Identification of European species of Maja ( Decapoda : Brachyura : Majidae ) : RFLP analyses of COI mtDNA and morphological considerations

Four species of crabs of the genus Maja have been described along the European coast: M. brachydactyla, M. squinado, M. goltziana and M. crispata. The commercially important species M. brachydactyla and M. squinado achieve the largest body sizes and are the most similar in morphology, and are therefore easily confused. The four species of Maja were identified using a novel morphometric index and a polymerase chain reaction followed by restriction fragment length polymorphism analysis (RFLP). The relationship between carapace length and the distance between the tips of antorbital spines was used to distinguish adults of M. brachydactyla and M. squinado. PCR-RFLP analysis of a partial sequence of the mitochondrial cytochrome oxidase type I (COI) revealed that the four species of the genus Maja can be unambiguously discriminated using the combination of restriction endonucleases enzymes HpyCH4V and Ase I. The molecular identification may be particularly useful in larvae, juvenile and young crabs, when the morphological differences found in adults are not applicable.

The larval stages of the Atlanto-Mediterranean species of Maja are morphologically very similar (Clark, 1986;Paula, 1988;Rodríguez, 2002;Guerao et al., 2008), with the zoea stages being virtually indistinguishable.The morphology of juveniles is also very similar; at least for M. brachydactyla (Guerao and Rotllant, 2009) and M. squinado (Guerao and Rotllant unpublished data).The possibility of correctly identifying all development stages (larvae, juveniles, adults) of a species is a prerequisite for all studies on population dynamics aimed at developing proper management of fisheries resources and their commercialization.
Among the different polymerase chain reaction (PCR) methods of genotype analysis, restriction fragment length polymorphism (RFLP) of PCR-amplified mitochondrial DNA (mtDNA) fragments has been recently used to identify different crustacean species (e.g.Bossier et al., 2004;Khamnamtong et al., 2005;Van Stappen et al., 2007).Sequence variation at the COI mtDNA barcode region has been shown to be effective for discriminating crustacean species (Costa et al., 2007).
The aim of this paper is to provide a useful molecular technique for identifying all the European species of the genus Maja at all stages of development, and also to provide new morphological and morphometric characters that allow easy differentiation between M. brachydactyla and M. squinado specimens.

MATERIALS AND METHODS
A total of 149 adult specimens of the four species of genus Maja collected between 2007 and 2010 in the Atlantic and Mediterranean Sea were used in morphological and/or molecular analyses (Table 1).One pereiopod of each individual was preserved in 94-100% ethanol for posterior DNA extraction.For the morphological study, additional individuals (carapaces) conserved in the laboratory were also measured (n=13 M. brachydactyla; n=8 M. squinado).The following measurements were taken: carapace length (CL) as the distance between the rostral margin (without rostral spines) and the posterior margin of the carapace (without intestinal spines); and antorbital spine length (ANSL), measured as the distance between the tips of the antorbital spines (the spine at the posterolateral corner of the supraorbital eave; see Figs. 1  and 2).The relationship between CL and ANSL was studied using least squares analysis of linear regression (log ANSL = log a + b log CL).Regression was performed using a SigmaStat 3 (Systat Software Inc., USA) software package.The significance of the correlation was tested with a t-test (H 0 : ρ=0, H 1 : ρ≠0), and the regression equations were compared with the F-test (H 0 : a 1 =a 2 , b 1 =b 2 ) (Cuadras, 1991).Total genomic DNA extraction was performed with muscle tissues using the QIAamp DNA Mini Kit (QIAGEN Inc).The concentration of DNA was estimated by spectrophotometry, using a GeneQuant pro.The 710 bp region of the mitochondrial cytochrome oxidase subunit I gene was amplified using universal primers LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G -3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAA TCA-3′) described by Folmer et al. (1994).Amplification was carried out with 80-100 ng of genomic DNA in a reaction containing 1U of Taq polymerase (Invitrogen), 1Χ buffer (Invitrogen), 2 µM of each primer and 800 µM dNTPs.The PCR thermal profile used was 95°C for 5 min for the initial denaturation, and 39 cycles at 94°C (20 s), 42°C (20 s), 72°C (30 s) with a final extension at 70°C for 7 min.In order to estimate the size of restriction fragments precisely, the mitochondrial COI gene was sequenced from 2-10 individuals for each restriction pattern obtained (Table 1).Sequencing reactions were performed with the ABI PRISM® BigDye TM Terminator Ready Reaction Cycle Sequencing kit, v3.1 (Applied Biosystems); the products were analyzed in an automated sequencer ABI PRISM 310 (Applied Biosystems).Sequencing reactions were carried out by an external laboratory (Sistemas Genómicos ® , Valencia, Spain).In addition, sequences of all four species of Maja from GenBank were analyzed (Table 1).To identify conserved nucleotide residues useful for an RFLP analysis, sequence alignment was performed using the BioEdit software (Hall, 1999).The amplification products were then incubated with two restriction enzymes, HpyCH4V (recognition site: 5′-TG′CA -3′/3′-AC′GT -5′) and Ase I (recognition site: 5′-AT´TAAT -3′/3′-TAAT′TA -5′) (New England Biolabs).Digestion was performed in a 20 µL mixture that contained 10 µL of PCR product, 25 U of HpyCH4V, 50 U Ase I and NEB digestion Buffer 2. Restriction enzyme digestions were incubated for 3.5 h at 37°C.Restriction fragments were electrophoretically separated on 1.5% agarose in 1X TBE buffer, stained with ethidium bromide and visualized under UV light.The number of individuals analyzed is shown in Table 1.

PCR-RFLP analyses
The size of the COI mtDNA fragment amplified in the four species was 710 bp.Restriction fragment length polymorphism analysis revealed that the four species can be unambiguously discriminated using the restriction enzymes HpyCH4V and Ase I (Fig. 3).As expected, digestion with both enzymes produced a specific pattern for M. crispata (9, 99, 227 and 375 bp), M. squinado (333 and 377 bp), M. goltziana (15, 122, 198 and 375 bp), and M. brachydactyla, which exhibited an alternative specific restriction pattern (the   1).In addition, the mutation at the enzyme recognition site related to pattern b can be observed in 14.3% of M. brachydactyla sequences from GenBank.None of the studied individuals of M. squinado and M. goltziana contained the recognition site for the Ase I enzyme (Fig. 4).It should be noted that M. squinado individuals may show one or two enzyme recognition sites for HpyCH4V; however, as both sites are very close (5 bp, see Fig. 4) the restriction patterns are indistinguishable.

DISCUSSION
The morphological differentiation of the European species in the genus Maja is sometime problematic due to a considerable amount of character overlap and ontogenetic variation.Maja brachydactyla and M. squinado reach the largest body sizes (exceeding 20 cm in carapace length) and they are the most similar species.Neumann (1998) reported that the different morphology of the male first gonopod is particularly useful for identifying the two species.Thus, the proximal margin of the distal region of the gonopod is rounded with gradual transition to the terminal process in M. brachydactyla, while it shows an angled outline and abrupt transition into a terminal process in M. squinado.However, the distinctive characters of the male first gonopod are subject to ontogenetic changes, making the identification of juvenile individuals very difficult or impossible.Moreover, the study of gonopods requires dissection and detailed microscope observations.In the present study, it was demonstrated that a simple relationship between CL and ANSL is a good morphometric index to differentiate adult individuals of both sexes (CL larger than 9.8 cm) of M. brachydactyla and M. squinado (see Figs. 1 and  2).However, early juveniles of the two species do not differ in this character and are nearly indistinguishable (Guerao andRotllant, 2009, 2010).The great similarity between the juveniles of related brachyuran species has been previously documented; species distinction is very difficult at the early postlarval stages, and phenotypic divergence increases with juvenile ontogeny (Ingle and Rice 1984;Martin et al., 1984;Felder et al., 1985;Neumann 1996).The adult individuals of M. crispata are smaller than adults of M. brachydactyla and M. squinado.This species can only be confused with juveniles of the two larger European species of Maja.Identification of crustacean species using PCR-RFLP analysis has been well documented (Power et al., 1999;Bossier et al., 2004;Khamnamtong et al., 2005;Hisar et al., 2008;Pascoal et al., 2008;Dharani et al., 2009).The PCR-RFLP technique, to be diagnostic, must be applied to DNA regions that are highly conserved within species but sufficiently variable between species.The COI mtDNA region has been shown to be a good marker for crustacean species (Costa et al., 2007).Importantly, mtCOI is sufficiently variable to be useful in identifying and discriminating even the most closely related species (Lefébure et al., 2006;Bucklin et al., 2007Bucklin et al., , 2009;;Darling and Tepolt, 2008).Sotelo et al. (2008) reported that the COI region displays diagnostic differences between species of the genus Maja; divergence between species was much higher than within them.The low level of intraspecific variation observed among the three species indicates that the COI gene fragment amplified utilizing the primers of Folmer et al. (1994) is a suitable marker for identifying Majidae species in the NE Atlantic and Mediterranean Sea.Using a combination of restriction enzymes (HpyCH4V and Ase I) proved useful for discriminating all four Maja European species.
Analysis of M. brachydactyla samples, from the eastern Atlantic (Spain, Galicia; and Morocco, Grand Casablanca) to SW Mediterranean (Spain, Ceuta), and M. squinado and M. crispata samples from the western (Spain, Catalonia) and central (Italy, Adriatic Sea) Mediterranean revealed identical restriction patterns among individuals of the same species.It should be noted that the specimens of Maja originating from Ceuta, on the Mediterranean coast (near the Strait of Gibraltar), have been unambiguously assigned to the species M. brachydactyla (Table 1).The presence of strictly eastern Atlantic coastal species in the Alboran Sea has been reported in several instances and related to the inflow of the less dense Atlantic surface water into the Mediterranean Sea through the Strait of Gibraltar (García Raso, 1984;García Muñoz et al., 2008;Lasram et al., 2008).
The molecular identification technique might be especially useful when dealing with larvae or juvenile individuals, which do not have the discriminating morphological characters of adults (Chow et al., 2006;Tang et al., 2009).The PCR-RFLP technique, which allows the four Maja species to be identified at any development stage, would contribute to the understanding of their life histories and hence to the management of their exploitation.It may also have forensic applications when fraud is suspected in the commerce of the two species (M.brachydactyla and M. squinado), as they are subject to different capture and marketing regulations.

Table 1 .
-Sampling locations and number of individuals for molecular and morphological studies of Maja.
Abbreviations: GB, GenBank accession number; MO, morphological studies; n, number of crabs; RF, RFLP studies; T, total studied individuals.