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).