Relaxation of purifying selection
Genetic drift can lead to relaxation of selection by lowering selection
efficacy. To evaluate the level of selection relaxation, we used the
descriptive model in RELAX (Wertheim et al., 2015) to infer a
relaxation parameter k for 7,835 orthologous genes between the
two spoonbill species and an outgroup, the crested ibis Nipponia
nippon (S. Li et al., 2014). A relaxation parameter k> 1 indicates that the gene was under intensified
selection, and k < 1 implies relaxed selection. We
counted the number of genes with a k < 1 for each
spoonbill species. First, we used the program Inparanoid v.
4.1(Remm, Storm, & Sonnhammer, 2001) to identify homologous genes
between the crested ibis and the black-faced spoonbill. Then, we usedgffread v. 0.11.4 (https://github.com/gpertea/gffread) to extract
the coding region sequences of each homologous gene from each
black-faced and royal spoonbill individual. We used MACSEv0.9b1(Ranwez, Harispe, Delsuc, & Douzery, 2011) to align the
homologous sequences from all three species. We removed sequences that
contained frameshifts and/or stop codons flagged by MACSE from the
subsequent analysis. Then we used RAxMAL8 v. 8.2.12(Stamatakis,
2014) to draw the maximum likelihood gene tree of all 5,657 well-aligned
homologous genes with the GTRGAMMA model. Gene trees of all orthologous
genes from all three species in focus were used for the RELAXanalysis. The likelihood ratio test in RELAX was used to estimatek for each homologous gene by comparing the model which fixedk =1 and the model which allowed k to be estimated. The
MRCA branch and each branch of each spoonbill species were alternatively
set as the test branch in the RELAX analysis.