Chromera velia, endosymbioses and the rhodoplex hypothesis--plastid evolution in cryptophytes, alveolates, stramenopiles, and haptophytes (CASH lineages)

Abstract

The discovery of Chromera velia, a free-living photosynthetic relative of apicomplexan pathogens, has provided an unexpected opportunity to study the algal ancestry of malaria parasites. In this work, we compared the molecular footprints of a eukaryote-to-eukaryote endosymbiosis in C. velia to their equivalents in peridinin-containing dinoflagellates (PCD) to reevaluate recent claims in favor of a common ancestry of their plastids. To this end, we established the draft genome and a set of full-length cDNA sequences from C. velia via next-generation sequencing. We documented the presence of a single coxI gene in the mitochondrial genome, which thus represents the genetically most reduced aerobic organelle identified so far, but focused our analyses on five "lucky genes" of the Calvin cycle. These were selected because of their known support for a common origin of complex plastids from cryptophytes, alveolates (represented by PCDs), stramenopiles, and haptophytes (CASH) via a single secondary endosymbiosis with a red alga. As expected, our broadly sampled phylogenies of the nuclear-encoded Calvin cycle markers support a rhodophycean origin for the complex plastid of Chromera. However, they also suggest an independent origin of apicomplexan and dinophycean (PCD) plastids via two eukaryote-to-eukaryote endosymbioses. Although at odds with the current view of a common photosynthetic ancestry for alveolates, this conclusion is nonetheless in line with the deviant plastome architecture in dinoflagellates and the morphological paradox of four versus three plastid membranes in the respective lineages. Further support for independent endosymbioses is provided by analysis of five additional markers, four of them involved in the plastid protein import machinery. Finally, we introduce the "rhodoplex hypothesis" as a convenient way to designate evolutionary scenarios where CASH plastids are ultimately the product of a single secondary endosymbiosis with a red alga but were subsequently horizontally spread via higher-order eukaryote-to-eukaryote endosymbioses.

Keywords: chromalveolate hypothesis; eukaryote-to-eukaryote endosymbioses; horizontal and endosymbiotic gene transfer; long-branch attraction artifacts; next-generation sequencing.

Fig. 1.—

Protein and gene structure of the nuclear-encoded plastid SBP3-HDR fusion protein from Chromera velia (KC899090; ARZB00000000). The putative N-terminal cleavage sites of the bipartite signal- and transit peptides are indicated with an “S” and “T,” respectively. A hinge region links the putative Calvin cycle SBP-3 enzyme of Chromera (green color) with the HDR enzyme that is essential for the plastid MEP-pathway for isoprenoid biosynthesis (red color). The exon–intron structure of the gene is shown below and introns are indicated with Roman numerals.

Fig. 2.—

Image detail of a phylogenetic ML analysis of PRK sequences focused on complex algae of the CASH lineages (blue box) and the novel sequence of C. velia (highlighted in yellow; KC899087). The complete RAxML analysis was performed with a LG + F + Γ4 model of 65 PRK sequences based on 247 amino acid positions. The tree is rooted on a cyanobacterial outgroup (see supplementary fig. S2a, Supplementary Material online). Species names are shown in red and green according to the rhodophycean or chlorophycean origin of their complex plastids.

Fig. 3.—

Phylogenetic ML RAxML analysis with a LG + F + Γ4 model of 80 SBP sequences based on 118 amino acid positions. The Calvin cycle-specific subtree of the CASH lineages is highlighted with a blue box. Distinct fungal and green plant subtrees have been merged; the complete phylogenetic tree is shown in supplementary figure S3, Supplementary Material online.

Fig. 4.—

Image detail of a phylogenetic ML analysis of GAPDH sequences rooted on the cytosolic GapC sequences from alveolates. The blue box highlights the bipartite subtree of plastid GapC-I sequences of CASH lineages, which are essential for the Calvin cycle. The complete RAxML analysis was performed with a LG + F + Γ4 model of 95 GAPDH sequences based on 194 amino acid positions (see supplementary fig. S8, Supplementary Material online). Species names are shown in red and green according to the rhodophycean or chlorophycean origin of their complex plastids, and species with heterotrophic plastids are shown in black. The number of asterisks at the species names indicates the current state of knowledge about the origin of their plastids via primary (*), secondary (**), or tertiary (***) endosymbiosis.

Fig. 5.—

Merged phylogenetic ML tree of HDR sequences of plastid-specific isoprenoid biosynthesis (MEP-pathway). The RAxML analyses of HDR subtrees I and II were performed with a LG + F + Γ4 model of 40 and 45 HDR sequences based on 288 and 257 amino acid positions, respectively (see supplementary fig. S9a and b, Supplementary Material online). The gray boxes indicate eukaryotic branches of the subtrees.

Fig. 6.—

Phylogenetic ML RAxML analysis with a LG + F + Γ4 model of 68 Cdc48 sequences of the ER-associated degradation (ERAD) system based on 618 amino acid positions. The subtree of the SELMA for protein transport through the periplastidal plastid membrane of CASH lineages is highlighted with a blue box. The complete phylogenetic tree is shown in supplementary fig. S6b, Supplementary Material online.

Fig. 7.—

(a) Origin of complex algae with red plastids via a single secondary endosymbiosis with a red alga and successive tertiary and quaternary endosymbioses. N: nucleus; M: mitochondrion; P: plastid. (b) Scenario of plastid evolution among CASH lineages according to the rhodoplex hypothesis. X-ray images of the Russian Matryoshka dolls indicate independent events of plastid endosymbioses. All CASH plastids originate from an initial engulfment of a rhodophyte (see [a]), but the genuine secondary endosymbiont and the order of subsequent endosymbioses remains to be determined (indicated by 2nd/3rd and 3rd/4th). The typical plastid of PCD may represent a reduced apicomplexan alga (see current study). The gain of rhodophycean plastids as well as the loss of photosynthesis/plastids is indicated by the red horizontal lines. With respect to stramenopiles, only a subset of separate lineages is shown. Micrograph courtesy of Peter Vontobel, Sven Gould, Woody Hastings, and Manfred Rohde.