1. Utilization of ancient horizontal gene transfer for phylogenetic reconstruction

We have developed a framework of utilizing ancient HGT events for phylogenetic reconstruction. These ancient HGT events not only may mark the recipient and descendent lineages as a monophyletic group, but also allow correlating the emerging sequence between the donor and the recipient (i.e., the donor must emerge at least as early as the recipient). Application of this principle has provided some strong and independent evidence for a common origin of animals and fungi and of red algae and green plants.

2. HGT and IGT in Cyanidioschyzon

An accurate estimation of foreign genes in the nuclear genome of a species is critical to understanding the role of gene transfer in genome evolution. Phylogenomic analyses of the red alga Cyanidioschyzon merolae were performed using PhyloGenie to screen for gene transfer candidates, followed by detailed phylogenetic analyses. About 320 genes that were likely acquired from various prokaryotic groups, chloroplasts or mitochondria, were identified. The exact number and the origins of these genes await further detailed phylogenetic analyses. Several genes apparently were acquired prior to the split of red algae and green plants. Topoisomerase VI β-subunit (TOP6B) has identifiable homologs only in archaea, red algae, green plants and a few δ-proteobacteria (Bdellovibrio and Anaeromyxobacter). The TOP6B protein sequences from red algae and green plants have the highest percent identities and share many conserved residues with those from crenarchaeotes. Phylogenetic analyses strongly support a common origin of TOP6B sequences from red algae and green plants and their close affinity with crenarchaeote homologs (Figure 1).

Figure 1. Phylogenetic analyses of topoisomerase VI b-subunit protein sequences. Numbers above the branches show posterior probabilities for Bayesian analyses and bootstrap values for maximum-likelihood analyses, respectively.

3. Ancient chlamydial endosymbiosis with primary photosynthetic eukaryotes

Chlamydiae are a group of obligate intracellular bacteria of uncertain evolutionary position. Many chlamydiae are important pathogens in humans and other animals whereas others are endosymbionts in amoebae and insects. Thus far, no chlamydial species has been reported in photosynthetic eukaryotes or plastid-containing lineages. In our preliminary analyses of Cyanidioschyzon using PhyloGenie, we systematically searched for genes related to chlamydial sequences. Our further very stringent phylogenetic analyses confirmed 12 genes of chlamydial origin in red algae, green plants and glaucophytes (Table 1). The sequences from primary photosynthetic eukaryotes always form a monophyletic group with chlamydial (often environmental Parachlamydia) sequences, instead with cyanobacterial sequences, which form a distinct, separate cluster (Figure 2). Another line of evidence supporting an ancient chlamydial endosymbiotic relationship comes from the phylogeny of enoyl-ACP reductase (fabI) (Figure 3). The fabI protein sequences from green plants, diatoms, chlorarachniophytes and apicomplexans contain a number of very conserved insertions and deletions and form a strongly supported group that is distinct from all other sequences. Interestingly, these other sequences also include cyanobacterial and red algal Cyanidioschyzon homologs, which group together as expected (Figure 3).

Figure 2. Phylogeny of aspartate transferase. Numbers above the branch show bootstrap values for maximum likelihood and distance analyses, respectively. Note that the Parachlamydia sequence groups with red algal and green plant homologs with strong support.

Figure 3. Phylogeny of enoyl-ACP reductase (fabI). Numbers above the branch show bootstrap values for maximum likelihood and distance analyses, respectively. Note that the sequences from green plants, diatoms, chlorarachniophytes and apicomplexans form a strongly supported clade that is distinct from all other sequences that include cyanobacterial and red algal Cyanidioschyzon homologs.

The coexistence of a heterotrophic host, cyanobacterium and Chlamydia with different biological requirements and capabilities might have offered an opportunity for some transient mutualistic interactions, and the availability of genes such as the ATP/ADP translocase from the chlamydial endosymbiont might have facilitated the successful endosymbiosis of cyanobacteria by allowing energy flux into the protoplastid organelle. Once a rich repertoire of transporters were in place and the plastid was fully established, transport of photosynthetic products and other metabolites across the photosynthetic organelle might be more easily adapted to the different membranes in secondary or tertiary endosymbiotic hosts.