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.