The genetic, cellular, and evolutionary basis of skin coloration in the highly polymorphic poison frog, Oophaga pumilio

Studies of phenotypic variation are undisputedly important to understanding the processes of evolution. Animal coloration is important in many ecological contexts, including thermoregulation, predator avoidance, male-male competition, and mate choice. Given its varied function, animal coloration is frequently characterized by phenotypic variation among and within species. The Central American strawberry poison frog, Oophaga pumilio, is known for exhibiting striking color polymorphism across the Bocas del Toro archipelago of Panama. Despite many studies that aim to understand the selective pressures that generated or maintain this variation, relatively little is known about its underlying genetic and morphological basis. Furthermore, the evolution of color variation in this species has been addressed by phylogenetics and population genetics studies, but they have been limited by the challenges associated with studying recently separated populations. To bridge this gap in our knowledge, I investigated the proximate mechanisms of color variation by leveraging the power of next-generation sequencing to find loci associated with color differences. I found that the genes involved in variation in dorsal background coloration are located across broad regions of the genome, rather than being co-localized in one or a few nearby regions. Next, I investigated the proximate mechanism of color variation by examining the presence and absence of pigment types, ratios of pigments, skin components, and chemical compositions of carotenoid-containing cells. My results suggest that large color variations in O. pumilio are explained by differences in pigment proportions and carotenoid suites, not by the absence of pigment cell types. Finally, I applied a multi-species coalescent approach to a SNP dataset derived from the extensive sampling of O. pumilio from Bocas del Toro, then perform ancestral character state reconstructions of dorsal coloration. These analyses provide a high-resolution estimate of relationships for O. pumilio, despite the challenges associated with recent timescales. The results suggest that vicariance explains pattern of genetic differentiation and support the convergent evolution of dull dorsal coloration. Overall, this dissertation provides an important first step toward understanding the proximate mechanisms of phenotypic variation and provides a much-needed phylogenetic backbone for future studies of this system

Genomic Takeover by Transposable Elements in the Strawberry Poison Frog

We sequenced the genome of the strawberry poison frog, Oophaga pumilio, at a depth of 127.5× using variable insert size libraries. The total genome size is estimated to be 6.76 Gb, of which 4.76 Gb are from high copy number repetitive elements with low differentiation across copies. These repeats encompass DNA transposons, RNA transposons, and LTR retrotransposons, including at least 0.4 and 1.0 Gb of Mariner/Tc1 and Gypsy elements, respectively. Expression data indicate high levels of gypsy and Mariner/Tc1 expression in ova of O. pumilio compared with Xenopus laevis. We further observe phylogenetic evidence for horizontal transfer (HT) of Mariner elements, possibly between fish and frogs. The elements affected by HT are present in high copy number and are highly expressed, suggesting ongoing proliferation after HT. Our results suggest that the large amphibian genome sizes, at least partially, can be explained by a process of repeated invasion of new transposable elements that are not yet suppressed in the germline. We also find changes in the spliceosome that we hypothesize are related to permissiveness of O. pumilio to increases in intron length due to transposon proliferation. Finally, we identify the complement of ion channels in the first genomic sequenced poison frog and discuss its relation to the evolution of autoresistance to toxins sequestered in the skin

Fitness consequences of infection by Batrachochytrium dendrobatidis in northern leopard frogs (Lithobates pipiens)

The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), has been linked to amphibian declines and extinctions worldwide. The pathogen has been found on amphibians throughout eastern North America, but has not been associated with mass die-offs in this region. In this study, we conducted laboratory experiments on the effects of Bd infection in a putative carrier species, Lithobates pipiens, using two estimators of fitness: jumping performance and testes morphology. Over the 8-week study period, peak acceleration during jumping was not significantly different between infected and uninfected animals. Peak velocity, however, was significantly lower for infected animals after 8 weeks. Two measures of sperm production, germinal epithelium depth, and maximum spermatic cyst diameter, showed no difference between infected and uninfected animals. The width, but not length, of testes of infected animals was significantly greater than in uninfected animals. This study is the first to show effects on whole-organism performance of Bd infection in post-metamorphic amphibians, and may have important long-term, evolutionary implications for amphibian populations co-existing with Bd infection

Transcriptomic analysis of cave, surface, and hybrid samples of the isopod Asellus aquaticus and identification of chromosomal location of candidate genes for cave phenotype evolution

Abstract Background Transcriptomic methods can be used to elucidate genes and pathways responsible for phenotypic differences between populations. Asellus aquaticus is a freshwater isopod crustacean with surface- and cave-dwelling ecomorphs that differ greatly in multiple phenotypes including pigmentation and eye size. Multiple genetic resources have been generated for this species, but the genes and pathways responsible for cave-specific characteristics have not yet been identified. Our goal was to generate transcriptomic resources in tandem with taking advantage of the species’ ability to interbreed and generate hybrid individuals. Results We generated transcriptomes of the Rakov Škocjan surface population and the Rak Channel of Planina Cave population that combined Illumina short-read assemblies and PacBio Iso-seq long-read sequences. We investigated differential expression at two different embryonic time points as well as allele-specific expression of F 1 hybrids between cave and surface individuals. RNAseq of F 2 hybrids, as well as genotyping of a backcross, allowed for positional information of multiple candidate genes from the differential expression and allele-specific analyses. Conclusions As expected, genes involved in phototransduction and ommochrome synthesis were under-expressed in the cave samples as compared to the surface samples. Allele-specific expression analysis of F 1 hybrids identified genes with cave-biased (cave allele has higher mRNA levels than the surface allele) and surface-biased expression (surface allele has higher mRNA levels than the cave allele). RNAseq of F 2 hybrids allowed for multiple genes to be placed to previously mapped genomic regions responsible for eye and pigmentation phenotypes. In the future, these transcriptomic resources will guide prioritization of candidates for functional analysis