Expression patterns of melanin-related genes are linked to crypsis and conspicuousness in a pumpkin toadlet.

Colour signals play pivotal roles in different communication systems, and the evolution of these characters has been associated with behavioural ecology, integumentary production processes and perceptual mechanisms of the species involved. Here, we present the first insight into the molecular and histological basis of skin colour polymorphism within a miniaturized species of pumpkin toadlet, potentially representing the lowest size threshold for colour polytypism in tetrapods. Brachycephalus actaeus exhibits a coloration ranging from cryptic green to conspicuous orange skin, and our findings suggest that colour morphs differ in their capability to be detected by potential predators. We also found that the distribution and abundance of chromatophores are variable in the different colour morphs. The expression pattern of coloration related genes was predominantly associated with melanin synthesis (including dct, edn1, mlana, oca2, pmel, slc24a5, tyrp1 and wnt9a). Up-regulation of melanin genes in grey, green and brown skin was associated with higher melanophore abundance than in orange skin, where xanthophores predominate. Our findings provide a significant foundation for comparing and understanding the diverse pathways that contribute to the evolution of pigment production in the skin of amphibians.

Importance of Genetic-Fitness Correlations for the Conservation of Amphibians.

Endangered animals suffer from isolation of their habitats. Isolation leads to a reduction in population size as well as a decrease in genetic diversity and a concomitant increase in the risk of extinction. Amphibians are the most endangered vertebrate class. Besides habitat loss, fragmentation and isolation, amphibians are threatened by emerging diseases e.g., chytrid fungus or Ranavirus. By employing experiments, researchers investigate whether changes in genetic diversity within or among isolated populations affect amphibian fitness. While genetic diversity estimates are based on molecular markers, typically microsatellites, fitness is mostly measured as tadpole performance in rearing experiments often under varying environmental conditions. Tadpole performances (e.g., body mass, growth rate and survival) have been found to be negatively affected by low genetic diversity, as several studies have found a positive association between genetic diversity and these fitness traits. Moreover, infection with pathogens also seems to be more likely in individuals or populations with lower genetic diversity. Overall, these genetic-fitness correlations seem to be more pronounced or detectable in smaller, declining populations but not in larger populations. Genomic studies, which sample a larger fraction of the genome, are still scarce in the conservation genetic literature on amphibians. These are likely to increase in upcoming years and may reveal adaptive variants that protect against dangerous pathogens or environmental changes. Altogether, genetic-fitness correlation studies should be a priority in order to develop effective management plans for the genetic rescue of isolated, imperilled amphibian populations.

Interactive, Browser-Based Graphics to Visualize Complex Data in Education of Biomedical Sciences for Veterinary Students.

In veterinary education, data from biomedical or natural sciences are mostly presented in the form of static or animated graphics with no or little amount of interactivity. These kinds of presentations are, however, often not sufficient to depict the complexity of the data or the presented topic. Interactive graphics, which allow to dynamically change data and related graphics, have rarely been considered as teaching tool in higher education of biomedical disciplines for veterinary education so far. In order to study the applicability and the usefulness of interactive graphics in biomedical disciplines for lecturers and students in veterinary education, three different courses from biomedical disciplines were exemplarily implemented as interactive graphics and evaluated in a pilot study by a survey amongst lecturers and students of our university. The interactive graphics were built using the Shiny environment, a web-based application framework for the statistic software R. The survey amongst lecturers and students was based on questionnaires covering questions on the handling and usefulness of the digital teaching tools. In total, n = 327 students and n = 5 lecturers participated in the evaluation study which revealed that the interactive graphics are easy to handle for lecturers and students, and that they can increase the motivation for either teaching or learning. In total, 71% of the students affirmed that interactive graphics led to an increased interest for the presented contents and 76% expressed the wish to get taught more topics with interactive graphics. We also provide a workflow that can be used as a guideline to develop interactive graphics. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-022-01613-x.

Evidence for coloration plasticity in the yellow-bellied toad, Bombina variegata.

Phenotypic adaptation in terms of background color matching to the local habitat is an important mechanism for survival in prey species. Thus, intraspecific variation in cryptic coloration is expected among localities with dissimilar habitat features (e.g., soil, vegetation). Yellow-bellied toads (Bombina variegata) display a dark dorsal coloration that varies between populations, assumed to convey crypsis. In this study, we explored I) geographic variation in dorsal coloration and II) coloration plasticity in B. variegata from three localities differing in substrate coloration. Using avian visual modeling, we found that the brightness contrasts of the cryptic dorsa were significantly lower on the local substrates than substrates of other localities. In experiments, individuals from one population were able to quickly change the dorsal coloration to match a lighter substrate. We conclude that the environment mediates an adaptation in cryptic dorsal coloration. We suggest further studies to test the mechanisms by which the color change occurs and explore the adaptive potential of coloration plasticity on substrates of varying brightness in B. variegata and other species.

Locality, time and heterozygosity affect chytrid infection in yellow-bellied toads.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) infects numerous amphibian species worldwide and is suggested to drive population declines and extinction events. We report a study of Bd infection at the northernmost distribution of the European yellow-bellied toad Bombina variegata. A total of 577 individuals from ponds in 16 study sites were sampled for DNA and Bd throughout the breeding season. Microsatellite genotyping revealed 3 genetic clusters for the host B. variegata with an overall low genetic diversity. One of the clusters displayed a low microsatellite heterozygosity, a high inbreeding coefficient as well as high Bd infection prevalence and intensities. Multi-model estimates identified site, time of sampling, and heterozygosity to be important predictors of an individual's Bd infection status, and identified a strong effect of site on individual Bd infection intensity. The study site effects are suggestive of localized infection peaks, and the increase of individual Bd infection probabilities towards the end of the sampling period suggests cumulative infection during the breeding season. This study highlights the need for regular monitoring of Bd infection variables at multiple localities and times to gain insights into Bd dynamics. Due to the detected relationship between individual Bd infection status and heterozygosity, conservation measures should focus on the maintenance of high genetic diversity and connectivity within and among amphibian populations.

Being red, blue and green: the genetic basis of coloration differences in the strawberry poison frog (Oophaga pumilio).

BACKGROUND: Animal coloration is usually an adaptive attribute, under strong local selection pressures and often diversified among species or populations. The strawberry poison frog (Oophaga pumilio) shows an impressive array of color morphs across its distribution in Central America. Here we quantify gene expression and genetic variation to identify candidate genes involved in generating divergence in coloration between populations of red, green and blue O. pumilio from the Bocas del Toro archipelago in Panama. RESULTS: We generated a high quality non-redundant reference transcriptome by mapping the products of genome-guided and de novo transcriptome assemblies onto a re-scaffolded draft genome of O. pumilio. We then measured gene expression in individuals of the three color phenotypes and identified color-associated candidate genes by comparing differential expression results against a list of a priori gene sets for five different functional categories of coloration - pteridine synthesis, carotenoid synthesis, melanin synthesis, iridophore pathways (structural coloration), and chromatophore development. We found 68 candidate coloration loci with significant expression differences among the color phenotypes. Notable upregulated examples include pteridine synthesis genes spr, xdh and pts (in red and green frogs); carotenoid metabolism genes bco2 (in blue frogs), scarb1 (in red frogs), and guanine metabolism gene psat1 (in blue frogs). We detected significantly higher expression of the pteridine synthesis gene set in red and green frogs versus blue frogs. In addition to gene expression differences, we identified 370 outlier SNPs on 162 annotated genes showing signatures of diversifying selection, including eight pigmentation-associated genes. CONCLUSIONS: Gene expression in the skin of the three populations of frogs with differing coloration is highly divergent. The strong signal of differential expression in pteridine genes is consistent with a major role of these genes in generating the coloration differences among the three morphs. However, the finding of differentially expressed genes across pathways and functional categories suggests that multiple mechanisms are responsible for the coloration differences, likely involving both pigmentary and structural coloration. In addition to regulatory differences, we found potential evidence of differential selection acting at the protein sequence level in several color-associated loci, which could contribute to the color polymorphism.

Author Correction: Sole coloration as an unusual aposematic signal in a Neotropical toad.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Toxicity and Alkaloid Profiling of the Skin of the Golfo Dulcean Poison Frog Phyllobates vittatus (Dendrobatidae).

Frogs in the genus Phyllobates are known for the presence of batrachotoxin, a highly toxic alkaloid, in their skin. Nevertheless, Phyllobates frogs from Costa Rica and Panama (P. lugubris and P. vittatus) are considered non-toxic, as they have been reported to harbor low concentrations of this alkaloid. However, the potential toxicity of Central American Phyllobates has not been assessed experimentally. Our goal was to determine the toxicity of the whole skin of P. vittatus, an endemic species from the Southeastern Pacific region of Costa Rica. We performed median lethal dose (LD50) tests in mice to determine general toxicity, and an irritant assay based on the behavioral responses of mice to subcutaneous injection, to determine differences in irritability, as a measure of toxicity, among three study localities. Using UPLC-ESI-QTOF, we obtained chemical profiles of the methanolic extract of frog skins. Due to the absence of mortality at the studied doses, we were unable to estimate LD50. However, we recorded a list of toxicity symptoms in mice that are consistent with cardiotoxic effects, and found that mice presented more symptoms at higher concentrations of skin extracts during the first hour of the LD50 assays, recovering completely at all doses by the end of the assay. On the other hand, we did not detect differences in irritability among studied localities. Additionally, we putatively identified three toxic alkaloids (Batrachotoxinin A, DHQ 251A and Lehm 275A). This study provides the first experimental data on the toxicity and associated symptoms in mice, as well as the chemical profile of the skin of P. vittatus. We suggest that the skin alkaloids of P. vitattus may confer a chemical defense towards predators.

Mate Choice versus Mate Preference: Inferences about Color-Assortative Mating Differ between Field and Lab Assays of Poison Frog Behavior.

Codivergence of mating traits and mate preferences can lead to behavioral isolation among lineages in early stages of speciation. However, mate preferences limit gene flow only when expressed as mate choice, and numerous factors might be more important than preferences in nature. In the extremely color polytypic strawberry poison frog (Oophaga pumilio), female mate preferences have codiverged with color in most allopatric populations tested. Whether these lab-assayed preferences predict mating (gene flow) in the wild remains unclear. We observed courting pairs in a natural contact zone between red and blue lineages until oviposition or courtship termination. We found color-assortative mating in a disturbed habitat with high population density but not in a secondary forest with lower density. Our results suggest color-assortative O. pumilio mate choice in the wild but also mating patterns that do not match those predicted by lab-assayed preferences.

Sole coloration as an unusual aposematic signal in a Neotropical toad.

Many animals have evolved remarkable strategies to avoid predation. In diurnal, toxic harlequin toads (Atelopus) from the Amazon basin, we find a unique colour signal. Some Atelopus populations have striking red soles of the hands and feet, visible only when walking. When stationary, the toads are hard to detect despite their yellow-black dorsal coloration. Consequently, they switch between high and low conspicuousness. Interestingly, some populations lack the extra colour display of the soles. We found comprehensive support that the red coloration can act as an aposematic signal directed towards potential predators: red soles are significantly more conspicuous than soles lacking red coloration to avian predators and the presence of the red signal significantly increases detection. Further, toads with red soles show bolder behaviour by using higher sites in the vegetation than those lacking this signal. Field experiments hint at a lower attack risk for clay models with red soles than for those lacking the signal, in a population where the red soles naturally occur. We suggest that the absence of the signal may be explained by a higher overall attack risk or potential differences of predator community structure between populations.

The conspicuous postmetamorphic coloration of fire salamanders, but not their toxicity, is affected by larval background albedo.

Experimental work in the early 20th century showed that background albedo experienced by larvae of the fire salamander (Salamandra salamandra) induce a durable morphological modification of the postmetamorphic color pattern, which needed confirmation due to the controversies regarding Paul Kammerer's experiments. Such a carry-over effect would be relevant as the black and yellow pattern of the alkaloid-containing adult fire salamanders has been suggested to serve as an aposematic signal. Hence, we hypothesized that (a) adult coloration is conspicuous to potential predators under light conditions at night, given the nocturnal activity of this species, and (b) a condition affecting the salamander's coloration pattern would also affect its toxicity to maintain a quantitatively honest aposematic signal. To test the first hypothesis, we used spectrometry to model the vision of potential avian and snake predators and confirmed that fire salamander's black-and-yellow pattern is contrasting enough against the forest leaf litter to be considered conspicuous at night. To test the second hypothesis, we first confirmed the background carry-over effect on black and yellow proportions in the dorsal skin of experimentally reared fire salamanders, using a rigorous experimental design. Then, we calculated the conspicuousness and determined the alkaloid profiles of these individuals. We did not find a correlation between conspicuousness and toxicity at the intrapopulation level. Moreover, there was no background carry-over effect on the alkaloid profile. We discuss our results in a physiological, ecological, evolutionary, and historical context.

Mating status correlates with dorsal brightness in some but not all poison frog populations.

Sexual signals are important for intraspecific communication and mate selection, but their evolution may be driven by both natural and sexual selection, and stochastic processes. Strawberry poison frogs (Oophaga pumilio) show strong color divergence among populations, but coloration also varies among individuals of the same population. The importance of coloration for female mate choice has been studied intensely, and sexual selection seems to affect color divergence in strawberry poison frogs. However, the effect of coloration on mating success under field conditions has received very little attention. Furthermore, few studies examined how phenotypic variation among individuals of the same color morph affects mate selection under natural conditions. We measured the spectral reflectance of courting and noncourting individuals and their background substrates in three geographically separated populations. In one population (Sarapiquí, Costa Rica), we found that naturally occurring courting pairs of males and females had significantly brighter dorsal coloration than individual males and females not engaged in courtship interactions. Our field observations suggest that, in the wild, females prefer brighter males while the reason for the higher courtship activity of brighter females remains unclear. Overall our results imply that brightness differences among individuals of the same color morph may actually affect reproductive success in some populations of strawberry poison frogs.

An Analysis of Predator Selection to Affect Aposematic Coloration in a Poison Frog Species.

Natural selection is widely noted to drive divergence of phenotypic traits. Predation pressure can facilitate morphological divergence, for example the evolution of both cryptic and conspicuous coloration in animals. In this context Dendrobatid frogs have been used to study evolutionary forces inducing diversity in protective coloration. The polytypic strawberry poison frog (Oophaga pumilio) shows strong divergence in aposematic coloration among populations. To investigate whether predation pressure is important for color divergence among populations of O. pumilio we selected four mainland populations and two island populations from Costa Rica and Panama. Spectrometric measurements of body coloration were used to calculate color and brightness contrasts of frogs as an indicator of conspicuousness for the visual systems of several potential predators (avian, crab and snake) and a conspecific observer. Additionally, we conducted experiments using clay model frogs of different coloration to investigate whether the local coloration of frogs is better protected than non-local color morphs, and if predator communities vary among populations. Overall predation risk differed strongly among populations and interestingly was higher on the two island populations. Imprints on clay models indicated that birds are the main predators while attacks of other predators were rare. Furthermore, clay models of local coloration were equally likely to be attacked as those of non-local coloration. Overall conspicuousness (and brightness contrast) of local frogs was positively correlated with attack rates by birds across populations. Together with results from earlier studies we conclude that conspicuousness honestly indicates toxicity to avian predators. The different coloration patterns among populations of strawberry poison frogs in combination with behavior and toxicity might integrate into equally efficient anti-predator strategies depending on local predation and other ecological factors.

Not everything is black and white: color and behavioral variation reveal a continuum between cryptic and aposematic strategies in a polymorphic poison frog.

Aposematism and crypsis are often viewed as two extremes of a continuum of visual conspicuousness to predators. Theory predicts that behavioral and coloration conspicuousness should vary in tandem along the conspicuousness spectrum for antipredator strategies to be effective. Here we used visual modeling of contrast and behavioral observations to examine the conspicuousness of four populations of the granular poison frog, Oophaga granulifera, which exhibits almost continuous variation in dorsal color. The patterns of geographic variation in color, visual contrast, and behavior support a gradient of overall conspicuousness along the distribution of O. granulifera. Red and green populations, at the extremes of the color distribution, differ in all elements of color, contrast, and behavior, strongly reflecting aposematic and cryptic strategies. However, there is no smooth cline in any elements of behavior or coloration between the two extremes. Instead populations of intermediate colors attain intermediate conspicuousness by displaying different combinations of aposematic and cryptic traits. We argue that coloration divergence among populations may be linked to the evolution of a gradient of strategies to balance the costs of detection by predators and the benefits of learned aversion.

Only distance matters - non-choosy females in a poison frog population.

BACKGROUND: Females have often been shown to exhibit preferences for certain male traits. However, little is known about behavioural rules females use when searching for mates in their natural habitat. We investigated mate sampling tactics and related costs in the territorial strawberry poison frog (Oophaga pumilio) possessing a lek-like mating system, where both sequential and simultaneous sampling might occur. We continuously monitored the sampling pattern and behaviour of females during the complete period between two successive matings. RESULTS: We found no evidence that females compared males by visiting them. Instead females mated with the closest calling male irrespective of his acoustic and physical traits, and territory size. Playback experiments in the natural home ranges of receptive females revealed that tested females preferred the nearest speaker and did not discriminate between low and high call rates or dominant frequencies. CONCLUSIONS: Our results suggest that females of O. pumilio prefer the closest calling male in the studied population. We hypothesize that the sampling tactic in this population is affected by 1) a strongly female biased sex ratio and 2) a low variance in traits of available males due to strong male-male competition, preventing low quality males from defending a territory and mating.

Ecological and genetic divergence between two lineages of middle American túngara frogs Physalaemus (= Engystomops) pustulosus.

BACKGROUND: Uncovering how populations of a species differ genetically and ecologically is important for understanding evolutionary processes. Here we combine population genetic methods (microsatellites) with phylogenetic information (mtDNA) to define genetic population clusters of the wide-spread Neotropical túngara frog (Physalaemus pustulosus). We measure gene flow and migration within and between population clusters and compare genetic diversity between population clusters. By applying ecological niche modeling we determine whether the two most divergent genetic groups of the túngara frog (1) inhabit different habitats, and (2) are separated geographically by unsuitable habitat across a gap in the distribution. RESULTS: Most population structure is captured by dividing all sample localities into two allopatric genetic lineages. The Northern genetic lineage (NW Costa Rica) is genetically homogenous while the Southern lineage (SW Costa Rica and Panama) is sub-divided into three population clusters by both microsatellite and mtDNA analyses. Gene flow is higher within the Northern lineage than within the Southern lineage, perhaps due to increased landscape heterogeneity in the South. Niche modeling reveals differences in suitable habitat between the Northern and Southern lineages: the Northern lineage inhabits dry/pine-oak forests, while the Southern lineage is confined to tropical moist forests. Both lineages seem to have had little movement across the distribution gap, which persisted during the last glacial maximum. The lack of movement was more pronounced for the Southern lineage than for the Northern lineage. CONCLUSIONS: This study confirms the finding of previous studies that túngara frogs diverged into two allopatric genetic lineages north and south of the gap in the distribution in central Costa Rica several million years ago. The allopatric distribution is attributed to unsuitable habitat and probably other unknown ecological factors present across the distribution gap. Niche conservatism possibly contributes to preventing movements across the gap and gene flow between both groups. Genetic and ecological data indicate that there is the potential for ecological divergence in allopatry between lineages. In this context we discuss whether the Northern and Southern lineages should be recognized as separate species, and we conclude that further studies of pre- and post-zygotic isolation are needed for a final assessment. Identified population clusters should motivate future behavioral and ecological research regarding within-species biodiversity and speciation mechanisms.

Multigene phylogenetic analysis of Lygodactylus dwarf geckos (Squamata: Gekkonidae).

The genus Lygodactylus is a group of small, diurnal geckos distributed mainly in Africa and Madagascar. A closely related, monotypic genus is Microscalabotes from Madagascar. We examined fragments of two moderate- to slow-evolving nuclear genes (RAG-1, RAG-2) and of two fast-evolving mitochondrial genes (16SrRNA, cytochrome b) to estimate phylogenetic relationships among 28 (of about 60) species (including Microscalabotes). Since cytochrome b provided little resolution for analysis of interspecific relationships in Lygodactylus, it was excluded from the concatenated Bayesian analysis. Our well-resolved Bayesian inference tree and its resulting clades confirm many previously suggested relationships among dwarf geckos based mainly on morphological characters, while several novel relationships lead us to regroup some species and to synonymize the genus Microscalabotes with the genus Lygodactylus. Our phylogenetic analysis well supports the monophyly of Lygodactylus (including Microscalabotes). However, it rejects monophyly of the Madagascan Lygodactylus. On the other hand, the data do not reject monophyly of the African forms. Though Africa is currently the main centre of diversity of Lygodactylus, our data suggest that the most parsimonious scenario would be a Madagascan origin, one dispersal to Africa, and one back to Madagascar.

Geographic variation of genetic and behavioral traits in northern and southern tüngara frogs.

We use a combination of microsatellite marker analysis and mate-choice behavior experiments to assess patterns of reproductive isolation of the túngara frog Physalaemus pustulosus along a 550-km transect of 25 populations in Costa Rica and Panama. Earlier studies using allozymes and mitochondrial DNA defined two genetic groups of túngara frogs, one ranging from Mexico to northern Costa Rica (northern group), the second ranging from Panama to northern South America (southern group). Our more fine-scale survey also shows that the northern and southern túngara frogs are genetically different and geographically separated by a gap in the distribution in central Pacific Costa Rica. Genetic differences among populations are highly correlated with geographic distances. Temporal call parameters differed among populations as well as between genetic groups. Differences in calls were explained better by geographic distance than by genetic distance. Phonotaxis experiments showed that females preferred calls of males from their own populations over calls of males from other populations in about two-thirds to three-fourths of the contrasts tested. In mating experiments, females and males from the same group and females from the north with males from the south produced nests and tadpoles. In contrast, females from the south did not produce nests or tadpoles with males from the north. Thus, northern and southern túngara frogs have diverged both genetically and bioacoustically. There is evidence for some prezygotic isolation due to differences in mate recognition and fertilization success, but such isolation is hardly complete. Our results support the general observation that significant differences in sexual signals are often not correlated with strong genetic differentiation.

Spatial distributions of male and female strawberry poison frogs and their relation to female reproductive resources.

In many species with a resource-based mating system, males defend resources to increase their attractiveness to females. In the strawberry poison frog, Dendrobates pumilio, suitable tadpole-rearing sites appear to be a limited resource for females. Territorial males have been suggested to defend tadpole-rearing sites to increase their access to females. In this study we investigate the spatial association between tadpole-rearing sites and the sexes as well as the spatial association of males and females. If strawberry poison frogs have resource defense polygyny, we expect males and females to be associated with tadpole-rearing sites and that females will deposit their offspring in tadpole-rearing sites inside the territories of their mates. To test this hypothesis, home range and core area sizes were calculated for both sexes and the association patterns were compared in two areas that differed in their abundance of tadpole-rearing sites. Home ranges and core areas of females were much larger than male home ranges. Females showed a clumped distribution in the vicinity of tadpole-rearing sites. Males were not clumped and were less associated with tadpole-rearing sites. Females generally did not use tadpole-rearing sites in the territory of their mates and we therefore conclude that males did not defend tadpole-rearing sites for females. Our data are consistent with the general assumption that female distribution is influenced by resource distribution and that male distribution depends on female distribution. Nevertheless, the distribution of D. pumilio females was also influenced by male spacing patterns. Males probably initially establish their core areas where female density is high and then females move among territories to sample males. Males compete vigorously for places with high female density, the defense of which is likely important for enhancing their mating success. In general, the spacing patterns did not differ between populations but the sex ratio was strongly female biased in the habitat with more tadpole-rearing sites, reflecting the direct reliance of females on these resources.