INTRODUCTIONTop

The spider crabs of the genus Maja Lamarck, 1801 (Majoidea: Majidae), with around 20 described species worldwide, are represented on European coasts by four species: M. brachydactyla (Balss, 1922), M. crispata (Risso, 1827), M. goltziana (D’Oliveira, 1888) and M. squinado (Herbst, 1788) (Neumann 1998Neumann V. 1998. A review of the Maja squinado (Crustacea: Decapoda: Brachyura) species-complex with a key to the eastern Atlantic and Mediterranean species of the genus. J. Nat. Hist. 32: 1667-1684., Ng et al. 2008Ng P.K.L., Guinot D., Davie P.J.F. 2008. Systema Brachyurorum: Part I. An annotated checklist of extant Brachyuran crabs of the world. Raffles Bull. Zool. 17 (Suppl.): 1-286., Sotelo et al. 2008Sotelo G., Morán P., Posada D. 2008. Genetic identification of the northeastern Atlantic spiny spider crab as Maja brachydactyla Balss, 1922. J. Crust. Biol. Biology 28: 76-81., 2009Sotelo G., Morán P., Posada D. 2009. Molecular phylogeny and biogeographic history of the European Maja spider crabs (Decapoda, Majidae). Mol. Phylogenet. Evol. 53: 314-319.). M. brachydactyla and M. squinado are the largest European species of the genus and are found from subtidal areas to about 90 m depth (De Kergariou 1984De Kergariou G. 1984. L’araignée de mer, H. Biologie et exploitation. La Pêche maritime 1279: 575-583., Števčić 1973Števčić Z. 1973. Les migrations de l’Araignée de mer. Rapport Commission International pour l’Exploration Scientifique de la Mediterranée 21: 597-598.). M. brachydactyla inhabits the eastern Atlantic from the British Isles to Senegal and has a high commercial value, while M. squinado inhabits the Mediterranean and is considered an endangered species (UNEP 1996UNEP 1996. Acta final de la reunión de plenipotenciarios sobre los anexos del protocolo referente a las zonas especialmente protegidas y la diversidad biológica en el Mediterráneo. UNEP(OCA)/MED IG 10/4, 2 diciembre 1996., Freire et al. 2002Freire J., Bernárdez C., Corgos A., et al. 2002. Management strategies for sustainable invertebrate fisheries in coastal ecosystems of Galicia (NW Spain). Aquat. Ecol. 36: 41-50., Martín et al. 2012Martín P., Maynou F., Stelzenmüller V., et al. 2012. A small-scale fishery near a rocky littoral marine reserve in the northwestern Mediterranean (Medes Islands) after two decades of fishing prohibition. Sci. Mar. 76: 607-618., Sotelo et al. 2008Sotelo G., Morán P., Posada D. 2008. Genetic identification of the northeastern Atlantic spiny spider crab as Maja brachydactyla Balss, 1922. J. Crust. Biol. Biology 28: 76-81., Guerao and Rotllant 2010Guerao G., Rotllant G. 2010. Development and growth of the early juveniles of the spider crab Maja squinado (Brachyura: Majoidea) in an individual culture system. Aquaculture 307: 105-110., Guerao et al. 2011Guerao G., Andree K.B., Froglia C., et al. 2011. Identification of european species of Maja (Decapoda, Brachyura, Majidae): RFLP analyses of COI mtDNA and morphological considerations. Sci. Mar. 75: 129-134., Abelló et al. 2014Abelló P., Guerao G., Salmerón F., et al. 2014. Maja brachydactyla (Brachyura: Majidae) in the western Mediterranean. Mar. Biodivers. Rec. 7: 1-5.), although it was abundant until the first half of the 20th century (Pons Muñoz 1994Pons Muñoz J.M. 1994. Menorca y su pesca. Arial.). Until recently, M. brachydactyla was considered a synonym of M. squinado (Zariquiey-Álvarez 1968Zariquiey-Álvarez R. 1968. Crustáceos Decápodos Ibéricos. Invest. Pesq. 32: 1-510., Neumann 1996Neumann V. 1996. Comparative investigations on the systematics and taxonomy of European Maja species (Decapoda, Brachyura, Majidae). Crustaceana 69: 821-852., 1998Neumann V. 1998. A review of the Maja squinado (Crustacea: Decapoda: Brachyura) species-complex with a key to the eastern Atlantic and Mediterranean species of the genus. J. Nat. Hist. 32: 1667-1684., Ng et al. 2008Ng P.K.L., Guinot D., Davie P.J.F. 2008. Systema Brachyurorum: Part I. An annotated checklist of extant Brachyuran crabs of the world. Raffles Bull. Zool. 17 (Suppl.): 1-286.), but molecular studies indicated that M. squinado is more closely related to M. crispata than to M. brachydactyla (Sotelo et al. 2009Sotelo G., Morán P., Posada D. 2009. Molecular phylogeny and biogeographic history of the European Maja spider crabs (Decapoda, Majidae). Mol. Phylogenet. Evol. 53: 314-319.). Molecular analysis also revealed that the divergence between M. brachydactyla and M. squinado-M. crispata clade occurred in the Miocene (9.5 mya) and the divergence between M. squinado and M. crispata, occurred in the Pliocene (3.9 mya). Accordingly, the large morphological resemblance between M. brachydactyla and M. squinado could be also due to convergent evolution (see Neumann 1996Neumann V. 1996. Comparative investigations on the systematics and taxonomy of European Maja species (Decapoda, Brachyura, Majidae). Crustaceana 69: 821-852., 1998Neumann V. 1998. A review of the Maja squinado (Crustacea: Decapoda: Brachyura) species-complex with a key to the eastern Atlantic and Mediterranean species of the genus. J. Nat. Hist. 32: 1667-1684., Sotelo et al. 2008Sotelo G., Morán P., Posada D. 2008. Genetic identification of the northeastern Atlantic spiny spider crab as Maja brachydactyla Balss, 1922. J. Crust. Biol. Biology 28: 76-81., 2009Sotelo G., Morán P., Posada D. 2009. Molecular phylogeny and biogeographic history of the European Maja spider crabs (Decapoda, Majidae). Mol. Phylogenet. Evol. 53: 314-319. for taxonomic status). A diagnostic tool was developed to identify the four species of Maja in Europe with morphological and molecular methods (Guerao et al., 2011Guerao G., Andree K.B., Froglia C., et al. 2011. Identification of european species of Maja (Decapoda, Brachyura, Majidae): RFLP analyses of COI mtDNA and morphological considerations. Sci. Mar. 75: 129-134.). Adults of M. brachydactyla and M. squinado are commercially exploited species and a simple relationship between carapace length (CL) and antorbital spine length could be used to differentiate the two species.

Marine brachyuran crabs are usually opportunistic consumers (Števčić 1967Števčić Z. 1967. Le complexe de la nourriture de l’araignée de mer Maja squinado. Helgolander Wiss. Meer. 15: 630-636., Choy 1986Choy C. 1986. Natural diet and feeding habits of the crabs Liocarcinus puber and L. holsatus (Decapoda, Brachyura, Portunidae). Mar. Ecol. Prog. Ser. 31: 87-99., Abelló and Cartes 1987Abelló P., Cartes J. 1987. Observaciones sobre la alimentación de Liocarcinus depurator (L.) (Brachyura: Portunidae) en el Mar Catalán. Invest. Pesq. 51(Supl. 1): 413-419., Abelló 1989Abelló P. 1989. Feeding habits of Macropipus tuberculatus (Brachyura, Portunidae) off the Catalan coast (NW Mediterranean). Misc. Zool. 13: 45-50.Abelló P., Cartes J. 1987. Observaciones sobre la alimentación de Liocarcinus depurator (L.) (Brachyura: Portunidae) en el Mar Catalán. Invest. Pesq. 51(Supl. 1): 413-419.) and this is also true for M. brachydactyla according to studies based on stomach contents (De Kergariou 1974De Kergariou G. 1974. Régime alimentaire de Maja squinado. International Council for the Exploration of the Sea 1974/K, 36, 6 pp., Bernárdez et al. 2000Bernárdez C., Freire J., González-Gurriarán E. 2000. Feeding of the spider crab Maja squinado in rocky subtidal areas of the Ría de Arousa (north-west Spain). J. Mar. Biol. Assoc. U.K. 80: 95-102.): M. brachydactyla feed on a large variety of preys, consuming 50% of algae and a mixture of animals (mollusks, crustaceans, echinoderms, etc.). De Kergariou (1974)De Kergariou G. 1974. Régime alimentaire de Maja squinado. International Council for the Exploration of the Sea 1974/K, 36, 6 pp. and Bernárdez et al. (2000)Bernárdez C., Freire J., González-Gurriarán E. 2000. Feeding of the spider crab Maja squinado in rocky subtidal areas of the Ría de Arousa (north-west Spain). J. Mar. Biol. Assoc. U.K. 80: 95-102. found differences in the diet between males and females and between juveniles and adults which they attributed to an uneven bathymetric distribution, a pattern typical of many large brachyuran crabs (e.g. Bennett and Brown 1983Bennett D.B., Brown C.G. 1983. Crab (Cancer pagurus) migrations in the English Channel. J. Mar. Biol. Assoc. U.K. 63: 371-398., Hines et al. 1987Hines A.H., Lipcius R.N., Haddon A.M. 1987. Population dynamics and habitat partitioning by size, sex, and molt stage of blue crabs Callinectes sapidus in a subestuary of central Chesapeake Bay. Mar. Ecol. Prog. Ser. 36: 55-64., Spivak et al. 1994Spivak E., Anger K., Luppi T., et al. 1994. Distribution and habitat preferences of two grapsid crab species in Mar Chiquita Lagoon (Province of Buenos Aires, Argentina). Helgolaender Meeresun. 48: 59-78.). However, recent research using stable isotopes has failed to find any differences between sexes in diet or habitat use patterns either in adults (Bodin et al. 2007Bodin N., Le Loc’h F., Hily C., et al. 2007. Variability of stable isotope signatures (δ¹³C and δ¹⁵N) in two spider crab populations (Maja brachydactyla) in Western Europe. J. Exp. Mar. Biol. Ecol. 343: 149-157.) or juveniles (Bodin et al. 2007Bodin N., Le Loc’h F., Hily C., et al. 2007. Variability of stable isotope signatures (δ¹³C and δ¹⁵N) in two spider crab populations (Maja brachydactyla) in Western Europe. J. Exp. Mar. Biol. Ecol. 343: 149-157., Freire et al. 2009Freire J., Carabel S., Verísimo P., et al. 2009. Patterns of juvenile habitat use by the spider crab Maja brachydactyla as revealed by stable isotope analyses. Sci. Mar. 73: 39-49.).

Stable isotope ratios of carbon and nitrogen are widely used in ecology for the study of trophic relationships and habitat use patterns, because the stable isotope ratios in a consumer’s tissues reflect those in the diet, which in turn depends on the relative availability of the light and heavy isotopes of each element for primary producers (Hobson 1999Hobson K.A. 1999. Tracing origins and migration of wildlife using stable isotopes: a review. Oecologia 120: 314-326., Koch 2008Koch P.L. 2008. Isotopic Study of the Biology of Modern and Fossil Vertebrates. In: Michener, R., Lajtha, K. (eds) Stable Isotopes in Ecology and Environmental Science, Oxford, Blackwell Publishing Ltd, pp. 99-154., Graham et al. 2010Graham B.S., Koch P.L., Newsome S.D., et al. 2010. Using isoscapes to trace the movements and foraging behavior of top predators in oceanic ecosystems. In: West J.B., Bowen G.J., Dawson T.E., et al. (eds), Isoscapes: Understanding Movement, Pattern and Process on Earth Through Isotope Mapping. Springer, New York, pp. 299-318.). Marine macrophytes are typically enriched in ¹³C compared with phytoplankton, but macrophytes occur only in shallow waters and hence the stable isotope ratio of carbon consumers typically decreases with depth in coastal areas (e.g. Cardona et al. 2007Cardona L., Revelles M., Sales M., et al. 2007. Meadows of the seagrass Posidonia oceanica are a significant source of organic matter for adjoining ecosystems. Mar. Ecol. Prog. Ser. 335: 123-131., Drago et al. 2009Drago M., Cardona L., Crespo E.A., et al. 2009. Ontogenic dietary changes in South American sea lions. J. Zool. 279: 251-261., Freire et al. 2009Freire J., Carabel S., Verísimo P., et al. 2009. Patterns of juvenile habitat use by the spider crab Maja brachydactyla as revealed by stable isotope analyses. Sci. Mar. 73: 39-49., Pinela et al. 2010Pinela A.M., Borrell A., Cardona L., et al. 2010. Stable isotope analysis reveals habitat partitioning among marine mammals off the NW African coast and unique trophic niches for two globally threatened species. Mar. Ecol. Prog. Ser. 416: 295-306.). Accordingly, the stable isotope ratio of carbon can be used to explore intraspecific differences in bathymetric distribution by sex or age, although tissue selection is critical because stable isotope turnover rates are tissue-dependent (Martínez del Río et al. 2009Martínez del Río C., Wolf N., Carleton S.A., et al. 2009. Isotopic ecology ten years after a call for more laboratory experiments. Biol. Res. 84: 91-111.).

Previous studies on M. brachydactyla using stable isotope ratios concluded that no sexual difference existed in diet or habitat use (Freire et al. 2009Freire J., Carabel S., Verísimo P., et al. 2009. Patterns of juvenile habitat use by the spider crab Maja brachydactyla as revealed by stable isotope analyses. Sci. Mar. 73: 39-49., Bodin et al. 2007Bodin N., Le Loc’h F., Hily C., et al. 2007. Variability of stable isotope signatures (δ¹³C and δ¹⁵N) in two spider crab populations (Maja brachydactyla) in Western Europe. J. Exp. Mar. Biol. Ecol. 343: 149-157.), in contrast with earlier studies based on stomach contents (De Kergariou 1974De Kergariou G. 1974. Régime alimentaire de Maja squinado. International Council for the Exploration of the Sea 1974/K, 36, 6 pp., Bernárdez et al. 2000Bernárdez C., Freire J., González-Gurriarán E. 2000. Feeding of the spider crab Maja squinado in rocky subtidal areas of the Ría de Arousa (north-west Spain). J. Mar. Biol. Assoc. U.K. 80: 95-102.). This incongruence may be due to the fact that they analysed tissues with a relatively high turnover rate sampled from specimens collected from the same habitat. This experimental design was useful to assess the existence of dietary differences between groups in the same habitat, but likely failed to detect any previous habitat segregation.

Tissues that remain inert after synthesis (e.g. feathers, hair, nails and arthropod exoskeleton) are a good choice for the study of habitat use patterns, as they record the unmodified stable isotope ratios of the habitat where they were formed (Hirons et al. 2001Hirons A., Schell D.M., St Aubin D.J. 2001. Growth rates of vibrissae of harbor seals (Phoca vitulina) and Steller sea lions (Eumetopias jubatus). Can. J. Zool. 79: 1053-1061., Cherel and Hobson 2005Cherel Y., Hobson K.A. 2005. Stable isotopes, beaks, and predators: a new tool to study the trophic ecology of cephalopods, including giant and colossal squids. Proc. R. Soc. B 272: 1601-1607.). The organic matter in the exoskeleton, a mixture of chitin and proteins, offers a good alternative, as the stable isotope ratios of arthropod exoskeleton remain stable after synthesis (Hobson et al. 1999Hobson K.A. 1999. Tracing origins and migration of wildlife using stable isotopes: a review. Oecologia 120: 314-326.) and spider crabs have a terminal moult, i.e. they do not moult after the moult of maturity. The last intermoult period is the longest representing several months (González-Gurriarán et al. 1995González-Gurriarán E., Freire J., Parapar J., et al. 1995. Growth at moult and moulting seasonality of the spider crab, Maja squinado (Herbst) (Decapoda: Majidae) in experimental conditions: implications for juvenile life history. J. Exp. Mar. Biol. Ecol. 189: 183-203., Corgos et al. 2007Corgos A., Sampedro M.P., González-Gurriarán E., et al. 2007. Growth at moult, intermoult period and moulting seasonality of the spider crab Maja brachydactyla: combining information from mark-recapture and experimental studies. J. Crust. Biol. 27: 255-262.). During the moulting process, energy reserves are used to form the exoskeleton of these crabs (Vernet and Charmantier 1994Vernet G., Charmantier-Daures M. 1994. Mue, Autotomie et régénèration. In: Masson (ed) Traité de Zoologie. Anatomie, Systématique, Biologie. Tome VII, Fascicule I: Crustacés. Morphologie, Physiologie, Reproduction, Systématique. Paris, France: pp. 107-155.), so the imprint of feeding during the last intermoult will be recorded in the carapace of the crabs. Thus, stable isotope ratios in the carapace are expected to represent a long-term signal of habitat use, compared to muscle and hepatopancreas tissues.

The aim of this study was to test the hypothesis that, within every population of M. brachydactyla and M. squinado, males and females forage at the same trophic level but differ in bathymetric distribution, and that this pattern is consistent across populations and species.

MATERIALS AND METHODSTop

Sampled individuals

In 2007-2009, 98 adult specimens of M. brachydactila and M. squinado (CL=141±20 mm) were collected by commercial fishery vessels in the Atlantic and Mediterranean Sea (Table 1, Fig. 1). Males and females were collected during the same sampling trials from all localities, except from Morocco, where only males were available.

Stable isotope analysis

Exoskeleton samples were collected from the propodus and dactylus segments without debris areas of the pereiopods of individuals from each population and sex. Exoskeletons were chosen to have at least a one-year signature integrating fed intake in a broad area of distribution where specimens lived and fed.

Samples were stored frozen at –20°C, thawed, and dried at 60°C for 48-72 h. Then, each sample was subdivided in two sub-samples. Lipids were removed from one of them by rinsing the powdered samples several times with a 2:1 chloroform:methanol solution (Bligh and Dyer 1959Bligh E.G., Dyer W.J. 1959. A rapid method of total lipid extraction and purification. Can. J, Biochem. Phys. 37: 911-917.). The same sub-samples were then treated for decarbonation with a 0.5 M hydrochloric acid (HCl) solution (e.g. Drago et al. 2009Drago M., Cardona L., Crespo E.A., et al. 2009. Ontogenic dietary changes in South American sea lions. J. Zool. 279: 251-261., Eder et al. 2012Eder E., Ceballos A., Martins S., et al. 2012. Foraging dichotomy in loggerhead sea turtles Caretta caretta off northwestern Africa. Mar. Ecol. Prog. Ser. 470: 113-122.). As lipid extraction and decarbonation with HCl may affect the δ¹⁵N values (Bunn et al. 1995Bunn S.E., Loneragan N.R., Kempster M.A. 1995. Effects of acid washing on stable isotope ratios of C and N in penaeid shrimp and seagrass: implications for food-web studies using multiple stable isotopes. Limnol. Oceanogr. 40: 622-625., Yokoyama et al. 2005Yokoyama H., Tamaki A., Harada K., et al. 2005. Variability of diet-tissue isotopic fractionation in estuarine macrobenthos. Mar. Ecol. Pro. Ser. 296: 115-128.), the untreated exoskeleton samples were used to measure δ¹⁵N. One milligram of sample was used for δ¹⁵N determination and 0.5-0.7 mg of sample was used for δ¹³C determination. The samples were weighed in tin cups (3.3-5 mm) and combusted in a continuous-flow isotope-ratio mass spectrometer (Flash 1112 IRMS Delta C Series EA, Thermo Finnigan, Bremen, Germany). The samples were processed at the Serveis Científics i Tecnològics of the University of Barcelona.

Isotopic composition was expressed in the standard δ notation in parts per thousand (‰) relative to predefined international standards, V-PDB (Vienna Pee Dee Belemnite) calcium carbonate for δ¹³C and atmospheric N₂ (air) for δ¹⁵N, according to:

δX = [(R_sample / R_standard) –1] 10³

where X is ¹³C or ¹⁵N, R_sample is the heavy-light isotope ratio of the sample (¹³C/¹²C or ¹⁵N/¹⁴N) and R_standard is the heavy-light isotope ratio in the reference standards. The conventional use of these internationally accepted standards with relatively low and high levels of ¹³C and ¹⁵N, respectively, results in negative carbon and positive nitrogen isotope values. The international stable isotope secondary standards of known ¹³C/¹²C ratios, as given by the International Atomic Energy Agency (IAEA, Vienna, Austria), namely polyethylene (IAEA CH₇, δ¹³C=–31.8‰), L-glutamic acid (IAEA USGS₄₀, δ¹³C=–26.3‰) and sucrose (IAEA CH₆, δ¹³C=–10.4‰), were used for calibration at a precision of 0.2‰. For nitrogen, international stable isotope secondary standards of known ¹⁵N/¹⁴N ratios, namely (NH₄)₂SO₄ (IAEA N1, δ¹⁵N=+0.4‰ and IAEA N2, δ¹⁵N=+20.3‰), L-glutamic acid (δ¹⁵N=–4.5‰), and caffeine (IAEA 600 δ¹⁵N=+.0‰), were used to a precision of 0.3‰.

Data analysis

Two-way analysis of variance (ANOVA) (sex x population) was used to test whether males and females differed in δ¹³C and δ¹⁵N values consistently across populations and species. As only males were available from Morocco, one-way ANOVA was used to analyse the effect of population on the stable isotope ratios of males.

We estimated the isotopic niche width using the convex hull area in the isotopic space defined by δ¹³C and δ¹⁵N (Layman et al. 2007Layman C.A., Arrington D.A., Montanä C.G., et al. 2007. Can stable isotope ratios provide for community-wide measures of trophic structure? Ecology 88: 42-48). However, the use of Euclidean methods, such as convex hulls to define the isotopic niche space of a species in a community (Layman et al. 2007Layman C.A., Arrington D.A., Montanä C.G., et al. 2007. Can stable isotope ratios provide for community-wide measures of trophic structure? Ecology 88: 42-48), is subject to sampling biases and sensitive to sample size (Jackson et al. 2011Jackson A.L., Parnell A.C., Inger R., et al. 2011. Comparing isotopic niche widths among and within communities: SIBER - Stable Isotope Bayesian Ellipses in R. J. Anim. Ecol. 80: 595-602.). The standard ellipse areas estimated by Bayesian inference can incorporate uncertainties such as sampling biases and small sample sizes into niche metrics (Jackson et al. 2011Jackson A.L., Parnell A.C., Inger R., et al. 2011. Comparing isotopic niche widths among and within communities: SIBER - Stable Isotope Bayesian Ellipses in R. J. Anim. Ecol. 80: 595-602.). Then we estimated the width of the isotopic niche at the population level using the Bayesian standard ellipse area by the function SIBER (Stable Isotope Bayesian Ellipses in R; Jackson et al. 2011Jackson A.L., Parnell A.C., Inger R., et al. 2011. Comparing isotopic niche widths among and within communities: SIBER - Stable Isotope Bayesian Ellipses in R. J. Anim. Ecol. 80: 595-602.) of the library SIAR (Stable Isotope Analysis in R; Parnell et al. 2010Parnell A.C, Inger R., Bearhop S., et al. 2010. Source Partitioning Using Stable Isotopes: Coping with Too Much Variation. PLoS One 5: e9672.) in the free software R (R Core Team 2013R Core Team. 2013. R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. URL: http://www.R-project.org). Furthermore, we calculated the magnitude of the isotopic overlap between sexes in each population based on 100000 posterior draws of the standard ellipse areas corrected for small sample size parameters (Jackson et al. 2011Jackson A.L., Parnell A.C., Inger R., et al. 2011. Comparing isotopic niche widths among and within communities: SIBER - Stable Isotope Bayesian Ellipses in R. J. Anim. Ecol. 80: 595-602.). This approach, based on Markov-chain Monte Carlo simulation, assigns measures of uncertainty to construct parameters of ellipses in a similar way to a bootstrap.

Discriminant Analysis was applied to δ¹³C and δ¹⁵N to test their reliability when used for individual assignation to the population of origin.

RESULTSTop

Figure 2 shows the δ¹³C and δ¹⁵N values of males and females of M. brachydactyla and M. squinado from the five considered populations. Males were consistently depleted in ¹³C when compared with females from the same population (ANOVA, model: F_7.64<0.001, sex: F_1.64<0.023, population, F_3.64<0.001). Furthermore, differences among populations were unrelated to species identity, as a Tukey post-hoc test revealed differences between the δ¹³C of M. brachydactyla from Ceuta and Galicia, but no differences between the δ¹³C of M. brachydactyla from Ceuta and that of M. squinado from Ancona or between the δ¹³C of M. squinado from Catalonia and the δ¹³C of M. brachydactyla from Galicia (Table 2). That geographic pattern did not change when the males from Morocco were incorporated into the analysis and all the females were removed (ANOVA, model: F_4.42<0.001), as a Tukey post-hoc test revealed differences between the δ¹³C of M. brachydactyla from Ceuta and those from Galicia and Morocco but no differences between the δ¹³C of M. squinado from Catalonia and the δ¹³C of M. brachydactyla from Galicia and those from Morocco.

Statistically significant differences were observed among populations for δ¹⁵N values, but differences between sexes were not consistent across populations, although results were on the verge of similarity (ANOVA, model: F_7.64<0.001, sex: F_1.64<0.083, population, F_3.64<0.001). Differences among populations were also unrelated to species identity, as a Tukey post-hoc test revealed differences between the δ¹⁵N of M. brachydactyla from Ceuta and Galicia but no differences between the δ¹⁵N of M. squinado from Catalonia and the δ¹⁵N of M. brachydactyla from Galicia (Table 2). The same was true when the females were removed from the analysis and the males from Morocco were incorporated, as only the males of M. squinado from Ancona were statistically different from the males of M. squinado from Catalonia and those of M. brachydactyla from Ceuta, Morocco and Galicia (ANOVA, model: F_4.42<0.001: Table 2).

The Bayesian ellipses of females were larger than those of males in the population of M. brachydactyla from Galicia and in the two populations of M. squinado from Catalonia and Ancona, whereas the opposite was true for the population of M. brachydactyla from Ceuta (Fig. 3). Everywhere, the overlap of the Bayesian ellipses of both sexes was highly asymmetrical. In the population of M. brachydactyla from Galicia and in the two populations of M. squinado from Catalonia and Ancona, the overlap area represented a larger fraction of the surface of the male ellipses than that of the female ellipses (42.1% vs. 11.3%, 74.3% vs. 52. 9% and 32.6% vs. 19.7% respectively), whereas the opposite was true for the population of M. brachydactyla from Ceuta (males, 13.8%; females, 53.5%).

Discriminant analysis ignoring species and sex classified correctly only 53.7% of the samples (Fig. 4), which is significantly higher than expected by chance but too low for traceability. Individual assignation improved when discriminant analysis was run independently for each species, as 81.6% of the samples of M. squinado and 66. 7% of those of M. brachydactyla were classified correctly. The highest accuracy was achieved when the discriminant analysis was run independently for the males and females of each species (Fig. 4). Figure 4 shows the accuracy of individual assignments for each species, sex and locality. The lowest accuracy was achieved for males of M. brachydactyla from Galicia (70%) and the highest for the females of the same species from Ceuta (100%) and males of M. squinado from Ancona (100%).

DISCUSSIONTop

Maja brachydactyla is an opportunistic species that consumes any range of food items that is available (Števčić 1967Števčić Z. 1967. Le complexe de la nourriture de l’araignée de mer Maja squinado. Helgolander Wiss. Meer. 15: 630-636., De Kergariou 1974De Kergariou G. 1974. Régime alimentaire de Maja squinado. International Council for the Exploration of the Sea 1974/K, 36, 6 pp., Bernárdez et al. 2000Bernárdez C., Freire J., González-Gurriarán E. 2000. Feeding of the spider crab Maja squinado in rocky subtidal areas of the Ría de Arousa (north-west Spain). J. Mar. Biol. Assoc. U.K. 80: 95-102.). Although there are no studies on the feeding habits of M. squinado, it seems quite possible that this species is also an opportunistic feeder. Both species have a complex biological cycle, with varying habitat requirements and changes in behaviour depending on the growth phase and the specific time period in the life history (Števčić 1973Števčić Z. 1973. Les migrations de l’Araignée de mer. Rapport Commission International pour l’Exploration Scientifique de la Mediterranée 21: 597-598., González-Gurriarán and Freire 1994González-Gurriarán E., Freire J. 1994. Movement patterns and habitat utilization in the spider crab Maja squinado (Herbst) (Decapoda, Majidae) measured by ultrasonic telemetry. J. Exp. Mar. Biol. Ecol. 184: 269-291., Hines et al. 1995Hines A.H., Wolcott T.G., González-Gurriarán E., et al. 1995. Movement Patterns and Migrations in Crabs: Telemetry of Juvenile and Adult Behaviour in Callinectes sapidus and Maja squinado. J. Mar. Biol. Assoc. U.K. 75: 27-42., González-Gurriarán et al. 2002González-Gurriarán E., Freire J., Bernárdez C. 2002. Migratory patterns of female spider crabs Maja squinado detected using electronic tags and telemetry. J. Crust. Biol. 22: 91-97.). The juvenile stage lasts approximately two years until a terminal moult, at which time sexual maturity is achieved (González-Gurriarán et al. 1995González-Gurriarán E., Freire J., Parapar J., et al. 1995. Growth at moult and moulting seasonality of the spider crab, Maja squinado (Herbst) (Decapoda: Majidae) in experimental conditions: implications for juvenile life history. J. Exp. Mar. Biol. Ecol. 189: 183-203., Hines et al. 1995Hines A.H., Wolcott T.G., González-Gurriarán E., et al. 1995. Movement Patterns and Migrations in Crabs: Telemetry of Juvenile and Adult Behaviour in Callinectes sapidus and Maja squinado. J. Mar. Biol. Assoc. U.K. 75: 27-42., Sampedro et al. 1999Sampedro M.P., González-Gurriarán E., Freire J., et al. 1999. Morphometry and sexual maturity in the spider crab Maja squinado (Decapoda, Majidae) in Galicia, Spain. J. Crust. Biol. 19: 578-592.). After the terminal moult, the adult individuals migrate to deeper waters (González-Gurriarán et al. 2002González-Gurriarán E., Freire J., Bernárdez C. 2002. Migratory patterns of female spider crabs Maja squinado detected using electronic tags and telemetry. J. Crust. Biol. 22: 91-97.), where mating probably occurs. Only the adult females return to the shallower waters to spawn. In terms of carbon and nitrogen stable isotopic composition, tissues with high turnover rates (muscle and hepatopancreas) reflect the diet in the short term, and could be indicative of differences in diet during the days or weeks previous to their capture and analysis (Freire et al. 2009Freire J., Carabel S., Verísimo P., et al. 2009. Patterns of juvenile habitat use by the spider crab Maja brachydactyla as revealed by stable isotope analyses. Sci. Mar. 73: 39-49.). However, the carapace provides an integrated signal of the animal assimilated diet on a long-term time scale (Hobson et al. 1999Hobson K.A., Wassena L.I., Taylo O.R. 1999. Stable isotopes (δD and δ¹³C) are geographic indicators of natal origins of monarch butterflies in eastern North America. Oecologia 120: 397-404.). In the present study, all animals were post-pubertal specimens and their exoskeleton was formed before the terminal moult (Sampedro et al. 1999Sampedro M.P., González-Gurriarán E., Freire J., et al. 1999. Morphometry and sexual maturity in the spider crab Maja squinado (Decapoda, Majidae) in Galicia, Spain. J. Crust. Biol. 19: 578-592.). Therefore, isotope signatures may provide insight into the trophic preferences during the last intermoult period before the animals reached sexual maturity (<3 months; Corgos et al. 2007Corgos A., Sampedro M.P., González-Gurriarán E., et al. 2007. Growth at moult, intermoult period and moulting seasonality of the spider crab Maja brachydactyla: combining information from mark-recapture and experimental studies. J. Crust. Biol. 27: 255-262.).

As expected, the stable isotope ratios of carbon and nitrogen in M. brachydactyla and M. squinado differed dramatically among populations, independently of species identity, hindering the capacity of stable isotopes for traceability unless species and sex are included in the analysis as priors. The relative abundance of ¹⁵N in marine primary producers and their consumers depends on the balance of fixed and recycled nitrogen and is usually very low in oligotrophic regions (Graham et al. 2010Graham B.S., Koch P.L., Newsome S.D., et al. 2010. Using isoscapes to trace the movements and foraging behavior of top predators in oceanic ecosystems. In: West J.B., Bowen G.J., Dawson T.E., et al. (eds), Isoscapes: Understanding Movement, Pattern and Process on Earth Through Isotope Mapping. Springer, New York, pp. 299-318, Somes et al. 2010Somes C.J., Schmittner A., Galbraith E.D., et al. 2010. Simulating the global distribution of nitrogen isotopes in the ocean. Global Biogeochem. Cy. 24, GB4019.), so it is lower in the Mediterranean than in the adjoining Atlantic (e.g. Borrell et al. 2006Borrell A., Aguilar A., Tornero V., et al. 2006. Organochlorine compounds and stable isotopes indicate bottlenose dolphin subpopulation structure around the Iberian Peninsula. Environ. Int. 32: 516-523., Gómez-Díaz and González-Solís 2007Gómez-Díaz E., González-Solís J. 2007. Geographic assignment of sea birds to their origin: combining morphologic, genetics, and biochemical analyses. Ecol. Appl. 17: 1484-1498., Giménez et al. 2013Giménez J., Gómez-Campos E., Borrell A., et al. 2013. Isotopic evidence of limited exchange between Mediterranean and eastern North Atlantic fin whales. Rapid Commun. Mass Spectrom. 27: 1801-1806.). Actually, the lowest δ¹⁵N values were observed in the specimens of M. squinado from Ancona, likely to be captured in the northern Ionian Sea, a region characterized by very low δ¹⁵N values in particulate organic matter (Pantoja et al. 2002Pantoja S., Repeta D.J., Sachs J.P., et al. 2002. Stable isotope constraints on the nitrogen cycle of the Mediterranean Sea water column. Deep-Sea Res. I 49: 1609-1621.). Moreover, the relative abundance of ¹³C in marine primary producers and their consumers depends on the availability of inorganic carbon within their boundary layer, which in turn depends on primary productivity, water turbulence, and plant architecture (Post 2002Post D.M. 2002. Using stable isotopes to estimate trophic position: models, methods, and assumption. Ecology 83: 703-718.). Interestingly, the highest values were observed in specimens from Morocco, Galicia and Catalonia, which are the most productive of the five regions considered (Longhurst 1998Longhurst A. 1998. Ecological geography of the sea. Academic Press., Bosc et al. 2004Bosc E., Bricaud A., Antoine D. 2004. Seasonal and interannual variability in algal biomass and primary production in the Mediterranean Sea, as derived from 4 years of SeaWiFS observations. Global Biogeochemical Cycles 18: GB1005.). This result suggests that the highest interpopulation variability in the δ¹³C values of the two species of crabs considered here depends on the intensity of the regional primary productivity, although other factors such as differences in the arrival of terrestrial allochthonous organic matter may also play a role.

The δ¹³C values within an area are also influenced by depth, because macrophytes are typically enriched in ¹³C when compared with phytoplankton in both the Atlantic (Freire et al. 2009Freire J., Carabel S., Verísimo P., et al. 2009. Patterns of juvenile habitat use by the spider crab Maja brachydactyla as revealed by stable isotope analyses. Sci. Mar. 73: 39-49.) and the Mediterranean (Cardona et al. 2007Cardona L., Revelles M., Sales M., et al. 2007. Meadows of the seagrass Posidonia oceanica are a significant source of organic matter for adjoining ecosystems. Mar. Ecol. Prog. Ser. 335: 123-131.), but macrophytes occur only in shallow waters. As a consequence, the δ¹³C values typically decrease in deeper, off-shore habitats as compared with shallow, on-shore habitats where the relevance of macrophytes as a source of organic carbon is higher (e.g. Cardona et al. 2007Cardona L., Revelles M., Sales M., et al. 2007. Meadows of the seagrass Posidonia oceanica are a significant source of organic matter for adjoining ecosystems. Mar. Ecol. Prog. Ser. 335: 123-131.). In this scenario, the consistent enrichment in ¹³C of females across populations and species suggests that female spider crabs use shallower habitats than males before the post-pubertal moult. Furthermore, Bayesian ellipses show that sexes differ dramatically in the breadth of their isotopic niches in all the populations studied and that females usually have a broader isotopic niche than males, except for M. brachydactyla in Ceuta.

These results are in agreement with the strong evidence that spatial and seasonal segregation exists by sex between adults and reproductive stage in this group of species (De Kergariou 1971De Kergariou G. 1971. L’araignée de mer, Maia squinado L., sur le littoral de Bretagne. Science et Pêche, Bulletin d’Institute des Pêches maritimes 205: 11-19., 1984De Kergariou G. 1984. L’araignée de mer, H. Biologie et exploitation. La Pêche maritime 1279: 575-583., Števčić 1973Števčić Z. 1973. Les migrations de l’Araignée de mer. Rapport Commission International pour l’Exploration Scientifique de la Mediterranée 21: 597-598., Rodhouse 1984Rodhouse D.M. 1984. Experimental fishing for the spider crab, Maia squinado: sea and laboratory trials. J. Mar. Biol. Assoc. U.K. 64: 251-259., González-Gurriarán and Freire 1994González-Gurriarán E., Freire J. 1994. Movement patterns and habitat utilization in the spider crab Maja squinado (Herbst) (Decapoda, Majidae) measured by ultrasonic telemetry. J. Exp. Mar. Biol. Ecol. 184: 269-291., González-Gurriarán et al. 2002González-Gurriarán E., Freire J., Bernárdez C. 2002. Migratory patterns of female spider crabs Maja squinado detected using electronic tags and telemetry. J. Crust. Biol. 22: 91-97., Corgos et al. 2007Corgos A., Sampedro M.P., González-Gurriarán E., et al. 2007. Growth at moult, intermoult period and moulting seasonality of the spider crab Maja brachydactyla: combining information from mark-recapture and experimental studies. J. Crust. Biol. 27: 255-262., Fahy and Carroll 2009Fahy E., Carroll J. 2009. Vulnerability of male spider crab Maja brachydactyla (Brachyura: Majidae) to a pot fishery in south-west Ireland. J. Mar. Biol. Assoc. U.K. 89: 1353-1366.). M. brachydactyla adults migrate to deep water in association with gonad maturation and the development of seminal receptacles in the females. They return to shallow inshore water to incubate eggs and release larvae. However, males migrate longer distances than females (Fahy and Carroll 2009Fahy E., Carroll J. 2009. Vulnerability of male spider crab Maja brachydactyla (Brachyura: Majidae) to a pot fishery in south-west Ireland. J. Mar. Biol. Assoc. U.K. 89: 1353-1366.). Our results suggested that males and females may be spatially segregated before the terminal (pubertal) moult, as indicated by δ¹³C values in their exoskeletons, indicating that pre-pubertal males migrate to deeper waters than females, though females may remain in shallow waters and migrate to deep water mainly for coupling. However, δ¹³C short-term isotopic signature values in the muscle of migrating males and females were similar in M. brachydactyla from western Brittany (Bodin et al. 2007Bodin N., Le Loc’h F., Hily C., et al. 2007. Variability of stable isotope signatures (δ¹³C and δ¹⁵N) in two spider crab populations (Maja brachydactyla) in Western Europe. J. Exp. Mar. Biol. Ecol. 343: 149-157.), indicating that both sexes co-habited and preyed on similar food items during migratory periods.

The δ¹⁵N values of individuals from the same species inhabiting the same area may also vary, depending on their trophic level (Post 2002Post D.M. 2002. Using stable isotopes to estimate trophic position: models, methods, and assumption. Ecology 83: 703-718.). In the present study, the absence of statistically significant differences between sexes for δ¹⁵N suggests that males and females did not differ consistently in their trophic level, although differences may exist in some populations. Experiments in captivity suggest that the exoskeleton of crustaceans has a negative ¹⁵N fractionation, in contrast with the positive fractionation of muscle (Yokoyama et al. 2005Yokoyama H., Tamaki A., Harada K., et al. 2005. Variability of diet-tissue isotopic fractionation in estuarine macrobenthos. Mar. Ecol. Pro. Ser. 296: 115-128.). These differences explain why the δ¹⁵N values reported here are much lower than those reported for the muscle of M. brachydactyla (Bodin et al. 2007Bodin N., Le Loc’h F., Hily C., et al. 2007. Variability of stable isotope signatures (δ¹³C and δ¹⁵N) in two spider crab populations (Maja brachydactyla) in Western Europe. J. Exp. Mar. Biol. Ecol. 343: 149-157., Freire et al. 2009Freire J., Carabel S., Verísimo P., et al. 2009. Patterns of juvenile habitat use by the spider crab Maja brachydactyla as revealed by stable isotope analyses. Sci. Mar. 73: 39-49.). Considering an average diet-to-exoskeleton fractionation of –2.4‰ reported by Yokohama et al. (2005)Yokoyama H., Tamaki A., Harada K., et al. 2005. Variability of diet-tissue isotopic fractionation in estuarine macrobenthos. Mar. Ecol. Pro. Ser. 296: 115-128., the δ¹⁵N values reported here for M. brachydactyla from Galicia suggest a diet with a δ¹⁵N value of approximately 7.4‰, consistent with an omnivorous diet with a strong plant component according to the regional isospace (Freire et al. 2009Freire J., Carabel S., Verísimo P., et al. 2009. Patterns of juvenile habitat use by the spider crab Maja brachydactyla as revealed by stable isotope analyses. Sci. Mar. 73: 39-49.). Likewise, the δ¹⁵N values reported here for M. squinado from the Mediterranean (2.5‰) suggest a diet with a δ¹⁵N value of approximately 4.9‰, also consistent with an omnivorous diet with a strong plant component according to the regional isospace (Cardona et al. 2007Cardona L., Revelles M., Sales M., et al. 2007. Meadows of the seagrass Posidonia oceanica are a significant source of organic matter for adjoining ecosystems. Mar. Ecol. Prog. Ser. 335: 123-131.).

Stomach content studies have shown that macroalgae were food items of importance in the diet of these species (Števčić 1967Števčić Z. 1967. Le complexe de la nourriture de l’araignée de mer Maja squinado. Helgolander Wiss. Meer. 15: 630-636., Bernárdez et al. 2000Bernárdez C., Freire J., González-Gurriarán E. 2000. Feeding of the spider crab Maja squinado in rocky subtidal areas of the Ría de Arousa (north-west Spain). J. Mar. Biol. Assoc. U.K. 80: 95-102.). Isotopic signals from the current study suggested that macroalgae seemed to be better digested and assimilated than other preys ingested (e.g. crustaceans, mollusks and echinoderms). This hypothesis is supported by the high level of α-amylase activities found in adult specimens held in captivity, which were one order of magnitude higher than total proteases (Rotllant et al. 2013Rotllant G., Roque A., Solé M., et al. 2013. Assessment of the effects of confinement in the spider crab Maja brachydactyla Blass, 1922 broodstock. Aqua. Res. 44: 412-426.). In the present study, differences between sexes for δ¹⁵N values were significant for populations of M. brachtylatyla from Galicia and M. squinado from Ancona. The low δ¹⁵N values observed in males indicate a higher capacity to digest organic material from vegetal origin, which is supported by their higher digestive enzyme capacities, in particular in α-amylase activity, observed in males compared to females caught in Galicia (Rotllant et al. 2013Rotllant G., Roque A., Solé M., et al. 2013. Assessment of the effects of confinement in the spider crab Maja brachydactyla Blass, 1922 broodstock. Aqua. Res. 44: 412-426.). In our study, higher δ¹⁵N values were observed in Atlantic populations than in Mediterranean ones, as previously reported for other species (e.g. Borrell et al. 2006Borrell A., Aguilar A., Tornero V., et al. 2006. Organochlorine compounds and stable isotopes indicate bottlenose dolphin subpopulation structure around the Iberian Peninsula. Environ. Int. 32: 516-523., Gómez-Díaz and González-Solís 2007Gómez-Díaz E., González-Solís J. 2007. Geographic assignment of sea birds to their origin: combining morphologic, genetics, and biochemical analyses. Ecol. Appl. 17: 1484-1498., Giménez et al. 2013Giménez J., Gómez-Campos E., Borrell A., et al. 2013. Isotopic evidence of limited exchange between Mediterranean and eastern North Atlantic fin whales. Rapid Commun. Mass Spectrom. 27: 1801-1806.).

The results reported here are useful for product traceability. In the Spanish market, during the whole year, but particularly during the spider crab closed season (May to November approximately, annual variations depending on total fishing quotas), the fisheries market is supplied by M. brachydactyla specimens caught in Ireland, France, the United Kingdom and/or Morocco (Mercasa 2007Mercasa 2007. El centollo. Distribución y Consumo 93: 86-89.), although crabs from Galicia fetch higher prices. In this study, stable isotope signatures for commercial Maja species allow their geographical origin of capture to be identified. Thus, this methodology could be used for fisheries management to identify the origin of the product, prevent fraud and guarantee its traceability along the commercial chain.

ACKNOWLEDGEMENTSTop

Financial support was provided by the Ministry of Science and Research to GG (post-doctoral fellowship; INIA). Bench fees were provided by JACUMAR (REPES project) to GR.

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