The evolutionary implications of diplonemids and their spliceosomal introns

The phylum Euglenozoa consists of three main groups: euglenoids, kinetoplastids and diplonemids (Simpson 1997). This phylum is unique in having three types of introns: nuclear trans-spliced "introns", nuclear conventional and "aberrant" introns. In order to determine the evolutionary history of the introns in this phylum, it is very important to know the general distributions of intron types within the phylum, and the likely phylogeny of the phylum. The nuclear genomes of euglenoids are known to contain all three types of introns, while only trans-spliced and conventional introns have been found in kinetoplastids. However, nothing is known about diplonemid introns, and the phylogenetic placement of diplonemids within the Euglenozoa is uncertain. Therefore, I looked for nuclear introns in diplonemids by sequencing four nuclear protein-coding genes (actin, alpha-tubulin, betatubulin, and GAPDH) from different diplonemids. I found 11 introns in nine of the twentynine newly obtained diplonemid nuclear protein-coding genes. They all have conventional 5'-GT-AG-3' splicing sites, but differ from well-studied eukaryotic conventional introns (mammalian introns) in several details. I have added these nuclear encoded sequences from diplonemids to the tubulin, actin and GAPDH alignments and then made global phylogenetic trees based on these protein alignments. The discrepancy between the tubulin trees and actin tree is whether the diplonemids are closer to kinetoplastids (tubulin trees) or euglenoids (actin tree). Taken together, I postulate that the GT-AG conventional introns were present in the euglenozoan ancestor and were largely lost in kinetoplastids and euglenoids. The "aberrant" intron is very likely a derived character restricted to euglenoids. The trans-spliced discontinuous "intron" is an ancestral character to this phylum and it is highly likely that it will be found in diplonemids as well. The phylogenetic position of the four newly sequenced diplonemid GAPDH sequences turned out to be very interesting. None of the four diplonemid GAPDH sequences branch with those of other euglenozoa. Instead, three of the four diplonemid-sequences branch with the gap3 of cyanobacteria with 100% bootstrap support, indicating a lateral gene transfer from bacteria to eukaryotes, and one GAPDH sequence branches in an uncertain position with other eukaryotic GAPDH sequences.Science, Faculty ofBotany, Department ofGraduat