Transcriptomic analysis reveals evidence for a cryptic plastid in the colpodellid Voromonas pontica, a close relative of chromerids and apicomplexan parasites

Abstract

Colpodellids are free-living, predatory flagellates, but their close relationship to photosynthetic chromerids and plastid-bearing apicomplexan parasites suggests they were ancestrally photosynthetic. Colpodellids may therefore retain a cryptic plastid, or they may have lost their plastids entirely, like the apicomplexan Cryptosporidium. To find out, we generated transcriptomic data from Voromonas pontica ATCC 50640 and searched for homologs of genes encoding proteins known to function in the apicoplast, the non-photosynthetic plastid of apicomplexans. We found candidate genes from multiple plastid-associated pathways including iron-sulfur cluster assembly, isoprenoid biosynthesis, and tetrapyrrole biosynthesis, along with a plastid-type phosphate transporter gene. Four of these sequences include the 5' end of the coding region and are predicted to encode a signal peptide and a transit peptide-like region. This is highly suggestive of targeting to a cryptic plastid. We also performed a taxon-rich phylogenetic analysis of small subunit ribosomal RNA sequences from colpodellids and their relatives, which suggests that photosynthesis was lost more than once in colpodellids, and independently in V. pontica and apicomplexans. Colpodellids therefore represent a valuable source of comparative data for understanding the process of plastid reduction in humanity's most deadly parasite.

Figure 1. Bayesian consensus phylogeny of small subunit rRNA sequences from alveolates representing the available diversity of colpodellids, chromerids, and related environmental sequences.

Support for nodes is indicated by % bootstrap support (out of 1000) in the ML analysis (RAxML GTRΓ)/Bayesian posterior probability (Phylobayes GTRCAT) where greater than 55 or 0.9. The subject of this study, Voromonas pontica, is indicated by white text on a black background. The photosynthetic chromerids Chromera velia and Vitrella brassicaformis are indicated by bold text. A question mark after accession AF372772 indicates a possible misidentification or chimeric sequence; this study also sampled a lake.

Figure 2. Maximum likelihood phylogeny of plastidic phosphate transporters.

Support for nodes is indicated by % bootstrap support (out of 1000) in the ML analysis and by posterior probabilities from two Bayesian analyses, one employing the LG model of amino acids substitution, and the other using the CAT model (RAxML LG+Γ+F/Phylobayes LG+Γ/Phylobayes CAT+Γ), where greater than 50% bootstrap support or 0.9 posterior probability. Black dots on branches indicate full support from all three analyses for the adjacent node, i.e. 100/1.0/1.0. The subject of this study, Voromonas pontica, is indicated by white text on a black background. Experimentally apicoplast-localized proteins are indicated by bold text. Hatch marks indicate a branch whose length has been reduced by half. Aside from the primary plastid pPTs enclosed by the lower shaded box, all sequences belong to secondary, rhodophyte-derived plastids.

Figure 3. Maximum likelihood phylogeny of SufB amino acids sequences.

Support for nodes is indicated by % bootstrap support (out of 1000) in the ML analysis and by posterior probabilities from two Bayesian analyses, one employing the LG model of amino acids substitution, and the other using the CAT model (RAxML LG+Γ+F/Phylobayes LG+Γ/Phylobayes CAT+Γ), where greater than 50% bootstrap support or 0.9 posterior probability. The subject of this study, Voromonas pontica, is indicated by white text on a black background. Hatch marks indicate branches whose lengths have been reduced by half.