Local fisherman_larvae knowledge

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Page 1: Local fisherman_larvae knowledge

Proof Delivery FormPlease return this form with your proof

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Using morphological and molecular tools toidentify megalopae larvae collected in thefield: the case of sympatric Cancer crabs

luis miguel pardo1

, david ampuero2,3

and david v�eliz4

1Laboratorio Costero Calfuco, Instituto de Biologia Marina, Universidad Austral de Chile, Casilla 567, Valdivia, Chile, 2Instituto deOceanologıa, Facultad de Ciencias del Mar, Universidad de Valparaıso, Casilla 13-D, Vina del Mar, Chile, 3Current address: 31469Motueka Valley Highway, Ngatimoti, Motueka, New Zealand, 4Instituto de Ecologıa y Biodiversidad (IEB), Departamento deCiencias Ecologicas, Facultad de Ciencias, Universidad de Chile. Casilla 653, Nunoa, Santiago, Chile

Studies of recruitment dynamics in meroplanktonic organisms are dependent on the correct identification of each ontogenicstage of each species. This is particularly difficult when studying the larval stages, which are not easy to identify due to theirlack of resemblance to conspecific adults and their high degree of similarity with congeneric at the same stage of development.This is the case with the crustacean megalopae of the genera Cancer along the coast of the south-eastern Pacific. This factrepresents a serious limitation on ecological studies of populations of these species which constitute a heavily exploitedlocal resource. In this study we describe in detail field collected megalopae larvae of three sympatric crab species of thegenera Cancer (C. edwardsii, C. setosus and C. coronatus). As a result of this analysis we were able to identify easilyvisible diagnostic characters which allow the species to be distinguished from one another. The megalopae were easily distin-guished by the form of the cheliped and the presence of spines on these. Cancer edwardsii has an elongated globulose cheliped,whereas C. coronatus has a subquadrate one. Both species possess a prominent ischial spine, which is absent in C. setosus. Wecorroborated the utility of these diagnostic characters by comparing the COI gene sequences of mitochondrial DNA of larvaeidentified by morphology with sequences taken from samples of the adults of all species of Cancer found in the region. Wediscuss the morphological variations between larvae found across the region (i.e. at sites separated by more than 800 km)and between megalopae obtained from the field versus those cultivated in the laboratory. We conclude that the simultaneoususe of morphological and molecular tools for identification of decapod larvae appears useful for the study of cryptic species.

Keywords: Decapoda, taxonomy, recruitment, estuary, Chile

Submitted 30 May 2008; accepted 20 October 2008

I N T R O D U C T I O N

Studies of the early ontogenic stages in benthic marine invert-ebrates are especially helpful for understanding the type ofpopulation control acting upon a species in a given area(Roughgarden et al., 1988). This is due to the larvae andearly juvenile stages are the most important for dispersaland they have the highest level of mortality at the populationlevel (Palmer et al., 1996; Gosselin & Qian, 1997; Hunt &Scheilbling, 1997). In addition, in the case of species thatcan be exploited commercially, these studies allow us tounderstand the fluctuations in stock and recruitment(Wahle, 2003), and thus form a biological baseline useful inthe sustainable management of the fishery (e.g. Eaton et al.,2003).

However, an important limitation for many field basedecological studies is the inability to correctly identify theearly stages of many species. Larval phases such as the zoea,decapoditae and megalopae of decapod crustaceans frequently

do not exhibit the specific diagnostic characteristics as evidentin adults, but have cryptic morphologies within the relatedgroup. As a result individuals are assessed and assigned tohigher taxa, genera or even families. However, this generatesambiguities and less precision in the interpretation of ecologi-cal data (e.g. recruitment events).

A classic tool for helping to identify larvae collected in thefield is to use complete descriptions of larvae obtained fromlaboratory cultures. These descriptions have proved usefulnot only in ecological studies but also for systematic and evol-utionary studies (Williamson, 1982; McHugh & Rouse, 1998;Feldmann, 2003). Despite the utility of this approach ingroups such as euphausids and decapods, the larval mor-phology may vary depending on environmental conditions(Anger, 2001). Thus, the controlled conditions (temperature,salinity, density and absence of predators) of laboratory culti-vation may reduce the morphological plasticity of the larvaecompared with those that develop in the field. Indeed import-ant morphological differences have been observed betweenbrachyuran larvae raised in the laboratory and those encoun-tered in the field. For example, Cuesta et al. (2002) describefield collected megalopae of the graspid Neohelice granulatawith morphological anomalies, in the form of additionalcephalothoracic spines and a reduced number of setae

Corresponding author:L.M. PardoEmail: [email protected]

1

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compared to megalopae raised in the laboratory. Anotherspecies, Pilumnoides perlatus, differs in the pattern of setationand also demonstrates a noticeable difference in size; megalo-pae larvae from the laboratory are 30% smaller than thoseencountered in the field (Ampuero, 2007).

When larval descriptions are not available, the stage ofinterest can be cultivated up to the identifiable stage (normallyearly juveniles) to generate a voucher collection which permitsthe rapid identification of the remaining individuals. Thistechnique has been successfully used in a number of studiesconcerning recruitment dynamics of decapods (Palma et al.,2006; Pardo et al., 2007; Negreiros-Fransozo et al., 2007).However, this form of larval identification is time consumingand carries with it a decided degree of uncertainty when thegenetic diversity of the sampling area is high and/or thespecies are only differentiated by morphological details ofappendages difficult to see without adequate dissection (i.e.mandible and maxilla).

The use of molecular markers has been demonstrated to bea powerful tool for the identification of cryptic species orontogenetic stages exhibiting little differentiation (Hebertet al., 2003a; Vences et al., 2005; Rao et al., 2006). Thisapplies especially to the sequencing of the COI gene of mito-chondrial DNA, as it has proved to be a useful character fordistinguishing between malacostran crustaceans (Knowlton& Weigt, 1998; Barber & Boyce, 2006), but is not clear inother zoological groups (e.g. France & Hoover, 2002;Shearer et al., 2002; Hebert et al., 2003b; Meier et al., 2006).Thus the molecular markers can be a good alternative forthe identification of cryptic larval stages of decapods,especially when various species of the same genera arecollected simultaneously.

The study of the megalopae of the genera Cancer is compli-cated by the problems outlined above, due to a high degree ofmorphological similarity between the species (Iwata &Konishi, 1981). Four species (Cancer edwardsii Bell, 1835,C. Coronatus Molina, 1782, C. setosus Molina, 1782 andC. porteri Rathbun, 1930) have overlapping geographical dis-tributions along the east coast of the South Pacific (Nations,1975) so their megalopa larvae can be encountered sympatri-cally. The first three have a latitudinal distribution whichextends from the equatorial region down to the Patagonianregion (Garth, 1957) and C. porteri has a discontinuous distri-bution in both hemispheres, except in the tropical region(Nations, 1975), with a southern limit at approximately 358S(Garth, 1957). Despite their high abundances and highlevels of commercial exploitation in the region (AnuarioChileno de Pesca, 2006)Q1 , little is known about their populationecology and even less about their recruitment dynamics (butsee Jesse & Stotz, 2003; Pardo et al., 2007).

One of the major difficulties in advancing research into thisgroup of crabs is the identification of the megalopa larvae,a key stage during which recruitment and the moult to thefirst juvenile stage takes place. There are two descriptionsavailable of megalopae for the genera Cancer along the coastof the south-eastern Pacific, C. edwardsii and C. setosus(Quintana, 1983; Quintana & Saelzer, 1986), both from lab-oratory cultures. These larvae are extremely similar and thecomparison of the general morphology presented in the pub-lished figures does not allow for adequate identification ofindividuals taken from the field. Additionally, all fourspecies can occur in the same habitat (Jesse & Stotz, 2003;Munoz et al., 2006) and three of the species (C. edwardsii,

C. setosus and C. coronatus) have been recorded with highjuvenile abundances in the estuaries of the north Patagonianregion (Pardo L.M., unpublished data). This increases thepossibility that megalopae of all three species may bepresent simultaneously in the same location. Without a cleardescription of these larvae taken from the natural environ-ment, it is difficult to advance our understanding of thespecific population ecology of these species throughout theirgeographical range.

Thus the objectives of this research are to: (1) describe themegalopa larvae of the three species found in the field;(2) determine the specific morphological characteristics(SMC) which permit rapid identification; (3) determinewhich of the SMC do not vary either spatially or temporally;and (4) corroborate, using molecular markers (the COIgene), the identification of specific megalopae which wereidentified by means of the SMC, contrasting them withadults of all the species of Cancer present in the region.

Taxonomic status of studied speciesAccording to Ng et al. (2008), some Pacific species of Cancerhave been reassigned to other genera: Cancer edwardsii asMetacarcinus edwardsii and Cancer setosus as Romaleon poly-odon. The criteria used by Ng et al. (2008) for this reassign-ment of both Metacarcinus and Romaleon were fully basedon the study carried out by Schweitzer & Feldmann (2000).This is a paleogeographical work, based only on the carapacemorphology without including other morphological or genetictraits. At present molecular evidence analysed by one of ourcollaborators in a phylogenetic study (Mantelatto F.L.M.,unpublished data) would not support new nominations atthis time and pending a further set of species. In this samecontext, Harrison & Crespi (1999) showed also some contro-versy among morphological and genetic Cancer species status.Therefore, with this non-consensual idea and possible contro-versy, we decided to keep the previous taxonomy and nomen-clature of this group. Additionally, we did not use Cancerplebejus (Poepping, 1836) and C. polyodon (Poepping, 1836)because they are considered as junior synonyms of C. corona-tus Molina, 1782 and C. setosus Molina, 1782 respectively. Inthis sense and considering that species studied here are com-mercially important we think that this is the best way, at thistime, to keep this name and avoid fisheries problems asrecently reported for shrimp species of the genus ‘Penaeus’(Flegel, 2008).

M A T E R I A L S A N D M E T H O D S

Collection of the megalopaeLarvae were collected in the subtidal of the San Carlos inlet inBahıa Corral, located on the south side of the mouth of the RioValdivia, Chile (39º 490S 73º 140W). In this area, the habitatconsists of a mixture of boulders and coarse sand. Sampleswere obtained by means of an airlift manipulated by diversat depths of between 8 and 10 m. Because crab megalopaewere found during spring and early autumn only, we analysedthe morphological characteristics of larvae collected duringOctober, December and April, in order to obtain a temporally

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representative analysis. In addition we analysed megalopae ofC. setosus obtained from Punta Tralca located in central Chile(338 350S 718 420W), some 800 km north of the principalsampling sites.

Description of the megalopaeBetween 10 and 20 specimens of each species from BahiaCorral were dissected and examined in detail to describelarvae morphology. Additionally, 10 specimens of C. setosusfrom central Chile (Pta de Tralca) were also analysed. Themegalopae and their appendages were dissected in glycerinon microscope slides under a stereo microscope. The figureswere made using a drawing tube mounted on a ZeissAXIOSKOP microscope. The descriptions were made follow-ing the scheme proposed by Clark et al. (1998). Typically themeasurement of the cephalothorax length (CL) in species ofCancer is made from the distal end of the rostral spine tothe mid-posterior margin of the carapace (DeBrosse et al.,1990). However, due to the damage found in the rostralspine on several individuals, in this study the crabs weremeasured from the base of the rostral spine to the mid-posterior margin of the cephalothorax. Cephalotorax width(CW) was measured on the midline of carapace.

DNA sequence based identificationAfter dissection of the megalopae, tissue samples from fourindividuals per species from Bahia Corral were preserved in95% ethanol for subsequent genetic analyses. Two additionalmegalopae were also sampled from Punta de Tralca only forC. setosus. As there is no baseline genetic information forthe species of the genera Cancer from Chile, we obtainedadult specimens from all Cancer crabs which inhabit theChilean coast to corroborate the genetic identification of thelarvae. Specifically adult specimens were collected fromBahia Corral (C. coronatus, N ¼ 1; C. edwardsii, N ¼ 2 andC. setosus, N ¼ 1), Caleta Lenga, Concepcion (36º 440 S 73º110W) (C. coronatus, N ¼ 1 and C. porterii, N ¼ 2) andPunta de Tralca (C. setosus, N ¼ 1). Additionally, one adultspecimen of the Platyxhantid crab Homalaspis plana(Milne-Edwards, 1834) from Bahia Corral was also analysed.From each adult specimen was extracted a sample of muscletissue from the chelipod, which was preserved in 95%ethanol for subsequent DNA extraction.

The DNA extraction, from both adults and megalopae, wasconducted using the method described by Aljanabi &Martinez (1997). The mitochondrial COI gene was amplifiedusing the protocol and primers described by Folmer et al.(1994) and the PCR (polymerase chain reaction) used the fol-lowing conditions: 1 � buffer (Invitrogen), 3.2 nM MgCl2,0.2 U/mL dNTP, 5 pmol forward and reverse primers and0.1 U U/mL of Taq polymerase. The PCR cycle consisted of3 minutes at 94ºC, 30 cycles of 30 seconds at 94ºC,90 seconds at 42ºC and 90 seconds at 72ºC with a finalelongation of 7 minutes at 72ºC. The PCR product wascleaned using QIAQuick columns (QIAGen, Mississaga,Ontario, Canada) and sequencing was performed atMacrogen Inc (www.macrogen.com). Later, sequences werealigned by eye using the ProSeq v.2.9 software (Filatov,2002). All haplotype was deposited in Genbank (AccessionNumbers: FJ155371 to FJ155382).

In order to determine the nucleotide relationship amongsamples (megalopae and adults crabs), a neighbour-joiningbased phylogenetic (NJ) analysis was performed using Mega4.0 software (Tamura et al., 2007). Using a bootstrap of10,000 replicates, the analysis tested the consistency of eachbranch in the tree, grouping sequences with similar nucleotidecomposition. Using this method, unidentified megalopaelarvae could be grouped with conspecific adults. Finally, toroot the tree, sequences of Homalaspis plana were used as out-group (Genbank Accession Number: FJ155383).

R E S U L T S

To avoid long and repetitive description, the megalopa ofCancer coronatus is described completely whereas only differ-ences are described in detail for the rest of species.

Cancer coronatus Molina, 1782Cephalotorax (Figure 1A): length to width ratio of 1.4, nar-rowing towards the anterior margin terminating in a ventrallydirected rostral spine. The posterior margin is smooth with amedial spine. The surface of the carapace is almost devoid ofsetae.

Antennule (Figure 2A): penduncle 3-segmented, with 2, 3,0 simple setae plus 2 plumose setae in the proximal segment.Unsegmented endopod with 2 subterminal and 2 terminalsimple setae. Exopod 4-segmented with 0, 6, 10, 3 and 0, 0,3, 1 simple setae; and 1 long terminal plumose seta on thedistal segment.

Antenna (Figure 2D): peduncle 3-segmented; setation 4, 2,4. Flagellum composed of 8 segments, with a setation patternof 0, 0, 4, 0, 5, 0, 4, 3 simple setae, proceeding from the prox-imal to distal segment.

Mandible (Figure 2G): a developed cutting plate.Three-segmented mandibular palp with 7 plumodenticulatecuspidate and 3 plumose setae on the distal segment.

Maxillule (Figure 2J): coxal endite with 11 simple, 13 plumo-denticulate cuspidate and 1 plumose seta. Basial endite with 8plumodenticulate cuspidate and 6 marginal simple setae.Endopod 2-segmented with 2 simple setae on the proximalsegment and 2 on the distal segment. Exopodal setae present.

Maxilla (Figure 3A): coxal endite bilobed. Proximal anddistal lobe with 4 þ 0 plumodenticulate cuspidate, 2 þ 1plumose setae and 3 þ 0 simple setae. Basial enditebilobed, the proximal and distal lobe with 7 þ 8 plumoden-ticulate cuspidate and 1 þ 1 simple setae. Endopod with 5plumose setae along the external margin. Scaphognathitewith 86 plumose and 2 simple marginal setae, plus 6 simplesetae on the internal surface.

First maxilliped (Figure 3D): triangular epipod at the prox-imal end narrowing rapidly towards the distal end, with 10evenly distributed long simple, and 5 shorter setae close tothe proximal end. Coxal and basial endite with 13 þ 22simple setae. Unsegmented endopod with 4 terminal simplesetae. Exopod 2-segmented with 4, 4 plumose setae.

Second maxilliped (Figure 3G): epipod elongated with 13long filiform setae. Gill present between the epipod andexopod. Endopod 4-segmented, where the proximal segmenthas an obvious suture line. Setation pattern of 3, 1, 7 8simple setae proceeding from the proximal to distalsegment. Exopod 2-segmented with 1 simple seta on the

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proximal segment and 5 terminal plumose setae on the distalsegment. Protopodite with 4 simple setae close to the base ofthe endopod.

Third maxilliped (Figure 4A): elongated epipod with 24evenly spread long simple setae and 2 plumose setae locatedat the proximal end. Protopod with 13 simple and 4plumose setae. Five-segmented endopod with 23 þ 9simple setae in the isquium and merus; and carpus, propodusand dactylus with 14 þ 13 þ 9 plumose setae. Exopod 2-segmented with 4 simple setae on the proximal segment and5 terminal plumose setae on the distal segment.

Chelipeds (Figure 4D): surface covered in a large numberof simple setae. Exhibits a prominant spine on the internalmargin of the isquium. Merus with an acute projection atthe distal end. Propodus subquadrate in form with a lengthto width ratio of 2.0, logitudinal grooves ill defined, internalchela fixed with shallow denticulation.

Second pereiopod (Figure 4G): surface covered in setae, themajority of which are simple in form. Coxa of second pereio-pod with a thick cuticular spine. Fifth pereiopod (Figure 1A)with 3 long terminal setae on the dactylus.

Abdomen (Figure 1A): composed of 6 somites plus thetelson, and dorsal surface with 2, 4, 6, 8, 8, 3 simple setae.Second and fifth segments possess biramous pleopods, fifthpleopod (Figure 5A) exopods have 21 terminal longplumose setae, endopods with 4 terminal cincinnuli. Sixth

segment with a pair of uniramous uropods with 1 plumoseseta on the external margin of the protopodite and 14 longplumose setae on the exopodite (Figure 5D).

Telson (Figure 5D): semicircular posterior margin, with 2pairs of simple setae on the ventral surface and a single pairon the dorsal surface.

Cancer edwardsii Bell, 1835Cephalothorax (Figure 1B): the ratio length/width wasapproximately 1.3. Sixty-two surface setae and 58 ventral mar-ginal setae.

Antennule (Figure 2B): penduncle with 4, 9, 2 simple setaeplus 8 plumose setae. Endopod has 5 simple setae. Exopodwith a setation of 0, 14, 10, 6 aesthetascs and 0, 0, 3, 1simple setae on each segment.

Antenna (Figure 2E): peduncle setation 4, 2, 4 (the interiorseta of the second segment is plumose); and flagellum with0, 0, 4, 0, 5, 0, 4, 3 simple setae per segment.

Mandible (Figure 2H): mandibular palp with 13 plumo-denticulate cuspidate setae on the distal segment.

Maxillule (Figure 2K): coxal and basial endite with 12(7 plumodenticulate cuspidate, 5 plumose) þ19 (plumodenti-culate cuspidate) setae and 5 þ 4 simple marginal setae.Epipodal and exopodal setae present.

Fig. 1. Comparative illustration of megalopae. General dorsal view (A) Cancer coronatus, (B) Cancer edwardsii and (C) Cancer setosus.

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Maxilla (Figure3B): proximal and distal lobe of the coxalendite with 7 þ 6 plumose setae. Proximal and distal lobeof the basial endite has 8 þ 10 plumodenticulate cuspidateand 1 þ 1 simple setae. Scaphognathite with 77 plumosesetae.

First maxilliped (Figure 3E): epipod with 12 very longsimple setae along the mid to distal exterior margin, and 2shorter simple setae on the proximal end. Coxal endite with14 plumose setae and the basial endite with 25 plumodenticu-late cuspidate setae. Endopod with additional plumose setae.Exopod with 4, 5 plumose setae and 1, 1 simple seta on theproximal and distal segment respectively.

Second maxilliped (Figure 3H): epipodite with 15 longsimple setae. Endopod with 5, 2 (1 simple, 1 plumose), 7 (2plumodenticulate cuspidate, 5 plumose), 4 (5 plumodenticu-late cuspidate, 4 plumose) setae. Exopod with 2 simple setaeon proximal segment and 5 long plumose setae on the distalsegment.

Third maxilliped (Figure 4B): epipod with 20 long simplesetae, and 6 plumose setae located on the proximal margin.Protopod with 25 plumose setae. Endopod, 27 (plus 6plumose), 7, 6, 12, 9 plumodenticulate cuspidate setae and

1, 4, 8, 0, 0 simple setae. Exopod with 5 simple setae on theproximal segment, and 8 long plumose setae on the distalsegment.

Chelipeds (Figure 4E): propodus globular in form with alength to width ratio of 1.5 and well defined longitudinalgrooves.

Abdomen (Figure 1B): somites dorsal surface with 4, 16, 14,14, 14, 6 simple setae. Biramous pleopods, fifth with exopodshaving 24 terminal long plumose setae (Figure 5B). Uropodwith 2 long plumose setae on the protopodite (Figure 5E).

Telson (Figure 5E): 2, 2 simple setae on the ventral anddorsal surface respectively.

Cancer setosus Molina, 1782Cephalothorax (Figure 1C): length to width ratio of 1.4. 32surface setae and 16 ventral marginal setae.

Antennule (Figure 2C): penduncle with 7 plumose setaeand 1 simple seta on the first segment: and 4 simple setaeon the second one. Endopod with 6 simple setae. Exopodwith 0, 18, 12, 10 aesthetascs.

Fig. 2. Comparative illustration of the cancridae megalopae appendages. Antennule, antenna, mandible and maxillule of Cancer coronatus (A, D, G, J); Canceredwardsii (B, E, H, K); Cancer setosus (C, F, I, L).

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Antenna (Figure 2F): peduncle with 4, 1, 5 setae (the setaon the second segment is plumose) and flagellum with 0, 0,4, 1, 5, 2, 3, 2 simple setae.

Mandible (Figure 2I): mandibular palp proximal segmentwith 1 plumose seta; distal segment with 15 plumodenticulatecuspidate setae.

Maxillule: (Figure 2L): coxal endite with 2 plumose, 7 plu-modenticulate cuspidate and 7 simple setae. Basial endite with24 plumodenticulate cuspidate and 5 simple setae.

Maxilla (Figure 3C): coxal endite proximal and distal lobewith 3 þ 5 plumodenticulate cuspidate, 3 þ 0 plumose and1 þ 1 simple seta. Basial endite proximal and distal lobe with10 þ 12 plumodenticulate cuspidate and 1 þ 1 simple seta.Endopod with 7 plumose setae. Scaphognathite with 95 mar-ginal plumose setae.

First maxilliped (Figure 3F): endopod with 22 long simplesetae. Coxal and basial endite with 21 þ 36 plumodenticulatecuspidate plumose setae. Endopod with 3 plumose setae and 5simple setae. Exopod with 4, 7 plumose setae.

Second maxilliped (Figure 3I): epipodite with 18 longsimple setae. Endopod with 2 (plus 2 plumose), 1, 8, 9 plumo-denticulate cuspidate, and 6, 1, 0, 0 simple setae. Exopod with

1, 7 plumose setae plus 2 simple setae. Protopod with 4simple setae and 4 plumose setae close to the base of theendopod.

Third maxilliped (Figure 4C): epipodite with 30 longsimple setae and 6 marginal plumose setae located near theproximal end. Protopod with 18 plumose setae and 2 simplesetae. Endopod with 37, 16, 20, 18, 9 plumodenticulate cuspi-date setae. Exopod with 3 simple setae on the proximalsegment and 10 plumose setae on the distal segment.

Chelipeds (Figure 4F): without isquial spine. Tubular pro-podus with a length to width ratio of 1.8 and several welldefined longitudinal grooves.

Abdomen (Figure 1C): somites dorsal surface with 4, 18,18, 18, 14, 6 simple setae. Biramous pleopods, fifth(Figure 5C) with exopods having 24 long and 3 short terminalplumose setae. Uropod with 2 long plumose setae on the pro-topod and 16 long plumose setae on the exopod (Figure 5F).

Telson (Figure 5F): 2, 8 simple setae on the ventral anddorsal surface respectively.

Intraregional variation: larvae collected around 800 kmbetween them did not show differences in SMC, but southernwere noticeably larger than northern megalopae (Table 1) .

Fig. 3. Comparative illustration of the cancridae megalopae appendages. Maxille, first maxilliped and second maxilliped of Cancer coronatus (A, D, G); Canceredwardsii (B, E, H); Cancer setosus (C, F, I).

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Molecular identificationThe sequences of 554 bp obtained from the COI gene of bothmegalopae and adults of the studied species of Cancer, exhib-ited a high level of differences between species. We observedaround 60 base pair differences between species and amaximum of 4 between individuals of the same species.These clear differences between the sequences of eachspecies were the basis for grouping the megalopae with theadults, without ambiguity, demonstrated by tree nodes withhigh support values between different species (Figure 6).

D I S C U S S I O N

Several characters have been used to identify megalopae of thegenera Cancer to the species level. Specific diagnostic charac-teristics that have been considered previously include: thenumber of plumose setae present on the uropods and cincin-nuli present on the pleopods (Poole, 1966; Trask, 1970), thecarapace length and presence or absence of a lateral knob(Orensanz & Gallucci, 1988), and the setae and spines over

the antenna and the endopod of the third maxilliped (Iwata& Konishi, 1981). However, we found a considerableamount of overlapping in these characteristics betweenspecies (Table 1).

The general morphology of the megalopae studied displaya high degree of intra-specific similarity. However, certainmorphological features in the chelipeds such as the presenceor absence of the isquial spine allow for a rapid and preciseidentification of each of the three species. These diagnosticcharacteristics have not been commented upon previously inthe descriptions of C. edwardsii and C. setosus (Quintana,1983; Quintana & Saelzer, 1986), based on laboratory rearedlarvae, and have for the first time been recorded for C. corona-tus. In addition, these features appear to be highly conservedon both the spatial and temporal scales. For example, megalo-pae of C. setosus collected at locations separated by more than800 km were identified to the species level and these were latercorroborated by the DNA analyses, despite differences in theirsize and date of collection.

The scarce morphological differentiation between themegalopae of the genus Cancer has been noted previously.DeBrosse et al. (1990) did not find invariable morphological

Fig. 4. Comparative illustration of the cancridae megalopae appendages. Third maxilleped, cheliped and second pereiopod of Cancer coronatus (A, D, G); Canceredwardsii (B, E, H); Cancer setosus (C, F, I).

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features which allowed for the identification of three sympa-tric species of Cancer (C. magister, C. oregonesis and C. pro-ductus) collected in the Puget Sound basin on the north-eastPacific coast. Such as our case, the megalopae presented ahigh degree of morphological similarity (except for the con-siderably larger size of C. magister) and it was not possibleto easily distinguish the larvae maintained in the laboratoryusing the published descriptions.

Another interesting finding was the differences in larvalmorphology between field and laboratory reared megalopae(Table 1). The larvae of C. edwardsii and C. setosus collectedin the field are considerably larger than those raised in the lab-oratory. In the case of C. coronatus there were no availabledescriptions for comparison. In addition to the size, thelarvae from the field exhibit a marked increase in thenumber of setae on their cephalothoracic appendages and in

general all the megalopae analysed appear to have moresetae than their conspecifics reared in the laboratory.Considerable variation has been described in the megalopaeof other species of the genus Cancer obtained in the field(Orensanz & Gallucci, 1988; DeBrosse et al., 1990) and alsoin other brachyuran species (Cuesta et al., 2002; Ampuero,2007).

The discrepancies between larvae collected in the field andthose cultivated in the laboratory could be explained by twonon-exclusive factors. First, the descriptions based on culti-vated larvae normally utilize a single or few reproductivefemales to provide the individuals described, reducing thegenotypic variation available for study. Second, the controlledconditions under which the larvae are cultivated (temperature,salinity and food availability) may restrict phenotypicexpression.

Fig. 5. Comparative illustration of the cancridae megalopae appendages. Second pleopod and telson of Cancer coronatus (A, D); Cancer edwardsii (B, E); Cancersetosus (C, F).

Table 1. Variation in biometric measures of Cancer megalopae from field and laboratory. All setal counts are listed from proximal to distal. CL ismeasured from base of rostral spine to middle of the posterior margin. From megalopae described in the literature (reared at the laboratory), CL andCW were estimated from illustrations, using the graphic scales provided by the authors (Quintana 1983; Quintana & Saelzer 1986). Abbreviations:

CL, cephalothorax length; CW, cephalothorax width; ND, no data. � indicate megalopae from Punta de Tralca.

Cancer edwardsii Cancer edwardsii Cancer setosus Cancer setosus Cancer coronatusLaboratory Field Laboratory Field Field

CephalothoraxCL (mm) 3.1 3.3 + 0.1 2.7 3.9 + 0.2–3.1 + 0.1� 3.01 + 0.1CW (mm) 2.05 2.6 + 0.1 1.9 2.6 + 0.1–2.3 + 0.1� 2.01 + 0.2

Antenna (peduncle) 2,2,3 4,1,4 3,2,4 4,1,5 4,2,4Antenna ( 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 0,0,4,0,5,0,4,3Third maxilliped

Endopodite 22,10,8,0,0 34,11,14,13,9 21,10,15,0,0 37,16,20,18,9 23,9,14,13,9Exopodite 5–8.5 5.8 4.5 3.1 4.5Epipodite 3 þ 19 6 þ 20 4 þ 16 6 þ 30 2 þ 24Protopodite 7 25 18 18 18

Pleopodal cincinnuli ND 4 ND 4 4Cheliped isquial spine Yes Yes Not Not Yes

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The analysis of molecular markers in larvae collected fromthe field are few, but with a high degree of certainty in terms ofidentification. For example, Webb et al. (2006) conductedgenetic analyses on Antarctic marine invertebrate larvae, butfound that the high resolution of their analysis was limitedby the general lack of genetic information for the adult organ-isms present in the area. In the present study, the lack of infor-mation on adults was not limiting for our analyses as they werecollected and analysed at the same time as the megalopae.Thus, we were able to assign the megalopae of the genusCancer on the coast of Chile to specific species using diagnosticmorphological features which were later corroborated with thesequencing of the COI gene of mitochondrial DNA.

This study affirms the necessity of including morphologicalvariation in the descriptions of larvae, and of analysing larvaecollected in the field, along with those raised under differentlaboratory conditions. Furthermore, the present study pro-vides clear evidence of the utility of molecular markers inthe study of larvae where previous descriptions are unavailableor where there are significant morphological differencesbetween the adults and the larvae of a species. These typesof techniques are also useful for identifying the differentstages of development of a species, and for those speciesthat exhibit little or no diagnostic characters (Burton, 1996;Neigel et al., 2007).

A C K N O W L E D G E M E N T S

We thank Elena Gessler, Constanza Ceroni, Juan PabloFuentes, Matthew Lee and Simon Cardyn for their active par-ticipation in this research, from sampling to drawing the mega-lopae. Thanks to Marcela Espinoza for helping with DNAextraction. L.M.P. thanks the Direction de Investigation andDevelopment (DID) of the Universidad Austral de Chile and

FONDECYT 110240071 for financial support. D.V. thanksthe Basal Grant PFB 023, CONICYT, Chile and IniciativaCientıfica Milenio Grant ICM P05-002. Thanks to two anon-ymous referees who have greatly helped us in improving theclarity of the presentation. All experimental work was con-ducted in Chile and complied with its existing laws.

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Cancer porteri 109–111 (19.86%) 108 (19.50%) 138–140 (25.09%)Cancer edwarsii 62–64 (11.37%) 96–99 (19.50%)Cancer coronatus 95–99 (19.50%)Cancer setosus

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Correspondence should be addressed to:L.M. PardoLaboratorio Costero Calfuco, Instituto de Biologia MarinaUniversidad Austral de Chile, Casilla 567, Valdivia, Chileemail: [email protected]

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