Diversity and Phylogenomic of Ascidiacea Based on Mitochondrial COI from Karimunjawa: Implications for Species Diversity and Antimicrobial Potential

Karimunjawa Marine National Park (MNP) is one of seven MNPs in Indonesia, yet its benthic biodiversity remains poorly documented. This study aimed to (i) inventory ascidian species from three shipwreck sites (Indonor, Kumbang, and Genteng), (ii) identify species using mitochondrial cytochrome oxidase I (COI) gene sequences, (iii) reconstruct their phylogenetic relationships, (iv) evaluate the antibacterial activity of ethyl-acetate (EtOAc) ascidian extracts against shrimp pathogenic bacteria, and (v) determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of active extracts. A total of 117 ascidian samples were collected in October 2022 and June 2023. Morphological and molecular analysis identified 32 species across eight families, with Didemnidae (37.5%), Styelidae (21.9%), and Clavelinidae (9.4%) being dominant. Eight species were new records in Indonesia or in Karimunjawa, including Botrylloides jacksonianum, Clavelina arafurensis, Ecteinascidia bandaensis, Ecteinascidia thurstoni, Eudistoma amplum, Eusynstyela hartmeyeri, Pseudodistoma fragile, and Rhopalaea macrothorax. Phylogenetic analysis was performed on 27 COI-confirmed samples shows relatedness with ascidian from Mediteranian Sea, UK, AustraliaPacific, and America. Six ascidian species from abundant families were tested for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, and Vibrio harveyii. Clavelina robusta exhibited the lowest MIC (3.9 µg/mL) against V. harveyii, outperforming the positive control, chloramphenicol (7.8 µg/mL). Didemnum molle showed the strongest overall antibacterial activity, with MIC and MBC values of 7.8 µg/mL against both E. coli and V. harveyii—comparable to chloramphenicol. These findings highlight the untapped biodiversity and bioactive potential of ascidians in Karimunjawa MNP and support further exploration for novel antimicrobial compounds from marine invertebrates.