Accumulation of deleterious mutations
Relaxation of selection can result in an accumulation of deleterious mutations in the genome. To evaluate whether the genome of the black-faced spoonbill accumulated a significant number of deleterious mutations, we counted the number of nonsynonymous substitutions in both spoonbill species. First, we used ANNOVAR (Wang, Li, & Hakonarson, 2010) to annotate substitutions in the coding region of both spoonbill species to two categories, synonymous and non-synonymous substitutions. Due to the lack of a suitable outgroup, we defined the dominant allele in a species as the ancestral allele, and the minor allele as the derived allele. Assuming that non-synonymous substitutions are deleterious, we counted the number of non-synonymous substitutions in each individual as accumulated deleterious mutations also known as genetic load. To further quantify the deleterious effect of non-synonymous substitutions, we used the Grantham score(Grantham, 1974) to measure their potential phenotypic effect. The Grantham score quantifies the differences in physiochemical similarity and evolutionary exchangeability between two amino acids, where a higher score indicates more physiochemical difference and lower evolutionary exchangeability (i.e. higher deleterious phenotypic effect of nonsynonymous substitution). Grantham scores can be classified into three categories(Chun & Fay, 2009), conservative changes (0-50), moderate changes (51-100) and radical changes (>100). Assuming conservative changes in amino acids to be neutral and different deleterious non-synonymous substitutions to have an independent and additive effect, we counted the accumulated deleterious effect of non-synonymous substitutions by summing up the Grantham scores categorized as moderate or radical changes for each individual.