Mine the Gaps Evolution of Eukaryotic Protein Indels and their Application for Testing Deep Phylogeny

Sammanfattning: Insertions/deletions (indels) are potentially powerful evolutionary markers, but little is known about their evolution and few tools exist to effectively study them. To address this, I developed SeqFIRE, a tool for automated identification and extraction of indels from protein multiple sequence alignments. The program also extracts conserved alignment blocks, thus covering all major steps in preparing multiple sequence alignments for phylogenetic analysis.I then used SeqFIRE to build an indel database, using 299 single copy proteins from a broad taxonomic sampling of mainly multicellular eukaryotes. A total of 4,707 indels were extracted, of which 901 are simple (one genetic event) and 3,806 are complex (multiple events). The most abundant indels are single amino acid simple indels. Indel frequency decreases exponentially with length and shows a linear relationship with host protein size. Singleton indels reveal a strong bias towards insertions (2.31 x deletions on average). These analyses also identify 43 indels marking major clades in Plantae and Fungi (clade defining indels or CDIs), but none for Metazoa.In order to study the 3806 complex indels they were first classified by number of states. Analysis of the 2-state complex and simple indels combined (“bi-state indels”) confirms that insertions are over 2.5 times as frequent as deletions. Three-quarters of the complex indels had three-nine states (“slightly complex indels”). A tree-assisted search method was developed allowing me to identify 1,010 potential CDIs supporting all examined major branches of Plantae and Fungi.Forty-two proteins were also found to host complex indel CDIs for the deepest branches of Metazoa. After expanding the taxon set for these proteins, I identified a total of 49 non-bilaterian specific CDIs. Parsimony analysis of these indels places Ctenophora as sister taxon to all other Metazoa including Porifera. Six CDIs were also found placing Placozoa as sister to Bilateria. I conclude that slightly complex indels are a rich source of CDIs, and my tree-assisted search strategy could be automated and implemented in the program SeqFIRE to facilitate their discovery. This will have important implications for mining the phylogenomic content of the vast resource of protist genome data soon to become available.