Morphology of the final stage phyllosoma larva of Scyllarus pygmaeus (Crustacea: Decapoda: Scyllaridae), identified by DNA analysis

Morphology of the final stage phyllosoma larva of Scyllarus pygmaeus (Crustacea: Decapoda: Scyllaridae), identified by DNA analysis FERRAN PALERO 1,3 *, GUILLERMO GUERAO 2 AND PERE ABELLÓ 3 1 DEPARTAMENT DE GENÈTICA (EVOLUCIÓ), FACULTAT DE BIOLOGIA (UB), AV. DIAGONAL 645, 08028 BARCELONA, CATALONIA, SPAIN, 2 IRTA, UNITAT DE CULTIUS EXPERIMENTALS, SANT CARLES DE LA RÀPITA, TARRAGONA, SPAIN AND 3 INSTITUT DE CIÈNCIES DEL MAR (CSIC), PASSEIG MARÍTIM DE LA BARCELONETA 37-49, 08003 BARCELONA, CATALONIA, SPAIN *CORRESPONDING AUTHOR: ferranpalero@ub.edu Received April 15, 2007; accepted in principle September 15, 2007; accepted for publication January 18, 2008; published online January 31, 2008 Corresponding editor: Roger Harris The morphology of the final phyllosoma larval stage of the slipper lobster Scyllarus pygmaeus is described and illustrated based on larvae captured from Mediterranean waters and compared with those described in other Scyllaridae. These larvae were always found in deep waters (.200 m). Nucleotide sequence analysis of a region of the nuclear 28S rdna gene identified these larvae as S. pygmaeus, the morphology of which had been previously un-described. An intensive review revealed a misidentification of Scyllarus arctus larvae in the literature during the last 180 years, since the identification of S. pygmaeus larvae as Chrysoma mediterraneum. Detailed examination indicated that the final-stage larvae examined belonged to a clearly defined phyllosoma larval group within the genus Scyllarus, morphologically very similar to some phyllosoma larvae collected from Japan, Hawaiian Islands, New Zealand or Juan Fernandez Islands. This constitutes the first complete description of a phyllosoma stage of a member of the family Scyllaridae, with the specific identity of the larva being validated with molecular techniques. INTRODUCTION The Achelata lobsters, belonging to the families Palinuridae Latreille, 1802, Scyllaridae Latreille, 1825, and Synaxidae Bate, 1881, constitute an important fishery resource with most research on these crustaceans being directed towards the adult benthic phase. All these families share a common and very characteristic type of larva, named the phyllosoma. Even though adult morphology is well described, studies on the planktonic phyllosoma phase have been comparatively neglected given its long duration, which has made it difficult to rear them in the laboratory (Kittaka, 1997). Despite the relatively large size of these larvae and their immediate recognition in plankton sorting, important identification problems due to the lack of detailed and specific morphological descriptions have precluded specific determination in many plankton samples. Furthermore, the effects of formalin fixation of plankton samples and the small amount of tissue in phyllosoma larvae have made it extremely difficult to obtain sufficient DNA suitable for polymerase chain reaction (PCR), preventing the specific identification of these larvae in most cases (Chase et al., 1997). A total of four species of Palinuridae (Palinurus elephas Fabricius, 1787, Palinurus mauritanicus Gruvel, 1911, Panulirus echinatus Smith, 1869 and Panulirus regius de Brito Capello, 1864) and four species of Scyllaridae (Scyllarides latus Latreille, 1803, Scyllarus arctus Linnaeus, 1758, Scyllarus posteli Forest, 1963 and Scyllarus pygmaeus doi:10.1093/plankt/fbn012, available online at www.plankt.oxfordjournals.org # The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Bate, 1888) occur in Mediterranean or adjacent eastern Atlantic waters (García-Raso, 1982; Holthuis, 1991). The complete larval development is only known for P. regius (Crosnier, 1971) and P. elephas (Bouvier, 1914; Santucci, 1926). The first phyllosoma stage, obtained from eggs hatched in the laboratory, is known for P. mauritanicus (Palero and Abelló, 2007), and a short description of the first phyllosoma stage of S. pygmaeus was provided by Mura and Pessani (1994). The eighth phyllosoma stage of P. echinatus was recently identified through molecular techniques and morphologically described (Konishi et al., 2006). However, no definitive identification of the later stages of any of the Scyllaridae species has been published yet. Nevertheless, the correct identification of phyllosoma larvae in plankton samples is essential for the study and comparison of the spatiotemporal distributions, population dynamics and reproductive strategies of the different species (Gonzalez- Gordillo et al., 2001). The phyllosoma larvae of the Scyllarinae are the most abundant larvae of the Achelata in coastal waters (Phillips et al., 1981; Webber and Booth, 2001), so a proper description of the Scyllarus larvae will facilitate ecological research in the future. Accurate identification of larvae and post-larvae usually requires rearing (Ito and Lucas, 1990) but molecular techniques can also be applied to the identification of larvae when a complete sampling of adults from every candidate species has been done. The development of the molecular phylogeny of Achelata lobsters from Mediterranean and eastern Atlantic waters will provide highly valuable species-specific markers for the correct identification of phyllosoma larvae collected from the plankton (Smith et al., 2001). Several final-stage phyllosoma larvae were collected in the western Mediterranean Sea and were provisionally identified as belonging to the family Scyllaridae and the genus Scyllarus. Detailed morphological observations, available descriptions and a thorough literature review indicated that the larvae would probably belong to S. arctus, but molecular analysis uncovered the real identity of the larvae. In this paper, we describe the morphology of the final phyllosoma stage of S. pygmaeus, and confirm the identification through molecular analysis using nuclear markers. This constitutes the first molecular identification of a phyllosoma stage for a species of the family Scyllaridae. METHOD Three final-stage phyllosoma larvae identified as belonging to the genus Scyllarus were caught by demersal trawling during fishery research surveys in the Table I: Stations where final-stage phyllosoma larvae of S. pygmaeus were found Accession code Station Date Latitude Longitude ICMD-65/ 2007 ICMD-66/ 2007 ICMD-67/ 2007 Depth (m) M02L59 5/26/2002 38804.49 N 0800.48 W 601 650 M04L48 5/16/2004 38806.08 N 0810.30 W 201 250 M04L68 5/20/2004 39833.98 N 0815.29 E 251 300 western Mediterranean in May 2002 and May 2004 (Table I). Each individual was preserved in 100% ethanol. DNA information was also obtained from adults of different Achelata species found in Mediterranean waters (S. arctus, S. posteli, S. pygmaeus, S. latus, P. elephas and P. mauritanicus). Nephrops norvegicus Linnaeus, 1758 (Decapoda: Astacidea: Nephropidae) and Polycheles typhlops Heller, 1862 (Decapoda: Eryonidea: Polychelidae) were used as outgroups. Total genomic DNA extraction was performed using the QIAamp DNA Mini Kit (QIAGEN Inc). A region of 450 500 bp of the 28S gene was amplified using newly developed universal primers for decapod crustaceans (NuriA 5 0 -GGTAAGCAGAACTGGCG CTGTGGG -35 0 ; NuriB 5 0 - GGGATCAGGCTTT CGCCTTGGG -35 0 ). Amplification was carried out with 30 ng of genomic DNA in a reaction containing 1 U of Taq polymerase (Amersham), 1 buffer (Amersham), 0.2 mm of each primer and 0.12 mm dntps. The PCR thermal profile used was 948C for 4 min for initial denaturation, followed by 30 cycles of 948C for 30 s, 508C for30s, 728C for 30 s and a final extension at 728C for4min. Amplified PCR products were purified with QIA-Quick PCR Purification Kit (QIAGEN Inc) prior to direct sequencing of the product. The sequences were obtained using the Big-Dye Ready-Reaction kit v3.1 (Applied Biosystems) on an ABI Prism 3770 automated sequencer from the Scientific and Technical Services of the University of Barcelona. A neighbour-joining (NJ) phylogenetic tree, based on Kimura s two-parameter model (K2P) and the maximum parsimony tree (MP) and associated bootstrap support values were obtained using MEGA version 3.1 (Kumar et al., 2004). A binocular microscope equipped with an ocular micrometer was used for dissections and measurements of individuals (all three phyllosomas were measured). A microscope was used for the determination of the setation and measurements of some appendages. The following measurements were taken: total length (TL) from the anterior margin of the cephalic shield between 484

F. PALERO ET AL. j MORPHOLOGY OF THE FINAL STAGE PHYLLOSOMA LARVA OF S. PYGMAEUS the eyes to the posterior margin of the telson; cephalic length (CL) from the anterior to the posterior margin of the cephalic shield; cephalic width (CW) measured as the widest part of the cephalic shield; thorax width (TW) measured as the widest part of the thorax; eye length (EL) from the base of the eyestalk to the tip of the eyes; antennular length (A1L) from the insertion point to the tip of the inner ramus; total antennal length (A2L) from the insertion point to the tip of inner ramus; abdomen length (AbdL) from the anterior margin of the abdomen to the posterior margin of the telson. The larvae are described using the basic malacostracan somite plan from anterior to posterior and appendage segments are described from proximal to distal, endopod then exopod (Clark et al., 1998). The three studied specimens of S. pygmaeus final phyllosoma stage have been deposited in the Biological Collections of Reference of the Institut de Ciències del Mar (CSIC) in Barcelona under accession code (Code: ICMD-65/2007, ICMD-66/2007 and ICMD-67/2007). RESULTS DNA analysis The length of the aligned data set for the 28S rdna gene was 415bp. The sequences have been deposited in GenBank with Accession numbers EU449504- EU449514. The 28S rdna data from the larvae studied were analysed together with those obtained in phylogenetic study on Achelata lobsters still in process (F. Palero, PhD thesis in progress). Since the tree topologies obtained by NJ and MP were in agreement, only the NJ tree is shown (Fig. 2). The phylogenetic tree clearly showed the actual identity of the final-stage phyllosoma larvae, with the clade formed by the phyllosoma specimens studied and the S. pygmaeus adult specimens providing a 100 bootstrap support. The distance (K2P) between larval samples and adult S. pygmaeus (0.000 + 0.000) was much smaller than those between the larvae and either S. arctus (0.014 + 0.006) or S. posteli (0.019 + 0.007). Therefore, we can conclude that the phyllosoma specimens belong to S. pygmaeus. A1L ¼ 0.39 + 0.02 cm; A2L ¼ 0.40 + 0.02 cm; TW ¼ 0.87 + 0.05 cm; AbdL ¼ 0.86 + 0.09 cm. Cephalic shield (Figs 1 and 3a): Subrectangular, 1.3 times wider than long and two times wider than thorax; eye longer than antennule and antenna. Antennule (Fig. 3a and b): Biramous, penduncle 3-segmented; inner ramus unsegmented with 2 setae, slightly longer than outer; outer ramus unsegmented with 13 14 rows of sensory setae. Antenna (Fig. 3a): Biramous, unsegmented and unarmed; similar in length to antennule. Mandibles (Fig. 4a): Asymetrical in dentition; incisor process and medial gnathal edge with a series of teeth and setae; molar process crowned by many denticules and papillae. Maxillule (Fig. 4b): Uniramous. Coxal endite with 10 setae; basial endite with 9 setae; palp (endopod) absent. Maxilla (Figs 3a and 4c): Endites and endopod not differentiated, with two proximal setae; scaphognathite without setae, flattened and considerably expanded to anterior and posterior. First maxilliped (Figs 3a and 4d): Unsegmented and unarmed; bilobed rudimentary bud. Morphological description Scyllarus pygmaeus (Bate, 1888): phyllosoma, final stage Dimensions: TL ¼ 2.52 + 0.12 cm; CL ¼ 1.44 + 0.06 cm; CW ¼ 1.91 + 0.09 cm; EL ¼ 0.52 + 0.02 cm; Fig. 1. Scyllarus pygmaeus. Last phyllosoma in dorsal view. Scale bar 5 mm. 485

Fig. 2. NJ phylogenetic tree based on K2P distances estimated from the nuclear 28S rdna sequence dataset, showing the position of the three phyllosoma specimens genetically analysed (M2L59, M4L48 and M4L68). All sequence data were obtained for this study. SARC: S. arctus, SPOS: S. posteli, SPYG: S. pygmaeus, SLAT: S. latus, PELE: P. elephas, PMAU: P. mauritanicus, NNOR: N. norvegicus, PTYP: P. typhlops. Fig. 3. Scyllarus pygmaeus. Phyllosoma. a, Ventral surface; b, antennule, distal part; b f, dactylus of pereiopods 1 4; g, left side of thorax, dorsal view. Scale bar ¼ 5 mm. Second maxilliped (Fig. 3a and 4e): Five-segmented, with 0,1,2,10,6 setae; with rudimentary and unarmed exopod bud. Third maxilliped (Fig. 3a): Six-segmented, exopod absent; with ventral coxal spine; distal part of propodus and dactylus densely setose. Pereiopods (Fig. 3a and c f): Pereiopods 1 4 biramous, with subexopodal spine, endopod four-segmented with two distal spines on isquio-merus and one distal spine on carpus, exopods flagellated distally with 24 23, 25 26, 22 23, 20 19 pairs of plumose setae respectively; pereiopod 5 uniramous, 5-segmented, not reaching Fig. 4. Scyllarus pygmaeus. Phyllosoma. a, Mandibles; b, maxillule; c, maxilla; d, first maxilliped; e, second maxilliped; d, telson, dorsla view. Scale bar of a d ¼ 0.5 mm, c ¼ 1 mm and d ¼ 2 mm. posterior margin of telson, with ventral coxal spine and one distal minute spine on isquio-merus. Thorax (Figs 1 and 3a and g): Dorsal thoracic spines present above pereiopods 1 4. Gills (Fig. 3g): Full complement of gill buds present: third maxilliped and pereiopod 1 with one pleurobranch, one arthrobranch and two podobranchs; pereiopods 2 4 with two pleurobranchs, one arthrobranch, two podobranchs; pereiopod 5 with one pleurobranch. Pleon (Figs 1, 3a and 4d): Segmented, with 6 somites; somites 2 5 with a pair of pleopods; pleopods biramous, 486

F. PALERO ET AL. j MORPHOLOGY OF THE FINAL STAGE PHYLLOSOMA LARVA OF S. PYGMAEUS unsegmented and unarmed; biramous uropods not outreaching posterior margin of telson; telson rounded posteriorly with strong postero-lateral processes. DISCUSSION The phyllosoma larvae studied in the present work are stage-10 larvae, with the presence of a complete set of gills, according to the 10-stage categories defined by Webber and Booth (2001). The key characteristics, useful for diagnosis, of the final stage phyllosoma larva of S. pygmaeus concern the shape of the cephalic shield, much wider than it is long, the absence of a rostrum, antennulae about the same length as the antennae, the absence of exopodite on the third maxilliped, the presence of strong dorsal thoracic spines and the long telson spines. The phyllosoma stages of Mediterranean and northeastern Atlantic Scyllarus species have been the focus of many studies since the early XIXth century, well before the phyllosoma was recognised as the larval phase of spiny and slipper lobsters (Risso, 1827; Richters, 1873). Nevertheless, the identities of these Scyllarus species remained uncertain until recently (Lindley et al., 2004). The overlapping distributions of the different species of Scyllarus made it difficult to assign larvae in plankton samples to a particular species, and therefore, specific identifications of larvae were taken from previous works with no further confirmation. Most of the research on the phyllosoma of S. arctus took Stephensen s (1923) work as a reference, and it was assumed that every Scyllarus phyllosoma found in the Mediterranean belonged to S. arctus. Furthermore, none of the larval stages of S. pygmaeus had been described with certainty, except for the short text description of the first phyllosoma stage by Mura and Pessani (1994). The identification through molecular techniques of the presently studied larvae, together with a thorough literature review has allowed us to describe in detail the phyllosoma of S. pygmaeus and to compare it with other Scyllarus phyllosomae described and/or illustrated in the literature. It is clear now that several species were mixed in the larval series attributed by Stephensen (1923) to S. arctus, and that both stages VIII and IX in his work are in fact phyllosomae of S. pygmaeus. Thus, what were previously thought to be phyllosoma stages of S. arctus belong in fact to S. pygmaeus. The final stage phyllosoma larvae described in this study showed a striking similarity with reported phyllosoma larvae collected from very distant localities. Morphologically similar larvae have been found in the Juan Fernandez Islands (Johnson, 1971), Indian Ocean (Prasad et al., 1975), W and SE Australia (Phillips et al., 1981) and Hawaii (Johnson, 1977). However, the phyllosoma obtained from Mediterranean waters differ from all those larvae by the absence of the exopod bud on the 3rd maxilliped, and by the telson fork being much more accentuated. The present work also provides further insight into the ecology and distribution of S. pygmaeus. Bathymetric distribution for S. pygmaeus is generally accepted to mainly cover the continental shelf, from 5 to 162 m (Holthuis, 1991; Mura and Pessani, 1994). However, Bouvier (1940) reported the occurrence of S. pygmaeus by the Travailleur Expedition in the Bay of Biscay at 1200-m depth and by the Talisman Expedition in Cape Verde islands at 398-m depth. The collection of some of these final stage phyllosoma larvae in waters deeper than 600 m may support Bouvier s findings and indicate that S. pygmaeus populations are also found in deeper waters. However, the ecology and behaviour of phyllosomae is far from being understood and it could well be that the occurrence of late stage larvae in relatively deep waters is actually a behavioural characteristic of these larvae and that the recruitment, performed by the nisto stage, takes mainly place in shallow rocky or maerl areas which seem to be the main habitat of the species. It is worth noting that the S. pygmaeus larvae are also the most commonly collected scyllarid larvae in Mediterranean and adjacent Atlantic waters (Stephensen, 1923; personal observations). This could also indicate that S. pygmaeus populations are larger than previously expected, or that the ecology and behaviour of the phyllosomae of S. pygmaeus and S. arctus are really different and those of S. arctus are difficult to sample with standard plankton or demersal trawl devices. This study has shown that molecular analysis coupled with morphological investigations may significantly contribute to refine species identification in planktonic larvae. The molecular markers developed in this study will help to accurately identify the phyllosoma larvae of S. pygmaeus. Work is in progress to develop these markers for every known species of Achelata lobster from Mediterranean and adjacent Atlantic waters. Finally, the definitive identification of the S. pygmaeus larvae also points out the fact that little is known about the ecology of both slipper lobster adults and larvae, and opens the door for new research on the life history of the members of the Scyllaridae family. ACKNOWLEDGEMENTS We wish to thank Dr Fátima Hernández, who kindly allowed us to study the phyllosoma collections at the 487

Museo de Ciencias Naturales in Tenerife; Dr Paul Clark for receiving us at the Natural History Museum in London and Dr Angelika Brandt for receiving us at the Zoologisches Institut und Zoologisches Museum of the University of Hamburg. Dr Lindley encouraged the completion of this study. We also wish to thank all participants in the MEDITS_ES 2002 and 2004 cruises on board the R/V Cornide de Saavedra, from which the studied larvae were collected. Drs Marta Pascual (University of Barcelona) and Enrique Macpherson (CSIC) provided helpful advice and guidance. FUNDING This work was supported by a pre-doctoral fellowship awarded by the Autonomous Government of Catalonia to F P (2006FIC-00082). Research was funded by project CGL2006-13423 from the Ministerio de Educación y Ciencia. The authors are part of the research groups 2005SGR-00191 and 2005SGR-00995 of the Generalitat de Catalunya. REFERENCES Bouvier, E. 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