Genomic and morphological evidence supports Rhopalodia californica sp. nov. as a divergent lineage within Rhopalodiales

The diatom Order Rhopalodiales comprises canal-bearing taxa with nitrogen-fixing cyanobacterial endosymbionts, but its genus-level systematics remains unresolved due to the paraphyly of Rhopalodia. We characterized two Northern California strains of Rhopalodia californica sp. nov. using valve morphology, complete organellar and spheroid body (SB) genomes, and multi-gene phylogenetics. Chloroplast (133,208-133,222 bp), mitochondrial (35,955-36,097 bp), and SB (3.064 Mbp) genomes were all structurally conserved and identical in gene content. Phylogenetic analysis of nuclear (18S, 28S), chloroplast (rbcL, psbC), and mitochondrial (cob) genes placed R. californica with Epithemia sp. GazosCreekCA in a strongly supported ‘californica’ clade, distinct from other clades of Epithemia and Rhopalodia species. Genome-wide nucleotide similarity was high within the clade (>97.6%) but markedly lower (<95%) when compared to other Rhopalodiales, a pattern that aligns with distinctions observed in the SB, chloroplast, and mitochondrial datasets. Evolutionary rate was fastest for the mitochondrion, followed by the SB and then the chloroplast. Synteny analysis revealed that gene order and structure were highly conserved between the two R. californica strains and remained well conserved even in comparison with Epithemia sp. GazosCreekCA. These results demonstrate that the ‘californica’ group is a genetically and morphologically cohesive lineage within Rhopalodia sensu lato, supporting its recognition in future taxonomic studies.