Skin and coat pigmentation are two of the best-studied examples of traits under natural selection given their quantifiable fitness interactions with the environment (e.g. camouflage) and signaling with other organisms (e.g. warning coloration). Previous morphological studies have found that skin pigmentation variation in the Virginia opossum (Didelphis virginiana) is associated with variation in precipitation and temperatures across its distribution range following Gloger’s rule (lighter pigmentation in temperate environments). To investigate the molecular mechanism associated with skin pigmentation variation, we used RNA-Seq and quantified gene expression of wild opossums from tropical and temperate populations. Using differential expression analysis and a coexpression network approach, we found that expression variation in genes with melanocytic and immune functions are significantly associated with the degree of skin pigmentation variation and may be underlying this phenotypic difference. We also found evidence suggesting that the Wnt/β-catenin signaling pathway might be regulating the depigmentation observed in temperate populations. Based on our study results, we present several alternative hypotheses that may explain Gloger’s rule pattern of skin pigmentation variation in opossum, including changes in pathogen diversity supporting a pathogen resistant hypothesis, thermal stress associated with temperate environments, pleiotropic and epistatic interactions between melanocytic and immune genes.
PCA input. cqn normalized and log2 transformed counts
Tab-delimited text file containing the cqn normalized and log2 transformed gene expression counts (using the conditional quantile normalization method) for 14,289 genes in 13 virginia opossum individuals. This normalized-transformed counts were used as input for the principal component analysis (PCA).
Nigenda-Morales_normalized_transfomed_counts_PCA_input.txt
PCA results
Tab-delimited text file containing the results of the principal component analysis (PCA). It shows the loadings of 13 principal components (PC) for 14,289 genes. The genes are ordered by the loadings of PC1 from most negative to most positive.
Nigenda-Morales_PCA_results.txt
WGCNA input. Phenotypic traits of Virginia opossum individuals
Tab-delimited text file with phenotypic trait measurements fro 13 Virginia opossum specimens that was used as input for the weighted gene coexpression network analysis (WGCNA). The traits that were used in the WGCNA analysis were PTP (proportion of tail pigmentation), PEP (proportion of ear pigmentation) and PTO (proportion of toe pigmentation).
Nigenda-Morales_Opossum_traits_WGCNAinput.txt
Raw sequenceing data, raw expression counts per gene after filtering, normalized counts and metadata
This links to the Gene Expression Omnibus (GEO) series accession number GSE112562, which contains the following data files that can be divided in 4 sections: 1) 26 raw sequencing data files (gzipped fastq files; two files per individual due to the pair-end sequencing strategy used), 2) a tab-delimited text file containing raw expression counts for 14,289 genes (HTSeq output) after filtering for low count numbers and muscle genes removal, 3) a tab-delimited text file containing the cqn normalized and log2 transformed counts for the 14,289 genes, and 4) associated metadata (excel file) for 13 Virginia opossum individuals with pigmented and depigmented phenotypes
WGCNA input. Normalized-transformed counts for the differentially expressed genes
Tab-delimited text file containing the cqn normalized and log2 transformed counts (using the conditional quantile normalization method) for the 1,186 differentially expressed genes between pigmented and depigmented opossums. This normalized-transformed counts were used as input for the weighted gene coexpression network analysis (WGCNA).
Nigenda-Morales_DEgenes_normalized-transformed_WGCNAinput.txt
Differential expression results and qvalue for multiple testing correction
Tab-delimited text file containing the results of the differential gene expression test (limma-voom results) between pigmented and depigmented Virginia opossums. To account for multiple hypothesis testing the false discovery rate (FDR) method was used as implemented in the qvalue package in R. Genes were considered differentially expressed if they had a qvalue < 0.1. In this file the genes are order by qvalue from lower to higher.
Nigenda-Morales_Differential_expression_results_qvalue.txt