Red and problematic green phylogenetic signals among thousands of nuclear genes from the photosynthetic and apicomplexa-related Chromera velia

Abstract

The photosynthetic and basal apicomplexan Chromera velia was recently described, expanding the membership of this otherwise nonphotosynthetic group of parasite protists. Apicomplexans are alveolates with secondary plastids of red algal origin, but the evolutionary history of their nuclear genes is still actively discussed. Using deep sequencing of expressed genes, we investigated the phylogenetic affinities of a stringent filtered set of 3,151 expressed sequence tag-contigs by generating clusters with eukaryotic homologs and constructing phylogenetic trees and networks. The phylogenetic positioning of this alveolate alga was determined and sets of phyla-specific proteins extracted. Phylogenetic trees provided conflicting signals, with 444 trees grouping C. velia with the apicomplexans but 354 trees grouping C. velia with the alveolate oyster pathogen Perkinsus marinus, the latter signal being reinforced from the analysis of shared genes and overall sequence similarity. Among the 513 C. velia nuclear genes that reflect a photosynthetic ancestry and for which nuclear homologs were available both from red and green lineages, 263 indicated a red photosynthetic ancestry, whereas 250 indicated a green photosynthetic ancestry. The same 1:1 signal ratio was found among the putative 255 nuclear-encoded plastid proteins identified. This finding of red and green signals for the alveolate mirrors the result observed in the heterokont lineage and supports a common but not necessarily single origin for the plastid in heterokonts and alveolates. The inference of green endosymbiosis preceding red plastid acquisition in these lineages leads to worryingly complicated evolutionary scenarios, prompting the search for other explanations for the green phylogenetic signal and the amount of hosts involved.

FIG. 1.—

Sequence logo of the BTS of nuclear-encoded plastid proteins. The logo was curated based on 255 sequences, which encode an N-terminal signal peptide followed by a transit peptide. The −20/+20 positions relative to the cleavage site (red arrow) between the two parts of the BTS are shown. Secretory and plastid proteins both encode an almost identical signal peptide but only in the latter case a transit peptide follows. The N-terminal part of the transit peptide is enriched in serine residues and the C-terminal end with positively charged arginine residues.

FIG. 2.—

Presence/absence pattern and identity of the nuclear-encoded Chromera velia ESTs compared with 34 organisms. (A) The 3,151 sequences are sorted by their specificity and frequency to other Apicomplexa sequences. One hundred and fifty-one sequences have homologs only in Apicomplexa, whereas 1,316 sequences had homologs only in organisms other than Apicomplexa. Note that outside the Apicomplexa, C. velia shares the highest amount of overall identity with Perkinsus marinus. In (B), the potential amount of proteins encoded within the genomes used in the analysis.

FIG. 3.—

Splits network of distances derived from a matrix representation of all splits from the 2,258 homolog cluster trees generated. The net places the apicomplexan Chromera velia between nonphotosynthetic organisms. Bottom right shows an enlargement of the two splits that on the one side unites C. velia’s nuclear gene phylogeny with the Apicomplexa (light red)—whereby C. velia shows a basal position—and on the other highlights the signal linking it with the nonphotosynthetic Perkinsus marinus (blue split). Not only is this seen in the phylogeny above but also clearly in the gene distribution pattern in figure 1. The question marks indicate the two cases (Ciliates and Goniomonas), where it is disputed whether they lost or never had a plastid.

FIG. 4.—

Comparison of the red and green signal of nuclear-encoded Chromera genes. Five hundred and thirteen phylogenetic trees contained genes of green and red origin and also an outgroup. Those part almost 50–50 into trees, in which the nearest neighbor of the Chromera velia homolog is either of red or green origin. In two splits networks combining all the red and green trees separately, the apicomplexan alga is unambiguously positioned among either the rhodophytes (A) or the chloroplastida (B).