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.

Phylogenomic analysis of evolutionary relationships in Ranitomeya poison frogs (Family Dendrobatidae) using ultraconserved elements.

The use of genome-scale data in phylogenetics has enabled recent strides in determining the relationships between taxa that are taxonomically problematic because of extensive morphological variation. Here, we employ a phylogenomic approach to infer evolutionary relationships within Ranitomeya (Anura: Dendrobatidae), an Amazonian lineage of poison frogs consisting of 16 species with remarkable diversity in color pattern, range size, and parental care behavior. We infer phylogenies with all described species of Ranitomeya from ultraconserved nuclear genomic elements (UCEs) and also estimate divergence times. Our results differ from previous analyses regarding interspecific relationships. Notably, we find that R. toraro and R. defleri are not sister species but rather distantly related, contrary to previous analyses based on smaller genetic datasets. We recover R. uakarii as paraphyletic, designate certain populations formerly assigned to R. fantastica from Peru as R. summersi, and transfer the French Guianan and eastern Brazilian R. amazonica populations to R. variabilis. By clarifying both inter- and intraspecific relationships within Ranitomeya, our study paves the way for future tests of hypotheses on color pattern evolution and historical biogeography.

Amphibians in Zootaxa: 20 years documenting the global diversity of frogs, salamanders, and caecilians.

Zootaxa is a mega-journal that since its inception, 20 years ago, has contributed to the documentation of the planet's biodiversity. Its role concerning terrestrial vertebrates has been crucial especially for amphibians, which are the most threatened class of vertebrates. As current editors of the Amphibia section, we reviewed the state of knowledge of taxonomic publications on amphibians over the last two decades (from 2001 to 2020). Our review reveals that 2,533 frogs, 259 salamanders, and 55 caecilians have been named in these 20 years, mainly in the tropical regions of South America, Asia, and Africa. More than half (57%) of these species descriptions were published in only 10 journals. At least 827 species of the new amphibians (29% of the total) were described in Zootaxa. This mega-journal has served also as a place of publication for monographs and systematic reviews, in addition to short articles documenting the vocalizations of anurans and the morphology of embryos and larvae. Its efficient evaluation process, the freedom of manuscript length, including full-color figures, and free of cost for the authors, has made Zootaxa a favorite for amphibian researchers. In an era of accelerating rates of biodiversity loss, documenting, describing, naming, and proposing evolutionary scenarios for species is, more than ever, an urgent task.

Mechanisms for Color Convergence in a Mimetic Radiation of Poison Frogs.

In animals, bright colors often evolve to mimic other species when a resemblance is selectively favored. Understanding the proximate mechanisms underlying such color mimicry can give insights into how mimicry evolves-for example, whether color convergence evolves from a shared set of mechanisms or through the evolution of novel color production mechanisms. We studied color production mechanisms in poison frogs (Dendrobatidae), focusing on the mimicry complex of Ranitomeya imitator. Using reflectance spectrometry, skin pigment analysis, electron microscopy, and color modeling, we found that the bright colors of these frogs, both within and outside the mimicry complex, are largely structural and produced by iridophores but that color production depends crucially on interactions with pigments. Color variation and mimicry are regulated predominantly by iridophore platelet thickness and, to a lesser extent, concentration of the red pteridine pigment drosopterin. Compared with each of the four morphs of model species that it resembles, R. imitator displays greater variation in both structural and pigmentary mechanisms, which may have facilitated phenotypic divergence in this species. Analyses of nonmimetic dendrobatids in other genera demonstrate that these mechanisms are widespread within the family and that poison frogs share a complex physiological "color palette" that can produce diverse and highly reflective colors.

A ketocarotenoid-based colour polymorphism in the Sira poison frog Ranitomeya sirensis indicates novel gene interactions underlying aposematic signal variation.

The accumulation of red ketocarotenoids is an important component of coloration in many organisms, but the underlying mechanisms are poorly understood. In some organisms, ketocarotenoids are sequestered from the diet and can accumulate when enzymes responsible for carotenoid breakdown are disrupted. In other organisms, ketocarotenoids are formed endogenously from dietary precursors via oxidation reactions carried out by carotenoid ketolase enzymes. Here, we study the genetic basis of carotenoid coloration in an amphibian. We demonstrate that a red/yellow polymorphism in the dendrobatid poison frog Ranitomeya sirensis is due to the presence/absence of ketocarotenoids. Using whole-transcriptome sequencing of skins and livers, we found that a transcript encoding a cytochrome P450 enzyme (CYP3A80) is expressed 3.4-fold higher in livers of red frogs versus yellow. As CYP3A enzymes are known carotenoid ketolases in other organisms, our results point to CYP3A80 as a strong candidate for a carotenoid ketolase in amphibians. Furthermore, in red frogs, the transcript encoding the carotenoid cleavage enzyme BCO2 is expressed at a low level or as a splice variant lacking key catalytic amino acids. This suggests that BCO2 function may be disrupted in red frogs, providing a mechanism whereby the accumulation of ketocarotenoids and their dietary precursors may be enhanced.

Phylogenetic relationships and systematics of the Amazonian poison frog genus Ameerega using ultraconserved genomic elements.

The Amazonian poison frog genus Ameerega is one of the largest yet most understudied of the brightly colored genera in the anuran family Dendrobatidae, with 30 described species ranging throughout tropical South America. Phylogenetic analyses of Ameerega are highly discordant, lacking consistency due to variation in data types and methods, and often with limited coverage of species diversity in the genus. Here, we present a comprehensive phylogenomic reconstruction of Ameerega, utilizing state-of-the-art sequence capture techniques and phylogenetic methods. We sequenced thousands of ultraconserved elements from over 100 tissue samples, representing almost every described Ameerega species, as well as undescribed cryptic diversity. We generated topologies using maximum likelihood and coalescent methods and compared the use of maximum likelihood and Bayesian methods for estimating divergence times. Our phylogenetic inference diverged strongly from those of previous studies, and we recommend steps to bring Ameerega taxonomy in line with the new phylogeny. We place several species in a phylogeny for the first time, as well as provide evidence for six potential candidate species. We estimate that Ameerega experienced a rapid radiation approximately 7-11 million years ago and that the ancestor of all Ameerega was likely an aposematic, montane species. This study underscores the utility of phylogenomic data in improving our understanding of the phylogeny of understudied clades and making novel inferences about their evolution.

Systematics of the Ameerega rubriventris complex (Anura: Dendrobatidae) with descriptions of two new cryptic species from the East-Andean versant of Peru.

We describe two new species of poison frog from central and southern Peru that have been referred to as Ameerega picta, A. hahneli, or A. altamazonica throughout the past thirty years. Our phylogenies generated with genomic data provide strong support that the two new species are successive sisters to two described taxa, A. rubriventris and A. altamazonica, and collectively comprise the Ameerega rubriventris complex. The first new taxon, Ameerega panguana sp. nov., can be distinguished from all other Ameerega by its combination of a unique white venter and an advertisement call of 1-2 notes per second. The second new taxon, Ameerega imasmari sp. nov., is the only cryptically colored Ameerega species that is disttributed across the Fitzcarrald Arch in Southern Peru which possesses a 'peep' advertisement call consisting of 3-4 notes per second and a dominant frequency of 4.3-4.5 kHz. Within the Ameerega rubriventris complex, we observed differences between species in their ventral coloration, tympanum diameter, and call, which suggest that these taxa are reproductively isolated from each other.

Mimetic Divergence and the Speciation Continuum in the Mimic Poison Frog Ranitomeya imitator.

While divergent ecological adaptation can drive speciation, understanding the factors that facilitate or constrain this process remains a major goal in speciation research. Here, we study two mimetic transition zones in the poison frog Ranitomeya imitator, a species that has undergone a Müllerian mimetic radiation to establish four morphs in Peru. We find that mimetic morphs are strongly phenotypically differentiated, producing geographic clines with varying widths. However, distinct morphs show little neutral genetic divergence, and landscape genetic analyses implicate isolation by distance as the primary determinant of among-population genetic differentiation. Mate choice experiments suggest random mating at the transition zones, although certain allopatric populations show a preference for their own morph. We present evidence that this preference may be mediated by color pattern specifically. These results contrast with an earlier study of a third transition zone, in which a mimetic shift was associated with reproductive isolation. Overall, our results suggest that the three known mimetic transition zones in R. imitator reflect a speciation continuum, which we have characterized at the geographic, phenotypic, behavioral, and genetic levels. We discuss possible explanations for variable progress toward speciation, suggesting that multifarious selection on both mimetic color pattern and body size may be responsible for generating reproductive isolation.

Number of genes controlling a quantitative trait in a hybrid zone of the aposematic frog Ranitomeya imitator.

The number of genes controlling mimetic traits has been a topic of much research and discussion. In this paper, we examine a mimetic, dendrobatid frog Ranitomeya imitator, which harbours extensive phenotypic variation with multiple mimetic morphs, not unlike the celebrated Heliconius system. However, the genetic basis for this polymorphism is unknown, and not easy to determine using standard experimental approaches, for this hard-to-breed species. To circumvent this problem, we first develop a new protocol for automatic quantification of complex colour pattern phenotypes from images. Using this method, which has the potential to be applied in many other systems, we define a phenotype associated with differences in colour pattern between different mimetic morphs. We then proceed to develop a maximum-likelihood method for estimating the number of genes affecting a quantitative trait segregating in a hybrid zone. This method takes advantage of estimates of admixture proportions obtained using genetic data, such as microsatellite markers, and is applicable to any other system where a phenotype has been quantified in an admixture/introgression zone. We evaluate the method using extensive simulations and apply it to the R. imitator system. We show that probably one or two, or at most three genes, control the mimetic phenotype segregating in a R. imitator hybrid zone identified using image analyses.

High levels of diversity uncovered in a widespread nominal taxon: continental phylogeography of the neotropical tree frog Dendropsophus minutus.

Species distributed across vast continental areas and across major biomes provide unique model systems for studies of biotic diversification, yet also constitute daunting financial, logistic and political challenges for data collection across such regions. The tree frog Dendropsophus minutus (Anura: Hylidae) is a nominal species, continentally distributed in South America, that may represent a complex of multiple species, each with a more limited distribution. To understand the spatial pattern of molecular diversity throughout the range of this species complex, we obtained DNA sequence data from two mitochondrial genes, cytochrome oxidase I (COI) and the 16S rhibosomal gene (16S) for 407 samples of D. minutus and closely related species distributed across eleven countries, effectively comprising the entire range of the group. We performed phylogenetic and spatially explicit phylogeographic analyses to assess the genetic structure of lineages and infer ancestral areas. We found 43 statistically supported, deep mitochondrial lineages, several of which may represent currently unrecognized distinct species. One major clade, containing 25 divergent lineages, includes samples from the type locality of D. minutus. We defined that clade as the D. minutus complex. The remaining lineages together with the D. minutus complex constitute the D. minutus species group. Historical analyses support an Amazonian origin for the D. minutus species group with a subsequent dispersal to eastern Brazil where the D. minutus complex originated. According to our dataset, a total of eight mtDNA lineages have ranges >100,000 km2. One of them occupies an area of almost one million km2 encompassing multiple biomes. Our results, at a spatial scale and resolution unprecedented for a Neotropical vertebrate, confirm that widespread amphibian species occur in lowland South America, yet at the same time a large proportion of cryptic diversity still remains to be discovered.

A review of Northern Peruvian glassfrogs (Centrolenidae), with the description of four new remarkable species.

Peru is well known for amphibian diversity and endemism, yet there have been relatively few field studies of glassfrog (Centrolenidae) diversity in this country. Research in Colombia and Ecuador indicates that centrolenid diversity is higher in the northern Andes. However, part of this trend appears to be due to sampling effort. We conducted fieldwork throughout northern Peru, and based on phylogenetic analysis of DNA sequences, combined with bioacoustic and morphological analyses of new and available material we now recognize 33 species from the country (versus 30 species prior to this work). Field surveys led to the discovery of four remarkable species: Centrolene charapita new species is a large, ornamented glassfrog that appears to be sister to Ce. geckoideum; Chimerella corleone new species represents the second-known member of the genus Chimerella; Cochranella guayasamini new species is the second-known member of the genus with humeral spines; and Hyalinobatrachium anachoretus new species occurs in the cloud forest of the east-Andean versant in Peru. In addition to the new species described here, we provide new country records, new localities including range extensions of up to 875 km, information on diagnostic characters and phylogenetic relationships, call and larval descriptions, and observations on natural history for several Peruvian centrolenids. Our results also revealed several taxonomic problems concerning species of the genus Rulyrana, and we conclude that R. croceopodes and R. tangarana are junior synonyms of R. saxiscandens. By implication of our phylogenetic analyses, we recognize the following new combinations: Espadarana audax new combination, Espadarana durrelorum new combination, and Espadarana fernandoi new combination. 

Reproductive isolation related to mimetic divergence in the poison frog Ranitomeya imitator.

In a mimetic radiation--when a single species evolves to resemble different model species--mimicry can drive within-species morphological diversification, and, potentially, speciation. While mimetic radiations have occurred in a variety of taxa, their role in speciation remains poorly understood. We study the Peruvian poison frog Ranitomeya imitator, a species that has undergone a mimetic radiation into four distinct morphs. Using a combination of colour-pattern analysis, landscape genetics and mate-choice experiments, we show that a mimetic shift in R. imitator is associated with a narrow phenotypic transition zone, neutral genetic divergence and assortative mating, suggesting that divergent selection to resemble different model species has led to a breakdown in gene flow between these two populations. These results extend the effects of mimicry on speciation into a vertebrate system and characterize an early stage of speciation where reproductive isolation between mimetic morphs is incomplete but evident.

Phenotypic and Genetic Divergence among Poison Frog Populations in a Mimetic Radiation.

The evolution of Müllerian mimicry is, paradoxically, associated with high levels of diversity in color and pattern. In a mimetic radiation, different populations of a species evolve to resemble different models, which can lead to speciation. Yet there are circumstances under which initial selection for divergence under mimicry may be reversed. Here we provide evidence for the evolution of extensive phenotypic divergence in a mimetic radiation in Ranitomeya imitator, the mimic poison frog, in Peru. Analyses of color hue (spectral reflectance) and pattern reveal substantial divergence between morphs. However, we also report that there is a "transition-zone" with mixed phenotypes. Analyses of genetic structure using microsatellite variation reveals some differentiation between populations, but this does not strictly correspond to color pattern divergence. Analyses of gene flow between populations suggest that, while historical levels of gene flow were low, recent levels are high in some cases, including substantial gene flow between some color pattern morphs. We discuss possible explanations for these observations.