Genetic demography of the blue and red shrimp, aristeus antennatus: A female-based case study integrating multilocus genotyping and morphometric data

Citation: Abras, A.; García-Marín, J.-L.; Heras, S.; Agulló, M.; Vera, M.; Planella, L.; Roldán, M.I. Genetic Demography of the Blue and Red Shrimp, Aristeus antennatus: A Female-Based Case Study Integrating Multilocus Genotyping and Morphometric Data. Genes 2022,13, 1186. https://doi.org/10.3390/ genes13071186 Academic Editor: Antonio Figueras Received: 26 May 2022 Accepted: 27 June 2022 Published: 1 July 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). genes G C A T T A C G G C A T Article Genetic Demography of the Blue and Red Shrimp, Aristeus antennatus: A Female-Based Case Study Integrating Multilocus Genotyping and Morphometric Data Alba Abras 1, Jose-Luis García-Marín1, Sandra Heras 1, Melania Agulló1, Manuel Vera 2, Laia Planella 1 and María Inés Roldán1,* 1Laboratori d’Ictiologia Genètica, Campus Montilivi, Universitat de Girona, 17003 Girona, Spain; [email protected] (A.A.); [email protected] (J.-L.G.-M.); [email protected] (S.H.); [email protected] (M.A.); [email protected] (L.P.) 2Departamento de Zoología, Genética e Antropología Física, Campus Lugo, Universidad de Santiago de Compostela, 27002 Lugo, Spain; [email protected] *Correspondence: marina.r[email protected]; Tel.: +34-630-758-063 Abstract: In this study, we quantified the three key biological processes, growth, recruitment, and dispersal pattern, which are necessary for a better understanding of the population dynamics of the blue and red shrimp Aristeus antennatus. This marine exploited crustacean shows sex-related distribution along the water column, being females predominate in the middle slope. The present study attempts to fill the existing gap in the females’ genetic demography, as scarce knowledge is available despite being the most abundant sex in catches. We analyzed morphometric data and genotyped 12 microsatellite loci in 665 A. antennatus females collected in two consecutive seasons, winter and summer 2016, at the main Mediterranean fishing ground as a model. Almost every female in summer was inseminated. Five modal groups were observed in both seasons, from 0+ to 4+ in winter and from 1+ to 5+ in summer. Commercial-sized sorting based on fishermen’s experience resulted in a moderate-to-high assertive method concerning cohort determination. Genetic data pointed out females’ horizontal movement between neighboring fishing grounds, explaining the low genetic divergence detected among western Mediterranean grounds. Our results could represent critical information for the future implementation of management measures to ensure long-time conservation of the A. antennatus populations. Keywords: deep-sea shrimp; genetic demography; horizontal movement; microsatellite loci; recruitment 1. Introduction The blue and red shrimp, Aristeus antennatus (A. antennatus) (Risso, 1816) (Crustacea, Aristeidae) (En—Blue and red shrimp; Fr—Crevette rouge; Sp—Gamba roja) is the eurybathic species that achieves the broadest vertical distribution in the Mediterranean Sea with reported records from 80 down to around 3000 m depth [ 1 , 2 ]. However, the species shows a demographic structure along the water column. The proportion of males and juveniles tends to increase with depth, whereas females are more abundant in the middle slope [ 3 , 4 ], with peaks of abundance around 700 m [ 1 ]. The mating activity starts from late winter to late spring (January–May) when adults of both sexes aggregate at 400–900 m depth, and after mating, males return to deeper waters [ 4 , 5 ]. Although females’ maturity begins in spring, they can store sperm to fertilize the eggs in the absence of males [ 5 , 6 ]. Hence, during the spawning season, shoals are composed mainly of mature adult females [ 4 , 7 , 8 ]. Then, in autumn and winter, there is a decrease in population density in the middle slope as reproductive shoals break up [9]. Larval dispersal by marine currents during the A. antennatus pelagic stage has been pointed out as the main responsible for the high level of genetic connectivity found across Genes 2022,13, 1186. https://doi.org/10.3390/genes13071186 https://www.mdpi.com/journal/genes Genes 2022,13, 1186 2 of 17 the Mediterranean Sea [ 10 – 17 ]. However, Guijarro et al. [ 18 ] reported a possible migration of juveniles and adults, and Agullóet al. [ 16 , 17 ] suggested adult migration as a contributor to the high genetic homogeneity found in the western Mediterranean populations. Genetic relatedness analyses showed that spermatophores attached to females collected at 400–700 m depth during the reproductive period came only partially from males captured simultaneously with the females, whereas a large proportion of spermatophores belonged to unsampled and less related males, likely from other spawning grounds [ 19 ]. Using genetic assignment analyses, Abras et al. [ 20 ] recently reported the horizontal dispersal of juvenile males. On the other hand, females’ horizontal displacements have only been proposed by a single tagging study [ 21 ] and, despite their relevant contribution to the fisheries of this species, there are no genetic studies inferring the demography and dispersal of A. antennatus females. At depths shallower than 1000 m, where A. antennatus fishery takes place, the species shows the highest abundance with a strong sex-biased ratio to females [ 4 , 22 ]. In the lowest status (>1500 m), where there is no fishing activity (virgin grounds), a sex ratio of 1:1 is reached [ 9 , 23 ]. Furthermore, peaks of juveniles were observed in March between 600 and 1000 m in depth [ 24 ], indicating the migration of juveniles from the bottom areas to the mid-slope and their subsequent incorporation into the fishery, which leads to the recovery of A. antennatus fishing stocks [2,25,26]. Fishermen sort by size A. antennatus individuals into commercial categories on board, resulting in four sizes: small (>70 pieces/Kg), medium (50–70 pieces/Kg), large ( 30–49 pieces/Kg ), and extra-large (<30 pieces/Kg). The average price ranks from 24.14 euros/Kg for small shrimps to 83.03 euros/Kg for the extra-large ones, but seasonal fluctuations are common according to catches and market demand [ 27 ]. The small size category is composed of males and females, whereas larger size groups are only represented by females [ 28 ]. Females are longer lived (with a life expectancy of 5/6 years) [ 25 , 29 ] and are the most pressured by fishing, especially in summer [ 4 , 7 ]. Although age estimations in this species are mostly based on body sizes (carapace length, e.g., [ 23 , 30 – 32 ]), there is no clear relationship between the aforementioned market sizes and cohorts [ 33 ]. Furthermore, the reduced growth rate for older individuals makes age determination difficult due to possible size overlapping between cohorts [34]. The blue and red shrimp fishery along the western Mediterranean coast constitutes a resource of high economic value [ 35 ]. However, to date, only a single regional management plan designed for A. antennatus exists at Palamós fishing ground (Spain, northwestern Mediterranean Sea). This plan includes meshing size regulations and a temporary cessation of trawling for a period of sixty working days per year [ 36 , 37 ]. Palamós is also one of the most important fishing harbors for A. antennatus of the western Mediterranean Sea and has its own quality brand [ 38 ]. For all these reasons, this fishing ground has been the focus of several studies based on the species [7,19,20,28,39,40]. In the present study, morphometric and genetic analyses have been integrated to investigate demographical aspects such as growth, recruitment, and horizontal displacements of A. antennatus females using the Palamós fishing ground as a model. This approach took advantage of previous genetic information in the same fishing ground on the breeding behavior of the species [ 19 ] and on the growth, recruitment, and geographical origin of A. antennatus males [ 20 ]. The results reported here fill the existing gap in the genetic demography of females, which is the most relevant sex in A. antennatus fishery [28]. 2. Materials and Methods 2.1. Biological Material A total of 665 A. antennatus females were collected on board the Nova Gasela commercial trawling vessel at Palamós fishing ground (41 ◦ 54 0 04 00 N, 3 ◦ 16 0 08 00 E) in two sampling campaigns, one on 3 March (winter) and the other on 7 July (summer) 2016. A single haul per campaign was conducted at 500 m depth, with an average cruising speed of 2 knots and an effective towing duration of 2 h. The codend had a mesh size of 40 mm stretched and Genes 2022,13, 1186 3 of 17 was covered by a codend cover with a mesh size of 12 mm stretched (see Abras et al. [ 20 ] for further details). As the codend cover is prohibited for commercial hauls, it was specifically authorized for these two scientific campaigns. Immediately after being captured, the crew sorted by eye all the individuals caught in the codend into four commercial sizes (small, medium, large and extra-large) according to their expertise. A random sample of around 100 adult females from each size group was kept for subsequent analyses, but due to limited availability, all captured females were retained for the largest sizes (Table 1). Females were identified through the presence of thelycum (morphological criteria [ 33 ]). In addition, one or more spermatophores adhered to the thelycum were detected in summer individuals, as July corresponds to the peak of the spawning season [ 19 ]. In winter, females in the codend cover were identified on board by members of the scientific team according to the morphological criteria. The presence of transparent gonads suggested they were juvenile females, and a sample of around 100 was randomly kept for analysis. In summer, there were no females captured in the codend cover. All kept individuals of each campaign were immediately stored on ice on board and quickly transported to the laboratory. Once there, the carapace length (CL) was measured from the orbital margin to the mid posterior edge of the carapace using a digital vernier caliper, and a piece of approximately 10 mg of the muscle of each individual was preserved in 70% ethanol and stored at room temperature for further genetic analyses. Table 1. Commercial size groups of Aristeus antennatus (A. antennatus) females, including sampling season, commercial category, mean carapace length (CL) with standard deviation (SD), number of females (N), number of females with one or more spermatophores adhered to the thelycum, and estimates of genetic diversity (excluding locus Aa818). Sampling Season Commercial Category Mean CL ±SD (mm) N Females with Spermatophore (Double) NAARHOHEFIS Winter Juvenile 118.98 ±1.53 105 - 9.7 5.32 0.46 0.63 0.27 Small 22.96 ±2.52 105 - 8.9 5.24 0.49 0.64 0.23 Medium 35.54 ±3.41 82 - 9.2 5.34 0.43 0.64 0.33 Large 42.03 ±1.97 22 - 5.7 4.64 0.43 0.60 0.28 * Extra-large 51.86 ±8.65 17 - 5.7 5.07 0.42 0.61 0.30 * Summer Small 27.65 ±2.37 100 99 9.7 5.40 0.51 0.63 0.19 * Medium 33.24 ±2.46 99 97 (3) 9.4 5.39 0.48 0.62 0.22 Large 44.88 ±3.78 91 91 (14) 9.4 5.36 0.51 0.63 0.19 * Extra-large 55.46 ±5.10 44 44 (9 + 1 triple) 7.8 5.14 0.45 0.62 0.27 * 1 Juvenile is not a commercial category. N A , Number of alleles; A R , allelic richness; H O , observed heterozygosity; H E , expected heterozygosity; F IS , inbreeding coefficient. * Significant departure from Hardy-Weinberg equilibrium after Bonferroni correction (α/99, p< 0.0005). 2.2. DNA Extraction and Molecular Markers Genomic DNA was extracted from the muscle tissue of each female following the phenol-chloroform protocol adjusted to A. antennatus by Fernández et al. [ 41 ]. Twelve polymorphic microsatellite loci developed for this species were amplified by means of three multiplex PCRs and one PCR singleplex: Aa123,Aa1255,Aa138,Aa1444,Aa496b,Aa667, Aa681,Aa751,Aa956,Aa1061,Aa1195, and Aa818 [ 19 , 42 ]. PCR products were analyzed in an ABI PRISM 3730 × l sequencer (Applied Biosystems, Foster City, CA, USA) at the Sequencing Unit of the Universidade de Santiago de Compostela (Campus Lugo, Lugo, Spain). Genotyping was carried out using GeneMapper 4.0 software with GeneScan TM 500 LIZ ®® size standard (Applied Biosystems). 2.3. Size Frequency Distributions, Cohort Identification, and Comparison with Commercial Categories The CL-frequency distributions in 1 mm class intervals of the females in winter and summer were studied through a modal progression analysis (MPA) implemented in the Genes 2022,13, 1186 4 of 17 FISAT II software [ 43 ]. The Bhattacharya method [ 44 ] was used for the visual identification of the different normal distributed size groups in the polymodal CL-frequency distributions. For each season, results were displayed as the computed mean of each size group with its standard deviation (SD) and a separation index (SI). The latter should be ≥ 2 between consecutive size groups to assume each one as a single age group; otherwise, it means substantial overlap between cohorts. Specific cohorts in our samples (0+, 1+, 2+, 3+, 4+, and 5+) were assigned, combining MPA results with CL-cohort relationships previously reported for western Mediterranean A. antennatus populations. A one-way ANOVA and Scheffe post-hoc tests included in the IBM SPSS Statistics version 25 package (Armonk, NY, USA) were performed to compare the mean CL in winter and summer of each size group present in both campaigns (small, medium, large, and extra-large). The same analysis was performed for the MPA-identified groups shared in the two sampling seasons (1+, 2+, 3+, and 4+). Cohen’s kappa coefficient (K) [ 45 ], which described the level of concordance among methods relating to the observed agreement and the agreement expected by chance, was calculated between the groups of females identified by the MPA and the categories derived from the commercial criteria, including juveniles, in each season. The interpretation of K values was as follows: 0 to 0.20 indicates slight agreement, 0.21 to 0.40 fair agreement, 0.41 to 0.60 moderate agreement or good agreement beyond chance, 0.61 to 0.80 substantial agreement, and 0.81 to 1 almost perfect agreement [ 45 ]. Calculations were performed with VassarStats, which is available online at http://www.vassarstats.net/kappa.html (accessed on 23 May 2022). 2.4. Genetic Analysis Genetic diversity at each female commercial size group (including winter juveniles caught in the codend cover) as well as at each age group was estimated as the number of alleles per locus (N A ) and allelic richness (A R ) calculated using FSTAT 2.9.3 [ 46 ], and observed (HO)and expected (HE) heterozygosities using GENEPOP 4.7.0 [47]. Deviations from Hardy-Weinberg equilibrium (HWE) were summarized using the inbreeding coefficient F IS and tested using the exact probability test of Guo & Thompson [ 48 ], implemented in GENEPOP 4.7.0. Bonferroni correction was used to adjust significance levels. Null allele frequency for each locus was assessed using Micro-Checker 2.2.3 [ 49 ]. Genetic distinctions between female commercial size groups and also between age groups in winter and summer were assessed by pairwise FST using the program FSTAT 2.9.3 [46]. As previously done with A. antennatus males in Abras et al. [ 20 ], the parental contribution of 2015 local spawners to the 2016 juvenile and small female groups were analyzed using assignment tests. Putative parental individuals from 2015 included adult females (F; N = 52; mean CL = 38.27 ± 3.76 mm), sympatric males captured with females (M; N = 61; mean CL = 22.78 ± 2.59 mm), and mating males inferred from spermatophores attached to females’ thelycum (S; N = 59) reported by Planella et al. [ 19 ]. HYBRIDLAB 1.0 software [ 50 ] was used to generate independent F1 offsprings from these 2015 parent genotypes involving mating between females and males (F × M F1) and between females and spermatophore genotypes (F × S F1). Ten sets of paired baselines of 100 simulated offspring individuals from the resulting females and spermatophores (F × S F1 baseline) and 100 individuals from the admixture of females and males (F × M F1 baseline) were used in subsequent assignations. Thus, 25 more independent sets of 100 individuals for each combination ( F×S F1 and F ×M F1 ) were also simulated and independently assigned to each of the 10 baseline sets, producing a total of 250 replicates to evaluate the accuracy of the assignments to simulated baselines. Thereafter, the 2016 female groups (winter juveniles, winter small, and summer small) were assigned to each of the 10 baselines. Assignments were carried out using the Bayesian Rannala and Mountain method [ 51 ], as implemented in GENECLASS 2 [ 52 ]. Individuals with an assignment probability < 0.01 to both baselines were considered as having another source. Comparisons of the results of these latter assignations with those from simulated data were performed using one-way ANOVA and Scheffe post-hoc tests included in IBM SPSS Statistics. Genes 2022,13, 1186 5 of 17 In addition, the geographical origin of juvenile and small females was analyzed through assignment tests. Winter juveniles and small females were used as a baseline together with two sets of small females collected during the same winter of 2016 at two nearby fishing grounds: one from Roses (Cap de Creus Canyon, n = 55) situated to the north of Palamós, and the second from Blanes (Blanes Canyon, n = 54), located to the south (Figure 1) [ 20 ]. Chi-square tests using VassarStats were computed to compare the assignment pattern of the summer females to winter baselines. Finally, assignment tests were also used to obtain the distribution to winter baselines of female summer groups up to 3+ derived from the MPA. Fisher exact probability tests were used to compare summer assignment patterns to winter baselines. Genes 2022, 13, x FOR PEER REVIEW 5 of 18 subsequent assignations. Thus, 25 more independent sets of 100 individuals for each combination (F × S F1 and F × M F1) were also simulated and independently assigned to each of the 10 baseline sets, producing a total of 250 replicates to evaluate the accuracy of the assignments to simulated baselines. Thereafter, the 2016 female groups (winter juveniles, winter small, and summer small) were assigned to each of the 10 baselines. Assignments were carried out using the Bayesian Rannala and Mountain method [51], as implemented in GENECLASS 2 [52]. Individuals with an assignment probability < 0.01 to both baselines were considered as having another source. Comparisons of the results of these latter assignations with those from simulated data were performed using one-way ANOVA and Scheffe post-hoc tests included in IBM SPSS Statistics. In addition, the geographical origin of juvenile and small females was analyzed through assignment tests. Winter juveniles and small females were used as a baseline together with two sets of small females collected during the same winter of 2016 at two nearby fishing grounds: one from Roses (Cap de Creus Canyon, n = 55) situated to the north of Palamós, and the second from Blanes (Blanes Canyon, n = 54), located to the south (Figure 1) [20]. Chi-square tests using VassarStats were computed to compare the assignment pattern of the summer females to winter baselines. Finally, assignment tests were also used to obtain the distribution to winter baselines of female summer groups up to 3+ derived from the MPA. Fisher exact probability tests were used to compare summer assignment patterns to winter baselines. Figure 1. Sampling locations of A. antennatus. Relief map: Sémhur/Wikimedia Commons 3. Results 3.1. Size-Age Composition of A. antennatus Females Mean CL was significantly lower in winter than in summer for all commercial female size groups (juveniles from codend cover removed) but the medium-sized category (Figure 2a). Practically all the females caught in summer were inseminated (331/334; 99.1%) and a small proportion of them (27/331; 8.2%) presented more than one spermatophore (Table 1). Blanes Palamós Roses Atlantic Ocean Mediterranean Sea 42º N 41º N 3º E 2º E Figure 1. Sampling locations of A. antennatus. Relief map: Sémhur/Wikimedia Commons. 3. Results 3.1. Size-Age Composition of A. antennatus Females Mean CL was significantly lower in winter than in summer for all commercial female size groups (juveniles from codend cover removed) but the medium-sized category ( Figure 2a ). Practically all the females caught in summer were inseminated (331/334; 99.1%) and a small proportion of them (27/331; 8.2%) presented more than one spermatophore (Table 1). The MPA from CL-frequency distributions showed that females in both winter and summer were composed of five separated (SI > 2) modal groups (Table 2, Figure 3a,b). Based on CL-age keys reported in previous studies on A. antennatus western Mediterranean fisheries [ 23 , 30 – 32 , 34 , 53 , 54 ] (Table 3), the age of each MPA group was assigned to cohorts from 0+ to 4+ in winter and from 1+ to 5+ in summer. The size of the first identified cohort (19.26 ± 1.77 mm) was slightly larger than the one reported by Carbonell [ 34 ] and looks similar to the smaller modal cohort indicated by D’Onghia et al. [ 23 ] for samples at the Ionian Sea collected in April and May. Though these latter authors did not use codend cover, but a stretched mesh of 20 mm in the codend and reduced proportions of collected females were in the range between 16 and 26 mm of CL, especially in April catches. Genes 2022,13, 1186 6 of 17 Genes 2022, 13, x FOR PEER REVIEW 6 of 18 Figure 2. Comparison of the mean carapace length (CL) of A. antennatus females based on commercial categories (a) and the groups derived from the modal progression analysis (b) in winter (filled circles) and summer (empty circles) for each group present in both campaigns. p < 0.05 obtained from one-way ANOVA and Post-hoc Scheffe test are indicated with an asterisk (*). The MPA from CL-frequency distributions showed that females in both winter and summer were composed of five separated (SI > 2) modal groups (Table 2, Figure 3a,b). Based on CL-age keys reported in previous studies on A. antennatus western Mediterranean fisheries [23,30–32,34,53,54] (Table 3), the age of each MPA group was assigned to cohorts from 0+ to 4+ in winter and from 1+ to 5+ in summer. The size of the first identified cohort (19.26 ± 1.77 mm) was slightly larger than the one reported by Carbonell [34] and looks similar to the smaller modal cohort indicated by D’Onghia et al. [23] for samples at the Ionian Sea collected in April and May. Though these latter authors did not use codend cover, but a stretched mesh of 20 mm in the codend and reduced proportions of collected females were in the range between 16 and 26 mm of CL, especially in April catches. Table 2. Description of the groups identified through the modal progression analysis of the carapace length (CL) frequency distributions of A. antennatus females, including sampling season, modal and age group, mean carapace length with standard deviation (SD), separation index (SI), number of females (N), and estimates of genetic diversity (excluding locus Aa818). Sampling Season Modal Group Age Group Mean CL ± SD (mm) SI N NA AR HO HE FIS Winter 1 0+ 19.26 ± 1.77 - 103 9.7 3.94 0.46 0.64 0.28 2 1+ 23.38 ± 2.23 2.06 69 8.3 3.88 0.49 0.64 0.24 * 3 2+ 33.51 ± 2.08 4.70 35 7.0 3.95 0.45 0.65 0.31 4 3+ 40.89 ± 2.54 3.19 39 7.2 3.71 0.41 0.63 0.34 5 4+ 49.12 ± 2.41 3.33 8 4.3 3.75 0.43 0.64 0.33 * Summer 1 1+ 27.26 ± 2.72 - 79 9.3 3.88 0.51 0.63 0.19 2 2+ 32.06 ± 1.97 2.05 76 9.2 3.95 0.51 0.63 0.20 3 3+ 42.53 ± 2.94 4.26 49 7.9 3.90 0.52 0.64 0.19 * 4 4+ 50.22 ± 2.97 2.60 41 7.7 3.83 0.47 0.62 0.24 * 5 5+ 62.09 ± 2.26 4.54 10 5.2 3.98 0.45 0.64 0.30 NA, Number of alleles; AR, allelic richness; HO, observed heterozygosity; HE, expected heterozygosity; FIS, inbreeding coefficient. * Significant departure from Hardy-Weinberg equilibrium after Bonferroni correction (α/110, p < 0.0005). Figure 2. Comparison of the mean carapace length (CL) of A. antennatus females based on commercial categories ( a ) and the groups derived from the modal progression analysis ( b ) in winter (filled circles) and summer (empty circles) for each group present in both campaigns. p< 0.05 obtained from one-way ANOVA and Post-hoc Scheffe test are indicated with an asterisk (*). Table 2. Description of the groups identified through the modal progression analysis of the carapace length (CL) frequency distributions of A. antennatus females, including sampling season, modal and age group, mean carapace length with standard deviation (SD), separation index (SI), number of females (N), and estimates of genetic diversity (excluding locus Aa818). Sampling Season Modal Group Age Group Mean CL ±SD (mm) SI N NAARHOHEFIS Winter 1 0+ 19.26 ±1.77 - 103 9.7 3.94 0.46 0.64 0.28 2 1+ 23.38 ±2.23 2.06 69 8.3 3.88 0.49 0.64 0.24 * 3 2+ 33.51 ±2.08 4.70 35 7.0 3.95 0.45 0.65 0.31 4 3+ 40.89 ±2.54 3.19 39 7.2 3.71 0.41 0.63 0.34 5 4+ 49.12 ±2.41 3.33 8 4.3 3.75 0.43 0.64 0.33 * Summer 1 1+ 27.26 ±2.72 - 79 9.3 3.88 0.51 0.63 0.19 2 2+ 32.06 ±1.97 2.05 76 9.2 3.95 0.51 0.63 0.20 3 3+ 42.53 ±2.94 4.26 49 7.9 3.90 0.52 0.64 0.19 * 4 4+ 50.22 ±2.97 2.60 41 7.7 3.83 0.47 0.62 0.24 * 5 5+ 62.09 ±2.26 4.54 10 5.2 3.98 0.45 0.64 0.30 N A , Number of alleles; A R , allelic richness; H O , observed heterozygosity; H E , expected heterozygosity; F IS , inbreeding coefficient. * Significant departure from Hardy-Weinberg equilibrium after Bonferroni correction (α/110, p< 0.0005). The comparison of the mean CL in winter and summer of each age group present in both campaigns (1+, 2+, 3+, and 4+) resulted in lower values in winter than in summer for all age groups except for the cohort 2+ but only significant in cohorts 1+ and 3+. In the case of the age group 2+ occurred the opposite; the mean CL was significantly higher in winter than in summer (Figure 2b). All females in the range of mean ± SD of each modal group was clearly considered as a member of the cohort and retained to have better baselines for individuals to make comparisons between cohorts and commercial sizes. Using this strategy, 77 out of 331 females captured in winter (23.3%) and 79 out of 334 of those caught in summer (23.7%) remained doubtful and were removed from cohort groups (Table 2). The agreement between these cohort baselines and the commercial sizes was substantial according to Cohen’s kappa either in winter samples (K = 0.75; 95% CI = 0.68–0.81) or in summer ones (K = 0.73; 95% CI = 0.67–0.80) (Table S1). Thus, each cohort baseline was mainly composed of females assigned to a single commercial size by the crew vessel, and only cohorts 3+ in winter and 4+ in summer were clear admixtures between two commercial size groups (Figure 3c,d). The results indicated that the medium commercial size in winter is composed of individuals from the 2+ and 3+ cohorts, and the large and extra-large summer females are also Genes 2022,13, 1186 7 of 17 admixtures of two consecutive cohorts (Figure 3a,b). The presence of cohort 0+ females in the small size group retained by the codend in winter indicated that a portion of these young individuals are captured even in a codend of a mesh size of 40 mm. The distribution of CL between each commercial category and the more related MPA group in winter is fairly coincident except for the extra-large females in summer that showed a mean CL value between the two older cohorts (Figure 4). Genes 2022, 13, x FOR PEER REVIEW 8 of 18 Figure 3. Size frequency distributions (carapace length, CL) of A. antennatus female groups in winter (a) and in summer (b) coupled with commercial size composition of each group derived from the modal progression analysis (MPA) in winter (c) and summer (d). Arrows indicate the CL mean computed for each age group by the MPA. All females in the range of mean ± SD of each modal group was clearly considered as a member of the cohort and retained to have better baselines for individuals to make comparisons between cohorts and commercial sizes. Using this strategy, 77 out of 331 females captured in winter (23.3%) and 79 out of 334 of those caught in summer (23.7%) remained doubtful and were removed from cohort groups (Table 2). The agreement between these cohort baselines and the commercial sizes was substantial according to Cohen’s kappa either in winter samples (K = 0.75; 95% CI = 0.68–0.81) or in summer ones (K = 0.73; 95% CI = 0.67–0.80) (Table S1). Thus, each cohort baseline was mainly composed of females assigned to a single commercial size by the crew vessel, and only cohorts 3+ in winter and 4+ in summer were clear admixtures between two commercial size groups (Figure 3c,d). The results indicated that the medium commercial size in winter is composed of individuals from the 2+ and 3+ cohorts, and the large and extra-large summer females are also admixtures of two consecutive cohorts (Figure 3a,b). The presence of cohort 0+ females in the small size group retained by the codend in winter indicated that a portion of these young individuals are captured even in a codend of a mesh size of 40 mm. The distribution of CL between each commercial category and the more related MPA group in winter is fairly coincident except for the extra-large females in summer that showed a mean CL value between the two older cohorts (Figure 4). Figure 3. Size frequency distributions (carapace length, CL) of A. antennatus female groups in winter ( a ) and in summer ( b ) coupled with commercial size composition of each group derived from the modal progression analysis (MPA) in winter ( c ) and summer ( d ). Arrows indicate the CL mean computed for each age group by the MPA. 3.2. Genetic Diversity and Genetic Divergence of A. antennatus Females A total of 665 A. antennatus females were genotyped for 12 microsatellite loci ( Table S2 ). Only locus Aa818 was ruled out from all subsequent analyses due to a null allele frequency > 0.2 in four commercial categories (Table S2) and six age groups (Table S3). Regarding commercial categories (Table 1), females with medium size reached the maximum mean A R (5.34, Table 1) among winter samples; meanwhile, the small size group displayed the highest H O and H E (0.492 and 0.644, respectively). The females from the large and extra-large size groups showed the lowest diversity values and were the only ones displaying significant departures from HWE (F IS = 0.284 and 0.303, respectively, Table 1). In summer, diversity indexes were fairly similar to winter ones, but small females achieved the highest value of A R (5.40) and large females showed the highest H O and H E (0.511 and 0.633, respectively). A significant departure from HWE was observed in the small, large, and extra-large females in summer (Table 1). These deviations were related to positive F IS values, indicating a deficit of heterozygosity, and affect commercial sizes composed of more than a cohort (Figure 3b). Genes 2022,13, 1186 8 of 17 Table 3. Comparison of the age groups of A. antennatus females with previous studies on Mediterranean populations. Reference Geographic Region Sampling Period Cohort 1 2 3 4 5 6 7 Present study Palamós (Spain, north-western Mediterranean Sea) Winter 2016 19.26 ±1.77 23.38 ±2.23 33.51 ±2.08 40.89 ±2.54 49.12 ±2.41 - - Summer 2016 - 27.26 ±2.72 32.06 ±1.97 42.53 ±2.94 50.22 ±2.97 62.09 ±2.26 García-Rodríguez (2003) [53] Gulf of Alicante (Spain, western Mediterranean Sea) January 1995–December 1998 - 25.65 33.66 43.19 51.16 - - Ibiza Channel (Spain, western Mediterranean Sea) January 1992–December 1994 - 29.18 37.56 45.75 54.1 - - Gulf of Vera (Spain, western Mediterranean Sea) January 1992–December 1994 - 29.1 - 43.8 52.6 - - Carbonell (2005) [34] Balearic Islands and other Mediterranean Sea regions and Atlantic Ocean data compilation 12 years 14–16 20–25 30 38–40 48–50 55–58 >60 Orsi-Relini et al. (2013) 1[32]Ligurian Sea (western Mediterranean Sea) 1994–2004 - 19–29 32–38 40–45 47–50 52 54 Vila-Gordillo (2001) [54]Alborán Sea (Spain, western Mediterranean Sea) June 1997 - 23.88 ±2.38 29.15 ±2.06 33.58 ±2.32 39.20 ±2.65 47.42 ±5.04 - Arculeo et al. (2011) [31] San Vito Lo Capo (Sicily, Italy, western Mediterranean Sea) June 2006–May 2007 - 25.25 36.19 46.44 57 - - Terrasini (Sicily, Italy, western Mediterranean Sea) June 2006–May 2007 - 23.91 32.29 42.86 51.85 - - D’Onghia et al. (2009) [23]North-western Ionian Sea (eastern Mediterranean Sea) April 2006 - 17.00 ±1.49 30.96 ±2.28 35.88 ±1.81 48.35 ±2.96 - - May 2006 - 18.16 ±2.59 31.00 ±2.39 39.71 ±2.85 45.69 ±2.56 52.37 ±2.06 - June 2006 - 20.79 ±2.63 30.05 ±1.50 - 42.10 ±4.45 51.43 ±1.60 - September 2006 - 24.35 ±2.71 - - 38.39 ±2.33 49.63 ±2.85 - Papaconstantinou and Kapiris (2001) [30] Greek Ionian Sea (eastern Mediterranean Sea) January–December 1997 - 30.0 - 41.0 49.0 54.0 - 1Data corresponds to the pooled data (MEDITS plus GRUND trawl surveys). Genes 2022,13, 1186 9 of 17 Genes 2022, 13, x FOR PEER REVIEW 9 of 18 Figure 4. Carapace length (CL) of A. antennatus female groups based on commercial categories (white boxes) and the modal progression analysis components (MPA, grey boxes) in winter (a) and summer (b). The line that cuts through the box indicates the median value. 3.2. Genetic Diversity and Genetic Divergence of A. antennatus Females A total of 665 A. antennatus females were genotyped for 12 microsatellite loci (Table S2). Only locus Aa818 was ruled out from all subsequent analyses due to a null allele frequency > 0.2 in four commercial categories (Table S2) and six age groups (Table S3). Regarding commercial categories (Table 1), females with medium size reached the maximum mean AR (5.34, Table 1) among winter samples; meanwhile, the small size group displayed the highest HO and HE (0.492 and 0.644, respectively). The females from the large and extra-large size groups showed the lowest diversity values and were the only ones displaying significant departures from HWE (FIS = 0.284 and 0.303, respectively, Table 1). In summer, diversity indexes were fairly similar to winter ones, but small females achieved the highest value of AR (5.40) and large females showed the highest HO and HE (0.511 and 0.633, respectively). A significant departure from HWE was observed in the small, large, and extra-large females in summer (Table 1). These deviations were related to positive FIS values, indicating a deficit of heterozygosity, and affect commercial sizes composed of more than a cohort (Figure 3b). In relation to age groups (Table 2), in winter, the highest mean AR and HE were achieved by group 2+ and the maximum HO by group 1+. Significant departures from HWE genotype expectations were observed in groups 1+ and 4+. In summer, group 5+ showed the maximum result of AR and HE, whereas the highest HO was achieved by group 3+. Significant departures from HWE were observed in groups 3+ and 4+. Pairwise FST between all-female commercial categories as well as age groups were low, ranging from 0.0000 to 0.0047 (Table 4). After Bonferroni correction, FST values between paired samples resulted in no significant differences. Figure 4. Carapace length (CL) of A. antennatus female groups based on commercial categories (white boxes) and the modal progression analysis components (MPA, grey boxes) in winter ( a ) and summer (b). The line that cuts through the box indicates the median value. In relation to age groups (Table 2), in winter, the highest mean A R and H E were achieved by group 2+ and the maximum H O by group 1+. Significant departures from HWE genotype expectations were observed in groups 1+ and 4+. In summer, group 5+ showed the maximum result of A R and H E, whereas the highest H O was achieved by group 3+. Significant departures from HWE were observed in groups 3+ and 4+. Pairwise F ST between all-female commercial categories as well as age groups were low, ranging from 0.0000 to 0.0047 (Table 4). After Bonferroni correction, F ST values between paired samples resulted in no significant differences. Table 4. Pairwise F ST among A. antennatus female groups (below diagonal) and p-values (above diagonal). Commercial Categories Win Juv Win S Win M Win L Win XL Sum S Sum M Sum L Sum XL Win Juv - 0.1600 0.8300 0.2100 0.4200 Sum S - 0.7750 0.1167 0.9083 Win S 0.0030 - 0.9350 0.0150 0.6650 Sum M 0.0000 - 0.2583 0.7083 Win M 0.0008 0.0000 - 0.5300 0.9250 Sum L 0.0039 0.0015 - 0.4583 Win L 0.0018 0.0003 0.0000 - 0.5050 Sum XL 0.0000 0.0000 0.0000 - Win XL 0.0047 0.0000 0.0000 0.0000 - Modal Progression Analysis Groups Win 0+ Win 1+ Win 2+ Win 3+ Win 4+ Sum 1+ Sum 2+ Sum 3+ Sum 4+ Sum 5+ Win 0+ - 0.3250 0.6100 0.3150 0.3250 Sum 1+ - 0.9250 0.0700 0.9650 0.8350 Win 1+ 0.0023 - 0.3250 0.0400 0.3200 Sum 2+ 0.0000 - 0.5270 1.0000 0.4250 Win 2+ 0.0000 0.0000 - 0.0850 0.6950 Sum 3+ 0.0000 0.0000 - 0.8400 0.5450 Win 3+ 0.0000 0.0017 0.0000 - 0.6800 Sum 4+ 0.0000 0.0000 0.0000 - 0.3900 Win 4+ 0.0000 0.0000 0.0000 - Sum 5+ 0.0000 0.0000 0.0000 0.0000 - Win, winter; Sum, summer; J, juveniles; S, small; M, medium; L, large; XL, extra-large. 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