An extended phylogenetic analysis reveals ancient origin of "non-green" phosphoribulokinase genes from two lineages of "green" secondary photosynthetic eukaryotes: Euglenophyta and Chlorarachniophyta

Background: Euglenophyta and Chlorarachniophyta are groups of photosynthetic eukaryotes harboring secondary plastids of distinct green algal origins. Although previous phylogenetic analyses of genes encoding Calvin cycle enzymes demonstrated the presence of genes apparently not derived from green algal endosymbionts in the nuclear genomes of Euglena gracilis (Euglenophyta) and Bigelowiella natans (Chlorarachniophyta), the origins of these "non-green" genes in "green" secondary phototrophs were unclear due to the limited taxon sampling. Results: Here, we sequenced five new phosphoribulokinase (PRK) genes (from one euglenophyte, two chlorarachniophytes, and two glaucophytes) and performed an extended phylogenetic analysis of the genes based on a phylum-wide taxon sampling from various photosynthetic eukaryotes. Our phylogenetic analyses demonstrated that the PRK sequences form two genera of Euglenophyta formed a robust monophyletic group within a large clade including stramenopiles, haptophytes and a cryptophyte, and three genera of Chlorarachniophyta were placed within the red algal clade. These "non-green" affiliations were supported by the taxon-specific insertion/deletion sequences in the PRK alignment, especially between euglenophytes and stramenopiles. In addition, phylogenetic analysis of another Calvin cycle enzyme, plastid-targeted sedoheptulose-bisphosphatase (SBP), showed that the SBP sequences from two genera of Chlorarachniophyta were positioned within a red algal clade. Conclusions: Our results suggest that PRK genes may have been transferred from a "stramenopile" ancestor to Euglenophyta and from a "red algal" ancestor to Chlorarachniophyta before radiation of extant taxa of these two "green" secondary phototrophs. The presence of two of key Calvin cycle enzymes, PRK and SBP, of red algal origins in Chlorarachniophyta indicate that the contribution of "non-green" algae to the plastid proteome in the "green" secondary phototrophs is more significant than ever thought. These "non-green" putative plastid-targeted enzymes from Chlorarachniophyta are likely to have originated from an ancestral red alga via horizontal gene transfer, or from a cryptic red algal endosymbiosis in the common ancestor of the extant chlorarachniophytes.