Monitoring temporal changes in population genetic diversity and effective population size can provide vital information on future viability. The dusky gopher frog, Lithobates sevosus, is a critically endangered species found only in coastal Mississippi, with low genetic variability as a consequence of isolation and population size reduction. Conservation management practices have been implemented, but their efficacy has not been addressed. We genotyped individuals collected 1997–2014 to determine temporal trends in population genetic variation, structure, and effective size. Observed and expected heterozygosity and allelic richness revealed temporally stable, but low, levels of genetic variation. Positive levels of inbreeding were found in each year. There was weak genetic structure among years, which can be attributed to increased effects of genetic drift and inbreeding in small populations. L. sevosus exhibited an increase in effective population size, and currently has an estimated effective size of 33.0–58.6 individuals, which is approximately half the census size. This large ratio could possibly be explained by genetic compensation. We found that management practices have been effective at maintaining and improving effective size and genetic diversity, but that additional strategies need to be implemented to enhance viability of the species.

Small, isolated populations often experience increased inbreeding and decreased heterozygosity, which increases the potential risk of inbreeding depression.The relationship between inbreeding and sperm health is well-documented in a variety of taxa, but has yet to be explored in amphibians. The dusky gopher frog, Lithobates sevosus, is a critically endangered species with years of documented inbreeding and low genetic variability as a consequence of isolation and population size reduction. This study investigated the effects of inbreeding on sperm quality in captive L. sevosus using an outbred, sister species (Lithobates pipiens) as a standard for comparison. We found L. sevosus to have severely reduced sperm quality in terms of total motility, forward progressive motility, concentration, and viability. Additionally, we observed a significant, negative relationship between total sperm motility and mean kinship within captive-bred individuals. These data serve to enhance our understanding of the role inbreeding plays in amphibians, and to provide valuable insight into new risk factors declining amphibian populations may face. K E Y W O R D S assisted reproductive technologies, captive breeding, dusky gopher frog, fertility, Lithobates sevosus

Novel conservation methods have become increasingly important in protecting our remaining biodiversity in the face of unprecedented rates of species declines. One method of maintaining genetic and species diversity is through germplasm cryopreservation. However, our knowledge of cryopreservation relies heavily on studies in domestic or aquaculture species. Moreover, most studies are narrowly focused on a single species. Consequently, the broader application of these methods to wildlife conservation is often unknown or assumed. Here, we examine the response of four anuran species from two families (Anaxyrus fowleri, Anaxyrus baxteri, Lithobates pipiens, and Lithobates sevosus) to three sperm cryopreservation treatments (5, 10, and 15% N,N-dimethylformamide with 10% trehalose). Within each family, we selected a congeneric species pair with one common, nonthreatened species and one endangered species. We found that endangered species had significantly lower initial sperm quality, though recovery rate of sperm after cryopreservation was not significantly different between nonthreatened and endangered species. Overall, responses to the three treatments were consistent across species, with 5% DMFA with 10% trehalose producing the highest recovery rates in all four species. In addition, cryopreserved sperm were used to successfully produce hatchlings in both of the nonthreatened species. Our results demonstrate that standardized methods developed for anurans can be applied more widely across families and can be transferred from model species to species of conservation concern.These findings form a basis for further exploration into cryopreservation as an effective tool for wildlife conservation in amphibians. K E Y W O R D Samphibian, assisted reproductive technology, Bufonidae, cryobiology, Ranidae, sperm motility

Amphibian populations are declining worldwide, and increased exposure to environmental stressors, including global climate change and pathogens like Batrachochytrium dendrobatidis (Bd), may be contributing to this decline. Our goal was to use a novel dermal swabbing method to measure glucocorticoid (GC) hormones and investigate the relationship among disease and environmental conditions in the critically endangered Wyoming toad (Anaxyrus baxteri). Our objectives were to (i) validate the use of dermal swabs to measure GCs using an adrenocorticotropic hormone (ACTH) challenge on eight captive toads (4 ACTH: 2 M, 2F and 4 saline as a control: 2 M, 2F), (ii) investigate stress physiology and disease status of toads across six reintroduction sites and (iii) compare dermal cortisol between reintroduced and captive toads. Dermal cortisol peaked immediately after the ACTH and saline injections. Faecal GC metabolites (FGMs) were significantly higher one week after the ACTH injection compared with the week before. Saline-injected toads had no change in FGM over time. Toads were only found in three reintroduction sites and dermal cortisol was similar across sites; however, reintroduced toads had higher dermal cortisol in August compared with June and compared with captive individuals. Bd status did not influence dermal cortisol concentrations. Dermal and faecal hormonal metabolite analyses can be used to study amphibian stress physiology and learn how environmental conditions are impacting population success.

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