Introduction
The inner ear of fish is an essential auditory sensor, containing three semicircular canals and paired otoliths. These otoliths are calcareous structures composed primarily of calcium carbonate, trace or vestigial elements, and trace amounts of organic matter (Borelli et al., Reference Borelli, Mayer-Gostan, Merle, De Pontual, Boeuf, Allemand and Payan2003). These dense otolith structures are the asterisci (otoliths of the lagena), lapilli (otoliths of the utriculus), and sagittae (otoliths of the sacculus) and are closely associated with the auditory sensory epithelium, function in the transmission of sound, and are also involved in maintaining a static and active balance (Parmentier et al., Reference Parmentier, Vandewalle and Lagardère2001). Of these otoliths, the sagittal otoliths or sagittae are the largest of them, found just behind the eyes and approximately level with them vertically, while the lapilli and asterisci are the smallest and are located within the utriculus and lagena chambers, respectively, which are continuous with three semi-circular canals (Payan et al., Reference Payan, Kossmann, Watrin, Mayer-Gostan and Boeuf1997; Schwarzhans et al., Reference Schwarzhans, Schulz-Mirbach, Lombarte and Tuset2017). Interestingly, the acoustic properties (density and elasticity) of fish tissue are very similar to the surrounding water, and when the fish are exposed to sound, the otoliths function as an accelerometer (Zhang et al., Reference Zhang, Tao, Zhou, Tang, Liu and Xu2021). Indeed, otoliths are denser than water, and their motion changes relative to water, creating a relative movement with auditory hair cells (Schulz-Mirbach et al., Reference Schulz-Mirbach, Ladich, Plath and Heß2019). When hair cells deviate with relative motion, neurotransmitters are released in the sensory epithelium to produce auditory responses (Popper and Lu, Reference Popper and Lu2000).
Sagittal otoliths are known to increase in size with increasing habitat depths down to about 1000 m (Tuset et al., Reference Tuset, Otero-Ferrer, Gómez-Zurita, Venerus, Stransky, Imondi, Orlov, Ye, Santschi, Afanasiev, Zhuang, Farré, Love and Lombarte2016) and decrease in size in fish fast-swimming (Volpedo et al., Reference Volpedo, Tombari and Echeverría2008), and are associated with feeding habitats (Lombarte et al., Reference Lombarte, Palmer, Matallanas, Gómez-Zurita and Morales-Nin2010; Tuset et al., Reference Tuset, Otero-Ferrer, Gómez-Zurita, Venerus, Stransky, Imondi, Orlov, Ye, Santschi, Afanasiev, Zhuang, Farré, Love and Lombarte2016). As a result, their morphology has long been used as a characterizing trait of species identification, i.e. species-specific features (Callicó Fortunato et al., Reference Callicó Fortunato, Benedito Durà and Volpedo2014; Avigliano et al., Reference Avigliano, Jawad and Volpedo2016; Ferri et al., Reference Ferri, Bartulin and Škeljo2018; Mejri et al., Reference Mejri, Bakkari, Allagui, Rebaya, Jmil, Mili, Shahin, Quignard, Trabelsi and Ben Faleh2022b). In addition, these otoliths have been documented to show variations in morphologies (shape and morphometry) at both the inter and intraspecific levels (Ferri et al., Reference Ferri, Bartulin and Škeljo2018; Tuset et al., Reference Tuset, Olivar, Otero-Ferrer, López-Pérez, Hulley and Lombarte2018; Ben Labidi et al., Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020b; Mejri et al., Reference Mejri, Trojette, Jmil, Ben Faleh, Chalh, Quignard and Trabelsi2020, Reference Mejri, Bakkari, Allagui, Rebaya, Jmil, Mili, Shahin, Quignard, Trabelsi and Ben Faleh2022b; Khedher et al., Reference Khedher, Mejri, Shahin, Quiganrd, Trabelsi and Ben Faleh2021; D'Iglio et al., Reference D'Iglio, Famulari, Albano, Carnevale, Di Fresco, Costanzo, Lanteri, Spanò, Savoca and Capillo2023) and their morphometry, in particular, has a close relationship to fish body size (Martucci et al., Reference Martucci, Pietrelli and Consiglio1993). In addition, it has been shown that these variations are influenced by several factors, including genetic (Vignon and Morat, Reference Vignon and Morat2010; Berg et al., Reference Berg, Almeland, Skadal, Slotte, Andersson and Folkvord2018), ontogenetic (Hüssy, Reference Hüssy2008; Capoccioni et al., Reference Capoccioni, Costa, Aguzzi, Menesatti, Lombarte and Ciccotti2011), physiological (Lombarte and Cruz, Reference Lombarte and Cruz2007; Schulz-Mirbach et al., Reference Schulz-Mirbach, Ladich, Plath and Heß2019), phylogeny (Campana and Neilson, Reference Campana and Neilson1985; Torres et al., Reference Torres, Lombarte and Morales-Nin2000), and exogenous factors, such as living depth (Wilson, Reference Wilson1985; Lombarte and Lleonart, Reference Lombarte and Lleonart1993), temperature (Lombarte and Lleonart, Reference Lombarte and Lleonart1993) and salinity of the water (Capoccioni et al., Reference Capoccioni, Costa, Aguzzi, Menesatti, Lombarte and Ciccotti2011), and food supplies (Gagliano and McCormick, Reference Gagliano and McCormick2004; Hüssy, Reference Hüssy2008; Bremm and Schulz, Reference Bremm and Schulz2014). Moreover, variations in otolith microchemistry have been used to estimate spatiotemporal migration and feeding behaviour between species (Lord et al., Reference Lord, Tabouret, Claverie, Ṕecheyran and Keith2011). Therefore, information on the shape and morphometry of fish sagittal otolith has long been considered an appropriate method for studying population structure and stock assessment (Pothin et al., Reference Pothin, González-Salas, Chabanet and Lecomte-Finiger2006; Gonzalez-Salas and Lenfant, Reference Gonzalez-Salas and Lenfant2007; Duarte-Neto et al., Reference Duarte-Neto, Lessa, Stosic and Morize2008; Rebaya et al., Reference Rebaya, Ben Faleh, Allaya, Khedher, Marsaoui, Chalh, Quignard and Trabelsi2016; Bose et al., Reference Bose, Adragna and Balshine2017; Mejri et al., Reference Mejri, Trojette, Allaya, Ben Faleh, Jmil, Chalh, Quignard and Trabelsi2018, Reference Mejri, Bakkari, Tazarki, Mili, Chalh, Shahin, Quignard, Trabelsi and Ben Faleh2022a; Mahé et al., Reference Mahé, Ider, Massaro, Hamed, Alba, Patricia, Aiketerini, Angelique, Chryssi, Romain, Zohir, Mahmoud, Rachid, Hélène and Bruno2019; Ben Labidi et al., Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020a; Khedher et al., Reference Khedher, Mejri, Shahin, Quiganrd, Trabelsi and Ben Faleh2021), species identification (Škeljo and Ferri, Reference Škeljo and Ferri2011; Bani et al., Reference Bani, Poursaeid and Tuset2013; Jawad et al., Reference Jawad, Hoedemakers, Ibáñez, Ahmed, Abu El-Regal and Mehanna2018), assessment of age and growth (Cardinale et al., Reference Cardinale, Doering-Arjes, Kastowsky and Mosegaared2004; Škeljo et al., Reference Škeljo, Brčić, Vuletin and Ferri2015), diet content (Lilliendahl and Solmundsson, Reference Lilliendahl and Solmundsson2006), ontogeny (Capoccioni et al., Reference Capoccioni, Costa, Aguzzi, Menesatti, Lombarte and Ciccotti2011), spatiotemporal migration (Lord et al., Reference Lord, Tabouret, Claverie, Ṕecheyran and Keith2011), and fisheries science and management (Vasconcelos et al., Reference Vasconcelos, Vieira, Sequeira, González, Kaufmann and Serrano Gordo2018; Rani et al., Reference Rani, Rai and Tyor2019). As the morphological variation of sagittal otoliths is influenced by genetic factors (Vignon and Morat, Reference Vignon and Morat2010), external factors, including depth (Lombarte and Lleonart, Reference Lombarte and Lleonart1993) and water temperature (Lombarte and Lleonart, Reference Lombarte and Lleonart1993; Hüssy, Reference Hüssy2008), salinity (Capoccioni et al., Reference Capoccioni, Costa, Aguzzi, Menesatti, Lombarte and Ciccotti2011), and food supply (Gagliano and McCormick, Reference Gagliano and McCormick2004; Hüssy, Reference Hüssy2008) also play a strong role in reshaping of otoliths (Vignon, Reference Vignon2012; Bremm and Schulz, Reference Bremm and Schulz2014).
Indeed, the study of inter and intraspecific variability in the sagittal otoliths’ shape has been performed using several techniques (for details, see Khedher et al., Reference Khedher, Mejri, Shahin, Quiganrd, Trabelsi and Ben Faleh2021), including elliptical Fourier analysis (EFA), which has been proven to be the most widely used and effective method to describe, characterize, and capture outlines information in a measurable manner (Lord et al., Reference Lord, Morat, Lecomte-Finiger and Leith2012).
As far as is known, mullet species of the Mugilidae family are widely distributed coastal marine species and are widespread in warm waters, including temperate, subtropical, and tropical regions (Kasımoğlu and Yılmaz, Reference Kasımoğlu and Yılmaz2012; Fortunato et al., Reference Fortunato, Benedito Durà, González-Castro and Volpedo2017), where they live on algae and detritus (Keith et al., Reference Keith, Le Bail and Planquette2000). In addition, these species have been assigned as highly euryhaline and eurythermal species and withstand wide-ranging salinities (González-Castro and Ghasemsadeh, Reference González-Castro, Ghasemsadeh, Crosetti and Blaber2015; González-Castro and Minos, Reference González-Castro, Minos, Crosetti and Blaber2015). Therefore, they inhabit various habitats, including shallow brackish and marine waters adjacent to lagoons, and consume part of their life cycle in coastal lagoons, lakes, and/or rivers, where they use these habitats for feeding and growth, refuge, spawning, and development (Papasotiropoulos et al., Reference Papasotiropoulos, Klossa-Kilia, Kilias and Alahiotis2002; González-Castro and Ghasemsadeh, Reference González-Castro, Ghasemsadeh, Crosetti and Blaber2015; González-Castro and Minos, Reference González-Castro, Minos, Crosetti and Blaber2015). Mugilids migrate to the Sea to breed after resting and maturing in various variable habitats (González-Castro et al., Reference González-Castro, Abachian and Perrotta2009, Reference González-Castro, Macchi and Cousseau2011; Whitfield, Reference Whitfield, Crosetti and Blaber2015). Some adults return to brackish water after breeding, whereas others remain in marine waters (Whitfield et al., Reference Whitfield, Panfili and Durand2012).
In Tunisian waters, Blel et al. (Reference Blel, Said and Durand2013) identified six species assigned to three genera of the family Mugilidae, including Mugil cephalus (Linnaeus, 1758), Chelon aurata (Risso, 1810), C. saliens (Risso, 1810), C. ramada (Risso, 1826), C. labrosus (Risso, 1827), and Oedalechilus labeo (Cuvier, 1829). Of these six species, the thin-lipped grey C. ramada is the most common of these species in Tunisian waters (Rebaya et al., Reference Rebaya, Ben Faleh, Allaya, Khedher, Marsaoui, Chalh, Quignard and Trabelsi2016). In addition, it has economic importance because it is one of the target species for commercial fishing along the Tunisian coast (Masmoudi et al., Reference Masmoudi, Romdhane, Kheriji and EL Cafsi2001) and its annual catch has increased significantly in recent years (Fehri-Bedoui et al., Reference Fehri-Bedoui, Gharbi and El Abed2002). According to the last data available by DGPA (2015), the overall annual landings of the Mugilidae species in Tunisia reached 3000 tons, accounting for 9.5% of total coastal landings and 2.26% of total Tunisian fisheries landings, with C. auratus alone accounting for around 1350 tonnes, which represents 45% of the total Mugilidae species landings (Romdhane et al., Reference Romdhane MS2019).
Geographically, the Tunisia coastline extends over 1300 km, with about 575 km of sandy beaches (Bounouh, Reference Bounouh2010), and various lagoons, including the Boughrara and El Bibane. Similar to Mediterranean lagoons, Tunisian lagoons experience significant seasonal and even daily variations in physicochemical parameters, such as temperature and salinity extremes (Afli et al., Reference Afli, Boufahja, Sadraoui, Ben Mustpha, Aissa and Mrabet2009). The most frequent group of species in the Boughrara lagoon is represented by the migratory species (sea to the lagoon and vice versa) of the families Sparidae, Soleidae, Moronidae, and Mugilidae. However, the most important species in the lagoon of El Bibane, regularly landing, are essentially those of the family of Moronidae, Sparidae, and Mugilidae. Among these species, the thin-lipped grey mullet C. ramada is one of the target fish species of the commercial fishery along the Tunisian coast (Masmoudi et al., Reference Masmoudi, Romdhane, Kheriji and EL Cafsi2001).
Because the variability in the sagittal otolith shape has been poorly studied in many species, including C. ramada, the present study was conducted for the first time to (1) compare the sagittal otolith shape and morphometry, particularly length (Lo), width (Wo), area (Ao), and perimeter (Po), between populations of this species inhabiting the Boughrara and El Bibane lagoons located in southeastern Tunisia and (2) assess the effect of potential fluctuating asymmetry (FA) in the sagittal otolith morphology (shape and morphometry) on the stock structure of C. ramada in the two lagoons to inform on appropriate management procedures.
Materials and methods
Study area
As shown in Figure 1, the Boughrara lagoon (32°28' 33°45''N, 10°45' 10°57''E) is located south of the Djerba island on the southern edge of the Gulf of Gabes and it ranks first among the Tunisian lagoons because it has an area of 50,000 ha (Guetat et al., Reference Guetat, Sellem, Akrout, Brahim, Atoui, Ben Romdhane and Daly Yahia2012). It communicates with the seawater of the Gulf of Gabes through the El Kantara in the northeastern part and the Ajim channels in the northwestern part. The climate of the Boughrara lagoon is dry and sunny, with strong easterly winds that carry particles into the sea (Brahim et al., Reference Brahim, Atoui, Sammari and Aleya2014, Reference Brahim, Atoui, Sammari and Aleya2015) and cause strong wind erosion. The surface water temperature varies strongly with the season, averaging 24.7 °C in summer and 11.2 °C in winter, increasing along a north-south gradient (Feki et al., Reference Feki, Hamza, Frossard, Abdennadher, Hannachi, Jacquot, Bel Hassen and Aleya2013). The salinity of the lagoon is higher than that of the Sea (Abdenadher et al., Reference Abdenadher, Hamza, Feki, Hannachi, Zouari-Belaa, Brada and Aleya2012) and mostly varies between 38 and 43%o (Sellem et al., Reference Sellem, Guetat, Enaceur, Ghorbel-Ouannes, Othman, Harki, Lakuireb and Rafrafi2019). However, it varies between 42.19 and 53.3%o during the summer season (Khedhri et al., Reference Khedhri, Djabou and Afli2015). The pH of the water fluctuates between an average of 7.92 in winter and 8.31 in summer (Ben Aoun et al., Reference Ben Aoun, Farhat, Chouba and Hadjali2007). In addition, the lagoon has in its southern part a distinctive harbour, making it suffers from organic discharges through harbour-related activities (Guetat et al., Reference Guetat, Sellem, Akrout, Brahim, Atoui, Ben Romdhane and Daly Yahia2012), as well as the pollution with sewage from the transportation of the surrounding ports and the entry of seawater loaded with phosphorous from the Gulf of Gabes (Sellem et al., Reference Sellem, Guetat, Enaceur, Ghorbel-Ouannes, Othman, Harki, Lakuireb and Rafrafi2019).
Figure 1. Chelon ramada Risso, 1826, the Boughrara and El Bibane lagoons sites (
) from which the individuals were collected in Tunisia.
The El Bibane lagoon is situated on the southern coast of Tunisia (33°15''N, 11°15''E). It has an area of 23,000 ha, a surface area of about 230 km2, and is separated from the sea by a limestone cordon of about 2.5 km long and is divided into nine small islands (Lemoalle, Reference Lemoalle1986) and it communicates with the sea through a footbridge of about 400 m in length. The surface water temperature is homogeneous but it increases in June in shallow areas and its average is 28 °C in summer and 14 °C in winter (Ben Abdeladhim, Reference Ben Abdeladhim2003). The salinity is relatively high and has an average of 44%o (Ben Abdeladhim, Reference Ben Abdeladhim2003). Along the central radial, it varies between 39.1 and 46.5%o, with a minimum at the end of winter and a maximum at the end of summer after intense evaporation of water (Zaouali, Reference Zaouali1985). The average dissolved oxygen saturation is 109% and can reach 133%, and the pH varies between 8.2 and 8.3 (Ben Abdeladhim, Reference Ben Abdeladhim2003; Akrout, Reference Akrout2012). These characteristics gave it an economic interest in Tunisian waters since fishing and aquaculture have been practiced there for a long time (Zaouali, Reference Zaouali1983).
Sampling
A total of 120 adult individuals of C. ramada were collected from the two lagoons (60 individuals each) between June and July 2018. All individuals were captured live by gillnets using artisanal coastal boats. Immediately after catching, individuals were examined visually for gonadal maturity, using the scale of Kesteven (Reference Kesteven1960) and Treasurer (Reference Treasurer1990), or microscopically in the case of small gonads, to confirm the maturity stage of all individuals. Afterwards, individuals were measured for total length (TL) using an ichthyometer, and the values were rounded to the nearest 0.1 mm (Table 1).
Table 1. The number of males and females (N) and mean of total length (TL) ± standard deviation (SD) of Chelon ramada examined from the Boughrara (B) and El Bibane (E) lagoons in Tunisia
Sagittae sampling and processing
Otolith extraction
The right and left sagittal otoliths (sagittae) were extracted according to the method of Panfili et al. (Reference Panfili, de Pontual, Troadec and Wright2002). After extraction, they were cleaned with distilled water and then air-dried at room temperature or in a low-temperature oven.
Otolith shape analysis
Otoliths were positioned onto a microscope slide with the sulcus directed down and the rostrum placed in the same direction to minimize distortion errors in the normalization process. Subsequently, each pair of otoliths was photographed under a binocular loupe using a Canon Ixus 185 high-performance digital camera with a resolution of 20 megapixels to obtain images that allow us to trace the outlines of the shape with full accuracy (Figure 2). The obtained images were then processed by Photoshop CS6 software, which transformed the original images of otoliths into binary images. Afterwards, images of the shapes were analysed using SHAPE 1.3 software (Iwata and Ukai, Reference Iwata and Ukai2002). The contour shape of each otolith was evaluated by EFA as previously described in detail by Ben Labidi et al. (Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020a, Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020b) and Khedher et al. (Reference Khedher, Mejri, Shahin, Quiganrd, Trabelsi and Ben Faleh2021) following the procedures suggested by Kuhl and Giardina (Reference Kuhl and Giardina1982).
Figure 2. Chelon ramada Risso, 1826, images of the left (L) and right (R) sagittal otoliths showing the length (LO) and width (WO) parameters examined among individuals collected from the (A) Boughrara and (B) El Bibane lagoons in Tunisia. Scale bar: 1 mm.
Morphometric parameters
Morphometric parameters of the otoliths, including length (Lo), width (Wo), area (Ao), and perimeter (Po), were determined using ImageJ software (Figure 2A, B). Before statistical analyses, one-way ANOVA was used to determine whether there were any significant differences between the mean values of Lo, Wo, Ao, and Po for the right and left otoliths. In addition, a two-way ANOVA was used to check whether there was a correlation between the otolith's morphometry and the geographic origin of the individuals. The mean values of the four parameters were analysed using the Student's t-test to determine the differences between the left and right and left-left and right-right otoliths within and among individuals of the two lagoons.
Fluctuating asymmetry measurements
The FA values between the right (r) and left (l) otoliths were calculated among individuals of the two lagoons for each morphometric parameter per individual (i) by applying the signed formula FA4 given by Palmer (Reference Palmer and Markow1994) and were expressed here as the FAi index:
where ri and li are the values of the parameter or trait on the right and left otoliths, respectively.
Then, the mean FA values were calculated for the right and left otoliths from all individuals for each morphometric parameter per lagoon. Besides, the inter-population differences in the mean values of the FA between the left and right otolith morphometric parameters between individuals of the two lagoons were tested using multivariate analysis of variance (MANOVA).
Data analysis
First, analysis of variance (ANOVA) was performed to evaluate the significance of differences in the mean values of TL among individuals of the two lagoons, and the values were tested for homogeneity (equality) and the normal distribution using Levene's and Shapiro–Wilks’ λ tests, respectively. Second, the differences in the contour shape of otoliths of all individuals from the two lagoons were revealed using discriminant function analysis (DFA) (Anderson and Robinson, Reference Anderson and Robinson2003). The effect of individuals on elliptical Fourier descriptors was first tested by MANOVA. Subsequently, all shape variable values were checked for being normal, and if the values did not follow a normal distribution, a Box-Cox (Box and Cox, Reference Box and Cox1964) transformation was performed. Finally, Levene's and Shapiro–Wilk's λ tests were applied to assess homogeneity (equality) and normality of variance in the variable values of otolith shapes. Afterwards, the DFA was performed by the normalized elliptical Fourier descriptors coefficients (77 coefficients per otolith) to illustrate the similarities and differences within and among individuals of the two lagoons. The objective of the DFA is to investigate the integrity of pre-determined groups of individuals of a particular population or lagoon and the percentage of their correct classification by finding linear combinations of descriptors that maximize Wilk's λ value. Wilk's λ test evaluates the performance of discriminant analyses. This statistic is the ratio between intra-population variation and total difference and provides an objective method for calculating the corrected percentage chance of agreement. In addition, the Fisher's (Reference Fisher1936) distance was also calculated to describe the differences in the shape of otolith within and among populations of the two lagoons. The results were interpreted using the data of Wilk's λ test, and barycenter projections were shown on graphs for the two lagoons. Besides, the MANOVA was used to test the significance of otolith shape values between individuals of the two lagoons. All these statistical analyses were performed using XLSTAT 2010.
Results
Otolith shape analysis
The Levene's and Shapiro–Wilks’ λ tests revealed that all values of the shape variance were equally and normally distributed with a P-value > 0.05. At the interpopulation level, the Wilks’ λ test showed a statistically significant shape difference (P = 0.0011), i.e. there was a bilateral asymmetry, between the right and left otoliths among individuals of the two populations (Table 2). Similarly, Fisher's (Reference Fisher1936) distances also showed significant shape bilateral asymmetry (P ⩽ 0.05) between the left and right otoliths, as well as between the left-left (L-L) and right-right (R-R) otoliths among individuals between the two lagoons (Table 3).
Table 2. Wilk's Lambda (λ) test of the left and right sagittal otoliths’ shape variance distance approximation values within and between populations of Chelon ramada collected from the Boughrara (B) and El Bibane (E) lagoons in Tunisia
Values marked in bold are statistically significant (P < 0.05).
Table 3. Pairwise Fisher's (Reference Fisher1936) distances (D) matrix (above diagonal) and their corresponding P-values (below diagonal) of the right (R) and left (L) sagittal otoliths within and between females (F) and males (M) of Chelon ramada samples collected from the Boughrara (B) and El Bibane (E) lagoons in Tunisia
-, not examined in this study; FLB, left otolith of Boughrara female; FRB, right otolith of Boughrara female; MLB, left otolith of Boughrara male; MRB, right otolith of Boughrara male; FLE, right otolith of El Bibane female; FRE, right otolith of El Bibane female; MLE, left otolith of El Bibane male; MRE, right otolith of El Bibane male. Values marked in bold are statistically significant (P < 0.05).
At the intrapopulation level, the Wilks’ λ test revealed a statistically significant shape difference (P < 0.0001), i.e. asymmetry, in the right and left otoliths among males and females of the Boughrara lagoon, i.e. there was a sexual dimorphism (Table 2). Conversely, a significant shape similarity (P > 0.0001), i.e. symmetry, was found in the right and left otoliths among the two sexes of the El Bibane lagoon (Table 2). In addition, the Fisher's (Reference Fisher1936) distances showed no significant differences (P > 0.05) in the left and right otoliths among males and females within each lagoon (Table 3).
Based on elliptical Fourier descriptors (EFDs) of the left and right otoliths contour shape from individuals of the two lagoons on the first two axes F1 and F2 of the DFA, the barycenter projection revealed that the first axis explained 26.91% and the second 18.45%, respectively, of the total variation, showing that otoliths from individuals tended to segregate along the F1 (Figure 3). Therefore, the two axes accounted for 45.36% of the total variance and confirmed the presence of shape variability in the left and right otoliths among individuals of the two populations, i.e. the presence of two distinct main groups of otoliths corresponding to the lagoons of Boughrara and El Bibane. The F1 axis separated the left and right otoliths of males and females of the Bibane lagoon on the positive part and those of the Boughrara lagoon on the negative part. The F2 axis discriminated between the left and right otoliths of males and females of the two populations on both the positive and negative parts (Figure 3).
Figure 3. Chelon ramada Risso, 1826, discriminant function analysis (DFA) showing the barycenter projection (
) of the left (L) and right (R) shape values of the sagittal otoliths of Chelon ramada collected from the Boughrara and El Bibane lagoons in Tunisia: FLB = left otoliths of Boughrara females, FRB, right otoliths of Boughrara females; MLB, left otoliths of Boughrara males; MRB, right otoliths of Boughrara males; FLE, left otoliths of El Bibane females; FRE, right otoliths of El Bibane females; MLE, left otoliths of El Bibane males; MRE, right otoliths of El Bibane males.
Morphometric analysis
The Student's t-test confirmed that there were no significant differences (P > 0.05), i.e. there was symmetry, between the left and right otoliths in Lo, Ao, and Po among males and females in the Boughrara lagoon, i.e. there was no sexual dimorphism (Table 4). However, a highly significant asymmetry (P = 0.0016) was detected in Wo between the left and right otoliths among males and females, i.e. there was a sexual dimorphism. In the El Bibane lagoon, Student's t-test revealed no significant differences (P > 0.05), i.e. there was symmetry, between the right and left otoliths in Lo, Wo, Ao, and Po among males and females (Table 4).
Table 4. Means ± standard deviation (SD) values and Student's t-test P-values between the right (R) and left (L) sagittal otoliths’ length (Lo), width (Wo), and area (Ao) measurements within and between Chelon ramada males and females collected from the Boughrara (B) and El Bibane (E) lagoons in Tunisia
P-values marked in bold are significant (P < 0.05).
Fluctuating asymmetry analysis
Estimates of the mean FA values of Lo, Wo, Ao, and Po between the right and left otoliths among individuals of the Boughrara lagoon showed significant FA (P < 0.05) only in Lo between the left and right otoliths among males, as well as between males and females, i.e. there was a sexual dimorphism (Table 5). On the contrary, significant FA differences (P < 0.05) were observed between the left and right otoliths in Wo and Po among males, in all parameters among females, and in Wo and Po between males and females of the El Bibane lagoon, i.e. there was a sexual dimorphism. However, at the interpopulation level, only significant FA differences were found in Lo between the left and right otoliths.
Table 5. Estimates of the mean values of fluctuating asymmetry (FA) between the left (L) and right (R) sagittal otoliths’ length (Lo), width (Wo), area (Ao), and perimeter (Po) of Chelon ramada males and females collected from the Boughrara (B) and El Bibane (E) lagoons in Tunisia
P-values marked in bold are significant (P < 0.05).
Discussion
Results of elliptic Fourier analysis (EFA) of the sagitta otolith shape revealed a statistically significant shape difference, i.e. asymmetry, between the left and right otoliths, as well as between the left-left (L-L) and right-right (R-R) otoliths, among populations of C. ramada collected from the Boughrara and El Bibane lagoons. Similar findings of the interpopulation variation of the left and right otoliths’ shape have been previously reported in a wide range of fish taxa for which data are available and occurring either in Tunisian waters or elsewhere worldwide (for details, see Ben Labidi et al., Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020a, Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020b; Khedher et al., Reference Khedher, Mejri, Shahin, Quiganrd, Trabelsi and Ben Faleh2021; Mejri et al., Reference Mejri, Bakkari, Tazarki, Mili, Chalh, Shahin, Quignard, Trabelsi and Ben Faleh2022a). In addition, significant intersexual shape difference, i.e. bilateral asymmetry, was only observed between the left and right otoliths in the Boughrara lagoon. This is consistent with the findings in Neobythites spp. (Schwarzhans, Reference Schwarzhans1994), Salvelinus namaycush (Simoneau et al., Reference Simoneau, Casselman and Fortin2000), D. anularis (Trojette et al., Reference Trojette, Ben Faleh, Fatnassi, Marsaoui, Mahouachi, Chalh, Quignard and Trabelsi2015), C. ramada (Rebaya et al., Reference Rebaya, Ben Faleh, Allaya, Kheder, Trojette, Marsaoui, Fatnassi, Chalh, Quignard and Trabelsi2017), P. erythrinus (Mejri et al., Reference Mejri, Trojette, Allaya, Ben Faleh, Jmil, Chalh, Quignard and Trabelsi2018, Reference Mejri, Trojette, Jmil, Ben Faleh, Chalh, Quignard and Trabelsi2020), and B. boops (Ben Labidi et al., Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020a).
Regarding the main reasons for such interpopulation bilateral asymmetry in the otolith shape, numerous investigations have reported that the variability in the otolith shape has been influenced by either genetic (Vignon and Morat, Reference Vignon and Morat2010; Berg et al., Reference Berg, Almeland, Skadal, Slotte, Andersson and Folkvord2018), ontogenetic (Hüssy, Reference Hüssy2008; Capoccioni et al., Reference Capoccioni, Costa, Aguzzi, Menesatti, Lombarte and Ciccotti2011), physiological (Lombarte and Cruz, Reference Lombarte and Cruz2007; Schulz-Mirbach et al., Reference Schulz-Mirbach, Ladich, Plath and Heß2019), phylogenetic (Campana and Neilson, Reference Campana and Neilson1985; Torres et al., Reference Torres, Lombarte and Morales-Nin2000), or environmental factors, such as living depth (Lombarte and Lleonart, Reference Lombarte and Lleonart1993), habitat types (Bautista-Vega et al., Reference Bautista-Vega, Letourneur, Harmelin-Vivien and Salen Picard2008), temperature and salinity of water (Panfili et al., Reference Panfili, Durand, Diop, Gourene and Simier2005; Hüssy, Reference Hüssy2008; Capoccioni et al., Reference Capoccioni, Costa, Aguzzi, Menesatti, Lombarte and Ciccotti2011; Mahé et al., Reference Mahé, Ider, Massaro, Hamed, Alba, Patricia, Aiketerini, Angelique, Chryssi, Romain, Zohir, Mahmoud, Rachid, Hélène and Bruno2019), and food supply (Hüssy, Reference Hüssy2008). In addition, Trojette et al. (Reference Trojette, Ben Faleh, Fatnassi, Marsaoui, Mahouachi, Chalh, Quignard and Trabelsi2015) mentioned that fertility, sexual maturity, survival, and growth are among the most important factors that play a role in otolith reshaping. Moreover, Ben Mohamed et al. (Reference Ben Mohamed, Mejri, Ben Faleh, Allaya, Jmil, Rebaya, Chalh, Quignard and Trabelsi2019), Jmil et al. (Reference Jmil, Ben Faleh, Rebaya, Allaya, Ben Mohamed, Trojette, Chalh, Quignard and Trabelsi2019), Ben Labidi et al. (Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020a, Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020b), Mejri et al. (Reference Mejri, Trojette, Jmil, Ben Faleh, Chalh, Quignard and Trabelsi2020, Reference Mejri, Bakkari, Tazarki, Mili, Chalh, Shahin, Quignard, Trabelsi and Ben Faleh2022a), and Khedher et al. (Reference Khedher, Mejri, Shahin, Quiganrd, Trabelsi and Ben Faleh2021) attributed the asymmetry in the otolith shape to the instability of development caused either by environmental stress related to the change in water temperature, salinity, living depth, feeding conditions, and pollutants that have led to abnormalities in the ontogenetic development of individuals or due to poor living conditions of larvae in an unfavourable environment. However, the intrapopulation significant shape bilateral asymmetry observed in the left and right otoliths among males and females within the Boughrara lagoon can be attributed to reproductive isolation between individuals (Wiff et al., Reference Wiff, Flores, Segura, Barrientos and Ojeda2020) that may lead to inter or even intraindividual variations (Panfili et al., Reference Panfili, Durand, Diop, Gourene and Simier2005) or the possibility of having intraindividual stress that has led to developmental abnormalities of individuals or poor larval living conditions (Ben Labidi et al., Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020b).
In addition to the environmental conditions, pollution can be a relevant factor in the two lagoons, where the Boughrara lagoon receives organic matter discharges through harbour-related activities (Guetat et al., Reference Guetat, Sellem, Akrout, Brahim, Atoui, Ben Romdhane and Daly Yahia2012), as well as sewage pollution from the traffic flow of the surrounding ports, entry of seawater carried with phosphorus from the Gulf of Gabes (Sellem et al., Reference Sellem, Guetat, Enaceur, Ghorbel-Ouannes, Othman, Harki, Lakuireb and Rafrafi2019), and industrial discharges from the Ghannouch chemical complex located on the shores of the Gulf of Gabes (DGPA, 2000; SCET-ERI, 2000). Moreover, the lagoon has been subjected to tidal phenomena with quite frequent eutrophication processes, often causing significant damage to the biota (Hamza, Reference Hamza1991, Daly Yahia et al., Reference Daly Yahia, Kefi and Romdhane1994; Daly Yahia and Romdhane, Reference Daly Yahia and Romdhane1996; Romdhane et al., Reference Romdhane MS2019; DGPA, 1999; Ben Rejeb Jenhani and Romdhane, Reference Ben Rejeb Jenhani and Romdhane2002). However, the El Biban lagoon receives pollution discharges from fishing and aquaculture activities (Zaouali, Reference Zaouali1983). As far as known, fish species are very sensitive to alteration in temperature by about 0.03 °C (Rebaya et al., Reference Rebaya, Ben Faleh, Allaya, Kheder, Trojette, Marsaoui, Fatnassi, Chalh, Quignard and Trabelsi2017), and the difference in the composition of otolith is associated with differences in fish responses to the effect of salinity reaction on temperature and the concentration of Cl, Mg, K, Na, and Ca (Martin and Wuenschel, Reference Martin and Wuenschel2006). Therefore, minor differences in the temperature, salinity, and pH between the two lagoons may directly affect the habitat and indirectly affect the chemical composition and shape of the otoliths in C. ramada. These interpretations are consistent with those of previous studies (Lombarte and Lleonart, Reference Lombarte and Lleonart1993; Martin and Wuenschel, Reference Martin and Wuenschel2006; Hüssy, Reference Hüssy2008; Capoccioni et al., Reference Capoccioni, Costa, Aguzzi, Menesatti, Lombarte and Ciccotti2011; Ben Labidi et al., Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020a, Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020b; Khedher et al., Reference Khedher, Mejri, Shahin, Quiganrd, Trabelsi and Ben Faleh2021; Mejri et al., Reference Mejri, Bakkari, Tazarki, Mili, Chalh, Shahin, Quignard, Trabelsi and Ben Faleh2022a, Reference Mejri, Bakkari, Allagui, Rebaya, Jmil, Mili, Shahin, Quignard, Trabelsi and Ben Faleh2022b). Additionally, Simoneau et al. (Reference Simoneau, Casselman and Fortin2000) reported that differences in the fish's age and sex may lead to a significant difference in the otolith shape in fish stocks. In this study, it is worth noting that the sampling was restricted to adult individuals to eliminate the effect of sexual maturity, which could affect the outline or contour shape of the otoliths (Cardinale et al., Reference Cardinale, Doering-Arjes, Kastowsky and Mosegaared2004). This is because it has been reported that the otolith shape is significantly different in juveniles than in adults due to differences in hearing function (Ferri et al., Reference Ferri, Bartulin and Škeljo2018), as well as to avoid the impact of confounding factors that can result from allometric growth in the two populations from the larval to the adult stage, where the shape is mostly stable (Santos et al., Reference Santos, Azevedo, Albuquerque and Araújo2017). Moreover, these interpopulation differences in the otolith shape detected here can be attributed to the differences in environmental factors between the two lagoons, such as water temperature and salinity, living depth, type of substrate, and food availability in terms of quantity and quality (Vignon and Morat, Reference Vignon and Morat2010; Capoccioni et al., Reference Capoccioni, Costa, Aguzzi, Menesatti, Lombarte and Ciccotti2011; Cañás et al., Reference Cañás, Stransky, Schlickeisen, Sampedro and Fariña2012; Sadighzadeh et al., Reference Sadighzadeh, Valinassab, Vosugi, Motallebi, Fatemi, Lombarte and Tuset2014; Pavlov, Reference Pavlov2016). As reported by Cardinale et al. (Reference Cardinale, Doering-Arjes, Kastowsky and Mosegaared2004), Galley et al. (Reference Galley, Wright and Gibb2006), Stransky et al. (Reference Stransky, Murta, Schlickeisen and Zimmermann2008), Rebaya et al. (Reference Rebaya, Ben Faleh, Allaya, Khedher, Marsaoui, Chalh, Quignard and Trabelsi2016), and Mejri et al. (Reference Mejri, Trojette, Jmil, Ben Faleh, Chalh, Quignard and Trabelsi2020), these environmental factors affect metabolism, which in turn affects somatic growth and thus the amount of material deposited on otoliths. Indeed, it has been declared by Fortunato et al. (Reference Fortunato, Benedito Durà, González-Castro and Volpedo2017) that the mugilids’ diet changes throughout their development, with larvae being planktivorous and juveniles feeding first through the water column, then changing to a benthic diet when they reach a total length of 20 to 30 mm. Regarding feeding behaviour, Rasheed et al. (Reference Rasheed, Saleh and Hamed2021) described that C. ramada feeds on a wide variety of prey types, including algae (36%), polychaetes (35%), diatoms (16.2%), crustaceans (mainly small prawns, crabs, copepods, and amphipod) (6.2%), sediment (3.4%), foraminifera (2.3%) and fish parts (1%), whose availability may be different between the Boughrara and El Biban lagoons due to the variation in the environmental characteristics, especially water temperature, salinity, and pollutants. Thus, we can suggest that this morphological bilateral asymmetry observed in the left and right otoliths contour shape between the two populations can be attributed either to the effect of the physico-chemical factors or pollutants present in the two lagoons.
On the other hand, examination of the morphometric dimensions of the left and right otoliths between and within individuals of the Boughrara and El Biban lagoons showed only significant asymmetry in Wo between the left and right otoliths among males and females within the Boughrara lagoon, i.e. there was a sexual dimorphism. Similar asymmetry in Wo has also been found in P. erythrinus (Mejri et al., Reference Mejri, Trojette, Jmil, Ben Faleh, Chalh, Quignard and Trabelsi2020), B. boops (Ben Labidi et al., Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020b) and D. vulgaris (Khedher et al., Reference Khedher, Mejri, Shahin, Quiganrd, Trabelsi and Ben Faleh2021). In addition, Wilk's test showed significant FA only in Lo between the right and left otoliths among individuals of the two populations. At the intrapopulation level, significant FA was observed only in Lo between the right and left otoliths among males and females, as well as among males, within the Boughrara lagoon. However, significant FA asymmetries were found between the left and right otoliths in Wo and Po among males, in all parameters among females, and in Wo and Po between males and females of the El Bibane lagoon. This differential significant asymmetry observed in the morphometric dimensions of the otoliths, as well as in FA, between and within males and females of the two populations may be largely attributed to the hypothesis that unfavourable environmental conditions may cause stress to the individuals, and thus leading to developing an asymmetry in both otoliths’ morphometry (Jawad et al., Reference Jawad, Al-Mamry, Hager, Al-Mamari, Al-Yarubi, Al-Busaidi and Al-Mamary2011; Jawad and Al-Sadighzadeh, Reference Jawad and Al-Sadighzadeh2013; Ben Labidi et al., Reference Ben Labidi, Mejri, Shahin, Quignard, Trabelsi and Ben Faleh2020b; Mejri et al., Reference Mejri, Trojette, Jmil, Ben Faleh, Chalh, Quignard and Trabelsi2020; Khedher et al., Reference Khedher, Mejri, Shahin, Quiganrd, Trabelsi and Ben Faleh2021). Indeed, environmental stress can arise from the contamination of seawater and sediments in the two lagoons with heavy metals, organic matter, and hydrocarbons discharged from harbour-related activities, as well as sewage pollution from the traffic flow of the surrounding ports and entry of seawater loaded with phosphorus from the Gulf of Gabes in the Boughrara lagoon (Guetat et al., Reference Guetat, Sellem, Akrout, Brahim, Atoui, Ben Romdhane and Daly Yahia2012; Sellem et al., Reference Sellem, Guetat, Enaceur, Ghorbel-Ouannes, Othman, Harki, Lakuireb and Rafrafi2019) or fishing and aquaculture activities in the El Biban lagoon (Zaouali, Reference Zaouali1983). Thus, the status of pollution in the Boughrara and El Biban lagoons may be responsible for the observed differential bilateral asymmetry in the otoliths’ contour shape, as well as FA detected in the otoliths’ morphometric parameters, within and among the two populations.
In conclusion, at the interpopulation level, analysis of the otolith shape showed a statistically significant difference (P = 0.0001), i.e. bilateral asymmetry, in the left and right otoliths’ shape between individuals of the two populations. In addition, a significant FA was detected only in Lo between the right and left otoliths among individuals of the two populations. At the intrapopulation level, a significant shape bilateral asymmetry was observed in the left and right otoliths among males and females, i.e. there was a sexual dimorphism, within the Boughrara lagoon. However, significant shape symmetry was observed in the left and right otoliths among individuals within the El Bibane lagoon. Moreover, a significant FA was found in Lo between the left and right otoliths only among males, as well as between males and females of the Boughrara lagoon. However, significant FA between the left and right otoliths was observed only in Wo among males and in all morphometric parameters among females, and in Wo between males and females of the El Bibane lagoon. DFA of the otoliths' contour shape confirmed the presence of two separate main stocks, one corresponding to the Boughrara lagoon and the other representing the El Bibane lagoon, which should be managed separately. The possible cause of morphological variation in the otolith shape and morphometry due to FA between the two populations of C. ramada can be attributed to the instability of development caused either by environmental stress associated with the variation in water temperature, salinity, living depth, feeding conditions and pollutants that have led to abnormalities in the development of individuals or by the poor living conditions of the larvae in an unfavourable environment. These findings contribute largely to the data on the otoliths' shape and morphometry, which has recently been considered a very important tool for identifying and discriminating fish stocks. In addition, the present study highlights the importance of potential FA in otolith morphometry for identifying fish stocks based on the otolith shape and morphometry and confirms that the C. ramada stocks from the two lagoons were discriminated from each other and, thus, should be managed separately.
Acknowledgements
The authors thank all the people and fishermen who helped us collect the C. ramada individuals from the Boughrara and El Bibane lagoons.
Data availability
Data supporting the findings of this study are available from the corresponding author upon request.
Authors’ contributions
All authors contributed to the conceptualization, discussion, and writing of this article. In addition, all authors read and approved the final version of the manuscript.
Financial Support
The authors declare that they did not receive any specific grant from funding agents for this work.
Competing interests
The authors declare that they have no competing financial interests or personal relationships that could appear to influence the work presented in the paper.
Ethical standards
The Laboratory of Ecology, Biology and Physiology of Aquatic Organisms, and Laboratory of Biodiversity, Biotechnology and Climate Change, Faculty of Sciences of Tunis, University of Tunis El Manar, Tunis, Tunisia, have approved this research. In addition, all procedures in this study were performed following the guidelines for the Proper Conduct of Animal Experiments outlined by the University of Tunis El Manar, Tunis, Tunisia (No. 1474 certificated on August 14th, 1995), as well as all applicable international, national, and/or institutional guidelines for the care and use of animals in Research.
