Flexible oviposition behavior enabled the evolution of terrestrial reproduction.

Among vertebrates, nearly all oviparous animals are considered to have either obligate aquatic or terrestrial oviposition, with eggs that are specialized for developing in those environments. The terrestrial environment has considerably more oxygen but is dry and thus presents both opportunities and challenges for developing embryos, particularly those adapted for aquatic development. Here, we present evidence from field experiments examining egg-laying behavior, egg size, and egg jelly function of 13 species of Central and South American treefrogs in the genus Dendropsophus, which demonstrates that flexible oviposition (individuals laying eggs both in and out of water) and eggs capable of both aquatic and terrestrial development are the likely factors which enable the transition from aquatic to terrestrial reproduction. Nearly half of the species we studied had previously undescribed degrees of flexible oviposition. Species with obligate terrestrial reproduction have larger eggs than species with aquatic reproduction, and species with flexible reproduction have eggs of intermediate sizes. Obligate terrestrial breeding frogs also have egg masses that absorb water more quickly than those with flexible oviposition. We also examined eight populations of a single species, Dendropsophus ebraccatus, and document substantial intraspecific variation in terrestrial oviposition; populations in rainy, stable climates lay fewer eggs in water than those in drier areas. However, no differences in egg size were found, supporting the idea that the behavioral component of oviposition evolves before other adaptations associated with obligate terrestrial reproduction. Collectively, these data demonstrate the key role that behavior can have in facilitating major evolutionary transitions.

Demographic consequences of foraging ecology explain genetic diversification in Neotropical bird species.

Despite evidence that species' traits affect rates of bird diversification, biogeographic studies tend to prioritise earth history in Neotropical bird speciation. Here we compare mitochondrial genetic differentiation among 56 co-distributed Neotropical bird species with varying ecologies. The trait 'diet' best predicted divergence, with plant-dependent species (mostly frugivores and nectivores) showing lower levels of genetic divergence than insectivores or mixed-diet species. We propose that the greater vagility and demographic instability of birds whose diets rely on fruit, seeds, or nectar  known to vary in abundance seasonally and between years  relative to birds that eat primarily insects, drives episodic re-unification of otherwise isolated populations, resetting the divergence 'clock'. Testing this prediction using coalescent simulations, we find that plant-dependent species show stronger signals of recent demographic expansion compared to insectivores or mixed-diet species, consistent with this hypothesis. Our study provides evidence that localised ecological phenomena scale up to generate larger macroevolutionary patterns.

Nothing as it seems: behavioural plasticity appears correlated with morphology and colour, but is not in a Neotropical tadpole.

In response to environmental stressors, organisms often demonstrate flexible responses in morphology, life history or behaviour. However, it is currently unclear if such plastic responses are coordinated or operate independently of one another. In vertebrates, this may partly result from studies examining population- or species-level mean responses, as opposed to finer grained analyses of individuals or families. We measured predator-specific morphological and coloration plasticity in 42 families of tadpoles of the treefrog Dendropsophus ebraccatus and behavioural plasticity from 18 of these families, allowing us to examine the correlation between three predator-induced plastic responses. For all three plastic responses, tadpoles showed strong opposing responses to each of two predators, providing the appearance of covariation in plasticity. However, the examination of individual families revealed a strong correlation between morphological and coloration plasticity, but no correlations between either morphology or colour and behavioural plasticity. Thus, our analysis shows that some aspects of the plastic phenotype develop together while others function independently. This highlights the importance of examining individual- and family-level variation for understanding the adaptive significance of developmental plasticity, which is crucial for a holistic appreciation of phenotypic plasticity and its importance in ecology and evolution.

Right phenotype, wrong place: predator-induced plasticity is costly in a mismatched environment.

Like many animals, tadpoles often produce different, predator-specific phenotypes when exposed to risk of predation. It is generally assumed that such plasticity enhances survival in the presence of the predator and is costly elsewhere, but evidence remains surprisingly scarce. We measured (1) the survival trade-off of opposing phenotypes developed by Dendropsophus ebraccatus tadpoles when exposed to different predators and (2) which specific aspects of morphology drive any potential survival benefit or cost. Tadpoles developed predator-specific phenotypes after being reared with caged fish or dragonfly predators for two weeks. In 24 h predation trials with either a fish or a dragonfly, survival was highest in the groups with their matched predator, and lowest among with those the mismatched predator, with predator-naive controls being relatively intermediate. Then, using a large group of phenotypically variable predator-naive tadpoles, we found that increased survival rates are directly related to the morphological changes that are induced by each predator. This demonstrates that induced phenotypes are indeed adaptive and the product of natural selection. Furthermore, our data provide clear evidence of an environmental cost for phenotypic plasticity in a heterogeneous environment. Such costs are fundamental for understanding the evolution and maintenance of inducible phenotypes.

Oviposition site choice under conflicting risks demonstrates that aquatic predators drive terrestrial egg-laying.

Laying eggs out of water was crucial to the transition to land and has evolved repeatedly in multiple animal phyla. However, testing hypotheses about this transition has been difficult because extant species only breed in one environment. The pantless treefrog, Dendropsophus ebraccatus, makes such tests possible because they lay both aquatic and arboreal eggs. Here, we test the oviposition site choices of D. ebraccatus under conflicting risks of arboreal egg desiccation and aquatic egg predation, thereby estimating the relative importance of each selective agent on reproduction. We also measured discrimination between habitats with and without predators and development of naturally laid aquatic and arboreal eggs. Aquatic embryos in nature developed faster than arboreal embryos, implying no cost to aquatic egg laying. In choice tests, D. ebraccatus avoided habitats with fish, showing that they can detect aquatic egg predators. Most importantly, D. ebraccatus laid most eggs in the water when faced with only desiccation risk, but switched to laying eggs arboreally when desiccation risk and aquatic predators were both present. This provides the first experimental evidence to our knowledge that aquatic predation risk influences non-aquatic oviposition and strongly supports the hypothesis that it was a driver of the evolution of terrestrial reproduction.

Putting µ/g in a new light: plasticity in life history switch points reflects fine-scale adaptive responses.

Life history theory predicts that organisms with complex life cycles should transition between life stages when the ratio of growth rate (g) to risk of mortality (µ) in the current stage falls below that in the subsequent stage. Empirical support for this idea has been mixed. Implicit in both theory and empirical work is that the risk of mortality in the subsequent stage is unknown. However, some embryos and larvae of both vertebrates and invertebrates assess cues of post-transition predation risk and alter the timing of hatching or metamorphosis accordingly. Furthermore, although life history switch points of prey have traditionally been treated as discrete shifts in morphology or habitat, for many organisms they are continuous transitional periods within which the timing of specific developmental and behavioral events can be plastic. We studied red-eyed treefrogs (Agalychnis callidryas), which detect predators of both larvae and metamorphs, to test if plastic changes during the process of metamorphosis could reconcile the mismatch between life history theory and empirical data and if plasticity in an earlier stage transition (hatching) would affect plasticity at a subsequent stage transition (metamorphosis). We reared tadpoles from hatching until metamorphosis in a full-factorial cross of two hatching ages (early- vs. late-hatched) and the presence or absence of free-roaming predators of larvae (giant water bugs) and metamorphs (fishing spiders). Hatching age affected the times from oviposition to tail resorption and from hatching to emergence onto land, but did not alter responses to predators or developmental stage at emergence. Tadpoles did not alter their age at emergence or tail resorption in response to larval or metamorph predators, despite the fact that predators reduced tadpole density by ~30%. However, developmental stage at emergence and time needed to complete metamorphosis in the terrestrial environment were plastic and consistent with predictions of the "minimize µ/g" framework. Our results demonstrate that likely adaptive changes in life history transitions occur at previously unappreciated timescales. Consideration of plasticity in the developmental timing of ecologically important events within metamorphosis, rather than treating it as a discrete switch point, may help to reconcile inconsistencies between empirical studies of predator effects and expectations of long-standing ecological theory.

Consequences of induced hatching plasticity depend on predator community.

Many prey species face trade-offs in the timing of life history switch points like hatching and metamorphosis. Costs associated with transitioning early depend on the biotic and abiotic conditions found in the subsequent life stage. The red-eyed treefrog, Agalychnis callidryas, faces risks from predators in multiple, successive life stages, and can hatch early in response to mortality threats at the egg stage. Here we tested how the consequences of life history plasticity, specifically early hatching in response to terrestrial egg predators, depend on the assemblage of aquatic larval predators. We predicted that diverse predator assemblages would impose lower total predation pressure than the most effective single predator species and might thereby reduce the costs of hatching early. We then conducted a mesocosm experiment where we crossed hatchling phenotype (early vs. normal hatching) with five larval-predator environments (no predators, either waterbugs, dragonflies, or mosquitofish singly, or all three predator species together). The consequences of hatching early varied across predator treatments, and tended to disappear through time in some predation treatments, notably the waterbug and diverse predator assemblages. We demonstrate that the fitness costs of life history plasticity in an early life stage depend critically on the predator community composition in the next stage.

Behavioral plasticity mitigates risk across environments and predators during anuran metamorphosis.

Most animals metamorphose, changing morphology, physiology, behavior and ecological interactions. Size- and habitat-dependent mortality risk is thought to affect the evolution and plastic expression of metamorphic timing, and high predation during the morphological transition is posited as a critical selective force shaping complex life cycles. Nonetheless, empirical data on how risk changes across metamorphosis and stage-specific habitats, or how that varies with size, are rare. We examined predator-prey interactions of red-eyed treefrogs, Agalychnis callidryas, with an aquatic predator (giant water bug, Belostoma) and a semi-terrestrial predator (fishing spider, Thaumasia) across metamorphosis. We manipulated tadpole density to generate variation in metamorph size and conducted predation trials at multiple developmental stages. We quantified how frog behavior (activity) changes across metamorphic development, habitats, and predator presence or absence. In aquatic trials with water bugs, frog mortality increased with forelimb emergence, as hypothesized. In semi-terrestrial trials, contrary to predictions, predation by spiders increased, not decreased, with tail resorption. In neither case did frog size affect mortality. Frogs reduced activity upon forelimb emergence in the water, and further with emergence into air, then increased activity with tail resorption. Longer-tailed metamorphs were captured more often in spider attacks, but attacked less, as most attacks followed prey movements. Metamorphs behaviorally compensated for poor escape performance more effectively on land than in water, thus emergence timing may critically affect mortality. The developmental timing of the ecological transition between environments that select for different larval and juvenile phenotypes is an important, neglected variable in studies of complex life cycles.

One of these things is not like the other: Mixed predator cues result in lopsided phenotypic responses in a Neotropical tadpole.

Many organisms have evolved to produce different phenotypes in response to environmental variation. Dendropsophus ebraccatus tadpoles develop opposing shifts in morphology and coloration when they are exposed to invertebrate vs vertebrate predators. Each of these alternate phenotypes are adaptive, conferring a survival advantage against the predator with which tadpoles were reared but imposing a survival cost with the mismatched predator. Here, we measured the phenotypic response of tadpoles to graded cues and mixed cues of both fish and dragonfly nymphs. Prey species like D. ebraccatus commonly co-occur with both of these types of predators, amongst many others as well. In our first experiment, tadpoles increased investment in defensive phenotypes in response to increasing concentrations of predator cues. Whereas morphology only differed in the strongest predation cue, tail spot coloration differed even at the lowest cue concentration. In our second experiment, tadpoles reared with cues from both predators developed an intermediate yet skewed phenotype that was most similar to the fish-induced phenotype. Previous studies have shown that fish are more lethal than dragonfly larvae; thus tadpoles responded most strongly to the more dangerous predator, even though the number of prey consumed by each predator was the same. This may be due to D. ebraccatus having evolved a stronger response to fish or because fish produce more kairomones than do dragonflies for a given amount of food. We demonstrate that not only do tadpoles assess predation risk via the concentration of predation cues in the water, they produce a stronger response to a more lethal predator even when the strength of cues is presumed to be identical.

A treefrog with reproductive mode plasticity reveals a changing balance of selection for nonaquatic egg laying.

Nonaquatic reproduction has evolved repeatedly, but the factors that select for laying eggs on land are not well understood. The treefrog Dendropsophus ebraccatus has plasticity in its reproductive mode, laying eggs that successfully develop in or out of water. This permits the first experimental comparison of the selective agents that shape adult oviposition behavior and embryo developmental capacity. I quantified the sources and strengths of arboreal and aquatic egg mortality and how mortality varies with weather patterns, and I assessed 39 years of daily rainfall patterns to infer historic levels of egg mortality and effects of climate change on the selective balance between aquatic and nonaquatic egg deposition. Aquatic predators and desiccation were the strongest selective agents in water and air, respectively. Egg mortality varied with weather such that aquatic oviposition was advantageous when rainfall was low but laying eggs out of water increased survival when rainfall was high. Additionally, I found that since 1972 there have been significant changes in the rainfall patterns in central Panama, and this has altered the selective landscape acting on egg-laying behavior. This work provides insight into the evolution and maintenance of adaptive phenotypic plasticity as well as historic and current selection on reproduction.

Estimating female malaria mosquito age by quantifying Y-linked genes in stored male spermatozoa.

Vector control strategies are among the most effective measures to combat mosquito-borne diseases, such as malaria. These strategies work by altering the mosquito age structure through increased mortality of the older female mosquitoes that transmit pathogens. However, methods to monitor changes to mosquito age structure are currently inadequate for programmatic implementation. Female mosquitoes generally mate a single time soon after emergence and draw down spermatozoa reserves with each oviposition cycle. Here, we demonstrate that measuring spermatozoa quantity in female Anopheles mosquitoes is an effective approach to assess mosquito age. Using multiplexed qPCR targeted at male spermatozoa, we show that Y-linked genes in female mosquitoes are exclusively found in the spermatheca, the organ that houses spermatozoa, and the quantity of these gene sequences significantly declines with age. The method can accurately identify mosquitoes more than 10 days old and thus old enough to potentially transmit pathogens harbored in the salivary glands during blood feeding. Furthermore, mosquito populations that differ by 10% in daily survivorship have a high likelihood of being distinguished using modest sample sizes, making this approach scalable for assessing the efficacy of vector intervention control programs.

Salinity increases growth and pathogenicity of water mold to cause mortality and early hatching in Rana sylvatica embryos.

Amphibian embryos often suffer increased mortality and altered hatching when exposed to road deicing salt runoff or pathogens such as water molds. However, the combined effects of such contaminants on embryos remain understudied. To test how pond salinization interacts with water mold (Saprolegniasp.) to influence hatching timing and survival, we first measured pond water conductivity and temperature and quantified the prevalence and abundance of water mold in four ponds in an ecological preserve. Second, we experimentally placed wood frog (Rana sylvatica) embryos in the presence or absence of water mold, crossed with environmentally realistic salt concentrations (100, 300 or 600 µS). Lastly, we quantified growth and colonization of water mold in this range of salinities. Our results demonstrate that salt had synergistic effects with water mold exposure that affected hatching time, though water mold had less of an effect at higher salinities. Water mold significantly reduced egg survival whereas salt did not. Higher salinities also increased water mold growth and colonization on new substrates. These results indicate that road salt runoff may enhance colonization of amphibian eggs by water molds increasing mortality and premature hatching of surviving embryos, which may in turn have detrimental effects on amphibian communities.

Amphibian embryo and parental defenses and a larval predator reduce egg mortality from water mold.

Water molds attack aquatic eggs worldwide and have been associated with major mortality events in some cases, but typically only in association with additional stressors. We combined field observations and laboratory experiments to study egg stage defenses against pathogenic water mold in three temperate amphibians. Spotted salamanders (Ambystoma maculatum) wrap their eggs in a protective jelly layer that prevents mold from reaching the embryos. Wood frog (Rana sylvatica) egg masses have less jelly but are laid while ponds are still cold and mold growth is slow. American toad (Bufo americanus) eggs experience the highest infection levels. They are surrounded by thin jelly and are laid when ponds have warmed and mold grows rapidly. Eggs of all three species hatched early when infected, yielding smaller and less developed hatchlings. This response was strongest in B. americanus. Precocious hatching increased vulnerability of wood frog hatchlings to invertebrate predators. Finally, despite being potential toad hatchling predators, R. sylvatica tadpoles can have a positive effect on B. americanus eggs. They eat water mold off infected toad clutches, increasing their hatching success.

Plastic hatching timing by red-eyed treefrog embryos interacts with larval predator identity and sublethal predation to affect prey morphology but not performance.

Many animals respond to predation risk by altering their morphology, behavior, or life-history. We know a great deal about the cues prey respond to and the changes to prey that can be induced by predation risk, but less is known about how plastic responses to predators may be affected by separate plastic responses occurring earlier in life, particularly during the embryonic period. Embryos of a broad array of taxa can respond to egg- or larval-stage risks by altering hatching timing, which may alter the way organisms respond to future predators. Using the red-eyed treefrog (Agalychnis callidryas), a model for understanding the effects of plasticity across life-stages, we assessed how the combined effects of induced variation in the timing of embryo hatching and variation in the larval predator community impacted tadpole morphology, pigmentation and swimming performance. We found that A. callidryas tadpoles developed deeper tail muscles and fins and darker pigmentation in response to fish predators, either when alone or in diverse community with other predators. Tadpoles altered morphology much less so to dragonfly naiads or water bugs. Interestingly, morphological responses to predators were also affected by induced differences in hatching age, with early and late-hatched tadpoles exhibiting different allometric relationships between tail height and body length in different predator environments. Beyond induced morphological changes, fish predators often damaged tadpoles' tails without killing them (i.e., sublethal predation), but these tadpoles swam equally quickly to those with fully intact tails. This was due to the fact that tadpoles with more damaged tails increased tail beats to achieve equal swimming speed. This study demonstrates that plastic phenotypic responses to predation risk can be influenced by a complex combination of responses to both the embryo and larval environments, but also that prey performance can be highly resilient to sublethal predation.

Prey responses to predator chemical cues: disentangling the importance of the number and biomass of prey consumed.

To effectively balance investment in predator defenses versus other traits, organisms must accurately assess predation risk. Chemical cues caused by predation events are indicators of risk for prey in a wide variety of systems, but the relationship between how prey perceive risk in relation to the amount of prey consumed by predators is poorly understood. While per capita predation rate is often used as the metric of relative risk, studies aimed at quantifying predator-induced defenses commonly control biomass of prey consumed as the metric of risk. However, biomass consumed can change by altering either the number or size of prey consumed. In this study we determine whether phenotypic plasticity to predator chemical cues depends upon prey biomass consumed, prey number consumed, or both. We examine the growth response of red-eyed treefrog tadpoles (Agalychnis callidryas) to cues from a larval dragonfly (Anax amazili). Biomass consumed was manipulated by either increasing the number of prey while holding individual prey size constant, or by holding the number of prey constant and varying individual prey size. We address two questions. (i) Do prey reduce growth rate in response to chemical cues in a dose dependent manner? (ii) Does the magnitude of the response depend on whether prey consumption increases via number or size of prey? We find that the phenotypic response of prey is an asymptotic function of prey biomass consumed. However, the asymptotic response is higher when more prey are consumed. Our findings have important implications for evaluating past studies and how future experiments should be designed. A stronger response to predation cues generated by more individual prey deaths is consistent with models that predict prey sensitivity to per capita risk, providing a more direct link between empirical and theoretical studies which are often focused on changes in population sizes not individual biomass.

Apomorphine-induced alterations in striatal and substantia nigra pars reticulata glutamate following unilateral loss of striatal dopamine.

We have reported time-dependent changes in extracellular glutamate within the striatum at 1 and 3 months following a unilateral lesion of the nigrostriatal pathway using the neurotoxin, 6-hydroxydopamine (6-OHDA) (Meshul, C.K., Emre, N., Nakamura, C.M., Allen, C., Donohue, M.K., Buckman, J.F., 1999. Time-dependent changes in striatal glutamate synapses following a 6-hydroxydopamine lesion. Neurosci. 88, 1-16.). The aim of the present study was to determine the effects of such a lesion on glutamate within the substantia nigra pars reticulata (SN-PR) and the effect of subchronic administration of the dopamine D-1/D-2 agonist, apomorphine, on extracellular glutamate within both the striatum and the SN-PR using in vivo microdialysis. One month after the lesion, there is an increase in extracellular glutamate within the striatum and apomorphine treatment leads to a further increase. Within the SN-PR, a loss of striatal dopamine leads to a decrease in extracellular glutamate, while apomorphine treatment leads to a further decrease in nigral glutamate. Three months after a 6-OHDA lesion, there is a decrease in extracellular striatal glutamate, with apomorphine administration leading to essentially no further change in glutamate. The loss of striatal dopamine increased extracellular glutamate within the SN-PR while apomorphine administration resulted in a decrease in extracellular glutamate back to the value observed in the control group. The data suggests that the increase in striatal glutamate 1 month following a 6-OHDA lesion alone or following subchronic apomorphine is consistent with the hypothesis that a decrease in glutamate within the SN-PR leads to activation of the thalamo-cortico-striatal pathway. The decrease in striatal glutamate 3 months after a nigrostriatal lesion is also consistent with the observed increase in extracellular glutamate within the SN-PR, thus leading to a decrease in output of the thalamo-cortico-striatal pathway.

Acute and subchronic MPTP administration differentially affects striatal glutamate synaptic function.

We previously reported that 1 month following unilateral loss (>95%) of striatal dopamine, there is an increase in striatal glutamate function as measured by in vivo microdialysis and quantitative immuno-gold electron microscopy, Neuroscience 88, 1-16). The goal of this study was to determine the effect of bilateral loss of striatal dopamine on striatal glutamate function following acute or subchronic administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57/B6J mice. Animals were administered either single injections (ip) of 30 mg/kg/day for 7 days (subchronically treated group) or 20 mg/kg x 4 doses every 2 h (acutely treated group) of the toxin or saline. One month following the first injection, there was a 44 and 65% loss in the relative density of tyrosine hydroxylase (TH) immunolabeling within the dorsolateral striatum in the subchronically and acutely MPTP-treated groups compared to the saline group, respectively. There was a decrease in the basal level of extracellular glutamate within the striatum in the subchronically MPTP-treated animals compared to an increase in the acutely treated group in relationship to the saline group. Ultrastructurally, only in the acutely MPTP-treated group was there a decrease in the density of glutamate immunolabeling within nerve terminals associated with an asymmetrical synaptic contact in the dorsolateral striatum compared to either the subchronic or saline groups. In addition, there was a decrease in the relative density of GluR-2/3 subunit immunolabeling within the dorsolateral striatum in the acute MPTP compared to the saline group. These data indicate that differences in striatal glutamate function appear to be associated with the dosing interval of MPTP administration and the variable loss of striatal TH immunolabeling.

Lesion of subthalamic or motor thalamic nucleus in 6-hydroxydopamine-treated rats: effects on striatal glutamate and apomorphine-induced contralateral rotations.

A unilateral lesion of the rat nigrostriatal pathway with 6-hydroxydopamine (6-OHDA) results in a decrease in the basal extracellular level of striatal glutamate, a nearly complete loss of tyrosine hydroxylase (TH) immunolabeling, an increase in the density of glutamate immunogold labeling within nerve terminals making an asymmetrical synaptic contact, and an increase in the number of apomorphine-induced contralateral rotations. [Meshul et al. (1999) Neuroscience 88:1-16; Meshul and Allen (2000) Synapse 36:129-142]. In Parkinson's disease, a lesion of either the subthalamic nucleus (STN) or the motor thalamic nucleus relieves the patient of some of the motor difficulties associated with this disorder. In this rodent model, either the STN or motor thalamic nucleus was electrolytically destroyed 2 months following a unilateral 6-OHDA lesions. Following a lesion of either the STN or motor thalamic nucleus in 6-OHDA-treated rats, there was a significant decrease (40-60%) in the number of apomorphine-induced contralateral rotations compared to the 6-OHDA group. There was a significant decrease (<30%) in the basal extracellular level of striatal glutamate in all of the experimental groups compared to the sham group. Following an STN and/or 6-OHDA lesion, the decrease in striatal extracellular levels was inversely associated with an increase in the density of nerve terminal glutamate immunolabeling. There was no change in nerve terminal glutamate immunogold labeling in either the motor thalamic or motor thalamic plus 6-OHDA lesion groups compared to the sham group. The decrease in the number of apomorphine-induced rotations was not due to an increase in TH immunolabeling (i.e., sprouting) within the denervated striatum. This suggests that alterations in striatal glutamate appear not to be directly involved in the STN or motor thalamic lesion-induced reduction in contralateral rotations.