Highly dynamic evolution of the chemosensory system driven by gene gain and loss across subterranean beetles.

Chemical cues in subterranean habitats differ highly from those on the surface due to the contrasting environmental conditions, such as absolute darkness, high humidity or food scarcity. Subterranean animals underwent changes to their sensory systems to facilitate the perception of essential stimuli for underground lifestyles. Despite representing unique systems to understand biological adaptation, the genomic basis of chemosensation across cave-dwelling species remains unexplored from a macroevolutionary perspective. Here, we explore the evolution of chemoreception in three beetle tribes that underwent at least six independent transitions to the underground, through a phylogenomics spyglass. Our findings suggest that the chemosensory gene repertoire varies dramatically between species. Overall, no parallel changes in the net rate of evolution of chemosensory gene families were detected prior, during, or after the habitat shift among subterranean lineages. Contrarily, we found evidence of lineage-specific changes within surface and subterranean lineages. However, our results reveal key duplications and losses shared between some of the lineages transitioning to the underground, including the loss of sugar receptors and gene duplications of the highly conserved ionotropic receptors IR25a and IR8a, involved in thermal and humidity sensing among other olfactory roles in insects. These duplications were detected both in independent subterranean lineages and their surface relatives, suggesting parallel evolution of these genes across lineages giving rise to cave-dwelling species. Overall, our results shed light on the genomic basis of chemoreception in subterranean beetles and contribute to our understanding of the genomic underpinnings of adaptation to the subterranean lifestyle at a macroevolutionary scale.

Groundwater is a hidden global keystone ecosystem.

Groundwater is a vital ecosystem of the global water cycle, hosting unique biodiversity and providing essential services to societies. Despite being the largest unfrozen freshwater resource, in a period of depletion by extraction and pollution, groundwater environments have been repeatedly overlooked in global biodiversity conservation agendas. Disregarding the importance of groundwater as an ecosystem ignores its critical role in preserving surface biomes. To foster timely global conservation of groundwater, we propose elevating the concept of keystone species into the realm of ecosystems, claiming groundwater as a keystone ecosystem that influences the integrity of many dependent ecosystems. Our global analysis shows that over half of land surface areas (52.6%) has a medium-to-high interaction with groundwater, reaching up to 74.9% when deserts and high mountains are excluded. We postulate that the intrinsic transboundary features of groundwater are critical for shifting perspectives towards more holistic approaches in aquatic ecology and beyond. Furthermore, we propose eight key themes to develop a science-policy integrated groundwater conservation agenda. Given ecosystems above and below the ground intersect at many levels, considering groundwater as an essential component of planetary health is pivotal to reduce biodiversity loss and buffer against climate change.

Evolution of endoglucanase genes in subterranean and surface isopod crustaceans from Central Western Australia.

Recent studies have identified a significant number of endogenous cellulase genes in various arthropods, including isopods, allowing them to process hydrocarbons efficiently as a food source. While this research has provided insight into underlying gene-level processes in cellulose decomposition by arthropods, little is known about the existence and expression of cellulase genes in species from cave environments where carbohydrates are sparse. To investigate whether endogenous cellulase genes are maintained in subterranean species, we sequenced the transcriptomes of two subterranean paraplatyarthrid isopod species from calcrete (carbonate) aquifers of central Western Australia and a related surface isopod species. Seven protein-coding open-reading frames associated with endoglucanase genes were identified in all species. Orthology inference analyses, using a wide range of cellulase sequences from available databases, supported the endogenous origin of the putative endoglucanase genes. Selection analyses revealed that these genes are primarily subject to purifying selection in most of the sites for both surface and subterranean isopod species, indicating that they are likely to encode functional peptides. Furthermore, evolutionary branch models supported the hypothesis of an adaptive shift in selective pressure acting on the subterranean lineages compared with the ancestral lineage and surface species. Branch-site models also revealed a few amino acid sites on the subterranean branches to be under positive selection, suggesting the acquisition of novel adaptations to the subterranean environments. These findings also imply that hydrocarbons exist in subsurface aquifers, albeit at reduced levels, and have been utilized by subterranean isopods as a source of energy for millions of years.

Parallel and convergent genomic changes underlie independent subterranean colonization across beetles.

Adaptation to life in caves is often accompanied by dramatically convergent changes across distantly related taxa, epitomized by the loss or reduction of eyes and pigmentation. Nevertheless, the genomic underpinnings underlying cave-related phenotypes are largely unexplored from a macroevolutionary perspective. Here we investigate genome-wide gene evolutionary dynamics in three distantly related beetle tribes with at least six instances of independent colonization of subterranean habitats, inhabiting both aquatic and terrestrial underground systems. Our results indicate that remarkable gene repertoire changes mainly driven by gene family expansions occurred prior to underground colonization in the three tribes, suggesting that genomic exaptation may have facilitated a strict subterranean lifestyle parallelly across beetle lineages. The three tribes experienced both parallel and convergent changes in the evolutionary dynamics of their gene repertoires. These findings pave the way towards a deeper understanding of the evolution of the genomic toolkit in hypogean fauna.

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.

eDNA in subterranean ecosystems: Applications, technical aspects, and future prospects.

Monitoring of biota is pivotal for the assessment and conservation of ecosystems. Environments worldwide are being continuously and increasingly exposed to multiple adverse impacts, and the accuracy and reliability of the biomonitoring tools that can be employed shape not only the present, but more importantly, the future of entire habitats. The analysis of environmental DNA (eDNA) metabarcoding data provides a quick, affordable, and reliable molecular approach for biodiversity assessments. However, while extensively employed in aquatic and terrestrial surface environments, eDNA-based studies targeting subterranean ecosystems are still uncommon due to the lack of accessibility and the cryptic nature of these environments and their species. Recent advances in genetic and genomic analyses have established a promising framework for shedding new light on subterranean biodiversity and ecology. To address current knowledge and the future use of eDNA methods in groundwaters and caves, this review explores conceptual and technical aspects of the application and its potential in subterranean systems. We briefly introduce subterranean biota and describe the most used traditional sampling techniques. Next, eDNA characteristics, application, and limitations in the subsurface environment are outlined. Last, we provide suggestions on how to overcome caveats and delineate some of the research avenues that will likely shape this field in the near future. We advocate that eDNA analyses, when carefully conducted and ideally combined with conventional sampling techniques, will substantially increase understanding and enable crucial expansion of subterranean community characterisation. Given the importance of groundwater and cave ecosystems for nature and humans, eDNA can bring to the surface essential insights, such as study of ecosystem assemblages and rare species detection, which are critical for the preservation of life below, as well as above, the ground.

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.

Rainfall as a trigger of ecological cascade effects in an Australian groundwater ecosystem.

Groundwaters host vital resources playing a key role in the near future. Subterranean fauna and microbes are crucial in regulating organic cycles in environments characterized by low energy and scarce carbon availability. However, our knowledge about the functioning of groundwater ecosystems is limited, despite being increasingly exposed to anthropic impacts and climate change-related processes. In this work we apply novel biochemical and genetic techniques to investigate the ecological dynamics of an Australian calcrete under two contrasting rainfall periods (LR-low rainfall and HR-high rainfall). Our results indicate that the microbial gut community of copepods and amphipods experienced a shift in taxonomic diversity and predicted organic functional metabolic pathways during HR. The HR regime triggered a cascade effect driven by microbes (OM processors) and exploited by copepods and amphipods (primary and secondary consumers), which was finally transferred to the aquatic beetles (top predators). Our findings highlight that rainfall triggers ecological shifts towards more deterministic dynamics, revealing a complex web of interactions in seemingly simple environmental settings. Here we show how a combined isotopic-molecular approach can untangle the mechanisms shaping a calcrete community. This design will help manage and preserve one of the most vital but underrated ecosystems worldwide.

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.

Evidence for speciation underground in diving beetles (Dytiscidae) from a subterranean archipelago.

Most subterranean animals are assumed to have evolved from surface ancestors following colonization of a cave system; however, very few studies have raised the possibility of "subterranean speciation" in underground habitats (i.e., obligate cave-dwelling organisms [troglobionts] descended from troglobiotic ancestors). Numerous endemic subterranean diving beetle species from spatially discrete calcrete aquifers in Western Australia (stygobionts) have evolved independently from surface ancestors; however, several cases of sympatric sister species raise the possibility of subterranean speciation. We tested this hypothesis using vision (phototransduction) genes that are evolving under neutral processes in subterranean species and purifying selection in surface species. Using sequence data from 32 subterranean and five surface species in the genus Paroster (Dytiscidae), we identified deleterious mutations in long wavelength opsin (lwop), arrestin 1 (arr1), and arrestin 2 (arr2) shared by a sympatric sister-species triplet, arr1 shared by a sympatric sister-species pair, and lwop and arr2 shared among closely related species in adjacent calcrete aquifers. In all cases, a common ancestor possessed the function-altering mutations, implying they were already adapted to aphotic environments. Our study represents one of the first confirmed cases of subterranean speciation in cave insects. The assessment of genes undergoing pseudogenization provides a novel way of testing modes of speciation and the history of diversification in blind cave animals.

Extreme genetic diversity among springtails (Collembola) in subterranean calcretes of arid Australia.

The subterranean islands hypothesis for calcretes of the Yilgarn region in Western Australia applies to many stygobitic (subterranean-aquatic) species that are "trapped" evolutionarily within isolated aquifers due to their aquatic lifestyles. In contrast, little is known about the distribution of terrestrial-subterranean invertebrates associated with the calcretes. We used subterranean Collembola from the Yilgarn calcretes to test the hypothesis that troglobitic species, those inhabiting the subterranean unsaturated (non-aquatic) zone of calcretes, are also restricted in their distribution and represent reciprocally monophyletic and endemic lineages. We used the barcoding fragment of the mtDNA cytochrome c oxidase subunit 1 (COI) gene from 183 individuals to reconstruct the phylogenetic history of the genus Pseudosinella Schäffer (Collembola, Lepidocyrtidae) from 10 calcretes in the Yilgarn. These calcretes represent less than 5% of the total possible calcretes in this region, yet we show that their diversity for subterranean Collembola comprises a minimum of 25 new species. Regionally, multiple levels of diversity exist in Pseudosinella, indicative of a complex evolutionary history for this genus in the Yilgarn. These species have probably been impacted by climatic oscillations, facilitating their dispersal across the landscape. The results represent a small proportion of the undiscovered diversity in Australia's arid zone.

Comparative analysis of morabine grasshopper genomes reveals highly abundant transposable elements and rapidly proliferating satellite DNA repeats.

BACKGROUND: Repetitive DNA sequences, including transposable elements (TEs) and tandemly repeated satellite DNA (satDNAs), collectively called the "repeatome", are found in high proportion in organisms across the Tree of Life. Grasshoppers have large genomes, averaging 9 Gb, that contain a high proportion of repetitive DNA, which has hampered progress in assembling reference genomes. Here we combined linked-read genomics with transcriptomics to assemble, characterize, and compare the structure of repetitive DNA sequences in four chromosomal races of the morabine grasshopper Vandiemenella viatica species complex and determine their contribution to genome evolution. RESULTS: We obtained linked-read genome assemblies of 2.73-3.27 Gb from estimated genome sizes of 4.26-5.07 Gb DNA per haploid genome of the four chromosomal races of V. viatica. These constitute the third largest insect genomes assembled so far. Combining complementary annotation tools and manual curation, we found a large diversity of TEs and satDNAs, constituting 66 to 75% per genome assembly. A comparison of sequence divergence within the TE classes revealed massive accumulation of recent TEs in all four races (314-463 Mb per assembly), indicating that their large genome sizes are likely due to similar rates of TE accumulation. Transcriptome sequencing showed more biased TE expression in reproductive tissues than somatic tissues, implying permissive transcription in gametogenesis. Out of 129 satDNA families, 102 satDNA families were shared among the four chromosomal races, which likely represent a diversity of satDNA families in the ancestor of the V. viatica chromosomal races. Notably, 50 of these shared satDNA families underwent differential proliferation since the recent diversification of the V. viatica species complex. CONCLUSION: This in-depth annotation of the repeatome in morabine grasshoppers provided new insights into the genome evolution of Orthoptera. Our TEs analysis revealed a massive recent accumulation of TEs equivalent to the size of entire Drosophila genomes, which likely explains the large genome sizes in grasshoppers. Despite an overall high similarity of the TE and satDNA diversity between races, the patterns of TE expression and satDNA proliferation suggest rapid evolution of grasshopper genomes on recent timescales.

Refining trophic dynamics through multi-factor Bayesian mixing models: A case study of subterranean beetles.

Food web dynamics are vital in shaping the functional ecology of ecosystems. However, trophic ecology is still in its infancy in groundwater ecosystems due to the cryptic nature of these environments. To unravel trophic interactions between subterranean biota, we applied an interdisciplinary Bayesian mixing model design (multi-factor BMM) based on the integration of faunal C and N bulk tissue stable isotope data (δ13C and δ15N) with radiocarbon data (Δ14C), and prior information from metagenomic analyses. We further compared outcomes from multi-factor BMM with a conventional isotope double proxy mixing model (SIA BMM), triple proxy (δ13C, δ15N, and Δ14C, multi-proxy BMM), and double proxy combined with DNA prior information (SIA + DNA BMM) designs. Three species of subterranean beetles (Paroster macrosturtensis, Paroster mesosturtensis, and Paroster microsturtensis) and their main prey items Chiltoniidae amphipods (AM1: Scutachiltonia axfordi and AM2: Yilgarniella sturtensis), cyclopoids and harpacticoids from a calcrete in Western Australia were targeted. Diet estimations from stable isotope only models (SIA BMM) indicated homogeneous patterns with modest preferences for amphipods as prey items. Multi-proxy BMM suggested increased-and species-specific-predatory pressures on amphipods coupled with high rates of scavenging/predation on sister species. SIA + DNA BMM showed marked preferences for amphipods AM1 and AM2, and reduced interspecific scavenging/predation on Paroster species. Multi-factorial BMM revealed the most precise estimations (lower overall SD and very marginal beetles' interspecific interactions), indicating consistent preferences for amphipods AM1 in all the beetles' diets. Incorporation of genetic priors allowed crucial refining of the feeding preferences, while integration of more expensive radiocarbon data as a third proxy (when combined with genetic data) produced more precise outcomes but close dietary reconstruction to that from SIA + DNA BMM. Further multidisciplinary modeling from other groundwater environments will help elucidate the potential behind these designs and bring light to the feeding ecology of one the most vital ecosystems worldwide.

Fundamental research questions in subterranean biology.

Five decades ago, a landmark paper in Science titled The Cave Environment heralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field.

Tracking down carbon inputs underground from an arid zone Australian calcrete.

Freshwater ecosystems play a key role in shaping the global carbon cycle and maintaining the ecological balance that sustains biodiversity worldwide. Surficial water bodies are often interconnected with groundwater, forming a physical continuum, and their interaction has been reported as a crucial driver for organic matter (OM) inputs in groundwater systems. However, despite the growing concerns related to increasing anthropogenic pressure and effects of global change to groundwater environments, our understanding of the dynamics regulating subterranean carbon flows is still sparse. We traced carbon composition and transformations in an arid zone calcrete aquifer using a novel multidisciplinary approach that combined isotopic analyses of dissolved organic carbon (DOC) and inorganic carbon (DIC) (δ13CDOC, δ13CDIC, 14CDOC and 14CDIC) with fluorescence spectroscopy (Chromophoric Dissolved OM (CDOM) characterisation) and metabarcoding analyses (taxonomic and functional genomics on bacterial 16S rRNA). To compare dynamics linked to potential aquifer recharge processes, water samples were collected from two boreholes under contrasting rainfall: low rainfall ((LR), dry season) and high rainfall ((HR), wet season). Our isotopic results indicate limited changes and dominance of modern terrestrial carbon in the upper part (northeast) of the bore field, but correlation between HR and increased old and 13C-enriched DOC in the lower area (southwest). CDOM results show a shift from terrestrially to microbially derived compounds after rainfall in the same lower field bore, which was also sampled for microbial genetics. Functional genomic results showed increased genes coding for degradative pathways-dominated by those related to aromatic compound metabolisms-during HR. Our results indicate that rainfall leads to different responses in different parts of the bore field, with an increase in old carbon sources and microbial processing in the lower part of the field. We hypothesise that this may be due to increasing salinity, either due to mobilisation of Cl- from the soil, or infiltration from the downstream salt lake during HR. This study is the first to use a multi-technique assessment using stable and radioactive isotopes together with functional genomics to probe the principal organic biogeochemical pathways regulating an arid zone calcrete system. Further investigations involving extensive sampling from diverse groundwater ecosystems will allow better understanding of the microbiological pathways sustaining the ecological functioning of subterranean biota.

Scratching the surface of subterranean biodiversity: Molecular analysis reveals a diverse and previously unknown fauna of Parabathynellidae (Crustacea: Bathynellacea) from the Pilbara, Western Australia.

Like other crustacean families, the Parabathynellidae is a poorly studied subterranean and aquatic (stygobiontic) group in Australia, with many regions of available habitat having not yet been surveyed. Here we used a combined approach of molecular species delimitation methods, applied to mitochondrial and nuclear genetic data, to identify putative new species from material obtained from remote subterranean habitats in the Pilbara region of Western Australia. Based on collections from these new localities, we delineated a minimum of eight and up to 24 putative new species using a consensus from a range of molecular delineation methods and additional evidence. When we placed our new putative species into the broader phylogenetic framework of Australian Parabathynellidae, they grouped with two known genera and also within one new and distinct Pilbara-only clade. These new species significantly expand the known diversity of Parabathynellidae in that they represent a 22% increase to the 109 currently recognised species globally. Our investigations showed that sampling at new localities can yield extraordinary levels of new species diversity, with the majority of species showing likely restricted endemic geographical ranges. These findings represent only a small sample from a region comprising less than 2.5% of the Australian continent.

The Dogma of Dingoes-Taxonomic status of the dingo: A reply to Smith et al.

Adopting the name Canis dingo for the Dingo to explicitly denote a species-level taxon separate from other canids was suggested by Crowther et al.  (2014) as a means to eliminate taxonomic instability and contention. However, Jackson et al.  (2017), using standard taxonomic and nomenclatural approaches and principles, called instead for continued use of the nomen C. familiaris for all domestic dogs and their derivatives, including the Dingo. (This name, C. familiaris, is applied to all dogs that derive from the domesticated version of the Gray Wolf, Canis lupus, based on nomenclatural convention.) The primary reasons for this call by Jackson et al.  (2017) were: (1) a lack of evidence to show that recognizing multiple species amongst the dog, including the Dingo and New Guinea Singing Dog, was necessary taxonomically, and (2) the principle of nomenclatural priority (the name familiaris Linnaeus, 1758, antedates dingo Meyer, 1793). Overwhelming current evidence from archaeology and genomics indicates that the Dingo is of recent origin in Australia and shares immediate ancestry with other domestic dogs as evidenced by patterns of genetic and morphological variation. Accordingly, for Smith et al.  (2019) to recognise Canis dingo as a distinct species, the onus was on them to overturn current interpretations of available archaeological, genomic, and morphological datasets and instead show that Dingoes have a deeply divergent evolutionary history that distinguishes them from other named forms of Canis (including C. lupus and its domesticated version, C. familiaris). A recent paper by Koepfli et al.  (2015) demonstrates exactly how this can be done in a compelling way within the genus Canis-by demonstrating deep evolutionary divergence between taxa, on the order of hundreds of thousands of years, using data from multiple genetic systems. Smith et al.  (2019) have not done this; instead they have misrepresented the content and conclusions of Jackson et al.  (2017), and contributed extraneous arguments that are not relevant to taxonomic decisions. Here we dissect Smith et al.  (2019), identifying misrepresentations, to show that ecological, behavioural and morphological evidence is insufficient to recognise Dingoes as a separate species from other domestic dogs. We reiterate: the correct binomial name for the taxon derived from Gray Wolves (C. lupus) by passive and active domestication, including Dingoes and other domestic dogs, is Canis familiaris. We are strongly sympathetic to arguments about the historical, ecological, cultural, or other significance of the Dingo, but these are issues that will have to be considered outside of the more narrow scope of taxonomy and nomenclature.

New species of Australian microgastrine parasitoid wasps (Hymenoptera: Braconidae: Microgastrinae) documented through the 'Bush Blitz' surveys of national reserves.

The braconid subfamily Microgastrinae are ecologically important parasitoids of larval lepidopterans, but are poorly studied in many regions of the world. In this study, we focus on describing new species of microgastrine wasps, in part from specimens collected on six different 'Bush Blitz' surveys of regional reserves in South Australia and Tasmania. Ten species of Microgastrinae are described as new and DNA barcodes of the genes COI and wingless are provided: three species in the genus Choeras Mason: C. bushblitz Fagan-Jeffries Austin sp. nov., C. parvoculus Fagan-Jeffries Austin sp. nov., and C. zygon Fagan-Jeffries Austin sp. nov.; six species in the genus Dolichogenidea Viereck: D. bonbonensis Fagan-Jeffries Austin sp. nov., D. brabyi Fagan-Jeffries Austin sp. nov., D. forrestae Fagan-Jeffries Austin sp. nov., D. garytaylori Fagan-Jeffries Austin sp. nov., D. kelleri Fagan-Jeffries Austin sp. nov., and D. lobesiae Fagan-Jeffries Austin sp. nov.; and one species from the genus Sathon Mason: S. oreo Fagan-Jeffries Austin sp. nov. These new species represent just a small fraction of the potential of 'Bush Blitz' surveys in regional Australia, which provide DNA-quality material allowing an integrative taxonomic approach and offer a window into the biodiversity of some of the least studied areas of the continent.

Cutaneous respiration by diving beetles from underground aquifers of Western Australia (Coleoptera: Dytiscidae).

Insects have a gas-filled respiratory system, which provides a challenge for those that have become aquatic secondarily. Diving beetles (Dytiscidae) use bubbles on the surface of their bodies to supply O2 for their dives and passively gain O2 from the water. However, these bubbles usually require replenishment at the water's surface. A highly diverse assemblage of subterranean dytiscids has evolved in isolated calcrete aquifers of Western Australia with limited/no access to an air-water interface, raising the question of how they are able to respire. We explored the hypothesis that they use cutaneous respiration by studying the mode of respiration in three subterranean dytiscid species from two isolated aquifers. The three beetle species consume O2 directly from the water, but they lack structures on their bodies that could have respiratory function. They also have a lower metabolic rate than other insects. O2 boundary layers surrounding the beetles are present, indicating that O2 diffuses into the surface of their bodies via cutaneous respiration. Cuticle thickness measurements and other experimental results were incorporated into a mathematical model to understand whether cutaneous respiration limits beetle size. The model indicates that the cuticle contributes considerably to resistance in the O2 cascade. As the beetles become larger, their metabolic scope narrows, potentially limiting their ability to allocate energy to mating, foraging and development at sizes above approximately 5 mg. However, the ability of these beetles to utilise cutaneous respiration has enabled the evolution of the largest assemblage of subterranean dytiscids in the world.