Glass-perchlets of the genus Ambassis (Teleostei; Ambassidae) form an important component of the brackish and marine coastal fish communities of tropical Asia. However, their species-level diversity is still poorly documented because of the absence of recent taxonomic revisions in this region and the limited availability of specimens for research. In addition, long-standing taxonomic and nomenclatural issues complicate the studies of this genus. Herein, we examine the diversity of Ambassis in Peninsular Malaysia using an integrative taxonomic approach and a large set of recently collected specimens from this region. Our initial morphological observations of 260 specimens revealed the presence of eight species, identified as Ambassis dussumieri, Ambassis interrupta, Ambassis kopsii, Ambassis macracanthus, Ambassis nalua, Ambassis octava, Ambassis urotaenia and Ambassis vachellii. We then sequenced the barcode fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene for 122 of our specimens, representing all eight morpho-species. Automatic species delimitation methods recovered nine Molecular Operational Taxonomic Units (MOTUs) because A. interrupta is made of two MOTUs. Morphological re-examination within A. interrupta detected variation at one character, congruent with molecular delimitation. Overall, our integrative approach unveiled rich species-level diversity within the genus Ambassis in Peninsular Malaysia, with the presence of nine species. Further comparisons between our COI dataset and the COI sequences archived in the Barcode of Life Data System (BOLD) from specimens of Ambassis broadly collected in tropical Asian regions, indicated regional-scale hidden diversity and identification conflicts, triggering the need for a complete taxonomic revision of this genus.
