Mitogenome structure and phylogenomics of the order Thoreales (Nemaliophycidae, Rhodophyta)

Organelle genomes offer a powerful tool for red algal evolutionary studies. Although the number of Rhodophyta genomes has increased substantially over the past 2 decades, only two mitochondrial genomes have been reported for the red algal order Thoreales. In this study, we generated 10 new complete mitochondrial genomes of Thoreales, representing the order's two genera and eight species, which, combined with additional mitogenomes of related orders of the subclass Nemaliophycidae, resulted in a comprehensive dataset of 20 mitogenomes. Thoreales mitogenomes exhibited little variation, ranging in length from 25,022 bp to 26,369 bp, with GC content from 25.6% to 28.3%, gene numbers from 46 to 54, and protein-coding genes from 21 to 25. Four genes were missing in one or more species (atp4, rpl20, sdh2, and sdh4), and no introns were detected. Synteny among members of Thoreales was highly conserved and similar to other orders of Nemaliophycidae. Phylogenomic analyses strongly supported the monophyly of Thoreales within Nemaliophycidae. Within the order, Nemalionopsis and Thorea as well as the relationship among the species received full support. Nemalionopsis shawii and N. parkeri formed an early-diverging clade that was sister to Thorea, which had species that resolved into two distinct lineages lacking evident geographic or morphological differentiation. By expanding the available dataset from two to 10 mitogenomes, this study provides a broad organellar perspective on Thoreales evolution, contributing to new insights into the systematics and diversification of freshwater red algae and, potentially, for clarifying inter-ordinal relationships within Nemaliophycidae.