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5. 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
e
10 1
Laboratorio Costero Calfuco, Instituto de Biologia Marina, Universidad Austral de Chile, Casilla 567, Valdivia, Chile, 2Instituto de
11 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, Chile
14
15
16 Studies of recruitment dynamics in meroplanktonic organisms are dependent on the correct identification of each ontogenic
17 stage of each species. This is particularly difficult when studying the larval stages, which are not easy to identify due to their
18 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 fact
20 represents a serious limitation on ecological studies of populations of these species which constitute a heavily exploited
21 local resource. In this study we describe in detail field collected megalopae larvae of three sympatric crab species of the
22 genera Cancer (C. edwardsii, C. setosus and C. coronatus). As a result of this analysis we were able to identify easily
23 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. We
26 corroborated the utility of these diagnostic characters by comparing the COI gene sequences of mitochondrial DNA of larvae
27 identified by morphology with sequences taken from samples of the adults of all species of Cancer found in the region. We
28 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 simultaneous
30 use of morphological and molecular tools for identification of decapod larvae appears useful for the study of cryptic species.
31
32
33 Keywords: Decapoda, taxonomy, recruitment, estuary, Chile
34
35 Submitted 30 May 2008; accepted 20 October 2008
36
37
38
39
40 INTRODUCTION do not exhibit the specific diagnostic characteristics as evident
41 in adults, but have cryptic morphologies within the related
42 Studies of the early ontogenic stages in benthic marine invert- group. As a result individuals are assessed and assigned to
43 ebrates are especially helpful for understanding the type of higher taxa, genera or even families. However, this generates
44 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 the
47 and they have the highest level of mortality at the population field is to use complete descriptions of larvae obtained from
48 level (Palmer et al., 1996; Gosselin Qian, 1997; Hunt laboratory cultures. These descriptions have proved useful
49 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 in
52 (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 conditions
54 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 larvae
57 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 between
59 brachyuran larvae raised in the laboratory and those encoun-
60 tered in the field. For example, Cuesta et al. (2002) describe
61 Corresponding author:
field collected megalopae of the graspid Neohelice granulata
62 L.M. Pardo with morphological anomalies, in the form of additional
63 Email: luispardo@uach.cl cephalothoracic spines and a reduced number of setae
1

6. 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 Cancer
103 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. In
106 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 this
108 (Nations, 1975), with a southern limit at approximately 358S time, to keep this name and avoid fisheries problems as
109 (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 population
112 ecology and even less about their recruitment dynamics (but
113 see Jesse Stotz, 2003; Pardo et al., 2007).
114 One of the major difficulties in advancing research into this MATERIALS AND METHODS
115 group of crabs is the identification of the megalopa larvae,
116 a key stage during which recruitment and the moult to the
117 first juvenile stage takes place. There are two descriptions
Collection of the megalopae
118 available of megalopae for the genera Cancer along the coast Larvae were collected in the subtidal of the San Carlos inlet in
119 of the south-eastern Pacific, C. edwardsii and C. setosus ´
Bahıa Corral, located on the south side of the mouth of the Rio
120 (Quintana, 1983; Quintana Saelzer, 1986), both from lab- Valdivia, Chile (39º 490 S 73º 140 W). In this area, the habitat
121 oratory cultures. These larvae are extremely similar and the consists of a mixture of boulders and coarse sand. Samples
122 comparison of the general morphology presented in the pub- were obtained by means of an airlift manipulated by divers
123 lished figures does not allow for adequate identification of at depths of between 8 and 10 m. Because crab megalopae
124 individuals taken from the field. Additionally, all four were found during spring and early autumn only, we analysed
125 species can occur in the same habitat (Jesse Stotz, 2003; the morphological characteristics of larvae collected during
126 ˜
Munoz et al., 2006) and three of the species (C. edwardsii, October, December and April, in order to obtain a temporally

7. identifying field collected cancer megalopae 3
127 representative analysis. In addition we analysed megalopae of In order to determine the nucleotide relationship among
128 C. setosus obtained from Punta Tralca located in central Chile samples (megalopae and adults crabs), a neighbour-joining
129 (338 350 S 718 420 W), some 800 km north of the principal based phylogenetic (NJ) analysis was performed using Mega
130 sampling sites. 4.0 software (Tamura et al., 2007). Using a bootstrap of
131 10,000 replicates, the analysis tested the consistency of each
132 branch in the tree, grouping sequences with similar nucleotide
133 Description of the megalopae composition. Using this method, unidentified megalopae
134 larvae could be grouped with conspecific adults. Finally, to
Between 10 and 20 specimens of each species from Bahia
135 root the tree, sequences of Homalaspis plana were used as out-
Corral were dissected and examined in detail to describe
136 group (Genbank Accession Number: FJ155383).
larvae morphology. Additionally, 10 specimens of C. setosus
137
from central Chile (Pta de Tralca) were also analysed. The
138
megalopae and their appendages were dissected in glycerin
139 RESULTS
on microscope slides under a stereo microscope. The figures
140
were made using a drawing tube mounted on a Zeiss
141 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 the
143 ences are described in detail for the rest of species.
measurement of the cephalothorax length (CL) in species of
144
Cancer is made from the distal end of the rostral spine to
145
146
the mid-posterior margin of the carapace (DeBrosse et al., Cancer coronatus Molina, 1782
1990). However, due to the damage found in the rostral
147 Cephalotorax (Figure 1A): length to width ratio of 1.4, nar-
spine on several individuals, in this study the crabs were
148 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 width
150 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 terminal
155 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 the
157 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 pattern
160 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 plumodenticulate
164 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 8
167 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 proximal
169 (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 and
171 tissue from the chelipod, which was preserved in 95% distal lobe with 4 þ 0 plumodenticulate cuspidate, 2 þ 1
172 ethanol for subsequent DNA extraction. plumose setae and 3 þ 0 simple setae. Basial endite
173 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 5
175 Martinez (1997). The mitochondrial COI gene was amplified plumose setae along the external margin. Scaphognathite
176 using the protocol and primers described by Folmer et al. with 86 plumose and 2 simple marginal setae, plus 6 simple
177 (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 10
180 0.1 U U/mL of Taq polymerase. The PCR cycle consisted of evenly distributed long simple, and 5 shorter setae close to
181 3 minutes at 94ºC, 30 cycles of 30 seconds at 94ºC, the proximal end. Coxal and basial endite with 13 þ 22
182 90 seconds at 42ºC and 90 seconds at 72ºC with a final simple setae. Unsegmented endopod with 4 terminal simple
183 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 13
185 Ontario, Canada) and sequencing was performed at long filiform setae. Gill present between the epipod and
186 Macrogen Inc (www.macrogen.com). Later, sequences were exopod. Endopod 4-segmented, where the proximal segment
187 aligned by eye using the ProSeq v.2.9 software (Filatov, has an obvious suture line. Setation pattern of 3, 1, 7 8
188 2002). All haplotype was deposited in Genbank (Accession simple setae proceeding from the proximal to distal
189 Numbers: FJ155371 to FJ155382). segment. Exopod 2-segmented with 1 simple seta on the

8. 4 luis miguel pardo et al.
190
191
192
193
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224 Fig. 1. Comparative illustration of megalopae. General dorsal view (A) Cancer coronatus, (B) Cancer edwardsii and (C) Cancer setosus.
225
226
227 proximal segment and 5 terminal plumose setae on the distal segment with a pair of uniramous uropods with 1 plumose
228 segment. Protopodite with 4 simple setae close to the base of seta on the external margin of the protopodite and 14 long
229 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 2
231 evenly spread long simple setae and 2 plumose setae located pairs of simple setae on the ventral surface and a single pair
232 at the proximal end. Protopod with 13 simple and 4 on the dorsal surface.
233 plumose setae. Five-segmented endopod with 23 þ 9
234 simple setae in the isquium and merus; and carpus, propodus
235 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, 1835
237 5 terminal plumose setae on the distal segment. Cephalothorax (Figure 1B): the ratio length/width was
238 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 setae
241 the distal end. Propodus subquadrate in form with a length plus 8 plumose setae. Endopod has 5 simple setae. Exopod
242 to width ratio of 2.0, logitudinal grooves ill defined, internal with a setation of 0, 14, 10, 6 aesthetascs and 0, 0, 3, 1
243 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 interior
245 majority of which are simple in form. Coxa of second pereio- seta of the second segment is plumose); and flagellum with
246 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 12
250 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.

9. identifying field collected cancer megalopae 5
253
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289
Fig. 2. Comparative illustration of the cancridae megalopae appendages. Antennule, antenna, mandible and maxillule of Cancer coronatus (A, D, G, J); Cancer
290
edwardsii (B, E, H, K); Cancer setosus (C, F, I, L).
291
292
293
294 Maxilla (Figure3B): proximal and distal lobe of the coxal 1, 4, 8, 0, 0 simple setae. Exopod with 5 simple setae on the
295 endite with 7 þ 6 plumose setae. Proximal and distal lobe proximal segment, and 8 long plumose setae on the distal
296 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 a
298 setae. length to width ratio of 1.5 and well defined longitudinal
299 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 exopods
302 14 plumose setae and the basial endite with 25 plumodenticu- having 24 terminal long plumose setae (Figure 5B). Uropod
303 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 and
305 proximal and distal segment respectively. dorsal surface respectively.
306 Second maxilliped (Figure 3H): epipodite with 15 long
307 simple setae. Endopod with 5, 2 (1 simple, 1 plumose), 7 (2
308 plumodenticulate cuspidate, 5 plumose), 4 (5 plumodenticu-
309 late cuspidate, 4 plumose) setae. Exopod with 2 simple setae
Cancer setosus Molina, 1782
310 on proximal segment and 5 long plumose setae on the distal Cephalothorax (Figure 1C): length to width ratio of 1.4. 32
311 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 setae
313 setae, and 6 plumose setae located on the proximal margin. and 1 simple seta on the first segment: and 4 simple setae
314 Protopod with 25 plumose setae. Endopod, 27 (plus 6 on the second one. Endopod with 6 simple setae. Exopod
315 plumose), 7, 6, 12, 9 plumodenticulate cuspidate setae and with 0, 18, 12, 10 aesthetascs.

10. 6 luis miguel pardo et al.
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347
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350
351
352 Fig. 3. Comparative illustration of the cancridae megalopae appendages. Maxille, first maxilliped and second maxilliped of Cancer coronatus (A, D, G); Cancer
353 edwardsii (B, E, H); Cancer setosus (C, F, I).
354
355
356
357 Antenna (Figure 2F): peduncle with 4, 1, 5 setae (the seta 1, 7 plumose setae plus 2 simple setae. Protopod with 4
358 on the second segment is plumose) and flagellum with 0, 0, simple setae and 4 plumose setae close to the base of the
359 4, 1, 5, 2, 3, 2 simple setae. endopod.
360 Mandible (Figure 2I): mandibular palp proximal segment Third maxilliped (Figure 4C): epipodite with 30 long
361 with 1 plumose seta; distal segment with 15 plumodenticulate simple setae and 6 marginal plumose setae located near the
362 cuspidate setae. proximal end. Protopod with 18 plumose setae and 2 simple
363 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 proximal
365 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 well
368 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, fifth
371 ginal plumose setae. (Figure 5C) with exopods having 24 long and 3 short terminal
372 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 and
375 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 km
377 simple setae. Endopod with 2 (plus 2 plumose), 1, 8, 9 plumo- between them did not show differences in SMC, but southern
378 denticulate cuspidate, and 6, 1, 0, 0 simple setae. Exopod with were noticeably larger than northern megalopae (Table 1) .

11. identifying field collected cancer megalopae 7
379
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416 Fig. 4. Comparative illustration of the cancridae megalopae appendages. Third maxilleped, cheliped and second pereiopod of Cancer coronatus (A, D, G); Cancer
417 edwardsii (B, E, H); Cancer setosus (C, F, I).
418
419
420 Molecular identification the antenna and the endopod of the third maxilliped (Iwata
421 Konishi, 1981). However, we found a considerable
422 The sequences of 554 bp obtained from the COI gene of both amount of overlapping in these characteristics between
423 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 display
425 around 60 base pair differences between species and a a high degree of intra-specific similarity. However, certain
426 maximum of 4 between individuals of the same species. morphological features in the chelipeds such as the presence
427 These clear differences between the sequences of each or absence of the isquial spine allow for a rapid and precise
428 species were the basis for grouping the megalopae with the identification of each of the three species. These diagnostic
429 adults, without ambiguity, demonstrated by tree nodes with characteristics have not been commented upon previously in
430 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 reared
432 larvae, and have for the first time been recorded for C. corona-
433 DISCUSSION tus. In addition, these features appear to be highly conserved
434 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 than
436 genera Cancer to the species level. Specific diagnostic charac- 800 km were identified to the species level and these were later
437 teristics that have been considered previously include: the corroborated by the DNA analyses, despite differences in their
438 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 the
440 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

12. 8 luis miguel pardo et al.
442
443
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465 Fig. 5. Comparative illustration of the cancridae megalopae appendages. Second pleopod and telson of Cancer coronatus (A, D); Cancer edwardsii (B, E); Cancer
466 setosus (C, F).
467
468
469 features which allowed for the identification of three sympa- general all the megalopae analysed appear to have more
470 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 megalopae
472 Pacific coast. Such as our case, the megalopae presented a of other species of the genus Cancer obtained in the field
473 high degree of morphological similarity (except for the con- (Orensanz Gallucci, 1988; DeBrosse et al., 1990) and also
474 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 and
477 Another interesting finding was the differences in larval those cultivated in the laboratory could be explained by two
478 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 reproductive
480 in the field are considerably larger than those raised in the lab- females to provide the individuals described, reducing the
481 oratory. In the case of C. coronatus there were no available genotypic variation available for study. Second, the controlled
482 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 phenotypic
484 number of setae on their cephalothoracic appendages and in expression.
485
486
487 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 and
488
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.
490
491 Cancer edwardsii Cancer edwardsii Cancer setosus Cancer setosus Cancer coronatus
492 Laboratory Field Laboratory Field Field
493 Cephalothorax
494 CL (mm) 3.1 3.3 + 0.1 2.7 3.9 + 0.2–3.1 + 0.1Ã 3.01 + 0.1
495 CW (mm) 2.05 2.6 + 0.1 1.9 2.6 + 0.1–2.3 + 0.1Ã 2.01 + 0.2
496 Antenna (peduncle) 2,2,3 4,1,4 3,2,4 4,1,5 4,2,4
497 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 0,0,4,0,5,0,4,3
498 Third maxilliped
499 Endopodite 22,10,8,0,0 34,11,14,13,9 21,10,15,0,0 37,16,20,18,9 23,9,14,13,9
500 Exopodite 5 –8.5 5.8 4.5 3.1 4.5
501 Epipodite 3 þ 19 6 þ 20 4 þ 16 6 þ 30 2 þ 24
Protopodite 7 25 18 18 18
502
Pleopodal cincinnuli ND 4 ND 4 4
503 Cheliped isquial spine Yes Yes Not Not Yes
504

13. identifying field collected cancer megalopae 9
505 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%)
508
509 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 setosus
512
513
514
515 FONDECYT 110240071 for financial support. D.V. thanks
516 the Basal Grant PFB 023, CONICYT, Chile and Iniciativa
517 ´
Cientıfica Milenio Grant ICM P05-002. Thanks to two anon-
518 ymous referees who have greatly helped us in improving the
519 clarity of the presentation. All experimental work was con-
520 ducted in Chile and complied with its existing laws.
521
522
523 REFERENCES
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614 Angeles County. Science Bulletin 23, 1 –104. Wahle R.A. (2003) Revealing stock–recruitment relationships in lobsters
615 Negreiros-Fransozo M.L., Wenner E.L., Knott D.M. and Fransozo A. and crabs:is experimental ecology the key? Fisheries Research 65, 3 –32.
616 (2007) The megalopa and early juvenile stages of Calappa tortugae Webb K.E., Barnes D.K.A., Clark M.S. and Bowden D.A. (2006) DNA
617 Rathbun, 1933 (Crustacea, Brachyura) reared in the laboratory from barcoding: a molecular tool to identify Antarctic marine larvae.
618 South Carolina neuston samples. Proceedings of the Biological Society Deep-Sea Research Part II—Topical Studies in Oceanography 53,
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620 Neigel J., Domingo A. and Stake J. (2007) DNA barcoding as a tool for and
621 coral reef conservation. Coral Reefs 26, 487 –499.
622 Williamson D.I. (1982) Larval morphology and diversity. In Abele L.G.
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623 extant brachyuran crabs of the world. The Raffles Bulletin of Zoology
624 and genetics. New York: Academic Press, pp. 43–110.
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625
Orensanz J.M. and Gallucci V.F. (1988) Comparative study of postlarval Correspondence should be addressed to:
626
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Brachyura: Cancridae). Journal of Crustacean Biology 8, 187–220. Laboratorio Costero Calfuco, Instituto de Biologia Marina
628
629 ´
Palma A.T., Pardo L.M., Veas R., Cartes C., Silva M., Manrıquez K., Universidad Austral de Chile, Casilla 567, Valdivia, Chile
630 ´ ˜
Dıaz A., Munoz C. and Ojeda F.P. (2006) Coastal brachyuran email: luispardo@uach.cl