Molecular Evolution Of Avian Diets Within A Phylogenetic Framework

As the largest group that is easy to be identified and has the highest species diversity in tetrapod vertebrates,birds possess several morphological characteristics that are distinct from other vertebrates,such as the cutin beak,feather,air sac,strong but lightweight skeleton,cariniform cartilage and synsacrum.Due to the rapid global expansion and radiation of birds after the mass extinction in the late Cretaceous(about 66 Mya),there is an abundant diversity in morphology,ecology and behavior of more than 10,000 existing birds(Neornithes),which is also a prerequisite for the avian dietary diversification.The Passerida,belonging to the order Passeriformes,are mainly composed of three major subclades: Passeroidea,Sylvioidea and Musicicapoidea.But the relationship between three subclades has been controversial.In addition,the taxonomic status of another group that was extensively studied,namely Paridae,and the relationship between Paridae and three major subclades are still unclear.In order to solve the problems mentioned above,we combined the genome data of available 13 Passerida birds with transcriptome data from mixed tissues of three Passerida birds(Aethopyga christinae,Garrulax canorus and Zosterops japonicus)sequenced by this study and a total of 3,342 orthologs have been found among 17 birds,with Corvus brachyrhynchos as outgroup.Because of the higher heterogeneity in gene trees,one coalescent method,the MP-EST was chosen to reconstruct the phylogeny of main groups among Passerida.Results showed that compared with Sylvioidea,Passeroidea is much closer to Musicicapoidea,and as the most ancient group,Paridae is the first clade diverging from Passerida and does not belong to any of three major subclades.Based on the phylogenetic tree reconstructed above,we hypothesized that genes relating to metabolism in 16 Passerida species are under adaptive evolution.With reference to the comprehensive avian dietary database,all birds were classified into two groups with higher and lower consumption of insects,seeds or nectar/friut.Selective pressure analyses were performed on the 3,443 orthologs.Results showed that most of genes related to proteins or lipids metabolism,such as AGK,GALNT7 and PHKB,are under rapid evolution in birds with higher insect consumption;for species with higher seed consumption,most genes related to starches or lipids metabolism,such as ALDH6A1,GPCPD1 and SLC16A1,have evolved relatively faster;For birds with higher nectar/fruit consumption,most genes related to carbohydrate metabolism,such as C1QL3,HYAL1 and PKM,have experienced accelerated evolution.All findings indicated that genes related to metabolism mentioned above may play important roles in the dietary diversification of Passerida.Additionally,with the aim to investigate whether differentially expressed genes related to dietary habits exist in the digestive glands or organs of birds,we selected two closely related birds(G.canorus and Z.japonicus)with different omnivorous degrees,performed transcriptome sequencing on their intestines and liver tissues,and finally obtained 6,428 and 5,549 orthologs from the intestine and the liver,respectively.Differential expression analyses showed that a total of 475(intestine of G.canorus),365(intestine of Z.japonicus),431(liver of G.canorus)and 348(liver of Z.japonicus)genes were highly expressed.GO enrichment indicated that genes with high expression in both the intestines and liver of G.canorus were mostly related to immunity,and then related to metabolism,while highly expressed genes in Z.japonicus were mostly related to metabolism and less related to immunity and biosynthesis.Considering the life history strategies of two species,we assumed that G.canorus and Z.japonicus tend to be K-selected and r-selected,respectively.Compared with r-selected organisms,K-selected organisms are normallyphysically strong and possess an enhanced immunity,G.canorus are thus more likely to have more immune-related highly expressed genes.Meanwhile,the greater complexity in food composition of Z.japonicus may be one of the reasons why most highly expressed genes enriched in its liver and gut are metabolism-related.Since digestive enzymes are closely related to energy acquisition and nutrition absorption,we next hypothesized that genes encoding avian digestive enzymes should be under adaptive evolution.We identified 15 digestive enzyme genes [four lipase genes(CYP7A1,LIPC,LIPF and PNLIP),three amylase genes(hAMY,pAMY and sAMY),two protease genes(CTRC and PGC),two lysozyme genes(LYZ and LYG),two chitinase genes(CHIA and CHIT)and two trehalase genes(ATHL1 and TREH)] from available genomes of 48 bird species.Synteny analysis suggests that four genes(CHIT,LIPF,Treh and sAMY)have been lost in the common ancestor of birds.Of the remaining 11 genes,seven are single-copied and four(CHIA,LYC,LYG and PGC)are multi-copied.The multi-copied PGC and LYG were predicted to have functional divergence among copies.Selective pressure analysis showed that positively selected sites were detected in each digestive enzyme gene,indicating functional importance of them in avian digestion,and that one amylase gene(pAMY)and one lipase gene(CYP7A1)evolved faster in species consuming more seeds and meat,respectively.Two lipase genes(LIPC and PNLIP)were found to be under relaxed selection in species with lower meat consumption.Together,we suggested that gene loss,gene duplication,functional divergence,positive selection and relaxed selection have collectively shaped the evolution of digestive enzymes in birds,which presumably linked to their dietary diversification.