Leopard Frog Research Articles

Spotless burnsi pattern in Northern Leopard Frog (<i>Lithobates pipiens</i>) in Maine

We document the spotless “burnsi” morph in Northern Leopard Frog (Lithobates pipiens) in Maine.

Perfluoroalkyl Substances Increase Susceptibility of Northern Leopard Frog Tadpoles to Trematode Infection

Per/polyfluoroalkyl substances (PFAS) are contaminants of emerging concern that can impair immune function, yet few studies have tested whether exposure increases infection risk. Using laboratory experiments, we found that exposure to 10 ppb of perfluorohexanesulfonic acid increased trematode (Echinoparyphium lineage 3) infections in larval northern leopard frogs (Lithobates pipiens). However, there was no effect of perfluorooctanesulfonic acid. Our results demonstrate that PFAS can potentially enhance infection risk in natural systems. Environ Toxicol Chem 2021;40:689-694. © 2020 SETAC.

Data for: Chronic PFAS-exposure under environmentally relevant conditions delays development in northern leopard frog (Rana pipiens) larvae

Data for: Chronic PFAS-exposure under environmentally relevant conditions delays development in northern leopard frog (Rana pipiens) larvae

Exposure to metals (Ca, K, Mn) and road salt (NaCl) differentially affect development and survival in two model amphibians

ABSTRACTHuman activity has led to widespread chemical alteration of natural environments. Aquatic ecosystems are especially susceptible to chemical changes, including those caused by runoff and invasive species. Here, we examined the effects of water chemistry, specifically sodium chloride as well as three metals (Ca, K, and Mn) known to differ between native and invasive wetland plant species’ leaf tissues, on the development of two model amphibians: the native Northern leopard frog, Lithobates pipiens, and the non-native African clawed frog, Xenopus laevis. We exposed frog eggs to metal treatment solutions and measured time to hatching (TTH), and following hatching, we exposed tadpoles to a lethal concentration of sodium chloride and recorded time to death (TTD). We found that increasing metal concentrations generally resulted in acceleration of TTH for Xenopus tadpoles, but had no effect on leopard frogs. However, increasing metal concentrations (Ca, Mn) increased leopard frog tadpole susceptibility to NaCl (decreased TTD), while increasing metal concentrations (Ca, K) generally resulted in decreased Xenopus tadpole susceptibility to NaCl. Overall, our data suggest that invasive amphibians may be more tolerant to chemical changes than native amphibians, including those driven by the introduction of invasive plant species.

Informing Amphibian Conservation Efforts with Abundance-Based Metapopulation Models

Science-based management strategies are needed to halt or reverse the global decline of amphibians. In many cases, sound management requires reliable models built using monitoring data. Historically, monitoring and statistical modeling efforts have focused on estimating occupancy using detection–nondetection data. Spatial occupancy models are useful for studying colonization–extinction dynamics, but richer insights can be gained from estimating abundance and density-dependent demographic rates. We developed an integrated abundance-based metapopulation model of the processes contributing to spatiotemporal variation in patch population density. We fit our model to a combination of detection–nondetection and count data from a 14-yr study of a reintroduced metapopulation of federally threatened Chiricahua Leopard Frogs (Lithobates chiricahuensis). Pond-specific population growth rate was influenced by pond hydroperiod and frog density, such that permanent and semipermanent ponds with low densities of adult frogs experienced the highest annual population growth rates. Immigration rate declined as the distance among ponds increased. After reintroduction in 2003, metapopulation-level abundance increased and appeared to stabilize around 1300 adult frogs (95% CI = 1192–1471) by year 2015. Further, changes in metapopulation abundance were driven mostly by changes in abundance at a few ponds. These high-density populations, which would not have been identifiable with traditional occupancy-based metapopulation models, are likely especially important for species recovery in the area. Abundance-based metapopulation models can be widely applied to inform conservation efforts, by providing higher quality information needed to prioritize habitat patches for management and can be used to make more accurate predictions of metapopulation extinction risk.

CO-OCCURRENCE OF CHIRICAHUA LEOPARD FROGS (LITHOBATES CHIRICAHUENSIS) WITH SUNFISH (LEPOMIS)

Invasive species are a major threat to the persistence of native species, particularly in systems where ephemeral aquatic habitats have been replaced by permanent water and predators, such as fish, have been introduced. Within the Altar Valley, Arizona, the invasive American bullfrog, Lithobates catesbeianus (formerly Rana catesbeianus), has been successfully eradicated to help recover Chiricahua leopard frogs (Lithobates chiricahuensis). However, other nonnative predators including sunfish (Lepomis) are present in some permanent water bodies. During four consecutive years (2014–2017), we detected both the federally threatened Chiricahua leopard frog and sunfish at one permanent water body in the Altar Valley. This suggests that despite the potential negative effect of predatory fish on amphibians, there may be conditions where the Chiricahua leopard frog can co-occur with this nonnative predator. A better understanding of rare situations of co-occurrence with nonnative predators may contribute to our understanding of why co-occurrence happens in some, but not all, systems and whether conservation strategies can be developed in situations where eradication of nonnative predators is infeasible.

A statistical forecasting approach to metapopulation viability analysis.

Conservation of at-risk species is aided by reliable forecasts of the consequences of environmental change and management actions on population viability. Forecasts from conventional population viability analysis (PVA) are made using a two-step procedure in which parameters are estimated, or elicited from expert opinion, and then plugged into a stochastic population model without accounting for parameter uncertainty. Recently developed statistical PVAs differ because forecasts are made conditional on models fitted to empirical data. The statistical forecasting approach allows for uncertainty about parameters, but it has rarely been applied in metapopulation contexts where spatially explicit inference is needed about colonization and extinction dynamics and other forms of stochasticity that influence metapopulation viability. We conducted a statistical metapopulation viability analysis (MPVA) using 11yr of data on the federally threatened Chiricahua leopard frog (Lithobates chiricahuensis) to forecast responses to landscape heterogeneity, drought, environmental stochasticity, and management. We evaluated several future environmental scenarios and pond restoration options designed to reduce extinction risk. Forecasts over a 50-yr time horizon indicated that metapopulation extinction risk was <4% for all scenarios, but uncertainty was high. Without pond restoration, extinction risk is forecasted to be 3.9% (95% CI 0-37%) by year 2066. Restoring six ponds by increasing their hydroperiod reduced extinction risk to <1% and greatly reduced uncertainty (95% CI 0-2%). Our results suggest that managers can mitigate the impacts of drought and environmental stochasticity on metapopulation viability by maintaining ponds that hold water throughout the year and keeping them free of invasive predators. Our study illustrates the utility of the spatially explicit statistical forecasting approach to MPVA in conservation planning efforts.

Amphibian Skin Microbiota Response to Variable Housing Conditions and Experimental Treatment across Space and Time

Host-associated microbial ecology research is becoming a popular tool in conservation. For amphibians, there is recognition that environmental factors and anthropogenic activities can alter the composition and function of skin microbiotas. Despite growing studies on this topic, the environmental conditions and experimental methods that amphibians are exposed to vary among studies, potentially influencing our ability to develop generalizations. Using Northern Leopard Frogs (Lithobates pipiens), we investigated how the movement of individuals between housing conditions affected the amphibian skin microbial communities. In addition, we evaluated whether variation in experimental venue (e.g., culture pools, mesocosms, or laboratory) and time influenced the effect of a common antibacterial (sulfadimethoxine; SDM) on the skin microbiota. We found that the microbial community diversity decreased when tadpoles were transferred from culture pools to mesocosms and subsequently increased between mesocosms and the laboratory. When comparing the effect of SDM exposure on the skin microbiota of tadpoles across experimental venues (mesocosm and lab), there was no effect of SDM on alpha diversity. However, we noted opposing patterns between the control and SDM-treated individuals within the mesocosm and laboratory groups. In the laboratory, there were differences in the abundances of operational taxonomic units (OTU) while in the mesocosm there were differences in OTU turnover. Finally, we found that SDM treatment on amphibian microbial communities was consistent across time in the laboratory. Because researchers are integrating microbial assessments into our understanding of conservation biology, our results underscore the importance of standard housing conditions and taking into consideration that experimental design may yield variable results.

LETHAL AND SUBLETHAL AMPHIBIAN HOST RESPONSES TO BATRACHOCHYTRIUM DENDROBATIDIS EXPOSURE ARE DETERMINED BY THE ADDITIVE INFLUENCE OF HOST RESOURCE AVAILABILITY

Host species may differ in their responses to pathogen exposures based on host energy reserves, which could be important for long-term trends in host population growth. Batrachochytrium dendrobatidis (BD) is a pathogen associated with amphibian population declines but also occurs without causing mass mortalities. The impact of BD in populations without associated declines is not well understood, and food abundance could play a role in determining the magnitude of its effects. We exposed American toad (Anaxyrus americanus), northern leopard frog (Lithobates pipiens), and cricket frog (Acris blanchardi) metamorphs to BD under low or high food treatments. Overall, anuran species responded differently to BD exposure and the combined effect of BD exposure and food abundance was additive. American toad survival was lowered by BD exposure and low food availability. Based on these results, we developed a population model for American toads to estimate how reductions in survival could influence population growth. We found that BD could reduce population growth by 14% with high food availability and 21% with low food availability. In contrast, survival of northern leopard frogs was high across all treatments, but their growth was negatively impacted by the additive effects of BD exposure and low food availability. Cricket frog growth and survival were unaffected by BD exposure, suggesting that this species is not sensitive to the effects of this pathogen in terms of growth and survival across environments of different quality in the time period examined. Our results showed that low food availability additively increased the species-specific lethal and sublethal impacts of BD on hosts, which could have implications for long-term host population dynamics.

Assessment of sublethal ecotoxicity of solvents on larvae of a model native amphibian (Lithobates pipiens).

Carrier solvents are used frequently in toxicity testing to assist hydrophobic chemicals into solution, but such solvents may have toxic effects on test subjects. Amphibians are model organisms in toxicity studies; however, little is known about the direct effects of solvents on native amphibians. Following modifications to standardized guidelines for native species, we used acute 96-hour exposures to assess the direct effects of three common solvents on survival, differences in morphology and occurrence of abnormalities of northern leopard frog larvae (Lithobates pipiens). The solvents, dimethyl sulfoxide (DMSO), ethanol (ETOH) and acetone (ACE) were used at nominal concentrations ranging from 1 to 100μL/L. We also conducted a 30-day exposure to assess the direct chronic effects of DMSO at 1 and 5μL/L, on larval growth, development and sex differentiation, but found no effects. Acute exposure to solvents also had no effect on the survival of larvae, but we found significant abnormalities in tadpoles acutely exposed to 100μL/L ACE. Acute exposure to DMSO and ETOH had further concentration-dependent effects on larval morphological traits. Our study suggests that DMSO and ETOH at ≤20μL/L may be used as solvents in amphibian ecotoxicological studies, but ACE should be limited to ≤50μL/L in ecotoxicity studies and perhaps much less (≤10μL/L) in studies with other amphibians, based on a review of existing literature. We emphasize pilot studies when using solvents on acute and chronic ecotoxicity tests, using native amphibians.

The non‐consumptive effects of predators and personality on prey growth and mortality

AbstractIndividual organisms vary in personality, and the ecological consequences of that variation can affect the strength of predator–prey interactions. Prey with bolder tendencies can mitigate the strength of species interactions by altering growth and initiating ontogenetic niche shifts (ONS). While the link between personality and growth has been established, recent research has highlighted the important interplay between ONS and predator cues in community ecology. The objective of this study was to evaluate the effects of prey personality and predator cues on prey growth and ONS. We predicted growth–mortality trade‐offs among personalities with higher survival, larger size, and accelerated ONS for bold individuals in comparison with shy individuals. To evaluate this objective, we conducted behavioral assays and a mesocosm experiment to test how southern leopard frog (Rana sphenocephala) tadpole personality and predatory fish (bluegill, Lepomis macrochirus) cues affects tadpole growth and metamorphosis. On average, bold tadpoles had higher mortality across all treatments in comparison with shy tadpoles. The effects of fish cues were dependent on tadpole personality with shy tadpoles metamorphosing significantly later than bold tadpoles. Bold tadpoles were larger than shy tadpoles at metamorphosis; however, that pattern reversed with fish cues as shy individuals metamorphosed larger than bold individuals. Our results suggest personality may be useful for predicting growth and life history for some prey species with predators. Specifically, the threat of predation can interact with personality to incur a benefit (earlier ONS) while also incurring a cost (size at metamorphosis). Hence by incorporating predator cues with personality, ecologists will be able to elucidate growth–mortality trade‐offs mediated by personality.

Population-Level Resistance to Chytridiomycosis is Life-Stage Dependent in an Imperiled Anuran.

Amphibian declines caused by chytridiomycosis have been severe, but some susceptible populations have persisted or even recovered. Resistance to the causal agent Batrachochytrium dendrobatidis (Bd) could result from alleles of the adaptive immune system. During metamorphosis, however, immune systems may not be fully functional, implying that an effective immune response to Bd may be life-stage dependent. We evaluated the susceptibility of the relict leopard frog (Rana onca) sourced from two areas where Bd was present or absent, and where the populations appeared to show differences in pathogen resistance. We evaluated whether population-level resistance manifested across life stages using challenge experiments with late-stage tadpoles (Gosner stage 31-38), metamorphs (stage 45-46), and juvenile frogs. We used three different Bd isolates including one from wild R. onca to challenge juvenile frogs and focused on the isolate from R. onca to challenge tadpoles and resulting metamorphs. We found that juveniles from the Bd exposed population were 5.5 times more likely to survive Bd infection and 10 times more likely to clear infections than those from the area without Bd. In contrast, and regardless of the source area, we observed 98% survivorship of tadpoles, but only 19% survivorship of resulting metamorphs following re-exposure. Given the low survivorship of exposed metamorphs in the laboratory, we speculate on how resistance characteristics, whether adaptive or innate, that do not manifest at each life stage could develop in the wild. We suggest that seasonal high temperatures during times when metamorphosis appears common may modulate the effects of the pathogen during this most susceptible life stage.

Amphibian Monitoring in Hardwood Forests: Optimizing Methods for Contaminant-Based Compensatory Restorations.

Amphibians such as frogs, toads, and salamanders provide important services in aquatic and terrestrial ecosystems and have been proposed as useful indicators of progress and success for ecological restoration projects. Limited guidance is available, however, on the costs and benefits of different amphibian monitoring techniques that might be applied to sites restored in compensation for contaminant injury. We used a variety of methods to document the amphibian communities present at 4 restored bottomland hardwood sites in Indiana, USA, and to compare the information return and cost of each method. For 1 method-automated recording units-we also modeled the effect of varying levels of sampling effort on the number of species detected, using sample-based rarefaction and Bayesian nonlinear (Michaelis-Menten) mixed effects models. We detected 13 amphibian species across the restored sites, including 2 species of conservation concern in Indiana-northern leopard frogs (Lithobates pipiens) and Blanchard's cricket frogs (Acris blanchardi). Sites across a range of restoration ages demonstrated encouraging returns of amphibian communities. Although more mature sites showed greater species richness, recently restored sites still provided important habitat for amphibians, including species of conservation concern. Among the 4 methods compared, amphibian rapid assessment yielded the highest number of species detected and the greatest catch per unit effort, with the lowest per-site cost. Our analysis of level-of-effort effects in the rarefied acoustic data found that number of nights sampled was a better predictor of observed species richness than the number of hours sampled within a night or minutes sampled within an hour. These data will assist restoration practitioners in selecting amphibian monitoring methods appropriate for their site characteristics and budget. Integr Environ Assess Manag 2019;00:1-15. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Parasite-induced vulnerability to predation in larval anurans.

Within communities, pathogens and parasites have the potential to indirectly influence predator-prey interactions. For instance, prey that exhibit pathology or altered traits (e.g. behavioral shifts) following infection could be more prone to predation, which is known as parasite-induced vulnerability to predation (PIVP). PIVP has been frequently documented for pathogens with trophic transmission, because predators are often critical in the pathogen's life cycle. However, for pathogens without trophic transmission, PIVP can lead to a healthy herds effect, thereby reducing transmission in the system. In this study, we explored whether the pathogen ranavirus (family Iridoviridae) enhances vulnerability of 4 species of larval amphibians (spring peepers Pseudacris crucifer, gray treefrogs Hyla versicolor, American toads Anaxyrus americanus, and northern leopard frogs Lithobates pipiens) to 2 common tadpole predators (larval green darners Anax junius [hereinafter Anax] and adult water bugs Belostoma flumineum [hereinafter Belostoma]). For each anuran species, we conducted short-term microcosm experiments to assess predation rates on individuals that were or were not exposed to virus. For 3 of the 4 species, we found that exposure to ranavirus decreased survival rates with Anax between 2- and 9-fold. However, we did not see the same trend with Belostoma, which indicates that predator identity is important in this interaction. More specifically, the higher efficiency of Anax in capturing and consuming prey, relative to Belostoma, may allow Anax to capitalize on trait changes induced by virus exposure and enhance the PIVP effect. Our results indicate that trait-mediated indirect effects could play a role in creating healthy herds in amphibian communities.

Effects of the Neonicotinoid Insecticide Clothianidin on Southern Leopard Frog (Rana sphenocephala) Tadpole Behavior.

Neonicotinoid insecticides are highly water soluble with relatively long half-lives, which allows them to move into and persist in aquatic ecosystems. However, little is known of the impacts of neonicotinoids on non-target vertebrates, especially at sublethal concentrations. We evaluated the effects of the neonicotinoid clothianidin on the behavior of southern leopard frog tadpoles (Rana sphenocephala) after a 96-h exposure at 6 concentrations, including 0 (control), 0.375, 0.75, 1.5, 3.0, 6.0µg/L. We quantified total displacement, mean velocity, maximum velocity, and time spent moving of tadpoles for 1h post-exposure. Total displacement and mean velocity of tadpoles decreased with clothianidin exposure. Maximum velocity decreased linearly with concentration, but there was no relationship between time spent moving and clothianidin concentration. Our results suggest exposure to clothianidin at sublethal concentrations can affect movementbehavior of non-target organisms such as tadpoles.

Assessment of Sublethal Effects of Neonicotinoid Insecticides on the Life-History Traits of 2 Frog Species.

Neonicotinoid insecticides are used extensively in agriculture and, as a consequence, are now detectable in nearby aquatic environments. Few studies have evaluated the effects of neonicotinoids on amphibians in these aquatic environments. In the present study, we examined the effects of 2 commercial formulations of neonicotinoids (active ingredients clothianidin and thiamethoxam) on survival and life‐history traits of wood frogs (Lithobates sylvaticus) and northern leopard frogs (Lithobates pipiens). We used artificial pond mesocosms to assess the effects of these neonicotinoids, at nominal concentrations of 2.5 and 250 µg/L, on amphibian larval development through metamorphosis. We found no differences between controls and neonicotinoid exposure for any of the endpoints assessed for either wood frogs or leopard frogs. The present study suggests that concentrations meeting or exceeding observed levels of clothianidin and thiamethoxam in surface waters will not directly affect metamorphosis in 2 amphibians. Environ Toxicol Chem 2019;38:1967–1977. © 2019 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Sub-chronic exposure to a neonicotinoid does not affect susceptibility of larval leopard frogs to infection by trematode parasites, via either depressed cercarial performance or host immunity.

Little information is available on the effects of neonicotinoid insecticides on vertebrates. Previous work using amphibians found chronic exposure to some neonicotinoids had no detrimental effects on fitness-relevant traits. However, there is some evidence of more subtle effects of neonicotinoids on immune traits and evidence that other pesticides can suppress tadpole immunity resulting in elevated levels of parasitism in the exposed tadpoles. The objective of our study was to assess whether neonicotinoid exposure affected tadpole immunometrics and susceptibility to parasitic helminths. We assessed northern leopard frog tadpole (Lithobates pipiens) levels of parasitism and leukocyte profiles following exposure to environmentally relevant concentrations of clothianidin and free-living infective cercariae of a helminth parasite, an Echinostoma sp. trematode. When comparing tadpoles from controls to either 1 or 100μg/L clothianidin treatments, we found similar measures of parasitism (i.e. prevalence, abundance and intensity of echinostome cysts) and similar leukocyte profiles. We also confirmed that clothianidin was not lethalfor cercariae; however, slight reductions in swimming activity were detected at the lowest exposure concentration of 0.23μg/L. Our results show that exposure to clothianidin during the larval amphibian stage does not affect leukocyte profiles or susceptibility to parasitism by larval trematodes in northern leopard frogs although other aspects such as length of host exposure require further study.

Identification of leopard frogs (Anura: Ranidae: Lithobates) distributed in some localities of the Southern Mexican Plateau using mitochondrial DNA sequences

Species of the genus Lithobates are difficult to identify, especially on the ‘Rana pipiens’ complex, because the morphological differences among the species are often subtle. In this study, we used 12S ribosomal RNA gene partial sequences to identify specimens of leopard frogs from some new localities (not sampled on previous studies) of the Southern Mexican Plateau, to carry out a phylogenetic analysis, and also a morphometric analysis of some morphological features were analyzed to evaluate their morphological variation. A phylogenetic analysis using partial sequences of 12S rDNA mitochondrial gene for 31 samples was carried out using Bayesian Inference, Maximum Parsimony, and Maximum Likelihood. In addition, 20 morphological lineal measurements from 97 specimens were analyzed by principal component analysis (PCA) to evaluate if the frogs studied are conspecific. Partial sequences of the 12S rDNA obtained from frogs distributed in the Southern Mexican Plateau show two haplotypes with a divergence of 0.4%. Phylogenetic hypothesis shows an exclusive group with a previous sequence of Lithobates montezumae. The PCA indicates that variables are not linearly correlated and specimens belong to a single group. Evidence found, let us consider that specimens studied belong to one species of the Lithobates montezumae subgroup, distributed principally in the Southern Mexican Plateau.

Healthy but smaller herds: Predators reduce pathogen transmission in an amphibian assemblage.

Predators and pathogens are fundamental components of ecological communities that have the potential to influence each other via their interactions with victims and to initiate density- and trait-mediated effects, including trophic cascades. Despite this, experimental tests of the healthy herds hypothesis, wherein predators influence pathogen transmission, are rare. Moreover, no studies have separated effects mediated by density vs. traits. Using a semi-natural mesocosm experiment, we investigated the interactive effects of predatory dragonfly larvae (caged or lethal [free-ranging]) and a viral pathogen, ranavirus, on larval amphibians (grey treefrogs and northern leopard frogs). We determined the influence of predators on ranavirus transmission and the relative importance of density- and trait-mediated effects on observed patterns. Lethal predators reduced ranavirus infection prevalence by 57%-83% compared to no-predator and caged-predator treatments. The healthy herds effect was more strongly associated with reductions in tadpole density than behavioural responses to predators. We also assessed whether ranavirus altered the responses of tadpoles to predators. In the absence of virus, tadpoles reduced activity levels and developed deeper tails in the presence of predators. However, there was no evidence that virus presence or infection altered responses to predators. Finally, we compared the magnitude of trophic cascades initiated by individual and combined natural enemies. Lethal predators initiated a trophic cascade by reducing tadpole density, but caged predators and ranavirus did not. The absence of a virus-induced trophic cascade is ostensibly the consequence of limited virus-induced mortality and the ability of infected individuals to continue interacting within the community. Our results provide support for the healthy herds hypothesis in amphibian communities. We uniquely demonstrate that density-mediated effects of predators outweigh trait-mediated effects in driving this pattern. Moreover, this study was one of the first to directly compare trophic cascades caused by predators and pathogens. Our results underscore the importance of examining the interactions between predators and pathogens in ecology.

SEASONALITY IN BATRACHOCHYTRIUM DENDROBATIDIS DETECTION IN AMPHIBIANS IN CENTRAL OKLAHOMA, USA.

Chytridiomycosis, an infectious disease caused by the fungus Batrachochytrium dendrobatidis (chytrid or Bd), has not been well studied in Oklahoma. This is of particular concern regarding the connection between seasonality and chytrid infection. To further investigate this connection, chytrid prevalence and infection load were quantified within amphibians in central Oklahoma from March to October, across two sites in Oklahoma Co. and two sites in Cleveland Co. The results show a trend between seasonality and chytrid, with spring and fall showing higher prevalence and summer showing lower prevalence, which coincides closely with the preferred chytrid growth temperatures. Additionally, periods of high rainfall in May 2015 are linked to increased chytrid prevalence, as has been suggested by other research. Additionally, species exhibiting high chytrid prevalence follow the results of previous studies: Blanchard's cricket frog (Acris blanchardi), American bullfrog (Rana catesbeiana), and southern leopard frog (Rana sphenocephala).