3.2 Genes under 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 , 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.
The HDMKPRF identified a similar number of genes under selection in the three Galerucella species (Fig. 1). In G. pusilla , 451 and 562 genes were identified as being under positive and negative selection, respectively. In G. calmariensis , 610 and 553 genes were identified as being under positive and negative selection, respectively. Finally, in G. tenella , 425 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 (e.g., immunity genes), we focus our analyses on gene function connected to 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 (Table S2-S4). 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). Third, unique gene categories under positive selection in G. pusilla included those coding for positive regulation of the innate immune response (e.g., positive regulation of small GTPase mediated signal transduction) and those coding for axoneme assembly (e.g., cilium movement, cilium organization). 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.