Page 1 of 39 Zoological Journal of the Linnean Society

1 2 3 1 DNA identification and larval morphology provide new evidence on the systematic 4 5 2 position of Ergasticus clouei A. Milne-Edwards, 1882 (Decapoda, Brachyura, 6 7 3 Majoidea) 8 9 10 4 11 1 2 1 3 12 5 Marco-Herrero, Elena , Torres, Asvin P. , Cuesta, José A. , Guerao, Guillermo , Palero, 13 14 6 Ferran 4, & Abelló, Pere 5 15 16 7 17 18 8 1Instituto de CienciasFor Marinas Review de Andalucía (ICMAN-C OnlySIC), Avda. República 19 20 21 9 Saharaui, 2, 11519 Puerto Real, Cádiz, Spain. 22 2 23 10 Instituto Español de Oceanografía, Centre Oceanogràfic de les Balears, Moll de Ponent 24 25 11 s/n, 07015 Palma, Spain. 26 27 12 3IRTA, Unitat de Cultius Aqüàtics. Ctra. Poble Nou, Km 5.5, 43540 Sant Carles de la 28 29 30 13 Ràpita, Tarragona, Spain. 31 4 32 14 Unitat Mixta Genòmica i Salut CSISP-UV, Institut Cavanilles Universitat de Valencia, 33 34 15 C/ Catedrático José Beltrán 2, 46980 Paterna, Spain. 35 36 16 5Institut de Ciències del Mar (CSIC), Passeig Marítim de la Barceloneta 37-49, 08003 37 38 17 Barcelona, Catalonia. Spain. 39 40 41 18 42 43 19 44 45 20 46 47 21 RUN TITLE: Larval evidence and the systematic position of Ergasticus clouei 48 49 50 22 51 52 53 54 55 56 57 58 59 60 Zoological Journal of the Linnean Society Page 2 of 39

1 2 3 23 ABSTRACT: The morphology of the complete larval stage series of the crab Ergasticus 4 5 24 clouei is described and illustrated based on larvae (zoea I, zoea II and megalopa) 6 7 25 captured from plankton samples taken in Mediterranean waters. Nucleotide sequence 8 9 10 26 analysis of a region of the 16S rDNA and Cox1 genes clearly identified these larvae as 11 12 27 Ergasticus clouei . The morphology of the second zoeal and megalopal stages, 13 14 28 previously unknown, is described in detail for the first time . Both t he analysis of DNA 15 16 29 sequences and the morphology of the larval stages did not support the current 17 18 30 assignment of ErgasticusFor to Reviewthe family Inachidae. In Onlycontrast, E. clouei larvae present a 19 20 21 31 set of morphological characters that do fit into the characteristics of the family 22 23 32 Oregoniidae. Particularly significant is the morphology of the antenna of the zoeal 24 25 33 stages of E. clouei , only found in Oregoniidae and Majidae. The molecular results 26 27 34 obtained further support the removal of Ergasticus from the Inachidae, and the 28 29 30 35 monophyly of the Oregoniidae + Ergasticus group. Finally, our results evidence that 31 32 36 developmental stages of brachyurans may provide reliable morphological 33 34 37 characteristics, independent from those of adults, to help resolving the phylogenetic 35 36 38 relationships among Majoidea genera. 37 38 39 39 40 41 40 42 43 41 44 45 42 46 47 43 48 49 50 44 51 52 45 53 54 46 ADDITIONAL KEYWORDS: DNA barcoding - Ergasticus clouei – Inachidae - larval 55 56 47 development - megalopa – Oregoniidae - spider crab – zoea. 57 58 59 60 Page 3 of 39 Zoological Journal of the Linnean Society

1 2 3 48 INTRODUCTION 4 5 49 Members of the crab superfamily Majoidea Samouelle, 1819 comprise one of the most 6 7 50 diversified groups within Brachyura (Ng, Guinot & Davie, 2008). The superfamily is 8 9 10 51 represented by approximately 950 species inhabiting regions from intertidal zones to 11 12 52 depths over 1000 meters (D'Udekem d'Acoz, 1999; De Forges & Poore, 2008; De Grave 13 14 53 et al. , 2009). Although most of the diversity of the group is restricted to the tropical 15 16 54 region, majoid species can be found all around the planet and show considerable 17 18 55 ecological diversity.For Probably Review due to this large mo Onlyrphological diversity, members of 19 20 21 56 Majoidea have had a confusing taxonomic history (Garth, 1958; Griffin & Tranter, 22 23 57 1986; Martin & Davis, 2001; Miers, 1879; Ng et al. , 2008). Familial and subfamilial 24 25 58 classifications in the Majoidea are generally based on adult morphology, especially 26 27 59 based on eyestalk or antennal shape and spination patterns (Garth, 1958; Griffin & 28 29 30 60 Tranter, 1986). However, recent taxonomic revisions of the group seem to suggest that 31 32 61 these adult morphological traits may in some cases be incongruent with larval characters 33 34 62 (Clark & Webber, 1991; Marques & Pohle, 2003). In the largest phylogenetic study 35 36 63 published to date, including sequences of both mitochondrial (16S, Cox1) and nuclear 37 38 64 (28S) markers for around 40 majoid species, Hultgren & Stachowicz (2008) found that 39 40 41 65 phylogenetic relationships inferred from genetic data are also incongruent with familial 42 43 66 relationships inferred from adult morphology. Most interestingly, the molecular-based 44 45 67 analyses corroborated phylogenetic relationships based on larval morphology (Hultgren 46 47 68 et al. , 2009). 48 49 50 69 Despite larval morphology provides a valuable set of characters to resolve 51 52 70 majoid systematics, the larval forms of many species are still undescribed. Most 53 54 71 plankton-captured larval stages are not identified to species level due to the scarceness 55 56 72 of full larval descriptions, and also on the high specialist level and time-consuming 57 58 59 60 Zoological Journal of the Linnean Society Page 4 of 39

1 2 3 73 identification work needed for visualizing the precise morphological features. Even 4 5 74 when larval descriptions are available, specific identifications based on morphological 6 7 75 criteria may still be impossible due to the low variability at intrageneric level observed 8 9 10 76 within Decapoda (Ingle, 1992). A valuable resource contributing to accurate species 11 12 77 identification has been made available in recent years thanks to the fast development of 13 14 78 new tools based on molecular analysis (DNA barcoding: Hebert, Ratnasingham & 15 16 79 Waard, 2003). One obvious advantage of DNA barcoding comes from the fact that 17 18 80 genetic markersFor do not changeReview during the ontogeny Only of the organism. Therefore, 19 20 21 81 molecular-based identification is most useful when there are no obvious means to match 22 23 82 adults with larval stages or when larval-rearing cannot be completed (Ampuero et al. , 24 25 83 2010; Palero, Guerao & Abelló, 2008). 26 27 84 Ergasticus clouei A. Milne-Edwards, 1882 is a rare majoid crab and the only 28 29 30 85 known species of the genus (Ng et al. , 2008). Specimens have been reported along the 31 32 86 western coasts of Africa and Europe, from the Cape Verde Islands to the Bay of Biscay, 33 34 87 including the Açores, Madeira and the Canary Islands as well as throughout the 35 36 88 Mediterranean (Guerao & Abelló, 2007; Manning & Holthui, 1981; Zariquiey Alvarez, 37 38 89 1968). Ergasticus has been recorded within a large bathymetric range, from 70 to 1000 39 40 41 90 m (D'Udekem d'Acoz, 1999), but it is mostly found between 250-800 m, i.e. from the 42 43 91 continental shelf break to the upper and middle slope (Abelló, Carbonell & Torres, 44 45 92 2002; Manning & Holthuis, 1981); Ramón et al., submitted). The precise biogeographic 46 47 93 range of the species is not yet fully understood due to the scarceness of adult captures in 48 49 50 94 benthic or epibenthic samples. Hardly anything is known about the biology and life 51 52 95 history of the species. As in most Majoidea, E. clouei shows a strong sexual 53 54 96 dimorphism in claw length, being much longer and stronger in adult males than in 55 56 97 females (Zariquiey Álvarez 1968; authors unpublished data). Even though no 57 58 59 60 Page 5 of 39 Zoological Journal of the Linnean Society

1 2 3 98 information is available on the reproductive biology of the species, ovigerous females 4 5 99 have been recorded in May, June and July (authors unpublished data). 6 7 100 The genus Ergasticus has been traditionally assigned to the family Inachidae 8 9 10 101 MacLeay, 1838 based in adult characters (Balss, 1957; Manning & Holthui, 1981; Ng et 11 12 102 al. , 2008); although Bouvier (1940) and Zariquiey Álvarez (1968) placed it in the 13 14 103 subfamily Pisinae. Inachid crabs are grouped together mostly for showing eyes without 15 16 104 orbits and eyestalks generally long, either non-retractile, or retractile against sides of 17 18 105 carapace, or againstFor an acute Review post-ocular spine affording Only no concealment (Garth, 1958; 19 20 21 106 Manning & Holthui, 1981). Despite adult Ergasticus fall within the Inachidae definition 22 23 107 given that their eyes are retractile against an acute post-ocular spine, the systematic 24 25 108 position of this species was questioned in a recent study based on the morphology of the 26 27 109 first zoeal stage (Guerao & Abelló, 2007), in which the overall features of the first zoea 28 29 30 110 was found to differ from the standard Inachidae zoeal morphology (Marques & Pohle, 31 32 111 2003). However, given the difficulties found in reaching further zoea and megalopa 33 34 112 stages through larval rearing, the results obtained in that study were limited and 35 36 113 prevented the assessment of a clearer phylogenetic position of the species. 37 38 114 The present study aims at resolving the uncertainties on the assignment of 39 40 41 115 Ergasticus clouei to the Inachidae by describing the complete morphology of all its 42 43 116 larval stages, identified by DNA analyses from plankton samples, comparing them to 44 45 117 previous descriptions of the larval stages of other majoid genera and carrying out a 46 47 118 complete phylogenetic analysis, including DNA sequences from representatives of other 48 49 50 119 Majoidea families. 51 52 120 53 54 121 55 56 122 MATERIAL AND METHODS 57 58 59 60 Zoological Journal of the Linnean Society Page 6 of 39

1 2 3 123 4 5 124 Sampling methods 6 7 125 Two multidisciplinary research surveys IDEADOS_2009 and 8 9 10 126 IDEADOS_2010, were conducted on board R/V “Sarmiento de Gamboa” off 11 12 127 the Balearic Archipelago (western Mediterranean; Fig 1) during late autumn 13 14 128 (29 November to 18 December 2009) and summer (11 to 30 July 2010). 15 16 129 These surveys aimed, among other objectives, at studying the meroplankton 17 18 130 communities foundFor at two Review stations over 200 mOnly and 900 m depth (shelf break 19 20 21 131 and middle slope, respectively). These stations were located west and south of 22 23 132 Mallorca Island (Balearic and Algerian sub-basins, respectively) and belong 24 25 133 to areas with distinct water masses and different environmental conditions 26 27 134 (López-Jurado, Marcos & Monserrat, 2008; Pinot, Lóez-Jurado & Riera, 28 29 30 135 2002). 31 32 136 A total of 218 depth-stratified mesozooplankton samples were collected using a 33 34 137 multi-net HYDRO-BIOS in 2009 and a Multiple Opening Closing Net and 35 36 138 Environmental Sensing System (MOCNESS) in 2010 (Olivar et al. , 2012). The mouth 37 38 139 opening of these nets was 0.25 and 1 m 2, respectively, and their mesh size was 333 m. 39 40 41 140 Both devices were towed at ~2 knots, performing oblique stratified hauls from near 42 43 141 bottom to the surface. A total of 66 macrozooplankton samples were collected using an 44 45 142 Isaac-Kidd mid-water trawl (IKMT) of 3 m 2 with a codend mesh size of 3 mm, the 46 47 143 fishing speed was 3 knots and the effective tow duration was 30 min. Immediately after 48 49 50 144 collection, IKMT samples were preserved in ethanol 96%, while the rest of samples 51 52 145 were stored and fixed in buffered 5% formalin, since mainly aimed at ichthyoplankton 53 54 146 studies. Once in the laboratory, decapod crustacean larvae were sorted and identified to 55 56 147 species level and developmental stage whenever possible, using available descriptions 57 58 59 60 Page 7 of 39 Zoological Journal of the Linnean Society

1 2 3 148 and keys (Dos Santos & Gonzalez-Gordillo, 2004; Dos Santos & Lindley, 2001; 4 5 149 Pessani, Tirelli & FlagellaL, 2004). In total, 2 zoeae I, 4 zoeae II and 2 megalopae 6 7 150 tentatively assigned to an unidentified majoid species were recorded. 8 9 10 151 Independently, samples from several adult majoid species were collected by 11 12 152 demersal trawling during a fishery research survey (MEDITS_ES_2003) carried out in 13 14 153 May 2003 along the western Mediterranean on board R/V “Cornide de Saavedra”. In 15 16 154 particular, two E. clouei individuals were collected off Almería near Cape Gata at 17 18 155 depths between For500-600 m andReview kept in 96° ethanol. AOnlynother adult specimen of E. clouei 19 20 21 156 was also collected by an epibenthic beam trawl off Mallorca during the 22 23 157 IDEADOS_2010 research survey in July 2010 on board F/V “Punta des Vent”. The E. 24 25 158 clouei adult specimens included in this study have been deposited at the Biological 26 27 159 Collections of Reference of the Institut de Ciències del Mar (CSIC) in Barcelona under 28 29 30 160 accession numbers #####, #####, ###### (pending) 31 32 161 33 34 162 Morphological descriptions 35 36 163 Drawings and measurements were made using a Wild MZ6 and Zeiss Axioskop 37 38 164 compound microscope with Nomarski interference, both equipped with a camera 39 40 41 165 lucida . All measurements were made using an ocular micrometer. Descriptions are 42 43 166 based on 2 zoeae I, 3 zoeae II and 2 megalopae, and measurements of different larval 44 45 167 stages are based on all specimens obtained. For the zoeal stages the following 46 47 168 measurements were taken: cephalothoracic dorsal spine length (DL) distance from base 48 49 50 169 to tip of the dorsal spine; cephalothoracic rostral spine length (RL) distance from base 51 52 170 to tip of the rostral spine; rostrodorsal length (RDL) distance from the tip of the rostral 53 54 171 spine to the the tip of the dorsal spine; cephalothorax length (CL) from between eyes 55 56 172 (base of the rostrum) to the postero-lateral cephalothorax margin; cephalothorax width 57 58 59 60 Zoological Journal of the Linnean Society Page 8 of 39

1 2 3 173 (CW) from the tip of one lateral spine to the tip of the other lateral spine. For the 4 5 174 megalopa: cephalothorax length (CL) measured from the base of rostrum to posterior 6 7 175 margin of cephalothorax; cephalothorax total length (CTL) measured from the tip of the 8 9 10 176 rostrum to posterior margin of cephalothorax and cephalothorax width (CW) as the 11 12 177 cephalothorax maximum width (excluding the hepatic protuberances). 13 14 178 The larvae are described using the basic malacostracan somite plan from anterior 15 16 179 to posterior and appendage segments are described from proximal to distal, endopod 17 18 180 then exopod (Clark,For Calazans Review & Pohle, 1998). All la rvalOnly specimens have been dissected 19 20 21 181 and used for descriptions with the exception of one zoea II that has been deposited at the 22 23 182 Centre Oceanogràfic de les Balears, in Palma de Mallorca (Spain), with catalogue 24 25 183 number ID2_0710_E9N2_ZII. 26 27 184 28 29 30 185 DNA extraction, amplification and sequencing 31 32 186 The identification of larval stages was based on partial sequences of the 16S rDNA and 33 34 187 Cox1 genes. Total genomic DNA was extracted from muscle tissue from 2 pereiopods 35 36 188 of one megalopa, from the pleon of one zoea II, and from one pereiopod of each of the 37 38 189 three adult specimens of Ergasticus clouei , and incubated for 1–24 hours in 300 l lysis 39 40 41 190 buffer at 65º C. Protein was precipitated by addition of 100 l of 7.5 M ammonium 42 43 191 acetate and subsequent centrifugation, and DNA precipitation was obtained by addition 44 45 192 of 300 l isopropanol and posterior centrifugation. The resulting pellet was washed with 46 47 193 ethanol (70 %), dried, and finally resuspended in Milli-Q destilled water. 48 49 50 194 Target mitochondrial DNA from the large subunit rRNA (16S) and Cox1 genes 51 52 195 was amplified with polymerase chain reaction (PCR) and the following cycling 53 54 196 conditions for reactions: 2 min at 95º C, 40 cycles of 20 s at 95º C, 20 s at 45-48º C, 45 55 56 197 s (16S) or 47 s (Cox1) at 72º C, and 5 min 72º C. Primers 1472 (5´- AGA TAG AAA 57 58 59 60 Page 9 of 39 Zoological Journal of the Linnean Society

1 2 3 198 CCA ACC TGG -3´) (Crandall & Fitzpatrick, 1996) and 16L2 (5´-TGC CTG TTT ATC 4 5 199 AAA AAC AT-3´) (Schubart, Cuesta & Felder, 2002) were used to amplify 540 bp of 6 7 200 16S, while primers COH6 (5´- TAD ACT TCD GGR TGD CCA AAR AAY CA -3´) 8 9 10 201 and COL6b (5´- ACA AAT CAT AAA GAT ATY GG -3´) (Schubart & Huber, 2006) 11 12 202 allowed amplification of 670 bp of Cox1. PCR products were sent to New Biotechnic 13 14 203 and Biomedal companies to be purified and then two-direction sequencing. 15 16 204 Sequences were edited using the software Chromas version 2.0. Adult and larval 17 18 205 sequences for bothFor genes Review are deposited in Genbank Only under the accession numbers 19 20 21 206 ########## (pending). The final sequences were compared with the sequences 22 23 207 obtained from adult specimens of several Iberian brachyuran crabs in the context of the 24 25 208 MEGALOPADN project or from the data available in public databases (see Table 1). 26 27 209 28 29 30 210 Phylogenetic analysis and hypothesis testing 31 32 211 In order to carry out a complete phylogenetic analysis, alignments of each gene data set 33 34 212 were conducted using the program Muscle v3.6 (Edgar, 2004) with default parameters. 35 36 213 To avoid alignment ambiguity for the 16S rDNA gene, gaps and hyper-variable regions 37 38 214 were excluded from further analysis using GBlocks software v0.91b (Castresana, 2000). 39 40 41 215 The combined selection of the best-fit partitioning scheme for the alignment and the 42 43 216 nucleotide substitution model for each partition was carried out using the new objective 44 45 217 method implemented in PartitionFinder (Lanfear et al. , 2012). The BEAST software 46 47 218 (Drummond & Rambaut, 2007) was used to infer phylogenetic relationships among 48 49 50 219 samples [two independent runs starting from a random tree; estimated base frequencies; 51 52 220 Yule tree prior; 50,000,000 generations, sampling every 1,000th tree with a 10 % burn- 53 54 221 in] and generated consensus data from the posterior trees. 55 56 57 58 59 60 Zoological Journal of the Linnean Society Page 10 of 39

1 2 3 222 It has been shown that the ‘‘uncorrelated relaxed-clock” models, in which the mutation 4 5 223 rates in each branch are allowed to vary within particular constraints, perform better 6 7 224 than strict molecular clock or the correlated models (Drummond et al. , 2006). 8 9 10 225 Therefore, we used here the Bayesian relaxed-clock uncorrelated lognormal approach as 11 12 226 implemented in BEAST v1.7.4 (Drummond & Rambaut, 2007) with the corresponding 13 14 227 model of sequence evolution previously inferred for each gene partition. 15 16 228 Besides the unconstrained search, BEAST runs were carried out using the same 17 18 229 conditions but includingFor a constrainedReview search in order Only to test the hypothesis of the genus 19 20 21 230 Ergasticus belonging to the same monophyletic clade as the other Inachidae genera 22 23 231 analyzed (i.e. Macropodia , Inachus , Podochela and Metoporaphis ). We employed the 24 25 232 Bayes factor to compare the different models (Nylander et al. , 2004), evaluating the 26 27 233 hypothesis (H0) that our constrained and unconstrained topologies explain the data 28 29 30 234 equally well, versus the alternative hypothesis (H1) that constrained BI searches provide 31 32 235 a poorer explanation of the data. We calculated the Bayes factor as twice the difference 33 34 236 in the harmonic mean 2 lnL scores (2 ln B01) between alternative hypotheses 35 36 237 (Brandley, Schmitz & Reeder, 2005) and compared these values to the framework 37 38 238 provided by Kass and Raftery (1995) where <0 is evidence against H1, 0–2 provides no 39 40 41 239 evidence for H1, 2–6 is positive support for H1, 6–10 is strong support for H1, and >10 42 43 240 is very strong support for H1 (see Nylander et al., 2004 and Brandley et al., 2005). 44 45 241 46 47 242 RESULTS 48 49 50 243 Among the decapod crustacean larvae found in the samples, those referred to 51 52 244 Ergasticus clouei were captured during summer (July) 2010 at the shelf break 53 54 245 station located south of Mallorca. The zoeal stages were captured with 55 56 57 58 59 60 Page 11 of 39 Zoological Journal of the Linnean Society

1 2 3 246 MOCNESS, between 250 and 100 m and the megalopa stage was captured 4 5 247 with IKMT from 272 m to surface. 6 7 248 Larval Description 8 9 10 249 The zoea I is completely redescribed and in subsequent stages only differences are 11 12 250 highlighted. 13 14 251 15 16 252 Ergasticus clouei A. Milne-Edwards, 1882 17 18 253 For Review(Figs. 3-8) Only 19 20 21 254 22 23 255 Zoea I 24 25 256 Size: RDL= 2.69-2.90 mm; CL= 1.05-1.06 mm; CW= 1.40 mm; RL= 0.61-0.66 mm; 26 27 257 DL= 1.33-1.27 mm; N=2. 28 29 30 258 Cephalothorax (Fig. 3A). With long dorsal spine, strongly curved distally backwards 31 32 259 and without setae; rostral spine long, slightly longer than antenna; lateral spines 33 34 260 present and well developed; each latero-ventral margin with 1 densely plumose 35 36 261 “anterior seta”, followed by 2 additional sparsely plumose setae; one pair of 37 38 262 antero-dorsal setae and one pair of postero-dorsal setae present; eyes sessile. 39 40 41 263 Antennule (Fig. 4A). Uniramous, smooth and conical; endopod absent; exopod 42 43 264 unsegmented with 4 terminal aesthetascs of different diameter/width and 1 minute 44 45 265 seta. 46 47 266 Antenna (Fig. 4D). Biramous, protopod very long with one row of 13-15 spinules of 48 49 50 267 different size; endopod bud present; one-segmented exopod shorter than the 51 52 268 spinous process, with 2 unequal subterminal setae. 53 54 269 Mandible. Incisor and molar processes differentiated; mandibular palp (endopod) 55 56 270 absent. 57 58 59 60 Zoological Journal of the Linnean Society Page 12 of 39

1 2 3 271 Maxillule (Fig. 5A). Coxal endite with 7 setae; basial endites with 7 setae (4 cuspidate); 4 5 272 endopod 2-segmented, proximal segment with 1 seta, distal segment with 2 6 7 273 medial, 2 subterminal and 2 terminal setae; exopodal seta absent. 8 9 10 274 Maxilla (Fig. 5D). Coxal endite bilobed with 4+4 setae; basial endite bilobed with 5+4 11 12 275 setae; unsegmented endopod not bilobed, with 6 terminal setae and microtrichia 13 14 276 on lateral margin; exopod (scaphognathite) margin with 9 plumose setae, 15 16 277 including distal process. 17 18 278 First maxillipedFor (Fig. 6A). Review Coxa with 1 seta; basis Only with 10 setae arranged 2+2+3+3; 19 20 21 279 endopod 5-segmented with 3, 2, 1, 2, 5 (1 subterminal and 4 terminal) setae, 22 23 280 respectively; exopod incipiently 2-segmented, distal segment with 4 terminal long 24 25 281 natatory plumose setae. 26 27 282 Second maxilliped (Fig. 6B). Coxa without setae; basis with 3 setae arranged 1+1+1; 28 29 30 283 endopod 3-segmented with 0, 1, 6 setae, respectively; exopod incipiently 2- 31 32 284 segmented, distal segment with 4 terminal long natatory plumose setae. 33 34 285 Third maxilliped. Present as small bud. 35 36 286 Pereiopods. Present as small buds. 37 38 287 Pleon (Figs. 3A, 7A). With 5 pleonites; pleonites 2 and 3 with one pair of dorso-lateral 39 40 41 288 processes; pleonites 3-5 with a pair of short postero-lateral processes; pleonite 1 42 43 289 without setae, pleonites 2-5 with one pair of postero-dorsal setae; pleopods absent. 44 45 290 Telson (Fig. 7a). Telson furcae with one pair of ventral and two pairs of dorsal spines; 46 47 291 inner margin with three pairs of serrulate setae. 48 49 50 292 51 52 293 Zoea II 53 54 294 Size: RDL= 3.34-3.97 mm; CL= 1.08-1.51 mm; CW= 1.71 mm; RL= 0.84-0.98 mm; 55 56 295 DL= 1.61-2.02 mm; N= 3. 57 58 59 60 Page 13 of 39 Zoological Journal of the Linnean Society

1 2 3 296 Cephalothorax (Fig. 3B). Antero-median region with five pairs of setae, one pair of 4 5 297 setae near the base of dorsal spine; each latero-ventral margin with 2 additional 6 7 298 setae (1 plumose + 1 sparsely plumose). Eyes stalked. 8 9 10 299 Antennule (Fig. 4B). Exopod with 7 aesthetascs; endopod bud present. 11 12 300 Antenna (Fig. 4E). Endopod longer, almost reaching middle of the length of protopod. 13 14 301 Protopod with one row of 20-22 spinules of different size. 15 16 302 Mandible. Palp bud present. 17 18 303 Maxillule (Fig. For 5B). Basial Review endite with 10 setae Only (5 cuspidate); one long plumose 19 20 21 304 exopodal seta on outer margin. 22 23 305 Maxilla (Fig. 5E). Basial endite with 5+5 setae; scaphognathithe with 19-20 marginal 24 25 306 plumose setae. 26 27 307 First and second maxillipeds. Exopod distal segment with 6 long and plumose natatory 28 29 30 308 setae. 31 32 309 Third maxilliped and pereiopods. More prominent buds than in first stage; cheliped 33 34 310 bilobed. 35 36 311 Pleon (Figs. 3B, 7B). With 6 pleonites, first pleonite with 2 long middorsal setae, 37 38 312 pleonites 2-4 with a pair of middorsal simple setae; pleonites 2-5 with long 39 40 41 313 pleopod buds, endopod buds present . 42 43 314 Telson (Fig. 7b). Inner margin with one pair of additional setae. 44 45 315 46 47 316 Megalopa 48 49 50 317 Size: CL =1.90-1.85 mm; CTL= 2.31-2.30 mm; CW =1.21-1.24 mm; N= 2 51 52 318 Cephalothorax (Figs. 3C, D). Longer than broad, with long, thin and straight rostrum; 53 54 319 hepatic regions with one anterior subacute tubercle; each protogastric region with 55 56 320 dorsally directed blunt process with 2 setae; one tubercle on mesogastric region 57 58 59 60 Zoological Journal of the Linnean Society Page 14 of 39

1 2 3 321 and posterodorsal margin; prominent long curved spine present on cardiac region; 4 5 322 setation as drawn. Dorsal organ present. Eyes stalked. 6 7 323 Antennule (Fig. 4C). Peduncle 3-segmented with 2, 1, 1 simple setae; unsegmented 8 9 10 324 endopod with 1 medial, 1 subterminal and 2 terminal simple setae; exopod 4- 11 12 325 segmented with 0, 1, 0, 1 simple setae, second segment with 11 and third segment 13 14 326 with 4 aesthetascs. 15 16 327 Antenna (Fig. 3F). Peduncle 3-segmented with 1, 0, 3 simple setae respectively, 17 18 328 proximal For segment withReview stout and ventrally-directed Only process; flagellum 5- 19 20 21 329 segmented with 0, 0, 4, 0, 3 simple setae respectively. 22 23 330 Mandible (Fig. 3G). Palp 2-segmented with 5 plumo-denticulate terminal setae on distal 24 25 331 segment. 26 27 332 Maxillule (Fig. 5C). Coxal endite with 5 subterminal plumose setae and 7 plumose setae 28 29 30 333 on margin; basial endite with 20 setae: 6 marginal cuspidate, 9 subterminal 31 32 334 plumo-denticulate, and 5 proximal plumose setae; endopod unsegmented with 2 33 34 335 terminal setae. 35 36 336 Maxilla (Fig. 5F). Coxal endite bilobed with 8 + 4 terminal plumose setae; basial endite 37 38 337 bilobed with 6 + 6 sparsely plumodenticulate setae; endopod unsegmented and 39 40 41 338 without setae; exopod (scaphognathite) with 34 marginal plumose setae plus 1 42 43 339 small simple seta on each lateral surface. 44 45 340 First maxilliped (Fig. 6C). Epipod triangular shaped without setae; coxal endite with 5 46 47 341 plumose setae; basial endite with 14 sparsely plumodenticulate setae; endopod 48 49 50 342 reduced, unsegmented and with 1 subterminal seta; exopod 2-segmented, with 4 51 52 343 terminal plumose setae on distal segment. 53 54 344 Second maxilliped (Fig. 6D). Epipod reduced without setae; protopod without setae. 55 56 345 Endopod 5-segmented with 0, 1 (simple), 1 (long simple), 5 (plumo-denticulate) 57 58 59 60 Page 15 of 39 Zoological Journal of the Linnean Society

1 2 3 346 and 5 (2 cuspidate, 3 plumo-denticulate) setae; exopod 2-segmented, with 1 4 5 347 medial simple seta on proximal segment and 4 terminal plumose setae on distal 6 7 348 segment. 8 9 10 349 Third maxilliped (Fig. 6E). Epipod relatively small with 1 subterminal and 3 terminal 11 12 350 long setae; protopod with 7 plumo-denticulate setae; endopod 5-segmented, with 13 14 351 14, 9, 3, 6, 4 setae respectively, ischium with denticulate margin; exopod 2- 15 16 352 segmented with1 medial simple setae on proximal segment and 4 terminal 17 18 353 plumose setaeFor on distal Review segment. Only 19 20 21 354 Pereiopods (Figs. 8A, B, b1, b2). Cheliped with a small proximal ventral tubercle on 22 23 355 coxa, setation as drawn; pereiopods II-V slender and setose, with dactyli 24 25 356 terminally very acute; ischium of pereiopods II-III with small spine; pereiopods 26 27 357 II-IV with inner margin of dactyli with 1 plumo-denticulate setae and 1 stout 28 29 30 358 serrulate spine. Setation as illustrated. 31 32 359 Sternum (Fig. 8C). Setation as illustrated. 33 34 360 Pleon (Fig. 3C, 7C). Six pleonites plus telson; pleonite 1 without setae; setation of 35 36 361 pleonites 2-6 as shown; sixth pleonite reduced. 37 38 362 Pleopods (Figs. 7D, E). Present on pleonites 2-5; endopods unsegmented with 3 39 40 41 363 cincinnuli; exopod unsegmented with 12 long plumose natatory setae. Uropods 2- 42 43 364 segmented, proximal segment without setae, distal segment with 2 terminal 44 45 365 plumose natatory setae. 46 47 366 Telson (Fig. 7C). Small with one pair of dorsal setae. 48 49 50 367 51 52 368 DNA analysis 53 54 369 The initial length of the aligned dataset for the rRNA (16S) and Cox1 genes was 446 55 56 370 and 611 bp, respectively. After running GBlocks, a total of 1027 positions were kept for 57 58 59 60 Zoological Journal of the Linnean Society Page 16 of 39

1 2 3 371 further analyses (97 % of the original 1057 positions). The best-fit partitioning scheme 4 5 372 for the alignment included 4 partitions (one per codon position within Cox1 and another 6 7 373 partition including the 16S gene region), and the nucleotide substitution models selected 8 9 10 374 for each partition were TrNef+G (COI_1st), HKY+I (COI 2nd), HKY+G (COI 3rd) and 11 12 375 GTR+I+G (16S). The performance of the BEAST runs was assessed using Tracer v1.5, 13 14 376 a graphical tool for visualization and diagnostics of MCMC output. The effective 15 16 377 sample size was >200 in all BEAST runs, indicating convergence of the MCMC. The 17 18 378 consensus phylogeneticFor tree Review (Figure 2) showed a higOnlyhly significant clustering of the 19 20 21 379 zoea and megalopa with the adult Ergasticus clouei , pointing out the real identity of the 22 23 380 larvae. 24 25 381 In order to test the statistical support for previously established hypotheses 26 27 382 (Ergasticus belongs to Inachidae), Bayes factors were computed comparing the tree 28 29 30 383 topology obtained under the unconstrained model against the constrained topologies. 31 32 384 The log-likelihood values obtained from the unconstrained tree (-8258.55 + 0.78) were 33 34 385 significantly larger than those obtained from the constrained tree (-8262.9 + 1.07). 35 36 386 According to the large Bayes factor obtained (BF = 8.70), it can be concluded that there 37 38 387 is strong support for the removal of Ergasticus from the Inachidae, and that its 39 40 41 388 clustering with the Oregoniidae is consistently supported by the genetic data. 42 43 389 44 45 390 DISCUSSION 46 47 391 The present study describes for the first time the complete larval development of 48 49 50 392 Ergasticus clouei thanks to the use of DNA barcoding methods on larvae collected from 51 52 393 the plankton. The genetically-identified larval stages of E. clouei show the general 53 54 394 characteristics listed by Rice (1980) for Majoidea larvae: presence of two zoeal stages, 55 56 395 with at least nine marginal setae on the scaphognathite in the first zoea, and with 57 58 59 60 Page 17 of 39 Zoological Journal of the Linnean Society

1 2 3 396 developed pleopods in the second zoea. However, the morphology of the larval stages of 4 5 397 E. clouei did not fit into the characteristic Inachidae (Marco-Herrero, Rodriguez & 6 7 398 Cuesta, 2012; Marques & Pohle, 2003; Rice, 1980). Clear differences were found, such 8 9 10 399 as the presence of 2 subterminal setae in the exopod of the antenna, rostral and lateral 11 12 400 carapace spines, additional spines on the telson furcae, subterminal setae on the distal 13 14 401 endopod segment of the maxillule, or the basis of first maxilliped with 2+2+3+3 setae 15 16 402 (see Table 2). 17 18 403 In relationFor with this, Reviewthe detailed description carOnlyried out in this study allowed us 19 20 21 404 to notice a remarkable character of E. clouei zoeal stages unnoticed by Guerao & Abelló 22 23 405 (2007): the presence of furcal spines in ventral position in the telson. After a thorough 24 25 406 review of the literature, this spine was found most likely to be homologous to the large 26 27 407 lateral spine present in some non-inachid Majoidea (Rice, 1980; Ingle, 1992). If this is the 28 29 30 408 case, the number of spines of the telson would be a character shared by E. clouei with the 31 32 409 Majidae Samouelle, 1819 family, which also presents 3 spines (one large and two small) 33 34 410 on each telson furca, but all in a lateral position (Guerao et al. , 2008; Rodriguez, 2002). 35 36 411 The fact that rostral and lateral spines are also present in Oregoniidae (Table 3), 37 38 412 Majidae, and some Pisinae Dana, 1851 (see Santana, Pohle & Marques, 2004), that the 39 40 41 413 setation pattern of the basis of the first maxilliped (2+2+3+3) is also present in 42 43 414 Oregoniidae and some Pisinae, and that the presence of three spines on the telson furcae 44 45 415 is also observed in Majidae (but not in Inachidae) further support the necessity to remove 46 47 416 Ergasticus from the Inachidae. Moreover, the morphology of the larval antenna is 48 49 50 417 particularly important in this regard. As described by Clark & Webber (1991) for the 51 52 418 Japanese giant spider crab Macrocheira kaempferi , the antennal type observed in 53 54 419 Ergasticus (with two subterminal setae on the exopod) is found in Oregoniidae and 55 56 420 Majidae genera, but not in Inachidae (see Pohle, 1991; Rodríguez, 2002). Finally, the fact 57 58 59 60 Zoological Journal of the Linnean Society Page 18 of 39

1 2 3 421 that Oregoniidae and E. clouei are the only Majoidea which show the presence of two 4 5 422 mid-dorsal setae on the fifth pleomere of the second zoea and the presence of a 5- 6 7 423 segmented antennal flagellum in the megalopa indicates that E. clouei should be placed 8 9 10 424 closer to Oregoniidae because it shares with them more than any other Majoidea family. 11 12 425 In agreement with the larval morphology and contrary to our expectations given 13 14 426 the current systematics of Majoidea, the molecular phylogenetic analysis did not show E. 15 16 427 clouei samples grouping with the remaining Inachidae genera ( Macropodia Leach, 1814; 17 18 428 Podochela Stimpson,For 1860; InachusReview Weber, 1795 and Only Metoporhaphis Stimpson, 1860). 19 20 21 429 Instead, both the larvae and adult E. clouei sequences clustered significantly with 22 23 430 Oregoniidae Garth, 1958 genera such as Chionoecetes Krøyer, 1838 and Hyas Leach, 24 25 431 1814. Our results using the Bayes Factor approach on alternative phylogenetic 26 27 432 hypotheses clearly indicates that there is strong support for the removal of Ergasticus 28 29 30 433 from the Inachidae, and that the clustering of Ergasticus with the Oregoniidae is 31 32 434 statistically significant. 33 34 435 As pointed out by Griffin & Tranter (1986) the limitations on the definition of 35 36 436 the Inachidae based on few adult traits has caused the family to become cluttered over 37 38 437 the years by various species with long eyestalks but not necessarily resembling other 39 40 41 438 inachids in other characteristics. Oh & Ko (2011), based on larval morphology (see 42 43 439 Table 2), have recently suggested that Platymaia wyvillethomsoni Miers, 1886 as well 44 45 440 as Pleistacantha sanctijohannis Miers, 1879 (larvae described by Kurata, 1969) are 46 47 441 closer to Macrocheira kaempferi than to any other Inachidae. They proposed that P. 48 49 50 442 wyvillethomsoni should not be placed within the Inachidae, although they did not 51 52 443 suggest a new placement, stating that “future investigations should check their 53 54 444 taxonomic status”. In a review of the Inachoididae Dana, 1851, Guinot (2012) has also 55 56 445 proposed changes in the generic composition of the Inachidae. She proposes to transfer 57 58 59 60 Page 19 of 39 Zoological Journal of the Linnean Society

1 2 3 446 Stenorhynchus from Inachidae to Inachoididae (resurrecting Stenorhynchinae Dana, 4 5 447 1851) and also suggests a reappraisal of Inachidae to reinstate some subfamilies 6 7 448 recognized in the past, like Inachinae, Podochelinae and Anomalopodine (presently 8 9 10 449 there are not subfamilial subdivisions within Inachidae). 11 12 450 During a recent visit to the Natural History Museum (NHM) in London, one of 13 14 451 the authors was able to review the adult morphology of several Inachidae genera 15 16 452 available in the NHM collections. In particular, the shape of the first gonopod in males, 17 18 453 which is commonlyFor used as a Reviewkey trait in Majoidea s ystematics,Only had never been described 19 20 21 454 in Ergasticus . The revision of adult morphology clearly showed that the three Inachidae 22 23 455 genera Ergasticus ; Bothromaia and Pleistacantha present a distinct type of gonopod, 24 25 456 bearing a subdistal papilla (see also Ahyong et al., 2005). All these three genera can also 26 27 457 be distinguished from other Inachidae by their long “rostral” horns, markedly divergent 28 29 30 458 and the strong spines at the base of the pseudorostrum and the supraorbital margin. Given 31 32 459 our results from larval morphology, genetic markers and the observations from adult 33 34 460 morphology, it is proposed to remove the three genera Ergasticus ; Bothromaia and 35 36 461 Pleistacantha from the Inachidae and place them within the Oregoniidae as a new 37 38 462 subfamily (Pleistacanthinae sub. nov.), that would correspond to the Pleistacanthini tribe 39 40 41 463 of Štev čić (2005). 42 43 464 The clear similarities in larval form between Ergasticus and Cyrtomaia , 44 45 465 Eurypodius or Platymaia indicate that these genera should also be removed from 46 47 466 Inachidae and placed within Oregoniidae. These observations would further support 48 49 50 467 Griffin & Tranter (1986), whom already mentioned “at least superficial resemblances to 51 52 468 Chionoecetes of the Oregoniinae” with regard to adult morphology of Cyrtomaia and 53 54 469 Platymaia species. Nevertheless, extending the assessment of the taxonomic position of 55 56 470 all the problematic genera (particularly all above mentioned such as Macrocheira , 57 58 59 60 Zoological Journal of the Linnean Society Page 20 of 39

1 2 3 471 Platymaia and Stenorhynchus ) would demand a more comprehensive review of the whole 4 5 472 family which goes beyond the purpose of this work. 6 7 473 In this study, the results obtained from both morphological information of all 8 9 10 474 larval stages as well as the analysis of DNA sequences (16S rDNA and Cox1 genes) 11 12 475 provided conclusive evidence to support the removal of Ergasticus from the family 13 14 476 Inachidae and its placement together with members of the family Oregoniidae. 15 16 477 Therefore, our results also evidence that developmental stages of brachyurans may 17 18 478 provide reliable Formorphological Review characteristics, independent Only from those of adults, to help 19 20 21 479 resolving the phylogenetic relationships among Majoidea genera. 22 23 480 24 25 481 ACKNOWLEDGEMENTS 26 27 482 The research was carried out within the framework of the projects 28 29 30 483 MEGALOPADN (CGL2009-11225) and IDEADOS (CTM2008-04489-C03), 31 32 484 funded by the “Ministerio de Economía y Competividad (MINECO)” Spanish 33 34 485 Plan R+D+I and FEDER. The authors are very grateful to all our colleagues 35 36 486 who participated in the IDEADOS surveys and to the crew of the R/V 37 38 487 Sarmiento de Gamboa . We also wish to thank all participants in the 39 40 41 488 MEDITS_ES_2003 cruise on board the R/V “Cornide de Saavedra”, from 42 43 489 which adult Ergasticus clouei were collected. We are especially grateful to 44 45 490 Antonina dos Santos who helped in the larvae identification and to Carlos 46 47 491 Sánchez Nieto for his assistance in the laboratory work. E. Marco-Herrero 48 49 50 492 and A. P. Torres acknowledge the pre-doctoral FPI Fellowships supported by 51 52 493 MINECO (BES-2010-033297) and Regional Government of the Balearic 53 54 494 Islands, Conselleria d’Educacció, Cultura i Universitats, selected as part of an 55 56 495 operational program co-financed by the Fondo Social Europeo, respectively. 57 58 59 60 Page 21 of 39 Zoological Journal of the Linnean Society

1 2 3 496 REFERENCES 4 5 497 Abelló P, Carbonell A, Torres P. 2002. Biogeography of epibenthic crustaceans on the 6 7 498 shelf and upper slope off the Iberian Peninsula Mediterranean coasts: 8 9 10 499 implications for the establishment of natural management areas. Scientia Marina 11 12 500 66: 183-198. 13 14 501 Ahyong ST, Chen H, Ng PKL. 2005. Pleisacantha stilipes , a new species of spider 15 16 502 crab from the South China Sea (Decapoda: Brachyura: Majidae). Zootaxa 822: 17 18 503 1-10. For Review Only 19 20 21 504 Ampuero D, T. PA, Veliz D, Pardo LM. 2010. Description, seasonal morphological 22 23 505 variation, and molecular identifcation of Paraxanthus barbiger megalopae 24 25 506 obtained from the natural environment. Helgoland Marine Research 64: 117- 26 27 507 123. 28 29 30 508 Balss H. 1957. Decapoda. VIII Systematik. In: Dr.H.G., ed. Bronn's, Klassen und 31 32 509 Ordnungen des Tierreichs . 1505-1672. 33 34 510 Bouvier EL. 1940. Déapodes Marcheurs . Paul Le Chevalier: Paris. 35 36 511 Brandley MC, Schmitz A, Reeder TW. 2005. Partitioned Bayesian Analyses, Partition 37 38 512 Choice, and the Phylogenetic Relationships of Scincid Lizards. Systematic 39 40 41 513 Biology 54: 373-390. 42 43 514 Campodonico IG, Guzman LM. 1972. Desarrollo larval de Eurypodius latreillei 44 45 515 Guerin en condiciones de laboratorio (Crustacea, Brachyura: Majidae, 46 47 516 Inachinae). Anales del Instituto de la Patagonia 3: 233-247. 48 49 50 517 Castresana J. 2000. Selection of conserved blocks from multiple alignments for their 51 52 518 use in phylogenetic analysis. Molecular Biology and Evolution 17: 540-552. 53 54 55 56 57 58 59 60 Zoological Journal of the Linnean Society Page 22 of 39

1 2 3 519 Clark PF, Calazans DK, Pohle GW. 1998. Accuracy and standardization of 4 5 520 brachyuran larval descriptions. Invertebrate Reproduction and Development 33: 6 7 521 127-144. 8 9 10 522 Clark PF, Webber WR. 1991. A redescription of Macrocheira kaempferi (Temminck, 11 12 523 1836) zoeas with a discussion of the classification of the Majoidea Samouelle, 13 14 524 1819 (Crustacea: Brachyura). Journal Natural History 25: 1259-1279. 15 16 525 Crandall K, A., Fitzpatrick JFJ. 1996. Crayfish molecular systematics: using a 17 18 526 combinationFor of procedures Review to estimate phylogeny. Only Systematic Biology 45: 1-26. 19 20 21 527 Christiansen ME. 1973. The complete larval development of Hyas araneus (Linnaeus) 22 23 528 and Hyas coarctatus Leach (Decapoda, Brachyura, Majidae) reared in the 24 25 529 laboratory. Norwegian Journal of zoology 21: 63-89. 26 27 530 D'Udekem d'Acoz C. 1999. Inventaire et distribution des crustacés décapodes de 28 29 30 531 l'Atlantique nord-oriental, de la Méditerranée et des eaux continentales 31 32 532 adjacentes au nord de 25°N. Muséum National d’Histoire Naturelle: Paris. 33 34 533 De Forges BR, Poore G. 2008. Deep-sea majoid crabs of the genera Oxypleurodon and 35 36 534 Rochinia (Crustacea: Decapoda: Brachyura: Epialtidae) from the continental 37 38 535 margin of Western Australia. Memoirs of the Museum of Victoria 65: 63-70. 39 40 41 536 De Grave S, Pentcheff ND, Ahyong ST, Chan T-Y, Crandall KA, Dworschak PC, 42 43 537 Felder DL, Feldmann RM, Fransen CJM, Goulding LYD, Lemaitre R, Low 44 45 538 M, Martin JW, Ng PKL, Schweitzer CE, Tan SH, Tshudy D, Wetzer R. 46 47 539 2009. A classification of living and fossil genera of Decapod Crustaceans. The 48 49 50 540 raffles bulletin of zoology 21: 1-109. 51 52 541 Dos Santos A, Gonzalez-Gordillo JI. 2004. Illustrated keys for the identification of the 53 54 542 Pleocyemata (Crustacea: Decapoda) zoeal stages, from the coastal region of 55 56 57 58 59 60 Page 23 of 39 Zoological Journal of the Linnean Society

1 2 3 543 south-western Europe. Journal of the Marine Biological Association of the 4 5 544 United Kingdom 84: 205-227. 6 7 545 Dos Santos A, Lindley JA. 2001. Crustacea Decapoda: Larvae II. Dendrobranchiata 8 9 10 546 (Aristeidae, Benthesicymidae, Penaeidae, Solenoceridae, Sicyonidae, 11 12 547 Sergestidae, and Luciferidae). Copenhagen. 13 14 548 Drummond AJ, Ho SYW, Phillips MJ, Rambaut A. 2006. Relaxed Phylogenetics 15 16 549 and Dating with Confidence. PLoS Biology 4: e88. 17 18 550 Drummond AJ,For Rambaut A.Review 2007. BEAST: Bayesian Only evolutionary analysis by 19 20 21 551 sampling trees. BMC Evolutionary Biology 7: 214. 22 23 552 Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high 24 25 553 throughput. Nucleic Acids Research 32: 1792-1797. 26 27 554 Garth JS. 1958. Brachyura of the Pacific coast of America: Oxyrhyncha. Garth, J.S. 28 29 30 555 21: 1-99. 31 32 556 González-Gordillo JI, Rodríguez A. 2001. The complete larval development of the 33 34 557 spider crab, Macropodia parva (Crustacea, Decapoda, Majidae) from laboratory 35 36 558 culture. Invertebrate Reproduction and Development 39: 135-142. 37 38 559 Griffin DJG, Tranter HA. 1986. The Decapoda Brachyura of the Siboga Expedition. 39 40 41 560 Part VIII. Majidae. Siboga- Expeditie Monograph 39: 1-35. 42 43 561 Guerao G, Abelló P. 2007. The first zoea of Inachus aguiarii , Inachus communissimus 44 45 562 and Ergasticus clouei (Decapoda, Brachyura, Majoidea) with implications for 46 47 563 the systematics of the family Inachidae. Zootaxa 1429: 55-68. 48 49 50 564 Guerao G, Pastor E, Martin JW, Andres M, Estevez A, Grau A, Duran J, Rotllant 51 52 565 G. 2008. The larval development of Maja squinado and M. brachydactyla 53 54 566 (Decapoda, Brachyura, Majidae) described from plankton collected and 55 56 567 laboratory-reared material. Journal Natural History 42: 2257-2276. 57 58 59 60 Zoological Journal of the Linnean Society Page 24 of 39

1 2 3 568 Guinot D. 2012. Remarks on Inachoididae Dana, 1851, with the description of a new 4 5 569 genus and the resurrection of Stenorhynchinae Dana, 1851, and recognition of 6 7 570 the inachid subfamily Podochelinae Neumann, 1878 (Crustacea, Decapoda, 8 9 10 571 Brachyura, Majoidea). Zootaxa 3416: 22-40. 11 12 572 Hebert PDN, Ratnasingham S, deWaard JR. 2003. Barcoding animal life: 13 14 573 cytochrome c oxidase subunit 1 divergences among closely related species. 15 16 574 Proceeding the Royal Society London 270: 96-99. 17 18 575 Hultgren KM, GueraoFor G, MarquesReview F, Palero F. 2009. Only Assessing the contribution of 19 20 21 576 molecular and larval morphological characters in a combined phylogenetic 22 23 577 analysis of the superfamily Majoidea. In: Martin JW, Crandall KA and Felder 24 25 578 DL, eds. Decapod crustacean phylogenetics : Crustacean Issues. 437-455. 26 27 579 Hultgren KM, Stachowicz JJ. 2009. Evolution of Decoration in Majoid Crabs: A 28 29 30 580 Comparative Phylogenetic Analysis of the Role of Body Size and Alternative 31 32 581 Defensive Strategies . The University of Chicago Press for The American Society 33 34 582 of Naturalists. 35 36 583 Ingle RW. 1977. The larval and post-larval development of the scorpion spider crab, 37 38 584 Inachus dorsettensis (Pennant) (Family: Majidae), reared in the laboratory. 39 40 41 585 Bulletin of the British Museum (Natural History), Zoology 30: 329-348. 42 43 586 Ingle RW. 1992. Larval stages of northeastern Atlantic crabs: An illustrated key . 44 45 587 Chapman & Hall (London & New York) 46 47 588 Iwata Y, Sugita H, Deguchi Y, Kamemoto FI. 1991. On the first zoea of Cyrtomaia 48 49 50 589 owstoni Terazaki (Decapoda, Majidae) reared in the laboratory. Researches on 51 52 590 crustacea : 17-21. 53 54 591 Kass RE, Raftery AE. 1995. Bayes Factors. Journal of the American Statistical 55 56 592 Association 90: 773-795. 57 58 59 60 Page 25 of 39 Zoological Journal of the Linnean Society

1 2 3 593 Kornienko ES, Korn OM. 2010. Illustrated Key fot thr idrntification of braquiuran 4 5 594 larvae in the northwestern Sea of Japan : Dalnauka. 6 7 595 Kurata H. 1969. Larvae of Decapoda Brachyura of Arasaki_Sagami Bay-IV. Bulletin 8 9 10 596 Tokai Regional Ficheries Research Laboratory 57: 81-127. 11 12 597 Lanfear R, Calcott B, Ho SYW, Guindon S. 2012. Partitionfinder: combined 13 14 598 selection of partitioning schemes and substitution models for phylogenetic 15 16 599 analyses. Molecular Biology and Evolution 29: 1695-1701. 17 18 600 López-Jurado JL,For Marcos ReviewM, Monserrat S. 2008. HydrographicOnly conditions affecting 19 20 21 601 two fishing grounds of Mallorca island (Western Mediterranean): during the 22 23 602 IDEA Project (2003-2004). Journal of Marine Systems 71: 303-315. 24 25 603 Manning BR, Holthui LB. 1981. West African Brachyuran Crabs (Crustacea: 26 27 604 Decapoda). Smithsonian Contribution to zoology 306: 1-379. 28 29 30 605 Marco-Herrero E, Rodriguez A, Cuesta JA. 2012. Morphology of the larval stages of 31 32 606 Macropodia czernjawskii (Brant, 1880) (Decapoda, Brachyura, Inachidae)reared 33 34 607 in the laboratory. Zootaxa : 1-31. 35 36 608 Marques F, Pohle G. 2003. Searching for larval support for majoid families 37 38 609 (Crustacea: Brachyura) with particular reference to Inachoididae Dana, 1851. 39 40 41 610 Invertebrate Reproduction & Development 43: 71-82. 42 43 611 Martin JW, Davis GE. 2001. An Updated Classification of the Recent Crustacea. 44 45 612 Natural History Museum of Los Angeles County 39: 1-24. 46 47 613 Miers EJ. 1879. On the classification of the Maioid Crustacea or Oxyrhyncha, with a 48 49 50 614 synopsis of the families, sub-families and genera. Journal of the Linnean Society 51 52 615 of London 14: 634-673, Plate 612-613. 53 54 616 Motoh H. 1973. Laboratory-reared zoeae and megalopae of Zuwai crab from the Sea of 55 56 617 Japan. Bulletin of the Japanese Society for the Science of Fish 39: 1223-1230. 57 58 59 60 Zoological Journal of the Linnean Society Page 26 of 39

1 2 3 618 Motoh H. 1976. The larval stages of Benizuwai-gani, Chionoecetes japonicus Rathbun 4 5 619 reared in the laboratory. Bulletin of the japanese Society of scientific fisheries 6 7 620 42: 533-542. 8 9 10 621 Ng PKL, Guinot D, Davie P. 2008. Systema Brachyurorum: Parte I. An annotated 11 12 622 checklist of extant brachyuran crabs of the world. Raffles Bulletin of Zoology 17: 13 14 623 1-286. 15 16 624 Nylander JAA, Ronquist F, Huelsenbeck JP, Nieves-Aldrey JL. 2004. Bayesian 17 18 625 PhylogeneticFor Analysis Review of Combined Data. Systematic Only Biology 53: 47-67. 19 20 21 626 Oh SM, Ko HS. 2011. The zoeal development of Platymaia wyvillethomsoni Miers, 22 23 627 1886 (Crustacea: Decapoda: Majoidea: Inachidae) described from laboratory 24 25 628 reared material. Invertebrate Reproduction & Development 56 . 26 27 629 Olivar MP, Bernal A, Moli B, Pena M, Balbin R, Castellon A, Miquel J, Massuti E. 28 29 30 630 2012. Vertical distribution, diversity and assemblages of mesopelagic fishes in 31 32 631 the western Mediterranean. Deep Sea Research Part I: Oceanographic Research 33 34 632 Papers 62: 53-69. 35 36 633 Palero F, Guerao G, Abelló P. 2008. Morphology of the final stage phyllosoma larva 37 38 634 of Scyllarus pygmaeus (Crustacea: Decapoda: Scyllaridae), identified by DNA 39 40 41 635 analysis. Journal of Plankton Research 30: 483-488. 42 43 636 Paula J, Cartaxana A. 1991. Complete larval development of the spider crab 44 45 637 Stenorhynchus lanceolatus (Brulle, 1837) (Decapoda, Brachyura, Majidae), 46 47 638 reared in the laboratory. Crustaceana 60: 113-122. 48 49 50 639 Pessani D, Tirelli T, FlagellaL S. 2004. Key for the identification of Mediterranean 51 52 640 brachyuran megalopae. Mediterranean Marine Science 5: 53-64. 53 54 55 56 57 58 59 60 Page 27 of 39 Zoological Journal of the Linnean Society

1 2 3 641 Pinot JM, López-Jurado JL, Riera M. 2002. The canals experiment (1996-1998). 4 5 642 Interannual, seasonal, and mesoscale variability of the circulation in the Balearic 6 7 643 channels. Progress In Oceanography 55: 335-370. 8 9 10 644 Pohle JW. 1991. Larval development of Canadian Atlantic oregoniid crabs (Brachyura: 11 12 645 Majidae), with enphasis on Hyas coarctatus alutaceus Brandt, 1851, and a 13 14 646 comparison with Atlantic and Pacific species. Canadian Journal of Zoology 69: 15 16 647 2717-2737. 17 18 648 Ramón M, AbellóFor P, Ordinas Review F, Serrano A., Massutí Only E. Submitted. Epibenthic 19 20 21 649 communities in two contrasting areas of the Balearic Islands. Journal of Marine 22 23 650 Systems 24 25 651 Rice AL. 1980. Crab zoeal morphology and its bearing on the classification of the 26 27 652 Brachyura. The Transactions of the Zoological Society of London 35: 271-372. 28 29 30 653 Rodriguez A. 2002. Larval development of Maja crispata Risso 1827 (Crustacea, 31 32 654 Decapoda, Majidae) reared in the laboratory. Journal Natural History 36: 1589- 33 34 655 1600. 35 36 656 Santana W, Pohle GW, Marques FPL. 2004. Larval development of Apiomithrax 37 38 657 violaceus (A. Milne Edwards, 1868) (Decapoda: Brachyura: Majoidea: Pisidae) 39 40 41 658 reared in laboratory conditions, and a review of larval characters of Pisidae. 42 43 659 Journal Natural History 38: 1773-1797. 44 45 660 Schubart CD, Cuesta JA, Felder DL. 2002. Glyptograpsidae, a new brachyuran 46 47 661 family from central america: Larval and adult morphology, and a molecular 48 49 50 662 phylogeny of the Grapsioidea. Journal of crustacean biology 22: 28-44. 51 52 663 Schubart CD, Huber MGJ. 2006. Genetic comparisons of German populations of the 53 54 664 stone crayfish, Austropotamobius torrentium (Crustacea: Astacidae). Bulletin 55 56 665 Français de la Péche et de la Pisciculture 380-381: 1019-1028. 57 58 59 60 Zoological Journal of the Linnean Society Page 28 of 39

1 2 3 666 Stevcic Z. 2005. The reclassification of brachyuran crabs (Crustacea: Decapoda: 4 5 667 Brachyura). Natura Croatica 14: 1-159. 6 7 668 Yang WT. 1967. A study of zoeal, megalopal, and early crab stages of some 8 9 10 669 oxyrhynchous crabs (Crustacea: Deacpoda). Unpublished: Doctoral 11 12 670 Dissertation, University of Miami. 13 14 671 Yang WT. 1976. Studies on the Western Atlantic arrow crab genus Stenorhynchus 15 16 672 (Decapoda Brachyura, Majidae): I. Larval characters of two species and 17 18 673 comparisonFor with other Review larvae of Inachinae. Crustaceana Only 31: 157-177. 19 20 21 674 Zariquiey Alvarez R. 1968. Crustáceos Decápodos Ibéricos . Investigación pesquera: 22 23 675 Barcelona. 24 25 676 26 27 677 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 29 of 39 Zoological Journal of the Linnean Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Table 1. Species used in the present study and gene sequences included in the phylogenetic analysis. Abbreviations; P, pending; ND, no data. 48 189x261mm (96 x 96 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 Zoological Journal of the Linnean Society Page 30 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Table 2. Morphological comparison between larval stages of selected Inachidae genera (according to Ng et 44 al., 2008). References: Achaeus tuberculatus (Kurata, 1969), Achaeus cranchii (Ingle, 1992), Cyrtomaia 45 owstoni (Iwata et al., 1991), Ergasticus clouei (Guerao & Abelló, 2007 and present study), Eurypodius 46 latreille (Campodonico & Guzman, 1972), Inachus thoracicus (Guerao et al., 2002), Macrocheira kaempferi 47 (Clark & Webber, 1991), Macropodia czernjwaskii (Marco-Herrero et al., 2012), Platymaia wyvillethomsoni 48 (Oh & Ko, 2011), Pleistacantha sanctijohannis (Kurata, 1969), Podochela riisei, (Yang, 1967), Stenorhynchus lanceolatus (Paula & Cartaxana, 1991) and Stenorhynchus seticornis (Yang, 1976). 49 Abbreviations: dlp, dorsolateral processes; l sp, lateral spine; mds, middorsal setae; nd, no data; rsp, rostral 50 spine; s, setae; se, segments; subt, subterminal; ss, serrulate setae. 51 239x261mm (96 x 96 DPI) 52 53 54 55 56 57 58 59 60 Page 31 of 39 Zoological Journal of the Linnean Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Table 3. Morphological comparison between larval stages of Ergasticus clouei, Oregoniidae (represented by the genera Chionocetes and Hyas) and Inachidae (represented by the genera Inachus and Macropodia) 48 showing only those characters shared with oregoniids and that differ in inachids (there were no characters 49 shared with inachids that would differ in oregoniids). References: Ergasticus clouei (Guerao & Abelló, 2007 50 and present paper); Chionocetes, C. opilio (Motoh, 1973), C. japonicus (Motoh, 1976); Hyas, H. araneus 51 (Christiansen, 1973; Pohle, 1991), H. ursinus (Kornienko & Korn, 2010); Inachus, I. dorsettensis (Ingle, 52 1977), I. longipes (Guerao et al., 2002); Macropodia, M. parva (González-Gordillo & Rodríguez, 2001), M. 53 czernjawskii (Marco-Herrero et al., 2012). Abbreviations: dlp, dorsolateral processes; lsp, lateral spine; 54 mds, middorsal setae; rsp, rostral spine; s, setae; se, segments, ss, serrulate setae. 55 189x261mm (96 x 96 DPI) 56 57 58 59 60 Zoological Journal of the Linnean Society Page 32 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Figure 1. Map of the study area (western Mediterranean) where both larvae (stars) and adult (triangles) of 43 Ergasticus clouei were collected. 44 243x261mm (96 x 96 DPI) 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 33 of 39 Zoological Journal of the Linnean Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Figure 2. Phylogenetic tree based on 16S rRNA and Cox1 gene sequence dataset, showing the position of 34 the larval specimens genetically analyzed. 35 329x261mm (96 x 96 DPI) 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Zoological Journal of the Linnean Society Page 34 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 3. Ergasticus clouei. General lateral view, A: zoea I; B: zoea II. Ventral margin detail, b: zoea II. Megalopa, C: dorsal view; D: lateral view. Scale bars = 0.5 mm. 48 1050x1406mm (96 x 96 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 Page 35 of 39 Zoological Journal of the Linnean Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 4. Ergasticus clouei. Antennule, A: zoea I; B: zoea II; C: megalopa. Antenna, D: zoea I; E: Zoea II; F: megalopa. Mandible, G: megalopa. Scale bars = 0.1 mm. 48 1050x1406mm (96 x 96 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 Zoological Journal of the Linnean Society Page 36 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 5. Ergasticus clouei. Maxillule, A: zoea I; B: zoea II; C: megalopa. Maxilla, D: zoea I; E: zoea II; F: megalopa. Scale bars = 0.1 mm. 48 1050x1406mm (96 x 96 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 Page 37 of 39 Zoological Journal of the Linnean Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 6. Ergasticus clouei. First maxilliped, A: zoea I; C: megalopa. Second maxilliped, B: zoea I; D: megalopa. Third maxilliped, E: megalopa. Scale bars = 0.1 mm. 48 1050x1406mm (96 x 96 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 Zoological Journal of the Linnean Society Page 38 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 7. Ergasticus clouei. Pleon, dorsal view, A: zoea I; B: zoea II; C: megalopa. Detail telson, a: zoea I; b: zoea II. Megalopa, D: 3rd pleopod; E: uropod. Scale A-C bars = 0.5 mm, D-E = 0.1mm. 48 1050x1406mm (96 x 96 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 Page 39 of 39 Zoological Journal of the Linnean Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 8. Ergasticus clouei. Megalopa, A: queliped; B: 3rd pereiopod, b1: dactylus II; b2: dactylus V; C: sternum. Scale bars = 0.5mm. 48 1050x1406mm (96 x 96 DPI) 49 50 51 52 53 54 55 56 57 58 59 60