Bottom-up and trait-mediated effects of resource quality on amphibian parasitism.

Leaf litter subsidies are important resources for aquatic consumers like tadpoles and snails, causing bottom-up effects on wetland ecosystems. Recent studies have shown that variation in litter nutritional quality can be as important as litter quantity in driving these bottom-up effects. Resource subsidies likely also have indirect and trait-mediated effects on predation and parasitism, but these potential effects remain largely unexplored. We generated predictions for differential effects of litter nutrition and secondary polyphenolic compounds on tadpole (Lithobates sylvatica) exposure and susceptibility to Ribeiroia ondatrae, based on ecological stoichiometry and community-ecology theory. We predicted direct and indirect effects on key traits of the tadpole host (rates of growth, development and survival), the trematode parasite (production of the cercaria infective stages) and the parasite's snail intermediate host (growth and reproduction). To test these predictions, we conducted a large-scale mesocosm experiment using a natural gradient in the concentrations of nutrients (nitrogen) and toxic secondary compounds (polyphenolics) of nine leaf litter species. To differentiate between effects on exposure vs. susceptibility to infection, we included multiple infection experiments including one with constant per capita exposure. We found that increased litter nitrogen increased tadpole survival, and also increased cercaria production by the snail intermediate hosts, causing opposing effects on tadpole per capita exposure to trematode infection. Increased litter polyphenolics slowed tadpole development, leading to increased infection by increasing both their susceptibility to infection and the length of time they were exposed to parasites. Based on these results, recent shifts in forest composition towards more nitrogen-poor litter species should decrease trematode infection in tadpoles via density- and trait-mediated effects on the snail intermediate hosts. However, these shifts also involve increased abundance of litter species with high polyphenolic levels, which should increase trematode infection via trait-mediated effects on tadpoles. Future studies will be needed to determine the relative strength of these opposing effects in natural wetland communities. [Correction added after online publication on 5 January 2017: wording changed to 'which should increase trematode infection via trait-mediated effects on tadpoles'.].

Ontogenetic changes in sensitivity to nutrient limitation of tadpole growth.

According to ecological stoichiometry (ES), the growth of a consumer with abundant resources should increase as body and resource stoichiometry become more similar. However, for organisms with complex life cycles involving distinct changes in biology, nutrient demands might change in response to ontogenetic changes in body stoichiometry. Tadpole growth and development has been found to be largely nitrogen (N) limited, as predicted for organisms developing N-rich tissues like muscle. However, tadpole metamorphosis includes periods of rapid development of phosphorus (P)-rich bones in preparation for a terrestrial lifestyle. We hypothesized that tadpole growth and development will exhibit variable nutrient demands during different stages of ontogeny, due to predictable changes in body tissue stoichiometry. To test this, we raised tadpoles on four diets with varying N:P ratios and assessed growth and developmental rates. Specifically, we predicted that tadpoles would be sensitive to N limitation throughout ontogeny (consistent with previous studies), but also sensitive to P limitation during the process of long-bone ossification. Consistent with our prediction, tadpole growth rates and development were sensitive to N limitation throughout ontogeny. Increased dietary N led to a shorter time to metamorphosis and a larger mass at metamorphosis. Also as predicted, growth rates were sensitive to both N and P during the period of peak bone ossification, indicative of co-limitation. These results indicate that P limitation changes through tadpole ontogeny consistent with, and can be predicted by, shifts in body tissue stoichiometry. Future studies should investigate whether ontogenetic shifts in tadpole P limitation lead to seasonal shifts in wetland nutrient cycling.

Leaf Litter Inhibits Growth of an Amphibian Fungal Pathogen.

Past studies have found a heterogeneous distribution of the amphibian chytrid fungal pathogen, Batrachochytrium dendrobatidis (Bd). Recent studies have accounted for some of this heterogeneity through a positive association between canopy cover and Bd abundance, which is attributed to the cooling effect of canopy cover. We questioned whether leaf litter inputs that are also associated with canopy cover might also alter Bd growth. Leaf litter inputs exhibit tremendous interspecific chemical variation, and we hypothesized that Bd growth varies with leachate chemistry. We also hypothesized that Bd uses leaf litter as a growth substrate. To test these hypotheses, we conducted laboratory trials in which we exposed cultures of Bd to leachate of 12 temperate leaf litter species at varying dilutions. Using a subset of those 12 litter species, we also exposed Bd to pre-leached litter substrate. We found that exposure to litter leachate and substrate reduced Bd spore and sporangia densities, although there was substantial variation among treatments. In particular, Bd densities were inversely correlated with concentrations of phenolic acids. We conducted a field survey of phenolic concentrations in natural wetlands which verified that the leachate concentrations in our lab study are ecologically relevant. Our study reinforces prior indications that positive associations between canopy cover and Bd abundance are likely mediated by water temperature effects, but this phenomenon might be counteracted by changes in aquatic chemistry from leaf litter inputs.

Stoichiometric implications of a biphasic life cycle.

Animals mediate flows of elements and energy in ecosystems through processes such as nutrient sequestration in body tissues, and mineralization through excretion. For taxa with biphasic life cycles, the dramatic shifts in anatomy and physiology that occur during ontogeny are expected to be accompanied by changes in body and excreta stoichiometry, but remain little-explored, especially in vertebrates. Here we tested stoichiometric hypotheses related to the bodies and excreta of the wood frog (Lithobates sylvaticus) across life stages and during larval development. Per-capita rates of nitrogen (N) and phosphorus (P) excretion varied widely during larval ontogeny, followed unimodal patterns, and peaked midway through development (Taylor-Kollros stages XV and XII, respectively). Larval mass did not increase steadily during development but peaked at stage XVII and declined until the termination of the experiment at stage XXII. Mass-specific N and P excretion rates of the larvae decreased exponentially during development. When coupled with population-biomass estimates, population-level excretion rates were greatest at stages VIII-X. Percent carbon (C), N, and C:N of body tissue showed weak trends across major life stages; body P and C:P, however, increased sixfold during development from egg to adult. Our results demonstrate that intraspecific ontogenic changes in nutrient contents of excretion and body tissues can be significant, and that N and P are not always excreted proportionally throughout life cycles. These results highlight the dynamic roles that species play in ecosystems, and how the morphological and physiological changes that accompany ontogeny can influence ecosystem-level processes.

Ecological stoichiometry quantitatively predicts responses of tadpoles to a food quality gradient.

Ecological stoichiometry (ES) uses elemental ratios and mass balance to explain organismal growth, an important parameter in ecological systems. In this study, we tested quantitative predictions of the ES "minimal model" for the growth rates of two tadpole species (wood frogs, Lithobates sylvaticus and American toads, Anaxyrus americanus), by manipulating light and the quality of a leaf litter mixture in a seminatural mesocosm experiment. We predicted that wood frogs, which consume leaf litter as a resource, would respond more strongly to leaf litter quality than toads, which forage on periphyton and algae. The ES minimal model, parameterized from literature values, provided strikingly accurate quantitative predictions of nonlinear wood frog growth patterns across gradients of leaf litter quality, both in this experiment and when applied to previously published data on wood frog growth responses to various leaf litter species. In contrast, toad growth was best explained by the biomass of periphyton, which was driven primarily by light availability and only indirectly influenced by litter-derived soluble polyphenols. This study demonstrates the power of ES to predict organism growth rates, and highlights potential applications of this theory to predicting population- and community-level responses to changing forest environments.

Leaf litter resource quality induces morphological changes in wood frog (Lithobates sylvaticus) metamorphs.

For organisms that exhibit complex life cycles, resource conditions experienced by individuals before metamorphosis can strongly affect phenotypes later in life. Such resource-induced effects are known to arise from variation in resource quantity, yet little is known regarding effects stemming from variation in resource quality (e.g., chemistry). For larval anurans, we hypothesized that variation in resource quality will induce a gradient of effects on metamorph morphology. We conducted an outdoor mesocosm experiment in which we manipulated resource quality by rearing larval wood frogs (Lithobates sylvaticus) under 11 leaf litter treatments. The litter species represented plant species found in open- and closed-canopy wetlands and included many plant species of current conservation concern (e.g., green ash, common reed). Consistent with our hypothesis, we found a gradient of responses for nearly all mass-adjusted morphological dimensions. Hindlimb dimensions and gut mass were positively associated with litter nutrient content and decomposition rate. In contrast, forelimb length and head width were positively associated with concentrations of phenolic acids and dissolved organic carbon. Limb lengths and widths were positively related with the duration of larval period, and we discuss possible hormonal mechanisms underlying this relationship. There were very few, broad differences in morphological traits of metamorphs between open- and closed-canopy litter species or between litter and no-litter treatments. This suggests that the effects of litter on metamorph morphology are litter species-specific, indicating that the effects of changing plant community structure in and around wetlands will largely depend on plant species composition.

Kin recognition by larval wood frogs (Rana sylvatica): effects of diet and prior exposure to conspecifics.

We investigated kin recognition by larval wood frogs (Rana sylvatica) in blind laboratory experiments using spatial affinity as a recognition assay. Tadpoles reared with full-sibs displayed a significant preference for familiar full-sibs over unfamiliar non-kin, but failed to discriminate between unfamiliar full-sibs and unfamiliar paternal half-sibs. Tadpoles reared in social isolation (with or without maternal egg jelly) from the two-celled embryonic stage displayed a significant preference for unfamiliar full-sibs over unfamiliar non-kin. Tadpoles reared on a meat diet with their full-sibs: 1) exhibited a significant preference for unfamiliar full-sibs fed meat over unfamiliar non-kin fed meat, 2) failed to discriminate between unfamiliar full-sibs fed lettuce and unfamiliar non-kin fed meat, 3) exhibited a significant preference for unfamiliar non-kin fed meat over unfamiliar non-lin fed lettuce, 4) failed to discriminate between unfamiliar full-sibs fed meat and unfamiliar full-sibs fed lettuce, and 5) displayed a significant spatial preference for odors associated with meat (a familiar food) over odors associated with lettuce (an unfamiliar food). Our results, together with those of Cornell et al. (1989), indicate that the recognition cue of larval R. sylvatica has both genetic and environmental (dietary) components. Our findings establish that previous exposure to maternal egg jelly, kin, or conspecifics is not necessary for the development of kin recognition ability in larval R. sylvatica. Our results are more consistent with the self-learning of recognition cues (a form of phenotype matching) than with a recognition mechanism that involves a genetically fixed recognition template. Finally, our results indicate that increasing similarity between the recognition template and perceived cue does not necessarily result in increasing spatial affinity for kin.

DISPERSAL IN THE WOOD FROG (RANA SYLVATICA): IMPLICATIONS FOR GENETIC POPULATION STRUCTURE.

Recapture of marked juvenile and adult wood frogs in five Appalachian Mountain ponds showed adults to be 100% faithful to the ponds in which they first bred, but approximately 18% of the juveniles dispersed to breed in ponds other than the one of origin. Effective population sizes were generally smaller than the population censuses and genetic neighborhoods had an average radius of 1,126 meters. Values of standardized genetic variance based on effective population size and mating success were relatively small. Genetic population structure estimated from the dispersal data suggested that ponds within about a 1,000 meter radius should show little genetic differentiation; ponds separated by a distance greater than 1,000 meters should experience little gene flow and show higher genetic differentiation. Wood frogs in these ponds do not show a meta-population structure as suggested for newts.

Kin recognition by tadpoles and froglets of the wood frog Rana sylvatica.

We investigated kin recognition by the wood frog Rana sylvatica in blind laboratory experiments using spatial proximity as a recognition assay. Tadpoles were tested for the ability to discriminate between: 1) familiar full-sibs and unfamiliar non-kin, 2) unfamiliar paternal half-sibs and unfamiliar non-kin, and 3) familiar and unfamiliar full-sibs. Tadpoles discriminated full- and paternal half-sibs from unrelated conspecifics, but did not discriminate between familiar and unfamiliar full-sibs. Froglets from the same laboratory population were tested for the ability to discriminate between 1) familiar full-sibs and unfamiliar non-kin, and 2) unfamiliar paternal half-sibs and unfamiliar non-kin. Froglets preferentially associated with full- and half-sibs over unrelated conspecifics. Our results show that familiarity, i.e., prior association, is not necessary for kin recognition in tadpoles and froglets. The ability of tadpoles and froglets to recognize unfamiliar paternal half-sibs demonstrates that a common maternal factor is not necessary for kin recognition, and indicates that the recognition cue has a genetic component. Our results add to the increasing evidence that a variety of vertebrate and invertebrate animals have the ability to recognize unfamiliar kin by using genetically specified recognition cues.

The relationship among egg size, density and food level on larval development in the wood frog (Rana sylvatica).

Although inter- and intraspecific variation in egg size among amphibians has been well documented, the relationship between egg size and fitness remains unclear. Recent attempts to correlate egg size intraspecifically with larval developmental patterns have been equivocal. In this study the development of larvae derived from large eggs and small eggs, from a single population in Maryland were compared under a range of food levels and larval population densities. Both food level and density had significant effects on the length of the larval period and size at metamorphosis. However, the response among larvae derived from different egg sizes was not additive. At low densities and high food levels, larvae from small eggs had longer larval periods and a larger size at metamorphosis than larvae derived from large eggs. In contrast, at high densities larvae from small eggs had longer developmental periods but were smaller at metamorphosis than larvae from large eggs. In addition, larvae from small eggs were more sensitive to density irrespective of food level. These results suggest that optimal egg size is correlated with environmental factors, which may explain the maintenance of both geographic and within population variation in egg size commonly observed in amphibians.

THE HERITABLE BASIS OF VARIATION IN LARVAL DEVELOPMENTAL PATTERNS WITHIN POPULATIONS OF THE WOOD FROG (RANA SYLVATICA).

This study compares the heritable basis of variation in larval developmental patterns of mountain and lowland populations of the wood frog, Rana sylvatica. Additive genetic variances, heritabilities, and genetic correlations for larval developmental time and size at metamorphosis are estimated from half-sib and full-sib crosses. Considerable additive-genetic variances and high heritabilities are revealed for developmental time in both the mountain and the lowland population. There was a high level of additive-genetic variance and high heritability for body size at metamorphosis in the mountain population, but these were very low in the lowland population. The genetic correlations between developmental rate and larval body size are negative for the mountain population and near zero for the lowland population. It is argued that the differences in genetic structure between these two populations reflect differences in the selective regimes of their respective environments.

The genetic basis of altitudinal variation in the wood frog Rana sylvatica II. An experimental analysis of larval development.

The variation in larval developmental patterns in the wood frog, Rana sylvatica, along an elevation gradient of 1,000 m was experimentally studied. Larval populations at high elevation ponds had lower growth rates, developmental rates and were larger at all stages (including metamorphic climax) than larval populations developing in low elevation ponds. There was considerable variation among ponds within each elevation in both the length of the larval period and size at metamorphic climax. Reciprocal transplant experiments and controlled laboratory experiments revealed that most of the observed variation between high and low elevation populations could be explained by the effects of temperature induction during ontogeny. Significant genetic differences in growth rates and non-genetic maternal effects on developmental rates between larvae of mountain origin and lowland origin were also demonstrated. Selection in both environments has acted to minimize the prevailing environmental effect of pond temperature on developmental rates, but has accentuated the prevailing environmental effects on larval body size. As a consequence mountain larvae were capable of completing metamorphosis sooner and at a larger size in all environments than lowland larvae.