INTRODUCTION
The Mediterranean Biodiversity Hotspot (MBH) is a widely renowned region for its significant environmental diversity and endangered wildlife (Myers et al., 2000; Darwall et al., 2014). The rivers of the Adriatic Sea Basin in Croatia are part of the MBH and Dalmatian freshwater ecoregions (Abell et al., 2008), and are especially rich in freshwater endemic fish, with 40 species, or almost 30% of the total Croatian ichthyofauna, endemic to this area (Myers et al., 2000; Kottelat & Freyhof, 2007; Ćaleta et al., 2015, 2019). This endemism is a feature of the habitats of the Dinaric karst that covers roughly 54% of Croatian territory, with the presence of numerous caves, sinkholes, cold seasonal wells, and underground rivers (Kutle et al. 1999; Mrakovčić et al., 2006).
Among these endemic fish species, Orsinigobius croaticus(Mrakovčić, Kerovec, Mišetić & Schneider, 1996) formerly described asKnipowitschia croatica , is a small benthic and short-lived (less than two years) sand goby confined to the Dinaric karst of the Dalmatian ecoregion. It can be found exclusively in the freshwaters of Croatia and Bosnia and Herzegovina (Abell et al., 2008; Zanella et al., 2011; Horvatić et al., 2017; Ćaleta et al., 2019; Tutman et al., 2020). In Croatia, this species inhabits the Eastern part of the Adriatic Basin, and has a naturally fragmented distribution range that includes the Neretva River, Matica River, the Vrgoračko Polje and Rastočko Polje fields, and Baćina Lakes, some of which are NATURA 2000 sites (Mrakovčić et al., 1996; Zanella et al., 2011, 2017; Ćaleta et al., 2015; see Horvatić et al., 2017 for the map with distribution area). In its natural habitat, O. croaticus occupies sandy bottoms with occasional stones/pebbles in karst rivers, slow-flowing streams and oligotrophic lakes (Zanella et al., 2011; Horvatić et al., 2017). During winter and early spring, O. croaticus thrives in small rivers and streams whereas during the summer period, this goby survives in small karst underground ponds and refuges, when the watercourses completely dry out (Miller, 2004; Mrakovčić et al., 2006). On the IUCN Red List,O. croaticus is assessed globally as vulnerable (VU, B2ab(iii); D2, ver. 3.1.), but regionally as endangered (EN) due to its highly limited/fragmented habitat and declining habitat quality (Crivelli, 2006, 2018; Mrakovčić et al., 2006; Horvatić et al., 2017). However, this endangered status is also due to a lack of knowledge regarding its biological traits. There is little published data on the ecology or biology of O. croaticus (Mrakovčić et al., 2006; Zanella et al., 2011, 2017; Horvatić et al., 2017). Like other sand gobies, O. croaticus is a polygamous multiple spawner species that achieves sexual maturity quite early (within its first year), with a reproduction season from March to November, although most spawning occurs from April to September (Mazzoldi & Rassotto, 2001; Kottelat & Freyhof, 2007; Zanella et al., 2011, 2017).
The sand gobies are a monophyletic gobiiform group (Gobionellidae, Gobiiformes) of about thirty species (in the generaKnipowitschia , Pomatoschistus , Economidichthys ,Ninnigobius and Orsinigobius ) (Thacker, 2009; Nelson, Grande & Wilson, 2016; Betancur-R et al., 2017; Thacker et al., 2018; Tougard et al. 2021). They inhabit intertidal marine and coastal freshwater ecosystems with muddy-to-pebble bottom across Europe, including the waters of the Mediterranean, Ponto-Caspian and Northeast Atlantic regions (Miller, 2004; Šanda & Kovačić, 2009; Kovačić & Patzner, 2011; Freyhof, 2011). Recent phylogenetic studies found evidence that sound production is widespread among actinopterygian fishes, suggesting that acoustic behaviour evolved independently multiple times in unrelated clades, and that there is a strong selection for the use of sound production as a behavioural trait across vertebrate evolution (Fine & Parmentier, 2015; Rice et al., 2020, 2022). Communicative sound emission in fish is usually linked to courtship and spawning or aggressive behaviour (Amorim, 2006; Myrberg & Lugli, 2006; Mann et al., 2008). In fish bioacoustics, acoustic signals associated with reproductive intersexual interactions have been the most commonly studied types of sounds (Amorim, 2006), since it is believed that these sounds serve to attract potential mates (Parmentier et al., 2010; Longrie et al., 2013), to synchronise spawning activities at aggregation sites (Lobel, 1992; Rowell et al., 2015; Erisman & Rowell, 2017; Jublier et al., 2019), or to synchronise gamete release by conspecifics (Hawkins & Amorim, 2000; Lobel 2002). Sand gobies are a common model group among soniferous actinopterygian fishes for sound production and have long been utilised in ethological and comparative bioacoustics studies. The acoustic abilities in sand gobies have been intensively investigated in the last 30 years, especially in the species of the genera Pomatoschistus ,Knipowitschia and Orsinigobius (Torricelli, Lugli & Pavan, 1990; Lugli et al., 1997; Malavasi et al., 2008, 2009; Amorim & Neves, 2007; Bloom et al., 2016; Zeyl et al., 2016; Parmentier et al., 2017). In eight sand goby species, two different types of acoustic signals (pulsatile and thump sounds) have been recorded to date (Amorim & Neves 2007; Blom et al. 2016; de Jong et al., 2016; Zeyl et al., 2016), while in Economidichthys pygmaeus (Holly, 1929), sounds were not detected during behavioural experiments (Gkenas et al., 2010). Most of our understanding regarding the acoustic abilities of Mediterranean sand gobies stems from the common, widely distributed and non-threatened species assigned to the least concern (LC; IUCN Red List) category (Amorim et al. 2013; Blom et al. 2016; de Jong et al., 2016; Zeyl et al., 2016). However, since O. croaticus was regionally classified as a vulnerable species with a very restricted distribution (Crivelli, 2006, 2018; Horvatić et al., 2017), this research is a first study of the acoustic communication of this endangered Mediterranean sand goby.
The main goal of this study was to empirically investigate acoustic communication of O. croaticus and the anatomy of its sound-producing mechanism. Specifically, our aims were to: i) investigate the sound production of captive O. croaticus males and quantify acoustic parameters of the sounds; ii) examine the reproductive behaviour of soniferous males and its association to sound production; iii) provide insight into the putative sound-producing mechanism by exploring the anatomy of the pectoral girdle, and iv) explore the acoustic diversification of soniferous sand gobies by quantitatively comparing acoustic signals between the study species and previously recorded Mediterranean sand gobies (generaNinnigobius , Pomatoschistus , Knipowitschia andOrsinigobius ).