Small population size and possible extirpation of the threatened Malagasy poison frog Mantella cowanii.

Amphibians are experiencing severe population declines, requiring targeted conservation action for the most threatened species and habitats. Unfortunately, we do not know the basic demographic traits of most species, which hinders population recovery efforts. We studied one of Madagascar's most threatened frog species, the harlequin mantella (Mantella cowanii), to confirm it is still present at historic localities and estimate annual survival and population sizes. We surveyed eleven of all thirteen known localities and were able to detect the species at eight. Using a naïve estimate of detection probability from sites with confirmed presence, we estimated 1.54 surveys (95% CI [1.10-2.37]) are needed to infer absence with 95% confidence, suggesting the three populations where we did not detect M. cowanii are now extirpated. However, we also report two new populations for the first time. Repeated annual surveys at three sites showed population sizes ranged from 13-137 adults over 3-8 years, with the most intensively surveyed site experiencing a >80% reduction in population size during 2015-2023. Annual adult survival was moderately high (0.529-0.618) and we recaptured five individuals in 2022 and one in 2023 first captured as adults in 2015, revealing the maximum lifespan of the species in nature can reach 9 years and beyond. Our results confirm M. cowanii is characterized by a slower life history pace than other Mantella species, putting it at greater extinction risk. Illegal collection for the international pet trade and continued habitat degradation are the main threats to the species. We recommend conservation efforts continue monitoring M. cowanii populations and reassess the International Union for Conservation of Nature (IUCN) Red List status because the species may be Critically Endangered rather than Endangered based on population size and trends.

The importance of reproductive isolation in driving diversification and speciation within Peruvian mimetic poison frogs (Dendrobatidae).

To explain how populations with distinct warning signals coexist in close parapatry, we experimentally assessed intrinsic mechanisms acting as reproductive barriers within three poison-frog species from the Peruvian Amazon belonging to a Müllerian mimetic ring (Ranitomeya variabilis, Ranitomeya imitator and Ranitomeya fantastica). We tested the role of prezygotic and postzygotic isolation barriers between phenotypically different ecotypes of each species, using no-choice mating experiments and offspring survival analysis. Our results show that prezygotic mating preference did not occur except for one specific ecotype of R. imitator, and that all three species were able to produce viable inter-population F1 hybrids. However, while R. variabilis and R. imitator hybrids were able to produce viable F2 generations, we found that for R. fantastica, every F1 hybrid males were sterile while females remained fertile. This unexpected result, echoing with Haldane's rule of speciation, validated phylogenetic studies which tentatively diagnose these populations of R. fantastica as two different species. Our work suggests that postzygotic genetic barriers likely participate in the extraordinary phenotypic diversity observed within Müllerian mimetic Ranitomeya populations, by maintaining species boundaries.

What Makes a Mimic? Orange, Red, and Black Color Production in the Mimic Poison Frog (Ranitomeya imitator).

Aposematic organisms rely on their conspicuous appearance to signal that they are defended and unpalatable. Such phenotypes are strongly tied to survival and reproduction. Aposematic colors and patterns are highly variable; however, the genetic, biochemical, and physiological mechanisms producing this conspicuous coloration remain largely unidentified. Here, we identify genes potentially affecting color variation in two color morphs of Ranitomeya imitator: the orange-banded Sauce and the redheaded Varadero morphs. We examine gene expression in black and orange skin patches from the Sauce morph and black and red skin patches from the Varadero morph. We identified genes differentially expressed between skin patches, including those that are involved in melanin synthesis (e.g. mlana, pmel, tyrp1), iridophore development (e.g. paics, ppat, ak1), pteridine synthesis (e.g. gch1, pax3-a, xdh), and carotenoid metabolism (e.g. dgat2, rbp1, scarb2). In addition, using weighted correlation network analysis, we identified the top 50 genes with high connectivity from the most significant network associated with gene expression differences between color morphs. Of these 50 genes, 13 were known to be related to color production (gch1, gmps, gpr143, impdh1, mc1r, pax3-a, pax7, ppat, rab27a, rlbp1, tfec, trpm1, xdh).

Genome Assembly of the Dyeing Poison Frog Provides Insights into the Dynamics of Transposable Element and Genome-Size Evolution.

Genome size varies greatly across the tree of life and transposable elements are an important contributor to this variation. Among vertebrates, amphibians display the greatest variation in genome size, making them ideal models to explore the causes and consequences of genome size variation. However, high-quality genome assemblies for amphibians have, until recently, been rare. Here, we generate a high-quality genome assembly for the dyeing poison frog, Dendrobates tinctorius. We compare this assembly to publicly available frog genomes and find evidence for both large-scale conserved synteny and widespread rearrangements between frog lineages. Comparing conserved orthologs annotated in these genomes revealed a strong correlation between genome size and gene size. To explore the cause of gene-size variation, we quantified the location of transposable elements relative to gene features and find that the accumulation of transposable elements in introns has played an important role in the evolution of gene size in D. tinctorius, while estimates of insertion times suggest that many insertion events are recent and species-specific. Finally, we carry out population-scale mobile-element sequencing and show that the diversity and abundance of transposable elements in poison frog genomes can complicate genotyping from repetitive element sequence anchors. Our results show that transposable elements have clearly played an important role in the evolution of large genome size in D. tinctorius. Future studies are needed to fully understand the dynamics of transposable element evolution and to optimize primer or bait design for cost-effective population-level genotyping in species with large, repetitive genomes.

Does testosterone underly the interplay between male traits and territorial behavior in neotropical poison frogs?

The ability of individual animals to defend a territory as well as various phenotypic and behavioral traits may be targets of sexual selection used by males to evaluate their competitors or by females to choose males. A frequent question in animal behavior is whether male traits and characteristics of their territory are correlated and what are the mechanisms that may mediate such associations when they exist. Because hormones link phenotype to behavior, by studying the role of testosterone in territoriality one may come closer to understanding the mechanisms mediating correlations or lack thereof between characteristics of territories and of males. We evaluated whether variation in characteristics of territories (size and quality) are correlated with variation in morphology, coloration, testosterone, heterozygosity, and calls in two species of poison frogs. The Amazonian frog Allobates aff. trilineatus exhibits male care and defends territories only during the breeding season, while the endangered frog Oophaga lehmanni displays maternal care and defends territories throughout the year. We found that morphological traits (body length, weight, thigh size), call activity, and testosterone levels correlated with size and various indicators of quality of the territory. However, the direction of these correlations (whether positive or negative) and which specific morphological, acoustic traits or testosterone level variables covaried depended on the species. Our findings highlight an endocrine pathway as part of the physiological machinery that may underlie the interplay between male traits and territorial behavior. We were able to identify some male traits related to territory attributes, but whether females choose males based on these traits requires further research.

Native lizards taught to avoid toxic toads by released toadlets.

Exposing monitor lizards to thousands of young cane toads helped them survive once the adult toads invaded.

Distribution and diversification of Adelphobates, emblematic poison frogs from Brazilian Amazonia.

Adelphobates contains three species, and the inaccurate identification of A. quinquevittatus and the scarcity of records of A. castaneoticus complicate inference of their distributions; the latter species occurs in sympatry with A. galactonotus. Our objective was to revise the distributions of Adelphobates by compiling data and modeling habitat suitability, as range limits may be shaped by landscape features and biotic interactions. We initially analyzed the existence of operational taxonomic units within the nominal species and subsequently inferred the observed and potential distributions, taking into account the possible independent lineages for the three species, and we also generated a molecular timetree to understand the chronology of interspecific diversification events. Adelphobates quinquevittatus was found to have a more easterly distribution than previously described, and specimens with phenotypic variation were found to occur in areas inconsistent with the modeling, and A. castaneoticus was concentrated in the Tapajós-Xingu interfluve, surrounded by A. galactonotus. Models indicated that the right bank of the Xingu River is suitable for both species, indeed, both were found there. Despite Adelphobates species having their distributions delimited by major Amazonian rivers, estimated divergence times predate the formation of the modern river network, suggesting that other mechanisms were involved in their diversification.

Toxic to the touch: The makings of lethal mantles in pitohui birds and poison dart frogs.

How do chemically defended animals resist their own toxins? This intriguing question on the concept of autotoxicity is at the heart of how species interactions evolve. In this issue of Molecular Ecology (Molecular Ecology, 2024, 33), Bodawatta and colleagues report on how Papua New Guinean birds coopted deadly neurotoxins to create lethal mantles that protect against predators and parasites. Combining chemical screening of the plumage of a diverse collection of passerine birds with genome sequencing, the researchers unlocked a deeper understanding of how some birds sequester deadly batrachotoxin (BTX) from their food without poisoning themselves. They identified that birds impervious to BTX bear amino acid substitutions in the toxin-binding site of the voltage-gated sodium channel Nav1.4, whose function is essential for proper contraction and relaxation of vertebrate muscles. Comparative genetic and molecular docking analyses show that several of the substitutions associated with insensitivity to BTX may have become prevalent among toxic birds through positive selection. Intriguingly, poison dart frogs that also co-opted BTX in their lethal mantles were found to harbour similar toxin insensitivity substitutions in their Nav1.4 channels. Taken together, this sets up a powerful model system for studying the mechanisms behind convergent molecular evolution and how it may drive biological diversity.

Deep divergences among inconspicuously colored clades of Epipedobates poison frogs.

Poison frogs (Dendrobatidae) are famous for their aposematic species, having a combination of diverse color patterns and defensive skin toxins, yet most species in this family are inconspicuously colored and considered non-aposematic. Epipedobates is among the youngest genus-level clades of Dendrobatidae that includes both aposematic and inconspicuous species. Using Sanger-sequenced mitochondrial and nuclear markers, we demonstrate deep genetic divergences among inconspicuous species of Epipedobates but relatively shallow genetic divergences among conspicuous species. Our phylogenetic analysis includes broad geographic sampling of the inconspicuous lineages typically identified as E. boulengeri and E. espinosai, which reveals two putative new species, one in west-central Colombia (E. sp. 1) and the other in north-central Ecuador (E. aff. espinosai). We conclude that E. darwinwallacei is a junior subjective synonym of E. espinosai. We also clarify the geographic distributions of inconspicuous Epipedobates species including the widespread E. boulengeri. We provide a qualitative assessment of the phenotypic diversity in each nominal species, with a focus on the color and pattern of inconspicuous species. We conclude that Epipedobates contains eight known valid species, six of which are inconspicuous. A relaxed molecular clock analysis suggests that the most recent common ancestor of Epipedobates is âˆ¼11.1 million years old, which nearly doubles previous estimates. Last, genetic information points to a center of species diversity in the Chocó at the southwestern border of Colombia with Ecuador. A Spanish translation of this text is available in the supplementary materials.

Thermal limits along tropical elevational gradients: Poison frog tadpoles show plasticity but maintain divergence across elevation.

Temperature is arguably one of the most critical environmental factors impacting organisms at molecular, organismal, and ecological levels. Temperature variation across elevation may cause divergent selection in physiological critical thermal limits (CTMAX and CTMIN). Generally, high elevation populations are predicted to withstand lower environmental temperatures than low elevation populations. Organisms can also exhibit phenotypic plasticity when temperature varies, although theory and empirical evidence suggest that tropical ectotherms have relatively limited ability to acclimate. To study the effect of temperature variation along elevational transects on thermal limits, we measured CTMAX and CTMIN of 934 tadpoles of a poison frog species, Epipedobates anthonyi, along two elevational gradients (200-1700 m asl) in southwestern Ecuador to investigate their thermal tolerance across elevation. We also tested if tadpoles could plastically shift their critical thermal limits in response to exposure to different temperatures representing the range of temperatures they experience in nature (20 °C, 24 °C, and 28 °C). Overall, we found that CTMAX did not change across elevation. In contrast, CTMIN was lower at higher elevations, suggesting that elevational variation in temperature influences this thermal trait. Moreover, all populations shifted their CTMAX and CTMIN according to treatment temperatures, demonstrating an acclimation response. Overall, trends in CTMIN among high, mid, and low elevation populations were maintained despite plastic responses to treatment temperature. These results demonstrate that, for tadpoles of E. anthonyi across tropical elevational gradients, temperature acts as a selective force for CTMIN, even when populations show acclimation abilities in both, CTMAX and CTMIN. Our findings advance our understanding on how environmental variation affects organisms' evolutionary trajectories and their abilities to persist in a changing climate in a tropical biodiversity hotspot.

Evidence for individual vocal recognition in a pair-bonding poison frog, Ranitomeya imitator.

Individually distinctive vocalizations are widespread in nature, although the ability of receivers to discriminate these signals has only been explored through limited taxonomic and social lenses. Here, we asked whether anuran advertisement calls, typically studied for their role in territory defense and mate attraction, facilitate recognition and preferential association with partners in a pair-bonding poison frog (Ranitomeya imitator). Combining no- and two-stimulus choice playback experiments, we evaluated behavioral responses of females to male acoustic stimuli. Virgin females oriented to and approached speakers broadcasting male calls independent of caller identity, implying that females are generally attracted to male acoustic stimuli outside the context of a pair bond. When pair-bonded females were presented with calls of a mate and a stranger, they showed significant preference for calls of their mate. Moreover, behavioral responses varied with breeding status: females with eggs were faster to approach stimuli than females that were pair bonded but did not currently have eggs. Our study suggests a potential role for individual vocal recognition in the formation and maintenance of pair bonds in a poison frog and raises new questions about how acoustic signals are perceived in the context of monogamy and biparental care.

The effects of preformed vitamin A and provitamin A carotenoid supplementation on tadpoles of the poison frog Phyllobates vittatus.

Understanding the nutritional requirements of captive animals is necessary for proper animal husbandry, however, the specific dietary requirements for many amphibian species commonly kept in captivity are unknown. Like most vertebrates, frogs cannot synthesize carotenoids and must therefore obtain these essential nutrients through diet. It is unclear if amphibians can cleave provitamin A carotenoids to form vitamin A metabolically within the body, so common practice is to supplement their captive diets with both preformed vitamin A and provitamin A carotenoids. We carried out a feeding experiment in tadpoles of Phyllobates vittatus, a commonly kept poison frog species, to test the effects of supplementing a fish flake diet with a provitamin A carotenoid (2.5 mg/g ß-carotene) and vitamin A (0.033-0.066 µg/mL retinyl acetate), both individually and in combination. Contrary to our expectations, supplementation had either no effect or adverse effects on tadpole growth and survivorship. Tadpoles reared under supplemented diets with vitamin A showed higher mortality rates, coupled with symptoms of hypervitaminosis A. Survivors had a smaller body size and mass at metamorphosis. ß-carotene supplementation alone had no detectable effect. The vitamin A and ß-carotene levels in our supplemented diet have been shown to be harmless or benefit tadpoles of other species, yet our results indicate that adding these amounts to what is found in a generalist fish flake mix can have detrimental effects on P. vittatus tadpoles. More broadly, this study highlights the importance of creating husbandry guidelines based on the specific physiological needs of the species (or species groups) being kept in captivity, rather than general ones for all amphibians, as is often done.

Water-borne testosterone levels predict exploratory tendency in male poison frogs.

Hormones play a fundamental role in mediating social behaviors of animals. However, it is less well understood to what extent behavioral variation between individuals can be attributed to variation in underlying hormonal profiles. The goal of the present study was to infer if individual androgen levels, and/or the modulation thereof, can explain among-individual variation in aggressiveness, boldness and exploration. We used as a model the dart-poison frog Allobates femoralis and took repeated non-invasive water-borne hormonal samples of individual males before (baseline) and after (experimental) a series of behavioral tests for assessing aggression, boldness, and exploratory tendency. Our results show that androgen levels in A. femoralis are quite stable across the reproductive season. Repeatability in wbT baseline levels was high, while time of day, age of the frog, and trial order did not show any significant impact on measured wbT levels. In general, experimental wbT levels after behavioral tests were lower compared to the respective baseline levels. However, we identified two different patterns with regard to androgen modulation in response to behavioral testing: individuals with low baseline wbT tended to have increased wbT levels after the behavioral testing, while individuals with comparatively high baseline wbT levels rather showed a decrease in hormonal levels after testing. Our results also suggest that baseline wbT levels are linked to the personality trait exploration, and that androgen modulation is linked to boldness in A. femoralis males. These results show that differences in hormonal profiles and/or hormonal modulation in response to social challenges can indeed explain among-individual differences in behavioral traits.

Slippery Porous-Liquid-Infused Porous Surface (SPIPS) with On-Demand Responsive Switching between "Defensive" and "Offensive" Antifouling Modes.

Slippery liquid-infused porous surfaces (SLIPS) have received widespread attention in the antifouling field. However, the reduction in antifouling performance caused by lubricant loss limits their application in marine antifouling. Herein, inspired by the skin of a poison dart frog which contains venom glands and mucus, a porous liquid (PL) based on ZIF-8 is prepared as a lubricant and injected into a silicone polyurethane (SPU) matrix to construct a new type of SLIPS for marine antifouling applications: the slippery porous-liquid-infused porous surface (SPIPS). The SPIPS consists of a responsive antifoulant-releasing switch between "defensive" and "offensive" antifouling modes to intelligently enhance the antifouling effect after lubricant loss. The SPIPS can adjust antifouling performance to meet the antifouling requirements under different light conditions. The wastage of antifoulants is reduced, thereby effectively maintaining the durability and service life of SLIPS materials. The SPIPS exhibits efficient lubricant self-replenishment, self-cleaning, anti-protein, anti-bacterial, anti-algal, and self-healing (97.48%) properties. Furthermore, it shows satisfactory 360-day antifouling performance in actual marine fields during boom seasons, demonstrating the longest antifouling lifespan in the field tests of reported SLIPS coatings. Hence, the SPIPS can effectively promote the development of SLIPS for neritic antifouling.

Poison in the nursery: Mercury contamination in the tadpole-rearing sites of an Amazonian frog.

Artisanal and small-scale gold mining (ASGM) has become a major threat for Neotropical forests. This technique for obtaining gold is a substantial driver of small-scale deforestation and the largest contributor of Hg emissions to both the atmosphere and freshwater systems globally. Previous studies have demonstrated the impacts of Hg accumulation on various aquatic ecosystems and organisms. However, its consequences in other, more discrete systems such as phytotelmata (water-holding plant structures), and the organisms therein, have so far gone unnoticed. Here, we show high concentrations of Hg (mean ± SD: 1.43 ± 2.19 ppm) in phytotelmata and other small pools, the aquatic microenvironments used by the Neotropical poison frog Dendrobates tinctorius as tadpole-rearing sites. In 17 % of the cases, we detected Hg concentrations above the severe effect level (SEL = 2 ppm) for freshwater sediments. Hg concentrations varied depending on pool characteristics and tended to increase in proximity to known ASGM sites. We did not find an effect of Hg concentration on the number of D. tinctorius tadpoles in a given pool. Tadpoles were found in pools with concentrations of up to 8.68 ppm, suggesting that D. tinctorius fathers do not avoid pools with high Hg levels for tadpole deposition. While further research is needed to determine the potential effects of Hg on tadpole development, we found an intriguing tendency for tadpoles in later developmental stages to have lower body condition when occurring in pools with higher Hg concentrations. Our findings provide evidence of relevant Hg concentrations in the terrestrial water systems used by phytotelm-breeding anurans, and highlight the need of further field and experimental studies investigating the implications of Hg contamination for tadpole development and behaviour and the overall conservation of Amazonian biodiversity.

Orientation by environmental geometry and feature cues in the green and black poison frog (Dendrobates auratus).

The ability to use environmental geometry when orienting in space reflects an animal's ability to use a global, allocentric framework. Therefore, understanding when and how animal's use geometry relative to other types of cues in the environment has interested comparative cognition researchers for decades. Yet, only two amphibians have been tested to date. We trained the poison frog Dendrobates auratus to find goal shelters in a rectangular arena, in the presence and absence of a feature cue, and assessed the relative influence of the two types of cues using probe trials. We chose D. auratus because the species has complex interactions with their physical and social environments, including parental care that requires navigating to and from distant locations. We found that, like many vertebrates, D. auratus are capable of using geometric information to relocate goals. In addition, the frogs preferentially used the more reliable feature cue when the location of the feature conflicted with the geometry of the arena. The frogs were equally successful at using the feature cue when it was proximal or distal to the goal shelter, consistent with prior studies that found that D. auratus can use distal cues in a flexible manner. Our results provide further evidence that amphibians can use environmental geometry during orientation. Future studies that examine when and how amphibians use geometry relative to other types of cues will contribute to a more complete picture of spatial cognition in this important, yet understudied, group.

Multiple Routes to Color Convergence in a Radiation of Neotropical Poison Frogs.

Convergent evolution is defined as the independent evolution of similar phenotypes in different lineages. Its existence underscores the importance of external selection pressures in evolutionary history, revealing how functionally similar adaptations can evolve in response to persistent ecological challenges through a diversity of evolutionary routes. However, many examples of convergence, particularly among closely related species, involve parallel changes in the same genes or developmental pathways, raising the possibility that homology at deeper mechanistic levels is an important facilitator of phenotypic convergence. Using the genus Ranitomeya, a young, color-diverse radiation of Neotropical poison frogs, we set out to 1) provide a phylogenetic framework for this group, 2) leverage this framework to determine if color phenotypes are convergent, and 3) to characterize the underlying coloration mechanisms to test whether color convergence occurred through the same or different physical mechanisms. We generated a phylogeny for Ranitomeya using ultraconserved elements and investigated the physical mechanisms underlying bright coloration, focusing on skin pigments. Using phylogenetic comparative methods, we identified several instances of color convergence, involving several gains and losses of carotenoid and pterin pigments. We also found a compelling example of nonparallel convergence, where, in one lineage, red coloration evolved through the red pterin pigment drosopterin, and in another lineage through red ketocarotenoids. Additionally, in another lineage, "reddish" coloration evolved predominantly through structural color mechanisms. Our study demonstrates that, even within a radiation of closely related species, convergent evolution can occur through both parallel and nonparallel mechanisms, challenging the assumption that similar phenotypes among close relatives evolve through the same mechanisms.

Binding and sequestration of poison frog alkaloids by a plasma globulin.

Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid-binding molecule in blood of poison frogs. Proteomic and biochemical studies establish this plasma protein to be a liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid-binding activity, ABG sequesters and regulates the bioavailability of 'free' plasma alkaloids in vitro. Unexpectedly, ABG is not related to saxiphilin, albumin, or other known vitamin carriers, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin-binding proteins. ABG represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points toward serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.