Sound production in the Ponto-Caspian goby Neogobius fluviatilis and acoustic affinities within the Gobius lineage: implications for phylogeny SVEN HORVATI  C 1 , FRANCESCO CAVRARO 2 , DAVOR ZANELLA 1 and STEFANO MALAVASI 2 * 1 Department of Zoology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000, Zagreb, Croatia 2 CEMAS – Centre for Estuarine and Coastal Marine Science, Department of Environmental Sciences, Informatics and Statistics, Universit  a Ca’ Foscari Venezia, Campo della Celestia, Castello 2737/b, 30122, Venice, Italy Received 12 June 2015; revised 25 August 2015; accepted for publication 25 August 2015 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. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 00, 000–000. ADDITIONAL KEYWORDS: acoustic properties – gobies – Messinian salinity crisis. INTRODUCTION Acoustic signals convey crucial information on spe- cies, sex and individual identity, individual motiva- tion and quality (Bradbury & Vehrencamp, 1998). The degree of similarity among acoustic signals in groups of closely related species could be related to phylogenetic relationships, as shown for anurans, insects, birds and mammals (Robillard et al., 2006; Tavares et al., 2006; Cap et al., 2008; Gingras et al., 2013). Among the vertebrates, the largest diversity of sound-generating mechanisms for acoustic commu- nication has evolved in fishes (Myrberg & Lugli, 2006; Fine & Parmentier, 2015). In comparison with tetrapods, fish have relatively simple central and peripheral vocal mechanisms and thus typically lack the ability to emit complex frequency-modulated calls (Rice & Bass, 2009). Sound production in a commu- nicative context has been documented in over 800 fish species representing 109 families (Kasumyan, 2008), although phylogenetic reconstruction based on acoustics has seldom been attempted, and the state *Corresponding author. E-mail: mala@unive.it 1 © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, , – Biological Journal of the Linnean Society, 2015,  , – . With 3 figures.