Variable effects of captivity on microbiomes in populations of IUCN-endangered Blanding's turtles (Emydoidea blandingii).

AIMS: Microbiome composition is increasingly considered in species reintroduction efforts and may influence survival and reproductive success. Many turtle species are threatened by anthropogenic pressures and are frequently raised in captivity for reintroduction efforts, yet little is known about turtle microbiome composition in either wild or captive settings. Here, we investigated trends in microbiome composition of captive and wild IUCN-endangered Blanding's turtles (Emydoidea blandingii). METHODS AND RESULTS: We amplified and sequenced the V4 region of the 16S rDNA locus from plastron, cloaca, and water samples of wild E. blandingii adults and two populations of captive E. blandingii juveniles being raised for headstarting. Plastron, cloaca, and water-associated microbiomes differed strongly from each other and were highly variable among captive sites and between captive and wild sites. Across plastron, cloaca, and water-associated microbial communities, microbial diversity changed over time, but not in a predictable direction between captive sites. Plastron beta diversity correlated with growth rate in captive samples, indicating that external microbiomes may correlate with individual fitness. CONCLUSIONS: Our results indicate that external and internal microbiomes vary between captive and wild turtles and may reflect differences in fitness of captive-raised individuals.

How turtles keep their cool: Seasonal and diel basking patterns in a tropical turtle.

Behavioural thermoregulation by ectotherms is an important mechanism for maintaining body temperatures to optimise physiological performance. Experimental studies suggest that nocturnal basking by Krefft's river turtles (Emydura macquarii krefftii) in the tropics may allow them to avoid high water temperatures, however, this hypothesis has yet to be tested in the field. In this study, we examined the influence of environmental temperature on seasonal and diel patterns of basking in E. m. krefftii in tropical north Queensland, Australia. Wildlife cameras were used to document turtle basking events for seven consecutive days and nights for each month over a year (April 2020-March 2021). Air and water temperatures were recorded simultaneously using temperature loggers. We used a negative binomial mixed effects model to compare mean basking durations (min) occurring among four environmental temperature categories based on population thermal preference (26 °C): 1) air temperature above and water temperature below preferred temperature; 2) air temperature below and water temperature above preferred temperature; 3) air and water temperatures both above preferred temperature; and 4) air and water temperatures both below preferred temperature. Basking behaviour was influenced significantly by the relationship between air and water temperature. During the day, turtles spent significantly less time basking when both air and water temperatures were above their preferred temperatures. Conversely, at night, turtles spent significantly more time basking when water temperatures were warm and air temperatures were cool relative to their preferred temperature. This study adds to the growing body of work indicating pronounced heat avoidance as a thermoregulatory strategy among tropical reptile populations.

The first linkage map for Australo-Papuan Treefrogs (family: Pelodryadidae) reveals the sex-determination system of the Green-eyed Treefrog (Litoria serrata).

Amphibians represent a useful taxon to study the evolution of sex determination because of their highly variable sex-determination systems. However, the sex-determination system for many amphibian families remains unknown, in part because of a lack of genomic resources. Here, using an F1 family of Green-eyed Treefrogs (Litoria serrata), we produce the first genetic linkage map for any Australo-Papuan Treefrogs (family: Pelodryadidae). The resulting linkage map contains 8662 SNPs across 13 linkage groups. Using an independent set of sexed adults, we identify a small region in linkage group 6 matching an XY sex-determination system. These results suggest Litoria serrata possesses a male heterogametic system, with a candidate sex-determination locus on linkage group 6. Furthermore, this linkage map represents the first genomic resource for Australo-Papuan Treefrogs, an ecologically diverse family of over 220 species.

Leech removal is not the primary driver of basking behavior in a freshwater turtle.

Leaving the water to bask (usually in the sun) is a common behavior for many freshwater turtles, with some species also engaging in "nocturnal basking." Ectoparasite removal is an obvious hypothesis to explain nocturnal basking and has also been proposed as a key driver of diurnal basking. However, the efficacy of basking, day or night, to remove leeches has not been experimentally tested. Therefore, we examined the number of leeches that were removed from Krefft's river turtles (Emydura macquarii krefftii) after experimentally making turtles bask at a range of times of day, durations, and temperatures. Turtles had high initial leech loads, with a mean of 32.1 leeches per turtle. Diurnal basking under a heat lamp for 3 hr at ~28°C significantly reduced numbers of leeches relative to controls. In diurnal trials, 90.9% of turtles lost leeches (mean loss of 7.1 leeches per turtle), whereas basking for 30 min under the same conditions was not effective (no turtles lost leeches, and all turtles were still visibly wet). Similarly, "nocturnal basking" at ~23°C for 3 hr was not effective at removing leeches. Only 18% of turtles lost leeches (one turtle lost one leech and another lost four leeches). Diurnal basking outdoors under direct sunlight for 20 min (mean temp = 34.5°C) resulted in a small reduction in leeches, with 50% of turtles losing leeches and an average loss of 0.7 leeches per turtle. These results indicate basking can remove leeches if temperatures are high or basking durations are long. However, it was only effective at unusually long basking durations in this system. Our data showed even the 20-min period was longer than 70.1% of natural diurnal basking events, many of which took place at cooler temperatures. Therefore, leech removal does not appear to be the purpose of the majority of basking events.

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.

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.

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.

Correcting for unequal catchability in sex ratio and population size estimates.

Wildlife populations often exhibit unequal catchability between subgroups such as males and females. This heterogeneity of capture probabilities can bias both population size and sex ratio estimates. Several authors have suggested that this problem can be overcome by treating males and females as separate populations and calculating a population estimate for each of them. However, this suggestion has received little testing, and many researchers do not implement it. Therefore, we used two simulations to test the utility of this method. One simulated a closed population, while the other simulated an open population and used the robust design to calculate population sizes. We tested both simulations with multiple levels of heterogeneity, and we used a third simulation to test several methods for detecting heterogeneity of capture probabilities. We found that treating males and females as separate populations produced more accurate population and sex ratio estimates. The benefits of this method were particularly pronounced for sex ratio estimates. When males and females were included as a single population, the sex ratio estimates became inaccurate when even slight heterogeneity was present, but when males and females were treated separately, the estimates were accurate even when large biases were present. Nevertheless, treating males and females separately reduced precision, and this method may not be appropriate when capture and recapture rates are low. None of the methods for detecting heterogeneity were robust, and we do not recommend that researchers rely on them. Rather, we suggest separating populations by sex, age, or other subgroups whenever sample sizes permit.