Microbial Communities Vary by Body Region but Remain Consistent Across Color Morphs in the Solitary Ascidian Rhopalaea abdominalis.

Ascidians are filter-feeding marine invertebrates that display inter- and intraspecific color variation and host diverse microbiomes. Although color variation has been used as an indicator of speciation and correlated with distinct microbiome structure in some colonial species, there is limited research on microbiome variation across color morphs and body regions in solitary ascidians. To address these knowledge gaps, three color morphs (purple, orange, and intermediate shades of pink) of the solitary Belizean ascidian Rhopalaea abdominalis were collected for phylogenetic analysis using the Cytochrome Oxidase I (COI) gene and for microbial characterization via sequencing the V4 region of the 16S rRNA gene. Microbial sequences derived from the ascidian branchial sac, gut, and tunic body regions were processed using both operational taxonomic unit (OTU) and amplicon sequence variant (ASV) pipelines to determine the resolution and consistency of sequence processing methods using mothur and QIIME 2 software, respectively. COI sequencing revealed two clades that were independent of color morphs. Microbiome characterization showed no significant differences in alpha- or beta-diversity across ascidian color morphs or COI clades but distinct microbiomes within each body region. The branchial sac harbored a higher number of core members (i.e., detected across all host individuals) that included known ascidian symbionts (e.g., Endozoicomonas), whereas the gut and tunic were colonized by taxa identified in seawater and across multiple classes of marine invertebrates. Results were highly congruent between OTU- and ASV-based pipelines, yielding statistically equivalent alpha-diversity metrics, consistent beta-diversity patterns, and similar core membership profiles. These findings reinforce the validity of both methods for studying microbial symbiont communities and corroborate previous research showing distinct microbiomes by body region and overall conservation when color morphs lack genetic differentiation.