Co-infecting pathogen lineages have additive effects on host bacterial communities.

Amphibian skin bacteria may confer protection against the fungus Batrachochytrium dendrobatidis (Bd), but responses of skin bacteria to different Bd lineages are poorly understood. The global panzootic lineage (Bd-GPL) has caused amphibian declines and extinctions globally. However, other lineages are enzootic (Bd-Asia-2/Brazil). Increased contact rates between Bd-GPL and enzootic lineages via globalization pose unknown consequences for host-microbiome-pathogen dynamics. We conducted a laboratory experiment and used 16S rRNA amplicon-sequencing to assess: (i) whether two lineages (Bd-Asia-2/Brazil and Bd-GPL) and their recombinant, in single and mixed infections, differentially affect amphibian skin bacteria; (ii) and the changes associated with the transition to laboratory conditions. We determined no clear differences in bacterial diversity among Bd treatments, despite differences in infection intensity. However, we observed an additive effect of mixed infections on bacterial alpha diversity and a potentially antagonistic interaction between Bd genotypes. Additionally, observed changes in community composition suggest a higher ability of Bd-GPL to alter skin bacteria. Lastly, we observed a drastic reduction in bacterial diversity and a change in community structure in laboratory conditions. We provide evidence for complex interactions between Bd genotypes and amphibian skin bacteria during coinfections, and expand on the implications of experimental conditions in ecological studies.

A new species of direct-developing frog of the genus Eleutherodactylus (Anura: Eleutherodactylidae) from the Pacific lowlands of Guerrero, Mexico.

We describe a new species of direct-developing frog of the genus Eleutherodactylus from the Pacific lowlands of Guerrero, Mexico. Eleutherodactylus erythrochomus sp. nov. is a saxicolous species that inhabit caves or crevices formed by groups of large granitic boulders, which create contrasting climatic characteristics to the surrounding habitat. This new species is geographically restricted to the Tierra Colorada region, a biologically important area that harbors microendemic taxa. This species is the first microendemic amphibian described from this region. The new species can be easily differentiated from congeners by having long and slender limbs, lacking a lumbo-inguinal gland, and expanded digital pads. Further exploration and conservation measures should be conducted at the type locality of this new species in order to preserve and understand the herpetofaunistic composition occurring therein.

Ecomorphological convergence in Eleutherodactylus frogs: a case of replicate radiations in the Caribbean.

Replicate radiations, the repeated multiplication of species associated with ecological divergence, have attracted much attention and generated as much debate. Due to the few well-studied cases, it remains unclear whether replicate radiations are an exceptional result of evolution or a relatively common example of the power of adaptation by natural selection. We examined the case of Eleutherodactylus frogs, which radiated in the Caribbean islands resulting in more than 160 species that occupy very diverse habitats. A time-calibrated phylogeny revealed that these frogs independently diversified on all larger islands producing species that occupy a broad range of microhabitats in different islands. Using phylogenetic comparative methods, we found an association between morphological traits and particular microhabitats, and for most microhabitats detected significant morphological convergence. Our results indicate Caribbean Eleutherodactylus are a novel example of replicate radiations, and highlight the predictability of evolutionary processes, as similar ecological opportunities can lead to similar outcomes.

The Chytrid Fungus, Batrachochytrium dendrobatidis, is Widespread Among Cuban Amphibians.

The fungus Batrachochytrium dendrobatidis (Bd) is a generalist amphibian pathogen responsible for chytridiomycosis. It was documented for the first time in Cuba in 2007, the apparent cause of the decline in one species of toad. In a recent survey, Bd was reported only for the highlands of Central Cuba. In the present study, we reexamined the geographic distribution and level of impact of Bd in Cuba by conducting an island-wide sampling in 10 localities and collecting skin swabs from 18 species and 28 environmental samples. We report detection of Bd in 60% of sampled sites and in 58% of sampled taxa. We show that Bd is associated with riparian, arboreal and terrestrial species, and it was estimated to occur in approximately 30% of the aquatic habitats we sampled. In addition, we confirmed that a dying individual of the species Eleutherodactylus casparii was severely infected with Bd. We also rise concern about the endanger toad Peltophryne longinasus and about three species of endemic riparian frogs that were not detected during our surveys. This study demonstrates that this pathogen is widespread throughout Cuba and provides relevant evidence to advance our understanding of its detection in amphibians and the aquatic environment in Cuba and about the occurrence of Bd in species with different ecologies. We provide valuable baseline information for Bd risk assessment and decision-making processes to mitigate its negative impact on Cuban amphibians.

Chromosome Banding in Amphibia. XXXVI. Multimorphic Sex Chromosomes and an Enigmatic Sex Determination in Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

A detailed cytogenetic study on the leaf litter frog Eleutherodactylus johnstonei from 14 different Caribbean islands and the mainlands of Venezuela and Guyana revealed the existence of multimorphic XY♂/XX♀ sex chromosomes 14. Their male sex determination and development depends either on the presence of 2 telocentric chromosomes 14 (XtYt), or on 1 submetacentric chromosome 14 (Xsm) plus 1 telocentric chromosome 14 (Yt), or on the presence of 2 submetacentric chromosomes 14 (XsmYsm). The female sex determination and development requires either the presence of 2 telocentric chromosomes 14 (XtXt) or 2 submetacentric chromosomes 14 (XsmXsm). In all individuals analyzed, the sex chromosomes 14 carry a prominent nucleolus organizer region in their long arms. An explanation is given for the origin of the (XtYt)♂, (XsmYt)♂, (XsmYsm)♂, (XtXt)♀, and (XsmXsm)♀ in the different populations of E. johnstonei. Furthermore, the present study gives detailed data on the chromosome banding patterns, in situ hybridization experiments, and the genome size of E. johnstonei.

Behavioural tuning in a tropical amphibian along an altitudinal gradient.

Males of the coqui treefrog, Eleutherodactylus coqui, produce a distinct two-note 'co-qui' advertisement call from sunset to midnight throughout most of the year. Previous work established that both the spectrotemporal aspects of the call and the frequency of highest inner-ear sensitivity change with altitude above sea level. These variations are such that the frequency of the emitted co-note closely matches the frequency to which the inner ear is most sensitive. Given this parallel variation, we expected that the call-evoked behavioural response of male coqui treefrogs would also show an altitude dependence, and hypothesized that males would produce their most robust acoustical territorial response to advertisement calls that match calls from their own altitude. We tested this hypothesis in the field by studying the vocal response behaviour of coquis to playbacks of synthetic, altitude-dependent conspecific calls, and indeed found that the most robust vocal responses were obtained using stimuli closely matching the calls from the same altitude.

Temperature variation, bacterial diversity and fungal infection dynamics in the amphibian skin.

Host-associated bacterial communities on the skin act as the first line of defence against invading pathogens. Yet, for most natural systems, we lack a clear understanding of how temperature variability affects structure and composition of skin bacterial communities and, in turn, promotes or limits the colonization of opportunistic pathogens. Here, we examine how natural temperature fluctuations might be related to changes in skin bacterial diversity over time in three amphibian populations infected by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Our focal host species (Eleutherodactylus coqui) is a direct-developing frog that has suffered declines at some populations in the last 20 years, while others have not experienced any changes. We quantified skin bacterial alpha- and beta-diversity at four sampling time points, a period encompassing two seasons and ample variation in natural infections and environmental conditions. Despite the different patterns of infection across populations, we detected an overall increase in bacterial diversity through time, characterized by the replacement of bacterial operational taxonomic units (OTUs). Increased frog body temperatures possibly allowed the colonization of bacteria as well as the recruitment of a subset of indicator OTUs, which could have promoted the observed changes in diversity patterns. Our results suggest that natural environmental fluctuations might be involved in creating opportunities for bacterial replacement, potentially attenuating pathogen transmission and thus contributing to host persistence in E. coqui populations.

Diversification and asymmetrical gene flow across time and space: lineage sorting and hybridization in polytypic barking frogs.

Young species complexes that are widespread across ecologically disparate regions offer important insights into the process of speciation because of their relevance to how local adaptation and gene flow influence diversification. We used mitochondrial DNA and up to 28 152 genomewide single nucleotide polymorphisms from polytypic barking frogs (Craugastor augusti complex) to infer phylogenetic relationships and test for the signature of introgressive hybridization among diverging lineages. Our phylogenetic reconstructions suggest (i) a rapid Pliocene-Pleistocene radiation that produced at least nine distinct lineages and (ii) that geographic features of the arid Central Mexican Plateau contributed to two independent northward expansions. Despite clear lineage differentiation (many private alleles and high between-lineage FST scores), D-statistic tests, which differentiate introgression from ancestral polymorphism, allowed us to identify two putative instances of reticulate gene flow. Partitioned D-statistics provided evidence that these events occurred in the same direction between clades but at different points in time. After correcting for geographic distance, we found that lineages involved in hybrid gene flow interactions had higher levels of genetic variation than independently evolving lineages. These findings suggest that the nature of hybrid compatibility can be conserved overlong periods of evolutionary time and that hybridization between diverging lineages may contribute to standing levels of genetic variation.

Climate change and frog calls: long-term correlations along a tropical altitudinal gradient.

Temperature affects nearly all biological processes, including acoustic signal production and reception. Here, we report on advertisement calls of the Puerto Rican coqui frog (Eleutherodactylus coqui) that were recorded along an altitudinal gradient and compared these with similar recordings along the same altitudinal gradient obtained 23 years earlier. We found that over this period, at any given elevation, calls exhibited both significant increases in pitch and shortening of their duration. All of the observed differences are consistent with a shift to higher elevations for the population, a well-known strategy for adapting to a rise in ambient temperature. Using independent temperature data over the same time period, we confirm a significant increase in temperature, the magnitude of which closely predicts the observed changes in the frogs' calls. Physiological responses to long-term temperature rises include reduction in individual body size and concomitantly, population biomass. These can have potentially dire consequences, as coqui frogs form an integral component of the food web in the Puerto Rican rainforest.

Population Declines of Mountain Coqui (Eleutherodactylus portoricensis) in the Cordillera Central of Puerto Rico.

The Mountain Coqui (Eleutherodactylus portoricensis) is a frog endemic to montane rainforests in the Cordillera Central and Luquillo Mountains of Puerto Rico. Classified as endangered by the IUCN Red List and as vulnerable by the Department of Natural and Environmental Resources of Puerto Rico, this species has undergone considerable decline in the Luquillo Mountains. To evaluate the population status of E. portoricensis across its entire range, we conducted ~87 hours of surveys at 18 historical localities and 25 additional localities that we considered suitable for this species. We generated occupancy models to estimate the probability of occurrence at surveyed sites and to identify geographic and climatic factors affecting site occupancy. We also constructed a suitability map to visualize population status in relation to the presence of land cover at elevations where the species has been documented, and determined the dates when populations were last detected at historical localities. Eleutherodactylus portoricensis was detected at 14 of 43 localities, including 10 of 18 historical localities, but it was not detected at any localities west of Aibonito (western Cordillera Central). Occupancy models estimated the probability of occurrence for localities in the western Cordillera Central as zero. Site occupancy was positively associated with montane cloud forest, and negatively associated with the western Cordillera Central, maximum temperature, and precipitation seasonality. The suitability map suggests that declines have occurred despite the presence of suitable habitat. We suggest upgrading the extinction risk of E. portoricensis and potentially developing a captive breeding program for this species.

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 µg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitroquinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 !g/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0 percent to 8 percent. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV < 10 percent). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.