Hidden in Plain Sight: A Novel Symbiotic Haplosclerid Sponge Species Revealed by its Mitochondrial Genome

An unusual third mitochondrial genome was discovered in the genomic data from the symbiotic association between two sponges, Haliclona plakophila (Haplosclerida, Demospongiae) and Plakortis symbiotica (Homoscleromorpha). This mitogenome exhibited an unusually high GC content, lacked the cox2 gene, and could not be reliably positioned among animals based on phylogenetic analysis of the sequence data. Nevertheless, conserved gene clusters and the presence of atp9 indicated that this species is a sponge, while phylogenetic analysis using 18S and 28S places it in the order Haplosclerida of Demospongiae. Since no tissue of the cryptic sponge was found in the investigated association, its relationship with other sponges remained unclear. Therefore, we sampled 10 additional samples assumed to be H. plakophila - P. symbiotica associations in the field to search for the mysterious third sponge. PCR analysis revealed that one of the collected samples contained the novel sponge, but not H. plakophila. A follow-up morphological investigation revealed that this sponge, Haplosclerida sp. nov., while superficially indistinguishable from H. plakophila, differs in skeletal organization and spicule composition. Here, we report the unusual mitochondrial genome of the new species, along with a phylogenetic analysis using 18S and 28S sequences, and demonstrate that Haplosclerida sp. nov. belongs to a new clade of Haplosclerida. We also report the mitogenome of Xestospongia deweerdtae, another haplosclerid species described in association with multiple Plakortis species, including P. symbiotica. Our results support multiple origins of Haplosclerida-Plakortis symbiosis and open new avenues for its investigation.