Endemic fish calling: Acoustics and reproductive behaviour of the Neretva dwarf goby .

The Neretva dwarf goby (Gobiiformes, Gobionellidae) is an endemic fish native to the freshwaters of the Adriatic Basin in Croatia and Bosnia and Herzegovina, a Mediterranean Biodiversity Hotspot. Due to its limited distribution range, specific karst habitat and endangered status, laboratory studies on reproductive biology are scarce but crucial. Herein, we investigated the sound production and acoustic behaviour of the endangered during reproductive intersexual laboratory encounters, utilising an interdisciplinary approach. We also performed dissections and micro-computed tomography (μCT) scanning of the pectoral girdle to explore its potential involvement in sound production. Finally, comparative acoustic analysis was conducted on sounds produced by previously recorded soniferous sand gobies to investigate whether acoustic features are species-specific. The endemic is a soniferous species. Males of this species emit pulsatile sounds composed of a variable number of short (15 ms) consecutive pulses when interacting with females, usually during the pre-spawning phase in the nest, but also during courtship outside the nest. Pulsatile sounds were low-frequency and short pulse trains (140 Hz, <1000 ms). Male visual behaviour rate was higher when co-occurring with sounds and females entered the male's nest significantly more frequently when sounds were present. Characteristic body movements accompanied male sound production, such as head thrust and fin spreading. Furthermore, μCT scans and dissections suggest that shares certain anatomical similarities of the pectoral girdle (i.e. osseous elements and arrangement of muscles) to previously studied sand gobies that could be involved in sound production. Multivariate comparisons, using sounds produced by eight soniferous European sand gobies, effectively distinguished soniferous (and sympatric) species based on their acoustic properties. However, the discrimination success decreased when temperature-dependent features (sound duration and pulse repetition rate) were excluded from the analysis. Therefore, we suggest both spectral and temporal features are important for the acoustic differentiation of sand gobies.