Hyla Versicolor Research Articles

Parallel changes in mate-attracting calls and female preferences in autotriploid tree frogs.

For polyploid species to persist, they must be reproductively isolated from their diploid parental species, which coexist at the same time and place at least initially. In a complex of biparentally reproducing tetraploid and diploid tree frogs in North America, selective phonotaxis--mediated by differences in the pulse-repetition (pulse rate) of their mate-attracting vocalizations--ensures assortative mating. We show that artificially produced autotriploid females of the diploid species (Hyla chrysoscelis) show a shift in pulse-rate preference in the direction of the pulse rate produced by males of the tetraploid species (Hyla versicolor). The estimated preference function is centred near the mean pulse rate of the calls of artificially produced male autotriploids. Such a parallel shift, which is caused by polyploidy per se and whose magnitude is expected to be greater in autotetraploids, may have facilitated sympatric speciation by promoting reproductive isolation of the initially formed polyploids from their diploid parental forms. This process also helps to explain why tetraploid lineages with different origins have similar advertisement calls and freely interbreed.

The role of body size on the outcome, escalation and duration of contests in the grey treefrog, Hyla versicolor

Aggressive interactions in animals are often resolved in favour of the individual with superior fighting ability, or resource-holding potential (RHP). A recent revival of studies of aggressive behaviour has focused on the assessment strategies used in animal contests. Strategies of dispute resolution through mutual or self-assessment of RHP differ in the predicted relationship between interaction duration and each competitor’s relative and absolute RHP. We studied potential components of RHP (mass, length, body condition) and their relationship to contest duration and the level of escalation in the grey treefrog, Hyla versicolor, using a novel method to stage aggressive interactions in the laboratory. Overall, large males were more likely to win than small males, but they only had an advantage in less escalated interactions and were not more successful in physical fights. There was limited evidence for an effect of body size on interaction duration or the level of escalation. Specifically, the body condition of both the smaller and larger contestant was weakly negatively related to the duration and level of escalation of contests. This relationship is the opposite of what would be expected under any assessment strategy. Given these data, coupled with the lack of relationships between other size measures and interaction duration, we conclude that assessment of body size does not occur in contests in H. versicolor. Other unmeasured components of RHP may play a role in determining interaction duration, and the relatively weak and ineffective fighting abilities of this species may limit the dominance of larger individuals. Relatively little is known about aggressive behaviour in frogs. Our method for staging aggressive interactions allows us to address predictions of game theory models in an important group for studies of animal communication.

Dip listening and the cocktail party problem in grey treefrogs: signal recognition in temporally fluctuating noise

Dip listening refers to our ability to catch brief "acoustic glimpses" of speech and other sounds when fluctuating background noise levels momentarily decrease. Exploiting dips in natural fluctuations of noise contributes to our ability to overcome the "cocktail party problem" of understanding speech in multi-talker social environments. We presently know little about how nonhuman animals solve analogous communication problems. Here, we asked whether female grey treefrogs (Hyla chrysoscelis) might benefit from dip listening in selecting a mate in the noisy social setting of a breeding chorus. Consistent with a dip listening hypothesis, subjects recognized conspecific calls at lower thresholds when the dips in a chorus-like noise masker were long enough to allow glimpses of nine or more consecutive pulses. No benefits of dip listening were observed when dips were shorter and included five or fewer pulses. Recognition thresholds were higher when the noise fluctuated at a rate similar to the pulse rate of the call. In a second experiment, advertisement calls comprising six to nine pulses were necessary to elicit responses under quiet conditions. Together, these results suggest that in frogs, the benefits of dip listening are constrained by neural mechanisms underlying temporal pattern recognition. These constraints have important implications for the evolution of male signalling strategies in noisy social environments.

Can Differences in Host Behavior Drive Patterns of Disease Prevalence in Tadpoles?

Differences in host behavior and resistance to disease can influence the outcome of host-pathogen interactions. We capitalized on the variation in aggregation behavior of Fowler's toads (Anaxyrus [ = Bufo] fowleri) and grey treefrogs (Hyla versicolor) tadpoles and tested for differences in transmission of Batrachochytrium dendrobatidis (Bd) and host-specific fitness consequences (i.e., life history traits that imply fitness) of infection in single-species amphibian mesocosms. On average, A. fowleri mesocosms supported higher Bd prevalences and infection intensities relative to H. versicolor mesocosms. Higher Bd prevalence in A. fowleri mesocosms may result, in part, from higher intraspecific transmission due to the aggregation of tadpoles raised in Bd treatments. We also found that, independent of species, tadpoles raised in the presence of Bd were smaller and less developed than tadpoles raised in disease-free conditions. Our results indicate that aggregation behavior might increase Bd prevalence and that A. fowleri tadpoles carry heavier infections relative to H. versicolor tadpoles. However, our results demonstrate that Bd appears to negatively impact larval growth and developmental rates of A. fowleri and H. versicolor similarly, even in the absence of high Bd prevalence.

Sperm depletion and a test of the phenotype-linked fertility hypothesis in Gray Treefrogs (Hyla versicolor)

The phenotype-linked fertility hypothesis (PLFH) predicts that ejaculate characteristics correlate with male phenotype, and that females may select for exaggerated phenotypes to guarantee high fertilization success. Female Gray Treefrogs ( Hyla versicolor LeConte, 1825) prefer males with long-duration calls and (or) calls produced at a fast rate. I tested the prediction that call duration or call rate were indicators of sperm number or viability. To assess whether female H. versicolor might benefit by identifying males with low sperm numbers, I examined the effect of mating history on sperm number and looked for evidence of sperm depletion. My study found no support for the PLFH in relation to call quality, as neither sperm number nor viability was related to call duration. I found that sperm stores are severely reduced after a single mating, and that sperm stores of males calling late in the season are often more similar to those of depleted males than unmated males. Thus, calling males may attempt to attract females without the sperm numbers necessary to fertilize the entire clutch. However, selection for females to identify males with high fertilization success may be low when all other factors that affect reproductive success (e.g., genetic benefits) are considered.

Biomass export of salamanders and anurans from ponds is affected differentially by changes in canopy cover

SUMMARY 1. Previous research shows that canopy-associated shifts from an algal to a detritus-based food web can affect anuran tadpoles negatively. This may not be true of salamander larvae, however, because they are predators. 2. To investigate the influence of canopy cover on the survival and growth of salamanders, and on the subsequent export of biomass from ponds, we conducted a mesocosm experiment examining effects of shading (high or low) and litter (leaves or grass) on Ambystoma maculatum (a forest specialist) and A. texanum (a habitat generalist). Additionally, we reanalysed data from Williams, Rittenhouse & Semlitsch (2008) to examine the effects of shading and litter on biomass export of three anurans: Rana sphenocephala, Pseudacris crucifer and Hyla versicolor. 3. In contrast to previous studies, we found that salamanders performed better in mesocosms with the characteristics of closed canopy ponds (high shade and leaf litter), whichresultedinagreaterexportof biomass.Salamandersgrewlargerunderclosedcanopy conditions, probably because of differences in prey abundance among treatments. Anurans responded differentlytocanopycoverthan caudates.The biomassexportofR. sphenocephala and P. crucifer was reduced under closed canopy conditions (although differently affected by litter and shading), while the biomass of H. versicolor was not affected. 4. This and other studies suggest that changes in canopy cover may induce a shift in the amphibians emerging from ponds, from primarily anurans in open canopy ponds to primarily salamanders in closed canopy ponds. Additional multispecies studies will determine whether these trends hold true for more diverse amphibian assemblages. Further investigation into the effects of canopy cover on salamanders will be important for understanding aquatic–terrestrial linkages.

Road Salt Stress Induces Novel Food Web Structure and Interactions

Freshwater salinization is an emerging global concern, and in northern latitudes can be largely attributed to road deicer applications during winter and spring. To investigate the effects of road salt contamination on pond food webs, we manipulated trophic structure and salt in experimental pond communities. In May 2008, we inoculated forty 600-L pond mesocosms with algae and zooplankton. Using a full-factorial design, we manipulated road salt presence (645 mg L−1 Cl−) and tadpoles (Hyla versicolor) in a food web that included two groups of primary producers (periphyton, phytoplankton) and two groups of primary consumers (tadpoles, zooplankton). Adult copepod density decreased significantly in the elevated chloride treatment (ANOVA P = 0.001). Tadpoles reared in the elevated chloride treatment grew larger (ANOVA P = 0.005) and metamorphosed sooner (ANCOVA P = 0.028) than those in the ambient treatment. Our results suggest there were more algal resources available to tadpoles as a result of declines in zooplankton, inducing a shorter time to and greater size at metamorphosis. While common pollutants such as road deicers are known to have direct effects on many taxa, the indirect effects on food web structure and interactions can be pronounced, even when pollutants occur at relatively low levels.

Finding Your Mate at a Cocktail Party: Frequency Separation Promotes Auditory Stream Segregation of Concurrent Voices in Multi-Species Frog Choruses

Vocal communication in crowded social environments is a difficult problem for both humans and nonhuman animals. Yet many important social behaviors require listeners to detect, recognize, and discriminate among signals in a complex acoustic milieu comprising the overlapping signals of multiple individuals, often of multiple species. Humans exploit a relatively small number of acoustic cues to segregate overlapping voices (as well as other mixtures of concurrent sounds, like polyphonic music). By comparison, we know little about how nonhuman animals are adapted to solve similar communication problems. One important cue enabling source segregation in human speech communication is that of frequency separation between concurrent voices: differences in frequency promote perceptual segregation of overlapping voices into separate “auditory streams” that can be followed through time. In this study, we show that frequency separation (ΔF) also enables frogs to segregate concurrent vocalizations, such as those routinely encountered in mixed-species breeding choruses. We presented female gray treefrogs (Hyla chrysoscelis) with a pulsed target signal (simulating an attractive conspecific call) in the presence of a continuous stream of distractor pulses (simulating an overlapping, unattractive heterospecific call). When the ΔF between target and distractor was small (e.g., ≤3 semitones), females exhibited low levels of responsiveness, indicating a failure to recognize the target as an attractive signal when the distractor had a similar frequency. Subjects became increasingly more responsive to the target, as indicated by shorter latencies for phonotaxis, as the ΔF between target and distractor increased (e.g., ΔF = 6–12 semitones). These results support the conclusion that gray treefrogs, like humans, can exploit frequency separation as a perceptual cue to segregate concurrent voices in noisy social environments. The ability of these frogs to segregate concurrent voices based on frequency separation may involve ancient hearing mechanisms for source segregation shared with humans and other vertebrates.

Dynamic regulation of aquaglyceroporin expression in erythrocyte cultures from cold‐ and warm‐acclimated cope's gray treefrog, Hyla chrysoscelis

Cope's gray treefrog, Hyla chrysoscelis,is a freeze-tolerant anuran which accumulates and distributes glycerol as a cryoprotectant before freezing. We hypothesize that HC-3, an aquaglyceroporin member of the MIP family of water pores, may play an important role in the process of freeze tolerance by mediating transmembrane passage of glycerol and water during cold-acclimation. The objectives of this study were two-fold: to examine HC-3 protein abundance and cellular localization in erythrocytes from cold- and warm-acclimated frogs and to develop and characterize an erythrocyte cell culture system for examining HC-3 gene regulation. Compared with warm-acclimated frogs, erythrocytes from cold-acclimated frogs had higher HC-3 protein expression and enhanced plasma membrane localization. Furthermore, erythrocytes from cold- and warm-acclimated frogs maintained in culture at 4 and 20°C exhibited time- and temperature-dependent regulation of HC-3 expression and an increase in the abundance of high molecular weight immunoreactive species within 24 hr of culture at 20°C. Deglycosylation of erythrocyte proteins resulted in the disappearance of the high molecular weight species, indicating that HC-3 is post-translationally modified by N-linked glycosylation. Erythrocytes cultured in media containing glycerol also showed an increased abundance of the high molecular weight bands and enhanced plasma membrane localization of HC-3, suggesting a role for glycerol in regulating HC-3 subcellular trafficking. Thus, the development of this erythrocyte cell culture system from H. chrysoscelis opened an opportunity to study the properties of cells with changing expression of an aquaglyceroporin, HC-3, and to explore the factors regulating that expression.

Increased Conductivity Affects Corticosterone Levels and Prey Consumption in Larval Amphibians

Abstract Exposure to altered conductivity can negatively impact many freshwater system inhabitants, including bacteria, plants, invertebrates, and vertebrates. Several studies have shown increased conductivity to affect amphibian behavior and ecology. Comparatively fewer studies have taken a physiological approach to understanding the effects of altered conductivity in amphibians. This study used laboratory experiments to assess conductivity effects on stress hormone (corticosterone) levels in Jefferson Salamander (Ambystoma jeffersonianum), Wood Frog (Rana sylvatica), and Gray Treefrog (Hyla versicolor) larvae. Prey consumption (only in A. jeffersonianum) and survival (in all species) also was examined after exposure to increased conductivity (0, 2,000, 4,000, and 8,000 ppb). There was a significant positive effect of increased conductivity exposure on baseline corticosterone levels in A. jeffersonianum after 1-week exposure. Exposure to increased conductivity did not influence baseline corticosterone le...

Endo-Porter–mediated delivery of phosphorodiamidate morpholino oligos (PMOs) in erythrocyte suspension cultures from Cope's gray treefrog Hyla chrysoscelis

Cope's gray treefrog, Hyla chrysoscelis, is a freeze-tolerant anuran that accumulates cryoprotective glycerol during cold acclimation. H. chrysoscelis erythrocytes express the aquaglyceroporin HC-3, which facilitates transmembrane glycerol and water movement. Aquaglyceroporins have no pharmacological inhibitors, and no genetic knockout tools currently exist for H. chrysoscelis. A phosphorodiamidate morpholino oligo (PMO)-mediated expression knockdown approach was therefore pursued to provide a model for testing the role of HC-3. We describe a novel procedure optimized for specific, efficient knockdown of HC-3 expression in amphibian erythrocyte suspensions cultured at nonmammalian physiological temperatures using Endo-Porter. Our protocol includes three critical components: pre-incubation at 37°C, two rounds of Endo-Porter and HC-3 PMO administration at ~23°C, and continuous shaking at 190 rpm. This combination of steps resulted in 94% reduction in HC-3 protein expression (Western blot), substantial decrease in HC-3 expression in >65% of erythrocytes, and no detectable expression in an additional 30% of cells (immunocytochemistry).

Estimating occupancy dynamics in an anuran assemblage from Louisiana, USA

AbstractEffective monitoring programs are designed to track changes in the distribution, occurrence, and abundance of species. We developed an extension of Royle and Kéry's (2007) single species model to estimate simultaneously temporal changes in probabilities of detection, occupancy, colonization, extinction, and species turnover using data on calling anuran amphibians, collected from 2002 to 2006 in the Lower Mississippi Alluvial Valley of Louisiana, USA. During our 5‐year study, estimates of occurrence probabilities declined for all 12 species detected. These declines occurred primarily in conjunction with variation in estimates of local extinction probabilities (cajun chorus frog [Pseudacris fouquettei], spring peeper [P. crucifer], northern cricket frog [Acris crepitans], Cope's gray treefrog [Hyla chrysoscelis], green treefrog [H. cinerea], squirrel treefrog [H. squirella], southern leopard frog [Lithobates sphenocephalus], bronze frog [L. clamitans], American bullfrog [L. catesbeianus], and Fowler's toad [Anaxyrus fowleri]). For 2 species (eastern narrow‐mouthed toad [Gastrophryne carolinensis] and Gulf Coast toad [Incilius nebulifer]), declines in occupancy appeared to be a consequence of both increased local extinction and decreased colonization events. The eastern narrow‐mouthed toad experienced a 2.5‐fold increase in estimates of occupancy in 2004, possibly because of the high amount of rainfall received during that year, along with a decrease in extinction and increase in colonization of new sites between 2003 and 2004. Our model can be incorporated into monitoring programs to estimate simultaneously the occupancy dynamics for multiple species that show similar responses to ecological conditions. It will likely be an important asset for those monitoring programs that employ the same methods to sample assemblages of ecologically similar species, including those that are rare. By combining information from multiple species to decrease the variance on estimates of individual species, our results are advantageous compared to single‐species models. This feature enables managers and researchers to use an entire community, rather than just one species, as an ecological indicator in monitoring programs. © 2011 The Wildlife Society.

Comparative feeding kinematics of temperate pond-dwelling tadpoles (Anura, Amphibia)

Several studies have explored various components of feeding kinematics in anuran larvae; however, a direct comparison of feeding kinematics among morphologically similar and sympatric taxa has not been undertaken. We used high-speed videography (500 frames/s) to capture feeding kinematics of Anaxyrus fowleri (Hinckley, 1882) (Fowler’s Toad), Hyla chrysoscelis (Cope, 1880) (Grey Treefrog), Scaphiopus holbrookii (Harlan, 1835) (Eastern Spadefoot Toad), and Lithobates sphenocephalus (Cope, 1889) (Southern Leopard Frog) tadpoles as they foraged from an algal-covered substrate. In total, we filmed 120 feeding sequences from 25 feeding bouts and quantified eight kinematic variables that were common among all four species. Despite relatively similar keratinized feeding structures among taxa, our videography data revealed fundamental differences in how the tadpoles used these structures. One specific difference was in the speed of the gape cycle. Among taxa, S. holbrookii tadpoles had the longest gape cycle and longest time to reach maximum gape, whereas A. fowleri and L. sphenocephalus tadpoles had shorter durations for both variables and did not differ between species. We also found species differences in the magnitude that tadpoles narrow their lower jaw sheath. Irrespective of gape size, the lower jaw sheath of S. holbrookii tadpoles narrowed by approximately 26% of its maximum width—a twofold difference from A. fowleri tadpoles, which narrowed only 13%. Our study revealed that tadpoles with similar oral structures feeding on the same substrate can exhibit major differences in feeding kinematics.

Effects of simultaneous polyandry on offspring fitness in an African tree frog

In externally fertilizing animals in which females mate with multiple males at the same time (simultaneous polyandry), the possibility that females accrue genetic benefits that improve offspring viability remains largely unexamined. Here, we investigate whether simultaneous polyandry influences offspring fitness in a wild population of the Grey Foam Nest Treefrog Chiromantis xerampelina. Simultaneous polyandry in this frog is the most extreme reported for any vertebrate, with more than 90% of females mating with 10 or more males during the deposition of a single clutch. We compared growth (using age and size at metamorphosis as proxies) and survival of offspring produced by females that naturally mated with either 1 male (monandrous females) or 10‐12 males (polyandrous females). Polyandry did not influence size or age at metamorphosis, but we found that offspring from polyandrous matings had both significantly higher mean survival and reduced variance in offspring survival. These findings implicate a genetic mechanism, but targeted cross-classified breeding experiments that control for both maternal and material effects will be required to conclusively determine whether elevated offspring viability is linked to enhanced genetic diversity, intrinsic sire effects, or genetic compatibility. Irrespective of the causation, the findings provide the first evidence that naturally formed polyandrous matings have increased offspring viability in an anuran amphibian. Key words: anuran amphibian, genetic benefits, offspring viability, polyandry, sexual selection. [Behav Ecol]

TESTS FOR CALL RESTORATION IN THE GRAY TREEFROG HYLA VERSICOLOR

ABSTRACT Phonemic restoration, a form of temporal induction, occurs when the human brain compensates for masked or missing portions of speech by filling in obscured or nonexistent sounds. We tested for temporal induction and related abilities in females of the Gray Treefrog Hyla versicolor. The number of pulses in calls is used by females for assessment of males. Accordingly, an ability to “restore” or interpolate between masked or otherwise sonically degraded portions of calls could help females during mate choice in noisy choruses. In phonotaxis experiments, we employed unmodified calls and those that had a centrally placed gap, a region overlapped by a portion of another call or filtered noise, or replaced with filtered noise. When offered call alternatives with equivalent numbers of clear pulses, we found that females discriminated against calls with gaps two or more times greater than the natural 25 ms interpulse interval. When a gap was replaced with a zone of call overlap or noise (so, again the call durations of the alternatives were unequal), females discriminated either in favour (overlap) of the modified stimuli or failed to discriminate (noise). However, when the unmodified and modified stimuli were the same duration, females discriminated against the latter. Normal calls were also chosen when paired against calls with multiple noise sections. Pulses formed from noise bursts were attractive, but less so than normal pulses. In single speaker tests, standardized rates of movement did not differ between calls containing noise segments of different duration. Our results therefore do not indicate that females of the Gray Treefrog employ a form of temporal induction that is fully restorative. However, the data indicate that acoustically anomalous sections of calls can retain attractive potential provided acoustic energy and pulses are present.

What Doesn't Kill You Makes You Sluggish: How Sublethal Pesticides Alter Predator–Prey Interactions

Pesticides commonly occur in ecological communities at relatively low concentrations, leading to growing interest in determining the sublethal effects of pesticides. Such effects should affect individuals and, in turn, alter interspecific interactions. We sought to determine how sublethal concentrations (0.1 and 1.0 mg/L) of two common pesticides (carbaryl and malathion) affected predator and prey behavior as well as subsequent predation rates. We conducted a series of experiments using three species of larval amphibians (Gray Treefrogs, Hyla versicolor; Green Frogs, Rana clamitans; and American Bullfrogs, R. catesbeiana) and three species of their predators (larval dragonflies, Anax junius; adult water bugs, Belostoma flumineum; and adult Red-spotted Newts, Notophthalmus viridescens). We found that the pesticides frequently reduced the activity of all three tadpole species. For the two invertebrate predators (Anax and Belostoma), the pesticides were lethal, precluding us from examining sublethal effects on predator–prey interactions. However, newt survival was high and the addition of the pesticides reduced the predation rates of newts in one of the three tadpole species. There were no effects of the pesticides on the striking frequency of the newts or on their prey capture efficiency. Thus, the mechanism underlying the pesticide-induced reduction in predation rates remains unclear. What is clear is that sublethal concentrations of pesticides have the potential to alter prey behavior and species interactions and thereby alter the composition of ecological communities.

Competitive stress can make the herbicide Roundup® more deadly to larval amphibians

Toxicity assessments on nontarget organisms have largely been addressed using short-term, single-species laboratory experiments. Although extremely helpful, these experiments inherently lack many pervasive ecological stressors found in nature. Though a substantial challenge, incorporating these ecological stressors in contaminant studies would shed light on potential synergistic effects. For the world's leading herbicide, glyphosate, we know little about how natural stressors affect the toxicity to nontarget organisms. To explore how the natural stress of competition might interact with a glyphosate-based herbicide, we used outdoor mesocosms containing three tadpole species that were exposed to a factorial combination of three glyphosate concentrations (0, 1, 2, or 3 mg acid equivalent (a.e.)/L of the commercial formulation Roundup Original MAX®) and three tadpole densities (low, medium, or high). We found that increased tadpole density caused declines in tadpole growth, but also made the herbicide significantly more lethal to one species. Whereas the median lethal concentration (LC50) values were similar across all densities for gray treefrogs (Hyla versicolor; 1.7-2.3 mg a.e./L) and green frogs (Rana clamitans; 2.2-2.6 mg a.e./L), the LC50 values for bullfrogs (R. catesbeiana) were 2.1 to 2.2 mg a.e./L at low and medium densities, but declined to 1.6 mg a.e./L at high densities. The large decrease in amphibian survival with increased herbicide concentration was associated with increases in periphyton abundance. We also found evidence that temperature stratification lead to herbicide stratification in the water column, confirming the results of a previous study and raising important questions about exposure risk in natural systems.

Living in the litter: the influence of tree leaf litter on wetland communities

Empirical research in streams has demonstrated that terrestrial subsidies of tree leaf litter influence multiple community factors including composition, diversity and growth of individuals. However, little research has examined the importance of tree litter species on wetlands, which are ubiquitous across the landscape and serve as important habitats for a unique and diverse community of organisms. Using outdoor mesocosms, we assessed the impact of 12 litter monocultures and three litter mixtures (from both broadleaf and conifer trees) on pond communities containing gray tree frog tadpoles Hyla versicolor, periphyton, phytoplankton and zooplankton. We found that leaf litter species had substantial and differential impacts on all trophic groups in the community including effects on algal abundance, zooplankton density and amphibian growth. In many instances, patterns of responses were specific to individual litter species yet some responses, including both pH values and periphyton biomass, were generalizable to broad taxonomic groups. In addition, while most responses of litter mixtures were additive, we found evidence for antagonistic effects of litter mixing among responses of periphyton and amphibian body mass. Our results highlight the potential impact of human and naturally driven changes in forest composition on wetland communities through associated changes in leaf litter.

Sub-optimal study design has major impacts on landscape-scale inference

Landscape-scale processes (e.g. habitat loss) are major drivers of the global biodiversity crisis, but the complexity and size of landscapes makes study design at this scale difficult. However, the impact of statistical problems associated with sub-optimal study design on inferences drawn from landscape-scale studies is poorly understood. Here, we examine how three common statistical ‘pitfalls’ associated with sub-optimal study design – (1) using landscapes that overlap in space; (2) using only a portion of the potential range of the landscape predictor variable(s) of interest; (3) failing to account for correlations among landscape predictor variables – affect the inferred relationships between the abundances of six species of anurans and the amount of forest in the landscape using a large ( n = 1141) empirical dataset from Wisconsin and Michigan, USA. We show that sub-optimal study design alone can be sufficient to cause a switch in the sign of the inferred relationship between a species response and landscape structure, and that using only a portion of the potential range of a predictor variable, and correlations between predictor variables, are particularly likely to affect inferences. Our results also provide the first evidence of a non-monotonic relationship between forest amount and gray treefrog abundance, and suggest that inconsistencies in the literature about the inferred relationships between anuran presence/abundance and forest amount in the Great Lakes basin are likely largely due to sampling design issues. Increased attention to study design is therefore necessary for the development of robust generalizations in landscape ecology.

Differential Patterns of Accumulation and Depuration of Dietary Selenium and Vanadium During Metamorphosis in the Gray Treefrog (Hyla versicolor)

Selenium (Se) and vanadium (V) are contaminants commonly found in aquatic systems affected by wastes derived from fossil fuels. To examine their effects on a widely distributed species of amphibian, we exposed gray tree frogs (Hyla versicolor) to Se (as SeO₂) or V (as NaVO₃) in their diet from the early larval period to metamorphosis. Concentrations of Se in Se-enriched food were 1.0 (Se control), 7.5 (Se low), and 32.7 (Se high) μg/g dw. Concentrations of V in V-enriched food were 3.0 (V control), 132.1 (V low), and 485.7 (V high) μg/g dw. Although we observed bioaccumulation of both metals throughout the larval period, no effects on growth, survival, metabolic rate, or lipid content were observed. Se concentrations in tissues did not vary among life stages, neither in Se low nor Se high treatments, such that maximum accumulation had occurred by the mid-larval period. In addition, there was no evidence of depuration of Se in either the Se low or the Se high treatments during metamorphosis. A strikingly different pattern of accumulation and depuration occurred in V-exposed individuals. In treatments V low and V high, maximum body burdens occurred in "premetamorphs" (i.e., animals with developed forelimbs but in which tail resorption had not begun), whereas body burdens in animals having completed metamorphosis were much lower and similar to those in larvae. These results suggest that compared with Se-exposed animals, V-exposed animals were able to depurate a substantial amount of accumulated V during the metamorphic period. In an ecologic context, it appears that amphibians exposed to Se during the larval period may serve as a vector of the metal to terrestrial predators, yet potential transfer of accumulated V to predators would largely be restricted to the aquatic habitat.