The transcriptomes of cave and surface populations of Gammarus minus (Crustacea: Amphipoda) provide evidence for positive selection on cave downregulated transcripts

Gammarus minus, a freshwater amphipod living in the cave and surface streams in the eastern USA, is an excellent model for investigating evolutionary adaptation to the subterranean environment. RNA-Seq was conducted on one pair of morphologically distinct sister populations inhabiting surface and cave habitats to identify genes that were differentially expressed in the two populations, as well as to compare levels and patterns of genetic variation within and between populations. Of the 104,630 transcripts identified in the transcriptome assembly, 57% had higher average levels of expression in the cave population. After Benjamini-Hochberg correction for multiple tests, 1517 and 551 transcripts were significantly upregulated or downregulated, respectively, in the cave population, indicating an almost three-fold enrichment of cave-upregulated genes. The average level of nucleotide diversity across all transcripts was significantly lower in the cave population. Within the cave population, where the average nucleotide diversity of cave-downregulated transcripts was 75% that of the cave-upregulated transcripts, a highly significant difference, whereas within the spring population the nucleotide diversities of cave-downregulated and cave-upregulated transcripts was virtually identical. Three lines of evidence suggest that the reduced variation in cave downregulated transcripts is due to positive selection in the cave population: 1) the average neutrality index of cave-downregulated genes was < 1, consistent with positive selection, and significantly less than that of cave-upregulated genes; 2) Tajima’s D was positively correlated with the cave:surface expression ratio, and 3) cave-downregulated transcripts were significantly more likely to be highly diverged from their surface homologs than cave-upregulated transcripts. Five transcripts had fixed premature termination codons in the cave population. The expression patterns and sequence variation in one such transcript, encoding the DNA repair protein photolyase, were examined in more detail and provide the first evidence for the relaxation of functional constraint in this light-dependent protein in a subterranean population.