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Journal of the Marine Biological Association of the United Kingdom, page 1 of 10. #2008 Marine Biological Association of the United Kingdom doi:10.1017/S0025315408003238 Printed in the United Kingdom 1 2 Using morphological and molecular tools to 3 4 5 identify megalopae larvae collected in the 6 7 ﬁeld: the case of sympatric Cancer crabs 8 9 luis miguel pardo1, david ampuero2,3 and david v liz4 e10 1 Laboratorio Costero Calfuco, Instituto de Biologia Marina, Universidad Austral de Chile, Casilla 567, Valdivia, Chile, 2Instituto de11 Oceanologıa, Facultad de Ciencias del Mar, Universidad de Valparaıso, Casilla 13-D, Vina del Mar, Chile, 3Current address: 31469 ´ ´ ˜12 Motueka Valley Highway, Ngatimoti, Motueka, New Zealand, 4Instituto de Ecologıa y Biodiversidad (IEB), Departamento de ´13 ´ ˜ ˜ Ciencias Ecologicas, Facultad de Ciencias, Universidad de Chile. Casilla 653, Nunoa, Santiago, Chile141516 Studies of recruitment dynamics in meroplanktonic organisms are dependent on the correct identiﬁcation of each ontogenic17 stage of each species. This is particularly difﬁcult when studying the larval stages, which are not easy to identify due to their18 lack of resemblance to conspeciﬁc adults and their high degree of similarity with congeneric at the same stage of development.19 This is the case with the crustacean megalopae of the genera Cancer along the coast of the south-eastern Paciﬁc. This fact20 represents a serious limitation on ecological studies of populations of these species which constitute a heavily exploited21 local resource. In this study we describe in detail ﬁeld collected megalopae larvae of three sympatric crab species of the22 genera Cancer (C. edwardsii, C. setosus and C. coronatus). As a result of this analysis we were able to identify easily23 visible diagnostic characters which allow the species to be distinguished from one another. The megalopae were easily distin-24 guished by the form of the cheliped and the presence of spines on these. Cancer edwardsii has an elongated globulose cheliped,25 whereas C. coronatus has a subquadrate one. Both species possess a prominent ischial spine, which is absent in C. setosus. We26 corroborated the utility of these diagnostic characters by comparing the COI gene sequences of mitochondrial DNA of larvae27 identiﬁed by morphology with sequences taken from samples of the adults of all species of Cancer found in the region. We28 discuss the morphological variations between larvae found across the region (i.e. at sites separated by more than 800 km)29 and between megalopae obtained from the ﬁeld versus those cultivated in the laboratory. We conclude that the simultaneous30 use of morphological and molecular tools for identiﬁcation of decapod larvae appears useful for the study of cryptic species.313233 Keywords: Decapoda, taxonomy, recruitment, estuary, Chile3435 Submitted 30 May 2008; accepted 20 October 20083637383940 INTRODUCTION do not exhibit the speciﬁc diagnostic characteristics as evident41 in adults, but have cryptic morphologies within the related42 Studies of the early ontogenic stages in benthic marine invert- group. As a result individuals are assessed and assigned to43 ebrates are especially helpful for understanding the type of higher taxa, genera or even families. However, this generates44 population control acting upon a species in a given area ambiguities and less precision in the interpretation of ecologi-45 (Roughgarden et al., 1988). This is due to the larvae and cal data (e.g. recruitment events).46 early juvenile stages are the most important for dispersal A classic tool for helping to identify larvae collected in the47 and they have the highest level of mortality at the population ﬁeld is to use complete descriptions of larvae obtained from48 level (Palmer et al., 1996; Gosselin Qian, 1997; Hunt laboratory cultures. These descriptions have proved useful49 Scheilbling, 1997). In addition, in the case of species that not only in ecological studies but also for systematic and evol-50 can be exploited commercially, these studies allow us to utionary studies (Williamson, 1982; McHugh Rouse, 1998;51 understand the ﬂuctuations in stock and recruitment Feldmann, 2003). Despite the utility of this approach in52 (Wahle, 2003), and thus form a biological baseline useful in groups such as euphausids and decapods, the larval mor-53 the sustainable management of the ﬁshery (e.g. Eaton et al., phology may vary depending on environmental conditions54 2003). (Anger, 2001). Thus, the controlled conditions (temperature,55 However, an important limitation for many ﬁeld based salinity, density and absence of predators) of laboratory culti-56 ecological studies is the inability to correctly identify the vation may reduce the morphological plasticity of the larvae57 early stages of many species. Larval phases such as the zoea, compared with those that develop in the ﬁeld. Indeed import-58 decapoditae and megalopae of decapod crustaceans frequently ant morphological differences have been observed between59 brachyuran larvae raised in the laboratory and those encoun-60 tered in the ﬁeld. For example, Cuesta et al. (2002) describe61 Corresponding author: ﬁeld collected megalopae of the graspid Neohelice granulata62 L.M. Pardo with morphological anomalies, in the form of additional63 Email: firstname.lastname@example.org cephalothoracic spines and a reduced number of setae 1
2 luis miguel pardo et al. 64 compared to megalopae raised in the laboratory. Another C. setosus and C. coronatus) have been recorded with high 65 species, Pilumnoides perlatus, differs in the pattern of setation juvenile abundances in the estuaries of the north Patagonian 66 and also demonstrates a noticeable difference in size; megalo- region (Pardo L.M., unpublished data). This increases the 67 pae larvae from the laboratory are 30% smaller than those possibility that megalopae of all three species may be 68 encountered in the ﬁeld (Ampuero, 2007). present simultaneously in the same location. Without a clear 69 When larval descriptions are not available, the stage of description of these larvae taken from the natural environ- 70 interest can be cultivated up to the identiﬁable stage (normally ment, it is difﬁcult to advance our understanding of the 71 early juveniles) to generate a voucher collection which permits speciﬁc population ecology of these species throughout their 72 the rapid identiﬁcation of the remaining individuals. This geographical range. 73 technique has been successfully used in a number of studies Thus the objectives of this research are to: (1) describe the 74 concerning recruitment dynamics of decapods (Palma et al., megalopa larvae of the three species found in the ﬁeld; 75 2006; Pardo et al., 2007; Negreiros-Fransozo et al., 2007). (2) determine the speciﬁc morphological characteristics 76 However, this form of larval identiﬁcation is time consuming (SMC) which permit rapid identiﬁcation; (3) determine 77 and carries with it a decided degree of uncertainty when the which of the SMC do not vary either spatially or temporally; 78 genetic diversity of the sampling area is high and/or the and (4) corroborate, using molecular markers (the COI 79 species are only differentiated by morphological details of gene), the identiﬁcation of speciﬁc megalopae which were 80 appendages difﬁcult to see without adequate dissection (i.e. identiﬁed by means of the SMC, contrasting them with 81 mandible and maxilla). adults of all the species of Cancer present in the region. 82 The use of molecular markers has been demonstrated to be 83 a powerful tool for the identiﬁcation of cryptic species or 84 ontogenetic stages exhibiting little differentiation (Hebert 85 et al., 2003a; Vences et al., 2005; Rao et al., 2006). This Taxonomic status of studied species 86 applies especially to the sequencing of the COI gene of mito- According to Ng et al. (2008), some Paciﬁc species of Cancer 87 chondrial DNA, as it has proved to be a useful character for have been reassigned to other genera: Cancer edwardsii as 88 distinguishing between malacostran crustaceans (Knowlton Metacarcinus edwardsii and Cancer setosus as Romaleon poly- 89 Weigt, 1998; Barber Boyce, 2006), but is not clear in odon. The criteria used by Ng et al. (2008) for this reassign- 90 other zoological groups (e.g. France Hoover, 2002; ment of both Metacarcinus and Romaleon were fully based 91 Shearer et al., 2002; Hebert et al., 2003b; Meier et al., 2006). on the study carried out by Schweitzer Feldmann (2000). 92 Thus the molecular markers can be a good alternative for This is a paleogeographical work, based only on the carapace 93 the identiﬁcation of cryptic larval stages of decapods, morphology without including other morphological or genetic 94 especially when various species of the same genera are traits. At present molecular evidence analysed by one of our 95 collected simultaneously. collaborators in a phylogenetic study (Mantelatto F.L.M., 96 The study of the megalopae of the genera Cancer is compli- unpublished data) would not support new nominations at 97 cated by the problems outlined above, due to a high degree of this time and pending a further set of species. In this same 98 morphological similarity between the species (Iwata context, Harrison Crespi (1999) showed also some contro- 99 Konishi, 1981). Four species (Cancer edwardsii Bell, 1835, versy among morphological and genetic Cancer species status.100 C. Coronatus Molina, 1782, C. setosus Molina, 1782 and Therefore, with this non-consensual idea and possible contro-101 C. porteri Rathbun, 1930) have overlapping geographical dis- versy, we decided to keep the previous taxonomy and nomen-102 tributions along the east coast of the South Paciﬁc (Nations, clature of this group. Additionally, we did not use Cancer103 1975) so their megalopa larvae can be encountered sympatri- plebejus (Poepping, 1836) and C. polyodon (Poepping, 1836)104 cally. The ﬁrst three have a latitudinal distribution which because they are considered as junior synonyms of C. corona-105 extends from the equatorial region down to the Patagonian tus Molina, 1782 and C. setosus Molina, 1782 respectively. In106 region (Garth, 1957) and C. porteri has a discontinuous distri- this sense and considering that species studied here are com-107 bution in both hemispheres, except in the tropical region mercially important we think that this is the best way, at this108 (Nations, 1975), with a southern limit at approximately 358S time, to keep this name and avoid ﬁsheries problems as109 (Garth, 1957). Despite their high abundances and high recently reported for shrimp species of the genus ‘Penaeus’110 levels of commercial exploitation in the region (Anuario (Flegel, 2008).111Q1 Chileno de Pesca, 2006), little is known about their population112 ecology and even less about their recruitment dynamics (but113 see Jesse Stotz, 2003; Pardo et al., 2007).114 One of the major difﬁculties in advancing research into this MATERIALS AND METHODS115 group of crabs is the identiﬁcation of the megalopa larvae,116 a key stage during which recruitment and the moult to the117 ﬁrst juvenile stage takes place. There are two descriptions Collection of the megalopae118 available of megalopae for the genera Cancer along the coast Larvae were collected in the subtidal of the San Carlos inlet in119 of the south-eastern Paciﬁc, C. edwardsii and C. setosus ´ Bahıa Corral, located on the south side of the mouth of the Rio120 (Quintana, 1983; Quintana Saelzer, 1986), both from lab- Valdivia, Chile (39º 490 S 73º 140 W). In this area, the habitat121 oratory cultures. These larvae are extremely similar and the consists of a mixture of boulders and coarse sand. Samples122 comparison of the general morphology presented in the pub- were obtained by means of an airlift manipulated by divers123 lished ﬁgures does not allow for adequate identiﬁcation of at depths of between 8 and 10 m. Because crab megalopae124 individuals taken from the ﬁeld. Additionally, all four were found during spring and early autumn only, we analysed125 species can occur in the same habitat (Jesse Stotz, 2003; the morphological characteristics of larvae collected during126 ˜ Munoz et al., 2006) and three of the species (C. edwardsii, October, December and April, in order to obtain a temporally
identifying field collected cancer megalopae 3127 representative analysis. In addition we analysed megalopae of In order to determine the nucleotide relationship among128 C. setosus obtained from Punta Tralca located in central Chile samples (megalopae and adults crabs), a neighbour-joining129 (338 350 S 718 420 W), some 800 km north of the principal based phylogenetic (NJ) analysis was performed using Mega130 sampling sites. 4.0 software (Tamura et al., 2007). Using a bootstrap of131 10,000 replicates, the analysis tested the consistency of each132 branch in the tree, grouping sequences with similar nucleotide133 Description of the megalopae composition. Using this method, unidentiﬁed megalopae134 larvae could be grouped with conspeciﬁc adults. Finally, to Between 10 and 20 specimens of each species from Bahia135 root the tree, sequences of Homalaspis plana were used as out- Corral were dissected and examined in detail to describe136 group (Genbank Accession Number: FJ155383). larvae morphology. Additionally, 10 specimens of C. setosus137 from central Chile (Pta de Tralca) were also analysed. The138 megalopae and their appendages were dissected in glycerin139 RESULTS on microscope slides under a stereo microscope. The ﬁgures140 were made using a drawing tube mounted on a Zeiss141 To avoid long and repetitive description, the megalopa of AXIOSKOP microscope. The descriptions were made follow-142 Cancer coronatus is described completely whereas only differ- ing the scheme proposed by Clark et al. (1998). Typically the143 ences are described in detail for the rest of species. measurement of the cephalothorax length (CL) in species of144 Cancer is made from the distal end of the rostral spine to145146 the mid-posterior margin of the carapace (DeBrosse et al., Cancer coronatus Molina, 1782 1990). However, due to the damage found in the rostral147 Cephalotorax (Figure 1A): length to width ratio of 1.4, nar- spine on several individuals, in this study the crabs were148 rowing towards the anterior margin terminating in a ventrally measured from the base of the rostral spine to the mid-149 directed rostral spine. The posterior margin is smooth with a posterior margin of the cephalothorax. Cephalotorax width150 medial spine. The surface of the carapace is almost devoid of (CW) was measured on the midline of carapace.151 setae.152 Antennule (Figure 2A): penduncle 3-segmented, with 2, 3,153 0 simple setae plus 2 plumose setae in the proximal segment.154 DNA sequence based identiﬁcation Unsegmented endopod with 2 subterminal and 2 terminal155 After dissection of the megalopae, tissue samples from four simple setae. Exopod 4-segmented with 0, 6, 10, 3 and 0, 0,156 individuals per species from Bahia Corral were preserved in 3, 1 simple setae; and 1 long terminal plumose seta on the157 95% ethanol for subsequent genetic analyses. Two additional distal segment.158 megalopae were also sampled from Punta de Tralca only for Antenna (Figure 2D): peduncle 3-segmented; setation 4, 2,159 C. setosus. As there is no baseline genetic information for 4. Flagellum composed of 8 segments, with a setation pattern160 the species of the genera Cancer from Chile, we obtained of 0, 0, 4, 0, 5, 0, 4, 3 simple setae, proceeding from the prox-161 adult specimens from all Cancer crabs which inhabit the imal to distal segment.162 Chilean coast to corroborate the genetic identiﬁcation of the Mandible (Figure 2G): a developed cutting plate.163 larvae. Speciﬁcally adult specimens were collected from Three-segmented mandibular palp with 7 plumodenticulate164 Bahia Corral (C. coronatus, N ¼ 1; C. edwardsii, N ¼ 2 and cuspidate and 3 plumose setae on the distal segment.165 C. setosus, N ¼ 1), Caleta Lenga, Concepcion (36º 440 S 73º ´ Maxillule (Figure 2J): coxal endite with 11 simple, 13 plumo-166 110 W) (C. coronatus, N ¼ 1 and C. porterii, N ¼ 2) and denticulate cuspidate and 1 plumose seta. Basial endite with 8167 Punta de Tralca (C. setosus, N ¼ 1). Additionally, one adult plumodenticulate cuspidate and 6 marginal simple setae.168 specimen of the Platyxhantid crab Homalaspis plana Endopod 2-segmented with 2 simple setae on the proximal169 (Milne-Edwards, 1834) from Bahia Corral was also analysed. segment and 2 on the distal segment. Exopodal setae present.170 From each adult specimen was extracted a sample of muscle Maxilla (Figure 3A): coxal endite bilobed. Proximal and171 tissue from the chelipod, which was preserved in 95% distal lobe with 4 þ 0 plumodenticulate cuspidate, 2 þ 1172 ethanol for subsequent DNA extraction. plumose setae and 3 þ 0 simple setae. Basial endite173 The DNA extraction, from both adults and megalopae, was bilobed, the proximal and distal lobe with 7 þ 8 plumoden-174 conducted using the method described by Aljanabi ticulate cuspidate and 1 þ 1 simple setae. Endopod with 5175 Martinez (1997). The mitochondrial COI gene was ampliﬁed plumose setae along the external margin. Scaphognathite176 using the protocol and primers described by Folmer et al. with 86 plumose and 2 simple marginal setae, plus 6 simple177 (1994) and the PCR (polymerase chain reaction) used the fol- setae on the internal surface.178 lowing conditions: 1 Â buffer (Invitrogen), 3.2 nM MgCl2, First maxilliped (Figure 3D): triangular epipod at the prox-179 0.2 U/mL dNTP, 5 pmol forward and reverse primers and imal end narrowing rapidly towards the distal end, with 10180 0.1 U U/mL of Taq polymerase. The PCR cycle consisted of evenly distributed long simple, and 5 shorter setae close to181 3 minutes at 94ºC, 30 cycles of 30 seconds at 94ºC, the proximal end. Coxal and basial endite with 13 þ 22182 90 seconds at 42ºC and 90 seconds at 72ºC with a ﬁnal simple setae. Unsegmented endopod with 4 terminal simple183 elongation of 7 minutes at 72ºC. The PCR product was setae. Exopod 2-segmented with 4, 4 plumose setae.184 cleaned using QIAQuick columns (QIAGen, Mississaga, Second maxilliped (Figure 3G): epipod elongated with 13185 Ontario, Canada) and sequencing was performed at long ﬁliform setae. Gill present between the epipod and186 Macrogen Inc (www.macrogen.com). Later, sequences were exopod. Endopod 4-segmented, where the proximal segment187 aligned by eye using the ProSeq v.2.9 software (Filatov, has an obvious suture line. Setation pattern of 3, 1, 7 8188 2002). All haplotype was deposited in Genbank (Accession simple setae proceeding from the proximal to distal189 Numbers: FJ155371 to FJ155382). segment. Exopod 2-segmented with 1 simple seta on the
4 luis miguel pardo et al.190191192193194195196197198199200201202203204205206207208209210211212213214215216217218219220221222223224 Fig. 1. Comparative illustration of megalopae. General dorsal view (A) Cancer coronatus, (B) Cancer edwardsii and (C) Cancer setosus.225226227 proximal segment and 5 terminal plumose setae on the distal segment with a pair of uniramous uropods with 1 plumose228 segment. Protopodite with 4 simple setae close to the base of seta on the external margin of the protopodite and 14 long229 the endopod. plumose setae on the exopodite (Figure 5D).230 Third maxilliped (Figure 4A): elongated epipod with 24 Telson (Figure 5D): semicircular posterior margin, with 2231 evenly spread long simple setae and 2 plumose setae located pairs of simple setae on the ventral surface and a single pair232 at the proximal end. Protopod with 13 simple and 4 on the dorsal surface.233 plumose setae. Five-segmented endopod with 23 þ 9234 simple setae in the isquium and merus; and carpus, propodus235 and dactylus with 14 þ 13 þ 9 plumose setae. Exopod 2-236 segmented with 4 simple setae on the proximal segment and Cancer edwardsii Bell, 1835237 5 terminal plumose setae on the distal segment. Cephalothorax (Figure 1B): the ratio length/width was238 Chelipeds (Figure 4D): surface covered in a large number approximately 1.3. Sixty-two surface setae and 58 ventral mar-239 of simple setae. Exhibits a prominant spine on the internal ginal setae.240 margin of the isquium. Merus with an acute projection at Antennule (Figure 2B): penduncle with 4, 9, 2 simple setae241 the distal end. Propodus subquadrate in form with a length plus 8 plumose setae. Endopod has 5 simple setae. Exopod242 to width ratio of 2.0, logitudinal grooves ill deﬁned, internal with a setation of 0, 14, 10, 6 aesthetascs and 0, 0, 3, 1243 chela ﬁxed with shallow denticulation. simple setae on each segment.244 Second pereiopod (Figure 4G): surface covered in setae, the Antenna (Figure 2E): peduncle setation 4, 2, 4 (the interior245 majority of which are simple in form. Coxa of second pereio- seta of the second segment is plumose); and ﬂagellum with246 pod with a thick cuticular spine. Fifth pereiopod (Figure 1A) 0, 0, 4, 0, 5, 0, 4, 3 simple setae per segment.247 with 3 long terminal setae on the dactylus. Mandible (Figure 2H): mandibular palp with 13 plumo-248 Abdomen (Figure 1A): composed of 6 somites plus the denticulate cuspidate setae on the distal segment.249 telson, and dorsal surface with 2, 4, 6, 8, 8, 3 simple setae. Maxillule (Figure 2K): coxal and basial endite with 12250 Second and ﬁfth segments possess biramous pleopods, ﬁfth (7 plumodenticulate cuspidate, 5 plumose) þ19 (plumodenti-251 pleopod (Figure 5A) exopods have 21 terminal long culate cuspidate) setae and 5 þ 4 simple marginal setae.252 plumose setae, endopods with 4 terminal cincinnuli. Sixth Epipodal and exopodal setae present.
identifying field collected cancer megalopae 5253254255256257258259260261262263264265266267268269270271272273274275276277278279280281282283284285286287288289 Fig. 2. Comparative illustration of the cancridae megalopae appendages. Antennule, antenna, mandible and maxillule of Cancer coronatus (A, D, G, J); Cancer290 edwardsii (B, E, H, K); Cancer setosus (C, F, I, L).291292293294 Maxilla (Figure3B): proximal and distal lobe of the coxal 1, 4, 8, 0, 0 simple setae. Exopod with 5 simple setae on the295 endite with 7 þ 6 plumose setae. Proximal and distal lobe proximal segment, and 8 long plumose setae on the distal296 of the basial endite has 8 þ 10 plumodenticulate cuspidate segment.297 and 1 þ 1 simple setae. Scaphognathite with 77 plumose Chelipeds (Figure 4E): propodus globular in form with a298 setae. length to width ratio of 1.5 and well deﬁned longitudinal299 First maxilliped (Figure 3E): epipod with 12 very long grooves.300 simple setae along the mid to distal exterior margin, and 2 Abdomen (Figure 1B): somites dorsal surface with 4, 16, 14,301 shorter simple setae on the proximal end. Coxal endite with 14, 14, 6 simple setae. Biramous pleopods, ﬁfth with exopods302 14 plumose setae and the basial endite with 25 plumodenticu- having 24 terminal long plumose setae (Figure 5B). Uropod303 late cuspidate setae. Endopod with additional plumose setae. with 2 long plumose setae on the protopodite (Figure 5E).304 Exopod with 4, 5 plumose setae and 1, 1 simple seta on the Telson (Figure 5E): 2, 2 simple setae on the ventral and305 proximal and distal segment respectively. dorsal surface respectively.306 Second maxilliped (Figure 3H): epipodite with 15 long307 simple setae. Endopod with 5, 2 (1 simple, 1 plumose), 7 (2308 plumodenticulate cuspidate, 5 plumose), 4 (5 plumodenticu-309 late cuspidate, 4 plumose) setae. Exopod with 2 simple setae Cancer setosus Molina, 1782310 on proximal segment and 5 long plumose setae on the distal Cephalothorax (Figure 1C): length to width ratio of 1.4. 32311 segment. surface setae and 16 ventral marginal setae.312 Third maxilliped (Figure 4B): epipod with 20 long simple Antennule (Figure 2C): penduncle with 7 plumose setae313 setae, and 6 plumose setae located on the proximal margin. and 1 simple seta on the ﬁrst segment: and 4 simple setae314 Protopod with 25 plumose setae. Endopod, 27 (plus 6 on the second one. Endopod with 6 simple setae. Exopod315 plumose), 7, 6, 12, 9 plumodenticulate cuspidate setae and with 0, 18, 12, 10 aesthetascs.
6 luis miguel pardo et al.316317318319320321322323324325326327328329330331332333334335336337338339340341342343344345346347348349350351352 Fig. 3. Comparative illustration of the cancridae megalopae appendages. Maxille, ﬁrst maxilliped and second maxilliped of Cancer coronatus (A, D, G); Cancer353 edwardsii (B, E, H); Cancer setosus (C, F, I).354355356357 Antenna (Figure 2F): peduncle with 4, 1, 5 setae (the seta 1, 7 plumose setae plus 2 simple setae. Protopod with 4358 on the second segment is plumose) and ﬂagellum with 0, 0, simple setae and 4 plumose setae close to the base of the359 4, 1, 5, 2, 3, 2 simple setae. endopod.360 Mandible (Figure 2I): mandibular palp proximal segment Third maxilliped (Figure 4C): epipodite with 30 long361 with 1 plumose seta; distal segment with 15 plumodenticulate simple setae and 6 marginal plumose setae located near the362 cuspidate setae. proximal end. Protopod with 18 plumose setae and 2 simple363 Maxillule: (Figure 2L): coxal endite with 2 plumose, 7 plu- setae. Endopod with 37, 16, 20, 18, 9 plumodenticulate cuspi-364 modenticulate cuspidate and 7 simple setae. Basial endite with date setae. Exopod with 3 simple setae on the proximal365 24 plumodenticulate cuspidate and 5 simple setae. segment and 10 plumose setae on the distal segment.366 Maxilla (Figure 3C): coxal endite proximal and distal lobe Chelipeds (Figure 4F): without isquial spine. Tubular pro-367 with 3 þ 5 plumodenticulate cuspidate, 3 þ 0 plumose and podus with a length to width ratio of 1.8 and several well368 1 þ 1 simple seta. Basial endite proximal and distal lobe with deﬁned longitudinal grooves.369 10 þ 12 plumodenticulate cuspidate and 1 þ 1 simple seta. Abdomen (Figure 1C): somites dorsal surface with 4, 18,370 Endopod with 7 plumose setae. Scaphognathite with 95 mar- 18, 18, 14, 6 simple setae. Biramous pleopods, ﬁfth371 ginal plumose setae. (Figure 5C) with exopods having 24 long and 3 short terminal372 First maxilliped (Figure 3F): endopod with 22 long simple plumose setae. Uropod with 2 long plumose setae on the pro-373 setae. Coxal and basial endite with 21 þ 36 plumodenticulate topod and 16 long plumose setae on the exopod (Figure 5F).374 cuspidate plumose setae. Endopod with 3 plumose setae and 5 Telson (Figure 5F): 2, 8 simple setae on the ventral and375 simple setae. Exopod with 4, 7 plumose setae. dorsal surface respectively.376 Second maxilliped (Figure 3I): epipodite with 18 long Intraregional variation: larvae collected around 800 km377 simple setae. Endopod with 2 (plus 2 plumose), 1, 8, 9 plumo- between them did not show differences in SMC, but southern378 denticulate cuspidate, and 6, 1, 0, 0 simple setae. Exopod with were noticeably larger than northern megalopae (Table 1) .
identifying field collected cancer megalopae 7379380381382383384385386387388389390391392393394395396397398399400401402403404405406407408409410411412413414415416 Fig. 4. Comparative illustration of the cancridae megalopae appendages. Third maxilleped, cheliped and second pereiopod of Cancer coronatus (A, D, G); Cancer417 edwardsii (B, E, H); Cancer setosus (C, F, I).418419420 Molecular identiﬁcation the antenna and the endopod of the third maxilliped (Iwata421 Konishi, 1981). However, we found a considerable422 The sequences of 554 bp obtained from the COI gene of both amount of overlapping in these characteristics between423 megalopae and adults of the studied species of Cancer, exhib- species (Table 1).424 ited a high level of differences between species. We observed The general morphology of the megalopae studied display425 around 60 base pair differences between species and a a high degree of intra-speciﬁc similarity. However, certain426 maximum of 4 between individuals of the same species. morphological features in the chelipeds such as the presence427 These clear differences between the sequences of each or absence of the isquial spine allow for a rapid and precise428 species were the basis for grouping the megalopae with the identiﬁcation of each of the three species. These diagnostic429 adults, without ambiguity, demonstrated by tree nodes with characteristics have not been commented upon previously in430 high support values between different species (Figure 6). the descriptions of C. edwardsii and C. setosus (Quintana,431 1983; Quintana Saelzer, 1986), based on laboratory reared432 larvae, and have for the ﬁrst time been recorded for C. corona-433 DISCUSSION tus. In addition, these features appear to be highly conserved434 on both the spatial and temporal scales. For example, megalo-435 Several characters have been used to identify megalopae of the pae of C. setosus collected at locations separated by more than436 genera Cancer to the species level. Speciﬁc diagnostic charac- 800 km were identiﬁed to the species level and these were later437 teristics that have been considered previously include: the corroborated by the DNA analyses, despite differences in their438 number of plumose setae present on the uropods and cincin- size and date of collection.439 nuli present on the pleopods (Poole, 1966; Trask, 1970), the The scarce morphological differentiation between the440 carapace length and presence or absence of a lateral knob megalopae of the genus Cancer has been noted previously.441 (Orensanz Gallucci, 1988), and the setae and spines over DeBrosse et al. (1990) did not ﬁnd invariable morphological
8 luis miguel pardo et al.442443444445446447448449450451452453454455456457458459460461462463464465 Fig. 5. Comparative illustration of the cancridae megalopae appendages. Second pleopod and telson of Cancer coronatus (A, D); Cancer edwardsii (B, E); Cancer466 setosus (C, F).467468469 features which allowed for the identiﬁcation of three sympa- general all the megalopae analysed appear to have more470 tric species of Cancer (C. magister, C. oregonesis and C. pro- setae than their conspeciﬁcs reared in the laboratory.471 ductus) collected in the Puget Sound basin on the north-east Considerable variation has been described in the megalopae472 Paciﬁc coast. Such as our case, the megalopae presented a of other species of the genus Cancer obtained in the ﬁeld473 high degree of morphological similarity (except for the con- (Orensanz Gallucci, 1988; DeBrosse et al., 1990) and also474 siderably larger size of C. magister) and it was not possible in other brachyuran species (Cuesta et al., 2002; Ampuero,475 to easily distinguish the larvae maintained in the laboratory 2007).476 using the published descriptions. The discrepancies between larvae collected in the ﬁeld and477 Another interesting ﬁnding was the differences in larval those cultivated in the laboratory could be explained by two478 morphology between ﬁeld and laboratory reared megalopae non-exclusive factors. First, the descriptions based on culti-479 (Table 1). The larvae of C. edwardsii and C. setosus collected vated larvae normally utilize a single or few reproductive480 in the ﬁeld are considerably larger than those raised in the lab- females to provide the individuals described, reducing the481 oratory. In the case of C. coronatus there were no available genotypic variation available for study. Second, the controlled482 descriptions for comparison. In addition to the size, the conditions under which the larvae are cultivated (temperature,483 larvae from the ﬁeld exhibit a marked increase in the salinity and food availability) may restrict phenotypic484 number of setae on their cephalothoracic appendages and in expression.485486487 Table 1. Variation in biometric measures of Cancer megalopae from ﬁeld and laboratory. All setal counts are listed from proximal to distal. CL is measured from base of rostral spine to middle of the posterior margin. From megalopae described in the literature (reared at the laboratory), CL and488 CW were estimated from illustrations, using the graphic scales provided by the authors (Quintana 1983; Quintana Saelzer 1986). Abbreviations:489 CL, cephalothorax length; CW, cephalothorax width; ND, no data. Ã indicate megalopae from Punta de Tralca.490491 Cancer edwardsii Cancer edwardsii Cancer setosus Cancer setosus Cancer coronatus492 Laboratory Field Laboratory Field Field493 Cephalothorax494 CL (mm) 3.1 3.3 + 0.1 2.7 3.9 + 0.2–3.1 + 0.1Ã 3.01 + 0.1495 CW (mm) 2.05 2.6 + 0.1 1.9 2.6 + 0.1–2.3 + 0.1Ã 2.01 + 0.2496 Antenna (peduncle) 2,2,3 4,1,4 3,2,4 4,1,5 4,2,4497 Antenna ( ﬂagellum) 0,0,2,0,5,0,4,4 0,0,4,0,5,0,4,3 0,0,4,0,4,0,3,5 0,0,4,1,5,2,3,2 0,0,4,0,5,0,4,3498 Third maxilliped499 Endopodite 22,10,8,0,0 34,11,14,13,9 21,10,15,0,0 37,16,20,18,9 23,9,14,13,9500 Exopodite 5 –8.5 5.8 4.5 3.1 4.5501 Epipodite 3 þ 19 6 þ 20 4 þ 16 6 þ 30 2 þ 24 Protopodite 7 25 18 18 18502 Pleopodal cincinnuli ND 4 ND 4 4503 Cheliped isquial spine Yes Yes Not Not Yes504
identifying field collected cancer megalopae 9505 Table 2. Summary of base pair difference between COI sequences (554 bp) showing maximum and minimum difference within and among species.506 The mean of differences is expressed in per cent.507 Cancer porteri (0) Cancer edwarsii (0–2) (0.36%) Cancer coronatus (0–2) (0.36%) Cancer setosus (0–3) (0.54%)508509 Cancer porteri 109 –111 (19.86%) 108 (19.50%) 138–140 (25.09%)510 Cancer edwarsii 62–64 (11.37%) 96–99 (19.50%)511 Cancer coronatus 95–99 (19.50%) Cancer setosus512513514515 FONDECYT 110240071 for ﬁnancial support. D.V. thanks516 the Basal Grant PFB 023, CONICYT, Chile and Iniciativa517 ´ Cientıﬁca Milenio Grant ICM P05-002. Thanks to two anon-518 ymous referees who have greatly helped us in improving the519 clarity of the presentation. All experimental work was con-520 ducted in Chile and complied with its existing laws.521522523 REFERENCES524525 Aljanabi S.M. and Martinez I. (1997) Universal and rapid salt-extraction526 Fig. 6. 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(1988) Comparative study of postlarval Correspondence should be addressed to:626 life-history schedules in four sympatric species of Cancer (Decapoda: L.M. Pardo627 Brachyura: Cancridae). Journal of Crustacean Biology 8, 187–220. Laboratorio Costero Calfuco, Instituto de Biologia Marina628629 ´ Palma A.T., Pardo L.M., Veas R., Cartes C., Silva M., Manrıquez K., Universidad Austral de Chile, Casilla 567, Valdivia, Chile630 ´ ˜ Dıaz A., Munoz C. and Ojeda F.P. (2006) Coastal brachyuran email: email@example.com