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The aim of this study was to describe the vocal repertoire of the Ponto-Caspian goby Neogobius fluviatilis and to compare the acoustic properties of this species with those of other soniferous Mediterranean gobies belonging to the Gobius lineage. Vocalizations and associated behaviours were recorded under controlled aquarium conditions in female and male N. fluviatilis. Sound emission was elicited by means of 'intruder tests', using an individual of the same or opposite sex as an intruder, and recording sounds using a hydrophone placed 20 cm from the shelter used as a nest for the resident fish. Five acoustic properties, including spectral and temporal properties, were measured from 13 individuals. The vocal repertoire of the species consisted of sequences of short vocalizations during both agonistic and reproductive intraspecific interactions. The wave form of each sound resolved in a pure sine wave composed of rapidly repeated pulses. Sounds lasted about 200 ms, showing an average fundamental frequency of about 80 Hz. Sound properties did not differ between reproductive and the aggressive contexts, and the general structure of sounds was highly stereotyped. The individual means of three acoustic independent traits characterizing the sounds of seven species of the Gobius lineage, including N. fluviatilis, were then entered in a discriminant function analysis to assess how well species could be differentiated on the basis of acoustics, and their degree of affinities. The results suggested that the pulse repetition rate of the sounds, i.e. the relative tonal/pulsatile nature of the sounds, was the most important property in differentiating the species, and that this trait may contain a high level of phylogenetic signal, as the species producing tonal sounds clustered together, in line with the results of recent molecular phylogenic studies. The results were discussed in light of the geological and phylogeographical events believed to have driven the diversification of European gobies.

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