475 Abstract–Lake Maracaibo, a large Lack of divergence between 16S mtDNA sequences Venezuelan estuarine lagoon, is report- edly inhabited by three species of the of the swimming crabs Callinectes bocourti genus Callinectes Stimpson, 1860 that are important to local fi sheries: C. sapi- and C. maracaiboensis (Brachyura: Portunidae) dus Rathbun, 1896, C. bocourti A. Milne from Venezuela* Edwards, 1879, and C. maracaiboensis Taissoun, 1969. Callinectes maracai- boensis, originally described from Lake Christoph D. Schubart Maracaibo and assumed endemic to Department of Biology those waters, has recently been reported University of Louisiana from other Caribbean localities and Lafayette, Louisiana 70504 Brazil. However, because characters Present address: Biologie I, Universität Regensburg sep arating it from the morphologically 93040 Regensburg, Germany similar C. bocourti are noted to be E-mail address: [email protected] vague, we have compared these spe- cies and several congeners by molec- ular methods. Among our specimens Jesús E. Conde from Lake Maracaibo and other parts Carlos Carmona-Suárez of the Venezuelan coast, those assign- able to C. bocourti and C. maracaiboen- Centro de Ecología sis on the basis of putatively diagnostic Instituto Venezolano de Investigaciones Científi cas (IVIC) characters in coloration and structural A. P. 21827 characteristics do not differ in their Caracas 1020-A, Venezuela 16S mtDNA sequences. These molec- ular results and our re-examination of supposed morphological differences Rafael Robles between these species suggest that C. Darryl L. Felder maracaiboensis is a junior synonym of Department of Biology C. bocourti, which varies markedly in University of Louisiana minor features of coloration and struc- Lafayette, Louisiana 70504 tural characteristics. Genetic relation- ships of this species to other species of swimming crabs of the genus Calli- nectes are also explored and presented as phylogenetic trees. Swimming crabs of the genus Calli- which are essential to fi shery-manage- nectes Stimpson, 1860 are widely dis - ment decisions. In particular, consistent tributed throughout the American trop- morphological distinction of the com- ics and subtropics, where many species monly encountered C. bocourti from C. are exploited commercially (Norse, 1977; maracaiboensis cannot be achieved with Williams, 1984). Members of this genus confi dence. play key trophic roles in coastal habitats Initially described as endemic from that range from sandy-mud bottoms to Lake Maracaibo, C. maracaiboensis seagrass meadows (Arnold, 1984; Orth has been reported from other Vene- and van Montfrans, 1987; Wilson et al., zuelan waters, as well as from sites 1987; Lin, 1991). Of the nine species in Jamaica, Curaçao, Colombia, and of Callinectes from the tropical western Brazil (Norse, 1977; Carmona-Suárez Atlantic, seven have been reported from and Conde, 1996; Sankarankutty et al., western Venezuela (Rodríguez, 1980; 1999). Although detailed descriptions Williams, 1984; Carmona-Suárez and have been provided by Taissoun (1969, Conde, 1996). However, knowledge of 1972) and Williams (1974) to diagnose these Venezuelan populations remains C. maracaiboensis, the same authors inadequate, despite the importance of have repeatedly emphasized its resem- several species in both large-scale com- blance to C. bocourti. Morphological mercial harvests and artisanal fi sh- distinction of the two species is based eries of coastal villages in the area upon postulated defi ning characters of (Oesterling and Petrocci, 1995; Ferrer- the frontal teeth, direction and form of Montaño, 1997; Conde and Rodríguez, 1999). Persistent diffi culty in identify- ing mixed samples of these species ham- * Contribution 75 of the University of Lou- Manuscript accepted 21 March 2001. pers understanding of their distribution, isiana Lafayette, Laboratory for Crusta- Fish. Bull. 99:475–481 (2001). abundance, and population dynamics, cean Research, Lafayette, LA 70504. 476 Fishery Bulletin 99(3) the anterolateral spines, convexity of the carapace, sur- product (547 bp excluding primer regions) from the mito- face granulation patterns, and varied morphometric mea- chondrial 16S rRNA gene was carried out by a polymerase- surements (Taissoun 1969, 1972). However, neither these chain-reaction (PCR) (35–40 cycles: 1 min at 94°C, 1 min at characters nor additional ones proposed by other authors 55°C, 2 min at 72°C (denaturing, annealing, and extension (Williams, 1974; Rodríguez, 1980) have proven to be of temperatures, respectively) with primers 16Sar (5′-CGCCT- consistent use in separating the species. Carapacial color GTTTATCAAAAACAT-3′), 16Sbr (5′-CCGGTCTGAACTCA- and granulation vary widely, and the outer frontal teeth GATCACGT-3′), 1472 (5′-AGATAGAAACCAACCTGG-3′), of many specimens cannot be classifi ed as a defi nitive tri- and 16L15 (5′-GACGATAAGACCCTATAAAGCTT-3′) (for angular (C. maracaiboensis) or obtuse (C. bocourti) shape. references to primers see Schubart et al., 2000). 16L15 is These concerns have led some authors to report only pos- an internal primer and, in combination with 16Sbr, was sible co-occurrence of C. maracaiboensis in their samples used for partial amplifi cation of the formalin-preserved of C. bocourti, as in records from Puerto Rico (Buchanan specimen. PCR products were purifi ed with Microcon-100® and Stoner, 1988). fi lters (Millipore Corp., Bedford, MA) and sequenced with Recently, molecular markers have proven particularly the ABI BigDye® Terminator Mix (PE Biosystems, Welles- valuable in decapod crustacean systematics. In addition to ley, MA) in an ABI Prism 310 Genetic Analyzer® (Applied reconstructing phylogenetic relationships, molecular se- Biosystems, Foster City, CA). Sequences were manually quences are of value in recognizing questionable species aligned with the multisequence-editing program ESEE separations (e.g. Geller et al., 1997; Sarver et al., 1998; (Cabot and Beckenbach, 1989). New sequences were ac- Schneider-Broussard et al., 1998; Schubart et al., 1998; cessioned to the European Molecular Biology Laboratory Schubart et al., in press). In our study, we used sequences (EMBL) genomic library (see Table 1). Sequences avail- of a mitochondrial gene (16S rRNA) to examine genetic able online (by electronic database accession numbers that differentiation between the morphologically similar spe- follow) were obtained for “Callinectes sapidus” (U75267), cies C. bocourti and C. maracaiboensis and to compare C. ornatus (U75268), C. similis (U75269), and C. sapidus these sequences to those of other swimming crabs of the (AJ130813). genus Callinectes in a phylogenetic analysis. Sequence divergence was analyzed by using Kimura 2-parameter distances, UPGMA cluster analysis, and neighbor-joining (NJ) distance analysis (Saitou and Nei, Materials and methods 1987) with the program MEGA (Kumar et al. 1993). Sta- tistical signifi cance of groups within inferred trees was Swimming crabs were caught by hand in shallows of the evaluated by the interior branch method (Rzhetsky and Golfete de Cuare (68°37′W, 10°06′N, State of Falcón, Vene- Nei, 1992). As a second phylogenetic method, a maximum zuela) in November–December 1998 and March 1999, and parsimony (MP) analysis was carried out with a heuristic in the shallows of El Moján, Lago de Maracaibo (71°39′W, search and random sequence addition (tree-bisection and 10°58′N, State of Zulia, Venezuela) in October 1999. Crabs reconnection as branch-swapping option) and by omission were transported to the Instituto Venezolano de Investig- of gaps with the program PAUP (Swofford, 1993). Signifi - aciones Científi cas, Caracas, in liquid nitrogen and then cance levels were evaluated with the same software and stored in a –70°C freezer. Two walking legs and one swim- the bootstrap method with 2000 replicates. ming leg were separated from each specimen, preserved individually in 85% ethanol, and shipped to the University of Louisiana, Lafayette (U.S.A.), for molecular analyses. Results Specimens were assigned to Callinectes bocourti or C. maracaiboensis according to morphological characters Sequencing of 15 specimens of Callinectes bocourti and provided by Taissoun (1969, 1972), Williams (1974, 1978), C. maracaiboensis from Golfete de Cuare and El Moján Fischer (1978), and Rodríguez (1980). Eight specimens of (both Venezuela) and the Río Sinú estuary (Colombia) C. bocourti and six of C. maracaiboensis from Venezuela revealed the existence of seven different haplotypes. Two were used for the molecular analysis. In addition, a short haplotypes (ht-1 and ht-5) clearly predominated (together fragment (~300 base pairs) of 16S mtDNA was obtained 64.3%) and were found in both species (ht-1 in two Cal- with an internal primer for one formalin-preserved speci- linectes bocourti and three C. maracaiboensis from Golfete men of C. bocourti from Colombia. These voucher speci- de Cuare, ht-5 in three C. bocourti and one C. maracai- mens, lacking limbs (which had been removed for analy- boensis from El Moján). The other fi ve haplotypes (ht-2, sis), were cataloged in collections of the Laboratorio de ht-3, ht-4, ht-6, ht-7) each differed from ht-1 or ht-5 in Ecología y Genética de Poblaciones, Instituto Venezolano not more than two positions and were found in only single de Investigaciones Científi cas (IVIC), and in the Univer- individuals (Fig. 1, Table 2). Consequently there is not sity of Louisiana Zoological Collection (Table 1). For out- a single diagnostic molecular character in our sequenced