Spatial variation in genomic signatures of local adaptation during the cane toad invasion of Australia.

Adaptive evolution can facilitate species' range expansions across environmentally heterogeneous landscapes. However, serial founder effects can limit the efficacy of selection, and the evolution of increased dispersal during range expansions may result in gene flow swamping local adaptation. Here, we study how genetic drift, gene flow and selection interact during the cane toad's (Rhinella marina) invasion across the heterogeneous landscape of Australia. Following its introduction in 1935, the cane toad colonised eastern Australia and established several stable range edges. The ongoing, more rapid range expansion in north-central Australia has occurred concomitant with an evolved increase in dispersal capacity. Using reduced representation genomic data of Australian cane toads from the expansion front and from two areas of their established range, we test the hypothesis that high gene flow constrains local adaptation at the expansion front relative to established areas. Genetic analyses indicate the three study areas are genetically distinct but show similar levels of allelic richness, heterozygosity and inbreeding. Markedly higher gene flow or recency of colonisation at the expansion front have likely hindered local adaptation at the time of sampling, as indicated by reduced slopes of genetic-environment associations (GEAs) estimated using a novel application of geographically weighted regression that accounts for allele surfing; GEA slopes are significantly steeper in established parts of the range. Our work bolsters evidence supporting adaptation of invasive species post-introduction and adds novel evidence for differing strengths of evolutionary forces among geographic areas with different invasion histories.

Constant-temperature predictions underestimate growth of a fungal amphibian pathogen under individual host thermal profiles.

Ectotherm body temperatures fluctuate with environmental variability and host behavior, which may influence host-pathogen interactions. Fungal pathogens are a major threat to ectotherms and may be highly responsive to the fluctuating thermal profiles of individual hosts, especially cool-loving fungi exposed to high host temperatures. However, most studies estimate pathogen thermal performance based on averages of host or surrogate environmental temperatures, potentially missing effects of short-term host temperature shifts such as daily or hourly heat spikes. We recorded individual thermal profiles of Australian rainforest frogs using temperature-sensitive radio-transmitters. We then reproduced a subset of individual thermal profiles in growth chambers containing cultures of the near-global amphibian pathogen Batrachochytrium dendrobatidis (Bd) to investigate how realistic host temperature profiles affect Bd growth. We focused on thermal profiles that exceed the thermal optimum of Bd because the effects of realistic heat spikes on Bd growth are unresolved. Our laboratory incubation experiment revealed that Bd growth varied in response to relatively small differences in heat spike characteristics of individual frog thermal profiles, such as a single degree or a few hours, highlighting the importance of individual host behaviors in predicting population-level disease dynamics. The fungus also grew better than predicted under the most extreme and unpredictable frog temperature profile, recovering from two days of extreme (nearly 32 °C) heat spikes without negative effects on overall growth, suggesting we are underestimating the growth potential of the pathogen in nature. Combined with the previous finding that Bd reduces host heat tolerance, our study suggests that this pathogen may carry a competitive edge over hosts in the face of anthropogenic climate change.

Microbiome diversity and composition varies across body areas in a freshwater turtle.

There is increasing recognition that microbiomes are important for host health and ecology, and understanding host microbiomes is important for planning appropriate conservation strategies. However, microbiome data are lacking for many taxa, including turtles. To further our understanding of the interactions between aquatic microbiomes and their hosts, we used next generation sequencing technology to examine the microbiomes of the Krefft's river turtle (Emydura macquarii krefftii). We examined the microbiomes of the buccal (oral) cavity, skin on the head, parts of the shell with macroalgae and parts of the shell without macroalgae. Bacteria in the phyla Proteobacteria and Bacteroidetes were the most common in most samples (particularly buccal samples), but Cyanobacteria, Deinococcus-thermus and Chloroflexi were also common (particularly in external microbiomes). We found significant differences in community composition among each body area, as well as significant differences among individuals. The buccal cavity had lower bacterial richness and evenness than any of the external microbiomes, and it had many amplicon sequence variants (ASVs) with a low relative abundance compared to other body areas. Nevertheless, the buccal cavity also had the most unique ASVs. Parts of the shell with and without algae also had different microbiomes, with particularly obvious differences in the relative abundances of the families Methylomonaceae, Saprospiraceae and Nostocaceae. This study provides novel, baseline information about the external microbiomes of turtles and is a first step in understanding their ecological roles.

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.

Host thermoregulatory constraints predict growth of an amphibian chytrid pathogen (Batrachochytrium dendrobatidis).

1. The course and outcome of many wildlife diseases are context-dependent, and therefore change depending on the behaviour of hosts and environmental response of the pathogen. 2. Contemporary declines in amphibian populations are widely attributed to chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis. Despite the thermal sensitivity of the pathogen and its amphibian hosts, we do not understand how host thermal regimes experienced by frogs in the wild directly influence pathogen growth. 3. We tested how thermal regimes experienced by the rainforest frog Litoria rheocola in the wild influence pathogen growth in the laboratory, and whether these responses differ from pathogen growth under available environmental thermal regimes. 4. Frog thermal regimes mimicked in the laboratory accelerated pathogen growth during conditions representative of winter at high elevations more so than if temperatures matched air or stream water temperatures. By contrast, winter frog thermal regimes at low elevations slowed pathogen growth relative to air temperatures, but not water temperatures. 5. The growth pattern of the fungus under frog thermal regimes matches field prevalence and intensity of infections for this species (high elevation winter > high elevation summer > low elevation winter > low elevation summer), whereas pathogen growth trajectories under environmental temperatures did not match these patterns. 6. If these laboratory results translate into field responses, tropical frogs may be able to reduce disease impacts by regulating their body temperatures to limit pathogen growth (e.g., by using microhabitats that facilitate basking to reach high temperatures); in other cases, the environment may limit the ability of frogs to thermoregulate such that individuals are more vulnerable to this pathogen (e.g., in dense forests at high elevations). 7. Species-specific thermoregulatory behaviour, and interactions with and constraints imposed by the environment, are therefore essential to understanding and predicting the spatial and temporal impacts of this global disease.

The return of the frogs: The importance of habitat refugia in maintaining diversity during a disease outbreak.

Recent decades have seen the emergence and spread of numerous infectious diseases, often with severe negative consequences for wildlife populations. Nevertheless, many populations survive the initial outbreaks, and even undergo recoveries. Unfortunately, the long-term effects of these outbreaks on host population genetics are poorly understood; to increase this understanding, we examined the population genetics of two species of rainforest frogs (Litoria nannotis and Litoria serrata) that have largely recovered from a chytridiomycosis outbreak at two national parks in the Wet Tropics of northern Australia. At the wetter, northern park there was little evidence of decreased genetic diversity in either species, and all of the sampled sites had high minor allele frequencies (mean MAF = 0.230-0.235), high heterozygosity (0.318-0.325), and few monomorphic markers (1.4%-4.0%); however, some recovered L. nannotis populations had low Ne values (59.3-683.8) compared to populations that did not decline during the outbreak (1,537.4-1,756.5). At the drier, southern park, both species exhibited lower diversity (mean MAF = 0.084-0.180; heterozygosity = 0.126-0.257; monomorphic markers = 3.7%-43.5%; Ne  = 18.4-676.1). The diversity patterns in this park matched habitat patterns, with both species having higher diversity levels and fewer closely related individuals at sites with higher quality habitat. These patterns were more pronounced for L. nannotis, which has lower dispersal rates than L. serrata. These results suggest that refugia with high quality habitat are important for retaining genetic diversity during disease outbreaks, and that gene flow following disease outbreaks is important for re-establishing diversity in populations where it was reduced.

Disentangling causes of seasonal infection prevalence patterns: tropical tadpoles and chytridiomycosis as a model system.

Identifying the factors that affect pathogen prevalence is critical to understanding the effects of wildlife diseases. We aimed to examine drivers of seasonal changes in the prevalence of infection by the amphibian chytrid fungus Batrachochytrium dendrobatidis in tadpoles. Because tadpoles may be important reservoirs for this disease, examining them will aid in understanding how chytridiomycosis affects entire amphibian populations. We hypothesized that temperature is a strong driver of prevalence of Bd in tadpoles, and the accumulation of infection as tadpoles become larger and older also drives prevalence in this system. We studied Litoria rheocola, a tropical rainforest stream frog with seasonal recruitment of annual tadpoles, and surveyed 6 streams in northeastern Queensland, Australia. Comparisons among models relating infection status to stream type, season, their interaction, tadpole age, and water temperature showed that age explained a large portion of the variance in infection status. Across sites and seasons, larger, older tadpoles had increased mean probabilities of infection, indicating that a large component of the variation among individuals was related to age, and thus to cumulative infection risk. Our results indicate that in systems with annual tadpoles, seasonal changes in infection prevalence may be strongly affected by seasonal patterns of tadpole growth and development in addition to stream type, season, and water temperature. These effects may then influence prevalence of infection in terrestrial individuals in species that have relatively frequent contact with water. This reinforces the need to integrate studies of the drivers of pathogen prevalence across all host life history stages.

Mixed population genomics support for the central marginal hypothesis across the invasive range of the cane toad (Rhinella marina) in Australia.

Understanding factors that cause species' geographic range limits is a major focus in ecology and evolution. The central marginal hypothesis (CMH) predicts that species cannot adapt to conditions beyond current geographic range edges because genetic diversity decreases from core to edge due to smaller, more isolated edge populations. We employed a population genomics framework using 24 235-33 112 SNP loci to test major predictions of the CMH in the ongoing invasion of the cane toad (Rhinella marina) in Australia. Cane toad tissue samples were collected along broad-scale, core-to-edge transects across their invasive range. Geographic and ecological core areas were identified using GIS and habitat suitability indices from ecological niche modelling. Bayesian clustering analyses revealed three genetic clusters, in the northwest invasion-front region, northeast precipitation-limited region and southeast cold temperature-limited region. Core-to-edge patterns of genetic diversity and differentiation were consistent with the CMH in the southeast, but were not supported in the northeast and showed mixed support in the northwest. Results suggest cold temperatures are a likely contributor to southeastern range limits, consistent with CMH predictions. In the northeast and northwest, ecological processes consisting of a steep physiological barrier and ongoing invasion dynamics, respectively, are more likely explanations for population genomic patterns than the CMH.

Condition-dependent reproductive effort in frogs infected by a widespread pathogen.

To minimize the negative effects of an infection on fitness, hosts can respond adaptively by altering their reproductive effort or by adjusting their timing of reproduction. We studied effects of the pathogenic fungus Batrachochytrium dendrobatidis on the probability of calling in a stream-breeding rainforest frog (Litoria rheocola). In uninfected frogs, calling probability was relatively constant across seasons and body conditions, but in infected frogs, calling probability differed among seasons (lowest in winter, highest in summer) and was strongly and positively related to body condition. Infected frogs in poor condition were up to 40% less likely to call than uninfected frogs, whereas infected frogs in good condition were up to 30% more likely to call than uninfected frogs. Our results suggest that frogs employed a pre-existing, plastic, life-history strategy in response to infection, which may have complex evolutionary implications. If infected males in good condition reproduce at rates equal to or greater than those of uninfected males, selection on factors affecting disease susceptibility may be minimal. However, because reproductive effort in infected males is positively related to body condition, there may be selection on mechanisms that limit the negative effects of infections on hosts.

Infection dynamics in frog populations with different histories of decline caused by a deadly disease.

Pathogens can drive host population dynamics. Chytridiomycosis is a fungal disease of amphibians that is caused by the fungus Batrachochytrium dendrobatidis (Bd). This pathogen has caused declines and extinctions in some host species whereas other host species coexist with Bd without suffering declines. In the early 1990s, Bd extirpated populations of the endangered common mistfrog, Litoria rheocola, at high-elevation sites, while populations of the species persisted at low-elevation sites. Today, populations have reappeared at many high-elevation sites where they presently co-exist with the fungus. We conducted a capture-mark-recapture (CMR) study of six populations of L. rheocola over 1 year, at high and low elevations. We used multistate CMR models to determine which factors (Bd infection status, site type, and season) influenced rates of frog survival, recapture, infection, and recovery from infection. We observed Bd-induced mortality of individual frogs, but did not find any significant effect of Bd infection on the survival rate of L. rheocola at the population level. Survival and recapture rates depended on site type and season. Infection rate was highest in winter when temperatures were favourable for pathogen growth, and differed among site types. The recovery rate was high (75.7-85.8%) across seasons, and did not differ among site types. The coexistence of L. rheocola with Bd suggests that (1) frog populations are becoming resistant to the fungus, (2) Bd may have evolved lower virulence, or (3) current environmental conditions may be inhibiting outbreaks of the fatal disease.

Patterns of Batrachochytrium dendrobatidis transmission between tadpoles in a high-elevation rainforest stream in tropical Australia.

The highly virulent fungal pathogen Batrachochytrium dendrobatidis (Bd) poses a global threat to amphibian biodiversity. Streams and other water bodies are central habitats in the ecology of the disease, particularly in rainforests where they may transport and transmit the pathogen and harbor infected tadpoles that serve as reservoir hosts. We conducted an experiment using larval green-eyed tree frogs Litoria serrata in semi-natural streamside channels to test the hypotheses that (1) the fungus can be transmitted downstream in stream habitats and (2) infection affects tadpole growth and mouthpart loss. Our results showed that transmission can occur downstream in flowing water with no contact between individuals, that newly infected tadpoles suffered increased mouthpart loss in comparison with controls that were never infected and that infected tadpoles grew at reduced rates. Although recently infected tadpoles showed substantial loss of mouthparts, individuals with longstanding infections did not, suggesting that mouthparts may re-grow following initial loss. Our study suggests that any management efforts that can reduce the prevalence of infections in tadpoles may be particularly effective if applied in headwater areas, as their effects are likely to be felt downstream.

Using pairs of physiological models to estimate temporal variation in amphibian body temperature.

Physical models are often used to estimate ectotherm body temperatures, but designing accurate models for amphibians is difficult because they can vary in cutaneous resistance to evaporative water loss. To account for this variability, a recently published technique requires a pair of agar models that mimic amphibians with 0% and 100% resistance to evaporative water loss; the temperatures of these models define the lower and upper boundaries of possible amphibian body temperatures for the location in which they are placed. The goal of our study was to develop a method for using these pairs of models to estimate parameters describing the distributions of body temperatures of frogs under field conditions. We radiotracked green-eyed treefrogs (Litoria serrata) and collected semi-continuous thermal data using both temperature-sensitive radiotransmitters with an automated datalogging receiver, and pairs of agar models placed in frog locations, and we collected discrete thermal data using a non-contact infrared thermometer when frogs were located. We first examined the accuracy of temperature-sensitive transmitters in estimating frog body temperatures by comparing transmitter data with direct temperature measurements taken simultaneously for the same individuals. We then compared parameters (mean, minimum, maximum, standard deviation) characterizing the distributions of temperatures of individual frogs estimated from data collected using each of the three methods. We found strong relationships between thermal parameters estimated from data collected using automated radiotelemetry and both types of thermal models. These relationships were stronger for data collected using automated radiotelemetry and impermeable thermal models, suggesting that in the field, L. serrata has a relatively high resistance to evaporative water loss. Our results demonstrate that placing pairs of thermal models in frog locations can provide accurate estimates of the distributions of temperatures experienced by individual frogs, and that comparing temperatures from model pairs to direct measurements collected simultaneously on frogs can be used to broadly characterize the skin resistance of a species, and to select which model type is most appropriate for estimating temperature distributions for that species.

Ecology: global warming and amphibian losses.

Is global warming contributing to amphibian declines and extinctions by promoting outbreaks of the chytrid fungus Batrachochytrium dendrobatidis? Analysing patterns from the American tropics, Pounds et al. envisage a process in which a single warm year triggers die-offs in a particular area (for instance, 1987 in the case of Monteverde, Costa Rica). However, we show here that populations of two frog species in the Australian tropics experienced increasing developmental instability, which is evidence of stress, at least two years before they showed chytrid-related declines. Because the working model of Pounds et al. is incomplete, their test of the climate-linked epidemic hypothesis could be inconclusive.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay.

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Context-dependent symbioses and their potential roles in wildlife diseases.

It is well known in ecology, evolution and medicine that both the nature (commensal, parasitic and mutualistic) and outcome (symbiont fitness, survival) of symbiotic interactions are often context-dependent. Less is known about the importance of context-dependence in symbioses involved in wildlife disease. We review variable symbioses, and use the amphibian disease chytridiomycosis to demonstrate how understanding context-dependence can improve the understanding and management of wildlife diseases. In chytridiomycosis, the host-pathogen interaction is context-dependent; it is strongly affected by environmental temperature. Skin bacteria can also modify the interaction; some bacteria reduce amphibians' susceptibility to chytridiomycosis. Augmentation of protective microbes is being considered as a possible management tool, but informed application of bioaugmentation requires understanding of how the interactions between host, beneficial bacteria and pathogen depend upon environmental context. The community-level response of the amphibian skin microbiota to environmental conditions may explain the relatively narrow range of environmental conditions in which past declines have occurred. Environmental context affects virulence and the protection provided by mutualists in other host-pathogen systems, including threatened bats and corals. Increased focus on context-dependence in interactions between wildlife and their symbionts is likely to be crucial to the future investigation and management of emerging diseases of wildlife.

Ontogenetic shifts in a prey's chemical defences influence feeding responses of a snake predator.

Foraging theory suggests that predator responses to potential prey should be influenced by prey chemical defences, but the effects of ontogenetic variation in such defences on prey vulnerability to predators remain unclear. Cane toads (Rhinella marina) are toxic to anurophagous snakes, including the keelback (Tropidonophis mairii, a natricine colubrid that occurs within the toads' invasive range in Australia). Toxin levels and diversity change through toad ontogeny, decreasing from the egg stage to metamorphosis, then increasing in postmetamorphic toads. If the toxin content of a prey item influences predator responses, we predict that keelbacks should exhibit selective predation on toads close to metamorphosis. The results of our laboratory trials on adult (field-collected, and thus toad-experienced) and hatchling (laboratory-incubated, and thus toad-naive) keelbacks supported this prediction. The snakes selectively consumed later-stage rather than earlier-stage tadpoles, and earlier-stage rather than later-stage metamorphs. Our data are thus consistent with the hypothesis that ontogenetic changes in toxin content can affect individuals' vulnerability to predation.

Prevalence of Batrachochytrium dendrobatidis infection is extremely low in direct-developing Australian microhylids.

The emerging infectious disease chytridiomycosis has been implicated in declines and disappearances of amphibian populations around the world. However, susceptibility to infection and the extent of pathological effects of infection vary among hosts, and species with life histories that include parental care of direct-developing terrestrial eggs may tend to be less susceptible. We examined samples from a total of 595 individuals of 9 species of direct-developing Australian frogs in the family Microhylidae for the presence of infection by Batrachochytrium dendrobatidis (Bd). Between 1995 and 2004, 336 samples were collected; 102 of these were analysed histologically and 234 were tissues stored in alcohol, which were examined using diagnostic quantitative PCR (qPCR). Swab samples were collected from 259 frogs from 2005 to 2008 and were examined using qPCR. None of the 595 samples showed evidence of infection by Bd. If these data are regarded as a single sample representative of Australian microhylids, the upper 95% binomial confidence limit for the prevalence of infection in frogs of this family is 0.0062 (<1%). Even if only the data from the more powerful diagnostic qPCR tests are used, the upper 95% confidence limit for prevalence is 0.0075 (<1%). Our data suggest that Australian microhylids have a very low prevalence of infection by Bd in nature, and thus are either not susceptible, or are only slightly susceptible, to chytridiomycosis. This could be due solely to, or in combination with, low rates of transmission and to factors that promote resistance to infection, including ecological or behavioural characteristics, innate immune functions such as antimicrobial skin peptides, or antimicrobial symbionts in skin flora.

Environmental refuge from disease-driven amphibian extinction.

Species that are tolerant of broad environmental gradients may be less vulnerable to epizootic outbreaks of disease. Chytridriomycosis, caused by the fungus Batrachochytrium dendrobatidis, has been linked to extirpations and extinctions of amphibian species in many regions. The pathogen thrives in cool, moist environments, and high amphibian mortality rates have commonly occurred during chytridiomycosis outbreaks in amphibian populations in high-elevation tropical rainforests. In Australia several high-elevation species, including the armored mist frog (Litoria lorica), which is designated as critically endangered by the International Union for the Conservation of Nature (IUCN), were believed to have gone extinct during chytridiomycosis outbreaks in the 1980s and early 1990s. Species with greater elevational ranges disappeared from higher elevations, but remained common in the lowlands. In June 2008, we surveyed a stream in a high-elevation dry sclerophyll forest and discovered a previously unknown population of L. lorica and a population of the waterfall frog (Litoria nannotis). We conducted 6 additional surveys in June 2008, September 2008, March 2009, and August 2009. Prevalences of B. dendrobatidis infection (number infected per total sampled) were consistently high in frogs (mean 82.5%, minimum 69%) of both species and in tadpoles (100%) during both winter (starting July) and summer (starting February). However, no individuals of either species showed clinical signs of disease, and they remained abundant (3.25 - 8.75 individuals of L. lorica and 6.5-12.5 individuals of L. nannotis found/person/100 m over 13 months). The high-elevation dry sclerophyll site had little canopy cover, low annual precipitation, and a more defined dry season than a nearby rainforest site, where L. nannotis was more negatively affected by chytridiomycosis. We hypothesize this lack of canopy cover allowed the rocks on which frogs perched to warm up, thereby slowing growth and reproduction of the pathogen on the hosts. In addition, we suggest surveys for apparently extinct or rare species should not be limited to core environments.