Charting the landscape of organellar genome evolution in eustigmatophyte algae

Organellar genomes are both a resource for reconstructing organismal phylogenies and interesting subjects for evolutionary studies. Herein, we focused on the organellar genomes of eustigmatophytes (eustigs), a class of the algal phylum Ochrophyta with a growing biotechnological potential, and massively expanded the existing limited sample by 51 new organellar genomes. Analyses of this large dataset provided a robustly resolved eustig phylogeny and important insights into the evolution of unique features of eustig organellar genomes. Eustig plastomes are rather stable in terms of the gene content, with only minor differences stemming from differential gene loss and rare lineage-specific gain. In contrast, eustig mitogenomes contain a very stable core of conserved genes accompanied by a broadly varying shell comprising “accessory” genes. Notably, the new data illuminated the origin of two mitochondrial genes previously deemed eustig-specific, namely orfX and orfY that were found to have evolved, respectively, by rps4 duplication and extreme divergence of an rps1 ortholog. Most interestingly, we identified five previously unrecognized orthogroups of mysterious mitochondrial orfs that are patchily distributed across eustigs yet likely evolved in the ancestor of this class. These orfs have no discernible homologs outside eustigmatophytes but are predicted to encode multipass membrane proteins with a soluble C-terminal domain. Our results also revise some of the previous conclusions regarding the mitochondrial translation in eustigs and suggest the recruitment of a group of unusual tRNAs for a translation-independent function in the genus Vischeria . Our study thus provides a glimpse into a “dark matter” of mitochondrial biology in eustigmatophytes. Significance statement Organellar genomes have been characterized for only a small subset of members of Eustigmatophyceae, a class of biotechnologically attractive coccoid microalgae within the phylum Ochrophyta. In this study, we present >50 newly determined organellar genome sequences from diverse eustigmatophytes, filling most major gaps in sampling across the eustigmatophyte phylogenetic tree and more than doubling the number of species represented. This greatly expanded dataset was analyzed to provide numerous new insights into phylogenetic relationships within the class, variation in organellar gene content, and the evolutionary processes and trends shaping eustigmatophyte organellar genomes. Most notably, our work reveals a surprisingly rich and evolutionarily dynamic repertoire of eustigmatophyte-specific mitochondrial genes, pointing to a functional “dark matter” within eustigmatophyte mitochondria.