Chloroplast and Mitochondrial Genomes of the Lichen-Symbiotic Green Alga Trebouxia Illuminate Evolutionary Relationships and Climate Associations and Yield New Phylogenetic Markers

The green-algal genus Trebouxia (Trebouxiophyceae, Chlorophyta) is the most common photosynthetic symbiont of lichens, displaying high phylogenetic diversity, and worldwide distribution across all climate zones. These single-celled terrestrial algae are valuable systems to study diversification, environmental adaptation and species interactions, yet genomic resources remain limited. We present over 30 new chloroplast and mitochondrial genomes of Trebouxia species, extracted from PacBio metagenomes of diverse Umbilicaria lichens from multiple climate zones. The genomes represent previously identified operational taxonomic units (OTUs) Trebouxia jamesii (A03), T. sp. (A04), T. incrustata (A06), T. vagua (A10), T. sp. (S02), T. sp. (S03), T. sp. (S04), T. suecica (S05), T. sp. (S08), T. angustilobata (S09), T. simplex (S10), T. sp. (S20) and T. barrenoae (S28); a newly designated OTU T. sp. (A57), and several Single-Occurrence Sequences (SOS) from clades A, I, and S. Up to four Trebouxia OTUs were found within a single thallus. Organelle genomes vary considerably in size and structure. The consensus phylogenies from chloroplast (77 genes) and mitochondrial (32 genes) genes are largely congruent with the nuclear ITS tree, differing mainly in the derived clade S sections. All genes are under purifying selection, with mitochondrial genes exhibiting higher nucleotide diversity and hence phylogenetic resolution than chloroplast genes. Certain gene and protein features correlate with temperature variability, and some (such as GC content, arginine, and valine content) mirror findings in mycobiont nuclear genomes from the same samples and highlight shared signatures of environmental adaptation. We designed primers for new, variable phylogenetic markers, including chloroplast genes ftsH and rpoC1, and mitochondrial genes ATP1, ATP6, and ND6. Overall, this study advances our understanding of organelle genome evolution in Trebouxia and provides valuable resources for future ecological and evolutionary research.