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.

A nanocarrier system that potentiates the effect of miconazole within different interkingdom biofilms.

BACKGROUND: Novel and new therapeutic strategies capable of enhancing the efficacy of existing antimicrobials is an attractive proposition to meet the needs of society. OBJECTIVE: This study aimed to evaluate the potentiating effect of a miconazole (MCZ) nanocarrier system, incorporated with iron oxide nanoparticles (IONPs) and chitosan (CS) (IONPs-CS-MCZ). This was tested on three representative complex interkingdom oral biofilm models (caries, denture and gingivitis). MATERIALS AND METHODS: The planktonic and sessile minimum inhibitory concentrations (MICs) of IONPs-CS-MCZ against different Candida albicans strains were determined, as well as against all represented bacterial species that formed within the three biofilm models. Biofilms were treated for 24 hours with the IONPs-CS nanocarrier system containing MCZ at 64 mg/L, and characterized using a range of bioassays for quantitative and qualitative assessment. RESULTS: MIC results generally showed that IONPs-CS-MCZ was more effective than MCZ alone. IONPs-CS-MCZ also promoted reductions in the number of CFUs, biomass and metabolic activity of the representative biofilms, as well as altering biofilm ultrastructure when compared to untreated biofilms. IONPs-CS-MCZ affected the composition and reduced the CFEs for most of the microorganisms present in the three evaluated biofilms. In particular, the proportion of streptococci in the biofilm composition were reduced in all three models, whilst Fusobacterium spp. percentage reduced in the gingivitis and caries models, respectively. CONCLUSION: In conclusion, the IONPs-CS-MCZ nanocarrier was efficient against three in vitro models of pathogenic oral biofilms, showing potential to possibly interfere in the synergistic interactions among fungal and bacterial cells within polymicrobial consortia.

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.

The tadpole of Ameerega boehmei in southeastern Bolivia.

To date, half (16 of 32) of the species of Ameerega have had their tadpoles described: A. altamazonica, A. bassleri, A. bilinguis, A. braccata, A. cainarachi, A. flavopicta, A. hahneli, A. macero, A. parvula, A. petersi, A. picta, A. rubriventris, A shihuemoy. A. silverstonei, A. smaragdina, and A. trivittata (Lescure, 1976; Silverstone, 1976; Duellman, 1978; Myers Daly, 1979; Rodriguez Myers, 1993; Haddad Martins, 1994; Lötters et al., 1997; Duellman, 2005; Costa et al., 2006; Twomey Brown, 2008; Brown Twomey, 2009; Poelman et al., 2010; Schulze et al., 2015). Ameerega boehmei is a putative member of a clade containing Ameerega braccata, A. flavopicta, A. berohoka, A. munduruku, all of which inhabit various parts of the 'dry diagonal' between the Amazon and Atlantic rainforests (Prado Gibbs, 1993). Adult frogs in this group are morphologically similar, generally dark-bodied with yellow dorsolateral stripes, orange flash marks and some also possessing bright-yellow dorsal spots. Despite considerable research on their breeding behavior, acoustics and systematics (Lötters et al., 2009; Forti et al., 2013), the tadpole of Ameerega boehmei, the southern-most and western-most distributed species in this tentative group, has not been described.

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.

Integrating statistical genetic and geospatial methods brings new power to phylogeography.

The field of phylogeography continues to grow in terms of power and accessibility. Initially uniting population genetics and phylogenetics, it now spans disciplines as diverse as geology, statistics, climatology, ecology, physiology, and bioinformatics to name a few. One major and recent integration driving the field forward is between "statistical phylogeography" and Geographic Information Systems (GIS) (Knowles, 2009). Merging genetic and geospatial data, and their associated methodological toolkits, is helping to bring explicit hypothesis testing to the field of phylogeography. Hypotheses derived from one approach can be reciprocally tested with data derived from the other field and the synthesis of these data can help place demographic events in an historical and spatial context, guide genetic sampling, and point to areas for further investigation. Here, we present three practical examples of empirical analysis that integrate statistical genetic and GIS tools to construct and test phylogeographic hypotheses. Insights into the evolutionary mechanisms underlying recent divergences can benefit from simultaneously considering diverse types of information to iteratively test and reformulate hypotheses. Our goal is to provide the reader with an introduction to the variety of available tools and their potential application to typical questions in phylogeography with the hope that integrative methods will be more broadly and commonly applied to other biological systems and data sets.

Tactical reproductive parasitism via larval cannibalism in Peruvian poison frogs.

We report an unusual example of reproductive parasitism in amphibians. Dendrobates variabilis, an Amazonian poison frog, oviposits at the surface of the water in small pools in plants and deposits tadpoles within the pools. Tadpoles are highly cannibalistic and consume young tadpoles if they are accessible. Deposition of embryos and tadpoles in the same pool is common. Genetic analyses indicate that tadpoles are frequently unrelated to embryos in the same pool. A pool choice experiment in the field demonstrated that males carrying tadpoles prefer to place them in pools with embryos, facilitating reproductive parasitism via cannibalism.

Genetic divergence and speciation in lowland and montane peruvian poison frogs.

Amazonia is famous for high biodiversity, and the highlands of the transition zone between the Andes and the lowlands of the Amazon basin show particularly high species diversity. Hypotheses proposed to explain the high levels of diversity in the highlands include repeated parapatric speciation across ecological gradients spanning the transition zone, repeated allopatric speciation across geographic barriers between the highlands and lowlands, divergence across geographic barriers within the transition zone, and simple lineage accumulation over long periods of time. In this study, we investigated patterns of divergence in frogs of the genus Epipedobates (family Dendrobatidae) using phylogenetic and biogeographic analyses of divergence in mitochondrial DNA (1778 aligned positions from genes encoding cyt b, 12S and 16S rRNA for 60 Epipedobates and 11 outgroup specimens) and coloration (measured for 18 specimens representing nine species in Epipedobates). The majority of phenotypic and species diversity in the poison frog genus Epipedobates occurs in the transition zone, although two morphologically conserved members of the genus are distributed across the lowlands of the Amazon basin. Phylogenetic analysis reveals that there is a single highland clade derived from an ancestral colonization event in northern Peru by a population of lowland ancestry. Epipedobates trivittatus, a widespread Amazonian species, is a member of the highland clade that reinvaded the lowlands. Comparative analyses of divergence in coloration and mtDNA reveals that divergence in coloration among populations and species in the highlands has been accelerated relative to the lowlands. This suggests a role for selection in the divergence of coloration among populations and species.