Dusky Gopher Frog Research Articles

Assessing factors that increase dusky gopher frog larval performance in open‐canopy wetlands

AbstractAmphibians 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.

Sperm Cryopreservation as a Tool for Amphibian Conservation: Production of F2 Generation Offspring from Cryo-Produced F1 Progeny

Sperm cryopreservation and biobanking are emerging as tools for supporting genetic management of small and threatened populations in amphibian conservation programs. However, there is little to no evidence demonstrating reproductive maturity and viability of offspring generated with cryopreserved sperm, potentially limiting widespread integration of these technologies. The purpose of this report is to demonstrate that amphibian sperm can be cryopreserved and thawed to successfully produce individuals of an F1 generation that can reach adulthood and reproductive maturity, to generating viable gametes and an F2 generation. Species-specific exogenous hormones were administered to both F0 and F1 adults to stimulate spermiation and oviposition in the eastern tiger salamander (Ambystoma tigrinum), dusky gopher frog (Lithobates sevosa), and Puerto Rican crested toad (Peltophryne lemur). Sperm cells collected non-lethally from F0 adults were cryopreserved, thawed, and used for in vitro fertilization (IVF) to produce F1 offspring. Individuals of the F1 generation are shown to reach adulthood, express viable gametes, and produce offspring through facilitated breeding, or IVF. The production of amphibian F2 generations shown here demonstrates that amphibian sperm collected non-lethally can be banked and used to generate reproductively viable animals of subsequent generations, thus maintaining valuable genetic linages and diversity in threatened amphibian species. The incredible value that cryopreservation of sperm has for long-term genetic management aids in the sustainability of both in situ and ex situ conservation efforts for this taxon.

Comment: Novak et al. (2021) overestimated the successes of species translocations and minimized their risks

Comment: Novak et al. (2021) overestimated the successes of species translocations and minimized their risks

First natural breeding of the endangered dusky gopher frog (Lithobates sevosus) in captivity.

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.

Effects of age on sperm quality metrics in endangered Mississippi gopher frogs (Lithobates sevosus) from captive populations used for controlled propagation and reintroduction efforts.

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.

Red Imported Fire Ants Reduce Invertebrate Abundance, Richness, and Diversity in Gopher Tortoise Burrows

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 >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.

Survival, movements and habitat use of captive‐bred and reintroduced dusky gopher frogs

AbstractDespite 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.

Inbreeding depression in sperm quality in a critically endangered amphibian.

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.

Time from injection of luteinizing hormone-releasing hormone analog affects sperm quality in the critically endangered Mississippi gopher frog (Lithobates sevosus).

The objective of this study was to investigate variation in sperm quality metrics (motility, velocity, and concentration) in the critically endangered Mississippi gopher frog (Lithobates sevosus) over three sampling time points after a luteinizing hormone-releasing hormone analog (LHRHa) induction injection. Sperm was repeatedly collected from 11 individuals over three sampling times (30, 60, and 120 min) after injection. Variation in sperm quality was investigated using a repeated-measures mixed model approach. Repeated measures analyses of variance showed a significant effect of sampling time for percent motility and velocity. Concentration was found to be marginally related to sampling time, while progressive motility was not significantly related to time after injection. Our findings are important for optimizing assisted reproduction-related fertilization success and increasing the successful propagation of endangered species of imperiled frogs in captive breeding programs.

Ultrasound imaging improves hormone therapy strategies for induction of ovulation and in vitro fertilization in the endangered dusky gopher frog (Lithobates sevosa).

Establishing captive breeding populations of amphibians is an important conservation strategy to safeguard against ongoing declines of wild populations and provide broodstock for reintroduction programs. The endangered dusky gopher frog (DGF) has never naturally reproduced in captivity and requires breeding intervention to sustain the population. Methods for inducing ovulation in female DGFs using hormone therapies have not been evaluated. To address this need, we tested four exogenous hormone treatments to induce ovulation in DGFs (n = 11/treatment), including: treatment (A) gonadotropin-releasing hormone agonist (GnRHa); (B) GnRHa with dopamine antagonist metoclopramide hydrochloride; (C) GnRHa and human chorionic gonadotropin (hCG) and (D) GnRHa with hCG following two low hCG priming doses. Treatments B, C and D resulted in a significantly greater (P < 0.0125) number of ovulating females compared to the control (no hormone); Treatment A was not different from control. For ovulating females, the number of eggs, relative fecundity and cleavage rates of eggs were compared between the four hormone treatments and initial ultrasound grade. Between treatments, there was no difference in number of eggs or relative fecundity; however, Treatments A and D resulted in higher (P < 0.05) cleavage rates than Treatment C, but were not different from Treatment B. Ultrasound imaging was used to assess the ovarian state of DGF females prior to and following hormone therapy. A grading scale (Grades 1-5) was developed to characterize ovarian states. Ultrasound grade was found to be a significant (P = 0.002) predictor for ovulation following hormone treatment, with only high-grade females (Grades 3-4) ovulating in response to hormones. Ultrasound grade did not influence egg numbers or cleavage rate (P > 0.05). Results demonstrate multiple hormone therapies are available for stimulating ovulation in female DGFs and ultrasonography is a valuable tool to inform hormone therapy. Ultimately, these reproductive technologies are critical to enhance breeding and reintroduction efforts for the DGF.

Initial founders of captive populations are genetically representative of natural populations in critically endangered dusky gopher frogs, Lithobates sevosus.

The rapid rate of decline in amphibian populations has urged many researchers and conservationists to establish captive, or ex situ, populations. Such populations are guarded against effects of habitat loss and degradation, and if actively managed, can serve as a reservoir for rare alleles that might be lost in the wild. Without proper management, ex situ population sizes can dwindle and will no longer perform this function. The dusky gopher frog, Lithobates sevosus, is a critically endangered species, imperiled by habitat loss and population isolation. To assist in recovery of the species and prevent further genetic erosion, a captive breeding program was initiated. We investigated how well natural genetic variation was captured within the ex situ population and determined relatedness within each ex situ population. We genotyped individuals from two natural populations and two founding, captive populations to compare metrics of genetic variation and relatedness. The data show the initial founder populations are genetically representative of the natural populations, although variation is low in each, and that relatedness values are similar. Therefore, founding captive populations were successful at capturing genetic variation in the wild. Future research should continue to compare genetic variation of captive and natural populations to monitor efficacy of their management programs. Zoo Biol. 35:378-384, 2016. © 2016 Wiley Periodicals, Inc.

Ranavirus could facilitate local extinction of rare amphibian species.

There is growing evidence that pathogens play a role in population declines and species extinctions. For small populations, disease-induced extinction may be especially probable. We estimated the susceptibility of two amphibian species of conservation concern (the dusky gopher frog [Lithobates sevosus] and boreal toad [Anaxyrus boreas boreas]) to an emerging pathogen (ranavirus) using laboratory challenge experiments, and combined these data with published demographic parameter estimates to simulate the potential effects of ranavirus exposure on extinction risk. We included effects of life stage during pathogen exposure, pathogen exposure interval, hydroperiod of breeding habitat, population carrying capacity, and immigration in simulations. We found that both species were highly susceptible to ranavirus when exposed to the pathogen in water at environmentally relevant concentrations. Dusky gopher frogs experienced 100% mortality in four of six life stages tested. Boreal toads experienced 100% mortality when exposed as tadpoles or metamorphs, which were the only life stages tested. Simulations showed population declines, greater extinction probability, and faster times to extinction with ranavirus exposure. These effects were more evident with more frequent pathogen exposure intervals and lower carrying capacity. Immigration at natural rates did little to mitigate effects of ranavirus exposure unless immigration occurred every 2years. Our results demonstrate that disease-induced extinction by emerging pathogens, such as ranavirus, is possible, and that threat may be especially high for species with small population sizes. For the species in this study, conservation organizations should incorporate ranavirus surveillance into monitoring programs and devise intervention strategies in the event that disease outbreaks occur.

Temporal trends in genetic data and effective population size support efficacy of management practices in critically endangered dusky gopher frogs (Lithobates sevosus).

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.

In vivo efficacy of anuran trypsin inhibitory peptides against staphylococcal skin infection and the impact of peptide cyclization.

Staphylococcus aureus is a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weak in vitro inhibitory activities against S. aureus, but several had strong antibacterial activities against S. aureus in an in vivo murine wound infection model. pYR, an immunomodulatory peptide from Rana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg(-1). Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

High susceptibility of the endangered dusky gopher frog to ranavirus.

Amphibians are one of the most imperiled vertebrate groups, with pathogens playing a role in the decline of some species. Rare species are particularly vulnerable to extinction because populations are often isolated and exist at low abundance. The potential impact of pathogens on rare amphibian species has seldom been investigated. The dusky gopher frog Lithobates sevosus is one of the most endangered amphibian species in North America, with 100-200 individuals remaining in the wild. Our goal was to determine whether adult L. sevosus were susceptible to ranavirus, a pathogen responsible for amphibian die-offs worldwide. We tested the relative susceptibility of adult L. sevosus to ranavirus (103 plaque-forming units) isolated from a morbid bullfrog via 3 routes of exposure: intra-coelomic (IC) injection, oral (OR) inoculation, and water bath (WB) exposure. We observed 100% mortality of adult L. sevosus in the IC and WB treatments after 10 and 19 d, respectively. Ninety-five percent mortality occurred in the OR treatment over the 28 d evaluation period. No mortality was observed in the control treatment after 28 d. Our results indicate that L. sevosus is susceptible to ranavirus, and if adults in the wild are exposed to this pathogen, significant mortality could occur. Additionally, our study demonstrates that some adult amphibian species can be very susceptible to ranavirus, which has been often overlooked in North American studies. We recommend that conservation planners consider testing the susceptibility of rare amphibian species to ranavirus and that the adult age class is included in future challenge experiments.

Survival to metamorphosis is positively related to genetic variability in a critically endangered amphibian species

AbstractIsolated small populations typically have reduced genetic variation and are more susceptible to local extinction. Examining the relationship between genetic variability and survival is important for conservation because it provides insight on evolutionary potential and population viability. We studied genetic associations of survival from egg to metamorphosis in the largest population of critically endangered dusky gopher frogs Lithobates sevosus. This approach allowed us to examine whether heterozygosity‐fitness correlations (HFCs) existed in an r‐selected species with large effect size, whereas previous research on HFCs has largely focused on k‐selected species often with low effect sizes. Our objectives were to determine if genetic‐fitness associations exist, if associations are a result of inbreeding and if natural selection against inbreeding has occurred. Specifically, we examined averaged population responses and HFC in relation to (1) mortality within egg clutches and (2) differential survival of offspring through metamorphosis. To do so, we used eight microsatellite DNA markers to compare genotypes across three life‐history stages from a single year in the following temporal order: adults, eggs and metamorphs. We discovered a strong genetic association with survival in early life stages. In terms of average population responses, we documented a within‐cohort decrease in FIS with life‐history stage progression (adults: FIS = 0.067; eggs: FIS = 0.063; metamorphs: FIS = −0.134), high FST between eggs and metamorphs (FST = 0.113), and a locus that had larger than expected FST. We found positive genetic‐fitness associations for survival of egg clutches and for survival to metamorphosis. We believe that inbreeding is the primary cause; however, we also found evidence suggesting existence of purging selection against a deleterious mutant linked to a microsatellite locus. Survival of individuals with greater genetic variability should prolong persistence of this isolated population. Our study underscores the importance of species management focused on preservation of genetic diversity.

120 THE DEVELOPMENTAL COMPETENCE OF TADPOLES PRODUCED IN VITRO FROM THE ENDANGERED DUSKY GOPHER FROG (RANA SEVOSA) USING EXOGENOUS HORMONE TREATMENT

Dusky gopher frogs once existed throughout the states of Mississippi, Alabama and Louisiana. Presently, the USA Fish and Wildlife Service estimates that there are less than 100 frogs left in the wild, with almost all of these residing in a single pond in Mississippi, making the dusky gopher frog America's most endangered frog species. Their habitat has been threatened by residential and forestry development, as well as from fire suppression and the decline of gopher tortoises, whose burrows the frogs use for shelter. The USA Fish and Wildlife Service brought the first dusky gopher frogs into captivity in 2001 and they have been kept at the Memphis Zoo since 2003 and at Omaha's Henry Doorly Zoo since 2004. Natural breeding attempts in captivity have been unsuccessful despite efforts to artificially mimic what is known to be the appropriate seasonal environmental (e.g. light, humidity, temperature) and social cues (e.g. calls from wild frogs during the breeding season) that stimulate reproduction in situ. Also, there was a concern about the incidences of abnormalities (e.g. spindly legs) from inadequate husbandry conditions (e.g. nutritional deficiencies) or diseases in tadpoles and froglets produced ex situ (e.g. dermomycoides). The objectives of this study were (1) to examine the developmental competence of tadpoles produced by exogenous hormone treatment for IVF to complete normal metamorphosis into viable froglets ex situ and (2) to determine the incidence of developmental abnormalities or dermomycoides in the froglets produced ex situ. Males and females were first separated by the identification of black male nuptial pads used during amplexus. Males and females were stimulated to spermiate and ovulate, respectively, using a variation of a standard amphibian hormonal stimulation protocol (Kouba et al. 2012 Reprod. Fertil. Dev. 24). As a result, more than 1460 viable tadpoles (61% of fertilized eggs) were produced from 18 segregated genetic pairings. To date, 602 (41%) of the tadpoles have completed normal metamorphosis into viable froglets (over 6 times the known existing population in situ), 51% were either culled to examine for dermomycoides or died for a variety of known (accidental) and unknown reasons (abnormal appendage development accounting for less than 7%) and 8% have yet to metamorphose after 9 months. The incidence of dermomycoides in the culled individuals was 100%; however, a study being conducted concurrently in their native habitat has confirmed that the organism does exist in situ with the dusky gopher frogs, which act as natural carriers. Efforts are currently in place to establish reintroduction programs for the dusky gopher froglets produced ex situ to increase the dwindling wild population now that tadpoles produced by IVF have been shown to be developmentally competent. In conclusion, IVF can be used to produce tadpoles of the dusky gopher frog with a low incidence of abnormalities, but the tadpoles were found to be carriers of the organism dermomycoides similar to their counterparts in the wild.

Comparative Morphometrics in Ranid Frogs (Subgenus Nenirana): Are Apomorphic Elongation and a Blunt Snout Responses to Small-bore Burrow Dwelling in Crawfish Frogs (Lithobates areolatus)?

The subgenus Nenirana of North American ranid frogs encompasses Pickerel Frogs (Lithobates palustris), Crawfish Frogs (L. areolatus), Gopher Frogs (L. capito), and Dusky Gopher Frogs (L. sevosus). All four species inhabit caves, crevices, stump holes, and/or burrows when not in breeding wetlands. Crawfish Frogs obligately inhabit crayfish burrows as their primary retreat sites, and in this study we examine whether the deep, small-bore crayfish burrows used by Crawfish Frogs have influenced Crawfish Frog morphology. Specimens of all four species of Nenirana were radiographed and snout–urostyle length, maximum headwidth, head length, femur length, and tibiofibula length were measured from films. Our results suggest that if Crawfish Frog morphology is a response to life in burrows, it is due in part to having the size characteristic of being the largest member of the clade and in part through the shape characteristic of generally exhibiting an intermediate morphology between Pickerel Frogs and the two Gopher Frog species. Not all shape metrics, however, are intermediate; among Nenirana, Crawfish Frogs have the longest hindlimbs and the relatively bluntest snouts. Further, Crawfish Frogs exhibit positive allometry in headwidth, a reversal of the ancestral pattern exhibited by Pickerel Frogs. None of the morphological features of Crawfish Frogs fit neatly into known or predicted functional/morphological cause-and-effect relationships associated with burrow occupancy. It may be that the ranid body plan is generalized enough to permit Crawfish Frogs to inhabit, despite being unable to dig, deep small-bore burrows without undergoing major morphological changes.

Genetic Consequences of Population Reduction and Geographic Isolation in the Critically Endangered Frog, Rana sevosa

Abstract Anthropogenic habitat fragmentation and reduction are major causes of population declines and extinction. As these processes intensify, our ability to rescue imperiled taxa is critically dependent on an understanding of historical, demographic, and genetic parameters of diminishing populations. We assessed the effects of recent geographic isolation and population reduction on genetic variability for endangered Dusky Gopher Frogs, Rana sevosa. Only two populations of R. sevosa exist, each is geographically isolated and restricted to a single breeding pond, and one of them may have gone locally extinct. Therefore, we studied the largest and perhaps only population of R. sevosa. The only option for comparison to non-isolated populations was of its ecologically similar sister species (other Gopher Frogs, R. capito) and of the sister species to R. sevosa and R. capito (Crawfish Frogs, R. areolata). Variation in seven microsatellite DNA loci was assessed for each population to determine the effects of ...

196 NONINVASIVE HORMONE MEASUREMENTS FOR GENDER IDENTIFICATION IN MONOMORPHIC ANURANS

Noninvasive techniques have been successful in distinguishing gender in dimorphic amphibian models (Szymanski DC et al. 2006 Zoo Biol. 25, 35-46). The aim of this study was to assess gender-specific fecal hormone metabolite concentrations in 2 dimorphic and 2 monomorphic, critically endangered species of amphibians to assist in ex situ breeding efforts. Fecal samples from 2 dimorphic anurans, the Puerto Rican Crested Toad (Bufo lemur) and the Wyoming Toad (Bufo baxteri) and 2 monomorphic species, the Mountain Chicken Frog (Leptodactylus fallax) and the Dusky Gopher Frog (Lithobates sevosa) were measured by enzyme immunoassay for sex-specific hormone differences (Student’s t-test; P &lt; 0.05) among 6 variables (concentrations of estradiol (E2), progesterone (P), and testosterone (T), and ratios of P:E2, T:E2, and P:T). Values are presented as means ± SEM. Mountain Chicken Frog samples were obtained from individuals of known sex. Dusky Gopher Frog samples were from confirmed females but males were presumed based on subtle physical characteristics. The E2 concentrations (ng g-1) were significantly higher in females than males (P &lt; 0.05) in all species except for the Dusky Gopher Frog. However, males in all 4 species, including the presumptive Dusky Gopher Frog males, exhibited significantly higher P:E2 and T:E2 ratios than females. Additionally, Wyoming Toad and Mountain Chicken Frog females had significantly higher P:T ratios than males (0.19 ± 0.05 v. 0.06 ± 0.00 and 0.34 ± 0.03 v. 0.20 ± 0.02, respectively). Similar to the American Toad (Szymanski et al. 2006), presumptive Dusky Gopher Frog males measured significantly higher T concentrations compared with females (376.4 ± 118.5 ng g-1 v. 174.0 ± 23.1 ngg-1, respectively). Although species-specific differences are apparent, fecal hormone measurements can be used to identify sex in monomorphic species and offer a potential tool for the captive breeding of endangered anurans. Table 1.