3.2 Codon based test of selection
The summary of synonymous and nonsynonymous polymorphisms and divergence across species indicates that singleton removal increased the estimated proportion of positively selected genes in all three species and reduced indices of negative selection (Table 1), indicating the presence of segregating weakly deleterious mutations. In G. calmariensis , we detected mainly positive selection (α > 0) and a higher proportion of positively selected genes. On the other hand, inG. tenella , weak negative selection was more common (α< 0) even after singleton removal and with a higher proportion of negatively selected genes. In the following sections, we only discuss inferences after removing singletons.
Out of the 4154 genes, the HDMKPRF identified a similar number of genes under selection in the three Galerucella species (Fig. 1). InG. pusilla , 469 and 562 genes were identified as being under positive and negative selection, respectively. In G. calmariensis , 665 and 598 genes were identified as being under positive and negative selection, respectively. Finally, in G. tenella , 442 and 466 genes were identified as being under positive and negative selection, respectively. Because genes under positive selection are more commonly associated with lineage-specific adaptive traits, we focus our analyses on genes under positive selection.
The gene set enrichment analysis found several functions that were enriched in genes under positive selection, some of which were common among the three beetle species and some that differed (Tables S3-S5). First, enriched functions common among the beetle species included functions involved in the formation of adult morphology, such as the imaginal disc pattern formation (forming the adult cuticle and appendage structures), the wing disc pattern formation (forming wing structures) and the dorsal/ventral pattern formation. Second, unique gene categories under positive selection in G. calmariensis included those coding for metabolic processes (e.g., processes related to carbohydrate derivatives, oligosaccharides, amino sugars, sulphur compounds and catechol-containing compounds) and those coding for processes in the nervous system (e.g., neuroblast proliferation, neuroblast differentiation, nervous system process) (Table S3). Third, unique gene categories under positive selection in G. pusilla included those coding for positive regulation of the innate immune response, including positive regulation of small GTPase mediated signal transduction, and those coding for axoneme assembly (e.g., cilium movement, cilium organization) (Table S5). Finally, unique gene categories under positive selection in G. tenella included genes coding for a range of biosynthetic processes (e.g., nucleobase-containing compound biosynthetic processes, heterocycle biosynthetic processes and aromatic compound biosynthetic processes), genes coding for lipid metabolic processes (e.g., sterol metabolism, membrane lipid biosynthesis, cellular lipid metabolism, sphingolipid metabolism and lipoprotein metabolism), but also GO pathways involving the activation of immune response, pigment metabolic process involved in pigmentation, peripheral nervous system development and response to oxidative stress (Table S4).