Anatomical analysis
For anatomical dissections, additional O . croaticusindividuals were collected in October 2020 from the same watercourse
near the village Brečići (43°7’11.30”N, 17°29’4.03”E) using
electrofishing. Five individuals were collected, of which three males
(40 – 50 mm LT) were immediately euthanised with an
overdose of MS-222 (tricaine methane sulphonate; Pharmaq, Overhalla,
Norway), and stored for one week in 7% formaldehyde fixative solution
and then transferred to 70% ethanol. Specimens were dissected and
examined with a Wild M10 binocular microscope (Leica Camera, Leica,
Wetzlar, Germany) equipped with a camera lucida to study the anatomy of
the putative sound producing mechanism. Since earlier research on gobies
highlighted the role of the pectoral girdle and (pectoral) fins in sound
production, dissections primarily addressed the muscles related to this
body part. The nomenclature used to designate muscular parts was based
on earlier research (Winterbottom, 1974; Adriaens et al., 1993;
Parmentier et al., 2013, 2017). Additionally, one specimen was subjected
to micro-computed tomography (μCT) scanning to visualise the fish
skeleton at the level of the neurocranium and pectoral girdle. Scanning
was completed using a RX EasyTom (RX Solutions, Chavanod, France;
http://www.rxsolutions.fr), with an aluminium filter. Images were
generated at 75 kV and 133 μA, with a frame rate of 12.5, 5 average
frames per image. This procedure generated 2897 images at a voxel size
of 10 μm. Reconstruction was performed using X-Act software from RX
Solutions. Segmentation, visualisation, and analysis were performed
using Dragonfly software (Object Research Systems (ORS) Inc, Montreal,
Canada, 2019; software available at
http://www.theobjects.com/dragonfly). Three-dimensional (3D) 16-bit
images were produced and subsequently converted into 8-bit voxels using
ImageJ (Abràmoff et al., 2014). Three-dimensional processing and
rendering were obtained after semi-automatic segmentation of the body
using a ‘generated surface’, according to the protocols described by
Zanette et al. (2014). Direct volume renderings (iso-surface
reconstructions) were used to visualise a subset of selected voxels of
the anterior skeleton in AMIRA 2019.2.