Mar Biol (2007) 150:1265–1274

DOI 10.1007/s00227-006-0437-7

RESEARCH ARTICLE

Molecular phylogeny of the American Callinectes Stimpson, 1860 (Brachyura: Portunidae), based on two partial mitochondrial genes

Rafael Robles · Christoph D. Schubart · Jesús E. Conde · Carlos Carmona-Suárez · Fernando Alvarez · José L. Villalobos · Darryl L. Felder

Received: 5 September 2005 / Accepted: 17 July 2006 / Published online: 24 August 2006© Springer-Verlag 2006

Abstract The genus Callinectes encompasses 16 spe-cies of commercially important swimming crabs. Most(13) occur on the PaciWc and Atlantic coasts of theAmericas. We compare mtDNA regions correspond-ing to 964 basepairs of the large (16S) and small (12S)ribosomal subunits among American Callinectes inorder to examine phylogenetic relationships. The sta-tus of Callinectes aVinis Fausto-Filho and Callinectesmaracaiboensis Taissoun is questioned, and C. mara-caiboensis is concluded to be a junior synonym of Calli-nectes bocourti A. Milne-Edwards, from which itcannot be consistently distinguished. We Wnd twomajor lineages, one of which includes C. aVinis, C.bocourti, Callinectes rathbunae Contreras, Callinectessapidus Rathbun, and Callinectes toxotes Ordway. Asecond lineage is comprised of Callinectes arcuatus

Ordway, Callinectes bellicosus (Stimpson), Callinectesdanae Smith, Callinectes exasperatus (Gerstaecker),Callinectes larvatus Ordway, Callinectes ornatus Ord-way, and Callinectes similis Williams. DeWnition ofthese clades is supported by previously described mor-phological diVerences in the length of the gonopodsand shared physioecological adaptations. A calibratedmolecular clock is used to estimate divergence of thetwo lineages near 13 mybp. Our analyses suggest thatC. ornatus is the closest relative of C. arcuatus, and thatC. aVinis is closest to C. bocourti.

Introduction

The portunid fauna of the Americas consists of »45 spe-cies, 13 of which belong to the genus Callinectes Stimp-son. The species of Callinectes are economicallyimportant with an annual catch of more than 89,000 and1,300 t for Callinectes sapidus and Callinectes danae,respectively (FAO 2004). Despite the ecological, evolu-tionary and economic importance of Callinectes, contro-versies remain regarding systematics of the genus(Williams 1974; Norse 1977). The systematics of thegroup has to date been based on morphological andphysioecological data. Recently, the validity of morpho-logical characters used to separate Callinectes maracai-boensis from Callinectes bocourti has been called intoquestion (Schubart et al. 2001). Also, morphological andcolor characters of Callinectes aVinis suggest to us thatthe separation of this species from C. bocourti should bequestioned, especially since C. bocourti is known to varyconspicuously in color pattern and morphology (Tais-soun 1972). These and other relationships within thegenus Callinectes can be tested with molecular data.

Communicated by P.W. Sammarco, Chauvin

R. Robles (&) · C. D. Schubart · D. L. FelderDepartment of Biology and Laboratory for Crustacean Research, University of Louisiana at Lafayette, Lafayette, LA 70504, USAe-mail: rxr8359@louisiana.edu

J. E. Conde · C. Carmona-SuárezCentro de Ecología, Instituto Venezolano de Investigaciones CientíWcas (IVIC), A.P. 21827, Caracas 1020-A, Venezuela

F. Alvarez · J. L. VillalobosColección Nacional de Crustáceos, Instituto de Biología, Universidad Nacional Autónoma de México, A.P. 70-153, 04510 México, D.F., México

Present Address:C. D. SchubartBiologie I, Universität Regensburg, 93040 Regensburg, Germany

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Molecular analyses are particularly useful in clarify-ing species status when analyses of morphological char-acters are not deWnitive (Knowlton 1993; Knowltonet al. 1993; Knowlton 2000). Mitochondrial DNA(mtDNA) has been used to clarify phylogenetic rela-tionships at varied taxonomic levels (Ballard et al.1992; Harrison and Crespi 1999). Three genes com-monly used are the 16S ribosomal RNA gene, a cyto-chrome oxidase gene (COI), and the 12S ribosomalRNA gene (e.g., Kocher et al. 1989; Ballard et al. 1992;Wägele and Stanjek 1995; Schubart et al. 2000a, b;Tudge and Cunningham 2000; Stillman and Reeb2001).

In this paper, we provide a phylogenetic analysis ofall the American species of Callinectes based on twomitochondrial genes, the large (16S) and the small(12S) ribosomal RNA. Our molecularly based phylo-genetic trees are compared to evolutionary groups for-merly proposed by Norse and Fox-Norse (1979) on thebasis of morphology and physioecology. While theseprevious workers were able to assign members of Calli-nectes to the “bocourti,” “danae,” “marginatus,” and“gladiator”-groups, there was no substantive attemptto explain relationships among the taxa comprisinggroups. Our analyses provide both an independentgenetic test of these groupings and further elaborationof relationships within them.

Our analyses include measures of genetic diVerencesbetween C. maracaiboensis, C. aVinis, and C. bocourti,along with assessments of their species status. Compar-isons between sister species of Callinectes from the

PaciWc and Atlantic coasts of America also allowed usto further test a proposed relationship between Calli-nectes arcuatus and C. danae and, on this basis, to cali-brate a molecular clock for the genus.

Materials and methods

Portunid swimming crabs of the genus Callinectes usedin this study were collected in Brazil (BRA), Honduras(HON), Mexico (MEX), Nicaragua (NIC), Louisiana(USA), and Venezuela (VEN), or were obtained asgifts or loans from museums (Table 1, Fig. 1). Speci-mens of the portunid crabs Arenaeus cribrarius(Lamarck) and Charybdis helleri (A. Milne-Edwards)were included in the analysis as outgroups for rootingof the phylogenetic analyses. Fresh specimens wereeither frozen at ¡70°C and transferred to 70% ethylalcohol (ETOH) or directly preserved in 70% ETOH.Crabs were identiWed on the basis of morphologicalcharacters (Taissoun 1969, 1972; Williams 1974; Fau-sto-Filho 1980).

Total genomic DNA was extracted from the third orfourth pereiopods. Muscle was ground and then incu-bated for 1–12 h in 600 �l of lysis buVer (100 mMEDTA, 10 mM tris pH 7.5, 1% SDS) at 65°C; proteinwas separated by the addition of 200 �l of 7.5 M ammo-nium acetate and subsequent centrifugation. DNA wasprecipitated by the addition of 600 �l of cold isopropa-nol followed by centrifugation (10 min at 20,800 g); theresulting pellet was rinsed in 70% ETOH, dried in a

Fig. 1 Collecting sites for species of Callinectes listed in Table 1, where three letter locality codes are interpreted

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speed vacuum system (DNA110 Speed Vac®) andresuspended in 10–20 �l of TE buVer (10 mM TRIS,1 mM EDTA).

Diluted total DNA was ampliWed by means of apolymerase chain reaction (PCR). The 16S and 12Sribosomal regions of mtDNA were ampliWed. Primersused for the 16S fragment were 16ar (5�-CGC CTGTTT ATC AAA AAC AT-3�), 16br (5�-CCG GTCTGA ACT CAG ATC ACG T-3�) (Palumbi et al.1991), 1472 (5�-AGA TAG AAA CCA ACC TGG-3�)(Crandall and Fitzpatrick 1996) and 16L2 (5�-TGCCTG TTT ATC AAA AAC AT-3�) (see Fratini et al.2005, derived from 16Sar). Primers used for the 12Sfragment were 12Sai (5�-AAA CTA GGA TTA GATACC CCT ATT AT-3�) (see Palumbi et al. 1991) and12H2 (5�-ATG CAC TTT CCA GTA CAT CTA C-3�)(see Fratini et al. 2005). Reactions were performed in25 �l volumes (200 �M each dntp, 1£ buVer, 0.5 �Meach primer, 1 unit Taq polymerase, 1 �l extractedDNA diluted to comprise between 20 and 30 ng). Ther-mal cycling was performed as follows: initial denatur-

ation for 10 min at 94–95°C followed by 40–42 cycles of1 min at 94–95°C, 1–1:30 min at 48°C and 1:30–2 min at72°C, with a Wnal extension of 10 min at 72°C. PCRproducts were puriWed using 100,000 MW Wlters(Microcon-100® Millipore Corp., Billerica, MA, USA)and sequenced with the ABI BigDye® terminator mix(PE Biosystems, Foster city, CA, USA). Both PCR andcycle sequence reactions were conducted on a Robocy-cler 96 cycler. Sequencing products were run on eithera 310 or 3100 Applied Biosystems (Foster City, CA,USA) automated sequencer. All sequences were con-Wrmed by sequencing both strands.

Sequences were aligned manually with the multise-quence editing program ESEE (Cabot and Becken-bach 1989). We conducted an incongruence lengthdiVerence (ILD) test or partition homogeneity test(Bull et al. 1993), as implemented in PAUP, to deter-mine whether the 16S and 12S genes could be consid-ered samples of the same underlying phylogeny. Themodel of evolution that best Wt the data was deter-mined with the software MODELTEST (Posada and

Table 1 List of species included in this study for which 16S mtDNA and 12S mtDNA were sequenced

ULLZ University of Louisiana, Lafayette Zoological Collection, CNCR Colección Nacional de Crustáceos, Instituto de Biología, UN-AM, IVIC-LEGP Instituto Venezolano de Investigaciones CientíWcas-Laboratorio de Ecología y Genética de Poblaciones, RMNH-LRijksmuseum Van Natuurlijke Historie, Leidena Schubart et al. (2000a)b Schubart et al. (2001)c Charybdis helleri 1 VEN corresponds to “Portunus ordwayi (Stimpson)” of Schubart et al. (2001), which was therein misidentiWed

Species with locality code (used in Figs. 2, 3)

Collection locality Catalog number GenBank accession numbers 16S/12S

Callinectes arcuatus NIC Nicaragua: Estero de Padre Ramos ULLZ 4370 DQ407668/DQ407687Callinectes arcuatus MEX México: Oaxaca CNCR 13 DQ407669/DQ407688Callinectes ornatus BRA Brazil: São Paulo ULLZ 4178 AJ298186b/DQ407703Callinectes ornatus VEN Venezuela: Falcón ULLZ 5185 DQ407679/DQ407704Callinectes similis USA USA: Louisiana ULLZ 4371 DQ407672/DQ407692Callinectes danae VEN Venezuela: Falcón IVIC-LEGP-C-1 AJ298184b/DQ407705Callinectes danae BRA Brazil: São Paulo ULLZ 4179 DQ407680/DQ407706Callinectes exasperatus VEN Venezuela: Isla Margarita ULLZ 4366 DQ407682/DQ407708Callinectes bellicosus MEX-1 México: Baja California ULLZ 4166 DQ407670/DQ407689Callinectes bellicosus MEX-2 México: Baja California CNCR 5021 DQ407671/DQ407690Callinectes larvatus VEN Venezuela: Falcón: Adícora ULLZ 5171 DQ407678/DQ407702Callinectes sapidus USA USA: Louisiana ULLZ 3895 AJ130813a/DQ407691Callinectes toxotes HON Honduras: Golfo de Fonseca ULLZ 5172 DQ407681/DQ407707Callinectes rathbunae MEX México: Tabasco CNCR 17056 DQ407673/DQ407693Callinectes bocourti VEN-1 Venezuela: Falcón IVIC-LEGP-C-30 = ULLZ 4180 AJ298170b/DQ407695Callinectes bocourti VEN-2 Venezuela: Zulia IVIC-LEGP-MZ5 AJ298180b/DQ407697Callinectes maracaiboensis VEN-1 Venezuela: Falcón IVIC-LEGP-C-40 = ULLZ 4181 AJ298171b/DQ407694Callinectes maracaiboensis VEN-2 Venezuela: Zulia IVIC-LEGP-MZ1 AJ298177b/DQ407696Callinectes maracaiboensis BRA-3 Brazil: Rio Grande Do Norte ULLZ 4419 DQ407674/DQ407698Callinectes aVinis BRA-1 Brazil: Fortaleza RMNH-L 32445 DQ407675/DQ407699Callinectes aVinis BRA-2 Brazil: Fortaleza RMNH-L 32445 DQ407676/DQ407700Callinectes aVinis BRA-3 Brazil: Rio Grande do Norte ULLZ 5170 DQ407677/DQ407701Charybdis helleri VEN-1c Venezuela: Falcón IVIC-LEGP-LV9 = ULLZ 4629c AJ298191b/DQ407685Charybdis helleri VEN-2 Venezuela: Falcón ULLZ 4630 DQ407665/DQ407683Charybdis helleri VEN-3 Venezuela: Falcón ULLZ 4631 DQ407666/DQ407684Arenaeus cribrarius VEN Venezuela: Falcón ULLZ 5173 DQ407667/DQ407686

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Crandall 1998) prior to conducting the Neighbor Join-ing (NJ) and Bayesian inference (BAY) analyses. Phy-logenetic and molecular evolutionary analyses wereconducted using MRBAYES for BAY analysis, andPAUP 4.0 beta 10 (SwoVord 2003) for Maximum Parsi-mony (MP) and NJ analyses.

The Bayesian analysis was performed by running aMarkov chain Montecarlo (MCMC) algorithm for2,000,000 generations with four parallel chains, sam-pling 1 tree every 100 generations starting with a ran-dom tree. A preliminary analysis showed that astationary distribution was reached at »20,000 genera-tions. Thus, we used this value as a burn-in for the tree-building analysis and discarded all previous trees. A50% majority rule consensus tree was obtained fromthe remaining saved trees. For the BAY analysis, val-ues were shown for posterior probabilities of therespective nodes obtained from the 19,800 remainingtrees after discarding 200 trees corresponding to theWrst 20,000 generations. Neighbor-joining analysis wascarried out with a maximum likelihood distance correc-tion set to parameters obtained from MODELTEST(Posada and Crandall 1998). The MP analysis was per-formed as a heuristic search with random sequenceaddition, including tree bisection and reconnectionas a branch swapping option. On the molecular trees,bootstrap conWdence values >50% were reported forboth NJ (2000 bootstraps) and MP (2000 bootstraps)analyses.

A second set of analyses with the same parametersas above was performed with the inclusion of only onespecimen of each recognized species. In this case, onlyfor the NJ tree, the rate of heterogeneity among lin-eages was tested with a likelihood ratio test (LRT)(Felsenstein 1981) as implemented in PAUP (SwoVord2003). Upon rejection of the molecular clock, nonpara-metric rate smoothing (NPRS) (Sanderson 1997) wasapplied in order to obtain an optimized ultrametrictree with the computer program TreeEdit Version 1alpha (Rambaut and Charleston 2000). Separation ofthe youngest trans-isthmian geminate species pair ofthis analysis was used to calibrate the molecular clockfor the optimized 16S and 12S rDNA data at around3.5 million years before present (mybp) (Knowlton1993; Knowlton et al. 1993; Knowlton and Weigt 1998)and to calculate divergence times for selected cladesamong the species of Callinectes. We also approxi-mated conWdence intervals (CI) for these dates bykeeping a Wxed tree and bootstrapping the data set 100times; divergence times for these 100 trees wereobtained as a mean with a 95% CI for each of theselected nodes. Sequences and complete alignmentwere accessioned to GenBank (Table 1).

Results

A total of 964 basepairs were aligned; 551 for the 16Sand 413 for the 12S sequences (excluding the primerregions). Alignment of both gene sequences was unam-biguous. The ILD test showed no signiWcant incongru-ence (P = 0.3680). Thus, all phylogenetic analyses wereperformed with a single database including the twogenes. The optimal model, selected with the LRT (AIC)in MODELTEST (Posada and Crandall 1998), was theHasegawa–Kishino–Yano model (Hasegawa et al. 1985)which accounted for invariable positions and unequalrates of substitutions under a gamma distribution(HKY + I + G) with the following parameters: assumednucleotide frequencies A = 0.3548, C = 0.1348, G = 0.1487,T = 0.3617; substitution model with a transition/trans-version ratio = 2.5962; proportion of invariable sitesI = 0.5809; variable sites followed a gamma distributionwith shape parameter = 0.6962.

In most of the clades, NJ, MP, and BAY analysessupported a single topology (Fig. 2). Overall, weobtained high support for the presence of two well-deWned evolutionary groups within Callinectes; one ofthem, hereafter called the “danae”-group (group A,Fig. 2), clusters C. arcuatus, Callinectes bellicosus, C.danae, Callinectes exasperatus, Callinectes larvatus, Cal-linectes ornatus, and Callinectes similis. A second clade,the “bocourti”-group (group B, Fig. 2), joined C. aVi-nis, C. bocourti, Callinectes rathbunae, C. maracaiboen-sis, C. sapidus, and Callinectes toxotes.

Within the “danae”-group we found a subgroup con-taining two pairs of sister taxa, C. ornatus/C. arcuatusand C. danae/C. similis. Callinectes exasperatus wasfound to be related to these two pairs of species. Whilethe three analyses consistently showed C. larvatus andC. bellicosus to fall within this major lineage, their rela-tionship to the other Wve species within the lineage wasnot resolved. The NJ analysis suggested that C. bellico-sus held a basal position within the “danae”-group, butthis was not conWrmed by the other two methods, asevident in the low bootstrap values on the consensustree (group A, Fig. 2).

Within the “bocourti”-group (group B, Fig. 2), C.bocourti and C. maracaiboensis had low genetic diVer-ences and shared at least one haplotype, which sug-gested that they belonged to the same species. Samplesof three specimens of C. aVinis from two localities allshared the same haplotype, which diVered slightly butconsistently from C. bocourti (one transversion in the16S sequence and two transitions in the 12S sequence).This level of diVerence, however, was comparable tothat between two specimens of C. arcuatus from diVer-ent populations, Mexico and Nicaragua (MEX, NIC).

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Callinectes rathbunae was positioned basal to the threeaforementioned species and exhibited seven mutationsseparating it from C. bocourti (four transitions and oneindel in the 16S sequence and three transitions and twotransversions in the 12S sequence). An additionaltrans-isthmian pair of related species in this group wascomprised of C. sapidus and C. toxotes.

A separate analysis that included only one repre-sentative of each species and one selected haplotypefor C. bocourti (ht1, from Schubart et al. 2001)showed the same relationships as described above

(Fig. 3). The tree resulting from BAY analysisgrouped C. bellicosus and C. larvatus, but there wasno support of this pairing by either MP or NJ analysesand only low support by BAY analysis. We used thisdata set to calculate radiation times within Callinectes.The LRT test (Felsenstein 1981) rejected rate con-stancy (P < 0.001; df = 12). Thus, an ultrametric treewas constructed under NPRS method with TreeEdit.Separation of the “bocourti” from the “danae”-groupwas calculated to have occurred about 13.22 § 0.75mybp (Table 2).

Fig. 2 Phylogeny for western Atlantic species of Callinectes in-ferred from NJ analysis of 964 basepairs (551 basepairs of 16Sand 413 basepairs of 12S rRNA) of mitochondrial DNA, from 26

specimens. Bootstrap values shown from top to bottom are forNJ, MP, and BAY, respectively: slash represents value equal to orlower than 50%. A,“danae”-group; B, “bocourti”-group

C. arcuatus NIC

C. arcuatus MEX

C. ornatus BRA

C. ornatus VEN

C. similis USA

C. danae VEN

C. danae BRA

C. exasperatus VEN

C. larvatus VEN

C. bellicosus MEX-1

C. bellicosus MEX-2

C sapidus USA

C. toxotes HON

C. rathbunae MEX

C. maracaiboensis VEN-1

C. bocourti VEN-1

C. maracaiboensis BRA-1

C. bocourti VEN-2

C. maracaiboensis VEN-2

C. affinis BRA-1

C. affinis BRA-2

C. affinis BRA-3

Charybdis helleri VEN-1

Charybdis helleri VEN-3

Arenaeus cribrarius VEN

0.01 substitutions/site

Charybdis helleri VEN-2100100100

52//

100100100

9798100

927391

99100100

647688

9399100

535870

100100100

9799100

100100100

809997

/5375

726893

879999

825593

A

B

100100100

638180

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Discussion

Relationships within the genus Callinectes have been asubject of debate (for review see Williams 1974). In aneVort to explain evolutionary relationships among allspecies of Callinectes, Norse and Fox-Norse (1979)proposed four evolutionary groups on the basis of mor-phology and physioecology (Table 3). They termedthese the “bocourti,” “danae,” “marginatus,” and“gladiator”-groups, although no attempt was made toexplain all evolutionary relationships within andamong these four groups. In the present study, we

included the same American species used by Norse andFox-Norse (1979) but also incorporated C. aVinis fromFortaleza, Brazil (Fausto-Filho 1980) and C. larvatus,the latter being the American sibling species of theAfrican C. marginatus (A. Milne-Edwards) (see Man-ning and Holthuis 1981).

Color of some specimens from Rio Grande doNorte, Brazil, has been interpreted as evidence that C.maracaiboensis occurs in northern Brazil (Sank-arankutty et al. 1999). However, Schubart et al. (2001)did not Wnd consistent molecular diVerences betweenC. maracaiboensis from Venezuela and C. bocourti on

Fig. 3 Phylogeny for western Atlantic species of Callinectes in-ferred from MP analysis of 964 basepairs (551 basepairs of 16Sand 413 basepairs of 12S rRNA) of mitochondrial DNA, ob-tained from 13 currently valid species. ConWdence values shown

from top to bottom are for NJ, MP, and BAY, respectively: slashrepresents value equal to or lower than 50%. A,“danae”-group;B, “bocourti”-group. Lower case letters a–h at selected nodes cor-respond to estimated divergence times in Table 2

C. arcuatus NIC

C. ornatus BRA

C. similis USA

C. danae VEN

C. exasperatus VEN

C. larvatus VEN

C. bellicosus MEX-1

C. sapidus USA

C. toxotes HON

C. rathbunae MEX

C. bocourti VEN-1

Charybdis helleri

Arenaeus cribrarius

748180

907688

10099

100

100100100

100100100

956096

1009996

929599

675476

/5870

A

B

100100100

C. affinis BRA-1

a

b

c

d

e

f

g

h

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the basis of 16S rDNA and assumed synonymy of thesetwo species. Thus, the specimens from Rio Grandewere assumed to represent either another color variantC. bocourti or yet another undescribed species of Calli-nectes. We obtained 10 swimming crabs from RioGrande do Norte, Brazil, and identiWed Wve as C. boco-urti, four as C. maracaiboensis and one as C. aVinis.One of four specimens from Rio Grande identiWed onmorphological bases as C. maracaiboensis shared acommon 12S-16S haplotype with our Venezuelan spec-imens of C. bocourti. Thus, on the basis of moleculardata, C. maracaiboensis cannot be separated from C.bocourti and should be regarded as a junior synonymof the latter species.

Sankarankutty et al. (1999) did not compare theirputative specimens of C. maracaiboensis to C. aVinisfrom Fortaleza, Brazil. While the morphological char-acters for diagnosis of C. aVinis are somewhat subjec-tive, they did apply relatively well to one specimen ofthe ten swimming crabs we obtained from Rio Grande,Brazil. Molecular comparisons of this specimen to thetwo type specimens of C. aVinis revealed no diVerencesamong the three specimens in either the 16S or the 12Smitochondrial genes. While the very similar morphol-ogy would suggest that this species, like C. maracaibo-ensis, is a synonym of C. bocourti, the three specimensthat we assign to C. aVinis all diVer from C. bocourti inthree base pair positions (one transversion in the 16Sand two transitions in the 12S). Even so, this same levelof diVerentiation can be observed between specimensassigned to C. arcuatus from Nicaragua and Mexico(present work) as well as between two haplotypes ofC. bocourti (ht1 and ht6) from Venezuela (Schubartet al. 2001).

We are tempted to assume that the morphologicaldiVerences reported for C. aVinis merely exemplify fur-ther variability of C. bocourti, much as alreadyobserved by Taissoun (1972), who recognized somesuch variants as C. maracaiboensis. We also note thatall three of these “species” occur sympatrically at leastat Rio Grande, Brazil, which makes it unlikely thatthey could represent reproductively isolated species.Nevertheless, our presently small sample of C. aVinisdoes exhibit the aforementioned consistent basepairdiVerences and is deWnable by morphological charac-ters, however subjective they may be. Thus, we electfor now to continue recognition of C. aVinis as a validspecies, even though we do relegate C. maracaiboensisto the synonymy of C. bocourti. Future analyses overlarger geographic areas and with larger samples willbe required to more deWnitively address the status ofC. aVinis.

We herein present a single phylogeny based on twomitochondrial genes, the 12S and the 16S. When deal-ing with such diVerent genes, there remains somedebate about how best to treat the resulting data.Three diVerent approaches are in general taken:“never combine,” “always combine,” or “conditionalcombination” (Cunningham 1997). Preliminary analy-sis of our data revealed that the major groups and mostof the internal clades were very similar for the separateanalyses. Following the conditional combinationapproach, our analysis was improved when weincreased the sample size by combining two genes thathave followed the same molecular evolutionary pro-cesses (Bull et al. 1993; Huelsenbeck et al. 1996; Cunn-ingham 1997), as proved by the ILD test.

Table 2 Estimated age of speciation at selected branch nodes forCallinectes, on the basis of nonparametric rate smoothing algo-rithms (NPRS values), and as obtained from 100 bootstraps of thedata for a Wxed tree (bootstrap means), the latter shown with 95%conWdence intervals (CI)

Letter indications for nodes correspond to their respective branchdivergences in Fig. 3

Node NPRS values (mybp)

Bootstrap means (mybp § 95% CI)

a 4.45 4.70 § 0.15b 2.02 2.11 § 0.23c 5.72 7.35 § 0.41d 13.36 13.22 § 0.75e 6.22 6.38 § 0.63f 6.78 8.21 § 0.77g 4.17 5.72 § 0.69h 1.78 2.66 § 0.37

Table 3 Table of evolutionary species groupings obtained withour analysis compared to those suggested by Norse and Fox-Norse (1979)

a African species

Species name Norse and Fox-Norse (1979)

Present analysis

Callinectes bocourti “bocourti” “bocourti”Callinectes maracaiboensis “bocourti” “bocourti”Callinectes rathbunae “bocourti” “bocourti”Callinectes toxotes “bocourti” “bocourti”Callinectes amnicolaa “bocourti” Not includedCallinectes sapidus “bocourti” “bocourti”Callinectes aVinis Not included “bocourti”Callinectes danae “danae” “danae”Callinectes arcuatus “danae” “danae”Callinectes similis “danae” “danae”Callinectes ornatus “danae” “danae”Callinectes bellicosus “danae” “danae”Callinectes marginatusa “marginatus” Not includedCallinectes larvatus “marginatus” “danae”Callinectes exasperatus “marginatus” “danae”Callinectes pallidusa “gladiator” Not included

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While our results agree with many Wndings of Norseand Fox-Norse (1979), separation of only two well-deWned groups is supported (80–100% bootstrap val-ues when including more than one specimen of thesame species, 96–100% bootstrap values when analyz-ing only one specimen per species) by analysis of ourcombined dataset of the 16S and the 12S mitochondrialgene sequences (Figs. 2, 3). Adopting the earliernomenclature used by Norse and Fox-Norse (1979),the “bocourti”-group clusters the Wve valid species C.aVinis, C. bocourti, C. rathbunae, C. sapidus, and C.toxotes (Fig. 2), while the “danae”-group joins C. arcu-atus, C. bellicosus, C. danae, C. exasperatus, C. larvatus,C. ornatus, and C. similis. Both C. larvatus and C. exa-speratus, thought to be part of the “exasperatus”-groupby Norse and Fox-Norse (1979), were clustered withinthe “danae”-group. Although we did not undertakemorphological or ecological analyses, it is evident thatsimilar physiological responses of species belonging tothe “marginatus” or “danae”-groups, noted by Norseand Fox-Norse (1979), are congruent with our Wndingthat all of them belong to a single phylogenetic unit. Incontrast to a suggestion by Norse and Fox-Norse(1979), we cannot conWrm that C. danae and C. arcua-tus are geminate trans-isthmian species. Instead, ourphylogenetic analysis suggests that C. ornatus is theclosest relative of C. arcuatus.

The presence of a long gonopod in all members ofthe “bocourti”-group (reaching the fourth thoracicsternite ventrally in mature and near mature males) isconsistent with our analysis. This character, previouslyused in an identiWcation key and thoroughly illustratedby Williams (1974: 721–722, Figs. 18–19), is clearly ofvalue for diagnosing major clades within the genus.Furthermore, based on this conclusion, we reckon thatboth Callinectes pallidus (De Rochebrune) and C. mar-ginatus, which were not included in our molecular anal-ysis, will likely Wt within the “danae”-group because oftheir morphology and physiology. Correspondingly,Callinectes amnicola (De Rochebrune) will probablycluster with other members of the “bocourti”-group.However, conWrmation of the phylogenetic position forthese African species awaits completion of a subse-quent analysis already in progress.

From our analysis, it is clear that C. aVinis is theclosest relative of C. bocourti. It also appears that C.rathbunae is the closest relative of what can be termedthe C. bocourti complex (C. bocourti–C. aVinis). Thisrelationship has not been reported previously, thoughit has been noted that there are few morphologicaldiVerences between C. rathbunae and C. bocourti (seeTaissoun 1972). Callinectes rathbunae represents yetanother example of an endemic within the Gulf of

Mexico that may have been derived as an isolate froman ancestral line shared with C. bocourti, widely dis-tributed in western Atlantic waters. Glacial advancesand retreats in North America have been invoked pre-viously to explain the origins of such endemics amongmarine decapods (Felder and Staton 2000; Felder2001) and could also account in varied ways for ende-mism of C. rathbunae in the southwestern Gulf of Mex-ico. Our molecular clock, discussed below, shows thatgenetic divergence of C. rathbunae occurred around4.17 mybp (Table 2), which is consistent with glacialXuctuations occurring during the Pliocene (Haq et al.1987). Also, change of ocean currents resulting fromthe gradual closing of the Panamanian Isthmus couldaccount for basin isolation, which has been proposed toexplain speciation among some alpheoid shrimps(Knowlton and Weigt 1998), as well as for Caribbeanforminiferal assemblages that appear to have devel-oped about 4.6 mybp (Haug and Tiedemann 1998),well before complete closure of the land bridge. How-ever early genetic divergence may have occurred, itmust be acknowledged that subsequent sorting of lin-eages and thus modern distributions should also reXecteVects of more recent events, such as recurrent epi-sodes of glaciation and retreat (Felder and Staton 1994;Felder 2001).

Major phylogenetic lineages of Callinectes likelydiverged before the closure of the Panamanian Isthmus(3.1 mybp) as representatives of both the “bocourti”and “danae”-groups, which today remain distributed assibling species on both sides of the Central Americanland bridge. Geological closure of the Panama landbridge (3.1 mybp) was used to calibrate a molecularclock for the evolution of Callinectes. Isolation amongmarine taxa separated by the Panamanian Isthmus hasbeen previously estimated to have occurred about3.5 mybp (Knowlton 1993; Knowlton et al. 1993;Knowlton and Weigt 1998), and this value was used inthe present study for molecular clock calibration. Inthe absence of rate consistency, we used the NPRS(Sanderson 1997), to calculate divergence times forspecies of Callinectes. Our molecular clock was cali-brated with two species, C. arcuatus and C. ornatus,which appear to be transisthmian siblings on the basisof our molecular analysis. We selected this pair of spe-cies, rather than the transisthmian sister taxa C. sapi-dus and C. toxotes, because of their lower moleculardivergence. Using pairs of species with higher molecu-lar divergence may not adequately consider eVects ofsome processes such as extinctions (Knowlton andWeigt 1998). The length of the NPRS at this node wasthen used to calibrate separation dates for other spe-cies of Callinectes.

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We estimate that divergence of the two major lin-eages within Callinectes occurred »13 mybp. Thisdivergence time is in relatively close agreement withthat of Weber et al. (2003), who report 12.5 mybp forthis event on the basis of morphology and allozymedata. The concordance of these estimates is remark-able given not only the diVerence in methodologies butalso that we take all American species of the genus intoaccount, while Weber et al. (2003) analyzed only threespecies found in southern Brazil. Our results are alsoconsistent with reports of fossil species of Callinectesfrom the Oligocene and Miocene (Rathbun 1919, 1933;Williams 1974); the most recent of these fossil formsappear very similar to the extant species, C. sapidus.

Acknowledgments We thank F. Mantelatto, C. Fransen, C.Sankarankutty, E. Castañeda, J. Bolaños, and G. Hernández forproviding some of the specimens used in our analyses. The man-uscript beneWted greatly from comments oVered by R. Bauer, S.Fredericq, R. Lemaitre, J. Neigel, and two anonymous reviewers.This study was supported under funding from US National Sci-ence Foundation (grant nos. DEB-0315995 and EF-0531603) andUS Department of Energy (grant no. DE-FG02-97ER12220) toD. L. Felder and the Venezuelan Institute for ScientiWc Researchto J. E. Conde. This is contribution number 115 from the Univer-sity of Louisiana Laboratory for Crustacean Research.

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