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Biodiversity knowledge is widely heterogeneous across the Earth’s biomes. Some areas, due to their remoteness and difficult access, present large taxonomic knowledge gaps. Mostly located in the tropics, these areas have frequently experienced a fast development of anthropogenic activities during the last decades and are therefore of high conservation concerns. The biodiversity hotspots of Southeast Asia exemplify the stakes faced by tropical countries. While the hotspots of Sundaland (Java, Sumatra, Borneo) and Wallacea (Sulawesi, Moluccas) have long attracted the attention of biologists and conservationists alike, extensive parts of the Sahul area, in particular the island of New Guinea, have been much less explored biologically. Here, we describe the results of a DNA‐based inventory of aquatic and terrestrial vertebrate communities, which was the objective of a multi‐disciplinary expedition to the Bird’s Head Peninsula (West Papua, Indonesia) conducted between October 17th and November 20th 2014. This expedition resulted in the assembly of 1,005 vertebrate DNA barcodes. Based on the use of multiple species‐delimitation methods (GMYC, PTP, RESL, ABGD), 264 Molecular Operational Taxonomic Units (MOTUs) were delineated, among which 75 were unidentified and an additional 48 were considered cryptic. This study suggests that the diversity of vertebrates of the Bird’s Head is severely underestimated and considerations on the evolutionary origin and taxonomic knowledge of these biotas are discussed.

DNA barcoding opens new perspectives on the way we document biodiversity. Initially proposed to circumvent the limits of morphological characters to assign unknown individuals to known species, DNA barcoding has been used in a wide array of studies where collecting species identity constitutes a crucial step. The assignment of unknowns to knowns assumes that species are already well identified and delineated, making the assignment performed reliable. Here, we used DNA‐based species delimitation and specimen assignment methods iteratively to tackle the inventory of the Indo‐Australian Archipelago grey mullets, a notorious case of taxonomic complexity that requires DNA‐based identification methods considering that traditional morphological identifications are usually not repeatable and sequences mislabeling are common in international sequence repositories. We first revisited a DNA barcode reference library available at the global scale for Mugilidae through different DNA‐based species delimitation methods to produce a robust consensus scheme of species delineation. We then used this curated library to assign unknown specimens collected throughout the Indo‐Australian Archipelago to known species. A second iteration of OTUs delimitation and specimen assignment was then performed. We show the benefits of using species delimitation and specimen assignment methods iteratively to improve the accuracy of specimen identification and propose a workflow to do so.