Plastid Genomes and Proteins Illuminate the Evolution of Eustigmatophyte Algae and Their Bacterial Endosymbionts

Abstract

Eustigmatophytes, a class of stramenopile algae (ochrophytes), include not only the extensively studied biotechnologically important genus Nannochloropsis but also a rapidly expanding diversity of lineages with much less well characterized biology. Recent discoveries have led to exciting additions to our knowledge about eustigmatophytes. Some proved to harbor bacterial endosymbionts representing a novel genus, Candidatus Phycorickettsia, and an operon of unclear function (ebo) obtained by horizontal gene transfer from the endosymbiont lineage was found in the plastid genomes of still other eustigmatophytes. To shed more light on the latter event, as well as to generally improve our understanding of the eustigmatophyte evolutionary history, we sequenced plastid genomes of seven phylogenetically diverse representatives (including new isolates representing undescribed taxa). A phylogenomic analysis of plastid genome-encoded proteins resolved the phylogenetic relationships among the main eustigmatophyte lineages and provided a framework for the interpretation of plastid gene gains and losses in the group. The ebo operon gain was inferred to have probably occurred within the order Eustigmatales, after the divergence of the two basalmost lineages (a newly discovered hitherto undescribed strain and the Pseudellipsoidion group). When looking for nuclear genes potentially compensating for plastid gene losses, we noticed a gene for a plastid-targeted acyl carrier protein that was apparently acquired by horizontal gene transfer from Phycorickettsia. The presence of this gene in all eustigmatophytes studied, including representatives of both principal clades (Eustigmatales and Goniochloridales), is a genetic footprint indicating that the eustigmatophyte-Phycorickettsia partnership started no later than in the last eustigmatophyte common ancestor.

Fig. 1.

—Eustigmatophyte phylogeny inferred from pt genome data. The tree shown was inferred using PhyloBayes-MPI v1.7 and the site-heterogeneous substitution model CAT + GTR from an alignment of 18,378 amino acid positions (derived from 68 conserved pt genome-encoded proteins). All branches received maximal support (posterior probability of 1.0). For simplicity, only the ochrophyte subtree is shown (omitting thus the outgroup comprising cryptophytes and haptophytes) and classes (other than eustigmatophytes) with multiple representatives are collapsed as triangles. Species (strains) with pt genomes sequenced in this study are highlighted in bold. Supplementary figure S10, Supplementary Material online, shows a full version of the essentially identical ML tree inferred from the same supermatrix using IQ-TREE v1.6.5. GenBank accession numbers of pt genomes employed in the phylogenomic analysis are listed in supplementary table S2 and in the legend to supplementary figure S10, Supplementary Material online. Light microphotographs are provided for the four sequenced strains whose appearance has been previously documented in the literature; scale bar: 10 μm.

Fig. 2.

—Major events in the evolution of the pt genomes in eustigmatophytes. The scheme shows inferred events of gene loss and gain along the eustigmatophyte phylogeny. The pt genome of the chrysophyte Ochromonas sp. CCMP 1393 (a representative of the presumed sister lineage of eustigmatophytes) is included to provide a broader phylogenetic context. Genes lost are in red, genes gained are in blue (“ebo” refers to gain or loss of the whole operon). Losses mapped before divergence of Ochromonas sp. and eustigmatophytes may have happened at various deep branches of the ochrophyte phylogeny. Possible homology of orf1_gon and orf1_eust genes is discussed and detail concerning the split sufB gene (or pseudogene?) are provided in the main text. Note labels of some genes (e.g., acpP) at multiple branches in the tree, indicating multiple independent losses during the eustigmatophyte evolution. Taxa printed in bold are those for which the pt genome sequence is reported in this study.

Fig. 3.

—Recurrent loss of 4.5S RNA (the RNA component of the plastidial SRP specified by the ffs gene) in ochrophytes. The figure shows a segment of a multiple sequence alignment of cpSRP54 (the protein component of the plastidial SRP) including two motifs critical for binding of the 4.5S RNA molecule. The presence of a discernible ffs gene in the pt genome of the respective species is indicated on the left by a red square. Note the perfect correlation between the conservation of the 4.5S RNA-binding motif in cpSRP54 (shown in red beneath the alignment) and the presence of the ffs gene.

Fig. 4.

—A novel pt-targeted paralog of the ribosomal protein L26 in eustigmatophytes. The ML tree was inferred from an alignment of 115 amino acid position using IQ-TREE v1.6.5 and the optimal substitution model selected by the program (LG + I + Γ4). Bootstrap values <75 are omitted from the figure. The tree displayed unveils the existence of a separate clade of L26-related sequences encoded by nuclear genomes of all eustigmatophytes investigated and possessing an N-terminal extension fitting the structure of a bipartite pt-targeting presequence (supplementary table S4, Supplementary Material online). This clade is clearly separated from conventional L26 proteins lacking the N-terminal targeting sequence and expected to function as components of the cytosolic ribosomes, as well as from the presumably independently evolved group of pt-targeted L26 proteins from euglenophytes. The tree was arbitrarily rooted between sequences from Archaea and eukaryotes.

Fig. 5.

—Phylogenetic analysis of ACP sequences. The ML tree was inferred from an alignment of 70 amino acid position using IQ-TREE v1.6.5 and the optimal substitution model selected by the program (LG + I + Γ4). Bootstrap values <75 are omitted from the figure. Four ACP types are distinguished by different abbreviations: ACP, bacterial; AcpP, plastid genome encoded; ptACP, nucleus-encoded plastid targeted; mtACP, nucleus-encoded mitochondrion targeted. For simplicity, several clades were collapsed and their composition is shown on the right. Tip labels of sequences from bacteria, except those from Rickettsiales, are omitted for simplicity, the respective branches are rendered in gray. Note the strongly supported relationship between ACP from P. trachydisci and the eustigmatophyte ptACP. Full version of the tree is provided as supplementary figure S16, Supplementary Material online. The right part of the figure shows a segment of a multiple alignment of the ACP sequences included into the phylogenetic analysis (listed in the same vertical order as they appear in the tree) that includes a characteristic insertion shared by ACP from P. trachydisci and ptACP from eustigmatophytes.