Evan Twomey

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.

Phytotelm size in relation to parental care and mating strategies in two species of Peruvian poison frogs

Summary Factors contributing to the evolution of reproductive strategies have been of great interest to evolutionary biologists. In tropical amphibians predation and competition have been suggested to play a major role. Poison frogs of the family Dendrobatidae display a trend towards the use of very small pools and increased parental care, particularly in the genus Dendrobates. Some species with female parental care, asymmetrical biparental care and biparental care, have evolved novel behaviors in association with the use very small phytotelmata. It has been hypothesized that selection pressure imposed by predation and competition favored the use of small phytotelmata, and this, in turn, produced selection for trophic egg provisioning to ameliorate the lack of available nutrients. To elucidate the ecological factors associated with the transition from uniparental male care to biparental care and associated changes in social behaviors, we evaluated key behavioral and ecological differences between Dendrobates imitator and D. variabilis. Dendrobates imitator used significantly smaller phytotelmata in different plant species than D. variabilis for tadpole and embryo deposition. The parental strategy of D. variabilis was limited to male parental care, whereas D. imitator exhibited biparental care. Males and females of D. variabilis were observed to have a promiscuous mating system with little mate fidelity. This contrasted with D. imitator, where paired males and females were observed interacting daily and were never observed courting additional mates. Our results are consistent with the hypothesis that a key ecological difference between these species, involving the size of pools typically used for reproduction, is strongly associated with the evolution of biparental care and monogamy in D. imitator.

Current State of Conservation Knowledge on Threatened Amphibian Species in Peru

This study documents the current state of conservation knowledge on threatened amphibian species in Peru. Following the International Union for the Conservation of Nature (IUCN) classification system, we considered species in the following categories: Critically Endangered, Endangered, Vulnerable, and Near Threatened. Even though only the first three categories are regarded as threatened by IUCN, we included the fourth category to make comparisons with the list of threatened species issued by the Peruvian government. We used the Global Amphibian Assessments database and the list issued in Peru for this comparison. We conducted separate field surveys in 17 regions of Peru to evaluate the presence/absence of threatened amphibian species and species that are potentially threatened. We also used the Declining Amphibian Database-DAPTF, to compare our results with previous assessments on population declines, and the World Wildlife Funds Wildfinder database, to determine in which Neotropical ecoregion each species occurs. We compiled data on 83 species, 44 of which are recognized as threatened by the IUCN and/or the Peruvian government. The remaining 39 species should be re-assessed as they face various threats. A re-evaluation of current estimates is needed as only 8% of all species recorded in Peru are recognized as threatened by the government, whereas the global estimate of threatened species is about 32%. In addition to using IUCN criteria, this re-assessment should follow national guidelines standardized in Peru and be in accordance with the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Because the habitat of almost 40% of threatened species reported herein still remains unprotected, and data on chytridiomycosis and other threats are lacking for most taxa, it is crucial to develop strategies for habitat conservation and research on disease dynamics in natural populations.

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.

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.

SPOTTED POISON FROGS: REDISCOVERY OF A LOST SPECIES AND A NEW GENUS (ANURA: DENDROBATIDAE) FROM NORTHWESTERN PERU

The Santiago poison frog, Adelphobates captivus, a species not seen in life since 1929, was recently rediscovered on an expedition to its type locality in northwestern Peru. The colors of this species, previously unknown, consist of a black dorsum with bright red-orange spots and yellow spots ventrally. We provide amendments to the original description as well as the first accounts of tadpole morphology, vocalization, and natural history. A Bayesian phylogenetic analysis suggests Adelphobates captivus and a species originally described as Dendrobates mysteriosus are sister species that form a monophyletic clade sister to Ranitomeya. We propose to clarify the taxonomic status of D. mysteriosus incertae sedis by erecting a new genus, Excidobates, to include mysteriosus and its sister taxon captivus. Members of this genus are distinguished from Ranitomeya by 11 site substitutions in their rrnS and rrnL sequences, well-developed first fingers, and pale spots on the ventral surfaces of the thighs.

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.

Intraspecific Call Variation in the Mimic Poison Frog Ranitomeya imitator

Abstract:  Calls are important premating isolating barriers in frogs; therefore, studying intraspecific variation in calls might allow the assessment of patterns of call divergence during the early stages of speciation. Ranitomeya imitator, a species of dart-poison frog (Dendrobatidae), has undergone extensive color-pattern diversification through a Müllerian mimetic radiation, establishing four distinct morphs in north-central Peru (striped, banded, varadero, and spotted). Partial reproductive isolation exists between certain color morphs, although the specific mechanisms responsible for this isolation are poorly understood. We conducted a species-wide analysis of variation in advertisement calls to investigate whether distinct mimetic morphs show advertisement call differences. We found that different color morphs generally show weak or no differences in advertisement calls, with two exceptions. First, call pulse rates differed between the striped and banded morphs, and that difference coincides geographically with the mimetic transition zone. Second, there is a difference in both note length and dominant frequency between the striped and varadero morphs, which is also geographically coincident with the mimetic transition zone. Future work in this system should attempt to measure the relative importance of different mating cues (color-pattern, body size, and advertisement calls) mediating mate choice in this species.