Being red, blue and green: the genetic basis of coloration differences in the strawberry poison frog (Oophaga pumilio)

Abstract

Background: Animal coloration is usually an adaptive attribute, under strong local selection pressures and often diversified among species or populations. The strawberry poison frog (Oophaga pumilio) shows an impressive array of color morphs across its distribution in Central America. Here we quantify gene expression and genetic variation to identify candidate genes involved in generating divergence in coloration between populations of red, green and blue O. pumilio from the Bocas del Toro archipelago in Panama.

Results: We generated a high quality non-redundant reference transcriptome by mapping the products of genome-guided and de novo transcriptome assemblies onto a re-scaffolded draft genome of O. pumilio. We then measured gene expression in individuals of the three color phenotypes and identified color-associated candidate genes by comparing differential expression results against a list of a priori gene sets for five different functional categories of coloration - pteridine synthesis, carotenoid synthesis, melanin synthesis, iridophore pathways (structural coloration), and chromatophore development. We found 68 candidate coloration loci with significant expression differences among the color phenotypes. Notable upregulated examples include pteridine synthesis genes spr, xdh and pts (in red and green frogs); carotenoid metabolism genes bco2 (in blue frogs), scarb1 (in red frogs), and guanine metabolism gene psat1 (in blue frogs). We detected significantly higher expression of the pteridine synthesis gene set in red and green frogs versus blue frogs. In addition to gene expression differences, we identified 370 outlier SNPs on 162 annotated genes showing signatures of diversifying selection, including eight pigmentation-associated genes.

Conclusions: Gene expression in the skin of the three populations of frogs with differing coloration is highly divergent. The strong signal of differential expression in pteridine genes is consistent with a major role of these genes in generating the coloration differences among the three morphs. However, the finding of differentially expressed genes across pathways and functional categories suggests that multiple mechanisms are responsible for the coloration differences, likely involving both pigmentary and structural coloration. In addition to regulatory differences, we found potential evidence of differential selection acting at the protein sequence level in several color-associated loci, which could contribute to the color polymorphism.

Keywords: Coloration genetics; Gene expression; Pigments; Poison frog; SNPs.

Fig. 1

Sampling scheme and general gene expression patterns. A) Geographic location of localities in the Bocas del Toro archipelago where Oophaga pumilio samples were obtained (AL, Almirante; AG, Aguacate; PO, Popa) and their associated color phenotypes (inset photos). B) Plot of the principal component analysis summarizing the expression pattern across samples of the three color phenotypes. The background map in A (© OpenStreetMap contributors) was created with open data cartography licensed under a Creative Commons Attribution-ShareAlike 2.0 license (CC BY-SA, https://www.openstreetmap.org/copyright)

Fig. 2

Expression patterns of genes in three color phenotypes of Oophaga pumilio classified into five functional groups of color-associated genes (pigment synthesis pathways, guanine synthesis in iridophores, and chromatophore differentiation). Each heat map plot was simplified by averaging expression values across all samples in each color morph to show the expression profiles of all genes in each group. DE genes are highlighted in bold except for the chromatophore differentiation where only DE genes are shown. Details of the gene sets are presented on the main text and SM

Fig. 3

Signatures of selection on single-nucleotide polymorphisms (SNPs) in the transcriptome of Oophaga pumilio. A) Graphical results of the BayeScan analysis, the plot shows, for each of the 398,910 bi-allelic SNPs tested, the Fst between the three color phenotypes and their corresponding q-values (SNPs under diversifying selection, q < 0.05 and α > 0, are highlighted in red). B) Pie chart showing the positions and predicted effects of the 370 outlier SNPs detected in the BayeScan analysis. C) Allelic frequencies of the outlier SNPs occurring in color-associated genes, each bar chart shows the frequency of alleles in each population