A male-specific sex marker for the endangered western sawshelled turtle (Myuchelys bellii) using in silico whole-genome subtraction

Artificial incubation of eggs for the mass release of hatchlings is a common conservation intervention for imperilled turtle species. Programs designed to reinforce wild populations need to ensure that they are releasing appropriate male to female ratios into the wild. In many turtle species, the sex of juveniles cannot be identified using external morphology until they approach sexual maturity. For the endangered western sawshelled turtle, Myuchelys bellii, sexual dimorphism does not occur until at least 6 years of age. We aimed to develop a molecular test to identify the sex of M. bellii during the life stages where they cannot be sexed morphologically-embryos, hatchlings and small juveniles. We used in silico whole-genome subtraction of a female M. bellii (XX) from a male (XY) to identify a Y chromosome-specific sequence which we characterized and developed into a PCR sex test. Our research is the first to use a whole-genome subtraction method in-silico to successfully establish sex chromosome markers in a freshwater turtle species. Developing this technology provides an opportunity for conservation programs to ensure that populations are supplemented with a proportionate number of male and female hatchlings. Further, it allows large scale measurement of naturally occurring sex ratios in hatchlings and small juveniles, which in turn enables estimates of sex ratios within wild populations free from age-at-maturity bias. The application of sex-specific marker technology also provides an opportunity to quantify the influence of sex on behaviour, movement and survival in the segment of populations that cannot be morphologically sexed

Spinal arthritis in cane toads across the Australian landscape

Loss of fitness can be a consequence of selection for rapid dispersal ability in invasive species. Increased prevalence of spinal arthritis may occur in cane toad populations at the invasion front as a cost of increased invasiveness, but our knowledge of the ecological drivers of this condition is lacking. We aimed to determine the factors explaining the prevalence of spinal arthritis in populations across the Australian landscape. We studied populations across a gradient of invasion histories. We collected 2415 toads over five years and determined the presence and size of spondylosis for each individual. We examined the effect of host size, leg length and invasion history on the prevalence of spondylosis. Host size was a significant predictor of spondylosis across populations. Contrary to our expectation, the overall prevalence of spondylosis was not positively related to invasion history and did not correlate with toad relative leg length. Rather than invasion age, the latitude at which populations were sampled provided an alternate explanation for the prevalence of spondylosis in cane toad populations and suggested that the incidence of this condition did not increase as a physiological cost of invasion, but is instead related to physical variables, such as climate

Interaction between temperature and sublethal infection with the amphibian chytrid fungus impacts a susceptible frog species

The amphibian chytrid fungus Batrachochytrium dendrobatidis is an emerging infectious pathogen present on every continent except Antarctica. It causes the disease chytridiomycosis in a subset of species but does not always result in disease or death for every host. Ambient temperature influences both amphibian metabolism and chytrid pathogenicity, however the interactive effects on host physiology is not well understood. We investigated the sublethal effect of B. dendrobatidis infection on a susceptible host, Litoria aurea to test (1) whether the infection load, metabolic activity, body fat and gonad size differed in L. aurea at either 24 degrees C or 12 degrees C ambient temperatures and (2) whether previous Bd infection caused long-term changes to body fat and gonad size. Litoria aurea in 12 degrees C treatments had higher infection loads of B. dendrobatidis and lower survivorship. Metabolic rate was higher and fat mass was lower in infected individuals and in animals in 24 degrees C treatments. Male L. aurea previously infected with B. dendrobatidis had smaller testes 5 months-post clearance of infection, an effect likely to translate to fitness costs in wild populations. These experiments demonstrate a physiological cost to sublethal B. dendrobatidis infection, which suggests a reduction in host fitness mediated by temperature in the host's environment regardless of whether infection leads to mortality

The interplay of fungal and bacterial microbiomes on rainforest frogs following a disease outbreak

Emerging infectious diseases are a serious threat to wildlife populations, and there is growing evidence that host microbiomes play important roles in infection dynamics, possibly even mitigating diseases. Nevertheless, most research on this topic has focused only on bacterial microbiomes, while fungal microbiomes have been largely neglected. To help fill this gap in our knowledge, we examined both the bacterial and fungal microbiomes of four sympatric Australian frog species, which had different population-level responses to the emergence of chytridiomycosis, a widespread disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). We sequenced 16,884 fungal amplicon sequence variants (ASVs) and 41,774 bacterial ASVs. Bacterial communities had higher richness and were less variable within frog species than were fungal communities. Nevertheless, both communities were correlated for both ASV richness and beta diversity (i.e., frogs with similar bacterial richness and community composition tended to also have similar fungal richness and community composition). This suggests that either one microbial community was having a large impact on the other or that they were both being driven by similar environmental factors. For both microbial taxa, we found little evidence of associations between Bd (prevalence or intensity) and either individuals' ASVs or beta diversity. However, there was mixed evidence of associations between richness (both bacterial and fungal) and Bd, with high richness potentially providing a protective effect. Surprisingly, the relative abundance of bacteria that have previously been shown to inhibit Bd was also positively associated with Bd infection intensity, suggesting that a high relative abundance of those bacteria provides poor protection against infection

The interplay of fungal and bacterial microbiomes on rainforest frogs following a disease outbreak

Emerging infectious diseases are a serious threat to wildlife populations, and there is growing evidence that host microbiomes play important roles in infection dynamics, possibly even mitigating diseases. Nevertheless, most research on this topic has focused only on bacterial microbiomes, while fungal microbiomes have been largely neglected. To help fill this gap in our knowledge, we examined both the bacterial and fungal microbiomes of four sympatric Australian frog species, which had different population-level responses to the emergence of chytridiomycosis, a widespread disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). We sequenced 16,884 fungal amplicon sequence variants (ASVs) and 41,774 bacterial ASVs. Bacterial communities had higher richness and were less variable within frog species than were fungal communities. Nevertheless, both communities were correlated for both ASV richness and beta diversity (i.e., frogs with similar bacterial richness and community composition tended to also have similar fungal richness and community composition). This suggests that either one microbial community was having a large impact on the other or that they were both being driven by similar environmental factors. For both microbial taxa, we found little evidence of associations between Bd (prevalence or intensity) and either individuals' ASVs or beta diversity. However, there was mixed evidence of associations between richness (both bacterial and fungal) and Bd, with high richness potentially providing a protective effect. Surprisingly, the relative abundance of bacteria that have previously been shown to inhibit Bd was also positively associated with Bd infection intensity, suggesting that a high relative abundance of those bacteria provides poor protection against infection

microDecon: a highly accurate read‐subtraction tool for the post‐sequencing removal of contamination in metabarcoding studies

Contamination is a ubiquitous problem in microbiome research and can skew results, especially when small amounts of target DNA are available. Nevertheless, no clear solution has emerged for removing microbial contamination. To address this problem, we developed the R package microDecon (https://github.com/donaldtmcknight/microDecon), which uses the proportions of contaminant operational taxonomic units (OTUs) or amplicon sequence variants (ASVs) in blank samples to systematically identify and remove contaminant reads from metabarcoding data sets. We rigorously tested microDecon using a series of computer simulations and a sequencing experiment. We also compared it to the common practice of simply removing all contaminant OTUs/ASVs and other methods for removing contamination. Both the computer simulations and our sequencing data confirmed the utility of microDecon. In our largest simulation (100,000 samples), using microDecon improved the results in 98.1% of samples. Additionally, in the sequencing data and in simulations involving groups, it enabled accurate clustering of groups as well as the detection of previously obscured patterns. It also produced more accurate results than the existing methods for identifying and removing contamination. These results demonstrate that microDecon effectively removes contamination across a broad range of situations. It should, therefore, be widely applicable to microbiome studies, as well as to metabarcoding studies in general

Infection dynamics, dispersal, and adaptation: understanding the lack of recovery in a remnant frog population following a disease outbreak

Emerging infectious diseases can cause dramatic declines in wildlife populations. Sometimes, these declines are followed by recovery, but many populations do not recover. Studying differential recovery patterns may yield important information for managing disease-afflicted populations and facilitating population recoveries. In the late 1980s, a chytridiomycosis outbreak caused multiple frog species in Australia's Wet Tropics to decline. Populations of some species (e.g., Litoria nannotis) subsequently recovered, while others (e.g., Litoria dayi) did not. We examined the population genetics and current infection status of L. dayi, to test several hypotheses regarding the failure of its populations to recover: (1) a lack of individual dispersal abilities has prevented recolonization of previously occupied locations, (2) a loss of genetic variation has resulted in limited adaptive potential, and (3) L. dayi is currently adapting to chytridiomycosis. We found moderate-to-high levels of gene flow and diversity (Fst range: <0.01-0.15; minor allele frequency (MAF): 0.192-0.245), which were similar to previously published levels for recovered L. nannotis populations. This suggests that dispersal ability and genetic diversity do not limit the ability of L. dayi to recolonize upland sites. Further, infection intensity and prevalence increased with elevation, suggesting that chytridiomycosis is still limiting the elevational range of L. dayi. Outlier tests comparing infected and uninfected individuals consistently identified 18 markers as putatively under selection, and several of those markers matched genes that were previously implicated in infection. This suggests that L. dayi has genetic variation for genes that affect infection dynamics and may be undergoing adaptation

Condition thresholds in Australia's threatened ecological community listings hinder conservation of dynamic ecosystems

Environmental degradation is threatening biodiversity and ecosystem function globally. Mandating ecosystem-level protection in policy and legislative frameworks is essential to prevent biodiversity loss. Australia’s Environment Protection and Biodiversity Conservation Act 1999 is the key legislative mechanism for supporting biodiversity at the national level, but has so far been ineffective at protecting habitat and ecological communities. Here we identify a major flaw in the current approach to listing threatened ecological communities (TECs): restrictive condition thresholds that threaten ecosystem function in dynamic ecosystems. Using two wetland TECs as a case study (Upland Wetlands and Coolibah-Black Box Woodlands), we argue that Australia’s environmental legislation should adopt a landscape-scale approach to TEC protection that acknowledges ecosystem function, accounts for different states in temporally dynamic systems, and sustains landscape connectivity of TEC distribution. We present a state-and-transition model for each TEC to show how human activities affect the reference-state continuum of wet and dry phases. We also show that the current listed condition thresholds do not acknowledge alternative ecosystem states and exclude areas that may be important for restoration and conservation of the TEC at the landscape-scale. Description of alternative and transitional states for dynamic systems, including how, when and why ecological communities shift between different states, should be formally integrated into the TEC listing process to protect Australia’s vulnerable ecosystems from further degradation and loss

Infection increases vulnerability to climate change via effects on host thermal tolerance

Unprecedented global climate change and increasing rates of infectious disease emergence are occurring simultaneously. Infection with emerging pathogens may alter the thermal thresholds of hosts. However, the effects of fungal infection on host thermal limits have not been examined. Moreover, the influence of infections on the heat tolerance of hosts has rarely been investigated within the context of realistic thermal acclimation regimes and potential anthropogenic climate change. We tested for effects of fungal infection on host thermal tolerance in a model system: frogs infected with the chytrid Batrachochytrium dendrobatidis. Infection reduced the critical thermal maxima (CTmax) of hosts by up to ~4 °C. Acclimation to realistic daily heat pulses enhanced thermal tolerance among infected individuals, but the magnitude of the parasitism effect usually exceeded the magnitude of the acclimation effect. In ectotherms, behaviors that elevate body temperature may decrease parasite performance or increase immune function, thereby reducing infection risk or the intensity of existing infections. However, increased heat sensitivity from infections may discourage these protective behaviors, even at temperatures below critical maxima, tipping the balance in favor of the parasite. We conclude that infectious disease could lead to increased uncertainty in estimates of species’ vulnerability to climate change

Remote sensing to characterize inundation and vegetation dynamics of upland lagoons

Understanding broad trends in the distribution and composition of wetlands is essential for making evidence-based management decisions. Determining temporal change in the extent of inundation in wetlands using remote sensing remains challenging and requires on-ground verification to determine accuracy and precision. Therefore, optimization and validation of remote sensing methods in threatened wetlands is a high priority for their conservation. Despite their ecological importance in the landscape, we have little knowledge of the variation in the spatial extent of inundation in upland lagoons, a threatened ecological community in New South Wales, Australia. Our project developed locally trained algorithms to predict the extent of water and emergent vegetation using imagery from the Landsat-5, -7, and -8 satellites. The best model for upland lagoons used shortwave infrared reflectance (performing better than normalized difference spectral indices), with model accuracy against validation transects greater than 95%. We applied the model to images from 1988 to 2020 across 58 lagoons to generate a dataset that demonstrates the variable water regime and vegetation change in response to local rainfall over 32 years such as in the lagoons. Our results reduce threats to a dynamic threatened ecological community by filling an important knowledge gap and demonstrate a valuable method to understand historical and current changes in the hydrology of dynamic wetland systems more broadly