Poison frogs sequester chemical defenses from arthropod prey, although the details of how arthropod diversity contributes to variation in poison frog toxins remains unclear. We characterized skin alkaloid profiles in the Little Devil poison frog, Oophaga sylvatica (Dendrobatidae), across three populations in northwestern Ecuador. Using gas chromatography/mass spectrometry, we identified histrionicotoxins, 3,5- and 5,8-disubstituted indolizidines, decahydroquinolines, and lehmizidines as the primary alkaloid toxins in these O. sylvatica populations. Frog skin alkaloid composition varied along a geographical gradient following population distribution in a principal component analysis. We also characterized diversity in arthropods isolated from frog stomach contents and confirmed that O. sylvatica specialize on ants and mites. To test the hypothesis that poison frog toxin variability reflects species and chemical diversity in arthropod prey, we (1) used sequencing of cytochrome oxidase 1 to identify individual prey specimens, and (2) used liquid chromatography/mass spectrometry to chemically profile consumed ants and mites. We identified 45 ants and 9 mites in frog stomachs, including several undescribed species. We also showed that chemical profiles of consumed ants and mites cluster by frog population, suggesting different frog populations have access to chemically distinct prey. Finally, by comparing chemical profiles of frog skin and isolated prey items, we traced the arthropod source of four poison frog alkaloids, including 3,5- and 5,8-disubstituted indolizidines and a lehmizidine alkaloid. Together, the data show that toxin variability in O. sylvatica reflects chemical diversity in arthropod prey.

We review the systematics of frogs of the Hyloscirtus larinopygion group. A new phylogenetic tree inferred frommitochondrial DNA (partial sequences of 12S rRNA, valine-tRNA, and 16S rRNA genes; ~2.3 kb) of eleven species ofthe H. larinopygion group is provided, based on maximum parsimony, maximum likelihood, and Bayesian analyses. Ourphylogeny confirms the close relationship of members of the H. larinopygion group with Andean relatives of the H.armatus group, which also occurs in the Andes. Hyloscirtus tapichalaca is placed as sister species to the rest of the H.larinopygion group, in which two clades (A+B) are evident. Although ingroup relationships are well supported, themonophyly of the H. larinopygion group and placement of H. tapichalaca require additional testing. Genetic divergencesamong species of the H. larinopygion group are shallow compared to those observed in many other anurans, with geneticdistance among sister species (H. princecharlesi and H. ptychodactylus) as low as 1.31%. However, this pattern isconcordant with radiations in other highland Andean lineages of anurans that show marked morphological or behavioraldifferentiation, but low divergence in mitochondrial markers. Divergence-time analyses (using BEAST) indicate that theHyloscirtus clade is a relatively ancient lineage that appeared in the Eocene, at a minimum age of 51.2 million years ago(MYA), while the H. larinopygion group originated in the Middle-Late Eocene at a minimum age of 40.9 MYA. Ourresults might suggest a rapid radiation of Hyloscirtus starting in the Miocene into the Pliocene, from at least 14.2 MYA tothe most recent divergence between sister taxa at ~2.6 MYA. We also describe two sympatric new species of Hyloscirtusfrom northwestern Ecuador: H. criptico sp. nov. and H. princecharlesi sp. nov. We diagnose them by their phylogeneticposition (they are not sister to each other), genetic divergence, and a unique combination of color patterns, and othermorphological features. Additionally, we describe the suctorial tadpoles and the extreme ontogenic color changes in H.larinopygion, H. lindae, H. pantostictus, H. princecharlesi, H. psarolaimus, and H. tigrinus. Furthermore, we describe theosteology of H. criptico, H. lindae, H. pacha, H. pantostictus, H. princecharlesi, H. psarolaimus, H. ptychodactylus, andH. staufferorum. We describe vocalizations of H. lindae, H. pacha, H. pantostictus, H. pasarolaimus, H. staufferorum, andH. tapichalaca. Hyloscirtus tigrinus is recorded for the first time in Ecuador and its range is extended 62.4 km (airlinedistance), from its southernmost locality record in Departamento de Nariño, Colombia. Most species of the H.larinopygion group are currently severely threatened by extinction, after surviving the catastrophic extinctions in the1980s and 1990s that led to the disappearance of many other sympatric anurans that bred in swiftly flowing water and hadlotic water tadpoles in the Andean highlands. Research and conservation actions are urgently needed for these species. Inorder to better call attention to these conservation issues, we name one of the new species in honor of Prince Charles ofWales, who is contributing significantly to the growth of awareness in the battle against tropical deforestation, climate change, and the catastrophic extinction of rainforest amphibians.

The generalist parasite Trypanosoma cruzi has two phylogenetic lineages associated almost exclusively with bats—Trypanosoma cruzi Tcbat and the subspecies T. c. marinkellei. We present new information on the genetic variation, geographic distribution, host associations, and potential vectors of these lineages. We conducted field surveys of bats and triatomines in southern Ecuador, a country endemic for Chagas disease, and screened for trypanosomes by microscopy and PCR. We identified parasites at species and genotype levels through phylogenetic approaches based on 18S ribosomal RNA (18S rRNA) and cytochrome b (cytb) genes and conducted a comparison of nucleotide diversity of the cytb gene. We document for the first time T. cruzi Tcbat and T. c. marinkellei in Ecuador, expanding their distribution in South America to the western side of the Andes. In addition, we found the triatomines Cavernicola pilosa and Triatoma dispar sharing shelters with bats. The comparisons of nucleotide diversity revealed a higher diversity for T. c. marinkellei than any of the T. c. cruzi genotypes associated with Chagas disease. Findings from this study increased both the number of host species and known geographical ranges of both parasites and suggest potential vectors for these two trypanosomes associated with bats in rural areas of southern Ecuador. The higher nucleotide diversity of T. c. marinkellei supports a long evolutionary relationship between T. cruzi and bats, implying that bats are the original hosts of this important parasite.

Poison frogs sequester small molecule lipophilic alkaloids from their diet of leaf litter arthropods for use as chemical defenses against predation. Although the dietary acquisition of chemical defenses in poison frogs is well documented, the physiological mechanisms of alkaloid sequestration has not been investigated. Here, we used RNA sequencing and proteomics to determine how alkaloids impact mRNA or protein abundance in the little devil frog (Oophaga sylvatica), and compared wild-caught chemically defended frogs with laboratory frogs raised on an alkaloid-free diet. To understand how poison frogs move alkaloids from their diet to their skin granular glands, we focused on measuring gene expression in the intestines, skin and liver. Across these tissues, we found many differentially expressed transcripts involved in small molecule transport and metabolism, as well as sodium channels and other ion pumps. We then used proteomic approaches to quantify plasma proteins, where we found several protein abundance differences between wild and laboratory frogs, including the amphibian neurotoxin binding protein saxiphilin. Finally, because many blood proteins are synthesized in the liver, we used thermal proteome profiling as an untargeted screen for soluble proteins that bind the alkaloid decahydroquinoline. Using this approach, we identified several candidate proteins that interact with this alkaloid, including saxiphilin. These transcript and protein abundance patterns suggest that the presence of alkaloids influences frog physiology and that small molecule transport proteins may be involved in toxin bioaccumulation in dendrobatid poison frogs.

Parental care has evolved repeatedly and independently across animals. While the ecological and evolutionary significance of parental behaviour is well recognized, underlying mechanisms remain poorly understood. We took advantage of behavioural diversity across closely related species of South American poison frogs (Family Dendrobatidae) to identify neural correlates of parental behaviour shared across sexes and species. We characterized differences in neural induction, gene expression in active neurons and activity of specific neuronal types in three species with distinct care patterns: male uniparental, female uniparental and biparental. We identified the medial pallium and preoptic area as core brain regions associated with parental care, independent of sex and species. The identification of neurons active during parental care confirms a role for neuropeptides associated with care in other vertebrates as well as identifying novel candidates. Our work is the first to explore neural and molecular mechanisms of parental care in amphibians and highlights the potential for mechanistic studies in closely related but behaviourally variable species to help build a more complete understanding of how shared principles and species-specific diversity govern parental care and other social behaviour.

Geographic barriers and elevational gradients have long been recognized as important in species diversification. Here, we illustrate an example where both mechanisms have shaped the genetic structure of the Neotropical rainfrog, Pristimantis ornatissimus, which has also resulted in speciation. This species was thought to be a single evolutionary lineage distributed throughout the Ecuadorian Chocó and the adjacent foothills of the Andes. Based on recent sampling of P. ornatissimus sensu lato, we provide molecular and morphological evidence that support the validity of a new species, which we name Pristimantis ecuadorensis sp. nov. The sister species are elevational replacements of each other; the distribution of Pristimantis ornatissimus sensu stricto is limited to the Ecuadorian Chocó ecoregion (< 1100 m), whereas the new species has only been found at Andean localities between 1450–1480 m. Given the results of the Multiple Matrix Regression with Randomization analysis, the genetic difference between P. ecuadorensis and P. ornatissimus is not explained by geographic distance nor environment, although environmental variables at a finer scale need to be tested. Therefore this speciation event might be the byproduct of stochastic historic extinction of connected populations or biogeographic events caused by barriers to dispersal such as rivers. Within P. ornatissimus sensu stricto, morphological patterns and genetic structure seem to be related to geographic isolation (e.g., rivers). Finally, we provide an updated phylogeny for the genus, including the new species, as well as other Ecuadorian Pristimantis.

20Poison frogs sequester chemical defenses from arthropod prey, although the details of how 21 arthropod diversity contributes to variation in poison frog toxins remains unclear. We 22 characterized skin alkaloid profiles in the Little Devil frog, Oophaga sylvatica (Dendrobatidae), 23 across three populations in northwestern Ecuador. Using gas chromatography mass 24 spectrometry, we identified histrionicotoxins, 3,5-and 5,8-disubstituted indolizidines, 25 decahydroquinolines, and lehmizidines as the primary alkaloid toxins in these O. sylvatica 26 populations. Frog skin alkaloid composition varied along a latitudinal gradient across 27 populations in a principal component analysis. We also characterized diversity in arthropods 28 isolated from frog stomach contents and confirmed O. sylvatica specialize on ants and mites. To 29 test the hypothesis that poison frog toxin diversity reflects species and chemical diversity in 30 arthropod prey, we (1) used liquid chromatography mass spectrometry to chemically profile 31 consumed ants and mites, and (2) used sequencing of cytochrome oxidase 1 to identify 32 individual prey specimens. We show that chemical profiles of consumed ants and mites cluster 33 by frog population, suggesting different frog populations have access to chemically distinct prey. 34We identified 45 ants and 9 mites isolated from frog stomachs, finding several undescribed 35 species. Finally, by comparing chemical profiles of frog skin and isolated prey items, we were 36 able to trace the arthropod source of four poison frog alkaloids, including 3,5-and 5,8-37 disubstituted indolizidines and a lehmizidine alkaloid. Together, our data shows the diversity of 38 alkaloid toxins found in O. sylvatica can be traced to chemical diversity in arthropod prey. 39 40

Some South American poison frogs (Dendrobatidae) are chemically defended and use bright aposematic colors to warn potential predators of their unpalatability. Aposematic signals are often frequency‐dependent where individuals deviating from a local model are at a higher risk of predation. However, extreme diversity in the aposematic signal has been documented in poison frogs, especially in Oophaga. Here, we explore the phylogeographic pattern among color‐divergent populations of the Little Devil poison frog Oophaga sylvatica by analyzing population structure and genetic differentiation to evaluate which processes could account for color diversity within and among populations. With a combination of PCR amplicons (three mitochondrial and three nuclear markers) and genome‐wide markers from a double‐digested RAD (ddRAD) approach, we characterized the phylogenetic and genetic structure of 199 individuals from 13 populations (12 monomorphic and 1 polymorphic) across the O. sylvatica distribution. Individuals segregated into two main lineages by their northern or southern latitudinal distribution. A high level of genetic and phenotypic polymorphism within the northern lineage suggests ongoing gene flow. In contrast, low levels of genetic differentiation were detected among the southern lineage populations and support recent range expansions from populations in the northern lineage. We propose that a combination of climatic gradients and structured landscapes might be promoting gene flow and phylogenetic diversification. Alternatively, we cannot rule out that the observed phenotypic and genomic variations are the result of genetic drift on near or neutral alleles in a small number of genes.