Comparative Plastid Genomics of Green-Colored Dinoflagellates Unveils Parallel Genome Compaction and RNA Editing

Abstract

Dinoflagellates possess plastids that are diverse in both pigmentation and evolutionary background. One of the plastid types found in dinoflagellates is pigmented with chlorophylls a and b (Chl a + b) and originated from the endosymbionts belonging to a small group of green algae, Pedinophyceae. The Chl a + b-containing plastids have been found in three distantly related dinoflagellates Lepidodinium spp., strain MGD, and strain TGD, and were proposed to be derived from separate partnerships between a dinoflagellate (host) and a pedinophycean green alga (endosymbiont). Prior to this study, a plastid genome sequence was only available for L. chlorophorum, which was reported to bear the features that were not found in that of the pedinophycean green alga Pedinomonas minor, a putative close relative of the endosymbiont that gave rise to the current Chl a + b-containing plastid. In this study, we sequenced the plastid genomes of strains MGD and TGD to compare with those of L. chlorophorum as well as pedinophycean green algae. The mapping of the RNA-seq reads on the corresponding plastid genome identified RNA editing on plastid gene transcripts in the three dinoflagellates. Further, the comparative plastid genomics revealed that the plastid genomes of the three dinoflagellates achieved several features, which are not found in or much less obvious than the pedinophycean plastid genomes determined to date, in parallel.

Keywords: Lepidodinium; Pedinophyceae; RNA editing; complex plastids; peDinoflagellates; plastid replacements; serial secondary endosymbiosis.

FIGURE 1

Plastid evolution in dinoflagellates. The cladogram of 22 dinoflagellates and Perkinsus marina was prepared by modifying the phylogenetic tree inferred from a 75-protein alignment analyzed in Sarai et al. (2020). The branches of Lepidodinium, strain TGD, and strain MGD are colored in green, as their non-canonical plastids were yielded independently from pedinophycean endosymbionts (serial secondary endosymbiosis; labeled as “s2°”). The clade of two kareniacean dinoflagellates, Karenia and Karlodinium, are colored in orange, as they possess the non-canonical plastids deduced from the endosymbiotic haptophyte (tertiary endosymbiosis; labeled as “3°”). The clade of the dinoflagellates bearing obligate diatom endosymbionts are highlighted by a yellow background. The Dinophysis branch is highlighted by a brown background, as some members of the order Dinophysiales retain and utilize the plastids of prey algae temporarily (kleptoplastidy). Non-photosynthetic species are shown by thin branches.

FIGURE 2

Circular map of the plastid genome of the peDinoflagellate strain TGD. Open reading frames (ORFs) encoding proteins involved in photosynthesis, translation, transcription, and other function are colored in green, brown, orange, and gray, respectively. Functionally unassigned ORFs are shown in white. Fused ORFs are highlighted in purple. rpoB in the TGD genome is considered as a pseudogene. The ORF for PsaA in the TGD genome was found to be interrupted by a frameshift (marked by a zigzag line). Ribosomal RNA genes and transfer RNA genes are colored in red and blue, respectively. The ORFs/genes, of which transcripts received RNA editing, are marked by stars. Red dots indicate the overlap of two neighboring ORFs/genes.

FIGURE 3

Circular map of the plastid genome of the peDinoflagellate strain MGD. The details of this figure are the same as Figure 1. ycf4 in the MGD genome is considered as a pseudogene. The precise nucleotide sequence between orf102 and rns, which is indicated by a dotted line in this figure, could not be determined because of repeat sequences. Star indicates the genes of which transcripts receive RNA editing.

FIGURE 4

Phylogeny of green algal plastids and green alga-derived plastids inferred from the alignment comprising 50 plastid-encoded proteins. The maximum-likelihood (ML) tree and non-parametric ML bootstrap support values (MLBP) are shown here. The overall tree topology inferred from Bayesian method agreed with the ML tree. Dots on branches indicate the bipartitions received MLBPs of 100% and Bayesian posterior probabilities (BPPs) of 1.0. Only MLBPs greater than 70% are shown. The bipartitions supported by BPPs of 1.0 are marked with diamonds. For the node uniting the three peDinoflagellates and pedinophycean green algae, the MLBPs calculated from the four ML bootstrap analyses are presented. “Full,” the analysis considering the three peDinoflagellates; “Lc only,” “MGD only,” and “TGD only,” those considering Lepidodinium chlorophorum, strain MGD, and strain TGD considering the sole representative of peDinoflagellates, respectively.

FIGURE 5

Comparison of branch length between peDinoflagellates and pedinophycean green algae. (A) The 5-taxon tree. The branch lengths were optimized based on each of the 50 single-protein alignments. We fixed the relationship among Pedinomonas minor (Pm), Pedinomonas tuberculata (Pt), Marsupiomonas sp. (Mars), Pedinophyceae sp. YPF-701 (YPF), and one of the three peDinoflagellates (peDino). For each tree with the optimum branch lengths, the length of the black branch was subtracted by the sum of the lengths of the grey branches (“branch length-ratio”). (B) Box plots of branch length-ratios between 30 “photosynthetic” proteins and 20 “non-photosynthetic” proteins. The ratios calculated based on the 5-taxon trees considering Lepidodinium chlorophorum, strain MGD, and strain TGD are shown left, center, and right, respectively. Wilcoxon rank-sum test rejected the null hypothesis of no difference in the median values of branch length-ratios calculated from the two categories was rejected with p < 0.01 (highlighted by double asterisks). The 5-taxon trees with the optimum branch lengths and branch length-ratios are provided as Supplementary Material.

FIGURE 6

Venn diagram for the functionally assignable ORFs in the three peDinoflagellate plastid genomes. Open reading frames (ORFs) encoding proteins involved in photosynthesis, translation, transcription, and other function are colored in green, brown, orange, and black, respectively. Notes, rpoC1 in Lepidodinium chlorophorum, rpoB in strain TGD, and ycf4 in strain MGD are pseudogenes and marked by asterisks. rpoC2 is highlighted by an exclamation mark as the N- and C-termini of RpoC2 are encoded in two distinct ORFs in strain TGD (see Figure 1).