Comparative Genomics and Positive selection analyses
To identify genes associated with adaptation to a pelagic lifestyle in the Procellariiformes, we performed a positive selection analysis across 12 species including eight Procellariiformes species applying the HyPhy aBSREL model. We identified the hallmark of positive selection in 20 (out of the 6,172 single-copy orthologs genes), after correcting for multiple testing (Table S5). The enriched GO analysis uncovered terms related with striated muscle cell differentiation, nutrient reservoir activity, response to starvation, visual learning, positive regulation of neural retina development, olfactory receptor activity or natriuresis (Table S6). We also performed an analysis to assess the global impact of natural selection in Procellariformes (both positive and negative selection), which uncovered a total of 310 genes (Table S7). The GO terms enriched in these genes include wound healing, response to wounding, inflammatory response, sensory perception of sound, smell and chemical stimulus, neurological system process, defense response, response to stress, camera-type eye development, renal system and chloride transport among others (Table S8).
Using OrthoFinder 2.3.8, we identified 182,487 N:N orthogroups across all genes identified in the 12 analysed genomes. This data, together with the estimated ultrametric tree, was used to estimate gene gains, losses, and number of genes in the ancestral nodes using BadiRate; for the analysis we selected the Free Rates (FR) model, since it was the best fitted branch model. The analysis was conducted including all orthogroups, and the minimum number of gains and losses per branch is represented in Figure 3. Our analysis showed a tendency to gain genes in Procellariiformes (+442/-34), while the branch leading to albatrosses (Diomedeidae) showed an opposite effect, with a noticeable loss of genes (+464/-3258); the branch leading to the rest of the Procellariiformes (+379/-15) is in the line of the general behavior of the tubenoses (Table S9). Within the order, families Oceanitidae and Hydrobatidae present the same trend, with the branch leading to H. thethyspresenting a stronger gene loss balance (+325/-966) than the branch leading to the ancestor of Oceanitidae (O. oceanicus and F. grallaria (+182/-414)).
We identified three gene families significantly expanded in the branch leading to the Procellariiformes (Table S9). These families encode zinc finger proteins (OG0000000), olfactory receptors (OG0000084) and avian histones (OG0000224).