuDNA markers are available, we recommend this species be managed as the following three subspecies: Potorous tridactylus tridactylus (southern Queensland and northern New South Wales); Potorous tridactylus trisulcatus (southern New South Wales and Victoria) Potorous tridactylus apicalis (Tasmania). Molecular dating estimated that divergences within Potorous occurred in the late Miocene through to the early Pliocene.

Phylogenetic relationships of rock-wallabies, Petrogale (Marsupialia: Macropodidae) and their biogeographic history within Australia.

The rock-wallaby genus Petrogale comprises a group of habitat-specialist macropodids endemic to Australia. Their restriction to rocky outcrops, with infrequent interpopulation dispersal, has been suggested as the cause of their recent and rapid diversification. Molecular phylogenetic relationships within and among species of Petrogale were analysed using mitochondrial (cytochrome oxidase c subunit 1, cytochrome b, NADH dehydrogenase subunit 2) and nuclear (omega-globin intron, breast and ovarian cancer susceptibility gene) sequence data with representatives that encompassed the morphological and chromosomal variation within the genus, including for the first time both Petrogale concinna and Petrogale purpureicollis. Four distinct lineages were identified, (1) the brachyotis group, (2) Petrogale persephone, (3) Petrogalexanthopus and (4) the lateralis-penicillata group. Three of these lineages include taxa with the ancestral karyotype (2n=22). Paraphyletic relationships within the brachyotis group indicate the need for a focused phylogeographic study. There was support for P. purpureicollis being reinstated as a full species and P. concinna being placed within Petrogale rather than in the monotypic genus Peradorcas. Bayesian analyses of divergence times suggest that episodes of diversification commenced in the late Miocene-Pliocene and continued throughout the Pleistocene. Ancestral state reconstructions suggest that Petrogale originated in a mesic environment and dispersed into more arid environments, events that correlate with the timing of radiations in other arid zone vertebrate taxa across Australia.

Diversity at the major histocompatibility complex Class II in the platypus, Ornithorhynchus anatinus.

The platypus (Ornithorhynchus anatinus) is the sole survivor of a previously widely distributed and diverse lineage of ornithorhynchid monotremes. Its dependence on healthy water systems imposes an inherent sensitivity to habitat degradation and climate change. Here, we compare genetic diversity at the major histocompatibility complex (MHC) Class II-DZB gene and 3 MHC-associated microsatellite markers with diversity at 6 neutral microsatellite markers in 70 platypuses from across their range, including the mainland of Australia and the isolated populations of Tasmania, King Island, and Kangaroo Island. Overall, high DZB diversity was observed in the platypus, with 57 DZB ß1 alleles characterized. Significant positive selection was detected within the DZB peptide-binding region, promoting variation in this domain. Low levels of genetic diversity were detected at all markers in the 2 island populations, King Island (endemic) and Kangaroo Island (introduced), with the King Island platypuses monomorphic at the DZB locus. Loss of MHC diversity on King Island is of concern, as the population may have compromised immunological fitness and reduced ability to resist changing environmental conditions.

Evolution: The evolutionary rat race in New Guinea and Australia.

A new molecular phylogeny of a remarkable radiation of New Guinean and Australian rodents indicates multiple transitions between biomes and biogeographical regions within the group, and suggests that a key role was played by the geological history of New Guinea.

AnimalTraits - a curated animal trait database for body mass, metabolic rate and brain size.

Trait databases have become important resources for large-scale comparative studies in ecology and evolution. Here we introduce the AnimalTraits database, a curated database of body mass, metabolic rate and brain size, in standardised units, for terrestrial animals. The database has broad taxonomic breadth, including tetrapods, arthropods, molluscs and annelids from almost 2000 species and 1000 genera. All data recorded in the database are sourced from their original empirical publication, and the original metrics and measurements are included with each record. This allows for subsequent data transformations as required. We have included rich metadata to allow users to filter the dataset. The additional R scripts we provide will assist researchers with aggregating standardised observations into species-level trait values. Our goals are to provide this resource without restrictions, to keep the AnimalTraits database current, and to grow the number of relevant traits in the future.