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\begin{document}

\title{Endemic fish calling: acoustics and reproductive behaviour of the
Neretva dwarf goby Orsinigobius croaticus}



\author[1]{Sven Horvatić}%
\author[2]{Eric Parmentier}%
\author[3]{Stefano Malavasi}%
\author[4]{Maria  Amorim}%
\author[4]{Paulo Fonseca}%
\author[1]{Davor Zanella}%
\affil[1]{University of Zagreb Faculty of Science}%
\affil[2]{University of Liège}%
\affil[3]{Universita Ca Foscari}%
\affil[4]{Universidade de Lisboa}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
The Neretva dwarf goby Orsinigobius croaticus (Gobiiformes,
Gobionellidae) is an endemic fish native to the freshwaters of the
Adriatic Basin in Croatia and Bosnia and Herzegovina. Due to its limited
distribution range, specific karst habitat and endangered status,
laboratory studies on reproductive biology are scarce. We investigated
the sound production and acoustic behaviour of this species during
reproductive intersexual laboratory encounters. We performed dissection
and micro-computed tomography (\selectlanguage{greek}μ\selectlanguage{english}CT) scanning of the pectoral girdle to
explore the anatomy of its putative sound producing mechanism. To study
interspecific acoustic differences and determine whether acoustic
features can discriminate among species, comparative analysis was
conducted on sounds produced by closely related soniferous sand gobies.
Our results indicate that males of the O. croaticus emit pulsatile
sounds composed of a variable number of short (\textasciitilde{} 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
(\textasciitilde{} 140 Hz, \textless{} 1000 ms), and spectro-temporal
parameters were correlated with physical traits and water temperature.
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. Male sound production was accompanied by movements
such as head thrust and fin spreading. \selectlanguage{greek}μ\selectlanguage{english}CT scans and dissections suggest
that O. croaticus shares certain anatomical similarities of the pectoral
girdle (osseous elements and arrangement of levator pectoralis muscles)
to previously studied sand gobies. Multivariate comparisons, using
sounds produced by eight soniferous European sand gobies, effectively
distinguished soniferous (and sympatric) species based on acoustic
properties. Discrimination success decreased when temperature-dependent
features (sound duration and pulse repetition rate) were excluded from
analysis. Therefore, we suggest both spectral and temporal features are
important for acoustic differentiation of sand gobies.%
\end{abstract}%



\sloppy


\textbf{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; \selectlanguage{polish}Ć\selectlanguage{english}aleta 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; Mrakov\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}et al.,
2006).

Among these endemic fish species, \emph{Orsinigobius
croaticus}(Mrakov\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć, \selectlanguage{english}Kerovec, Mi\selectlanguage{polish}š\selectlanguage{english}eti\selectlanguage{polish}ć \selectlanguage{english}\& Schneider, 1996) formerly
described as\emph{Knipowitschia 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; Horvati\selectlanguage{polish}ć \selectlanguage{english}et al., 2017; \selectlanguage{polish}Ć\selectlanguage{english}aleta 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 Vrgora\selectlanguage{polish}č\selectlanguage{english}ko Polje and
Rasto\selectlanguage{polish}č\selectlanguage{english}ko Polje fields, and Ba\selectlanguage{polish}ć\selectlanguage{english}ina Lakes, some of which are NATURA 2000
sites (Mrakov\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}et al., 1996; Zanella et al., 2011, 2017; \selectlanguage{polish}Ć\selectlanguage{english}aleta et
al., 2015; see Horvati\selectlanguage{polish}ć \selectlanguage{english}et al., 2017 for the map with distribution
area). In its natural habitat, \emph{O. croaticus} occupies sandy
bottoms with occasional stones/pebbles in karst rivers, slow-flowing
streams and oligotrophic lakes (Zanella et al., 2011; Horvati\selectlanguage{polish}ć \selectlanguage{english}et al.,
2017). During winter and early spring, \emph{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; Mrakov\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}et al., 2006).
On the IUCN Red List,\emph{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; Mrakov\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}et al., 2006; Horvati\selectlanguage{polish}ć \selectlanguage{english}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 \emph{O. croaticus} (Mrakov\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}et al.,
2006; Zanella et al., 2011, 2017; Horvati\selectlanguage{polish}ć \selectlanguage{english}et al., 2017). Like other
sand gobies, \emph{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 genera\emph{Knipowitschia}
, \emph{Pomatoschistus} , \emph{Economidichthys} ,\emph{Ninnigobius} and
\emph{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; \selectlanguage{polish}Š\selectlanguage{english}anda \& Kova\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć, \selectlanguage{english}2009; Kova\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}\& 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 \emph{Pomatoschistus}
,\emph{Knipowitschia} and \emph{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 \emph{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 \emph{O. croaticus} was regionally
classified as a vulnerable species with a very restricted distribution
(Crivelli, 2006, 2018; Horvati\selectlanguage{polish}ć \selectlanguage{english}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 \emph{O. croaticus} and the anatomy of its
sound-producing mechanism. Specifically, our aims were to: i)
investigate the sound production of captive \emph{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 (genera\emph{Ninnigobius}
, \emph{Pomatoschistus} , \emph{Knipowitschia} and\emph{Orsinigobius} ).

\textbf{MATERIAL AND METHODS}

\textbf{Fish sampling, laboratory housing and experimental design}

\emph{Orsinigobius croaticus} was sampled using electrofishing (Hans
Grassl, model: EL65 IIGI, power: 13 kW) from a boat during spring 2019.
Sampling was performed on the Matica River in Croatia (near the village
Vina 43\selectlanguage{ngerman}°10'30.33'' N, 17°23'12.36'' E). In total, we collected 25
individuals (15 males and 10 females) from the main river channel at a
depth of 0.5 -- 2 m. Fish were transferred to large plastic water
containers equipped with aerators and transported to the laboratory. At
the laboratory, fish were sexed based on urogenital papilla and body
coloration (Miller, 1984) and housed in appropriate community
rectangular tanks (120 L). After an acclimatization period of 5 -- 8
days, prospective soniferous males were chosen for subsequent laboratory
acoustic-visual recordings based on coloration (complete or partially
darken body, fins and head; Zanella et al., 2011), aggressiveness and
territoriality. Eight males (\selectlanguage{english}\emph{x} \selectlanguage{ngerman}± \emph{s.d.} = 49.21 ± 0.8 mm
total length, L\textsubscript{T}; range: 48.07 -- 50.06 mm; 41.07 ± 1.01
mm standard length, L\textsubscript{S}; range: 38.96 -- 42.29 mm; 1.24 ±
0.11 g weight, W; range: 1.10 -- 1.40 g) exhibiting typical reproductive
behaviour were chosen for the experiments. Females (N = 5; 36.75 ± 5.10
mm L\textsubscript{S}; range: 28.75 -- 41.27 mm) were chosen for the
recording sessions according to yellow belly coloration, luminescent
green spot on the first dorsal fin and dark eyes, all indicators of
female readiness for spawning (Zanella et al., 2011; Bloom et al.,
2016). Measurements were made using digital callipers CD-15APX with a
precision of 0.01 mm (Mitutoyo, Japan) and a digital scale (0.1 g
precision). As a metric of male body condition, we calculated the
condition factor {[}Fulton's \emph{K} , where \emph{K} =
(W/L\textsubscript{S}\textsuperscript{3}) *10\textsuperscript{5}{]}.

We followed the acoustic-visual recording protocol established by
previous authors (Amorim \& Neves, 2007; Amorim et al., 2013; Pedroso et
al., 2013), where experimental tanks were divided into three
compartments separated by removable partitions. Each lateral compartment
housed one territorial male with a nest (artificial pvc shelter), while
the middle section (``arena'') was occupied by a ripe female. The female
compartment was not provided with a nest. Briefly, our experimental
rectangular tanks were 120 L (60 x 48 x 35 cm) in volume and were
divided into three unequal sections by means of two opaque removable
acrylic partitions to prevent intersexual (visual) interactions. The
tanks had a 5 cm thick layer of fine sand or gravel and each male in
each section was provided with a water pump system and aeration. The
experiments were performed from mid-April to October, at random times.
The reproductive behaviour of resident males was elicited by introducing
one ripe female into the ``arena''. Before experiments, each female was
left 12 -- 24 h in the experimental tank for acclimatization. Eight
territorial males were kept in the experimental tanks until the end of
the recording sessions in October, after which they were returned to
male community tanks. Prior to recordings (approx. 15 min), electricity,
water pumps and aeration were switched off to minimise ambient noise.
Between recording sessions of the same resident male, 15 -- 30 min
pauses were included. In addition, to reduce substrate-born noise, the
experimental tanks were placed onto three cm thick rubber foam shock
absorbers. Fish were kept at natural photoperiod and fed daily \emph{ad
libitum} with \emph{Daphnia} . Water temperature, monitored with a
thermometer (AQUATERRA, Gare\selectlanguage{polish}š\selectlanguage{english}nica, Croatia), was maintained between 18
-- 22\selectlanguage{ngerman}°C. The male-female trials lasted approximately 30 min, and began
by removing one of the lateral partitions, allowing intersexual
interaction. To eliminate the noise from the room light system,
recording sessions were performed in the dark, with the experimental
tank illuminated by LED light from the side of the aquarium. This
procedure had no noticeable effects on fish behaviour.

\textbf{Acoustic recordings and sound analysis}

Acoustic experiments lasted from mid-April to October, which overlaps
the natural reproduction season of \emph{O. croaticus} . During acoustic
recording sessions, a hydrophone (H2A-XLR hydrophone, Aquarian Audio \&
Scientific, Anacortes, WA, USA; sensitivity: -180 dB re. 1 V
\selectlanguage{greek}µ\selectlanguage{english}Pa\textsuperscript{-1}; frequency range \selectlanguage{ngerman}± 4 dB from 0.01 to 100 kHz),
was placed in the centre of the experimental tank above the shelter, and
connected to a IRIG PRE preamplifier (Aquarian Audio \& Scientific).
Sounds were recorded using a ZOOM H4n portable digital audio recorder
(16 bit/44.1 kHz sample rate; ZOOM, Tokyo, Japan). The hydrophone was
placed within the attenuation distance from the emitter (less than five
cm), and we obeyed the laboratory protocol for minimum resonant
frequency for small glass tanks (\emph{e.g.} , 2.7 kHz for 170 L tanks,
according to Akamatksu et al., 2002). Sounds, monitored with headphones
and noted by the observer, were stored on the recorder memory card as
.wav files. Recordings were later band-pass filtered (0.05 -- 3 kHz) to
improve S/N ratio and subsampled at 4 kHz, and further amplified (10 dB)
for better auditory and visual inspection of the audio tracks.
Digitalised sounds were analysed using Avisoft - SASLab Pro 5.2 Software
(1024-point FFT, FlatTop window; 100\% frame; Avisoft Bioacoustics,
Berlin, Germany). Ten audio recordings (2.5 per male, each lasting
approx. 30 min) were aurally and visually inspected. Each \emph{O.
croaticus} sound was labelled using the ``insert label'' function of
Avisoft - SASLab Pro. In this study, we recorded 367 pulsatile sounds
from four soniferous males, but not all sounds presented a good signal
to noise ratio (S/N) for acoustic analysis. From ten recordings
presenting the best S/N ratio, we analysed 20 randomly selected sounds.
Temporal features were measured from oscillograms, while
frequency-related variables were obtained from the logarithmic power
spectra (FlatTop window, 512-points FFT, 96.87\% overlap; resolution 8
Hz). For sounds, we measured the following acoustic properties
(\textbf{Figure A1} ): (1) sound rate (SR, number of sounds emitted in 1
min); (2) sound duration (DUR, total length of the call, measured in
milliseconds); (3) number of pulses (NP); (4) pulse repetition rate
(PRR; NP divided by DUR and multiplied by 1000; Hz); (5) pulse duration
(PD; ms); (6) pulse period (PP; average peak-to-peak interval of
consecutive pulses, ms); (7) fatigue (FAT; ratio between the average PP
of the last three and first three pulses representing the decrease in
pulse emission rate possibly due to muscle fatigue, following Amorim et
al., 2013); (8) frequency modulation (FM, after the sound has been
divided into three temporally identical sections, FMi - initial, FMm -
middle and FMf - final - see \textbf{Figure A1} , frequency modulation
was calculated as the difference between the final and initial pulse
repetition rate and expressed in Hz; FMi, pulse repetition rate of the
initial section of a sound and FMf, pulse repetition rate of the final
section of a sound); (9) peak frequency (PF, the peak with the highest
energy from the logarithmic power spectrum function, Hz). In order to
follow the previous recording protocols as closely as possible (Amorim
\& Neves, 2007; Amorim et al., 2013), we also calculated the vocal
activity parameters per male: (I) sound rate (number of sounds produced
per min), (II) maximum sound rate (maximum number of sounds emitted in 1
min) and (III) calling effort (percentage of time spent calling, i.e.,
seconds of sound production divided by the duration of the recording in
seconds). Despite the fact that the variables PP and PRR indicate the
pulse repetition pattern, they were deliberately indicated separately
here in order to facilitate comparisons with the goby literature on
sound production.

\textbf{Video recordings and ethological analysis of behavioural
categories}

During acoustic-visual recordings, a second hydrophone (HTI-96-Min, High
Tech Inc., Long Beach, MS, USA, sensitivity: -201 dB re. 1V
\selectlanguage{greek}µ\selectlanguage{english}Pa\textsuperscript{-1}, frequency response 2 Hz to 30 kHz), placed less
than three cm from the nest opening, was connected directly to a video
camcorder (Canon Legria FS200, 41x digital zoom, 25 frames/sec, Tokyo,
Japan) to directly synchronise acoustic and visual signals into a
uniform dataset (.mod format) for subsequent analysis. By including or
excluding different females into a male's territory, we could control
the experimental variables of interest under standardised conditions and
study multimodal communication in males. The camcorder was mounted on a
stand and positioned approx. 40 cm from the front of the experimental
glass tanks. Quantitative ethological analysis of the courtship,
pre-spawning and spawning phases was performed by calculating the number
of sounds co-occurring with each behavioural category. Courtship
behaviour began when the females entered the male territory at a
distance of \textless{}5 cm from the male's nest, while the pre-spawning
phase was observed when the ripe female entered the male's nest.
Spawning was observed beginning with the female turning upside-down in
the nest numerous times in short succession (assuming\emph{oviposition}
) and started circling the ceiling. Male behaviours and the associated
sound emissions were observed in four soniferous males during seven
recording sessions and analysed using Solomon Coder (ver. beta
19.08.02). Again, the ripe females were chosen for the recording
sessions according to two indicators of their readiness for spawning
(belly and eye coloration). Behavioural categories (and behavioural acts
per category) expressed by the males were classified and scored
according to our observations and the literature (Amorim \& Neves, 2007,
2008; Amorim et al., 2013; Malavasi et al., 2009). In Solomon Coder, two
datasets were analysed separately and then compared. Firstly,
behavioural coding was performed by re-watching the video recordings
with sound production involving five females (N = 5; 37.23 \selectlanguage{ngerman}± 4.29 mm
L\textsubscript{S}; range: 31.62 -- 43.76 mm) and documenting the
frequency (\emph{n} min\textsuperscript{-1}) and duration (in sec) of
behavioural categories, calculating their total occurrence and
percentage. In addition, we analysed eight video recordings (two per
male) containing the behaviours of the same four tested males, but
performed with six different females (N = 6; 37.23 ± 4.29; range: 31.62
-- 43.76 mm) when they did not co-occur with sound production
(\emph{i.e.,} males were mute for the entire recording period). By
having these two datasets, we investigated the differences in
frequencies of behavioural categories in males when they engaged in
sound production and when they did not. In total, we used a total of
eight males in our experiments, but four were unresponsive (\emph{i.e.}
, did not perform courtship behaviour or sounds), resulting in
insufficient data for their further analyses. We analysed videos using
Solomon Coder for the following behavioural parameters: male behaviour
rate (the total number of behavioural acts per min) (1) co-occurring
with sounds or (2) not co-occurring with sounds; number of times a
female entered the male's nest accompanied with sounds (3) or (4)
without sounds; (5) total behaviours (number of behavioural categories
per video recording).

\textbf{Anatomical analysis}

For anatomical dissections, additional \emph{O} .
\emph{croaticus}individuals were collected in October 2020 from the same
watercourse near the village Bre\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć\selectlanguage{english}i (43\selectlanguage{ngerman}°7'11.30''N, 17°29'4.03''E)
using electrofishing. Five individuals were collected, of which three
males (40 -- 50 mm L\textsubscript{T}) 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 (\selectlanguage{greek}μ\selectlanguage{english}CT) 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 \selectlanguage{greek}μ\selectlanguage{english}A, with a frame rate of 12.5, 5 average
frames per image. This procedure generated 2897 images at a voxel size
of 10 \selectlanguage{greek}μ\selectlanguage{english}m. 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 (Abr\selectlanguage{ngerman}àmoff 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.

\textbf{Acoustic comparison among soniferous sand gobies}

The sounds of seven soniferous sand gobies, \emph{Knipowitschia
panizzae} Verga, 1841, \emph{Ninnigobius canestrinii} (Ninni
1883)\emph{, Orsinigobius punctatissimus} (Canestrini
1864),\emph{Pomatoschistus marmoratus} (Risso 1810), \emph{P. pictus}
(Malm, 1865), \emph{P. microps} (Krøyer, 1838\emph{)} and \emph{P.
minutus}(Pallas 1770), were previously recorded and characterised (Lugli
et al., 1995; Lugli \& Torricelli, 1999; Malavasi et al., 2008; Amorim
et al., 2013, 2018; Bolgan et al., 2013; Pedroso et al., 2013). However,
these acoustic data were never combined into a single phylogenetic
dataset and analysed interspecifically. Therefore, we studied their
interspecific acoustic variability (\emph{P. marmoratus} was separated
geographically into two populations, Italian and Portuguese). Briefly,
the species were sampled either from brackish habitats in north Adriatic
Sea (\emph{K. panizzae} , \emph{P. marmoratus} and \emph{N. canestrinii}
), from freshwaters of north-west part of Reggio Emilia Romagna, Italy
(\emph{O. punctatissimus} ) (Lugli et al., 1995, 1997; Lugli \&
Torricelli, 1999; Lindström \& Lugli, 2000), from Portuguese
marine/brackish waters (Amorim et al., 2013, 2018; Bolgan et al., 2013)
or the west coast of Sweden (Pedroso et al., 2013). Sound recordings
gathered from the previously conducted laboratory experiments were
re-analysed to allow for interspecific comparison with a minimal
measurement experimental error. All investigated sand gobies produced
pulsatile sounds, thus enabling acoustic interspecific comparisons. The
dataset was composed of 36 individuals of eight soniferous sand gobies
including \emph{O. croaticus} (min -- max: 3 -- 5 individuals, except
for a single individual of \emph{P. microps} ), with at least three
sounds recorded per individual. In total we calculated the means for
five acoustic variables (temporal: DUR in ms, NP, PRR in Hz; spectral:
PF and FM, both in Hz) for each individual. Since gobies included in the
current study were recorded at different water temperatures (range: 15.8
-- 22.6°C) and it is well known that the ambient water temperature
affects fish acoustic signals (Vicente et al., 2015; Ladich, 2018), we
conducted two separate multivariate analyses: the first involving the
complete dataset (all five acoustic features for each species), and the
second excluding the temporal features (DUR and PRR) known to be
influenced by water temperature (Lugli et al., 1996; Vicente et al.,
2015).

\textbf{Statistical analysis}

Each sound produced by a different \emph{O. croaticus} male was assumed
to be an independent acoustic unit, and the statistical analyses were
performed by combining the sounds from multiple individuals into a
single dataset. In the intraspecific analyses, the \emph{O.
croaticus}individuals were utilised as a grouping variable, to explore
acoustic variation between males. For each spectral and temporal
variable of the sound, the descriptive statistics are presented.
Outliers and extremes were detected visually from the boxplot and were
eliminated from the dataset if necessary. In order to test the
assumption of normality, we initially performed Shapiro-Wilk normality
test on a raw intraspecific dataset. Since the assumption of normality
was not met for some variables, the overall acoustic dataset was then
transformed (either using log or square root functions) followed by a
Box-cox function to estimate the transformation parameter by maximum
likelihood estimation. The acoustic dataset was re-examined for
distribution fitting, and since assumptions of normality (Shapiro - Wilk
test, \emph{P} \textless{} 0.05) and homoscedasticity (Bartlett test,
\emph{P} \textless{} 0.05) of the variances were not achieved, we
continued with non-parametric tests. For pairwise comparisons between
soniferous \emph{O. croaticus} males, we employed the non-parametric
Kruskal-Wallis rank sum test \emph{H}followed by pairwise Dunn's
multiple comparison test with Bonferroni correction for the \emph{P}
values. To investigate the mutual relationship between mean individual
acoustic variables, we utilised non-parametric \emph{Spearman}
correlation tests. The association between acoustic variables with body
characteristics (L\textsubscript{S}, \emph{W} and Fulton's \emph{K} )
and water temperature (T, in °C) was performed with \emph{Spearman}
correlations. Additionally, the \emph{Chi} -square
(\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}) was used to test for independence of behaviour
(expressed as behavioural categories) from sound production. In this
test, the residuals from the \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} were used to determine
which behaviours were positively related to sound production.
Kruskal-Wallis \emph{H} test was used to compare the mean behavioural
variables (calling rate, behaviour rate, n. of female nest entrances)
between soniferous males. Wilcoxon signed-rank test was performed to
compare the two dependent samples,\emph{i.e.,} mean behavioural
variables (behaviour rate and female nest entrance) of males when they
produced sounds and when they were mute. Additionally, Wilcoxon test was
used to compare the frequency and duration of courtship and pre-spawning
phases between males.

For the interspecific comparisons, the means of individual acoustic
properties of soniferous sand gobies were compared with the
non-parametric Kruskal-Wallis \emph{H} test. To quantify interspecific
acoustic variability among the soniferous sand gobies
(genera\emph{Knipowitschia} , \emph{Orsinigobius} ,
\emph{Pomatoschistus} and\emph{Ninnigobius} ), we used a multivariate
approach. Principal component analysis (PCA) is a commonly used method
in the bioacoustics literature for detecting the variables that explain
the most variance among soniferous fish species or populations. PCA,
based on the correlation matrix, was performed on transformed and
standardised individual means of five sound variables (temporal: DUR,
NP, PRR; spectral: PF and FM) to assess overall acoustic variability
between sand gobies, and additionally to recognise acoustic variables
explaining the observed variance. To assess the percentage of successful
classification of the sounds assigned to the correct goby species, and
to maximise the separability among taxa, we used linear discriminant
analysis (LDA). Two different LDAs were performed, first with the
complete dataset (five acoustic variables for each species) and then
removing the temperature-dependent features (DUR and PRR). Our results
were presented as means (\emph{x} \selectlanguage{english}) \selectlanguage{ngerman}± standard deviation (\emph{s.d.} ),
while the level of significance for inter- and intraspecific comparisons
was 5\% (\selectlanguage{greek}α \selectlanguage{english}= 0.05). Statistical analyses were performed in
STATISTICA\selectlanguage{ngerman}\textsuperscript{®} (v. 13.6.0., TIBCO, USA), Past (v. 4.11)
and R Studio (2022.07.0) software.

\textbf{Permits}

\emph{Orsinigobius croaticus} is legally protected by law as an
endangered taxon in Croatia (Official Gazette of the Republic of
Croatia, 2016). In addition, it is an endemic species with very limited
distribution. As a result, the number of individuals employed in the
laboratory experiments was kept to a minimum (less than 15) to prevent
possible effects on the natural population of this species. The sampling
by electrofishing for scientific purposes in the natural habitat was
approved by the Ministry of Agriculture (permit number
525-13/0545-19-2), while all laboratory experimental protocols were
approved by the Bioethics and Animal Welfare Committee of the Faculty of
Science, University of Zagreb (permit number 251-58-10617-21-147).

\textbf{RESULTS}

\textbf{Sound production and intraspecific sound signal structure}

Males of \emph{O. croaticus} produced a single type of acoustic signal,
named pulsatile sound, during intersexual (male-female) interactions
conducted within the reproductive season (April -- October). Four
resident males (\selectlanguage{english}\emph{x} \selectlanguage{ngerman}± \emph{s.d.} = 49.1 ± 0.8; range: 48.0 -- 50.0
mm L\textsubscript{T}; 40.9 ± 1.8; range: 38.9 -- 42.2 mm
L\textsubscript{S}; 1.2 ± 0.1; range: 1.1 -- 1.4 g \emph{W} ; 1.7 ± 0.1;
range: 1.5 -- 1.9 Fulton's \emph{K} ) produced sounds when interacting
with females, while the other four males remained mute and did not
court. We recorded 372 sounds produced by the four males (mean 93.0
sounds per male). These signals were reminiscent of brief felid purrs.
They were produced in a relatively irregular repetition pattern, at a
rate of 4.7 sounds per min, while the maximum recorded rate was 10
sounds per min (7.7 ± 1.4; range: 6 -- 10) (\textbf{Figure 1, Table 1}
). Structurally, the sounds are short duration signals, lasting around
450 ms (442.0 ±132.6; range: 156.8 -- 952.8 ms), and composed of a
variable number of short pulses (14.2 ± 4.0; range: 5 -- 32) of around
15 ms (14.5 ± 1.9; range: 9.7 -- 22.9 ms) (\textbf{Figure 2a-e, Table 1}
). The individual pulse unit structure differed markedly between sounds,
exhibiting one to three peaks with variable amplitude. Generally, the
amplitude of a sound changed gradually, first increasing and then
steadily decreasing throughout the sound, with the first two or three
pulses being the loudest (\textbf{Figure 2a-e} ). The pulse repetition
rate varied from 26.0 to 38.0 Hz (32.5 ± 1.6 Hz), while the sound pulse
period (PP) averaged 32 ms (31.9 ± 1.4; range: 27.6 -- 37.5 ms). PP
changed with water temperature, with higher values occurring at lower
temperatures (\textbf{Figure 3} ). Fatigue averaged 1.1 ± 0.08 (range:
0.9 -- 1.4). The sound peak frequency varied from 89 to 340 Hz (137.4 ±
38.3 Hz), although several higher frequency components were also
present, especially in the range 0.5 - 1.5 kHz. Energy extended from
0.05 to 2 kHz (\textbf{Figure 2a-e, Table 1} ), with most of the sound
energy within 0.05 -- 0.6 kHz. Frequency modulation of the sounds ranged
from 0.7 -- 1.1 Hz. Additionally, the calling effort varied between
males from 0.37 to 0.60 (0.49 ± 0.09), indicating that some individuals
emitted sounds more frequently than others (\textbf{Table 1} ).
Interestingly, sounds were never organised in bursts, which are usually
composed of several consecutive sounds produced with regular inter-sound
interval, as observed in some sand gobies.

Intraspecifically, soniferous \emph{O. croaticus} males differed
significantly in all acoustic features (Kruskal-Wallis \emph{H} test,
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 8.53 -- 41.97; d.f. = 3; N = 80;
\emph{P}\textless{} 0.05) except for calling effort (Kruskal-Wallis
\emph{H}test, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 5.67; d.f. = 3; N = 7;
\emph{P}\textgreater{} 0.05) (\textbf{Table 1} ). Some acoustic features
were significantly correlated (\emph{Spearman} rank correlations;
\emph{rS} = - 0.90 -- 0.97; \emph{P} \textless{} 0.05), while the
strongest correlation was observed between NP and DUR (\emph{rS} =
0.97), PRR with FMf (\emph{rS} = 0.7), and PRR with PP (\emph{rS} =
-0.9). Note that for pairwise correlation between sound variables, we
only considered\emph{rS} \textgreater{} 0.7 due to the limited sample
size. These relations are expected as DUR results in part from NP, PRR
and PP are redundant metrics, and FMf is the PRR of the initial part of
the sound. In addition, some acoustic features were also correlated with
physical features L\textsubscript{S} (PF and FMi, \emph{rS} = -0.53;
\emph{P}\textless{} 0.05 for both variables), \emph{W} (DUR and NP,
\emph{rS} = 0.53 and 0.55; \emph{P} \textless{} 0.05), and water
temperature (PRR, PP, PF and FMi, \emph{rS} = -0.67 -- 0.69; \emph{P}
\textless{} 0.05) (\textbf{Figure 4} ).

\textbf{Reproductive ethology and association with sounds}

We identified nine male behavioural categories within three distinct
reproductive phases in \emph{O. croaticus} : Chase, Lead, Approach and
Circling (courtship phase), Nest display, Frontal display, Nest rubbing,
Pre-mating (pre-spawning phase) and Spawning (spawning phase). The
category Pre-mating included all behaviours that occurred after the
female entered the male nest and prior to belly-up position
(\emph{oviposition} ). Spawning was not observed, but we observed the
female's repeated upside-down or belly-up position (an indication
of\emph{oviposition} ), during which she probably laid eggs on the nest
ceiling. Briefly, the behaviours Chase, Lead, Approach and Circling were
performed by the male outside the nest (recognised as the ``courtship''
phase), while Nest display, Frontal display, Nest rubbing, Pre-mating
and Spawning were performed within the nest (``pre-spawning'' and
``spawning'' phases). In some cases, Nest display and Frontal display
were performed by the male occupying the nest with or without a female
inside. However, Nest rubbing, Pre-mating and Spawning were always
performed by the male when the female was inside the nest. The
frequency, duration and overall percentage of these categories were
scored during ethological analysis. The first dataset included seven
video recordings (210 min) where at least one sound occurred per
recording. Overall, we observed 410 behavioural acts (102.5 per male).
The behavioural categories Nest display (29.3\%), Pre-mating (22.7\%)
and Approach (19.1\%) were most frequently observed, while Circling
(1.2\%), Chase (1.0\%) and Lead (0.7\%) were rarely recorded
(\textbf{Figure 5a-d} ). Concerning the association of sound production
with behavioural acts, of the 410 behavioural acts, 99 acts (24.1\%)
were accompanied by sound production. The behaviours Pre-mating (303
sounds), Nest display (27 sounds), Frontal display (20 sounds), Nest
rubbing (16 sounds) and Spawning (5 sounds) were coincided with sounds,
while Approach, Circling and Lead never co-occurred with sound
production (\textbf{Figure 5a} ). During four Chase acts, only one sound
was recorded. Only one spawning act (Spawning) was observed in this
study, during which five sounds were produced. Unfortunately, spawning
sounds were not used in the comparative purposes due to their limited
occurrence. The chi-square (\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}) test of independence
indicated that behavioural categories Nest display and Pre-mating were
significantly associated with sound production (\selectlanguage{greek}\emph{χ}
\selectlanguage{english}\textsuperscript{2} = 138.3; d.f. = 5; \emph{P} \textless{} 0.05;
residual score: 1.5 and 41.5, respectively), while other categories
failed to support this hypothesis (\textbf{Figure 5a} ).

To compare male behaviour when soniferous or mute, a second dataset of
eight video recordings was considered (190.5 min) of the same four males
but in which no sound production was documented. In these recordings, we
have observed 324 male behavioural acts (averaging 81.0 per male), of
which Approach (38.3\%), Lead (31.8\%) and Nest display (20.1\%) were
the most frequent categories (\textbf{Figure 5b} ). Contrary, Chase,
Circling, Pre-mating or Spawning were not documented within these
recording sessions. In general, there is an obvious dissimilarity
between the frequency of the behavioural categories that were or were
not accompanied by sounds. Specifically, Pre-mating, one of the two
behaviours significantly associated with sound production, decreased
from an average of 22.7\% in the trials with sounds to 0\% in the trials
without sound. In addition, Nest display, Nest rubbing and Frontal
display acts produced during sound emission decreased from 29.3\%,
13.9\% and 12\%, to 20.1\%, 3.7\% and 6.2\% in frequency in experiments
without the sounds, respectively. On the other hand, Approach and Lead
were more frequent during the mute behaviours (31\% and 38\%,
respectively) than during sound production (19\% and 0.7\%,
respectively) (\textbf{Figure 5b} ). Overall, the behavioural rate
decreased from 55.8\% to 44.1\% when males produced sounds in comparison
to when they were mute (soniferous \emph{vs.} mute males: means
2.79\emph{vs.} 1.55), though the differences were not significant
(\emph{Wilcoxon} signed-rank test, \emph{P} \textgreater{} 0.05)
(\textbf{Figure 6a} ). Importantly, the number of times the females
entered the male nest differed significantly between the two datasets
(means 3.71 \emph{vs} 0.71), as female nest entrance was more frequent
when males produced sound than when they were mute
(\emph{Wilcoxon}signed-rank test, \emph{P} \textless{} 0.05)
(\textbf{Figure 6b} ).

When producing sounds, the occurrence frequency between the courtship
(Chase, Lead, Approach and Circling) and pre-spawning (Nest display,
Frontal display, Nest rubbing, Pre-mating) phases of reproduction did
not differ between soniferous males (\emph{Wilcoxon} signed-rank
test,\emph{P} \textgreater{} 0.05), though their duration did
(\emph{Wilcoxon} signed-rank test, \emph{P} \textless{} 0.05).
Generally, males exhibited courtship-related behaviours less frequently
and for a shorter period compared to pre-spawning behaviours (\emph{x} \selectlanguage{english}=
22.5 \emph{vs.} 79.7 for frequency and \emph{x} \selectlanguage{english}= 173.4 \emph{vs.}2393.3
for duration).

\textbf{Anatomical findings and movements during sound production}

The pectoral girdle of \emph{O. croaticus} was subjected to \selectlanguage{greek}µ\selectlanguage{english}CT scanning
and anatomical dissection to identify the various osseous structures and
muscles that may be involved in sound generation. From \selectlanguage{greek}μ\selectlanguage{english}CT scans, three
functional units were distinguished in the skeletal part of the pectoral
girdle of \emph{O. croaticus} : the shoulder girdle (composed of the
post-temporal, the supracleithrum and the cleithrum bones) dorsally
attached to the neurocranium, the shoulder plate (i.e., four large
radials), and the fin plate, made up of fin rays articulated with the
shoulder plate (\textbf{Figure 7a-b} ). On the dorsal tip of the
cleithrum, anterior and posterior processes are present. The
supracleithrum articulates with the post-temporal and the cleithrum,
connecting with the cleithrum bone at its dorsal tip. The post-temporal
is made up of a basal plate and two rostrally oriented processes (a
``fork'') with dorsal and lateral attachments to the neurocranium. The
rostral tip of the dorsal process is flattened and firmly attached to
the epiotic bone. Putative sound producing muscles were observed during
the dissection and were found originating on the neurocranium and
inserting on the pectoral girdle (\textbf{Figure 7a-b} ).
The\emph{levator pectoralis} muscle is divided into two bundles:
the\emph{pars lateralis} and the \emph{pars medialis} . The \emph{pars
lateralis} originates on the posterior part of the pterotic and inserts
on the anterior dorsal process of the cleithrum. The \emph{parsmedialis}
is the thicker of the two muscles. It originates on the posterior part
of the basioccipital and inserts on the medial part of the posterior
dorsal process of the cleithrum.

Moreover, video recordings allowed to highlight characteristic fish
movements during sound production, especially concerning the head region
and fins. During sound production, soniferous males would usually stop
swimming and would suspend the body on the fused pelvic fins. In
addition, the pectoral fins were abducted, and the rays spread during
sound emission. The male performed a lateral body quiver starting from
the head to the tail (including dorsal fins), while the dorsal fins
(both first and second) were erected prior to the production of the
first pulse. Then the male would rapidly elevate the head and perform
lateral head motions, while spreading the buccal and opercular cavities.
The mouth was closed during the period of emission, though the anterior
part of the branchial basket was slightly uplifted.

\textbf{Interspecific acoustic diversity in soniferous sand gobies}

Eight soniferous sand gobies, namely \emph{Ninnigobius canestrinii}
,\emph{Pomatoschistus marmoratus} (two populations; five individuals
originating from Venice lagoon, Italy, and three from Portugal),\emph{P.
minutus} , \emph{P. microps} , \emph{P. pictus} ,\emph{Orsinigobius
punctatissiumus} , \emph{O. croaticus} and\emph{Knipowitschia panizzae}
, produce pulsatile sounds, thus enabling acoustic interspecific
comparisons. Interspecific pairwise comparisons revealed interspecific
differences in the acoustic features DUR, NP, PRR, and FM
(Kruskal-Wallis \emph{H} test, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 15.97~ \selectlanguage{english}30.19; d.f.
= 8; N = 36; \emph{P} \textless{} 0.05), while they did not differ in PF
(Kruskal-Wallis \emph{H} test, \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 11.54; d.f. = 8; N
= 36; \emph{P} \textgreater{} 0.05 (\textbf{Figure 8a-f} ). On average,
\emph{P. marmoratus} (Portuguese), \emph{O. punctatissimus} , \emph{P.
microps} and \emph{K. panizzae} were the smallest in size (34~ \selectlanguage{english}43 mm
L\textsubscript{T}), while \emph{P. minutus} , \emph{N. canestrinii} and
\emph{P. marmoratus} (Italian) were the largest species (50~ \selectlanguage{english}59 mm
L\textsubscript{T}). In most cases,\emph{K. panizzae} differed
significantly from other species, especially in DUR and NP (Dunn's
multiple comparison test, \emph{P} \textless{} 0.05). Regarding PF,
\emph{P. microps} had the highest mean values, alongside with \emph{K.
panizzae} (Dunn's multiple comparison test,\emph{P} \textless{} 0.05).
Finally, \emph{P. marmoratus} (Italian population) and \emph{P. pictus}
differed significantly from the rest of the species having lower values
of FM, while other species presented upward or downward modulated sounds
(Dunn's multiple comparison test,\emph{P} \textless{} 0.05)
(\textbf{Figure 8a-f} ).

Results of multivariate statistics were summarised by a PCA and LDA,
based on the five transformed (log\textsubscript{10} or square root) and
standardised acoustic features (DUR, NP, PRR, PF and FM), highlighting
the acoustic differences (heterogeneity) between species according to
sound feature. Due to the FM's negative raw values, we introduced a
positive factor so that we could use this feature in the analyses. In
PCA, the first two principal components of the PCA explained
cumulatively 69.79\% of the variation, with PC1 and PC2 explaining
39.28\% and 30.51\% of the variation, respectively. On the PC1 and PC2
scatterplot, although several species are clearly separated based on the
acoustic features of their sounds, most of the plots overlap
(\textbf{Figure A2} ). PC1 was strongly associated with DUR (-0.69) and
NP (-0.67), while PRR (-0.65) and PF (0.58) mostly contributed to PC2.
We performed two LDA analysis, first with the complete dataset (five
acoustic variables DUR, NP, PRR, PF and FM) and the second excluding the
temperature-dependent features (DUR and PRR), to test for sound
classification into correct groups (\emph{i.e.} , species). In the first
LDA, the first two axes accounted for a discrimination of 83.36\%, with
LD1 accounting for 61.09\% and LD2 for 22.27\%. LDA successfully
attributed the most sounds of a sand goby to the correct species
according to five acoustic parameters, with a correct interspecific
classification rate of 86.11\%. For some goby species, a contingency
table supports the 100\% level of correct classification of sounds
(\emph{N. canestrinii} , \emph{O. croaticus} , \emph{O. punctatissimus,
P. pictus} and \emph{P. microps} ), while for the remaining species
lower levels were achieved (67\% for \emph{K. panizzae} , 80\% for
Italian and 67\% for Portuguese \emph{P. marmoratus} , 60\% for \emph{P.
minutus} ). In the LDA bi-plot, species clusters overlap, but not
significantly, with some taxa occupying relatively isolated positions
along the LD axes (\textbf{Figure 9} ). LD1 supports differentiation of
\emph{N. canestrinii} and \emph{P. marmoratus} (Italian) from the rest
of the species within this acoustic space, while LD2 separated \emph{K.
panizzae} and \emph{P. pictus} from the other taxa. Plots of other
species means show overlapping patterns (\textbf{Figure 9} ).
Interestingly, two populations of \emph{P. marmoratus} (Italian and
Portuguese) overlap substantially on the scatterplot, even though the
LDA successfully separated the species. LD1 was significantly loaded
with FM (0.32), while LD2 with PRR (-0.47) and PF (-0.28). To explore
the effect of water temperature on the interspecific acoustic
classification success by LDA, we carried out a second LDA, including
only the three acoustic features that are known to be unaffected by
water temperature, namely NP, PF and FM. In this second LDA, axis 1 and
2 accounted for 95.49\% of discrimination, with LD1 axis accounting for
66.88\% and LD2 for 28.61\%. However, the second LDA was less successful
than the first LDA in accurately classifying the sounds of sand gobies,
with a 69.44\% rate of correct interspecific classification. Again, some
species (\emph{K. panizzae} , \emph{P. microps} and \emph{N.
canestrinii} ) achieved 100\% classification, while the remaining
species were misidentified in different percentages in comparison to
first LDA (75\% for \emph{O. croaticus} and 80\% \emph{O.
punctatissimus} , 66\% for \emph{K. panizzae} , 40\% for Italian and
33\% for Portuguese\emph{P. marmoratus} , 40\% \emph{P. minutus} and
80\% for \emph{P. pictus} ).

\textbf{DISCUSSION}

Freshwater sand gobies are considered important indicators for the
conservation of Mediterranean inland aquatic ecosystems due to their
wide range of habitats and high level of endemism (Vanhove, Kova\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć \selectlanguage{english}\&
Zogaris, 2016). Our study investigated, for the first time, the sound
production and reproductive intersexual behaviour of a freshwater
endemic Mediterranean goby, the Neretva dwarf goby \emph{Orsinigobius
croaticus,} under laboratory conditions. In addition, we investigated
multimodal signalling, using both acoustic and visual signals, in this
protected and geographically restricted sand goby. The results of this
study are consistent with prior research suggesting that gobies employ
sound production, along with visual or chemical communication, as an
important sensory modality for intraspecific communication (Lugli et
al., 1995; Amorim \& Neves, 2007; Malavasi et al., 2009; Amorim et al.,
2013; Bolgan et al., 2013; Blom et al., 2016). \emph{O. croaticus} is
the ninth acoustically investigated European sand goby,
excluding\emph{Economidichthys pygmaeus} , which was mute during the
experiments. However, it is the only one with a high IUCN Red List
classification status, listed as vulnerable (Crivelli, 2006, 2018).

Males of \emph{O. croaticus} produced pulsatile sounds when interacting
with females, during courtship, pre-spawning and spawning phases of the
reproductive behaviour. Males did not produce sounds in all trials and
calling rate varied between males and with female proximity. When males
were in close contact with females or the prospective female
approached/entered the nest, the calling rate would significantly
increase from a few up to 10 sounds per min. Structurally, the pulsatile
sounds in sand gobies are composed from a variable number (range 5 --
32) of pulses (organised in pulse trains), which are considered the
fundamental units of this acoustic signal (Lindstr\selectlanguage{ngerman}öm \& Lugli, 2000;
Zeyl et al., 2016). \emph{Orsinigobius croaticus} acoustic signals are
short and low-frequency sounds (\textless{} 500 ms, \textasciitilde{}
140 Hz) composed from a short number of sound pulses with an average
duration and period of around 15 ms and 32 Hz, respectively.

In this study, PD and PP differed significantly among males. In pulsed
acoustic signals, PD can be related to body size and condition (Amorim
et al., 2010) or temperature (Vicente et al., 2015), while PP is often
dependent on temperature, but also reflects phylogenetic affinities in
fish groups such as pomacentrids, cichlids, and sand gobies (Myrberg et
al., 1978; Amorim et al., 2008, 2013; Vicente et al., 2015). Here, PP
in\emph{O. croaticus} increased with decreasing water temperature
(\textasciitilde{} 37 ms at 19°C \emph{vs} . \textasciitilde{} 29 ms at
21.1°C). In \emph{P. pictus} water temperature significantly influenced
pulse period and explained 83\% of its variability (Amorim et al.,
2013). In ectothermic animals (such as fish), temperature-dependence in
sound-producing central and peripheral mechanisms is corroborated, since
it results from muscle activation (Bennett, 1985; Feher et al., 1998;
Rome \& Lindstedt, 1998; Kéver, Boyle, Parmentier, 2015; Vicente et al.,
2015; Ladich, 2018).

Pulsatile sounds of \emph{O. croaticus} males differed in all acoustic
features except calling effort. These acoustic differences amongst
soniferous males highlights the unique intraspecific acoustic
variability of their reproductive sounds. In addition, several acoustic
features were shown to be correlated to physical characteristics. We
found an inverse effect of male size on sound frequency, since these two
features were significantly and negatively related. This effect has
previously been recognised in acoustic studies on sand gobies (Lindström
\& Lugli, 2000; Malavasi et al., 2008; Amorim et al., 2013). In terms of
relationships, DUR and NP were highly and positively correlated in our
study, whereas PRR and PP had a negative association. The strong
correlation between DUR and NP suggests that the sound-producing
mechanism is based on a fixed motor pattern (Parmentier \& Lecchini,
2022).

In this study, \emph{O. croaticus} males exhibited nine (visual)
behavioural acts, confined to three distinct reproductive phases. The
sound production in males was mostly associated with pre-spawning
behaviours. Also, males exhibited courtship-related behaviours less
frequently and for a shorter period then pre-spawning behaviours. These
findings imply that the sound production is key in the mating process
in\emph{O. croaticus} and that it is likely efficient in transmitting
information at only short-range distances (within one body length).
Regarding the multimodal communication, soniferous \emph{O.
croaticus}males differed in the frequency and occurrence of displayed
behavioural categories (and their acts) when producing sounds and when
they were mute, since most of the categories in the mute experiments
were related to the courtship phase (outside the nest). Some behavioural
acts, such as Pre-mating, Chase, Circling and Spawning, were completely
absent from mute experiments. When producing sounds, Pre-mating and Nest
display were the most frequent categories, indicating that males
modulate their behaviour according to mate attraction investment. These
findings could indicate that the multimodal signals, as produced by
\emph{O. croaticus}males, could convey a wider set of information to the
prospective breeding females, rather than using only one signal type.
Indeed, males of different species, such as \emph{P. pictus} , make a
suite of signals from one or more modalities that females may use in
mating decisions (Amorim \& Neves, 2007; Amorim et al., 2013;
Bro-Jørgensen, 2010). Multimodal signals, which are used by many species
to communicate, contain components that can be analysed by multiple
sensory channels (Otovic \& Partan, 2009). Fish communicate through
visual, chemical and acoustic signals often operating simultaneously to
improve the chances of mating success, by indicating the physical
quality or the motivation of the emitter (e.g., Levine, Lobel \&
MacNichol, 1980; Liley, 1982; Heuschele et al., 2009; Amorim et al.,
2013). It has been suggested that this acoustic modality is highly
advantageous for territorial species, in which the nest site is
frequently hidden, and the male is out of sight from the prospective
mate (Myrberg, 1981).

Another significant finding from the current study is that females
entered the male's territory, particularly the nest hollow, more
frequently when accompanied by sound production than when the males were
mute. In this study, the two males who received the most female entries
were the largest. These two males exhibited the sounds with highest
values of NP, FMi and PRR, suggesting that these acoustic features might
be used to communicate important information to potential mates. Other
studies suggest that different acoustic traits or morphological features
could advertise male quality (genetic or phenotypic), serving as honest
signals of different aspects of male quality in sand gobies (Knapp \&
Kovach, 1991; Amorim et al., 2013). According to Amorim et al. (2013),
successful breeding \emph{P. pictus} males produced more sounds and with
a higher number of pulses than unsuccessful males.

Our findings indicate there are anatomical similarities in the
musculo-skeletal system of the pectoral girdle between the previously
studied \emph{Pomatoschistus} gobies and \emph{O. croaticus} (Adriaens
et al., 1993; Parmentier et al., 2017). Our study provided the first
anatomical dissections and \selectlanguage{greek}μ\selectlanguage{english}CT scans of the \emph{O. croaticus} pectoral
girdle and neurocranium. However, it is hypothesised that the Bauplan of
soniferous gobies does not show deep significant modifications, meaning
that the anatomy of soniferous species appears to be comparable to that
of their mute relatives (Parmentier \& Fine, 2016). To investigate the
anatomy of the sound producing mechanism in gobies, Parmentier et al.
(2013, 2017) undertook two empirical studies in two European gobies,
gobiid \emph{Gobius paganellus} (Gobiidae) and sand goby \emph{P.
pictus} (Gobionellidae), with the goal of testing the hypothesis of
contraction of the pectoral girdle muscles. These multidisciplinary
studies suggested strong similarities between the two gobies, and that
sounds might be generated by the contraction of the \emph{levator
pectoralis} muscle. These results suggested that the pectoral girdle is
most likely involved in sound production. It is worth noting that sound
production was coupled with nodding in \emph{G. paganellus} or with
lateral head movements in \emph{P. pictus} (Parmentier et al., 2013,
2017). However, this does not mean head movements are responsible for
the sound production. In this study, the pectoral girdle of \emph{O.
croaticus} consists of three functional osseous parts, with main
elements present as in other dissected sand gobies (Adriaens et al.,
1993; Parmentier et al., 2013, 2017). In addition, the \emph{levator
pectoralis} muscles, divided into two bundles (\emph{pars lateralis}
and\emph{pars medialis} ), were also found in \emph{O. croaticus} ,
originating on the neurocranium and inserting onto the pectoral girdle.
Four large radial bones were also present, forming the shoulder plate
in\emph{O. croaticus} . Lastly, the males performed lateral head
movements during sound emission. Some authors suggest that certain sound
characteristics are positively correlated with temperature if pulses are
directly based on sonic muscle contractions (Ladich, 2018). Although our
study did not include methodologies such as muscle histology, high-speed
video, or electromyography to fully corroborate the findings from
earlier research, we believe there is sufficient evidence to hypothesise
that the assumed sound producing mechanism in \emph{O. croaticus} could
be related with the contractions of the \emph{levator
pectoralis}(\emph{pars lateralis and medialis} ) muscles. Our
assumptions are based on: 1) the observed anatomical similarities (i.e.,
muscle organization) between \emph{O. croaticus} and other tested sand
gobies, 2) prominent temperature-dependence of the peripheral (muscular)
part of the sonic mechanism (as seen from the correlation of acoustic
features with water temperature, and PP variation), and 3) head lateral
movements observed during sound emission. Interestingly, in some
situations, males were observed to perform body movements (lateral
movements, head uplift, erection of fins), but without sound production,
indicating that sound production requires more than just body movements.
This supports the hypothesis that sounds are intentional and not only a
by-product of other activities such as breathing, feeding or swimming.

Sand gobies are highly similar morphologically (Kova\selectlanguage{polish}č\selectlanguage{english}i\selectlanguage{polish}ć, \selectlanguage{english}2008) and
frequently live in sympatry (Miller, 1986), making their discrimination
difficult. Several discrimination techniques have previously been
proposed for gobioids, such as mitochondrial/nuclear DNA markers
(Agorreta et al., 2013; Vanhove et al., 2012; Thacker et al., 2018),
otoliths in the inner ear (Lombarte et al., 2018) and behaviour
(Malavasi et al., 2012). Recently, the sounds (and their acoustic
features) have become a useful parameter in determining the phylogenetic
relationships in fish (Rice \& Bass, 2009; Parmentier et al., 2009;
M\selectlanguage{ngerman}élotte et al., 2016; Bolgan et al., 2020), particularly in gobies
(Malavasi et al., 2008; Horvati\selectlanguage{polish}ć \selectlanguage{english}et al., 2021). The aim of this study
was not to infer the phylogenetic relationships between sand gobies, but
rather to investigate how the species can be separated according to
their acoustic features, and how well the sounds can be classified for
each taxon. However, qualitatively, the pulsatile sounds of soniferous
sand gobies from this study are similar in that they are composed of a
series of pulses (\textbf{Figure 9} ), though when examined
quantitatively (using a multivariate approach), they were discriminated
according to their spectral and temporal acoustic parameters. In the
present study, we found interspecific differences among the sand gobies
species based on acoustic properties. The LDA assigned each sound
produced by sand gobies to the correct species with a discrimination
rate of 86\%. On the scatterplot, acoustic variables NP and FM
contributed to the separation of species in the negative direction,
while DUR, PF and PRR separated species in the positive direction. Note,
however, that species were recorded at different temperatures and
results should be taken with caution. The observed interspecific
differences, although based on a limited dataset, shed light on the
taxonomic position and affinities of the genera \emph{Orsinigobius}
and\emph{Ninnigobius} , relative to the rest of the sand
gobies.\emph{Ninnigobius canestrinii} and \emph{K. panizzae} , along
with\emph{P. pictus} , were the species most separated from the other
taxa on the LDS bi-plot. Some authors have opposed the separation of
\emph{O. croaticus} and \emph{O. punctatissimus} into the
genus\emph{Orsinigobius} , and the isolation of \emph{N. canestrinii}
from the genus \emph{Pomatoschistus} (Thourgard et al., 2021). On the
LDS bi-plot, the two \emph{Orsinigobius} taxa were closely
situated.\emph{Pomatoschistus} taxonomy is currently complicated, but
\emph{P. minutus} from our study was in the close proximity of the
two\emph{Orsinigobius} taxa. Interestingly, the hulls of the two
populations of \emph{P. marmoratus} overlapped in LDA, despite the fact
they encompass individuals from a wide geographic area (the Po River
delta in Italy and Parede/Arr\selectlanguage{ngerman}ábida in Portugal). However, the Italian
population appeared partially isolated from the rest of the species.
When applying the reduced dataset, the classification rate in LDA
decreased from 86\% to 69\%, which is not an unacceptable outcome,
though it implies that interspecific discrimination becomes more
difficult without certain acoustic features, such as
temperature-dependent DUR and PRR in our case.

\textbf{CONCLUSION}

In conclusion, our study demonstrates that the threatened and
geographically restricted freshwater sand goby, \emph{Orsinigobius
croaticus} , produces pulsatile sounds during intersexual laboratory
experiments. These sounds were produced during courtship, pre-spawning
and spawning phases of the reproduction interactions with females. In
addition, our results suggest that \emph{levator pectoralis} muscles
could be responsible for pulse emission. Finally, at the interspecific
level, acoustic signals produced by soniferous sand gobies appear to be
sufficiently different and species-specific to enable the discrimination
of species.

\textbf{AUTHOR CONTRIBUTIONS}

\selectlanguage{polish}\textbf{Sven Horvatić} : \selectlanguage{english}conceptualization (lead); data curation (lead);
formal analysis (equal); investigation (equal); methodology (equal);
visualization (lead); software (lead); writing - original draft (lead);
writing - review \& editing (equal). \textbf{Eric Parmentier} : formal
analysis (equal); investigation (equal); methodology (equal);
visualization (equal); software (equal); writing - original draft
(equal); writing - review \& editing (equal). Stefano Malavasi:
conceptualization (equal); data curation (equal); writing - original
draft (equal); supervision (equal); writing - review \& editing
(equal).\textbf{Maria P.} \textbf{Clara Amorim} : conceptualization
(equal); data curation (equal); writing - original draft (equal);
writing - review \& editing (equal). \textbf{Paulo J. Fonseca} :
conceptualization (equal); data curation (equal); writing - original
draft (equal); writing - review \& editing (equal). \textbf{Davor
Zanella} : data curation (equal); supervision (equal); writing -
original draft (equal); writing - review \& editing (equal); resources
(lead).

\textbf{ACKNOWLEDGMENTS}

We thank Sini\selectlanguage{polish}š\selectlanguage{english}a Vajdi\selectlanguage{polish}ć, \selectlanguage{english}Roman Karlovi\selectlanguage{polish}ć \selectlanguage{english}and Lucija Onorato for their help
during field sampling and fish maintenance in laboratory conditions. In
addition, we thank Linda Zanella for proofreading and for constructive
comments on the manuscript.

\textbf{CONFLICT OF INTEREST STATEMENT}

The authors declare that they have no competing interests.

\textbf{FUNDING INFORMATION}

This study was supported by Ministry of Agriculture, Croatia.

\textbf{DATA AVAILABILITY STATEMENT}

Audio files and Excel tables are available from the Figshare data
repository (https://doi.org/10.6084/m9.figshare.22787093.v1 and
https://doi.org/10.6084/m9.figshare.22786952.v1).

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