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, Kovačić &
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 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). O. croaticus is
the ninth acoustically investigated European sand goby, excludingEconomidichthys 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 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 (Lindström & Lugli, 2000;
Zeyl et al., 2016). Orsinigobius croaticus acoustic signals are
short and low-frequency sounds (< 500 ms, ~
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 inO. croaticus increased with decreasing water temperature
(~ 37 ms at 19°C vs . ~ 29 ms at
21.1°C). In 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 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, 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 inO. croaticus and that it is likely efficient in transmitting
information at only short-range distances (within one body length).
Regarding the multimodal communication, soniferous O. croaticusmales 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 O. croaticusmales, 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 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 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 Pomatoschistus gobies and O. croaticus (Adriaens
et al., 1993; Parmentier et al., 2017). Our study provided the first
anatomical dissections and μCT scans of the 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 Gobius paganellus (Gobiidae) and sand goby 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 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 G. paganellus or with
lateral head movements in 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 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 levator
pectoralis muscles, divided into two bundles (pars lateralis andpars medialis ), were also found in O. croaticus ,
originating on the neurocranium and inserting onto the pectoral girdle.
Four large radial bones were also present, forming the shoulder plate inO. 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 O. croaticus could
be related with the contractions of the levator pectoralis(pars lateralis and medialis ) muscles. Our assumptions are based
on: 1) the observed anatomical similarities (i.e., muscle organization)
between 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 (Kovačić, 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;
Mélotte et al., 2016; Bolgan et al., 2020), particularly in gobies
(Malavasi et al., 2008; Horvatić 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 (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 Orsinigobius andNinnigobius , relative to the rest of the sand gobies.Ninnigobius canestrinii and K. panizzae , along withP. pictus , were the species most separated from the other taxa on
the LDS bi-plot. Some authors have opposed the separation of O.
croaticus and O. punctatissimus into the genusOrsinigobius , and the isolation of N. canestrinii from the
genus Pomatoschistus (Thourgard et al., 2021). On the LDS
bi-plot, the two Orsinigobius taxa were closely situated.Pomatoschistus taxonomy is currently complicated, but P.
minutus from our study was in the close proximity of the twoOrsinigobius taxa. Interestingly, the hulls of the two
populations of P. marmoratus overlapped in LDA, despite the fact
they encompass individuals from a wide geographic area (the Po River
delta in Italy and Parede/Arrábida 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.