Dietary Analysis of Commercial Fish (Families Mullidae and Sparidae) from Bay of Cádiz, Southern Spain: An Integrative Approach

Abstract

A combination of stomach contents analysis (SCA) and nitrogen (δ¹⁵N) and carbon (δ¹³C) stable isotope analysis (SIA) was used to assess the trophic structure of nine fish species (two belonging to the family Mullidae, Mullus barbatus and Mullus surmuletus, and seven belonging to the family Sparidae, Diplodus sargus, Diplodus vulgaris, Pagellus acarne, Pagellus erythrinus, Pagrus auriga, Pagrus pagrus, and Sparus aurata) with high commercial value in the Bay of Cádiz, Southern Spain. A total of 91 different food items were identified in the stomachs, mainly belonging to four animal phyla (Arthropoda, Mollusca, Annelida, and Echinodermata). Crustaceans (primarily decapods and amphipods) were the most common prey consumed by the species of Mullus, Pagrus, and Pagellus, whereas macroalgae, polychaetes, and molluscs were dominant in D. sargus, D. vulgaris, and S. aurata stomachs, respectively. Diet composition and isotopic signature differed among fish species, indicating food partitioning among coexisting species. Some discrepancies appeared when comparing fish trophic level using SCA versus SIA, since SCA provides information on recently consumed items, while SIA generates data about source utilization over a period of several months. Integration of both approaches offers a more comprehensive understanding of feeding strategies. Dietary studies shed light on the trophic ecology of commercial fish species, being the baseline for future ecological modelling and long-term management of marine resources.

1. Introduction

Baseline information on the trophic ecology of commercial fish is needed for decision-making, not only regarding respectful catch management but also for resource valorization at the nutritional level or selection of adequate taxa for aquaculture [1]. This knowledge is especially relevant in overexploited areas, such as the Gulf of Cádiz (North-eastern Atlantic, Spain), where proper fisheries management is mandatory [2]. Fishery in this area has an important economic and employment role due to the high diversity and productivity of exploited species [3,4]. Nevertheless, over the last decade, the landings of fish stocks in the Gulf of Cádiz have been declining [5], making it necessary to increase basic knowledge on the biology and trophic ecology of fish species. Despite the socio-economic importance of the Bay of Cádiz within the Gulf of Cádiz as an exploited area, there is a lack of studies focused on food web and trophic structure [5,6].

Comparison of food habits and diet overlap of coexisting species is useful for understanding the mechanisms controlling diet variation and food consumption across species and ecosystems [7,8]. Historically, the traditional method to characterize diet and determine food niche breadth has been the stomach content analysis (hereafter SCA) [9,10], and much of the existing information on trophic ecology in the literature is based on this approach [11]. Although it may achieve high taxonomic resolution, SCA does not always provide accurate estimation in describing diet composition [12] due to a large set of drawbacks and limitations of this method: (i) stomach content only represents a snapshot of very recent feeding by consumers [13]; (ii) food items in the gut can be highly variable based on variation in digestion rates, feeding habits, seasonal or diel collection times, fish size, and individual dietary whims ([9] and references therein); (iii) empty stomachs may make trophic characterization difficult [14]; and (iv) food contents can be laborious to identify, requiring an important input of taxonomical expertise [15,16]. On the other hand, carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope ratios have been successfully used to improve understanding of the trophic functioning of marine systems. When consuming a prey, a predator integrates the C and N isotopic ratios of its prey into its own tissues [15]. Carbon isotope composition usually provides information on the origin of the ingested organic matter, and nitrogen isotope signature can be used to define the trophic level of organisms ([17] and references therein). Regarding fish trophic ecology, scientists have increasingly used stable isotope analysis (hereafter SIA) in order to provide a wider understanding that counters some of the limitations of SCA [18]. Indeed, stable isotope ratios have been widely used to assess trophic patterns in fish communities, sometimes replacing time-consuming SCA [19,20,21,22]. Although SIA cannot provide the taxonomic resolution of SCA, it allows for long-term integrated measures of assimilated diet over time [23], revealing contributions of different energy sources, e.g., pelagic and benthic [24], and allowing the estimation of consumer trophic position and food web length [25]. However, although δ¹⁵N is traditionally considered an indicator of the trophic position of consumers [26], the single comparison of δ¹⁵N values of organisms does not necessarily reflect their trophic level and diet composition [15]. The isotopic values measured in a consumer result not only from the isotopic ratios of its prey but also from other factors, such as trophic baseline, isotopic fractionation factor, and consumer metabolism, which may bias direct trophic level interpretation [27,28,29,30,31,32]. In fact, due to biases linked with isotopic ratios of the trophic baseline, some authors call for a cautious use of δ¹⁵N as a direct indicator of trophic level [32]. Thus, the correct interpretation of trophic relationships of organisms based on stable isotopic signatures is a complex task, which does not preclude the knowledge of their biology and feeding behaviors [15].

A combination of SCA and SIA makes a more holistic understanding of fish diets possible. This integration helps to obtain valuable information on behavior and food source used by sympatric species, providing more accurate representations of consumers’ diet and food resource use [17,33,34,35,36,37]. Therefore, a joint use of the two methods allows an accurate quantification of resource overlap between species, assesses the effects of fish management decisions, evaluates the potential impacts of invasive species on natural food webs and estimates trophic redundancy [38,39]. Despite the interest in the combination of stable isotope and gut content analyses, most of the trophic studies in fish use only one of the two methodologies and deal with a single or very few fish species. Therefore, a comprehensive comparison of the two methods based on the diet of a significant number of cohabiting species is lacking [11]. Furthermore, [10] pointed out the remarkably low number of experimental comparisons among methods, species, and ecosystems and underlined the urgent need for further investigation to fill this significant knowledge gap. Consequently, the main objective of the present study is to characterize the diet of nine commercial fish species coexisting in the Bay of Cádiz using SCA and SIA and to compare trophic diversity, niche breadth, and dietary overlap among species. Given differences in the scope, accuracy, and temporal resolution [40], both approaches are compared to identify their strengths and limitations.

2. Materials and Methods

2.1. Study Area and Fish Sampling

The Gulf of Cádiz (Figure 1) is located in the southwest of the Iberian Peninsula. It is a dynamic area, with a continuous exchange of water masses between the Atlantic Ocean and the Mediterranean Sea [41]. Several factors, such as bathymetric characteristics of its continental shelf and slope, warm-temperature climate, and enrichment produced by river outflows, explain the abundance of marine resources and contribute to making the Gulf of Cádiz a spawning, breeding, and juvenile area for many important fishery species [5,42]. Artisanal fisheries in the Gulf of Cádiz show a clear multi-species and multi-gear pattern. More than 50 species are landed, and catches are made with a large number of diverse fishing gears, including trammel nets (trasmallos), gillnets (pargueras, volantás), hand-jigs (chivos, pulperas), longlines (palangres, voraceras), traps (nasas, alcatruces), among others [43]. Within the Gulf of Cádiz, the Bay of Cádiz (Figure 1) comprises a zone where demographic concentration and development of economic activities alternates with the conservation status of a protected area (Bay of Cádiz Natural Park), which represents one of the largest areas of salt marshes on the Spanish South Atlantic coast [44,45]. The Bay of Cádiz is a natural nursery habitat for several fish species of commercial interest, being an important area for local professional fishing [46,47,48,49].

The present study was conducted in the outer Bay of Cádiz and nearby areas (from Rota to Conil) (Figure 1). The sampling was focused on two families, Mullidae and Sparidae, which comprise commercially important species in the area. Mullids are among the most important fishery targets throughout the Mediterranean coasts and nearby Atlantic [50,51,52,53]. Sparids, with more than 100 species worldwide, have a peak of diversity in the northeast Atlantic and the Mediterranean, where 24 species have been described [54]. Many of these species have high economic value and are prioritized in fish catches, being exploited and farmed for human consumption, and even fished for recreational purposes [55,56].

Nine species with high commercial value were selected for the present study (

Table 1. Characteristics of fish specimens used in this study. SE: standard error of the mean, M: muddy, S: sandy, R: rocky, N₁: number of fish used for stomach content analysis (SCA), N₂: number of samples used for stable isotope analysis (SIA).

				Length (cm)		Weight (g)
	Bottom	Depth (m)	Collection Date	Range	Mean ± SE	Range	Mean ± SE	N1	N2
Fish species:
Diplodus sargus	R	5–10	10 February–13 March 2015	15.3–26.2	20.2 ± 0.4	97–295	138 ± 8	40	20
Diplodus vulgaris	M, S, R	20–30	9 June–3 September 2015	18.5–27.4	23.8 ± 0.3	153–332	230 ± 6	52	20
Mullus barbatus	M	10–20	10 February–23 July 2015	17.0–26.0	19.7 ± 0.3	46–195	97 ± 5	56	20
Mullus surmuletus	R	15–40	6 May–9 September 2015	17.0–24.0	21.3 ± 0.2	70–187	130 ± 4	54	20
Pagellus acarne	S, R	20–40	9 June–9 September 2015	20.5–27.7	24.0 ± 0.2	112–305	202 ± 7	40	20
Pagellus erythrinus	M, S, R	14–30	20 May 2015–1 February 2016	18.4–29.8	24.5 ± 0.3	82–326	199 ± 7	61	20
Pagrus auriga	M, S, R	15–30	13 March–3 September 2015	18.1–38.7	23.2 ± 0.3	117–437	232 ± 8	74	20
Pagrus pagrus	M	15–40	24 April–30 June 2015	23.3–29.2	26.8 ± 0.2	246–420	321 ± 6	51	20
Sparus aurata	S	10–15	2 September 2015–1 February 2016	19.3–29.9	21.9 ± 0.4	193–362	269 ± 8	32	20

), two belonging to the family Mullidae (Mullus barbatus Linnaeus, 1758, and Mullus surmuletus Linnaeus, 1758) and seven belonging to the family Sparidae (Diplodus sargus (Linnaeus, 1758), Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817), Pagellus acarne (Risso, 1826), Pagellus erythrinus Linnaeus, 1758, Pagrus auriga (Valenciennes, 1843), Pagrus pagrus Linnaeus, 1758, and Sparus aurata Linnaeus, 1758). Fish were collected by local fishermen using gillnet fishing, from 10 February 2015 to 1 February 2016, at a depth ranging from 5 to 40 m along muddy, sandy, and rocky bottoms (Table 1). The anonymity of the exact locations where the fish were collected has been preserved, according to the wishes of the local fishermen [57]. All fish specimens were individually placed into polyethylene bags, labeled, and transported to the laboratory in an icebox. Upon arrival at the laboratory, the total length and wet weight of each fish were recorded.

2.2. Stomach Contents Procedure

Preserved stomachs of all collected fish specimens (Table 1) were opened, and the contents examined and clustered according to major taxonomic groups. A total of 460 stomachs were examined. The number of stomachs analyzed per species ranged from 32 (S. aurata) to 74 (P. auriga) (Table 1). Identification of food items was carried out to the lowest possible taxonomy level [54]. Dietary items (macroalgae and macrofauna) (Table S1) were weighted (precision 0.0001 g) after removal of surface water by blotting paper [58]. The wet weight of each food item was used as a quantification approach (gravimetric method), as it is considered the most convenient measure [9]. Indeed, the number of individuals (numerical method) is frequently considered not adequate, and it was not used in the present study because (i) in many cases, only pieces of prey can be detected and mastication of the food can make the counting difficult [9,59]; (ii) it can overemphasize the importance of small prey items taken in large numbers [9]; and (iii) food items such as macroalgae or bryozoans do not occur in discrete units and, therefore, measure of number of individuals is not applicable [60,61]. Hence, to obtain a single data matrix with quantitative data of all the food items (belonging to macroalgae and macrofauna), wet weight was always used.

2.3. Analytical Methods for Stable Isotope Measurements

Muscle samples of 180 specimens (20 of each species selected randomly from the total available) were used for stable isotopes. Fish were rinsed with sterile distilled water and immediately dissected with aseptic plastic forceps and a knife. Dorsal white muscle was selected, as this tissue gives the most reliable values for stable isotope analysis [62]. Muscle samples were kept frozen at −80 °C. Prior to analyses, samples were freeze-dried for 48 h in an ilShin Biobase Europe lyophilicer model FD8512 (ilShin Biobase Europe, Ede, The Netherlands) to constant weight. Freeze-dried samples were milled to a fine powder using a ball mill, Retsch MM400 (Retsch, Haan, Germany). Subsamples of powdered material were weighed to the nearest 0.300 mg with an error of ±0.002 mg and placed into tin capsules for δ¹³C and δ¹⁵N determinations. All samples were combusted at 1020 °C using a continuous-flow isotope ratio mass spectrometry system by means of a Flash HT Plus elemental analyzer coupled to a Delta-V Advantage isotope ratio mass spectrometer via a CONFLO IV interface (Thermo Fisher Scientific, Bremen, Germany).

2.4. Data Analyses

Regarding stomach contents, the importance of different food items was evaluated by calculating the frequency of occurrence, defined as %O = 100 × [number of stomachs containing food item i/total number of stomachs containing food items], and percentage weight (percentage gravimetric composition), defined as %W = 100 × [wet weight of food item i/total wet weight of all food items] [9,58]. The vacuity index (VI = 100 × [number of empty stomachs/total number of stomachs analyzed]) was used to calculate the proportion of empty stomachs [58,63]. The trophic diversity and evenness were estimated by the number of taxa found in each stomach (S); the Shannon–Wiener’s diversity index (H′), defined as H′ = −∑p_ilnp_i, where p_i is the proportion of wet weight (W) of food item i [64,65,66]; and the Pielou’s evenness index [67], defined as E = H′/H′max, where H′max is the maximum value of H′ for a given number of food items. H′ does not usually exceed 5.0 for biological communities [68], while E ranges from 0 to 1 (maximum evenness in the contribution of food items). The dietary niche breadth was calculated using standardized Levins’ index (B_A), according to the formula B_A = (B − 1)/(n − 1), where B = 1/∑p_i², p_i is the proportion of item i in the diet (based on %W), and n is the number of food items. The values of this index range from 0 to 1, where low values indicate a diet specialized in a few prey items and high values imply a generalist diet [63,68,69,70]. Food overlap (%Ov) among the species was assessed with Schoener’s dietary overlap index (%Ov_xy), defined as %Ov_xy = 100 × [1 − 0.5(∑ |p_xi − p_yi|)], where %Ov_xy is the percentage overlap between species diets of species x and y, p_xi is the proportion of food category i in the diet of species x, and p_yi is the proportion of food category i in the diet of species y (based on %W) [71,72]. The overlap index ranges from 0 to 100, with the overlap being greatest when the index is close to 100%. It is generally considered to be biologically significant when the value of the index exceeds 60% [73,74,75,76]. Non-parametric multidimensional scaling analyses (nMDSs) were carried out to show the relationship among species according to the diet items using frequency of occurrence (%O) and percentage weight (W%), based on the Bray–Curtis similarity. The relative level of acceptable stress for nMDS was checked following [77]. Differences among fish species according to %W were tested by permutational multivariate analysis of variance (PERMANOVA) [78]. Analyses were based on the Bray–Curtis similarity matrix. Significant p-values were obtained by computing 9999 permutations under a model of unrestricted permutation of raw data, which is recommended when there is only a single factor [78]. Pairwise comparisons were then used.

Differences in bivariate isotope space (δ¹³C and δ¹⁵N) among fish species were tested using a permutational multivariate analysis of variance (PERMANOVA) based on Euclidean distance, under the same design described for the diet composition analysis (see above). To further explore isotopic niche structure, we used the R package SIBER v. 2.1.9 [79]. Core isotopic niches were estimated for each species using standard ellipse areas corrected for small sample size (SEAc), which represent approximately 40% of the data. In addition, Bayesian standard ellipse areas (SEAb) were calculated to incorporate uncertainty and reported as the mode with 95% credible intervals. We assessed the assumption of bivariate normality underlying SEAc/SEAb with Royston’s multivariate Shapiro test and Mardia’s skewness and kurtosis tests (R package MVN, version 6.2). In addition, we estimated isotopic niches using non-parametric kernel utilization distributions (KUD; 50% isopleth) with the R package rKIN v. 1.0.4 [80]. Finally, metrics proposed by [81] to describe variation in isotopic niche between species were calculated. These metrics include δ¹³C range (CR), δ¹⁵N range (NR), mean distance to centroid (CD), mean nearest-neighbor distance (M-NND), and standard deviation of the nearest-neighbor distance (SD-NND). Briefly, CR is indicative of niche diversification at the base of food webs. NR is a representation of vertical structure in a food web, and larger ranges suggest more trophic levels and a greater degree of trophic diversity. CD provides a measure of the average degree of trophic diversity within a food web. M-NND represents trophic redundancy. Finally, SD-NND is a measure of the evenness of the food web, and large values suggest more diversification of trophic niches (see [81], for more details).

Multivariate analyses were carried out using the PRIMER v.6 plus PERMANOVA package [82,83].

3. Results

3.1. Food Items

The vacuity index (VI) ranged from 5.5% (M. surmuletus) to 78.7% (P. erythrinus) (

Table 2. Diet metrics based on food items found in the stomachs of the nine species studied. N: number of stomachs studied, n: number of stomachs containing food items, VI: vacuity index, S: number of taxa, H′: Shannon–Wiener diversity index, E: Pielou evenness index, B_A: standardized Levins index.

	n/N	VI	S	H′	E	BA
Diplodus sargus	27/40	32.5	30	2.58	0.76	0.32
Diplodus vulgaris	21/52	59.6	29	2.33	0.69	0.17
Mullus barbatus	51/56	8.9	29	1.80	0.53	0.08
Mullus surmuletus	51/54	5.5	49	2.17	0.56	0.09
Pagellus acarne	29/40	27.5	49	2.83	0.72	0.22
Pagellus erythrinus	13/61	78.7	13	1.33	0.52	0.16
Pagrus auriga	46/74	37.8	31	1.67	0.48	0.06
Pagrus pagrus	29/51	43.1	31	2.03	0.58	0.13
Sparus aurata	13/32	59.4	10	1.54	0.67	0.28

). In fact, only 13 of 61 stomachs analyzed in P. erythrinus had food remains. Diplodus vulgaris and S. aurata also displayed a high proportion of empty stomachs (with VI exceeding 50%).

Stomach content analysis allowed the identification of 91 different food items, mainly belonging to four animal phyla (Arthropoda: 29 items, Mollusca: 21, Annelida: 14, Echinodermata: 8) (Table S1). Macroalgae, represented by nine taxa, were the dominant component of D. sargus’ diet (Figure 2 and Table S1); the large vegetation percentage was present in all fish specimens regardless of their size. Conversely, the weight contribution of macroalgae to the diet of the remaining species was unimportant. Although Polychaeta (mainly Aphroditidae) and Mollusca (primarily Varicorbula gibba (Olivi, 1792), Abra sp., and Ringicula sp.) dominated the diet of D. vulgaris and S. aurata, respectively (Table S1), Crustacea was the most frequent and abundant group in most of the fish species (Figure 2). Among Crustacea, Decapoda (mostly Brachyura) and Amphipoda were the most common prey items consumed by fish (Table S1). Although Peracarida, represented by Amphipoda, Cumacea, Isopoda, Mysida, and Tanaidacea, were very important in terms of frequency of occurrence (%O), their contribution in terms of weight percentage (%W) was low in comparison with Decapoda abundances. Concerning Echinodermata, the species Echinocardium cordatum (Pennant, 1777) and Amphiura chiajei Forbes, 1843, were found in most of the fish species. Cheilostomatida Bryozoa, Hydrozoa, and Ascidiacea were also common prey in the present study (Table S1).

Regarding diet metrics (Table 2), the most diverse diet in terms of the number of food items corresponded to M. surmuletus and P. acarne (S = 49). Indeed, P. acarne was the species with the highest Shannon–Wiener diversity (H′ = 2.83), since the Pielou evenness was also high (E = 0.72), indicating an equitable distribution in the contribution of each food item to the total stomach content. As evenness was also high in D. sargus (E = 0.76) and D. vulgaris (E = 0.69), these two species also exhibited high trophic diversity according to the Shannon–Wiener index (H′ = 2.58 and H′ = 2.33, respectively). As expected, dietary niche breadth was higher in those species with the highest trophic diversity (H′), such as P. acarne (B_A = 0.22), D. sargus (B_A = 0.32), and D. vulgaris (B_A = 0.17), with a more generalist diet. Interestingly, a high value was also measured in S. aurata (B_A = 0.28). In spite of having a very low number of food items (S = 10) and low trophic diversity (H′ = 1.54), the high evenness (E = 0.67), due to the lack of dominant food items, contributed to a high dietary niche breadth in this species. Conversely, M. surmuletus (with a diverse diet) was characterized by a low value B_A = 0.09, which indicated a diet specialized in a few prey items, due to the high contribution in weight of the polychaetes Sternaspis aculeata (Ranzani, 1817) or Brachyura. The dominant contribution of Decapoda in the diet of M. barbatus and P. auriga also determined low dietary niche breadth for these two species.

According to PERMANOVA based on wet weight data (%W), diet composition differed among fish species. Apart from P. auriga and P. pagrus, which had a similar diet, all species displayed significant differences (

Table 3. Summary of the one-way PERMANOVA results based on stomach contents (percentage of wet weight, %W) and stable isotopes. Asterisks indicate significant differences, p < 0.001. MS: mean square, Dsa: Diplodus sargus, Dvu: Diplodus vulgaris, Mba: Mullus barbatus, Pac: Pagellus acarne, Per: Pagellus erythrinus, Pau: Pagrus auriga, Ppa: Pagrus pagrus.

Source of Variation	Df	MS	Pseudo-F	p	Unique Permutations
Stomach contents (%W)
Species	8	26,931	7.802	0.0001 ***	9809
Residual	271	3452
Total	279
Pairwise tests	All pairwise different except for Pau = Ppa
Stable isotopes
Species	8	24.305	12.243	0.0001 ***	9918
Residual	171	1.9852
Total	179
Pairwise tests	All pairwise different except for Dsa = Per = Pau Dvu = Mba = Per = Pau Pac = Ppa

). Schoener’s dietary overlap index (%Ov) totally agreed with this result since the only significant dietary overlap between species was recorded for P. auriga and P. pagrus (62.6%). Values of this index above 50% were also measured between M. surmuletus and P. acarne (56.1%) and M. barbatus and M. surmuletus (52.9%) (Table S2). nMDSs supported the patterns obtained with PERMANOVA. The analysis based on %W was more discriminant than nMDS based on frequency of occurrence (%O), which only separated three species, D. sargus, P. erythrinus, and S. aurata, from the others (Figure 3). In any case, both MDSs agreed in revealing that the diet of these three species clearly differed from the diet of the remaining ones.

3.2. Stable Isotopes

Fish species showed a wide range in their carbon and nitrogen isotopic signatures (

Table 4. Layman’s metrics. NR, Nmin, Nmax: nitrogen range, minimum and maximum; CR, Cmin, Cmax: carbon range, minimum and maximum; CD: distance to centroid; M-NND: mean of nearest-neighbor distance; SD-NND: standard deviation of the nearest-neighbor distance (‰); SEAc, standard ellipse area corrected (‰²), analyzed in muscle of the nine fish species studied.

	NR	Nmin	Nmax	CR	Cmin	Cmax	CD	M-NND	SD-NND	SEAc
Diplodus sargus	5.19	10.61	15.80	4.21	−20.90	−16.69	1.99	0.52	0.45	5.90
Diplodus vulgaris	3.29	11.09	14.38	1.16	−18.05	−16.89	0.80	0.32	0.18	1.01
Mullus barbatus	3.95	10.81	14.76	3.11	−19.36	−16.25	1.32	0.34	0.16	2.37
Mullus surmuletus	1.41	10.56	11.97	1.37	−18.86	−17.49	0.51	0.21	0.12	0.54
Pagellus acarne	2.51	10.91	13.42	1.19	−18.02	−16.83	0.61	0.19	0.09	0.68
Pagellus erythrinus	2.95	11.34	14.29	1.46	−18.16	−16.70	0.76	0.30	0.18	0.94
Pagrus auriga	6.07	10.33	16.40	6.94	−20.19	−13.25	2.02	0.91	1.02	10.32
Pagrus pagrus	2.91	10.38	13.29	1.69	−18.24	−16.55	0.79	0.27	0.13	1.00
Sparus aurata	3.10	11.92	15.02	4.51	−18.21	−13.70	1.07	0.51	0.61	2.75

and Figure 4). PERMANOVA results showed differences in the bivariate isotopic space among species (Table 3). Differences were significant for most of the pairwise, except for a few groups of species, such as (i) D. sargus, P. erythrinus, and P. auriga; (ii) D. vulgaris, M. barbatus, P. erythrinus, and P. auriga; and (iii) P. acarne and P. pagrus (Table 3).

P. auriga exhibited the widest nitrogen range, followed by D. sargus. Additionally, carbon ranges were also greater in P. auriga, followed by S. aurata and D. sargus. P. auriga, together with D. sargus, also displayed the highest CD values. Indeed, P. auriga showed the largest NND, followed by D. sargus, S. aurata, and M. barbatus (Table 4).

Bayesian and standard ellipse areas (SEAb and SEAc) indicated that P. auriga, D. sargus, and S. aurata exhibited the widest isotopic trophic niches (Table 4 and Figure 4). In contrast, M. surmuletus, P. acarne, and P. erythrinus showed the smallest ellipse areas. Although PERMANOVA revealed significant differences in isotopic composition among species, the standard ellipses showed that D. vulgaris, M. barbatus, P. acarne, P. erythrinus, and P. pagrus overlapped partially with the ellipses of D. sargus and P. auriga (Figure 4). The ellipses of D. vulgaris and P. erythrinus, as well as P. acarne and P. pagrus, also displayed a high degree of overlap. Conversely, the standard ellipses of S. aurata, P. auriga, and D. sargus exhibited limited overlap with those of the remaining species.

Royston’s test indicated deviations from normality in four species, but only Sparus aurata failed both Royston and Mardia tests. Kernel-based estimates produced slightly different rankings of niche size, but the qualitative interpretation of differences among species remained consistent. Isotopic niches using non-parametric kernel distributions (KUD; 50% isopleth) are included in Figure S1.

4. Discussion

4.1. Diet Characterization and Resource Partitioning

Crustaceans (primarily decapods) were the main prey consumed by most fish species, whereas macroalgae, polychaetes, and molluscs were dominant in D. sargus, D. vulgaris, and S. aurata stomachs, respectively. Although peracarids (mostly amphipods) were also abundant in terms of frequency of occurrence (%O), their importance in weight (%W) was lower due to the larger size of decapods in comparison with peracarids. Despite sharing some of the most abundant food items, statistical analyses based on SCA and SIA showed significant diet differences among fish species and, consequently, low diet overlap, supporting the existence of resource partitioning. Indeed, it is well known that although the high taxonomic groups of the major food can be similar in coexisting fish species, these can prey on different families or genera of the same family; therefore, food overlap and, presumably, competition are rare. Resource partition sensu lato can include food, habitat, and/or time segregation (e.g., [72,73,74,75,76,77,78,79,80,81,82,83,84]) and has been very well documented among fishes in temperate rocky reefs (e.g., [85,86,87,88]).

In the present study, the resource partitioning has been mainly approached through the food segregation, which seems to play a more important role than habitat or temporal separations within many fish assemblages [89]. Indeed, the use of different food resources can enable the coexistence of similar species within the same ecosystem [90], as in the present study.

Regarding habitat segregation, the diverse nature of substrate (rocky, gravel, sand, and mud bottoms) within a few kilometers over the Gulf of Cádiz shelf can also contribute to the coexistence of different fish species inhabiting different bottoms of the same area [5]. In any case, the habitats where the specimens were collected (listed in Table 1) do not necessarily represent the whole species habitat in the present study. The studied material depended on fish available from fishermen and was not based on a systematic collection with equal sampling efforts in all habitats and periods. Therefore, interpretations related to habitat segregation should be taken with caution.

Time segregation cannot be properly approached either due to these sampling design limitations. Although sampling was conducted concurrently, several species were not captured in every period or under fully overlapping temporal conditions. Therefore, some species may be using the area in a seasonally alternating manner rather than truly coexisting in space and time. Given that prey diversity and availability are subject to strong seasonal variation, and that the diet of a fish may differ depending on its age and biological state (e.g., reproduction and overwintering), such temporal partitioning in habitat use could influence diet composition and overlap estimates. These sampling constraints and ontogenetic effects should be considered to interpret the results prudently.

The majority of trophic studies on fish conducted in the Mediterranean and nearby areas have focused on Mullus spp. within the family Mullidae and Diplodus spp. and Sparus aurata within the family Sparidae. [91] reported that M. surmuletus had a higher diet diversity than M. barbatus in the Gulf of Cádiz (53 vs. 23 taxa), with a dominance of crustaceans (mainly decapods and peracarids) and also a remarkable contribution of the annelid Sternaspis scutata. This study confirms a more diverse diet for M. surmuletus (49 vs. 29 taxa). The importance of crustaceans and polychaetes for M. barbatus and M. surmuletus species has also been reported by other authors in the Mediterranean [50,51,53,92,93,94,95,96]. Due to this dominance of some prey in the diet, both species, M. barbatus and M. surmuletus, were characterized by low values of the B_A (0.08 and 0.09, respectively), which are usually associated with a specialized diet [63]. Some authors have pointed out a segregation of the feeding niche between the two mullet species facilitated by morphological differences in their feeding apparatus, which contributes to a reduction in interspecific competition [92,95]. Indeed, M. barbatus is usually associated with soft bottoms, where M. surmuletus inhabits rocky bottoms (see Table 1). Although these sympatric species feed on a similar wide range of prey, the relative proportion of their items differs between species and throughout their ontogeny [51]. In the same way, although SCA in the present study reveals a similar diet composition for both species, specialized in decapods, and some polychaetes such as S. scutata, differences in the contribution of some prey were measured (e.g., Brachyura or some Amphipoda). The segregation is supported by SIA, with M. barbatus showing higher values of δ¹⁵N than M. surmuletus (Figure 4). Previous studies have reported that subsurface deposit-feeding and carnivorous polychaetes, shrimps, and brachyurans present higher δ¹⁵N values than bivalves, small crustaceans, and ophiurids [17]. Although in the present study the pattern is not clear for polychaetes and brachyurans, the contribution of small crustaceans (e.g., peracarids) and echinoderms was more important in M. surmuletus, supporting the agreement between stomach contents and stable isotopes. Furthermore, the isotopic signature shows that M. barbatus has a wider niche and higher values of Layman’s metrics (Table 4) than M. surmuletus. Probably, the sediment where M. barbatus feeds on is characterized by a more diverse invertebrate community and with a higher contribution of carnivores than rocky patches where M. surmuletus inhabits. In any case, we must consider that the composition of the prey consumed in Mullus spp. can vary with fish size, season, and depth [17,50,52,93].

Within the family Sparidae, resource partitioning and competition become more significant for co-occurring species, especially under scarcity of food [76]. Indeed, the present study reinforces this partition, based on low diet overlap. According to SCA, only P. auriga and P. pagrus showed a significant overlap of trophic niche. According to SIA, notable overlap was observed between D. vulgaris and P. erythrinus, as well as P. acarne and P. pagrus.

Diplodus sargus was the only species in this study with a high contribution of macroalgae to the diet, indicating mainly an herbivorous feeding habit. The high values of δ¹⁵N measured in some specimens during the present study (Figure 4) could reflect the importance of algae in the diet of this species, since benthic primary producers classically present higher δ¹⁵N than phytoplankton [97]. Algae were also found to be a major item in the diet of the D. sargus inhabiting other Atlantic and Mediterranean regions [90,98,99,100]. [90] found that algae were the most frequent food in the stomachs of both D. sargus and D. vulgaris in the Mediterranean rocky shore, although molluscs (mostly bivalves) dominated in weight. Seabreams may ingest algae to profit from their epiphyte diatoms [98], to extract nutrients directly from the algae [101], or to optimize the digestion of animals [102]. Conversely, in some studies, the algae contribution to the diet of this species is not so important. For example, [76] found that in the North Aegean Sea, the diet of D. sargus was mostly based on invertebrates to avoid dietary overlap with the co-occurring Spondyliosoma cantharus (Linnaeus, 1758), which, in this case, adopted the herbivorous strategy based on a diet consisting mainly of algae. In the present study, a clear resource partitioning between the two Diplodus species, D. sargus and D. vulgaris, was revealed by the diet based on SCA and SIA. Diplodus vulgaris was the species in this study with the highest proportion of echinoderms in the diet (Figure 2). Other authors (e.g., [91,92,93,94,95,96,97,98,99,100,101,102,103]) also found an important contribution of echinoderms in this species and pointed out that the consumption of significant quantities of echinoderms, usually with low nutritional content, could be used by D. vulgaris, in part, to help break down the rest of the ingested trophic components. This “grinding” strategy would increase digestion, allowing greater feeding rates [104]. The present study also reveals the presence of sponge as part of the diet of D. vulgaris, with this species being the only one in the present study to have this food item. Several authors have found sponges as a feeding resource for Diplodus, especially for D. sargus ([105], and references therein). Future research is needed to understand the nutritional contributions of sponges and their role in the feeding ecology of seabreams.

Concerning the gilthead seabream Sparus aurata, this species is an opportunistic feeder and can adapt its diet to the food items available in the habitat ([58], and references therein). In agreement with stomach contents found in S. aurata in the present study, previous works on diet characterization of this fish revealed that primary prey items are mollusc bivalves, followed by gastropods and crustacean decapods [58,61,106,107,108,109]. In some Mediterranean populations of the species, diet can comprise mainly arthropods, followed by molluscs (e.g., [110]).

Unlike Mullus spp. and sparids such as Diplodus spp. or S. aurata, the number of studies regarding the diet of the sparid genera Pagellus and Pagrus is very scarce [91]. Regarding Pagellus, we found differences in the diet between P. erythrinus and P. acarne (Table 3, Figure 3). [111] in a study conducted in the southern Tyrrhenian Sea, western Mediterranean, found that P. erythrinus mainly preyed on strictly benthic organisms (polychaetes, brachyuran crabs, and benthic crustaceans), while P. acarne preferred suprabenthic prey such as peracarid crustaceans from the benthic boundary layer, a few metres above the bottom. These results agree with our study, since an important contribution of peracarids (mainly amphipods) was measured in P. acarne, whereas they were not present in the diet of P. erythrinus, which was dominated by large benthic decapods, such as Nephropos norvergicus and Upogebia, among others, and Gastropoda. Dominance of decapods and polychaetes in the diet of P. erythrinus has also been measured in Mediterranean populations of this species ([112], and references therein). The resource partitioning between the two Pagellus species revealed by the diet in the present study was also reflected in the isotopic signature, with higher values of δ¹⁵N in P. erythrinus. Ref. [111] also found higher δ¹⁵N in this species, probably related to a more marked benthic behavior and higher predation rate on carnivore prey measured in P. erythrinus. Regarding diet diversity, Pagellus acarne showed the highest number of taxa in its diet (49) and the highest values of Shannon–Wiener index (H′) and evenness (E), indicating a generalist diet. In a study conducted in Tunisia, [113] found a total of 36 different food items, coinciding in that Arthropoda was the most important prey group. On the other hand, the diet of Pagellus erythrinus showed a very low number of taxa (13) with the lowest value of trophic diversity (H′) in this study (1.33). The low number of taxa could be related to the low number of specimens studied due to the high vacuity index (VI), ca. 80%, which importantly reduced the stomach contents sampling size for this species in the present study (n = 13). Previous research (i.e., [91]) has also shown high VI values for P. erythrinus (ca. 50%). Empty stomachs in fish may result from autecological factors (e.g., differences in gastric evacuation rates, feeding habits, rate of gut clearance, presence of a non-feeding life stage), individual fish health, environmental conditions (e.g., prey encounter rate, temperature), or sampling artifacts (e.g., contents regurgitated on capture or digested after capture) [14]. Our study reflects specialization of P. erythrinus in some taxa, such as Nephrops norvegicus (Linnaeus, 1758) or Upogebia sp. Therefore, a lower prey availability could explain the high percentage of empty stomachs. Anyway, we cannot discard that a higher rate of regurgitating food during the capture for this species could also be involved in the large number of empty stomachs. In any case, regurgitation effects on the underestimation of stomach contents could be a source of bias affecting all species examined, not only P. erythrinus.

Regarding the genus Pagrus, there is also a lack of diet studies, despite its abundance and popularity as a commercial fish. Similarly to this study, decapods dominated the diet of P. pagrus from Greece [114]. Conversely, fish that are abundant in the diet of Greek populations were absent in specimens from the Gulf of Cádiz. [114] pointed out that, differently from other related species, piscivory diminishes with an increase in size in P. pagrus. Considering the higher length of fish studied in the Mediterranean populations (up to 48.5 cm) in comparison with the Gulf of Cádiz (up to 29.2 cm), differences in prey fish consumed are probably related to other factors other than fish size (e.g., prey availability or seasonal variations). To our knowledge, this study represents the first comprehensive approach to the diet of P. auriga, which shows a remarkable contribution of decapods, suggesting specialized feeding. Indeed, the very high weight proportion of Brachyura in the diet (58.5%) probably contributed to the decreased values of H′, E, and B_A despite showing a high number of food items (31). P. auriga has the broadest length range, varying between 18.1 and 38.7 cm (Table 1). Ontogenetic (size-related) dietary shifts are well documented in demersal fishes, with different size classes often exploiting distinct habitats and prey resources. This may explain the large niche breadth observed for this species and highlights the importance of considering size-related dietary shifts.

In the present study, SCA revealed a significant overlap between P. auriga and P. pagrus. Indeed, it was the only significant dietary overlap obtained in the present study according to the Schoener index. The ellipses based on stable isotopes also pointed to a certain degree of overlap between the two species, although the niche breadth was considerably higher in P. auriga.

Although some species did not fully meet the assumption of bivariate normality, particularly S. aurata, SEAc/SEAb were retained in the main text because they remain the most widely applied metrics for comparing isotopic niche size in ecological studies. For this species, niche metrics should therefore be interpreted with caution. Kernel-based analyses (50% isopleth) (Figure S1) showed that the overall conclusions were robust, despite slight differences in absolute niche size and species ranking.

4.2. Discrepancies Between the Two Methods

Ref. [10] demonstrated that quantitative diet estimation using SCA and SIA techniques align well in cases with less than six dietary resources (ca. 65% similarity) but can diverge considerably when applied to more complex diet mixtures (<30% similarity). Therefore, constructing mixing models that integrate information from both methods should be performed with caution, especially when there are more than six diet items, as is the case in this work. Although some authors have already drawn attention to the discrepancies in the two methods within fish trophic ecology (e.g., [11,15,40,115,116]), more experimental comparisons among dietary approaches are necessary [10].

In the present study, SIA confirmed the results obtained through SCA in most cases. However, some dissimilarities were observed, which probably reflect the strengths and weaknesses of the two approaches. SCA provides information on recently consumed items that can often be taxonomically identified, whereas SIA provides data about source use over a period of several weeks or months [11]. The most evident discrepancies between the two methods revealed by our data were as follows: (i) P. auriga showed the lowest trophic niche breadth according to SCA and the highest based on SIA, and (ii) the only significant diet overlap based on SCA was measured for P. auriga and P. pagrus, while ellipses of SIA indicated overlapping between D. vulgaris and P. erythrinus, and P. acarne and P. pagrus. Despite the higher degree of overlap measured by SIA in this study, several authors have pointed out that, among ecologically similar species, SIA tends to show less dietary overlap among species than SCA [115,117,118,119].

The discrepancies between methods in dietary trophic niche and diet overlapping revealed by the present study could be related to variation in the trophic discrimination factor, diet changes on a temporal scale, different sample size, or rapid digestion of some prey, among others.

During assimilation, the lighter stable isotope is preferentially excreted, meaning that consumers normally become isotopically enriched relative to their prey [120], a phenomenon known as trophic discrimination (TD). The offset between the isotope ratios of a consumer and its prey is termed the trophic discrimination factor (TDF) [10]. Therefore, the isotopic values measured in a consumer result not only from the isotopic ratios of its prey but also from TDF ([15], and references therein). TDF can fluctuate between habitats, environmental conditions, size/age, and nutritional status of consumer, taxonomic groups, individuals, tissues, and resources, especially when diet components have different fatty acid profiles than their consumers ([10,121], and references therein). These TDF fluctuations could partially explain the different results obtained by SCA and SIA regarding dietary trophic breadth and diet overlap.

Discrepancies measured in the present study could also reflect diet variation on a time scale (weeks, months, or seasons). Although some fish species can focus their foraging on particularly abundant prey items on a day-to-day basis, they can feed on a wider range of taxa over longer periods (from weeks to months) [40]. These authors highlighted that, in commercially valuable fish species, information regarding this dietary flexibility could aid in fisheries management through a better understanding of the role that intermittently open habitats (e.g., estuaries) play as nursery areas for species of interest.

Differences in trophic niche breadth and diet overlapping obtained by the two methods in this study could also be partially due to the different sampling size for SCA and SIA; although 20 specimens were always used for SIA, the number of available stomachs containing food items for SCA ranged from 13 (P. erythrinus and S. aurata) to 51 (M. barbatus and M. surmuletus). The degree to which a small variation in sample size could affect comparisons between methods should be further investigated in future studies.

The quick digestion of some food items could prevent their detection in SCA, conditioning the values of trophic descriptors, and explaining differences between SCA and SIA output, as pointed out by some authors (e.g., [115]). Effectively, SCA could underestimate prey which have been rapidly digested, as reflected in our study by the high proportion of crustaceans, molluscs, and echinoderms. However, other soft-bodied organisms, such as polychaetes, were also detected, since hard structures can remain longer in the digestive tract. Nevertheless, SCA is essential for taxonomic discrimination of prey and for better distinguishing between similar sources that exhibit overlapping isotopic signatures that hinder their differentiation through SIA.

In this context, the combined use of SCA and SIA provides a more complete picture of trophic ecology, as each method addresses different temporal and taxonomic scales. In our case, SCA results are consistent with previous studies in the area, whereas isotopic ellipses revealed broader trophic niches and degrees of overlap that would not be evident from SCA alone. The use of SIBER adds further value, offering a standardized framework to represent isotopic niche width and overlap. While it assumes a bivariate normal distribution in δ¹³C–δ¹⁵N space [79], which may not fully capture complex or multimodal structures, the Bayesian approach (SEAb) accounts for uncertainty in niche area estimates, facilitating more robust comparisons even with small sample sizes.

5. Conclusions

Integration of methods provides new possibilities to understand complex trophic relationships, which are difficult to untangle when using only a single method. Both approaches are complementary but should be interpreted with caution. Comprehensive dietary studies, combined with more empirical testing of methods and the establishment of open data repositories for dietary data, are necessary baselines to properly conduct future ecological modelling and improve long-term management of marine resources.