DNA barcoding potentially offers scientists who are not expert taxonomists a powerful tool to support the accuracy of field studies involving taxa that are diverse and difficult to identify. The taxonomy of rays has received reasonable attention in Australia, although the fauna in remote locations such as Ningaloo Reef, Western Australia is poorly studied and the identification of some species in the field is problematic. Here, we report an application of DNA-barcoding to the identification of 16 species (from 10 genera) of tropical rays as part of an ecological study. Analysis of the dataset combined across all samples grouped sequences into clearly defined operational taxonomic units, with two conspicuous exceptions: the Neotrygon kuhlii species complex and the Aetobatus species complex. In the field, the group that presented the most difficulties for identification was the spotted whiptail rays, referred to as the ‘uarnak’ complex. Two sets of problems limited the successful application of DNA barcoding: (1) the presence of cryptic species, species complexes with unresolved taxonomic status and intra-specific geographical variation, and (2) insufficient numbers of entries in online databases that have been verified taxonomically, and the presence of lodged sequences in databases with inconsistent names. Nevertheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to highlight species complexes where taxonomic uncertainty might confound ecological data.

We investigate the presence of cryptic species among three highly variable nereidid polychaetes commonly found in Australian coral reefs − Nereis denhamensis Augener, 1913, Perinereis suluana (Horst, 1924) and Pseudonereis anomala Gravier, 1901 − based on morphological and molecular data (mitochondrial cytochrome c oxidase subunit I, COI, and nuclear histone H3). DNA extracted and sequenced from 70 specimens from northern Australia and the Philippines indicated the existence of eight species: three matched the types of existing species; four are newly described (Nereis heronensis, sp. nov., N. lizardensis, sp. nov., Perinereis pictilis, sp. nov. and Pseudonereis anomalopsis, sp. nov.) from NE Australia, and one species is described but not named due to lack of material. Nereis denhamensis, N. heronensis, sp. nov. and N. lizardensis, sp. nov. are distinguished from each other by proboscidial paragnath number and morphology of the metamorphosed female. Perinereis suluana can only be separated from P. pictilis, sp. nov. by colour pattern, while Pseudonereis anomala, P. anomalopsis, sp. nov. and P. sp. differ in colour pattern and the number and arrangement of paragnaths. Nereis (Lycoris) tydemani from Maluku, Indonesia, is newly synonymised with P. anomala. Divergence times estimated using COI indicated that speciation in all three groups occurred in the mid Miocene (20–17 ± 7 mya), which corresponds to a period of restricted east–west dispersal as Australia collided with the Indo-Malay archipelago, followed by range expansion opportunities in NE Australia as a result of flourishing coral reefs responding to warming seas and rising sea levels.

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