A new species of barred frog, Mixophyes (Anura: Myobatrachidae) from south-eastern Australia identified by molecular genetic analyses.

Mixophyes are large ground-dwelling myobatrachid frogs from eastern Australia and New Guinea. Several of the species found in mid-eastern and south-eastern Australia are listed as threatened, due largely to declines presumably caused by the amphibian disease chytridiomycosis. Given the wide distribution of several of these species and that their distributions cross well-known biogeographic boundaries that often correspond to deep genetic breaks or species boundaries among closely related vertebrates, we undertook a molecular genetic assessment of population structure across the range of each species to determine the presence of undescribed species. Of the four species of Mixophyes subject to molecular population genetic analyses, one, the Stuttering Frog (Mixophyes balbus), showed a level of diversity consistent with the presence of two species. Morphometric, meristic and bioacoustic analyses corroborate these distinctions, and a new species is described for the populations south of the Macleay River valley in mid-eastern New South Wales to east Gippsland in Victoria. Applying the IUCN Red List threat criteria the new species meets the conservation status assessment criteria for Endangered 2B1a,b because its extent of occupancy and area of occupancy are below the threshold value and it has declined and disappeared from the southern two thirds of its distribution over the past 30 years.

Differential transcriptomic responses to heat stress in surface and subterranean diving beetles.

Subterranean habitats are generally very stable environments, and as such evolutionary transitions of organisms from surface to subterranean lifestyles may cause considerable shifts in physiology, particularly with respect to thermal tolerance. In this study we compared responses to heat shock at the molecular level in a geographically widespread, surface-dwelling water beetle to a congeneric subterranean species restricted to a single aquifer (Dytiscidae: Hydroporinae). The obligate subterranean beetle Paroster macrosturtensis is known to have a lower thermal tolerance compared to surface lineages (CTmax 38 °C cf. 42-46 °C), but the genetic basis of this physiological difference has not been characterized. We experimentally manipulated the thermal environment of 24 individuals to demonstrate that both species can mount a heat shock response at high temperatures (35 °C), as determined by comparative transcriptomics. However, genes involved in these responses differ between species and a far greater number were differentially expressed in the surface taxon, suggesting it can mount a more robust heat shock response; these data may underpin its higher thermal tolerance compared to subterranean relatives. In contrast, the subterranean species examined not only differentially expressed fewer genes in response to increasing temperatures, but also in the presence of the experimental setup employed here alone. Our results suggest P. macrosturtensis may be comparatively poorly equipped to respond to both thermally induced stress and environmental disturbances more broadly. The molecular findings presented here have conservation implications for P. macrosturtensis and contribute to a growing narrative concerning weakened thermal tolerances in obligate subterranean organisms at the molecular level.

Parallel decay of vision genes in subterranean water beetles.

In the framework of neutral theory of molecular evolution, genes specific to the development and function of eyes in subterranean animals living in permanent darkness are expected to evolve by relaxed selection, ultimately becoming pseudogenes. However, definitive empirical evidence for the role of neutral processes in the loss of vision over evolutionary time remains controversial. In previous studies, we characterized an assemblage of independently-evolved water beetle (Dytiscidae) species from a subterranean archipelago in Western Australia, where parallel vision and eye loss have occurred. Using a combination of transcriptomics and exon capture, we present evidence of parallel coding sequence decay, resulting from the accumulation of frameshift mutations and premature stop codons, in eight phototransduction genes (arrestins, opsins, ninaC and transient receptor potential channel genes) in 32 subterranean species in contrast to surface species, where these genes have open reading frames. Our results provide strong evidence to support neutral evolutionary processes as a major contributing factor to the loss of phototransduction genes in subterranean animals, with the ultimate fate being the irreversible loss of a light detection system.

A new species of Philoria (Anura: Limnodynastidae) from the uplands of the Gondwana Rainforests World Heritage Area of eastern Australia.

The six species of mountain frogs (Philoria: Limnodynastidae: Anura) are endemic to south-eastern Australia. Five species occur in headwater systems in mountainous north-eastern New South Wales (NSW) and south-eastern Queensland (Qld), centred on the Gondwana Rainforests of Australia World Heritage Area. A previous molecular genetic analysis identified divergent genetic lineages in the central and western McPherson Ranges region of Qld and NSW, but sampling was inadequate to test the species status of these lineages. With more comprehensive geographic sampling and examination of the nuclear genome using SNP analysis, we show that an undescribed species, P. knowlesi sp. nov., occurs in the central and western McPherson Ranges (Levers Plateau and Mount Barney complex). The new species is not phylogenetically closely related to P. loveridgei in the nuclear data but is related to one of two divergent lineages within P. loveridgei in the mtDNA data. We postulate that the discordance between the nuclear and mtDNA outcomes is due to ancient introgression of the mtDNA genome from P. loveridgei into the new species. Male advertisement calls and multivariate morphological analyses do not reliably distinguish P. knowlesi sp. nov. from any of the Philoria species in northeast NSW and southeast Qld. The genetic comparisons also enable us to define further the distributions of P. loveridgei and P. kundagungan. Samples from the Lamington Plateau, Springbrook Plateau, Wollumbin (Mt Warning National Park), and the Nightcap Range, are all P. loveridgei, and its distribution is now defined as the eastern McPherson Ranges and Tweed caldera. Philoria kundagungan is distributed from the Mistake Mountains in south-eastern Qld to the Tooloom Scrub on the Koreelah Range, southwest of Woodenbong, in NSW, with two subpopulations identified by SNP analysis. We therefore assessed the IUCN threat category of P. loveridgei and P. kundagungan and undertook new assessments for each of its two subpopulations and for the new taxon P. knowlesi sp. nov., using IUCN Red List criteria. Philoria loveridgei, P. kundagungan (entire range and northern subpopulation separately) and P. knowlesi sp. nov. each meet criteria for Endangered (EN B2(a)(b)[i, iii]). The southern subpopulation of P. kundagungan, in the Koreelah Range, meets criteria for Critically Endangered (CE B2(a)(b)[i, iii]). These taxa are all highly threatened due to the small number of known locations, the restricted nature of their breeding habitat, and direct and indirect threats from climate change, and the potential impact of the amphibian disease chytridiomycosis. Feral pigs are an emerging threat, with significant impacts now observed in Philoria breeding habitat in the Mistake Mountains.

Comparison of Reptilian Genomes Reveals Deletions Associated with the Natural Loss of γδ T Cells in Squamates.

T lymphocytes or T cells are key components of the vertebrate response to pathogens and cancer. There are two T cell classes based on their TCRs, αß T cells and γδ T cells, and each plays a critical role in immune responses. The squamate reptiles may be unique among the vertebrate lineages by lacking an entire class of T cells, the γδ T cells. In this study, we investigated the basis of the loss of the γδ T cells in squamates. The genome and transcriptome of a sleepy lizard, the skink Tiliqua rugosa, were compared with those of tuatara, Sphenodon punctatus, the last living member of the Rhynchocephalian reptiles. We demonstrate that the lack of TCRγ and TCRδ transcripts in the skink are due to large deletions in the T. rugosa genome. We also show that tuataras are on a growing list of species, including sharks, frogs, birds, alligators, and platypus, that can use an atypical TCRδ that appears to be a chimera of a TCR chain with an Ab-like Ag-binding domain. Tuatara represents the nearest living relative to squamates that retain γδ T cells. The loss of γδTCR in the skink is due to genomic deletions that appear to be conserved in other squamates. The genes encoding the αßTCR chains in the skink do not appear to have increased in complexity to compensate for the loss of γδ T cells.

Taxonomic revision of south-eastern Australian giant burrowing frogs (Anura: Limnodynastidae: Heleioporus Gray).

The rarely encountered giant burrowing frog, Heleioporus australiacus, is distributed widely in a variety of sclerophyll forest habitats east of the Great Dividing Range in south-eastern Australia. Analyses of variation in nucleotide sequences of the mitochondrial ND4 gene and thousands of nuclear gene SNPs revealed the presence of two deeply divergent lineages. Multivariate morphological comparisons show the two lineages differ in body proportions with > 91% of individuals being correctly classified in DFA. The two lineages differ in the number and size of spots on the lateral surfaces and the degree by which the cloaca is surrounded by colour patches. The mating calls are significantly different in number of pulses in the note. The presence of a F2 hybrid in the area where the distribution of the two taxa come into closest proximity leads us to assign subspecies status to the lineages, as we have not been able to assess the extent of potential genetic introgression. In our sampling, the F2 hybrid sample sits within an otherwise unsampled gap of ~90km between the distributions of the two lineages. The nominate northern sub-species is restricted to the Sydney Basin bioregion, while the newly recognised southern subspecies occurs from south of the Kangaroo Valley in the mid-southern coast of New South Wales to near Walhalla in central Gippsland in Victoria. The habitat of the two subspecies is remarkably similar. Adults spend large portions of their lives on the forest floor where they forage and burrow in a variety of vegetation communities. The southern subspecies occurs most commonly in dry sclerophyll forests with an open understory in the south and in open forest and heath communities with a dense understory in the north of its distribution. The northern subspecies is also found in dry open forests and heaths in association with eroded sandstone landscapes in the Sydney Basin bioregion. Males of both taxa call from both constructed burrows and open positions on small streams, differing from the five Western Australian species of Heleioporus where males call only from constructed burrows. Using the IUCN Red List process, we found that the extent of occupancy and area of occupancy along with evidence of decline for both subspecies are consistent with the criteria for Endangered (A2(c)B2(a)(b)).

Development and evaluation of a custom bait design based on 469 single-copy protein-coding genes for exon capture of isopods (Philosciidae: Haloniscus).

Transcriptome-based exon capture approaches, along with next-generation sequencing, are allowing for the rapid and cost-effective production of extensive and informative phylogenomic datasets from non-model organisms for phylogenetics and population genetics research. These approaches generally employ a reference genome to infer the intron-exon structure of targeted loci and preferentially select longer exons. However, in the absence of an existing and well-annotated genome, we applied this exon capture method directly, without initially identifying intron-exon boundaries for bait design, to a group of highly diverse Haloniscus (Philosciidae), paraplatyarthrid and armadillid isopods, and examined the performance of our methods and bait design for phylogenetic inference. Here, we identified an isopod-specific set of single-copy protein-coding loci, and a custom bait design to capture targeted regions from 469 genes, and analysed the resulting sequence data with a mapping approach and newly-created post-processing scripts. We effectively recovered a large and informative dataset comprising both short (<100 bp) and longer (>300 bp) exons, with high uniformity in sequencing depth. We were also able to successfully capture exon data from up to 16-year-old museum specimens along with more distantly related outgroup taxa, and efficiently pool multiple samples prior to capture. Our well-resolved phylogenies highlight the overall utility of this methodological approach and custom bait design, which offer enormous potential for application to future isopod, as well as broader crustacean, molecular studies.

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.

The critical thermal maximum of diving beetles (Coleoptera: Dytiscidae): a comparison of subterranean and surface-dwelling species.

Thermal tolerance limits in animals are often thought to be related to temperature and thermal variation in their environment. Recently, there has been a focus on studying upper thermal limits due to the likelihood for climate change to expose more animals to higher temperatures and potentially extinction. Organisms living in underground environments experience reduced temperatures and thermal variation in comparison to species living in surface habitats, but how these impact their thermal tolerance limits are unclear. In this study, we compare the thermal critical maximum (CTmax) of two subterranean diving beetles (Dytiscidae) to that of three related surface-dwelling species. Our results show that subterranean species have a lower CTmax (38.3-39.0°C) than surface species (42.0-44.5°C). The CTmax of subterranean species is ∼10°C higher than the highest temperature recorded within the aquifer. Groundwater temperature varied between 18.4°C and 28.8°C, and changes with time, depth and distance across the aquifer. Seasonal temperature fluctuations were 0.5°C at a single point, with the maximum heating rate being ∼1000x lower (0.008°C/hour) than that recorded in surface habitats (7.98°C/hour). For surface species, CTmax was 7-10°C higher than the maximum temperature in their habitats, with daily fluctuations from ∼1°C to 16°C and extremes of 6.9°C and 34.9°C. These findings suggest that subterranean dytiscid beetles are unlikely to reach their CTmax with a predicted warming of 1.3-5.1°C in the region by 2090. However, the impacts of long-term elevated temperatures on fitness, different life stages and other species in the beetle's trophic food web are unknown.

The tuatara genome reveals ancient features of amniote evolution.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2-is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Publisher Correction: The tuatara genome reveals ancient features of amniote evolution.

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

A draft genome assembly of the eastern banjo frog Limnodynastes dumerilii dumerilii (Anura: Limnodynastidae).

Amphibian genomes are usually challenging to assemble due to their large genome size and high repeat content. The Limnodynastidae is a family of frogs native to Australia, Tasmania and New Guinea. As an anuran lineage that successfully diversified on the Australian continent, it represents an important lineage in the amphibian tree of life but lacks reference genomes. Here we sequenced and annotated the genome of the eastern banjo frog Limnodynastes dumerilii dumerilii to fill this gap. The total length of the genome assembly is 2.38 Gb with a scaffold N50 of 285.9 kb. We identified 1.21 Gb of non-redundant sequences as repetitive elements and annotated 24,548 protein-coding genes in the assembly. BUSCO assessment indicated that more than 94% of the expected vertebrate genes were present in the genome assembly and the gene set. We anticipate that this annotated genome assembly will advance the future study of anuran phylogeny and amphibian genome evolution.

Horizontal transfer of BovB and L1 retrotransposons in eukaryotes.

BACKGROUND: Transposable elements (TEs) are mobile DNA sequences, colloquially known as jumping genes because of their ability to replicate to new genomic locations. TEs can jump between organisms or species when given a vector of transfer, such as a tick or virus, in a process known as horizontal transfer. Here, we propose that LINE-1 (L1) and Bovine-B (BovB), the two most abundant TE families in mammals, were initially introduced as foreign DNA via ancient horizontal transfer events. RESULTS: Using analyses of 759 plant, fungal and animal genomes, we identify multiple possible L1 horizontal transfer events in eukaryotic species, primarily involving Tx-like L1s in marine eukaryotes. We also extend the BovB paradigm by increasing the number of estimated transfer events compared to previous studies, finding new parasite vectors of transfer such as bed bug, leech and locust, and BovB occurrences in new lineages such as bat and frog. Given that these transposable elements have colonised more than half of the genome sequence in today's mammals, our results support a role for horizontal transfer in causing long-term genomic change in new host organisms. CONCLUSIONS: We describe extensive horizontal transfer of BovB retrotransposons and provide the first evidence that L1 elements can also undergo horizontal transfer. With the advancement of genome sequencing technologies and bioinformatics tools, we anticipate our study to be a valuable resource for inferring horizontal transfer from large-scale genomic data.

DNA barcoding of microgastrine parasitoid wasps (Hymenoptera: Braconidae) using high-throughput methods more than doubles the number of species known for Australia.

The Microgastrinae are a hugely diverse subfamily of endoparasitoid wasps of lepidopteran caterpillars. They are important in agriculture as biological control agents and play a significant ecological role in the regulation of caterpillar populations. Whilst the group has been the focus of intensive rearing and DNA barcoding studies in the Northern Hemisphere, the Australian fauna has received little attention. In total, 99 species have been described from or have been introduced into Australia, but the real species diversity for the region is clearly much larger than this. In this study, museum ethanol samples and recent field collections were mined for hundreds of specimens of microgastrine wasps, which were then barcoded for the COI region, ITS2 ribosomal spacer and the wingless nuclear genes, using a pooled sequencing approach on an Illumina Miseq system. Full COI sequences were obtained for 525 individuals which, when combined with 162 publicly available sequences, represented 417 haplotypes, and a total of 236 species were delimited using a consensus approach. By more than doubling the number of known microgastrine wasp species in Australia, our study highlights the value of DNA barcoding in the context of employing high-throughput sequencing methods of bulk ethanol museum collections for biodiversity assessment.

DNA profiling reveals Neobenedenia girellae as the primary parasitic monogenean in global fisheries and aquaculture.

Accurate identification of parasite species and strains is crucial to mitigate the risk of epidemics and emerging disease. Species of Neobenedenia are harmful monogenean ectoparasites that infect economically important bony fishes in aquaculture worldwide, however, the species boundaries between two of the most notorious taxa, N. melleni and N. girellae, has been a topic of contention for decades. Historically, identifications of Neobenedenia isolates have overwhelmingly been attributed to N. melleni, and it has been proposed that N. girellae is synonymous with N. melleni. We collected 33 Neobenedenia isolates from 22 host species spanning nine countries and amplified three genes including two nuclear (Histone 3 and 28S rDNA) and one mitochondrial (cytochrome b). Four major clades were identified using Maximum Likelihood and Bayesian inference analyses; clades A-D corresponding to N. girellae, N. melleni, N. longiprostata and N. pacifica, respectively. All unidentified isolates and the majority of Neobenedenia sequences from GenBank fell into clade A. The results of this study indicate that N. girellae is a separate species to N. melleni, and that a large proportion of previous samples identified as N. melleni may be erroneous and a revision of identifications is needed. The large diversity of host species that N. girellae is able to infect as determined in this study and the geographic range in which it is present (23.8426°S and 24.1426°N) makes it a globally cosmopolitan species and a threat to aquaculture industries around the world.

Superior ab initio identification, annotation and characterisation of TEs and segmental duplications from genome assemblies.

Transposable Elements (TEs) are mobile DNA sequences that make up significant fractions of amniote genomes. However, they are difficult to detect and annotate ab initio because of their variable features, lengths and clade-specific variants. We have addressed this problem by refining and developing a Comprehensive ab initio Repeat Pipeline (CARP) to identify and cluster TEs and other repetitive sequences in genome assemblies. The pipeline begins with a pairwise alignment using krishna, a custom aligner. Single linkage clustering is then carried out to produce families of repetitive elements. Consensus sequences are then filtered for protein coding genes and then annotated using Repbase and a custom library of retrovirus and reverse transcriptase sequences. This process yields three types of family: fully annotated, partially annotated and unannotated. Fully annotated families reflect recently diverged/young known TEs present in Repbase. The remaining two types of families contain a mixture of novel TEs and segmental duplications. These can be resolved by aligning these consensus sequences back to the genome to assess copy number vs. length distribution. Our pipeline has three significant advantages compared to other methods for ab initio repeat identification: 1) we generate not only consensus sequences, but keep the genomic intervals for the original aligned sequences, allowing straightforward analysis of evolutionary dynamics, 2) consensus sequences represent low-divergence, recently/currently active TE families, 3) segmental duplications are annotated as a useful by-product. We have compared our ab initio repeat annotations for 7 genome assemblies to other methods and demonstrate that CARP compares favourably with RepeatModeler, the most widely used repeat annotation package.

Characterisation of major histocompatibility complex class I transcripts in an Australian dragon lizard.

Characterisation of squamate major histocompatibility complex (MHC) genes has lagged behind other taxonomic groups. MHC genes encode cell-surface glycoproteins that present self- and pathogen-derived peptides to T cells and play a critical role in pathogen recognition. Here we characterise MHC class I transcripts for an agamid lizard (Ctenophorus decresii) and investigate the evolution of MHC class I in Iguanian lizards. An iterative assembly strategy was used to identify six full-length C. decresii MHC class I transcripts, which were validated as likely to encode classical class I MHC molecules. Evidence for exon shuffling recombination was uncovered for C. decresii transcripts and Bayesian phylogenetic analysis of Iguanian MHC class I sequences revealed a pattern expected under a birth-and-death mode of evolution. This work provides a stepping stone towards further research on the agamid MHC class I region.

LINEs between Species: Evolutionary Dynamics of LINE-1 Retrotransposons across the Eukaryotic Tree of Life.

LINE-1 (L1) retrotransposons are dynamic elements. They have the potential to cause great genomic change because of their ability to 'jump' around the genome and amplify themselves, resulting in the duplication and rearrangement of regulatory DNA. Active L1, in particular, are often thought of as tightly constrained, homologous and ubiquitous elements with well-characterized domain organization. For the past 30 years, model organisms have been used to define L1s as 6-8 kb sequences containing a 5'-UTR, two open reading frames working harmoniously in cis, and a 3'-UTR with a polyA tail. In this study, we demonstrate the remarkable and overlooked diversity of L1s via a comprehensive phylogenetic analysis of elements from over 500 species from widely divergent branches of the tree of life. The rapid and recent growth of L1 elements in mammalian species is juxtaposed against the diverse lineages found in other metazoans and plants. In fact, some of these previously unexplored mammalian species (e.g. snub-nosed monkey, minke whale) exhibit L1 retrotranspositional 'hyperactivity' far surpassing that of human or mouse. In contrast, non-mammalian L1s have become so varied that the current classification system seems to inadequately capture their structural characteristics. Our findings illustrate how both long-term inherited evolutionary patterns and random bursts of activity in individual species can significantly alter genomes, highlighting the importance of L1 dynamics in eukaryotes.