Evidence for the retention of two evolutionary distinct plastids in dinoflagellates with diatom endosymbionts

Abstract

Dinoflagellates harboring diatom endosymbionts (termed "dinotoms") have undergone a process often referred to as "tertiary endosymbiosis"--the uptake of algae containing secondary plastids and integration of those plastids into the new host. In contrast to other tertiary plastids, and most secondary plastids, the endosymbiont of dinotoms is distinctly less reduced, retaining a number of cellular features, such as their nucleus and mitochondria and others, in addition to their plastid. This has resulted in redundancy between host and endosymbiont, at least between some mitochondrial and cytosolic metabolism, where this has been investigated. The question of plastidial redundancy is particularly interesting as the fate of the host dinoflagellate plastid is unclear. The host cytosol possesses an eyespot that has been postulated to be a remnant of the ancestral peridinin plastid, but this has not been tested, nor has its possible retention of plastid functions. To investigate this possibility, we searched for plastid-associated pathways and functions in transcriptomic data sets from three dinotom species. We show that the dinoflagellate host has indeed retained genes for plastid-associated pathways and that these genes encode targeting peptides similar to those of other dinoflagellate plastid-targeted proteins. Moreover, we also identified one gene encoding an essential component of the dinoflagellate plastid protein import machinery, altogether suggesting the presence of a functioning plastid import system in the host, and by extension a relict plastid. The presence of the same plastid-associated pathways in the endosymbiont also extends the known functional redundancy in dinotoms, further confirming the unusual state of plastid integration in this group of dinoflagellates.

Keywords: dinotom; redundancy; relict plastid; tertiary endosymbiosis.

Fig. 1.—

Schematic representation of a dinotom cell. The host cytosol contains a spherical nucleus, mitochondria and the triple membrane-bound eyespot. The cytosol of the endosymbiont contains a large, multilobed nucleus, mitochondria and four membrane-bound plastids, where the outermost chloroplast membrane is continuous with the outer membrane of the nucleus (Pienaar et al. 2007).

Fig. 2.—

Taxonomic affinities of dinotom transcripts based on BLAST analyses. A bar chart shows the proportions of transcripts with top BLAST hits to various taxa for the transcriptome data sets of Durinskia baltica (Db), Glenodinium foliaceum (Gf), and Kryptoperidinium foliaceum (Kf) after filtering for peptides greater than 100 amino acids. Bars marked with an asterisk indicate subgroups deviating from the general pattern: (A) “light” data sets and (B) “dark” data sets.

Fig. 3.—

Phylogeny of hemE/UROD as inferred by ML (LG + Γ model), depicting several dinoflagellate- and diatom-derived hemE/UROD paralogs with different evolutionary origins: Cyanobacterial, from the primary algal nucleus (nucleus of a primary alga that may have been taken up as secondary endosymbiont), and/or the secondary algal nucleus (nucleus of a heterotrophic eukaryote that has taken up an alga). Black dots correspond to greater than 95% ML bootstrap support. Numbers at nodes represent bootstrap supports of greater than 50%. Shaded boxes indicate clades of interest. Blue and green texts indicate dinoflagellate- and diatom-derived transcripts, respectively; the numbers of the transcripts correspond to the original contig numbering of NCGR. The black box surrounding the paralog of “Pseudonitzschia multiseries 3” indicates the possibility of an alphaproteobacterial-derived form of hemE/UROD in this organism. The numbers after species names indicate different paralogs and are numbered according to node order. Similarly, the diatom clades of cyanobacterial origin are numbered according to node order. The scale bar represents the estimated number of amino acid substitutions per site.

Fig. 4.—

Phylogeny of ispD as inferred by ML (LG + Γ model), depicting dinoflagellate- and diatom-derived ispD homologs, with the Durinskia baltica homolog missing in both, the dinoflagellate- as well as the diatom-derived, clade. Dinoflagellate- and diatom-derived genes cluster with plastid-bearing eukaryotes but not with cyanobacteria. Black dots correspond to greater than 95% ML bootstrap support. Numbers at nodes represent bootstrap supports of greater than 50%. Shaded boxes indicate clades of interest. Blue and green texts indicate dinoflagellate- and diatom-derived transcripts, respectively; the numbers of the transcripts correspond to the original contig numbering of NCGR. The scale bar represents the estimated number of amino acid substitutions per site.

Fig. 5.—

GC contents of dinoflagellate-derived and diatom-derived transcripts do not support endosymbiotic gene transfer for the MEP/DOXP and heme pathway in dinotoms. A histogram shows the frequency distribution of the GC contents of all dinoflagellate-derived and diatom-derived transcripts present in the phylogenetic analyses.

Fig. 6.—

Dinoflagellate-derived transcripts contain characteristic dinoflagellate-plastid targeting sequences. (A) Class I transit peptides, containing a transmembrane domain: Manual alignment of N-terminal regions of MEP/DOXP and heme pathway transcripts at the “FVAP” motif and their transmembrane regions, respectively. The average hydrophobicity score for each column in the transmembrane and arginine-rich domain is plotted above the alignment. The “FVAP” motif for every sequence is displayed next to the alignment. Note: The phenylalanine in the Durinskia baltica ispG was substituted by tyrosine, another hydrophobic residue. An asterisk next to the transcript name indicates a truncated signal peptide. Amino acid color code: Yellow, hydrophobic; blue, polar; green, negatively charged; red, positively charged. (B) Class II transit peptide: The dinoflagellate-derived presequence lacks a transmembrane domain in the transit peptide but contains the characteristic “FVAP” motif at the signal peptide cleavage site.

Fig. 7.—

Phylogeny of Tic110 as inferred by ML (LG + Γ model), depicting dinoflagellate- and diatom-derived Tic110 homologs. Black dots correspond to greater than 95% ML bootstrap support. Numbers at nodes represent bootstrap supports of greater than 50%. Shaded boxes indicate clades of interest. Blue and green texts indicate dinoflagellate- and diatom-derived transcripts, respectively; the numbers of the transcripts correspond to the original contig numbering of NCGR. The scale bar represents the estimated number of amino acid substitutions per site.