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.

Specimen collection is essential for modern science.

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

Selection on Visual Opsin Genes in Diurnal Neotropical Frogs and Loss of the SWS2 Opsin in Poison Frogs.

Amphibians are ideal for studying visual system evolution because their biphasic (aquatic and terrestrial) life history and ecological diversity expose them to a broad range of visual conditions. Here, we evaluate signatures of selection on visual opsin genes across Neotropical anurans and focus on three diurnal clades that are well-known for the concurrence of conspicuous colors and chemical defense (i.e., aposematism): poison frogs (Dendrobatidae), Harlequin toads (Bufonidae: Atelopus), and pumpkin toadlets (Brachycephalidae: Brachycephalus). We found evidence of positive selection on 44 amino acid sites in LWS, SWS1, SWS2, and RH1 opsin genes, of which one in LWS and two in RH1 have been previously identified as spectral tuning sites in other vertebrates. Given that anurans have mostly nocturnal habits, the patterns of selection revealed new sites that might be important in spectral tuning for frogs, potentially for adaptation to diurnal habits and for color-based intraspecific communication. Furthermore, we provide evidence that SWS2, normally expressed in rod cells in frogs and some salamanders, has likely been lost in the ancestor of Dendrobatidae, suggesting that under low-light levels, dendrobatids have inferior wavelength discrimination compared to other frogs. This loss might follow the origin of diurnal activity in dendrobatids and could have implications for their behavior. Our analyses show that assessments of opsin diversification in across taxa could expand our understanding of the role of sensory system evolution in ecological adaptation.

2b or not 2b? 2bRAD is an effective alternative to ddRAD for phylogenomics.

Restriction-site-associated DNA sequencing (RADseq) has become an accessible way to obtain genome-wide data in the form of single-nucleotide polymorphisms (SNPs) for phylogenetic inference. Nonetheless, how differences in RADseq methods influence phylogenetic estimation is poorly understood because most comparisons have largely relied on conceptual predictions rather than empirical tests. We examine how differences in ddRAD and 2bRAD data influence phylogenetic estimation in two non-model frog groups. We compare the impact of method choice on phylogenetic information, missing data, and allelic dropout, considering different sequencing depths. Given that researchers must balance input (funding, time) with output (amount and quality of data), we also provide comparisons of laboratory effort, computational time, monetary costs, and the repeatability of library preparation and sequencing. Both 2bRAD and ddRAD methods estimated well-supported trees, even at low sequencing depths, and had comparable amounts of missing data, patterns of allelic dropout, and phylogenetic signal. Compared to ddRAD, 2bRAD produced more repeatable datasets, had simpler laboratory protocols, and had an overall faster bioinformatics assembly. However, many fewer parsimony-informative sites per SNP were obtained from 2bRAD data when using native pipelines, highlighting a need for further investigation into the effects of each pipeline on resulting datasets. Our study underscores the importance of comparing RADseq methods, such as expected results and theoretical performance using empirical datasets, before undertaking costly experiments.

Overcoming Language Barriers in Academia: Machine Translation Tools and a Vision for a Multilingual Future.

Having a central scientific language remains crucial for advancing and globally sharing science. Nevertheless, maintaining one dominant language also creates barriers to accessing scientific careers and knowledge. From an interdisciplinary perspective, we describe how, when, and why to make scientific literature more readily available in multiple languages through the practice of translation. We broadly review the advantages and limitations of neural machine translation systems and propose that translation can serve as both a short- and a long-term solution for making science more resilient, accessible, globally representative, and impactful beyond the academy. We outline actions that individuals and institutions can take to support multilingual science and scientists, including structural changes that encourage and value translating scientific literature. In the long term, improvements to machine translation technologies and collective efforts to change academic norms can transform a monolingual scientific hub into a multilingual scientific network. Translations are available in the supplemental material.

A set of principles and practical suggestions for equitable fieldwork in biology.

Field biology is an area of research that involves working directly with living organisms in situ through a practice known as "fieldwork." Conducting fieldwork often requires complex logistical planning within multiregional or multinational teams, interacting with local communities at field sites, and collaborative research led by one or a few of the core team members. However, existing power imbalances stemming from geopolitical history, discrimination, and professional position, among other factors, perpetuate inequities when conducting these research endeavors. After reflecting on our own research programs, we propose four general principles to guide equitable, inclusive, ethical, and safe practices in field biology: be collaborative, be respectful, be legal, and be safe. Although many biologists already structure their field programs around these principles or similar values, executing equitable research practices can prove challenging and requires careful consideration, especially by those in positions with relatively greater privilege. Based on experiences and input from a diverse group of global collaborators, we provide suggestions for action-oriented approaches to make field biology more equitable, with particular attention to how those with greater privilege can contribute. While we acknowledge that not all suggestions will be applicable to every institution or program, we hope that they will generate discussions and provide a baseline for training in proactive, equitable fieldwork practices.

A review of chemical defense in harlequin toads (Bufonidae: Atelopus).

Toads of the genus Atelopus are chemically defended by a unique combination of endogenously synthesized cardiotoxins (bufadienolides) and neurotoxins which may be sequestered (guanidinium alkaloids). Investigation into Atelopus small-molecule chemical defenses has been primarily concerned with identifying and characterizing various forms of these toxins while largely overlooking their ecological roles and evolutionary implications. In addition to describing the extent of knowledge about Atelopus toxin structures, pharmacology, and biological sources, we review the detection, identification, and quantification methods used in studies of Atelopus toxins to date and conclude that many known toxin profiles are unlikely to be comprehensive because of methodological and sampling limitations. Patterns in existing data suggest that both environmental (toxin availability) and genetic (capacity to synthesize or sequester toxins) factors influence toxin profiles. From an ecological and evolutionary perspective, we summarize the possible selective pressures acting on Atelopus toxicity and toxin profiles, including predation, intraspecies communication, disease, and reproductive status. Ultimately, we intend to provide a basis for future ecological, evolutionary, and biochemical research on Atelopus.

Trade-offs between cost of ingestion and rate of intake drive defensive toxin use.

Animals that ingest toxins can become unpalatable and even toxic to predators and parasites through toxin sequestration. Because most animals rapidly eliminate toxins to survive their ingestion, it is unclear how populations transition from susceptibility and toxin elimination to tolerance and accumulation as chemical defence emerges. Studies of chemical defence have generally focused on species with active toxin sequestration and target-site insensitivity mutations or toxin-binding proteins that permit survival without necessitating toxin elimination. Here, we investigate whether animals that presumably rely on toxin elimination for survival can use ingested toxins for defence. We use the A4 and A3 Drosophila melanogaster fly strains from the Drosophila Synthetic Population Resource (DSPR), which respectively possess high and low metabolic nicotine resistance among DSPR fly lines. We find that ingesting nicotine increased A4 but not A3 fly survival against Leptopilina heterotoma wasp parasitism. Further, we find that despite possessing genetic variants that enhance toxin elimination, A4 flies accrued more nicotine than A3 individuals, likely by consuming more medium. Our results suggest that enhanced toxin metabolism can allow greater toxin intake by offsetting the cost of toxin ingestion. Passive toxin accumulation that accompanies increased toxin intake may underlie the early origins of chemical defence.

An insight into molecular taxonomy of bufonids, microhylids, and dicroglossid frogs: First genetic records from Pakistan.

The current study was focused on documentation of amphibian assemblage in North Punjab and Islamabad Capital Territory, Pakistan, by using mitochondrial gene sequences of 16S rRNA. Our study entailed 37% of the known amphibian species of the country. We provided a phylogenetic analysis based on 74 newly generated mitochondrial 16S rRNAs from nine species of genus Microlyla, Duttaphrynus, Allopaa, Nanorana, Sphaerotheca, Minervarya, Hoplobatrachus, and Euphlyctis. We employed the maximum-likelihood inference and Bayesian analysis to assess the taxonomic status of the samples obtained from Pakistan, with respect to other congeneric species from surrounding regions. Our findings confirmed the taxonomic status of South Asian anuran species Duttaphrynus stomaticus, Duttaphrynus melanostictus, Microhyla nilphamariensis, Allopaa hazarensis, Nanorana vicina, Sphaerotheca maskeyi (synonym: S. pashchima), Minervarya pierrei, Hoplobatrachus tigerinus, and Euphlyctis kalasgramensis in Pakistan. We have reported new country records of genus Minervarya ( M. pierrei). Minervarya pierrei was previously misidentified as Fejervarya limnocharis, due to dearth of genetic information. We provided the first genetic records of our endemic species N. vicina. The results revealed the taxonomic placement of N. vicina with respect to its congeners and validated the taxonomic status of N. vicina from its type locality (Murree) for the first time. The findings of the present study also indicated the paraphyletic relationship of A.- hazarensis with Nanorana species. So, based on our phylogenetic inferences, morphological characters, and habitat preferences, validity of generic status of A. hazarensis is undecided. As our data were not enough to resolve this issue, we suggest sequencing of additional mitochondrial and nuclear genes in the future studies to get a better resolution. We recommend carrying out extensive surveys throughout the country for proper scientific documentation of amphibians of Pakistan. Many new species, some of them might be endemic to Pakistan, are expected to be discovered, and taxonomic status of other species would be resolved.

A Sucker for Taste.

Biology is entering a new era in which techniques honed in model systems can be applied to the expanding array of organisms with sequenced genomes. In this issue of Cell, van Giesen et al. (2020) characterize the molecular foundation of the touch-taste sensory system in octopus suckers.

Diversity within diversity: Parasite species richness in poison frogs assessed by transcriptomics.

Symbionts (e.g., endoparasites and commensals) play an integral role in their host's ecology, yet in many cases their diversity is likely underestimated. Although endoparasites are traditionally characterized using morphology, sequences of conserved genes, and shotgun metagenomics, host transcriptomes constitute an underused resource to identify these organisms' diversity. By isolating non-host transcripts from host transcriptomes, individual host tissues can now simultaneously reveal their endoparasite species richness (i.e., number of different taxa) and provide insights into parasite gene expression. These approaches can be used in host taxa whose endoparasites are mostly unknown, such as those of tropical amphibians. Here, we focus on the poison frogs (Dendrobatidae) as hosts, which are a Neotropical clade known for their bright coloration and defensive alkaloids. These toxins are an effective protection against vertebrate predators (e.g., snakes and birds), bacteria, and skin-biting ectoparasites (e.g., mosquitoes); however, little is known about their deterrence against eukaryotic endoparasites. With de novo transcriptomes of dendrobatids, we developed a bioinformatics pipeline for endoparasite identification that uses host annotated RNA-seq data and set of a priori parasite taxonomic terms, which are used to mine for specific endoparasites. We found a large community of helminths and protozoans that were mostly restricted to the digestive tract and a few systemic parasites (e.g., Trypanosoma). Contrary to our expectations, all dendrobatid frogs regardless of the presence of alkaloid defenses have endoparasites, with their highest species richness located in the frog digestive tract. Some of these organisms (e.g., roundworms) might prove to be generalists, as they were not found to be co-diversifying with their frog hosts. We propose that endoparasites may escape poison frogs' chemical defenses by colonizing tissues with fewer alkaloids than the frog's skin, where most toxins are stored.

Interacting amino acid replacements allow poison frogs to evolve epibatidine resistance.

Animals that wield toxins face self-intoxication. Poison frogs have a diverse arsenal of defensive alkaloids that target the nervous system. Among them is epibatidine, a nicotinic acetylcholine receptor (nAChR) agonist that is lethal at microgram doses. Epibatidine shares a highly conserved binding site with acetylcholine, making it difficult to evolve resistance yet maintain nAChR function. Electrophysiological assays of human and frog nAChR revealed that one amino acid replacement, which evolved three times in poison frogs, decreased epibatidine sensitivity but at a cost of acetylcholine sensitivity. However, receptor functionality was rescued by additional amino acid replacements that differed among poison frog lineages. Our results demonstrate how resistance to agonist toxins can evolve and that such genetic changes propel organisms toward an adaptive peak of chemical defense.

The birth of aposematism: High phenotypic divergence and low genetic diversity in a young clade of poison frogs.

Rapid radiation coupled with low genetic divergence often hinders species delimitation and phylogeny estimation even if putative species are phenotypically distinct. Some aposematic species, such as poison frogs (Dendrobatidae), have high levels of intraspecific color polymorphism, which can lead to overestimation of species when phenotypic divergence primarily guides species delimitation. We explored this possibility in the youngest origin of aposematism (3-7 MYA) in poison frogs, Epipedobates, by comparing genetic divergence with color and acoustic divergence. We found low genetic divergence (2.6% in the 16S gene) despite substantial differences in color and acoustic signals. While chemical defense is inferred to have evolved in the ancestor of Epipedobates, aposematic coloration evolved at least twice or was lost once in Epipedobates, suggesting that it is evolutionarily labile. We inferred at least one event of introgression between two cryptically colored species with adjacent ranges (E. boulengeri and E. machalilla). We also find evidence for peripheral isolation resulting in phenotypic divergence and potential speciation of the aposematic E. tricolor from the non-aposematic E. machalilla. However, we were unable to estimate a well-supported species tree or delimit species using multispecies coalescent models. We attribute this failure to factors associated with recent speciation including mitochondrial introgression, incomplete lineage sorting, and too few informative molecular characters. We suggest that species delimitation within young aposematic lineages such as Epipedobates will require genome-level molecular studies. We caution against relying solely on DNA barcoding for species delimitation or identification and highlight the value of phenotypic divergence and natural history in delimiting species.

Convergent Substitutions in a Sodium Channel Suggest Multiple Origins of Toxin Resistance in Poison Frogs.

Complex phenotypes typically have a correspondingly multifaceted genetic component. However, the genotype-phenotype association between chemical defense and resistance is often simple: genetic changes in the binding site of a toxin alter how it affects its target. Some toxic organisms, such as poison frogs (Anura: Dendrobatidae), have defensive alkaloids that disrupt the function of ion channels, proteins that are crucial for nerve and muscle activity. Using protein-docking models, we predict that three major classes of poison frog alkaloids (histrionicotoxins, pumiliotoxins, and batrachotoxins) bind to similar sites in the highly conserved inner pore of the muscle voltage-gated sodium channel, Nav1.4. We predict that poison frogs are somewhat resistant to these compounds because they have six types of amino acid replacements in the Nav1.4 inner pore that are absent in all other frogs except for a distantly related alkaloid-defended frog from Madagascar, Mantella aurantiaca. Protein-docking models and comparative phylogenetics support the role of these replacements in alkaloid resistance. Taking into account the four independent origins of chemical defense in Dendrobatidae, phylogenetic patterns of the amino acid replacements suggest that 1) alkaloid resistance in Nav1.4 evolved independently at least seven times in these frogs, 2) variation in resistance-conferring replacements is likely a result of differences in alkaloid exposure across species, and 3) functional constraint shapes the evolution of the Nav1.4 inner pore. Our study is the first to demonstrate the genetic basis of autoresistance in frogs with alkaloid defenses.

Divergence in coloration and ecological speciation in the Anolis marmoratus species complex.

Adaptive divergence in coloration is expected to produce reproductive isolation in species that use colourful signals in mate choice and species recognition. Indeed, many adaptive radiations are characterized by differentiation in colourful signals, suggesting that divergent selection acting on coloration may be an important component of speciation. Populations in the Anolis marmoratus species complex from the Caribbean island of Guadeloupe display striking divergence in the colour and pattern of adult males that occurs over small geographic distances, suggesting strong divergent selection. Here we test the hypothesis that divergence in coloration results in reduced gene flow among populations. We quantify variation in adult male coloration across a habitat gradient between mesic and xeric habitats, use a multilocus coalescent approach to infer historical demographic parameters of divergence, and examine gene flow and population structure using microsatellite variation. We find that colour variation evolved without geographic isolation and in the face of gene flow, consistent with strong divergent selection and that both ecological and sexual selection are implicated. However, we find no significant differentiation at microsatellite loci across populations, suggesting little reproductive isolation and high levels of contemporary gene exchange. Strong divergent selection on loci affecting coloration probably maintains clinal phenotypic variation despite high gene flow at neutral loci, supporting the notion of a porous genome in which adaptive portions of the genome remain fixed whereas neutral portions are homogenized by gene flow and recombination. We discuss the impact of these findings for studies of colour evolution and ecological speciation.