# Linking courtship calls and observed behaviours of Epinephelus marginatus in the wild offers new insights into its reproduction and conservation

Elena Desiderà, Carlotta Mazzoldi, Augusto Navone, Rémi Blandin, Antonio Calò, Cédric Gervaise, Pieraugusto Panzalis, Egidio Trainito, Paolo Guidetti, Lucia Di Iorio

► To cite this version:

Elena Desiderà, Carlotta Mazzoldi, Augusto Navone, Rémi Blandin, Antonio Calò, et al. Linking courtship calls and observed behaviours of Epinephelus marginatus in the wild offers new insights into its reproduction and conservation. Aquatic Conservation: Marine and Freshwater Ecosystems, 2023, 34 (1), 10.1002/aqc.4037. **hal-04738232**

HAL Id: hal-04738232

https://univ-perp.hal.science/hal-04738232v1

Submitted on 8 Dec 2024

**HAL** is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire **HAL**, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 Research paper

2 Linking *Epinephelus marginatus*' courtship calls and observed behaviours in

3 the wild offers new insights into its reproduction and conservation

4

5 Elena Desiderà¹,²*, Carlotta Mazzoldi²,³, Augusto Navone⁴, Rémi Blandin⁵, Antonio Calò⁶, Cèdric

6 Gervaise⁷, Pieraugusto Panzalis⁴, Egidio Trainito¹, Paolo Guidetti¹,⁸,⁹, and Lucia Di Iorio⁷,¹⁰

7

8 ¹Department of Integrative Marine Ecology (EMI), Genoa Marine Centre (GMC), Stazione Zoologica Anton

9 Dohrn-National Institute of Marine Biology, Ecology and Biotechnology, Villa del Principe, Piazza del
Principe 4, 16126 Genoa, Italy

10

11 ²Department of Biology, University of Padova, Via U. Bassi 58/B, 35121 Padova, Italy

12 ³CoNISMa, Interuniversity National Consortium for Marine Sciences, Piazzale Flaminio 9, 00196 Rome,

13 Italy

14 ⁴Marine Protected Area of Tavolara-Punta Coda Cavallo, Via Dante 1, 07026 Olbia, Italy

15 ⁵Institute of Acoustics and Speech Communication, TU Dresden, 01062 Dresden, Germany

16 ⁶Department of Earth and Marine Sciences (DiSTeM), University of Palermo, Via Archirafi 20-22, 90123,

17 Palermo, Italy

18 ⁷CHORUS Institute, Phelma Minatec, 3 Parvis Louis Néel, 38016 Grenoble, France

19 ⁸National Research Council, Institute for the Study of Anthropic Impact and Sustainability in the Marine

20 Environment (CNR-IAS), Via de Marini 6, 16149 Genoa, Italy

21 ⁹NBFC, National Biodiversity Future Center, Palermo 90133, Italy

22 ¹⁰UMR 5110 CEFREM, CNRS, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan, France

23

24 * Corresponding author, at: Department of Integrative Marine Ecology (EMI), Genoa Marine Centre (GMC),

25 Stazione Zoologica Anton Dohrn-National Institute of Marine Biology, Ecology and Biotechnology, Villa del
Principe, Piazza del Principe 4, 16126 Genoa, Italy.

27 E-mail address: elena.desidera@szn.it
28 **ABSTRACT**

29 Groupers are marine fishes particularly vulnerable to overexploitation chiefly because
30 of their reproduction-related traits. The dusky grouper *Epinephelus marginatus* is
31 classified as 'Endangered' in the Mediterranean Sea, where protection measures have
32 proved critical for allowing population recovery and persistence of the species.
33 However, knowledge gaps remain regarding its reproductive behaviours and spawning
34 sites. As other grouper species, the dusky grouper is known to produce courtship-
35 associated sounds, which were validated in captivity, but no study has ever
36 established a link between sound production and visual behaviours recorded in the
37 wild. This study aims to characterise, both visually and acoustically, the behavioural
38 patterns of wild dusky grouper and to link visual courtship behaviours to the
39 associated sound production. The study was conducted over two consecutive
40 summers (2017 and 2018) using visual observations and passive acoustic monitoring
41 at two presumptive spawning sites within a Mediterranean Marine Protected Area in
42 the North-Western Mediterranean Sea. Results showed that courtship calls were
43 mostly recorded during evening hours at both study sites and were significantly
44 associated with visual reproductive behaviours. Results also indicated a temporal
45 partitioning in the species acoustic activity suggesting different behaviours occurring
46 at different times of the day. These findings have important implications for identifying
47 and monitoring dusky grouper spawning sites using passive acoustic methods,
48 therefore providing valuable information for the development and implementation of
49 effective conservation measures.

50

51 **Keywords:** Dusky grouper, sound production, passive acoustics, spawning, MPA,

52 Mediterranean Sea.
54 **1. INTRODUCTION**

55 The increasing global demand for seafood is driving the overexploitation of many
56 marine fish stocks (FAO, 2020), with some species being more vulnerable than others
57 due to their life history and behavioural traits (Jennings et al., 1998; Reynolds et al.,
58 2005). In particular, reproductive behavioural traits are crucial determinants of species
59 susceptibility to overfishing and their recovery capacity from exploitation (Biggs et al.,
60 2021). Species with large size, long lifespan, late sexual maturity, sequential
61 hermaphroditism and aggregating spawning behaviour, such as groupers, are at high
62 risk (Erisman et al., 2013; Rowe and Hutchings, 2003). Groupers (Epinephelidae, Smith
63 and Craig, 2007) are ecologically and economically valuable fishes known to mate in
64 aggregations of several tens up to thousands of individuals occurring at predictable
65 times and places (Prato et al., 2013; Sadovy de Mitcheson and Colin, 2012; Sala et al.,
66 2001). Such aggregating behaviour makes them highly susceptible to overfishing,
67 leading to a global population decline of over 80% (Sadovy de Mitcheson et al., 2020).
68 Identifying and characterizing grouper spawning aggregations (i.e., in terms of fish
69 densities, sizes, sex-ratio, and timing of spawning) is crucial for the proper
70 conservation of these species (Sadovy de Mitcheson, 2016).

71 Directly identifying spawning sites in the wild is, however, challenging due to the
72 significant effort required to monitor large areas over long periods and the elusive
73 behaviour of grouper species toward divers. In this regard, passive acoustic
74 monitoring (PAM) can be coupled with observational methods to non-intrusively assess
75 temporal patterns of reproductive activity and help identify reproductive sites (Schärer
76 et al., 2014, 2012a, 2012b).

77 Groupers are, in fact, known to produce sounds, some of which have been associated
78 with visual courtship behaviours (Bertucci et al., 2015; Koenig et al., 2016; Rowell et
79 al., 2019). Courtship calls are species-specific sounds emitted during courtship activity
80 and can be used to spatially and temporally localise the occurrence of spawning
81 aggregations (Locascio and Burton, 2015; Mann et al., 2010; Wall et al., 2014). PAM
82 can be particularly valuable when monitoring reproductive behaviours that occur at
83 multiple spawning sites simultaneously, in remote areas or where diving conditions
84 are constrained (e.g., deep water, strong currents) or the visibility is limited (e.g.,
85 turbid water, at dusk or nighttime).

86 The widespread and systematic implementation of PAM for spawning site
87 identification, however, relies on the accurate detection of courtship calls in acoustic
88 recordings (Ibrahim et al., 2018), which requires a field-verified sound-behaviour
89 pairing. In situ surveys combining direct behavioural observations with passive
90 acoustic recordings are thus critical to link species-specific visual courtship behaviours
91 to their associated calls. However, such surveys have been limited in number, greatly
92 limiting the reliability of PAM for spawning site detection (Colin, 1990; Mann et al.,
93 2010; Rowell et al., 2019). This is especially true in the Mediterranean Sea, where no
94 studies have yet linked wild grouper behaviours with concurrent sound production,
95 thereby preventing the use of PAM for identifying spawning aggregations and
96 ultimately hindering the implementation or reinforcement of protection measures at
97 critical spawning sites.

98 The dusky grouper *Epinephelus marginatus* is an iconic and endangered
99 Mediterranean grouper species that has received special conservation attention
100 through time, being protected under the Barcelona and the Bern Conventions (Pollard
101 et al., 2018). Effective conservation actions include restrictions or bans on fishing,

102 mainly achieved through well-enforced Marine Protected Areas (MPAs), especially

103 where fishing is prohibited (i.e., fully protected areas; Giakoumi et al., 2017). Despite

104 its endangered status, knowledge about its biology, particularly reproduction, is

105 limited (Condini et al., 2017).

106 Our study aimed to characterise the behavioural patterns of wild dusky grouper, using

107 both visual and acoustic approaches. The specific aims included: 1) characterising the

108 species' sound production through PAM, 2) visually documenting behaviours and

109 associated colour patterns during the reproductive season, and 3) validating the link

110 between courtship calls and visual courtship behaviours at presumptive spawning

111 sites within a Mediterranean MPA. Ultimately, our study contributes to the

112 identification of spawning sites and supports the implementation of effective

113 protection measures therein.

114

## 2. MATERIALS AND METHODS

### 2.1. Study species

117 The dusky grouper forms spawning aggregations of tens of individuals in the

118 Mediterranean Sea during summer (June-September) (Hereu et al., 2006; Zabala et al.,

119 1997a). However, to our knowledge, evidence of these aggregations has only been

120 collected in six locations: the Medes Islands, Cerbère-Banyuls, Port-Cros, Lavezzi

121 Islands, La Revellata and Lampedusa Island (Culioli and Quignard, 1999; Hereu et al.,

122 2006; Louisy and Culioli, 1999; Marinaro et al., 2005; Marino et al., 2001; Pelaprat,

123 1999; Zabala et al., 1997a). During the spawning season, dusky grouper display

124 distinctive colour patterns indicating their reproductive conditions (Zabala et al.,

125 1997b). Males exhibit a silver streaked colour pattern during courtship and territorial

126 behaviours (Hereu et al., 2006; Zabala et al., 1997a, 1997b). Males are also known to

127 emit two main types of sounds: single and serial low-frequency pulses (< 200 Hz),

128 previously defined as “booms”, both validated in captivity with the latter associated

129 with courtship displays (Bertucci et al., 2015). Additionally, a low-frequency down-

130 sweeping sound (< 200 Hz), previously defined as “growl”, was recorded in the wild at

131 two known spawning sites and attributed to the dusky grouper (Bertucci et al., 2015).

132

### 2.2. Study sites

134 The study took place in the Tavolara-Punta Coda Cavallo Marine Protected Area

135 (hereafter TPCC MPA), located in the Western Mediterranean Sea (Sardinia, Italy). It

136 was established in 1997 but became effective around 2003-2004. The TPCC MPA is

137 divided into three zones with different protection levels (Fig. 1).

138 As part of a larger study, two unprotected and two protected sites were identified as

139 potential dusky grouper spawning sites based on local ecological knowledge and

140 scientific literature (Desiderà, 2020). Underwater Visual Census (UVC) data collected

141 at the four sites indicated that only the protected ones hosted significant grouper

142 densities, suggesting the occurrence of spawning aggregations (Desiderà, 2020).

143 Therefore, the present work was conducted at these two sites, namely “Secche Papa”

144 (SP) and “Molarotto” (MOL) (Fig. 1).
145 SP features two adjacent series of limestone pinnacles where diving is permitted while
146 fishing is forbidden. MOL is a granitic rocky outcrop located in the fully protected zone
147 of the TPCC MPA.

148

149 **2.3. Acoustic data collection**

150 The audio data were recorded from July 5th to July 31st, 2017, and July 18th to
151 September 23rd, 2018. Recorders were moored at depths of 25-40 m using sandbags,
152 with hydrophones situated 1.5 m above the bottom. One hydrophone was deployed at
153 MOL, while two to three hydrophones were used at SP to account for the influence of
154 bottom topography on sound propagation and signal detection. Despite using multiple
155 hydrophones, SP was treated as a single study site due to the close proximity of the
156 pinnacles. Recording cycles varied between sites depending on the recorders used. At
157 MOL in 2017, a recorder connected to a battery pack allowed for nearly continuous
158 recording. However, adverse weather conditions in the same year prevented
159 hydrophone recovery and deployment at SP, causing a gap in recordings. See
160 Supplementary Material for additional information on the recording equipment and
161 sampling schedule (Fig. S1).

162

163 **2.4. Acoustic data processing**

164 Audio recordings were down-sampled to 4 kHz since dusky grouper calls are low in
165 frequency (<450 Hz, Bertucci et al., 2015). Low-frequency pulses and downsweeping
166 sounds were a priori identified as known dusky grouper sound types (Bertucci et al.,
167 2015). After inspecting a full day of audio recordings from MOL (July 7th, 2017) using
168 Raven PRO 1.5 software (The Cornell Lab of Ornithology), we found that these sound
169 types could occur individually or in combinations. To classify these sounds, we defined
170 five main sound types (Fig. 2, Audio Files S1, S2): (1) Low-frequency (always referred
to as with a peak frequency <200 Hz) single Pulses (LP), (2) Low-frequency Pulse
171 Series (LPS) made up of at least three LP sounds, (3) Low-frequency Down-sweeping
172 Sounds (LDS), (4) Low-frequency Series of Down-sweeping Sounds (LDSS) made up of
173 at least three LDS sounds, and (5) a combination of Low-frequency Pulses and Down-
174 sweeping sounds (LPLDS).

175 Using the same custom-built MATLAB interface and procedure described in Desiderà et
176 al. (2022), audio recordings were audio-visually inspected to quickly identify and
177 attribute detected sounds to one of the aforementioned five sound types. Sound
178 selections were summarized in .csv output files and used for further analysis as
detailed in the following sections.

181
182 **2.5. Behavioural observations**

183 Dusky grouper spawning was reported to occur around sunset (Hereu et al., 2006;
184 Zabala et al., 1997a), but no systematic monitoring had been conducted at other
185 times of the day. To investigate the timing of dusky grouper spawning and collect data
186 on reproductive behaviours, direct observations were conducted at the study sites
187 during the summers of 2017 and 2018 (from June to late September-early October).
188 Observations were made during three time slots: after sunrise (04:00-07:00 hours
189 Universal Time Coordinated, UTC), during daytime (07:01-11:00 hours UTC), and
190 before sunset (15:00-19:00 hours UTC). Three SCUBA divers, one observer per dive,
191 recorded in situ observations of dusky grouper behaviours onto an underwater board,
192 noting the start and end times of each survey and the time of observations.
193 Additionally, a high-definition camera (Sony Cyber-shot DSC-RX100) was used to
194 record dusky grouper behaviours.

195 The sex of individuals was inferred from their behaviours and colour patterns. Males
196 were identified by their silver streaked colour pattern and females were identified
197 when being courted by males (Zabala et al., 1997b). If there was insufficient evidence,
198 sex was left unspecified.

199 Observed behaviours were classified as territorial or reproductive, following the
terminology described in the literature for the dusky grouper (Table 1).

201

202 **2.6. Analysis of sound production**

203 To describe the temporal patterns of dusky grouper sound production at the study
204 sites, the audio recordings collected in July 2017 were analysed. This period was
205 selected based on the intense patrolling and courtship activity observed, along with
206 previous studies reporting a peak in spawning during July in the Western
207 Mediterranean (Balearic Islands, Reñones et al., 2010). Because fish emit sounds
208 mainly at night (Bertucci et al., 2015; Parsons et al., 2016), and nighttime sound
209 production is less masked by anthropogenic noise, only data recorded from 16:00 to
210 6:00 hours UTC were processed as per section 2.4. Furthermore, the chosen period
211 encompasses dusky grouper reproductive activity (Hereu et al., 2006; Zabala et al.,
212 1997a). Diel 14-hour intervals (from 16:00 to 6:00 hours UTC) were divided into three
213 subgroups encompassing sunset (16-20), midnight (20-24), and sunrise (00-06) to test
214 for differences in sound abundance across these time intervals.

215 Acoustic data were analysed using the following approaches:

216 1) Visualising sound production patterns: The hourly number of detected sounds was
217 rescaled to the range of 0-1 by sound type and study site (min-max normalization), to
218 ensure data comparability. Normalized data were used to create bar plots and heat
219 maps, which visualized sound production patterns over the analysed 14-hour interval
220 and across different days, respectively.

221 2) Testing the relationship between hourly sound abundance and sound type across
time periods: Hourly sound abundance (response variable) was modelled as a function
223 of sound type (categorical with five levels: LP, LPS, LDS, LDSS, LPLDS), study site
224 (categorical with two levels: SP and MOL), and time period (categorical with three
225 levels: 16-20, 20-24, 00-06).

226 Data exploration followed the protocol by Zuur et al. (2010). A zero-inflated negative
227 binomial (ZINB) model was used to account for the high percentage of zeros and
228 overdispersion. To account for differences in recording cycles, an offset (natural
229 logarithm of the amount of minutes effectively recorded per hour) was included in the
230 model. Model selection was based on the Akaike Information Criteria (AIC) after model
231 testing using the package "glmmTMB" (Brooks et al., 2017). The model used for this
232 study was the following ZINB GLM model (in words):

233 *count part of the model:* Sound.abundance ~ Site + Sound.type + Time.period +
234 Site:Time.period + Sound.type:Time.period + Site:Sound.type +
235 offset(LogMinutes) + (1 | Date)

236 *binary part of the model:* ~ Site + Sound.type + Time.period

237 The notation ':' indicates the interaction between categorical explanatory variables.
238 Date was treated as a random effect ("1 | Date") to handle pseudo-replication
239 (repeated measurements from the same sites).

240 Model validation was conducted using the package "DHARMa" (Hartig, 2022) by
241 plotting residuals versus fitted values and versus each covariate in the model, and by
242 verifying that the model could cope with the number of zeros in the dataset (Figs S2,
243 S3, S4). Residuals were also assessed for temporal correlation. Post-hoc tests were
244 performed using the package "emmeans" (Lenth, 2022).

245

246 **2.7. Analysis of behavioural observations**

247 To examine the relationship between the behaviours observed during the summers of
248 2017 and 2018 and their dependence on time, the number of behaviours observed per
249 dive (response variable) was modelled as a function of behavioural category
250 (categorical with five levels: stimulation, courtship, patrol, chase, bumping) and time
251 of the day (categorical with three levels: after sunrise, daytime, before sunset). The
252 same protocol explained in section 2.6 was used for data exploration, model selection
253 and model validation (Figs S6, S7, S8). To account for differences in observation
254 duration, an offset (natural logarithm of the actual observation time per dive) was
255 included in the model, while site was excluded due to the small number of
256 observations per site by time period. The model used was the following GLM model
257 with negative binomial distribution (in words):

258 Behaviour.abundance ~ Category + Time.period + offset(LogMinutes) + (1 | Date)

260 Date was treated as a random effect ("1 | Date") to handle pseudo-replication.

261 In both this section and the previous one (2.6), all calculations were performed using R
262 version 4.2.1 (R Core Team, 2022), along with the support file by Zuur et al. (2009)
263 and the specific R packages listed in the Supplementary Material.

264
265
266

## 2.8. Relationship between observed courtship activity and acoustic behaviour

267 To characterize and validate sound production activity associated with reproductive behaviours, only dives with concomitant acoustic recordings were analysed. Dives were categorised as either C ("Courtship") or NC ("No-Courtship") based on the presence or absence of courtship behaviours, respectively. Only audio files recorded within a three-hour interval overlapping each dive were considered. The audio recordings were labelled as C or NC corresponding to their respective dives and processed using the MATLAB interface previously mentioned. The number of sounds by sound type was assessed for each three-hour interval and differences in the weighted number of sounds (i.e., number of sounds per effective hour of recording) were tested between C and NC recordings. Considering the non-normal distribution of the data, Kruskal-Wallis tests were used to investigate if specific sound types were associated with courtship behaviour in R version 4.2.1 (R Core Team, 2022).

279

280

281

# 3. RESULTS

282

## 3.1. Acoustic and visual behaviours

283 The audio recordings collected at the two study sites during July 2017 yielded a total of 4353 dusky grouper sounds (Table S1). The courtship call (LPS) was the most abundant sound type at both study sites.

286 Regarding observations, a total of 156 behaviours were documented across 117 dives (n=35 in 2017, n=82 in 2018) (Table S2), categorised as follows: after sunrise (n=32 dives), during daytime (n=23 dives), and before sunset (n=62 dives). In 53% of these dives, at least one reproductive behaviour was observed (n=17 in 2017, n=45 in 2018). Courtship was the most commonly observed behavioural category at both study sites (Table S2).

292

293

## 3.2. Temporal patterns of sound production

15

16
294 Temporal patterns in sound production were comparable between the two study sites
295 although a clear site-specific trend was observed (Figs 3-4).

296

297 While all sound types were emitted throughout the investigated period, production
298 rates of each sound type varied over time and between study sites (Fig. 3). Peaks in
299 sound production were not synchronized between study sites.

300 Dusky grouper were acoustically active from evening to morning hours, with different
301 sound types detected at different times of the day (Fig. 4). LPS (courtship call) was
302 significantly more prevalent during the evening (16-20 UTC) compared to other time
303 periods and sound types (Fig. S5). LP showed a similar temporal pattern to LPS sounds
304 (Figs 3-4). In contrast, LDS, LDSS, and LPLDS were more abundant later at night and in
305 the early morning at both study sites.

306

307 **3.3. Temporal patterns of behavioural observations**

308 Among the observed behaviours, courtship was the most frequently documented,
309 particularly before sunset compared to other times of the day (Fig. S9). Statistically
310 significant differences were found between courtship before sunset and all other
311 behavioural categories and times, except for courtship after sunrise and chase before
312 sunset (Table S4).

313

314 **3.4. Relationship between observed courtship activity and acoustic**
315 **behaviour**

316 Courtship was observed in 19 out of the 37 dives with simultaneous acoustic
317 recordings. Analyses showed that LPS sounds were significantly more common during
318 dives with courtship (C) compared to dives without courtship (NC) (N=37, $\chi^2$ = 6.6, p =
319 0.01), confirming their classification as courtship calls (Fig. 5). The other sound types,
320 except for LP, did not significantly differ between C and NC dives (all p > 0.05),
321 suggesting that they are not directly related to courtship but possibly associated with
322 other behaviours, such as resource defence (Fig. 5).

323

324 **4. DISCUSSION**
In this study, we showed that the dusky grouper courtship call (LPS) was the most frequently recorded sound type at both sites and was associated with visual courtship behaviour in the wild.

Dusky grouper courtship calls (LPS) were more frequently recorded at dusk (16-20 hours UTC) at both study sites. However, peaks in acoustic activity were not synchronized between sites, possibly due to the asynchronous reproductive cycle of the species at the population level (i.e., different stages of gonadal development across individuals) (Reñones et al., 2010). This spawning asynchronicity was also documented by direct observations in the Medes Islands, where different monitored males did not spawn on the same days (Hereu et al., 2006).

In our study, dusky grouper courtship behaviours were predominantly observed before sunset (15-19 hours UTC), coinciding with the peak detection of courtship calls. This result provides strong indications on the timing of reproductive behaviours. Additionally, both LPS and LP sound types were significantly associated with the observation of courtship behaviours, suggesting that the study sites are dusky grouper spawning sites, although no direct observation of spawning was made. Further investigations are needed to verify if peaks in LPS production indicate actual spawning events. If so, passive acoustic recordings could be used to monitor the spatio-temporal occurrence of dusky grouper spawning events, as demonstrated in other grouper species (Rowell et al., 2019).

Unlike LPS calls, the presumptive dusky grouper sound types (LDS, LDSS and LPLDS) were mostly recorded later at night, corroborating previous findings on the "growl-like sounds" (Bertucci et al., 2015). This nocturnal acoustic activity challenges the prevailing notion that the dusky grouper is primarily a diurnal species (Azzurro et al., 2013; Koeck et al., 2014; Pastor et al., 2009). Grouper species have a diverse vocal repertoire as they can emit pulse and tonal sounds individually, in series or in combination (Mann et al., 2010; Rowell et al., 2019; Schärer et al., 2012a, 2012b). However, most studies have mainly focused on identifying courtship-associated sounds and their temporal patterns, rather than the entire repertoire of grouper sounds (but see Santiago et al., 2020). Here we found a clear temporal partitioning in dusky grouper acoustic activity, with distinct sound types produced at different times of the day, suggesting the occurrence of different behaviours at different times. Validating this temporal partitioning in the wild requires identifying the species' visual nocturnal behaviours and associated sound types, which can be challenging due to their timing. Possible methods include nocturnal diving, although it can impair fish behaviour ("light shock effect", (Reebs, 2002)), or using fixed cameras with infrared illumination or acoustic cameras (Mallet and Pelletier, 2014; Martignac et al., 2015) for a less invasive behavioural monitoring.

Despite differences in sampling effort, potentially influencing the number and temporal distribution of detected sounds, our findings suggest that the recording cycles used in this study effectively capture both short-term (diel 14-h periods) and long-term trends (about one month) in dusky grouper acoustic activity. The most comprehensive acoustic activity pattern was recorded at MOL, despite recent evidence showing a lower dusky grouper abundance at MOL compared to SP, as determined through underwater visual census and photo-identification during the same investigated period (Desiderà et al., 2021). This suggests that the findings at MOL are likely attributed to continuous recordings and minimal human-made noise. In fact, anthropogenic noise from SCUBA diving and boat traffic is higher at SP than at MOL, potentially affecting sound detection and dusky grouper behaviour at that site. Tourism in the study area peaks in July-August (Hogg et al., 2017), exposing the species to increased motorboat noise during the spawning season and potentially impacting its behaviours and reproductive success at SP (Amorim et al., 2022; Popper and Hawkins, 2019). In fact, our findings highlight the importance of acoustic
378 communication for the dusky grouper and potential vulnerability to anthropogenic
379 noise, which should be further investigated.

380 Knowledge on the timing of reproductive activity can aid environmental managers in
381 mitigating anthropogenic disturbances at spawning sites. Implementing noise
382 reduction measures, such as reducing vessel speed and restricting SCUBA diving
383 during critical hours in summer, may enhance the species reproductive success at
384 critical habitats (Nedelec et al., 2022). Such conservation measures would not only
385 benefit the dusky grouper but also other species aggregating in the same areas
386 (Erisman et al., 2017; Sadovy de Mitcheson, 2016). Indeed, multiple grouper species
387 are known to spawn either simultaneously or at different times at the same sites, as
388 observed in the Mediterranean Sea (Aronov and Goren, 2008) and specifically within
389 the TPCC MPA (Desiderà et al., 2022).

390 In conclusion, our study provides valuable insights into the reproductive biology of the
391 dusky grouper and highlights the effectiveness of passive acoustic monitoring (PAM) in
392 studying its reproduction globally, including the Mediterranean Sea. By reliably
393 identifying reproductive activity through dusky grouper courtship calls (LPS), we can
394 determine the timing and location of spawning, which is crucial for conserving critical
395 habitats often shared by multiple ecologically valuable species. Protecting such
396 habitats is paramount, as it ensures grouper long-term survival, preserves
397 biodiversity, and maintains the ecological functioning of the marine ecosystem.
398 Therefore, prioritizing the protection of these high-quality reproductive habitats
399 becomes more important than focusing solely on expanding the protected ocean
400 surface area.

401

402 **Funding**

403 This work was conducted as part of the PhD project of E. Desiderà and the Citizen
404 Science Project "TavolaraLab", both funded by the Tavolara-Punta Coda Cavallo
405 Marine Protected Area, Ministero dell'Ambiente e della Sicurezza Energetica (MASE,
406 previously known as Ministero dell'Ambiente e della Tutela del Territorio e del Mare,
407 MATTM). Additional support was provided by Ministero dell'Istruzione, dell'Università e
408 della Ricerca (MIUR) and Consorzio Nazionale Interuniversitario per le Scienze del Mare
409 (CoNISMa). The funders have no role in the study design, data collection and analysis,
410 decision to publish, or preparation of the manuscript.

411

412 **CRediT authorship contribution statement**

413 ED: Conceptualization, Investigation, Data curation, Formal analysis, Project
414 administration, Validation, Visualization, Writing - original draft and review & editing;
415 CM: Conceptualization, Supervision, Writing - review & editing; AN: Funding
416 acquisition, Project administration, Writing - review & editing; RB: Data curation,
417 Visualization, Writing - review & editing; AC: Formal analysis, Writing - review &
418 editing CG: Resources, Software, Data curation, Writing - review & editing; PP:
419 Investigation, Writing - review & editing; ET: Investigation, Resources, Writing - review
420 & editing; PG: Conceptualization, Supervision, Writing - review & editing; LDI:
421 Conceptualization, Resources, Supervision, Formal analysis, Writing - review & editing.

422

423 **Declaration of competing interest**

424 The authors have no conflict of interest to declare.

425

426 **Acknowledgements**

21

22
427 We wish to thank Luana Magnani for her invaluable help in the field.

428

429 **References**

430 Amorim, M.C.P., Vieira, M., Meireles, G., Novais, S.C., Lemos, M.F.L., Modesto, T., Alves, D., Zuazu, A., Lopes, A.F., Matos, A.B., Fonseca, P.J., 2022. Boat noise impacts *Lusitanian toadfish* breeding males and reproductive outcome. Sci. Total Environ. 830. https://doi.org/10.1016/j.scitotenv.2022.154735

434 Aronov, A., Goren, M., 2008. Ecology of the Mottled Grouper (*Mycteroperca rubra*) in the Eastern Mediterranean. Electron. J. Ichthyol. 2, 43-55.

436 Azzurro, E., Aguzzi, J., Maynou, F., Chiesa, J., Savini, D., 2013. Diel rhythms in shallow Mediterranean rocky-reef fishes: a chronobiological approach with the help of trained volunteers. J. Mar. Biol. Assoc. UK 93, 461-470. https://doi.org/10.1017/S0025315412001166

440 Bertucci, F., Lejeune, P., Payrot, J., Parmentier, E., 2015. Sound production by dusky grouper *Epinephelus marginatus* at spawning aggregation sites. J. Fish Biol. 87, 400-421. https://doi.org/10.1111/jfb.12733

443 Biggs, C.R., Heyman, W.D., Farmer, N.A., Kobara, S., Bolser, D.G., Robinson, J., Lowerre-Barbieri, S.K., Erisman, B.E., 2021. The importance of spawning behavior in understanding the vulnerability of exploited marine fishes in the U.S. Gulf of Mexico. PeerJ 9, 1-33. https://doi.org/10.7717/peerj.11814

447 Brooks, M.E., Kristensen, K., Van Benthem, K.J., Magnusson, A., Berg, C.W., Nielsen, A., Skaug, H.J., Maechler, M., Bolker, B.M., 2017. glmmTMB Balances Speed and Flexibility Among Packages for Zero-Inflated Generalized Linear Mixed Modeling. The R Journal 9 (2): 378-400. https://journal.r-project.org/archive/2017/RJ-2017-066/index.html.

452 Colin, P.L., 1990. Preliminary investigations of reproductive activity of the jewfish, *Epinephelus itajara* (Pisces: Serranidae), in: Proceedings of the 43rd Gulf Caribbean Fisheries Institute. Miami, USA, pp. 138-147.

455 Condini, M.V., García-Charton, J.A., Garcia, A.M., 2017. A review of the biology, ecology, behavior and conservation status of the dusky grouper, *Epinephelus marginatus* (Lowe 1834). Rev. Fish Biol. Fish. 28, 301-330. https://doi.org/10.1007/s11160-017-9502-1

459 Culioli, J.-M., Quignard, J.-P., 1999. Suivi de la démographie et du comportement territorial des mâles de mérous bruns *Epinephelus marginatus* (Lowe, 1834) (Pisces, Serranidae) du site du Pellu (Réserve naturelle des Bouches de Bonifacio, Corse, Méditerranée N.O.) 9, 3-9.

463 Desiderà, E., 2020. Reproductive behaviours of groupers (Epinephelidae) in the Tavolara-Punta Coda Cavallo Marine protected area (NW Mediterranean Sea). Unpublished doctoral dissertation. University of Padova, Université Côte d'Azur.

466 Desiderà, E., Mazzoldi, C., Navone, A., Panzalis, P., Gervaise, C., Guidetti, P., Di Iorio, L., 2022. Reproductive Behaviours and Potentially Associated Sounds of the Mottled Grouper *Mycteroperca rubra*: Implications for Conservation. Diversity 14, 1-20. https://doi.org/https://doi.org/10.3390/d14050318

470 Desiderà, E., Trainito, E., Navone, A., Blandin, R., Magnani, L., Panzalis, P., Mazzoldi, C., Guidetti, P., 2021. Using complementary visual approaches to investigate residency, site fidelity and movement patterns of the dusky grouper (*Epinephelus marginatus*) in a Mediterranean marine protected area. Mar. Biol. 168, 1-16. https://doi.org/10.1007/s00227-021-03917-9
475 Erisman, B., Heyman, W., Kobara, S., Ezer, T., Pittman, S., Aburto-Oropeza, O.,
476 Nemeth, R.S., 2017. Fish spawning aggregations: Where well-placed management
477 actions can yield big benefits for fisheries and conservation. Fish Fish. 18, 128-
478 144. https://doi.org/10.1111/faf.12132

479 Erisman, B.E., Petersen, C.W., Hastings, P.A., Warner, R.R., 2013. Phylogenetic
480 Perspectives on the Evolution of Functional Hermaphroditism in Teleost Fishes.
481 Integr. Comp. Biol. 53, 736-754. https://doi.org/10.1093/icb/ict077

482 FAO. 2020. The State of World Fisheries and Aquaculture 2020. Sustainability in action.
483 Rome. https://doi.org/10.4060/ca9229en

484 Giakoumi, S., Scianna, C., Plass-Johnson, J., Micheli, F., Grorud-Colvert, K., Thiriet, P.,
485 Claudet, J., Di Carlo, G., Di Franco, A., Gaines, S.D., García-Charton, J.A.,
486 Lubchenco, J., Reimer, J., Sala, E., Guidetti, P., 2017. Ecological effects of full and
487 partial protection in the crowded Mediterranean Sea: A regional meta-analysis.
488 Sci. Rep. 7, 1-12. https://doi.org/10.1038/s41598-017-08850-w

489 Hartig, F. 2022. DHARMA: Residual Diagnostics for Hierarchical (Multi-Level / Mixed)
490 Regression Models. https://CRAN.R-project.org/package=DHARMA.

491 Hereu, B., Diaz, D., Pasqual, J., Zabala, M., Sala, E., 2006. Temporal patterns of
492 spawning of the dusky grouper *Epinephelus marginatus* in relation to
493 environmental factors. Mar. Ecol. Prog. Ser. 325, 187-194.
494 https://doi.org/10.3354/meps325187

495 Hogg, K., Noguera-Méndez, P., Semitiel-García, M., 2017. Lessons from three north-
496 western Mediterranean MPAs: A governance analysis of Port-Cros National Park,
497 Tavolara Punta-Coda Cavallo and Ustica. Mar. Policy 1-11.
498 https://doi.org/10.1016/j.marpol.2017.10.034

499 Ibrahim, A.K., Chérubin, L.M., Zhuang, H., Umpierre, M.T.S., Dalgleish, F., Ouyang, B.,
500 Dalgleish, A., Zhuang, H., Sch, M.T., 2018. An approach for automatic
501 classification of grouper vocalizations with passive acoustic monitoring. J. Acoust.
502 Soc. Am. 143, 666-676. https://doi.org/10.1121/1.5022281

503 Jennings, S., Reynolds, J.D., Mills, S.C., 1998. Life history correlates of responses to
504 fisheries exploitation. Proc. R. Soc. B Biol. Sci. 265, 333-339.
505 https://doi.org/10.1098/rspb.1998.0300

506 Koeck, B., Pastor, J., Saragoni, G., Dalias, N., Payrot, J., Lenfant, P., 2014. Diel and
507 seasonal movement pattern of the dusky grouper Epinephelus marginatus inside a
508 marine reserve. Mar. Environ. Res. 94, 38-47.
509 https://doi.org/10.1016/j.marenvres.2013.12.002

510 Koenig, C.C., Bueno, L.S., Coleman, F., Ellis, R.D., Kingon, K., Locascio, J. V., Murie, D.J.,
511 Stallings, C.D., 2016. Diel, lunar, and seasonal spawning patterns of the Atlantic
512 goliath grouper, Epinephelus itajara, off Florida, United States. Bull. Mar. Sci. 92,
513 1-16. https://doi.org/10.5343/bms.2016.1013

514 Lenth, R.V. 2022. emmeans: Estimated Marginal Means, Aka Least-Squares Means.
515 https://CRAN.R-project.org/package=emmeans.

516 Locascio, J. V., Burton, M.L., 2015. A passive acoustic survey of fish sound production
517 at Riley's Hump within Tortugas South Ecological Reserve: implications regarding
518 spawning and habitat use. Fish. Bull. 114, 103-116.
519 https://doi.org/10.7755/FB.114.1.9

520 Louisy, P., Culioli, J.-M., 1999. Synthèse des observations sur l'activité reproductrice du
521 mérou brun *Epinephelus marginatus* (Lowe, 1834) en Méditerranée nord-
522 occidentale. Mar. Life 9, 47-57.

523 Mallet, D., Pelletier, D., 2014. Underwater video techniques for observing coastal
524 marine biodiversity: A review of sixty years of publications (1952-2012). Fish. Res.
525 154, 44-62. https://doi.org/10.1016/j.fishres.2014.01.019

526 Mann, D., Locascio, J., Schärer, M., Nemeth, M., Appeldoorn, R., 2010. Sound
527 production by red hind *Epinephelus guttatus* in spatially segregated spawning
528 aggregations. Aquat. Biol. 10, 149-154. https://doi.org/10.3354/ab00272

529 Marinaro, J.Y., Roussel, E., Lawson, J., Crech'hriou, R., Planes, S., 2005. First record of
530 effective reproduction of the dusky grouper, *Epinephelus marginatus* (Lowe,
531 1834), in the Cerbere-Banyuls (France) marine protected area. Cybium 29, 198-
532 200.

533 Marino, G., Azzurro, E., Massari, A., Finoia, M.G., Mandich, A., 2001. Reproduction in
534 the dusky grouper from the southern Mediterranean. J. Fish Biol. 58, 909-927.
535 https://doi.org/10.1006/jfbi.2000.1502

536 Martignac, F., Daroux, A., Bagliniere, J.L., Ombredane, D., Guillard, J., 2015. The use of
537 acoustic cameras in shallow waters: New hydroacoustic tools for monitoring
538 migratory fish population. A review of DIDSON technology. Fish. Fish. 16, 486-510.
539 https://doi.org/10.1111/faf.12071

540 Nedelec, S.L., Radford, A.N., Gatenby, P., Davidson, I.K., Velasquez Jimenez, L., Travis,
541 M., Chapman, K.E., McCloskey, K.P., Lamont, T.A.C., Illing, B., McCormick, M.I.,
542 Simpson, S.D., 2022. Limiting motorboat noise on coral reefs boosts fish
543 reproductive success. Nat. Commun. 13, 1-9. https://doi.org/10.1038/s41467-022-
544 30332-5

545 Parsons, M.J.G., Salgado Kent, C.P., Recalde-Salas, A., McCauley, R.D., 2016. Fish
546 choruses off Port Hedland, Western Australia. Bioacoustics 4622, 1-18.
547 https://doi.org/10.1080/09524622.2016.1227940

548 Pastor, J., Verdoit-Jarraya, M., Astruch, P., Dalias, N., Nelva Pasqual, J.S., Saragoni, G.,
549 Lenfant, P., 2009. Acoustic telemetry survey of the dusky grouper (*Epinephelus
550 marginatus*) in the Marine Reserve of Cerbère-Banyuls: informations on the
551 territoriality of this emblematic species. Comptes Rendus - Biol. 332, 732-740.
552 https://doi.org/10.1016/j.crvi.2009.03.010

553 Pelaprat, C., 1999. Observations on the spawning behaviour of the dusky grouper
554 *Epinephelus marginatus* (Lowe, 1834) in the north of Corsica (France). Mar. Life 9,
555 59-65.

556 Pollard, D.A., Afonso, P., Bertoncini, A.A., Fennessy, S., Francour, P., Barreiros, J., 2018.
557 *Epinephelus marginatus*, Dusky Grouper [WWW Document]. IUCN Red List Threat.
558 Species 2018. URL https://dx.doi.org/10.2305/IUCN.UK.2018-
559 2.RLTS.T7859A100467602.en (accessed 9.23.19).

560 Popper, A.N., Hawkins, A.D., 2019. An overview of fish bioacoustics and the impacts of
561 anthropogenic sounds on fishes. J. Fish Biol. 94, 692-713.
562 https://doi.org/10.1111/jfb.13948

563 Prato, G., Guidetti, P., Bartolini, F., Mangialajo, L., Francour, P., 2013. The importance
564 of high-level predators in marine protected area management: Consequences of
565 their decline and their potential recovery in the Mediterranean context. Adv.
566 Oceanogr. Limnol. 4, 176-193. https://doi.org/10.1080/19475721.2013.841754

567 R Core Team. 2022. R: A Language and Environment for Statistical Computing. Vienna,
568 Austria: R Foundation for Statistical Computing. https://www.R-project.org/.

569 Reebs, S.G., 2002. Plasticity of diel and circadian activity rhythms in fishes. Rev. Fish
570 Biol. Fish. 12, 349-371. https://doi.org/10.1023/A:1025371804611

571 Reñones, O., Grau, A., Mas, X., Riera, F., Saborido-Rey, F., 2010. Reproductive pattern
572 of an exploited dusky grouper *Epinephelus marginatus* (Lowe 1834) (Pisces:
573 Serranidae) population in the western Mediterranean. Sci. Mar. 74, 523-537.

574 https://doi.org/10.3989/scimar.2010.74n3523

575 Reynolds, J.D., Dulvy, N.K., Goodwin, N.B., Hutchings, J.A., 2005. Biology of extinction

576 risk in marine fishes. Proc. R. Soc. B Biol. Sci. 272, 2337-2344.

577 https://doi.org/10.1098/rspb.2005.3281

578 Rowe, S., Hutchings, J.A., 2003. Mating systems and the conservation of commercially

579 exploited marine fish. Trends Ecol. Evol. 18, 567-572.

580 https://doi.org/10.1016/j.tree.2003.09.004

581 Rowell, T.J., Aburto-Oropeza, O., Cota-Nieto, J.J., Steele, M.A., Erisman, B.E., 2019.

582 Reproductive behaviour and concurrent sound production of Gulf grouper

583 *Mycteroperca jordani* (Epinephelidae) at a spawning aggregation site. J. Fish Biol.

584 1-20. https://doi.org/10.1111/jfb.13888

585 Sadovy de Mitcheson, Y., 2016. Mainstreaming Fish Spawning Aggregations into

586 Fishery Management Calls for a Precautionary Approach. Bioscience 66, 295-306.

587 https://doi.org/10.1093/biosci/biw013

588 Sadovy de Mitcheson, Y., Colin, P.L., 2012. Reef fish spawning aggregations: biology,

589 research and management, Fish and F. ed, Fish & Fisheries 35. Springer.

590 Sadovy de Mitcheson, Y., Linardich, C., Barreiros, J.P., Ralph, G.M., Aguilar-Perera, A.,

591 Afonso, P., Erisman, B.E., Pollard, D.A., Fennessy, S.T., Bertoncini, A.A., Nair, R.J.,

592 Rhodes, K.L., Francour, P., Brulé, T., Samoilys, M.A., Ferreira, B.P., Craig, M.T.,

593 2020. Valuable but vulnerable: Over-fishing and under-management continue to

594 threaten groupers so what now? Mar. Policy 116.

595 https://doi.org/10.1016/j.marpol.2020.103909

596 Sala, E., Ballesteros, E., Starr, R.M., 2001. Rapid Decline of Nassau Grouper Spawning

597 Aggregations in Belize: Fishery Management and Conservation Needs. Fisheries

598 26, 23-30.

599 Santiago, C.M.Z., Appeldoorn, R.S., Schärer-Umpierre, M.T., Cruz-Motta, J.J., 2020. Red

600 hind *Epinephelus guttatus* vocal repertoire characterization, temporal patterns

601 and call detection with micro accelerometers. Gulf Caribb. Res. 31.

602 Schärer, M.T., Nemeth, M.I., Mann, D., Locascio, J., Appeldoorn, R.S., Rowell, T.J.,

603 2012a. Sound production and reproductive behavior of yellowfin grouper,

604 *Mycteroperca venenosa* (Serranidae) at a spawning aggregation. Copeia 1, 135-

605 144. https://doi.org/10.1643/CE-10-151

606 Schärer, M.T., Nemeth, M.I., Rowell, T.J., Appeldoorn, R.S., 2014. Sounds associated

607 with the reproductive behavior of the black grouper (*Mycteroperca bonaci*). Mar.

608 Biol. 161, 141-147. https://doi.org/10.1007/s00227-013-2324-3

609 Schärer, M.T., Rowell, T.J., Nemeth, M.I., Appeldoorn, R.S., 2012b. Sound production

610 associated with reproductive behavior of Nassau grouper *Epinephelus striatus* at

611 spawning aggregations. Endanger. Species Res. 19, 29-38.

612 https://doi.org/10.3354/esr00457

613 Smith, W.L., Craig, M.T., 2007. Casting the Percomorph Net Widely: The Importance of

614 Broad Taxonomic Sampling in the Search for the Placement of Serranid and Percid

615 Fishes. Copeia 35-55. https://doi.org/10.1643/0045-8511(2007)7

616 Wall, C.C., Simard, P., Lindemuth, M., Lembke, C., Naar, D.F., Hu, C., Barnes, B.B.,

617 Muller-Karger, F.E., Mann, D.A., 2014. Temporal and spatial mapping of red

618 grouper *Epinephelus morio* sound production. J. Fish Biol. 85, 1470-1488.

619 https://doi.org/10.1111/jfb.12500

620 Zabala, M., Garcia-Rubies, A., Louisy, P., Sala, E., 1997a. Spawning behaviour of the

621 Mediterranean dusky grouper *Epinephelus marginatus* (Lowe, 1834) (Pisces,
622 Serranidae) in the Medes Islands Marine Reserve (NW Mediterranean, Spain). Sci.

623 Mar. 61, 65-77.

624 Zabala, M., Louisy, P., García-Rubies, A., Gracia, V., 1997b. Socio-behavioural context

625 of reproduction in the Mediterranean dusky grouper *Epinephelus marginatus*

626 (Lowe, 1834) (Pisces, Serranidae) in the Medes Islands Marine Reserve (NW

627 Mediterranean, Spain). Sci. Mar. 61, 79-89.

628 Zuur, A.F., Ieno, E.N., Elphick, C.S., 2010. A protocol for data exploration to avoid

629 common statistical problems. Methods Ecol. Evol. 1, 3-14.

630 https://doi.org/10.1111/j.2041-210x.2009.00001.x

631 Zuur, A.F., Ieno, E.N., Walker, N.J., Saveliev, A.A., Smith, G.M., 2009. Mixed Effects

632 Models and Extensions in Ecology with R. Springer, New York, NY.

633 https://doi.org/10.1007/978-0-387-87458-6
<table><caption>634 Table 1 Descriptions of dusky grouper observed behaviours.</caption><thead><tr><td>Behaviour</td><td>Category</td><td>Description</td></tr></thead><tbody><tr><td>Patrol</td><td>Territorial</td><td>Males swim rapidly within 1-2 m off the bottom throughout their territory, typically below the thermocline. Patrols are particularly intense during evening hours.</td></tr><tr><td>Chase</td><td>Territorial</td><td>When encountering another male within its territory, the male rapidly chases the intruder, even covering relatively long distances (several meters), before returning to patrolling. Chases have been observed between suspected females and dusky groupers targeting individuals of other grouper species, such as Epinephelus aeneus and Mycteroperca rubra.</td></tr><tr><td>Bumping</td><td>Territorial</td><td>Male-male as well as female-female aggressive interactions involve the raising of the dorsal fin and striking each other's sides by tilting the body, typically near the bottom. Females have been observed displaying an "aggressive zebra colour pattern" as mentioned by Zabala et al. (1997b). Subsequent to these interactions, one individual moves away (Video S1).</td></tr><tr><td>Stimulation</td><td>Reproductive</td><td>A single individual, presumed to be male, was observed nudging the abdominal area of a presumed female, possibly to sense her readiness to spawn through pheromone release occurring at the vent (personal communication, Y. Sadovy). This behaviour was displayed by a presumptive male exhibiting a light colour pattern and a straight dorsal fin, while the presumed female showed a bulging belly and a uniformly dark livery. This behaviour suggests that spawning is imminent, likely within 24 hours. A video capturing this behaviour is available (Video S2)</td></tr><tr><td>Courtship (Approach and Display)</td><td>Reproductive</td><td>A single male approaches a single female from behind, tilts to a horizontal position, and shakes its posterior part over the female (referred to as "ritualized caudal flapping" by Zabala et al., 1997a). Concurrently with the caudal flapping, males emit low-frequency pulse-series (Bertucci et al., 2015). These sounds were once heard in situ by one of the authors (ED) (Video S3).</td></tr></tbody></table>

635

636
<figure><img src="image_2.png" /><figcaption>**Fig. 1 Map of the Tavolara-Punta Coda Cavallo Marine Protected Area (TPCC MPA) showing study sites and zonation.** A zones are fully protected (no-take, no-access). B and C zones are partially protected, with specific restrictions on human activities. In B zones, licensed local artisanal fishing and diving are allowed, while in C zones, recreational fishing is also allowed. Spearfishing and grouper fishing are prohibited throughout the MPA.</figcaption></figure>

637

638

639

640

641

642
<figure><img src="image_3.png" /><figcaption>b)</figcaption></figure>

<figure><img src="image_4.png" /></figure>

**Fig. 2** Spectrograms and oscillograms of dusky grouper sound types. a) *LP*: Low-frequency Pulse; *LPS*: Low-frequency Pulse Series. b) *LDS*: Low-frequency Downsweeping Sound. LP and LPS are confirmed dusky grouper sounds recorded in captivity and in the wild, with LPS sounds linked to courtship activity in captivity; LDS sounds were only recorded in the wild and attributed to the species (Bertucci et al., 2015).

643

644

645

646

647

648
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Normalized (0-1) values of dusky grouper sounds detected by sound type and study site per hour interval from July 5th to 31st, 2017. On July 7th, the entire day's audio recordings were analysed at MOL. Grey areas indicate gaps in acoustic data (unrecorded or unanalysed data).</figcaption></figure>

649

650

651

652

653

39

40
<figure><img src="image_6.png" /></figure>

Fig. 4 Cumulative diel variation of normalized sound abundances (0-1) of the five sound types recorded at the two sites over the month of July from 16:00 to 6:00 hours UTC. MOL: Molarotto, SP: Secche del Papa.

<figure><img src="image_7.png" /></figure>

Fig. 5 Mean (± SE) number of sounds per sound type during dives with dusky grouper courtship behaviour (C) and without (NC) (N=37, C=19 and NC=18 dives), across both study sites. * p < 0.05, ** p < 0.01.