Captive-release programs are an increasingly popular conservation strategy to combat wild extinctions. However, it is critical to determine if translocating animals from captive colonies ("source populations") leads to the establishment of new wild populations that are both stable and self-sustaining. To fill this knowledge gap, we provide a case study from the dusky gopher frog (Lithobates sevosus) reintroduction program to serve as an example for other critically endangered amphibians. In this study, we provide quantitative information on the reintroduction and survivorship of zoo-bred individuals that are released into the wild. This unique opportunity is the culmination of close to 20 years of collective efforts across multiple agencies. By taking advantage of the key monitoring window shortly after initial releases, we can formally declare the first successfully reintroduced, breeding population of dusky gopher frogs founded solely from a captive-bred colony.

Abstract Amphibians are declining worldwide, and research on their habitats and ecology is important for their conservation. The endangered dusky gopher frog (Rana sevosa) breeds exclusively in isolated, open canopy wetlands, usually with extensive herbaceous growth. Larvae of the dusky gopher frog have higher growth and survival under open canopy than under closed canopy conditions. The mechanisms whereby this occurs are poorly understood, however. In the summer of 2021 in Harrison County, Mississippi, USA, we conducted a complete factorial experiment in mesocosms to compare the relative influence of factors differing between open and closed canopy ponds on larvae. A heat and light treatment consisted of one third of tanks being exposed to full sunlight, one third of tanks being shaded with 70% shadecloth, and the final third being shaded with 70% shadecloth but heated to mimic the temperature of the full sun tanks. In addition, tanks received a closed canopy tree leaf litter mixture or an open canopy herbaceous vegetation mixture, and vertical vegetation‐like structure made of polypropylene rope or not. Cool shaded tanks and heated shaded tanks had identical survival to metamorphosis at 74.7% and produced frogs with an average mass of 2.21 g and 2.09 g, respectively, while tanks in full sunlight achieved significantly higher 93.4% survival to metamorphosis and an average mass of 2.64 g. The open canopy vegetation mixture yielded an average tank survival of 88.3% and mass of 2.94 g, compared to closed canopy vegetation tanks with a significantly lower survival of 73.5% and average mass of 1.61 g. Added structure had no effect on survival or mass. These results indicate that sunlight (not heat alone) and herbaceous plants are important in increasing dusky gopher frog survival and mass in open canopy conditions and suggest that management for these characteristics receive priority in captive‐rearing programs and habitat restoration.

Abstract An inherent challenge in managing rare or cryptic species is data deficiency. For this reason, ancillary data is a potentially valuable resource for generating key population estimates for priority species. We compiled ancillary commensal data collected between 1982 and 2020 during surveys of gopher tortoise (Gopherus polyphemus) burrows to estimate the terrestrial distribution of gopher frogs (Rana capito) from potential breeding wetlands. Gopher frogs were detected in gopher tortoise burrows 30‒3,879 m from identified wetlands. A global model of all records from all sites indicated that the probability of a gopher frog residing in a gopher tortoise burrow declined with increasing distance from a wetland. This pattern also held for 4 of 5 sites with a sufficient number of gopher frog detections to model independently. Based on the full data set, we estimated that 50%, 90%, and 99% of gopher frog observations occurred within 392 m, 1,019 m, and 2,752 m of the nearest wetland, respectively. Our results indicate a higher proportion of gopher frogs emigrate longer distances from wetlands than was previously reported using other methods, such as radio‐telemetry. This information can directly assist with management decisions, notably the spatial extent for application of habitat management surrounding breeding wetlands. More generally, this study illustrates the capacity of ancillary data to fill data deficiencies for a rare and cryptic species and highlights the importance of these data.

Among mobile terrestrial animals, movement among microsites can allow individuals to behaviorally moderate their body temperatures and rates of water loss, which can have important consequences for activity times, growth, fecundity, and survival. Ground-layer vegetation can change the availability and variability of microclimates; however, gaps in our understanding of how individuals interact with the microclimates created by vegetation limit our ability to inform management actions for wildlife. Amphibians can simultaneously balance operant body temperatures and water loss and the availability of heterogeneous microclimates should moderate how effectively they are able to do so. However, relatively few studies have attempted to mechanistically demonstrate how ground vegetation-driven effects on microclimatic variation may affect amphibian performance and survival. Agent-based modeling (ABM) can incorporate behavior and other mechanisms to understand how animals interact with their environments to result in larger scale patterns. They are effective for exploring alternative scenarios and representing the uncertainty in systems. Here, we use ABMs to integrate field and laboratory measurements of movement behavior, physiology, and plant effects on near-ground microclimate to explore how ground vegetation and the availability of terrestrial refugia may affect the survival and terrestrial distributions of juvenile gopher frogs (Rana capito) under two weather regimes. We also examine how assumptions regarding micro-scale movement (< 1 m2) affect the influence of ground vegetation on survival and settlement within refugia. While all variables affected settlement and survival, our models predict that inter-annual variation in weather and the density and spatial distribution of permanent refugia likely have the greatest influence on juvenile survival. The benefit of increased ground vegetation was dependent on the reasonable assumption that gopher frogs exhibit microclimate habitat selection throughout the day and night to limit water loss. Our models suggest that vegetation would be most beneficial to amphibians under warmer weather regimes provided there is sufficient rainfall.

Delineating genetically distinct population segments of threatened species and quantifying population connectivity are important steps in developing effective conservation and management strategies aimed at preventing extinction. The gopher frog (Rana capito) is a xeric-adapted, pond-breeding species endemic to the Gulf and Atlantic coastal plains of the southeastern United States. This species has experienced extensive habitat loss and fragmentation in the formerly widespread longleaf pine-wiregrass savanna where it lives, resulting in individual abundance declines and population extinctions throughout its range. We used individual-based clustering methods along with Bayesian inference of historical migration based on almost 1500 multilocus microsatellite genotypes to examine genetic structure in this taxon. Clustering analyses identified panhandle and peninsular populations in Florida as distinct genetic clusters separated by the Aucilla River, consistent with the division between the Coastal Plain and peninsular mitochondrial lineages, respectively. Analysis of historical migration indicated an east–west population divergence event followed by immigration to the east. Together, our results indicate that the genetically distinct Coastal Plain and peninsular Florida lineages should be considered separately for conservation and management purposes.

Translocation is increasingly used to move animals of conservation concern away from sites where habitat will be destroyed (mitigation translocation), but outcomes have rarely been adequately monitored, particularly for amphibians. We used radiotelemetry monitoring to assess survival and movement of 23 experimentally translocated, adult Gopher Frogs (Lithobates capito) at a recipient site in north-central Florida, USA. Although posttranslocation monitoring was our primary goal, we also compared our results with those of 24 nontranslocated frogs that were monitored in three previous efforts, conducted at different locations and times. For both translocated and nontranslocated frogs, movement was the most important predictor of mortality, with translocated frogs having a significantly higher probability of movement and higher mortality during the first month following release. However, there was no effect of translocation on survival after controlling for probability of movement because movement was dangerous for both translocated and nontranslocated frogs. Movement by translocated frogs was likely a behavioral response to the translocation experience, whereas movement by nontranslocated frogs was associated with breeding pond visitation, which was not observed within the translocated group. Survival was high for both groups once they settled into underground refugia and movement declined. Despite the comparatively high mortality of translocated frogs immediately following release, more than half survived to the end of monitoring and remained in the vicinity of the release site, meeting an early benchmark of translocation success.

The Gopher Frog (Rana capito) is a threatened species native to the southeastern longleaf pine ecosystem. Although once much more widespread across the southeastern United States, they now occur in North Carolina at only a handful of disjunct sites in the Coastal Plain and Sandhills regions of the state. The long-term persistence of these populations is thus a concern, as is a loss of genetic variation over time. We used mitochondrial and microsatellite markers to better understand the spatial structure of genetic variation and levels of genetic variability across these remaining populations in order to inform conservation and management decisions. Eight unique mitochondrial haplotypes were found, but these were all genetically similar to one another. Levels of genetic diversity based on the microsatellite analyses were similar across populations, but inbreeding coefficients in two populations were significant, suggesting a potential vulnerability to inbreeding depression. All disjunct populations showed significant genetic differentiation, which was not related to geographic distance. Conversely, within populations, the genetic relatedness of individuals between ponds decreased as distance between ponds increased. This kinship pattern is likely driven by strong breeding philopatry (individuals returning to the same ponds across years) and indicates that conservation actions at the scale of <1 km would primarily affect kin groups of Gopher Frogs, whereas conservation actions at scales ‡1.5 km would be needed to capture more distantly related individuals. Management efforts should thus focus on local metapopulation dynamics by maintaining multiple breeding ponds at each location, and by enhancing connectivity between these breeding ponds.

Population viability analysis for a pond-breeding amphibian under future drought scenarios in the southeastern United States

Abstract Populations of amphibians that breed in isolated, ephemeral wetlands may be particularly sensitive to breeding and recruitment rates, which can be influenced by dynamic and difficult-to-predict extrinsic factors. The gopher frog Rana capito is a declining species currently proposed for listing under the U.S. Endangered Species Act, as well as one of many pond-breeding amphibians of conservation concern in the southeastern United States. To represent gopher frog breeding dynamics, we applied an occupancy modeling framework that integrated multiple data sets collected across the species' range to 1) estimate the influence of climate, habitat, and other factors on wetland-specific seasonal breeding probabilities; and 2) use those estimates to characterize seasonal, annual, and regional breeding patterns over a 10-y period. Breeding probability at a wetland was positively influenced by seasonal precipitation (Standardized Precipitation Index) and negatively influenced by fish presence. We found some evidence that the amount of suitable habitat surrounding a wetland was positively correlated with breeding probability during drought conditions. The percentage of sampled wetlands (N = 192) predicted to have breeding varied seasonally, annually, and regionally across the study. Within-year temporal patterns of breeding differed across the range: in most locations north of Florida, peaks of breeding occurred in winter and spring months; whereas breeding was more dispersed throughout the year in Florida. Peaks of breeding across the 10-y period often occurred during or in the season following high rainfall events (e.g., hurricanes). These results have direct applications for site-level management that aims to increase successful breeding opportunities of gopher frogs and other associated pond-breeding amphibians, including monitoring protocol and intensity, removal of fish, and improving terrestrial habitat conditions surrounding wetlands (e.g., via tree or shrub removal and prescribed fire). The results also have implications for better-informed management through the closer alignment of breeding activity monitoring with predicted seasonal peaks. Furthermore, estimates of breeding frequency can be incorporated into population viability analyses to inform forthcoming assessments of extinction risk and designation of the species' conservation status by the U.S. Fish and Wildlife Service.

A persistent 2-month long outbreak of Ranavirus in a natural community of amphibians contributed to a mass die-off of gopher frog tadpoles (Lithobates capito) and severe disease in striped newts (Notophthalmus perstriatus) in Florida. Ongoing mortality in L. capito and disease signs in N. perstriatus continued for 5weeks after the first observation. Hemorrhagic disease and necrosis were diagnosed from pathological examination of L. capito tadpoles. We confirmed detection of a frog virus 3 (FV3)-like Ranavirus via quantitative PCR in all species. Our findings highlight the susceptibility of these species to Rv and the need for long-term disease surveillance during epizootics.

To date, all captive breeding of the dusky gopher frog, Lithobates sevosus, a federally listed endangered species, has been accomplished using in vitro fertilization (IVF). Here, we describe multievent and highly fecund captive reproduction of dusky gopher frogs driven solely by natural environmental factors. Six pairs of L. sevosus were kept in a 3.7 × 4.4 m2 outdoor enclosure designed to resemble their natural breeding habitat, which included a pool and three artificial burrows. Modifications to the enclosure that simulated temperatures and conditions within their natural range during winter were added in October and removed in late February. Following a warm, rainy period, five egg masses were laid between March 5 and 11, 2020. The number of oocytes per egg mass was 2300 ± 409 (range = 1341–3565), with the total across all five egg masses being 11,501. Of these oocytes, the hatching rate was 68.58 ± 10.05% (range = 37.53%–95.59%), with a total of 7887 successful hatchlings overall. Clutch sizes were similar to those in the wild and greater than those typically produced using IVF; thus, natural breeding can substantially increase the number of frogs available for reintroduction programs. Although assisted reproductive technologies such as IVF will continue to be useful for ensuring the success of strategic genetic pairings of captive L. sevosus, the new tool of nonassisted reproduction in specifically designed outdoor enclosures is an important advancement for the conservation and recovery of this endangered species.

Military installations are valuable in global biodiversity conservation as they secure representative ecosystems from land conversion and protect many threatened or endangered species. Selecting suitable areas for biodiversity conservation within military installations is a challenging problem as this must not impede military training activities. The issue gets more complicated when considering multiple cohabiting species in a metacommunity with species dependency. In this paper, we present an example for the conservation of two cohabiting species, Gopher Tortoise (GT) and Gopher Frog (GF), located within the boundaries of a military installation, Fort Stewart, Georgia, United States. The GF depends on both locations of GT habitat (burrows) and ephemeral vernal ponds (for breeding). We develop a model that identifies the cost-efficient areas for the conservation of these two species while taking into account the dependency of GF on GT burrows. The model selects a specified number of conservation areas for the two species, where each GF conservation area covers an adequate number of vernal ponds for the GFs to accommodate their reproduction, and each GT conservation area provides adequate habitat quality to sustain a viable GT population. The model also requires each GF site to be located close to GT sites so that the GFs could find refuge after they leave the water. We use the total distance of selected sites to the main roads in the military installation as a proxy for the conservation cost. We achieve contiguity of each conservation area by selecting sites that are adjacent to a central site of the conservation area to ensure undisrupted travel for both the GFs and the GTs. Using the model, we generated alternative configurations of conservation areas that could be considered by the land managers of Fort Stewart. Our methods are general and can be applied to other reserve site selection and land management problems with cohabiting interrelated species.

Abstract Isolated wetlands embedded within longleaf pine savannas support a high proportion of regional biodiversity including many amphibian species. Today, remnant isolated wetlands are often overgrown and hydrologically altered due to fire exclusion or incompatible, cool season fire regimes. In the absence of warm season fires when wetlands are dry, shrubs and trees succeed herbaceous plants, which alters wetland productivity via effects on light and detritus quality. We used a factorial aquatic mesocosm study to test the effects of altered detritus and shade on the growth, development, and survival of tadpoles of two priority amphibian species: gopher frogs (Rana capito) and ornate chorus frogs (Pseudacris ornata). Gopher frog survival was higher among maidencane, sedge, and pine treatments compared to oak and sweetgum treatments. While gopher frog larval periods were lowest in the sedge treatment, there was a nominal general effect of litter type on gopher frog larval periods, growth rates, and mass at metamorphosis. Shading had a nominal and inconsistent effect on gopher frog growth rates, but did extend larval periods in all litter treatments, decreased survival in all litter treatments except oak, and decreased mass at metamorphosis in all litter treatments except pine and sweetgum. Ornate chorus frog survival was minimally affected by shading and litter treatments, but growth rates and mass at metamorphosis were highest in maidencane and sedge treatments, and larval periods were extended with shading in all litter treatments. Shading also decreased growth rates in maidencane and sedge litters and decreased mass at metamorphosis in pine and sweetgum litters. Our results demonstrate that succession of isolated wetlands can reduce tadpole performance for two priority species both through changes in leaf litter and shading, though the effect on survival, larval growth, larval period, and size at metamorphosis can differ between species. These results support management recommendations to restore and maintain open canopy, grassy conditions in isolated wetlands for conservation of priority amphibian species.

Longleaf pine savannas historically supported abundant ground cover maintained by frequent fire but little other disturbance. Ground cover creates microclimates with lower temperatures, higher humidity, and increased soil moisture that may benefit wildlife, particularly small vertebrates such as amphibians. Today, most historical pine savannas have had extensive soil disturbance and altered fire regimes resulting in reduced ground cover and altered soil fauna communities including predatory invertebrates. We used a factorial terrestrial cage study to test the effects of native wiregrass (Aristida spp.) cover and the exclusion of a native predatory ant (Dorymyrmex smithi) on the survival of post‐metamorphic Ornate chorus frogs (Pseudacris ornata) and Gopher frogs (Rana capito). Although we were unable to achieve full ant exclusion, ant reduction in exclusion treatments and plant cover had an interactive effect on metamorph survival. Ant exclusion tended to increase Gopher frog survival and this effect was more pronounced when wiregrass was present. Within ant treatments, survival of Gopher frogs increased slightly with increasing wiregrass cover. Ornate chorus frogs had a high probability of survival (&gt;95%) in all ant exclusion treatments regardless of wiregrass cover; however, in treatments without ant exclusion, survival increased with increasing wiregrass cover. Our results demonstrate that high abundances of a native ant species and low coverage of native wiregrass, which are legacies of historical soil disturbance and altered fire regimes, interact to elevate mortality of juvenile amphibians. Minimizing soil disturbance and restoring native ground cover are likely important for amphibian habitat management within historical southeastern pine savannas.

A decline in sperm quality with age is a common prediction of senescence-based hypotheses and empirical studies. While widely studied across taxa, there is little known on the effect of ageing on sperm quality in amphibians, especially in captive populations used for controlled propagation and reintroduction efforts. Here, we investigated variation in sperm quality metrics (i.e., motility, concentration, and morphology) in the endangered Mississippi gopher frog (Lithobates sevosus) among males of three age categories using individuals from captive breeding populations housed at three different zoological institutions. Different aged males across the species expectant lifespan (1-9, 1-2, 3-4, and 8-9-year-old subcategories) were chosen in an attempt to identify an optimal breeding age relevant for captive breeding programs. Moreover, we explored and statistically controlled for potential differences in sperm quality which may be attributed to the type of induction hormones and source populations that differed among institutions. Results indicated that males of different ages did not differ in sperm motility or concentration. However, we did find that older males (8-9 years old) had significantly longer sperm than other age categories and younger males (1-2 years old) had significantly more atypical sperm than other age categories. Furthermore, we found no significant differences in any sperm quality metrics between the different induction hormones or source populations used at the different institutions. Within a captive breeding program, this information is especially valuable as our results indicate that males that have only recently sexually matured may not be ready to breed, while older males maintain sperm quality metrics presumably related to fertilization success.

Gopher Tortoise (Gopherus polyphemus) burrows support diverse commensal invertebrate communities that may be of special conservation interest. We investigated the impact of red imported fire ants (Solenopsis invicta) on the invertebrate burrow community at 10 study sites in southern Mississippi, sampling burrows (1998–2000) before and after bait treatments to reduce fire ant populations. We sampled invertebrates using an ant bait attractant for ants and burrow vacuums for the broader invertebrate community and calculated fire ant abundance, invertebrate abundance, species richness, and species diversity. Fire ant abundance in gopher tortoise burrows was reduced by &gt;98% in treated sites. There was a positive treatment effect on invertebrate abundance, diversity, and species richness from burrow vacuum sampling which was not observed in ant sampling from burrow baits. Management of fire ants around burrows may benefit both threatened gopher tortoises by reducing potential fire ant predation on hatchlings, as well as the diverse burrow invertebrate community. Fire-ant management may also benefit other species utilizing tortoise burrows, such as the endangered Dusky Gopher Frog and Schaus swallowtail butterfly. This has implications for more effective biodiversity conservation via targeted control of the invasive fire ant at gopher tortoise burrows.

Abstract The gopher frog Lithobates capito is one of the most terrestrial frogs in the southeastern United States and often inhabits gopher tortoise burrows Gopherus polyphemus outside of the breeding season. Gopher frog populations have declined, and the species is under review for listing as threatened or endangered under the U.S. Endangered Species Act. Much of our knowledge on the status of gopher frogs is based on detections of larvae at breeding wetlands, which can be challenging because of environmental variability and provides no information on the terrestrial life stages of the species. Therefore, an alternative method is called for. We recorded observations of gopher frogs during gopher tortoise surveys at four conservation lands in Florida and used line-transect distance sampling to estimate frog abundance. We also recorded burrow size, incidence of frog co-occupancy with tortoises, and distance from frog-occupied burrows to breeding wetlands. We observed 274 gopher frogs in 1,097 tortoise burrows at the four sites. The proportion of burrows occupied by gopher frogs among sites ranged from 0.17 to 0.25. Gopher frog abundance in tortoise burrows was 742 (512–1,076 95% CL) at Etoniah Creek State Forest, 465 (352–615) at Ft. White Wildlife Environmental Area, 411 (283–595) at Mike Roess Gold Head Branch State Park, and 134 (97–186) at Watermelon Pond Wildlife Environmental Area. We observed up to four frogs in a single burrow. The proportion of frogs detected in burrows occupied by a gopher tortoise ranged from 0.46 to 0.79 among sites, and overall, gopher frogs preferred burrows occupied by tortoises over unoccupied burrows (χ2 = 15.875; df = 3; P = 0.001). Gopher frogs used burrows from 7 to 43 cm in width; mean width of frog-occupied burrows did not differ from that of unoccupied burrows (F1,3 = 0.049, P = 0.825). Distance from frog-occupied tortoise burrows to the nearest breeding wetland ranged from 141 to 3,402 m. Our data on gopher frogs collected in conjunction with gopher tortoise monitoring efforts using line-transect distance sampling and burrow cameras provided novel information on frog abundance in their terrestrial habitat and required no additional effort. However, the extent to which frogs use tortoise burrows relative to other available refuges (small mammal burrows, stumps, or other structures) is unknown; thus, our estimates should be considered conservative. We suggest that terrestrial abundance estimates for gopher frogs can complement efforts to monitor breeding activity to provide a more comprehensive means of monitoring population trends in this cryptic species.

Conservation planning often involves multiple species occupying large areas including habitat sites with varying characteristics. For a given amount of financial resources, designing a spatially coherent nature reserve system that provides the best possible protection to targeted species is an important ecological and economic problem. In this paper, we address this problem using optimization methods. Incorporating spatial criteria in an optimization framework considering spatial habitat needs of multiple species poses serious challenges because of modeling and computational complexities. We present a novel linear integer programming model to address this issue considering spatial contiguity and compactness of the reserved area. The model uses the concept of path in graph theory to ensure contiguity and minimizes the sum of distances between selected sites and a central site in individual reserves to promote compactness. We test the computational efficiency of the model using randomly generated data sets. The results show that the model can be solved quite efficiently in most cases. We also present an empirical application of the model to simultaneous protection of two cohabiting species, Gopher Tortoise and Gopher Frogs, in a military installation in Georgia, USA.

Abstract Despite the growing roles of assisted reproduction and captive breeding in reintroductions, we have a limited understanding of the viability of these animals once released. We radiotracked endangered dusky gopher frogs Lithobates sevosus for up to 24 days after they were released into the wild to (1) assess the viability of juvenile frogs produced using artificial fertilization and raised in captivity until four weeks post‐metamorphosis, (2) investigate survival rates, movement patterns, and habitat use and (3) examine whether it is more beneficial to release frogs at the pond edge or into burrows in the uplands. Released frogs had a relatively high survival rate (76% overall, with mortality ceasing after 14 days) and behaved as expected, moving fairly long distances (up to 153 m daily and 426 m total) and locating suitable shelters, including burrows and stump holes. Frogs that sheltered underground had a 22% higher survival rate than frogs that did not, highlighting the importance of these habitat features. Compared to frogs released into burrows, frogs released at the pond had a 33% lower survival rate, spent less time underground and moved farther and more often, likely while searching for shelter. Frogs selected habitat characteristics that are associated with effects of fire, including an open canopy and abundant ground vegetation. Our study demonstrates that juvenile dusky gopher frogs successfully transitioned to their natural habitat, and that any effects of artificial fertilization or captivity on juvenile viability appear to be minimal. Releasing frogs into areas with abundant underground refuges and using frequent prescribed fire to maintain the habitat should benefit dusky gopher frog populations and improve the success of reintroductions.

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.