Morphology of the First Zoeal Stages of Five Species of the Portunid Genus Callinectes (decapoda, Brachyura) Hatched at the Laboratory

The genus Callinectes Stimpson, 1860 currently consists of 16 species, six of which are reported in Brazilian coast. In the present study, the first zoeal stages of Callinectes bocourti, C. danae, C. exasperatus, C. ornatus and C. sapidus from Brazil were obtained from ovigerous females. The morphological and meristic characters of all these larval stages are described and illustrated. Those of C. bocourti, C. danae and C. sapidus are redescribed and compared with the previous descriptions, and differences are listed. Larval characters of these species were examined for interspecific differences, as well as larval features to distinguish the genus Callinectes within Portunidae. In addition, other portunid genera and species with a known first zoeal stage are compared, with special attention to those species present in the same geographical area. Our findings concord with some previous molecular studies, and we discuss the complexity within the group.


INTRODUCTION
Swimming crabs of the genus Callinectes Stimpson, 1860 are ubiquitous representatives of the portunid fauna in tropical and subtropical waters.Under present systematic treatments, 16 species of Callinectes are currently recognized for this genus worldwide (compiled from Rathbun 1930, Stephenson and Campbell 1959, Williams 1974, 1984, Melo 1996, Schubart et al. 2001, Robles et al. 2007, Ng et al. 2008).Six are reported in Brazilian waters: Callinectes affinis Fausto-Filho 1980, C. Despite the ecological, evolutionary and economic importance of Callinectes, controversies remain regarding the systematic arrangement of the genus (Williams 1974, Norse 1977), which has been based up the present on morphological and physioecological data.The validity of morphological characters was tested by molecular tools only recently (Schubart et al. 2001, Robles et al. 2007).However, despite the unquestionable importance of larval morphology for the study http://dx.doi.org/10.1590/0001-3765201420130030755-767 FERNANDO L. MANTELATTO, ÁLVARO L.D. REIGADA, ALINE C.R. GATTI and JOSÉ A. CUESTA of phylogenetic relationships (Rice 1980), larval descriptions are not available for most species and many genera of portunids; and many existing descriptions are incomplete or erroneous.Larval information is available for less than 8% of all known portunid crab species.
Considering this promising scenario for taxonomic investigation and the potential for zoeal morphology to provide helpful information, we describe and illustrate here the first zoeal stages of five species (Callinectes bocourti, C. danae, C. exasperatus, C. ornatus and C. sapidus) hatched in the laboratory, and compare their morphology with described zoeae of other portunid species.The morphology of the zoea I of Callinectes exasperatus and C. ornatus is described for the first time, and the zoeae of C. bocourti, C. danae and C. sapidus are redescribed and compared with the previous descriptions by Lopes et al. (2000), Sankarankutty et al. (1999) and Costlow and Bookhout (1959), respectively.The differences in morphology are listed.

MATERIALS AND METHODS
Ovigerous females were obtained by trawling in two regions (São Vicente and Ubatuba) of the coast of São Paulo, Brazil, during 2002 and2003.Females were transported to the laboratory, and the ovigerous females were isolated in aquaria with aerated sea water at a salinity of 34 psu and constant temperature (24 ± 1°C) until hatching.Newly hatched zoea stages were fixed in a 1 : 1 solution of 70% ethanol and glycerin.The first zoeae were dissected for detailed examination under a stereoscope, and mounted on semipermanent slides.Measurements and morphological characters were checked using a compound microscope equipped with a camera lucida.A minimum of 10 specimens were used for measurements, and 20 specimens for descriptions.
For the zoeae I the following measurements were taken: cephalothoracic dorsal spine length (DL), distance from the base to tip of the dorsal spine; cephalothoracic rostral spine length (RL), distance from the base to tip of the rostral spine; rostrodorsal length (RDL), distance from the tip of the rostral spine to the tip of the dorsal spine; cephalothorax length (CL), from between the eyes (base of the rostrum) to the postero-lateral cephalothorax margin.The descriptions follow the standard proposed by Clark et al. (1998).The long terminal plumose natatory setae on distal exopod segments of the first and second maxillipeds are drawn truncated.We followed the seta and setal classifiation proposed by Garm (2004).Voucher samples of the parental female and zoea I of all species were deposited in the Crustacean Collection of the Biology Department (CCDB) of the Faculty of Philosophy, Sciences and Letters of Ribeirão Preto (FFCLRP) at the University of São Paulo (USP) under accession numbers CCDB 4256-4271.The collections of specimens conducted in this study complied with current applicable state and federal laws of Brazil (permanent license to FLM for collection of Zoological Material No. 11777-1 MMA/IBAMA/SISBIO).

DESCRIPTION
A complete detailed description of the zoea I of the type species of the genus, Callinectes sapidus, as well as of the previously unknown zoea I of C. exasperatus and C. ornatus are provided.For the zoeae I of Callinectes bocourti and C. danae only the differences with regards to that of C. sapidus are mentioned.
Telson (Fig. 1D-F).Telson furcae with one pair of well-developed lateral spines, one pair of small lateral simple setae just below the lateral spines, and one pair of dorsal spines; inner margin with 3 pairs of serrate setae.
Antenna (Fig. 6E).Protopod very long, with 2 rows of 14-16 spinules of different sizes, increasing toward the tip, the last 3 spines not paired, in the basal part 3 medium-sized spinules; one-segmented exopod, shorter than the spinous process, approximately 1/8 of protopod length.
Antenna (Fig. 6G).Protopod very long, with 2 unequal rows of 10 and 5 spinules of similar sizes, all spinules not paired, in the basal part a group of Mandible.Incisor and molar processes differentiated; mandibular palp absent.
Telson (Fig. 7C).Telson furcae with one pair of well-developed lateral spines, one pair of small lateral simple setae just below the lateral spines, and one pair of dorsal spines; inner margin with 3 pairs of serrate setae.Slight differences in spinulation of serrulate setae respect to Callinectes sapidus.
Telson (Fig. 7D).Telson furcae with one pair of well-developed lateral spines, one pair of small lateral simple setae just below the lateral spines, and one pair of dorsal spines; inner margin with 3 pairs of serrate setae.Slight differences in spinulation of serrulate setae respect to Callinectes sapidus.

DISCUSSION
The zoea I of the five species of Callinectes described here has very similar morphology, and only a combination of some slight differences allows them to be distinguished.Main differences are found in the antennule and antenna formulae, the ratio between the antennal exopod and protopod length (see Table I), the degree of curvature of the dorsal spine, the spinulation of the cephalothoracic lateroventral margin, and the spinulation of the serrulate setae of the telson.
Regarding the previous descriptions of C. bocourti, C. danae and C. sapidus, some commonly overlooked differences in the setae or other structures were detected (see Table II), but must be corrected to prevent confusion and not taken to be real differences rather than errors.
Among the portunids reported from the southwestern Atlantic, those of the subfamily Portuninae are characterized by possessing: antennal protopod as long as rostral spine (in some cases longer), lateral and dorsal spines on protuberance; each lateroventral margin with a few strongly acute denticles, without setae; one pair of posterodorsal simple setae present; eyes sessile.
Second maxilliped.the furca, and relatively small zoeal stages.Only a combination of several anatomical characters would allow routine separation of zoea I from these species.The first zoeal stages of Callinectes species described here have the common features known for this subfamily, suggesting that this taxonomic unit is phenetically coherent based on larval morphology.We can therefore infer that all species of Callinectes have a proportionally shorter exopod than those of Portuninae (see Table I), which could be a potential character to differentiate larvae.In addition, the cohesiveness of the clade formed by larvae of members of Callinectes is unquestionable, and supports the previous phylogeny of the group based on molecular tools (Schubart et al. 2001, Robles et al. 2007).
Our description of larval morphology provided more information to support recent results obtained from molecular analysis (Mantelatto et al. 2007(Mantelatto et al. , 2009)).These inferences led to the proposal of taxonomic changes for Cronius and some members of Portunus (Mantelatto et al. 2009), in combination with important differences noted previously in the larval morphology of Cronius species (Fransozo et al. 2002) I) and a basal position in the molecular phylogeny of Mantelatto et al. (2007Mantelatto et al. ( , 2009)).The zoeal subgroups correspond perfectly with the groups obtained by 16S mtDNA analyzed by these latter authors; only members of the newly defined Achelous have an antennal exopod length equal to or exceeding 1/3 the protopod length and a maxillule endopod setation formula of 0,6.
While far from being complete, significant progress has been made in recent decades in the knowledge of decapod larvae of the southwestern Atlantic (Pohle et al. 1999).Determining phylogenetic relationships of this subfamily based on zoea descriptions alone is premature; but as descriptions of zoea morphology become available for more species of Portuninae, as well as other taxonomically useful information (e.g., cladistic analysis, molecular sequences, genetic, spermatophore morphology and fossil evidence), it could be possible to determine the phylogeny and evolution of the anatomically diverse Portunoidea.We argue here in favor of new descriptions, especially of genera and species for which larval morphology is still unknown, in order to gain a more complete overview of this topic and use it in a phylogenetic context.

TABLE II Comparison of differences between previous and present descriptions of zoeae I of Callinectes bocourti, C. danae and C. sapidus. Abbreviations: a, aesthetascs, s, setae; ps, plumose setae; (-) character without differences.
*(these internal lateral spines could not exist).
. Zoeae with relatively long antennal exopods are typical of the presently assigned Achaelous tumidulus, Achaelous gibbesii, and Achaelous spinicarpus (sensu Mantelatto et al. 2009), while those with short antennal exopods occur in Cronius ruber, Arenaeus cribrarius, and some members of Callinectes and Charybdis.The exception is Scylla serrata, which holds a somewhat intermediate position in terms FERNANDO L. MANTELATTO, ÁLVARO L.D. REIGADA, ALINE C.R. GATTI and JOSÉ A. CUESTA of larval morphology (as previously mentioned byFransozo et al. 2002: Table