Taxonomic revision of the Mediterranean Plakina Schulze (Porifera, Demospongiae, Homoscleromorpha)

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1 Zoological Journal of the Linnean Society (1998), 124: With 16 figures Article ID: zj Taxonomic revision of the Mediterranean Plakina Schulze (Porifera, Demospongiae, Homoscleromorpha) GUILHERME MURICY 1,2, NICOLE BOURY-ESNAULT 2, CHANTAL BÉZAC 2 AND JEAN VACELET 2 1 Depto. de Zoologia, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Cidade Universitaria, CCS, bloco A, Rio de Janeiro, RJ, Brazil 2 Centre d Océanologie de Marseille, Université de la Méditerranée, UA-CNRS 41, Station Marine d Endoume, rue de la Batterie des Lions, Marseille, France Received July 1996; accepted for publication April 1997 Only three species of the sponge genus Plakina Schulze have been described from the Mediterranean since 1880, in spite of a large amount of allegedly intraspecific variability in morphological characters. However, recent genetic studies based on electrophoretic techniques have revealed extensive cryptic speciation in north-western Mediterranean Plakina, demonstrating that most of this variation was interspecific rather than intraspecific. We describe in detail the morphology and anatomy of four new species of Plakina from the Mediterranean P. crypta, P. weinbergi, P. endoumensis and P. jani of which the latter two were discovered through allozyme electrophoresis. Plakina monolopha Schulze and P. trilopha Schulze are redescribed, and their morphological and geographical limits are discussed along with those of P. dilopha Schulze. Accurate analysis of the internal anatomy and of the shape and ramification pattern of lophose spicules in scanning electron microscopy provides new, powerful morphological criteria for species discrimination in Plakina. More widespread use of such new taxonomic characters should provide evidence against the alleged cosmopolitanism of some Plakina species, thus generating an increase in estimates of the biodiversity of plakinids The Linnean Society of London ADDITIONAL KEY WORDS: sponges Plakinidae systematics morphology spicules scanning electron microscopy. CONTENTS Introduction Abbreviations Material and methods Correspondence to Dr G. Muricy. Present address: Depto. de Invertebrados, Museu Nacional, Universidade Federal do Rio de Janeiro. Quinta da Boa Vista, s/n o,são Cristóvão, Rio de Janeiro, R.J, Brazil. muricy@acd.ufrj.br /98/ $30.00/ The Linnean Society of London

2 170 G. MURICY ET AL. Results Plakina monolopha Schulze Plakina dilopha Schulze Plakina crypta sp. nov Plakina endoumensis sp. nov Plakina trilopha Schulze Plakina jani sp. nov Plakina weinbergi sp. nov Discussion Key to the Mediterranean species of Plakina Acknowledgements References INTRODUCTION The cosmopolitan sponge genus Plakina Schulze, 1880 is considered to contain only eight species, characterized by the presence of lophose calthrops (Diaz & van Soest, 1994). These are modifications of tetractinal spicules (calthrops) in which one, two, three or all four actines ramify in two to five rays, sometimes ramifying again close to the extremities. Schulze (1880) originally described three species from the Adriatic, distinguished by the presence of spicules with one, two or three ramified (= lophose) actines: respectively, P. monolopha, P. dilopha and P. trilopha. So far, no other Plakina species have been described from the Mediterranean. Despite the five other species described from other regions [from Azores P. brachylopha Topsent, 1927; from the Caribbean coast of Panama P. elisa (de Laubenfels, 1936); from Madagascar P. corticioides Vacelet, Vasseur & Lévi, 1976; from New Caledonia P. corticolopha Lévi, 1983; from Japan P. tetralopha Tanita & Hoshino, 1989], most records of the genus worldwide are of the allegedly cosmopolitan species P. monolopha and P. trilopha (e.g. de Laubenfels, 1951; Bergquist, 1968; Thomas, 1970; Vacelet & Vasseur, 1971; Lévi& Lévi, 1989). In most studies in which polymorphic or allegedly cosmopolitan sponge species have been analysed with modern taxonomic methods (e.g. estimation of genetic divergence through allozyme electrophoresis), at least two species emerged from a complex previously known under a unique specific name (Solé-Cava & Thorpe, 1986; Hooper et al., 1990; Solé-Cava et al., 1991; Boury-Esnault, Solé-Cava & Thorpe, 1992). Reinterpretations of species boundaries may also be necessary when more accurate information is obtained from morphological traits (e.g. spicule shape) through the scanning electron microscope (SEM). This may highlight subtle differences in spicular morphology, which can sometimes suggest an homoplastic origin for spicules previously interpreted as homologous (Fromont & Bergquist, 1990; Boury-Esnault, Pansini & Uriz, 1993). The present study arose from the discovery of several different morphotypes of Plakina during a survey for plakinids in caves and vertical walls on the coast of Provence, France (Western Mediterranean). Four of these morphotypes were analysed through allozyme electrophoresis, which subsequently revealed that they were genetically divergent and not conspecific (Muricy et al., 1996a). Genetic divergence in this species group was closely correlated with variation in cytological data (Muricy, 1996). This paper describes the morphology and anatomy of four new Mediterranean species of Plakina, two of which were discovered through allozyme electrophoresis.

3 MEDITERRANEAN PLAKINA 171 Endoume Marseille Cassis Jarre * Riou 2 km Grand Congloue Mediterranean Sea * La Ciotat 3PP Cave N 5 35 E 30 E Mediterranean Sea Cyprus 35 N Figure 1. Map of the Mediterranean Sea showing the sites of collection of Plakina species: (Β) P. monolopha; ( ) P. trilopha; (Ε) P. jani; (Α) P. endoumensis; ( ) P. crypta; ( ) P. weinbergi. The two allegedly cosmopolitan species, P. monolopha and P. trilopha, are redescribed and a holotype and a neotype were respectively designated for them. An analysis of morphological, anatomical and especially of spicular characteristics using SEM is made, in order to evaluate the relative importance of the different characters for species discrimination in the genus Plakina. ABBREVIATIONS bc basal cavity; cc choanocyte chamber; e embryo; ex exhalant canal; in inhalant canal; os oscule; sc subectosomal cavity. MATERIAL AND METHODS Samples were collected by SCUBA from August 1990 to July 1995 in different localities along the Mediterranean coast of France and in Cyprus (Fig. 1). Colour

4 172 G. MURICY ET AL. codes refer to Kornerup & Wanscher (1978). Specimens were fixed in formaldehyde or in glutaraldehyde as previously described (Boury-Esnault et al., 1984): glutaraldehyde 2.5% in a mixture of 0.4 M cacodylate buffer and sea water (4 vol.: 5 vol.; 1120 mosm) and postfixed in 2% osmium tetroxide in sea water. For light microscopy (LM) the specimens were embedded in Araldite. Semi-thin sections were stained with toluidine blue. The ratio between the volume occupied by the mesohyl and that occupied by choanocyte chambers (called here proportion of mesohyl to chambers ) was determined by linear integration in semi-thin sections observed in LM (Nourtier, 1971). Measurements were taken only in the choanosome. The volume occupied by pinacocytes, ectosome and canals was excluded from calculations, but choanocytes were included in chamber volume. Spicule dimensions given are: length minimum-average-maximum/thickness minimum-maximum (number of spicules measured). Micrographs of clean dehydrated spicules sputter-coated with gold-palladium were taken through a Hitachi S570 scanning electron microscope. The patterns of ramification of lophose actines as seen in SEM micrographs were briefly described using a simple alphanumerical code, which takes into account the number of rounds of ramification in lophose actines (1 or 2), their location along the actine ( p for proximal, m for medial, d for distal), and the presence of terminal spines (ts). In this system, the ramification pattern of a spicule with only one round of ramification close to the base of the lophose actines and bearing tiny terminal spines at their extremities is coded lp, ts. A spicule with one round of ramification at the middle of the length of the actine, a second round of ramification close to the extremity of the secondary ray, and bearing no terminal spines is coded lm, 2d, and so on. Specimens were deposited in the sponge collections of the Muséum National d Histoire Naturelle, Paris, France (MNHN); British Museum of Natural History, London, UK (BMNH); Station Marine d Endoume, Marseille, France (SME); and Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (UFRJPOR). RESULTS Phylum Porifera Grant, 1836 Class Demospongiae Sollas, 1885 Subclass Homoscleromorpha Lévi, 1973 Diagnosis (from Muricy et al., 1996b). Demospongiae viviparous with typical cinctoblastula larvae, and both exo-and endopinacocytes flagellated. A basement membrane-like collagen layer lines both pinacoderm and choanoderm. Skeleton, when present, formed by a combination of relatively small calthrops and/or derivatives (diods and triods), generally arranged uniformly in the sponge body, usually surrounding the aquiferous system in an irregular reticulation. Choanocyte chambers are spherical, μm in diameter, usually eurypylous, occasionally aphodal or diplodal. A system of large, basal exhalant cavities is often present. Order Homosclerophorida Dendy, 1905 Diagnosis as for the subclass. Family Plakinidae Schulze, 1880 Diagnosis as for the subclass.

5 MEDITERRANEAN PLAKINA 173 os cc ex e bc Figure 2. Schematic drawing of a section through Plakina monolopha, showing the general organization of the skeleton and aquiferous system. Plakina crypta and P. endoumensis share the same anatomy, but have also dilophose (P. crypta), trilophose and tetralophose calthrops (P. endoumensis). Spicule density has been reduced for clarity. Scale bar =100 μm (lobes and oscule are out of scale). bc, basal cavity; cc, choanocyte chamber; e, embryo; ex, exhalant canal; os, oscule. Genus Plakina Schulze, 1880 Plakoosa de Laubenfels, 1936 Type-species: Plakina monolopha Schulze, 1880 Diagnosis (emended after Diaz & van Soest, 1994). Thinly to massively encrusting Plakinidae with a spiculation of diods, triods, and calthrops, and with lophocalthrops with one, two, three or four lophate rays. Lophocalthrops are usually concentrated at the sponge surface. Development of ectosome is variable, and subectosomal cavities may be present. Proportion of mesohyl to chambers varies between 0.7 and 1.8:1. Choanocyte chambers are eurypylous or aphodal, usually with a radial arrangement around incurrent and excurrent canals. Plakina monolopha Schulze, 1880 (Figs 2 4, Appendix 1) Material examined Holotype. BMNH , in spirit, Naples, Italy. Other material. UFRJPOR 4350 (slides C.47, C.48), 4351 (slides C.49, C.50), 4352 (slide C.51), Grand Congloue Island, Marseille, France, N, 5 20 E, 15 m deep, coll. G. Muricy, Diagnosis (Schulze, 1880). Thin, ornate crusts, irregularly round or lobate, sometimes broken by rounded holes. Colour white or rose. Surface finely rugose, bordered by a ring canal with the external borders short and smooth, upon which protrude one or more oscular tubes. The basal portion, thin and flat, turn up at the borders making a hollow ring that surrounds the upper portion of the body, deep and strongly rugose, which contains the flagellated chambers. Exo- and endopinacocytes

6 174 G. MURICY ET AL. C D A A A B B C D Figure 3. Camera lucida drawings of Plakina monolopha spicules. A, diods; B, triods, C, calthrops, D, monolophose calthrops. Scale bar =13 μm (A C) or 6 μm (D). Figure 4. Spicules of Plakina monolopha (SEM). A, diod and triod; B, monolophose calthrops (upper view); C, monolophose calthrops (side view). Scale bar =17 μm (A), 5 μm (B), or 6 μm (C). are flagellated. Candelabra have only one ramified actine [monolophose calthrops], perpendicular to the surface of the sponge, directed outwards. Reproductive period: summer and fall. Hermaphroditic. Larvae are vibrating, rose, uniform, ciliated, and possess the posterior pole small, dark, with a short region of small flagella. Type locality: Trieste, Lesina and Naples. (translated from German) Description. Specimens from the coast of Provence are very similar to the original description and to the holotype. They are small, thin crusts up to 2 2 cm wide by 1 5 mm thick. Shape is discoidal or irregular, with smooth, elevated borders forming a ring canal around the sponge body. Body is attached to the substratum only by thin filaments. Surface is microlobate, with rugose, irregular lobes mm in

7 MEDITERRANEAN PLAKINA 175 diameter and in height, which may fuse together. Colour in vivo and in spirit is white or cream. Consistency is soft, fragile. General organization (Fig. 2). Ectosome is reduced to a μm thick layer of mesohyl, indistinct from the choanosomal mesohyl and without subectosomal cavities. The spaces between lobes form open inter-digitations μm wide; inhalant canals are absent. Ostia lead almost directly to the chambers. The aquiferous system is sylleibid-like, with spherical, eurypylous choanocyte chambers μm in diameter. Exhalant canals lead to a system of basal cavities and then to the oscula, located at the borders of the sponge. Proportion of mesohyl to choanocyte chambers varies from 0.5:1 to 1.2:1 (mean 0.8:1). Skeleton (Fig. 2). The skeleton is organized in close relationship with the aquiferous system. Diods, triods and calthrops are arranged in a dense, confuse reticulation around the choanocyte chambers, canals and basal cavity. Lophose calthrops are concentrated at the surface of the sponge, most with their lophose actines pointing outward, but a few are dispersed in the choanosome or line canals and basal cavities. Lophose and non-ramified spicules may form a sort of dense, unorganized palisade at the surface, μm thick. Spicules (Figs 3, 4). Diods (Figs 3A, 4A), common, slender, irregular, sinuous, with actines gradually pointing to sharp ends. Actines often irregularly spined, with variable number and size of spines. The central swelling shows the s-bent shape characteristic of the subclass but may also be centrotylote or almost smooth: / μm (n =26). Triods (Figs 3B, 4A), abundant, often with one or two ill-developed or malformed actines. The central swelling, actine size and angle between actines vary widely. Actines / μm (n =21). Calthrops (Fig. 3C), rare, same general shape and variations, one actine usually reduced to a small button. Actines / μm (n =19). Monolophose calthrops (Figs 3D, 4B, C), common, irregular, lophose actine ramifies once at the middle of its length in two to six slender, cylindrical rays, which usually show two or three tiny terminal spines (a pattern of ramification coded 1 m, ts). Total length: / μm (n =16). Reproduction. The specimens studied, collected in August, incubated hollow cinctoblastula larvae typical of the Homoscleromorpha (Lévi & Porte, 1962; Boury-Esnault et al., 1995). Larvae are 120 μm in diameter, pink or red in vivo. The species is reported to reproduce in summer and fall. Habitat. In this study, P. monolopha was found associated to sciaphilic algae in vertical rocky substrate, m deep, at Grand Congloue Island, Marseille (Fig. 1). It has been reported from sciaphilic environments such as submarine caves and undersides of boulders, from the intertidal up to 540 m deep (Appendix 2). Distribution. Mediterranean. The species is reported to be cosmopolitan, but most records from outside the Mediterranean were probably misidentified (see Appendix 2). Remarks. Schulze (1880) designated no holotype for this species. In accordance with articles 73 and 74 of the International Code of Zoological Nomenclature (ICZN Ride et al., 1985), the only specimen found from the original series studied by

8 176 G. MURICY ET AL. Schulze is designated as the holotype (BMNH , from Naples). Plakina monolopha shares with P. endoumensis and P. crypta a simple anatomy with folded surface and reduced mesohyl, a sylleibid-like aquiferous system with eurypylous choanocyte chambers, a ring canal at the borders of the body, and the absence of subectosomal cavities. These species have small, discoidal bodies fixed on the substrate only by thin filaments, a character also shared by P. brachylopha from the Azores (Topsent, 1927). P. monolopha differs from all other Plakina species by the possession of monolophose calthrops with a simple ramification pattern (1 m, ts) as the only lophose spicules. Plakina dilopha Schulze, 1880 Diagnosis (Schulze, 1880). White crusts irregularly rounded and shiny, whose surface, just broken by rounded holes of varying sizes, has no hollow ring at the borders. The oscular tubes do not protrude at the borders. The simple basal portion, thin and flat, has no major turns on the sides of the voluminous body. The flagellated chambers are contained in the principal part of the body. Candelabra possess two ramified actines [dilophose calthrops], diagonally inclined outwards, directed against the surface. Reproductive period: fall. Larvae are vibrating, rose, ciliated, oval and elongated, almost ellipsoidal. In the dark, small posterior pole, there is a round zone of pigmented grains, and a short region of small flagella. Type locality: Trieste. (Translated from German) Distribution. NW Mediterranean: Trieste (Schulze, 1880), Naples (Balduzzi et al., 1989), Marseille (Pouliquen, 1972), Bandol (Topsent, 1895). According to Diaz and van Soest (1994), the records from the Caribbean (Topsent, 1937; Pulitzer-Finali, 1986) probably belong to P. elisa (de Laubenfels, 1936). Remarks. No specimens could be found for reexamination of this rare species, despite extensive search both in the principal sponge collections in American and European museums and along the coast of Provence, France (NW Mediterranean). Both Schulze (1880) and Topsent (1895) described P. dilopha as having a smooth surface, no ring canal at the borders, and no subectosomal cavities. According to Schulze s (1880) original description and drawings, the species is characterized by the possession of dilophose calthrops as the only ramified spicule type. The pattern of ramification of lophose actines appears to be a simple one, with only one, medial ramification point and sometimes also terminal spines (1 m, ts). This ramification pattern is a distinctive feature between P. dilopha and P. elisa from the Caribbean (de Laubenfels, 1936), which upon reexamination of the holotype (USNM 22237) showed a more complex ramification pattern (1 m, 2d, ts). Plakina crypta sp. nov. (Figs 2, 5, 6, Appendix 1) Material examined Holotype. MNHN D.NBE.1467, in spirit, 3PP Cave, La Ciotat, France, N, 5 35 E, 22 m deep, coll. J. Vacelet,

9 MEDITERRANEAN PLAKINA 177 Figure 5. Plakina crypta sp. nov. A, living specimen in situ (colour white; photo J. Vacelet); B, monolophose calthrops (SEM); C, dilophose calthrops (SEM). Scale bar =0.5 cm (A) or 5 μm (B, C). Paratypes. UFRJPOR T-14, , T-15, T-16, in spirit, 3PP Cave, La Ciotat, France, N, 5 35 E, 22 m deep, coll. J. Vacelet, Description (Fig. 5A). Thinly encrusting, discoidal sponge, 1 2 cm 2 by 1 5 mm thick, morphologically similar to P. monolopha. It is loosely attached to the substrate by thin filaments extending from its basal surface. The basal region of the sponge is made by a thin, delicate and transparent membrane covering a system of basal cavities, which is elevated at the borders of the sponge body forming a hollow exhalant ring canal. The upper surface is microlobate, with many 1 4 mm wide and high lobes, separated by irregular depressions. Lobes often anastomose in an irregular reticulation. The surface of lobes is irregular, slightly rugose, with dispersed inhalant openings. One or two irregularly round oscula, 2 mm in diameter, are located over the elevated borders of the sponge, and are surrounded by a delicate, transparent oscular rim. Colour in vivo is white, cream in spirit. Consistency is soft, fragile.

10 178 G. MURICY ET AL. B A A A B B D C D E Figure 6. Camera lucida drawings of the spicules of Plakina crypta sp. nov. A, diods; B, triods; C, calthrops; D, monolophose calthrops; E, dilophose calthrops. Scale bar =12 μm (A C) or 6 μm (D, E). General organization (Fig. 2). Ectosome is thin, unspecialized, reduced to a 5 10 μm thick layer of mesohyl between the outermost chambers and the exopinacoderm. The aquiferous system is organized as in P. monolopha. There are no subectosomal cavities, and the eurypylous chambers are μm in diameter. Proportion of mesohyl to choanocyte chambers varies from 0.4:1 to 1.2:1 (mean 0.7:1). Skeleton (Fig. 2). The skeleton shows the typical organization of the genus, as described for P. monolopha, with lophocalthrops concentrated at the surface. Spicules (Figs 5B, C, 6). Diods (Fig. 6A), abundant, irregular, straight or sinuous, with actines smooth or rugose, rarely spined, and with sharp ends. Central swelling is S-bent, tuberose, or almost smooth: / μm (n =22). Triods (Fig. 6B), abundant, same general shape and variations than diods, often with one or two ill-developed or malformed actines. The central swelling, actine size and angle between actines vary widely. Actines: /1.5 3 μm (n = 16). Calthrops (Fig. 6C), common, with one actine usually reduced to a small button. Actines: / μm (n =21). Monolophose calthrops (Figs 5B, 6D), common, uniform, with an unique pattern

11 MEDITERRANEAN PLAKINA 179 of ramification: lophose actines ramify close to the distal end in 3 5 short rays that ramify once again distally in usually three smaller rays, which often show 2 4 terminal spines (ramification pattern 1d, 2d, ts). Total length / μm (n =15). Dilophose calthrops (Figs 5C, 6E), very rare, same shape and pattern of ramification. Total length / μm (n =10). Lophose spicules are quite uniform in shape and pattern of ramification, but vary in the number of ramifications in different lophose actines of the same spicule. Reproduction. Specimens collected in September 1992 and July 1994 incubated typical cinctoblastula larvae, about 150 μm in diameter. Habitat and distribution. This species has been collected so far only at obscure zones in 3PP Cave (La Ciotat, France), where a moderately dense population colonizes the walls and ceiling of the cave m from its entrance, 22 m deep. Etymology. The species name is derived from its cryptic habitat. Remarks. Plakina crypta shares with P. monolopha and P. endoumensis a simple anatomy with folded surface, reduced mesohyl, sylleibid-like aquiferous system with eurypylous choanocyte chambers, a ring canal at the borders of the body, no subectosomal cavities, and a small discoidal body fixed on the substrate only by thin filaments. Plakina crypta differs from P. monolopha by having larger lobes, a more complex pattern of ramification of lophose calthrops (1d, 2d, ts Appendix 1; Fig. 5B, C), and by the presence of dilophose calthrops. It can be distinguished from P. endoumensis by the shape of its spicules as well as by the absence of both trilophose and tetralophose calthrops. In contrast to P. dilopha, P. crypta has a microlobate surface, monolophose calthrops, and a more complex ramification pattern of lophocalthrops. P. crypta differs from all other Mediterranean Plakina species by its external morphology, spicular composition and by the ramification pattern of its lophose spicules. Plakina endoumensis sp. nov. (Figs 2, 7, 8, Appendix 1) Material examined Holotype. MNHN D.NBE.1465, in spirit, Endoume Cave, Marseille, France, N, 5 23 E, 3 m deep, coll. G. Muricy, Paratypes. UFRJPOR T-18, T-19, all in spirit, Endoume Cave, Marseille, France, N, 5 23 E, 3 m deep, coll. G. Muricy, Other material. UFRJPOR 4353 (slide C.14), 4354 (slide C.15), 4355 (slide C.20), 4356 (slide C.23), Endoume Cave, Marseille, France, N, 5 23 E, 3 5 m deep, coll. G. Muricy, Description. Small, thinly encrusting sponge, which covers surfaces of 1 4 cm 2 and is 2 5 mm thick. Its morphology is similar to that of P. monolopha and P. crypta. Shape is discoidal or irregular, with smooth, elevated borders forming a ring canal. It is loosely attached to the substratum by thin filaments. Lobes are regular, very small, mm wide and high, giving a corrugated appearance to the surface. Inhalant openings, μm in diameter, are randomly dispersed. One to three oscula, cm in diameter, are located at the borders of the sponge, and are surrounded

12 180 G. MURICY ET AL. C B A A B D D E G F F Figure 7. Camera lucida drawings of the spicules of Plakina endoumensis sp. nov. A, diods; B, triods; C, calthrops; D, monolophose calthrops; E, dilophose calthrops; F, trilophose calthrops; G, tetralophose calthrops. Scale bar =11 μm (A C), 7 μm (D, E), or 5 μm (F, G). by a transparent oscular rim. Colour in vivo is white and in spirit cream. Consistency is delicate, fragile. General organization (Fig. 2). The internal anatomy follows the same pattern of P. monolopha and P. crypta, with thin ectosome, eurypylous chambers μm in diameter, and without inhalant canals or subectosomal cavities. Proportion of mesohyl to choanocyle chambers varies from 0.6:1 to 1.5:1 (mean 0.9:1). Skeleton. The skeleton shows the typical organization of the genus, as described for P. monolopha.

13 MEDITERRANEAN PLAKINA 181 Figure 8. SEM micrographs of Plakina endoumensis sp. nov. spicules. A, diod, triods and calthrops; B, simple and monolophose calthrops; C, dilophose calthrops; D, E, trilophose calthrops; F, triod and tetralophose calthrops; G, tetralophose calthrops. Scale bar =24 μm (A), 11 μm (B, F), 6 μm (C, E, G), or 7.5 μm (D). Spicules (Figs 7, 8). Diods (Figs 7A, 8A) abundant, regular, straight or slightly curved, smooth, with sharp ends. Central swelling is S-bent or tuberose. Actines: / μm (n =31).

14 182 G. MURICY ET AL. Triods (Figs 7B, 8A, F), abundant, same general shape of diods. Actines have a thick basis, and become gradually thinner towards the extremity. Actine size and angle between actines are very regular. Each actine: / μm (n =33). Calthrops (Figs 7C, 8A, B), common, smooth, same general shape of diods and triods. Actines: / μm (n =28). Monolophose calthrops (Figs 7D, 8B), rare, regular, lophose actine ramifying in three or four secondary rays. Total length / μm (n =8). Dilophose calthrops (Figs 7E, 8C), abundant, uniform in shape, lophose actines ramifying into two to four points. Total length / μm (n = 28). Trilophose calthrops (Figs 7F, 8D, E), common, 2 5 ramifications by lophose actine. The smooth actine can be simple or bifurcated. Total length / μm (n =25). Tetralophose calthrops (Figs 7G, 8F, G), rare, 2 5 ramifications by actine. Total length / μm (n =11). Intermediates between categories and malformations are rare. Lophose actines ramify close to the base in 2 5 conical rays which usually bear 2 4 small terminal spines at the extremity (ramification pattern 1p, ts). The number of ramifications often varies in different actines of the same spicule. Reproduction. All specimens examined, collected in August 1993, incubated typical cinctoblastula larvae, μm in diameter. Habitat and distribution. This species is known only from the walls and ceiling of Endoume Cave (Marseille), in semi-obscure zones, at depths of 2 5 m. Etymology. The species name comes from the only site where it has been collected, Endoume Cave (Marseille). Remarks. Plakina endoumensis shares the small discoidal habit and simple internal anatomy with P. monolopha and P. crypta, from which it differs by the simultaneous presence of mono-, di-, tri- and tetralophose calthrops. It differs from all other Mediterranean Plakina by its external morphology, the particularly stout and uniform general shape of its spicules and the pattern of ramification of its lophose calthrops. Plakina trilopha Schulze, 1880 (Figs 9 11, Appendix 1) Material examined Neotype. MNHN D.NBE.1468, , 3PP Cave, La Ciotat, France, N, 5 35 E, 15 m deep, coll. N. Boury-Esnault. Other material. UFRJPOR 4335, T-03, , coll. G. Muricy, , , coll. J. Vacelet, all in spirit, 3PP Cave, La Ciotat, France, 15 m deep. UFRJPOR 4332, 4333, 4337, 4360 (slide B.79), 4361 (slide B.81), 4362 (slide B.95), , T.05, , 4363 (slide B.88), , T.04, 4357 (slides B.76, B.91), 4358 (slides B.89, B.92), 4359 (slides B.93, B.94), , Jarre Cave, Marseille, 15 m, coll. G. Muricy (see also Appendix 3).

15 MEDITERRANEAN PLAKINA 183 Figure 9. Plakina trilopha. A, in situ close-up of a living specimen (colour white or pinkish-white; photo J. Vacelet); B F, SEM micrographs of spicules. B, C, trilophose calthrops; D, E, tetralophose calthrops (E ill-formed). Scale bar =1 cm (A), 5 μm (B, D, E), or 6 μm (C). Diagnosis (Schulze, 1880). Irregular crusts, which in alcohol appear yellowish white, without ring canal at the borders. The basal part possesses a system of lacunae and no flagellated chambers. In the free surface of the sponge there is a cortex impregnated with pores and free of flagellated chambers, but with subdermal lacunae. Candelabra have at least three ramified actines [trilophose calthrops], obliquely directed to the surface. Hermaphroditic. Type locality: Naples. (translated from German) Description (Fig. 9A). Thinly encrusting, lobate sponge, which covers surfaces up to 200 cm 2 and is 2 10 mm thick. It is firmly attached to the substratum, and has smooth, irregular borders. Lobes are irregular, 5 10 mm long and thick, and appear as low undulations in the surface. The surface of the lobes is smooth, perforated by closely spaced inhalant openings, μm in diameter, arranged in a microscopically alveolar pattern. A few oscules, cm in diameter, are located at the apex of some lobes, and are surrounded by a transparent oscular rim. Colour in vivo is white or slightly pinkish-white, and in spirit whitish-yellow to cream. Consistency is firm but compressible.

16 184 G. MURICY ET AL. sc in cc ex bc Figure 10. Schematic drawing of the internal anatomy of Plakina trilopha and P. jani. Scale bar =80 μm (spicules are out of scale). Abbreviations: bc, basal cavity; cc, choanocyte chamber; e, embryo; ex, exhalant canal; in, inhalant canal; sc, subectosomal cavity. General organization (Fig. 10). Ectosome is μm thick, well-differentiated, separated from the choanosome by inhalant subectosomal cavities, μm in diameter. Ostia open in inhalant canals or subectosomal cavities. The organization of the aquiferous system is transitional between sylleibid and leuconoid types. Choanocyte chambers are eurypylous or aphodal, roughly spherical, μm in diameter. Exhalant canals lead to a system of large exhalant basal cavities, which communicate with the oscula. Proportion of mesohyl to choanocyte chambers varies from 1:1 to 2:1 (mean 1.5:1). Skeleton (Fig. 10). The skeleton shows the organization typical of the genus, as described for P. monolopha, with lophose calthrops concentrated at the surface of the sponge, with their ramified actines pointing outwards. Spicules (Figs 9B F, 11). Diods (Fig. 11A), abundant, irregular, straight or sinuous, with smooth or rugose actines which become thinner gradually. The central swelling is variable: S-bent shape, tuberose or almost smooth: / μm (n =25). Triods (Fig. 11B), abundant, same general shape and variations than diods, often with one or two ill-developed or malformed actines. The central swelling, actine size and angle between actines vary widely. Actines: / μm (n =24). Calthrops (Fig. 11C), common to rare, same general shape and variations. Actines: / μm (n =19). Monolophose calthrops (Fig. 11D), rare, irregular, lophose actine with two to six points. Total length / μm (n =11). Dilophose calthrops (Fig. 11E), rare, irregular. Total length / μm (n =8). Trilophose calthrops (Figs 9B, C, 11F), abundant, with variable shape. The

17 MEDITERRANEAN PLAKINA 185 C A B A A A B C D G E F F H Figure 11. Camera lucida drawings of Plakina trilopha spicules. A, diods; B, triods; C, calthrops; D, monolophose calthrops; E, dilophose calthrops; F, trilophose calthrops; G, tetralophose calthrops; H, ill-formed spicule. Scale bar =12 μm (A C) or 7 μm (D H). smooth actine can be simple or bifurcated. Total length / μm (n =25). Tetralophose calthrops (Figs 9D E, 11G), common, variable shape. Total length / μm (n =18). Spicules are irregular, variable, with many intermediates and malformations (Figs 9E, 11H). Lophose actines divide once medially or at about two-thirds of their length in two or three slightly conical rays, which may terminate in blunt ends or bear one to four terminal spines (pattern of ramification 1 md, ts Appendix 1). Reproduction. This species incubates hollow cinctoblastula larvae typical of the subclass. It is reported to reproduce in summer and fall. Embryos were found in specimens collected from June to August. Habitat. This is a moderately sciophilous species, found in this survey only in semiobscure zones of caves, 5 to 20 m from the entrance of 3PP Cave, La Ciotat (Fig. 1), at depths of m, and m from the entrance at Jarre Cave, Marseille (Fig. 1), 15 m deep. It is usually found on the walls and ceilings of the caves, or in epibiosis on other sponges. It has been reported from the undersides of rocks and

18 186 G. MURICY ET AL. boulders at the shallow sublittoral to 920 m, but it is doubtful whether the deeper records have been correctly identified (Appendix 3). Distribution. Plakina trilopha is reported to be cosmopolitan, but most records outside the Mediterranean are doubtful (Appendix 3). Remarks. We were unable to find any specimens from the original series of this species, despite an extensive search in several important sponge collections. In accordance with article 75 of the ICZN (Ride et al., 1985), and to reduce the present confusion about the identification and distribution of this species, we have designated a neotype from the Marseille area (see Material examined ), chosen because of very close similarity to the original description and drawings of the species (Schulze, 1880). The concept of this species should be restricted to such Mediterrancan populations until additional extra-limital material should prove otherwise. Plakina trilopha is most closely related to P. jani sp. nov., from which it differs by its white colour, by the general spicule shape, which are rarely spiny, and by the less complex pattern of ramification of its lophose calthrops, in which lophose actines often have only one round of ramification. It differs from all other Mediterranean Plakina by its anatomy and spicule shape. Plakina jani sp. nov. (Figs 10, 12, 13, Appendix 1.) Material examined Holotype. MNHN D.NBE.1464, , 3PP Cave, La Ciotat, France, N, 5 35 E, 16 m deep, coll. J. Vacelet. Paratypes. UFRJPOR T-01, T-02, in spirit, 3PP Cave, La Ciotat, France, 15 m deep, coll. G. Muricy, Other material. UFRJPOR 4368 (B.36 54), , 4365 (slide B.29), , , 4388, 4389, coll. J. Vacelet, 4330, 4331, 4364 (slide B.27), 4366 (slide B.30), 4367 (slide B.33), , coll. G. Muricy, 3PP Cave, 15 m deep, La Ciotat, France. Description (Fig. 12A). Thinly to thickly encrusting, lobate sponge, which covers surfaces up to 600 cm 2 and is 5 20 mm thick. The general appearance, consistency, distribution of oscula and ostia are quite similar to those of P. trilopha. Lobes are however more compressed onto each other, thus giving a more irregular appearance to the surface. Colour in vivo is yellow to yellowish-brown, becoming white, whitishyellow or light brown in spirit. Consistency is firm but compressible. General organization (Fig. 10). The aquiferous system is transitional between sylleibid and leuconoid types, and is organized as in P. trilopha. The ectosome is μm thick, and subectosomal cavities are μm in diameter. Choanocyte chambers are eurypylous or aphodal, roughly spherical, μm in diameter. Exhalant canals lead to large basal exhalant cavities. Proportion of mesohyl to choanocyte chambers varies from 1:1 to 2:1 (mean 1.6:1). Skeleton (Fig. 10). The skeleton shows the typical arrangement of the genus, as described for P. monolopha, with lophocalthrops concentrated at the surface of the sponge.

19 MEDITERRANEAN PLAKINA 187 Figure 12. Plakina jani sp. nov. A, in situ close-up of a living specimen (colour yellow, yellowishbrown; photo J. Vacelet); B H, SEM micrographs of spicules. B, diods, triods and calthrops; C, D, simple trilophose calthrops; E, bifurcated trilophose calthrops; F, tetralophose calthrops; G, trilophose triod (malformation?); H, ill-formed spicules. Scale bar =1.3 cm (A), 27 μm (B), 7.5 μm (C, D), 6 μm (E, F, G), or 5 μm (H).

20 188 G. MURICY ET AL. B C A A A A B D E F F G G H H Figure 13. Camera lucida drawings of spicules of Plakina jani sp. nov. A, diods; B, triods; C, calthrops; D, monolophose calthrops; E, dilophose calthrops; F, trilophose calthrops; G, tetralophose calthrops; H, ill-formed spicules. Scale bar =12 μm (A C) or 8 μm (D H). Spicules (Figs 12B H, 13). Diods (Figs. 12B, 13A), abundant, irregular, sinuous, with actines gradually pointing to sharp ends. Actines often irregularly acanthose, with variable number and size of spines. The central swelling is typically s-bent, but the spicules may be centrotylote or almost smooth: / μ (n = 53). Triods (Figs 12B, 13B), abundant, same general shape and variations than diods, often with one or two ill-developed or malformed actines. The central swelling, actine size, and angle between actines vary widely. Actines: / μ (n =54). Calthrops (Figs. 12B, 13C), common, same general shape and variations, often with one actine reduced to a small button. Actines: / μ (n = 39).

21 MEDITERRANEAN PLAKINA 189 Monolophose calthrops (Fig. 13D), rare, irregular, lophose actine with two to six points. Total length / μ (n =13). Dilophose calthrops (Fig. 13E), rare, irregular. Total length / μm (n =9). Trilophose calthrops (Figs 12C E, 13F), common, with ramification pattern variable. The smooth actine can be simple, bifurcated or trifurcated; in the latter case, the spicule could be also interpreted as tetralophose calthrops. Total length / μm (n =43). Tetralophose calthrops (Figs 12F, 13G), common, often ill-formed. Total length / μm (n =29). All spicule types are irregular and variable in shape. Intermediates between categories (e.g. trilophose triods Fig. 12G) and ill-formed spicules (Figs 12H, 13H) are abundant. The pattern of ramification of lophocalthrops is distinctive: lophose actines have a relatively constant thickness, and ramify medially in 2 5 rays, which often ramify again distally in 2 3 short secondary rays. In SEM, secondary rays frequently show 2 4 tiny spines at the extremity. This pattern of ramification is coded 1 m, 2 d, ts. Any of the points of ramification may be lacking in some actines of the same spicule, making up complex forms which are difficult to ascribe to traditional categories. Reproduction. Embryos were found in specimens collected in June and August. This species incubates hollow cinctoblastula larvae typical of the subclass, μm in diameter. Habitat and distribution. This is a moderately sciophilous species, found only in a semiobscure zone, 5 20 m from the entrance of 3PP Cave (La Ciotat, Fig. 1), at depths of m. It is usually found on the walls and ceiling of the cave, but sometimes it grows in epibiosis over other sponges. Etymology. The species name comes from Janus, the guardian of portals in Roman mythology, in reference to the only habitat it has been found so far, close to the entrance of 3PP Cave. Remarks. P. jani shares with P. trilopha an undulated, smooth surface, abundant ostia arranged in an alveolar pattern, dispersed elevated oscula, well-developed mesohyl, and an aquiferous system transitional between sylleibid and leuconoid types, with inhalant subectosomal cavities. Eurypylous and aphodal choanocyte chambers may be seen in the same specimen. Both species are irregularly spreading, attached to the substrate through their whole bases. Morphologically, P. jani differs from P. trilopha by its yellow colour, the general spicule shape, more often spiny, and by the more complex pattern of ramification of its lophose calthrops, in which lophose actines often have two rounds of ramification. It differs from all other Mediterranean Plakina by its colour, large spreading habit, and spicule shape. The ramification pattern of its trilophose calthrops (1 m, 2 d, ts) is shared with P. corticioides from the Indian Ocean (Vacelet et al., 1976), which however has also peculiar monolophose calthrops with short, conical actines ramifying in the proximal portion (a pattern coded 1p ).

22 190 G. MURICY ET AL. Plakina weinbergi sp. nov. (Figs 14 16, Appendix 1) Material examined Holotype. MNHN D.NBE.1466, in spirit, Agios Georgios Island, Cape Arnaoutis, Cyprus, N, E, 9 m deep, coll. S. Weinberg, Slides and a fragment of the holotype have been deposited in the sponge collection of the Universidade Federal do Rio de Janeiro, Brazil (UFRJPOR T-06). Description (Fig. 14A). Thickly encrusting to massive, lobate sponge, which covers a surface area of up to 1000 cm 2 and is 5 20 mm thick. It is firmly attached to the substrate, and has long lobes, 3 5 cm long by 5 10 mm in diameter, pendant from the ceiling of the cave. Surface is smooth, with irregular depressions and abundant, dispersed ostia. Oscula are round, 5 mm in diameter, elevated at the top or at the sides of the lobes. Colour in vivo and in spirit is cream. Consistency is cartilaginous. General organization (Fig. 15). Ectosome is μm thick, separated from the choanosome by irregular inhalant sub-ectosomal cavities, μm wide. A large (up to 1 mm in diameter), round, central exhalant canal traverses the choanosome of each lobe. Basal cavities could not be observed in the somewhat damaged specimen studied, but a zone devoid of choanocyte chambers and with irregular lacunae μm wide surrounds the central canal. Aquiferous system is leuconoid, and choanocyte chambers are spherical or ovoid, aphodal, μm in diameter. Proportion of mesohyl to choanocyte chambers is 1.8:1. Skeleton (Fig. 15). Lophose spicules of all kinds are haphazardly dispersed throughout the mesohyl. Diods, triods and calthrops are rare, and the reticulation around choanocyte chambers typical of the genus is absent. The spiculation is denser in the ectosome, forming an unorganized palisade μm thick. Spicules (Figs 14B H, 16). Diods (Figs 14B, 16A), contorted, irregular, smooth or spined, often dilophose, smooth forms rare. Total length: / μm (n =11). Triods (Figs 14C, 16B), contorted, irregular, often with basal spines or trilophose, common to rare. Actines: / μm (n =17). Calthrops (Figs 14B, C, 16C), contorted, irregular, often with actines biphid, rare. Actines: / μm (n =13). Monolophose calthrops (Figs 14D, 16D), common, irregular, basally spined, 3 5 ramifications by actine. Total length: / μ (n =14). Dilophose calthrops (Figs 14E, 16E), common, irregular, spined, 2 4 ramifications by actine. Total length / μm (n =11). Trilophose calthrops (Figs 14C, F, 16F), abundant, irregular, spined, 2 5 ramifications by actine. Basal (non-ramified) actine is sometimes bifurcated. Total length / μm (n =17). Tetralophose calthrops (Figs 14H, 16G), abundant, irregular, smooth or spined, 2 5 ramifications by actine. Total length / μm (n =16). Intermediates between categories, such as dilophose diods μm long (Fig. 16H), and trilophose triods μm long (Fig. 16I), are common, as well as tetralophose pentactines μm long (Fig. 14G). Lophose actines usually ramify at the middle of their lengths in 2 5 conical rays, which terminate in blunt ends.

23 MEDITERRANEAN PLAKINA 191 Figure 14. Plakina weinbergi sp. nov. A, panoramic view of the population in a cave at Agios Georgios Island (colour cream; photo S. Weinberg); B H, SEM micrographs of spicules. B, diod and calthrops with three biphid actines; C, triod, calthrops and trilophose calthrops; D, monolophose calthrops; E, dilophose calthrops; F, trilophose calthrops; G, tetralophose pentactine (?); H, tetralophose calthrops. Scale bar =5 cm (A), 17 μm (B, C), 8.5 μm (D, E), 6 μm (F, G), or 7.5 μm (H). Lophose actines of mono- and dilophose calthrops ramify only distally in 2 4 short rays which also have blunt ends. This ramification pattern is coded 1 m/1 d (first ramification medial or distal, without terminal spines).

24 192 G. MURICY ET AL. in ex Figure 15. Schematic drawing of a transverse section through a lobe of Plakina weinbergi sp. nov., showing the arrangement of the skeleton and the leuconoid aquiferous system. Abbreviations: cc, choanocyte chamber; ex, exhalant canal; in, inhalant canal; sc, subectosomal cavity. Scale bar = 120 μm (choanocyte chambers are out of scale). Reproduction. The specimen studied, collected in August 1990, showed no reproductive elements. Habitat and distribution. This species inhabits a cave in Agios Georgios Island, Cape Arnaoutis, Cyprus. It is found pendant from the ceiling of the cave like a stalactite, about 9 10 m deep. Etymology. The species name refers to its collector, Dr Steven Weinberg, who kindly sent us a specimen and in situ photographs of the living population in Agios Georgios Island. Remarks. Plakina weinbergi is atypical of the genus. It is relatively massive, its long lobes have a large central exhalant canal, and the mesohyl is well developed with a leuconoid aquiferous system and aphodal choanocyte chambers. Apart from its peculiar habit and anatomy, it does not show the reticulation of diods, triods and calthrops around choanocyte chambers typically found in Plakina species. Its spiculation is characterized by the extreme rarity of diods, triods, and calthrops, and by the great variability of lophose spicules. There is a continuum of variation from almost non-ramified to complex tetralophose calthrops, with different degrees of spination and ramification. The rarity of non-ramified spicules is shared with P. corticioides and P. corticolopha from New Caledonia (Vacelet et al., 1976; Lévi & Lévi, 1983), from which P. weinbergi differs greatly by the shape of its lophose spicules. DISCUSSION Our results show that the biodiversity of the genus Plakina in the Mediterranean has been greatly underestimated, as was that of the closely-related genus Oscarella (Boury-Esnault et al., 1992; Muricy et al., 1996b). Most species are, however,

25 MEDITERRANEAN PLAKINA 193 B C A A B C E D D E F F G G H I Figure 16. Camera lucida drawings of spicules of Plakina weinbergi sp. nov. A, diods; B, triods; C, calthrops; D, monolophose calthrops; E, dilophose calthrops; F, trilophose calthrops; G, tetralophose calthrops; H, dilophose diod; I, trilophose triod. Scale bar =11 μm. morphologically similar sibling species which can be differentiated only after careful examination of morphological and anatomical characters, whenever possible in connection with biochemical or cytological analyses. External morphology coupled with anatomical characters (degree of ectosome development, arrangement of the aquiferous system) can be used to define subgroups in Mediterranean Plakina. A

26 194 G. MURICY ET AL. simple anatomy with folded surface, reduced mesohyl, a sylleibid-like aquiferous system with eurypylous choanocyte chambers and no subectosomal cavities, is shared by P. monolopha, P. endoumensis and P. crypta. These species have small, discoidal bodies fixed on the substrate only by thin filaments, a character also shared by P. brachylopha from the Azores (Topsent, 1927). The borders of the body are elevated, with extensions of the basal cavities (called ring canal by Schulze, 1880), and bear the elevated oscula. A second group composed of P. jani and P. trilopha shares an undulated, smooth surface, abundant ostia arranged in an alveolar pattern, dispersed elevated oscula, well-developed mesohyl, and an aquiferous system transitional between sylleibid and leuconoid, with inhalant subectosomal cavities. Eurypylous and aphodal choanocyte chambers may be seen in the same specimen. Both species are irregularly spreading, attached to the substrate through their whole bases. According to Schulze (1880) and Topsent (1895), P. dilopha shows an intermediate ectosomal thickness, a smooth surface, no ring canal at the borders, and subectosomal cavities are absent, appearing to be an intermediary between these two groups. Unfortunately no specimens could be found for reexamination to confirm this observation. Finally, Plakina weinbergi is relatively massive, its long lobes have a large central exhalant canal, and the mesohyl is well developed with a leuconoid aquiferous system and aphodal choanocyte chambers. It is difficult to allocate other species of the genus within these subgroups without reexamination of relevant type specimens, as most published descriptions do not provide sufficient detail on anatomical characters to differentiate closely related sibling species. Plakina jani, P. trilopha and P. endoumensis have been previously subjected to allozyme electrophoresis to ascertain their specific status (morphotypes A, B and C, respectively, in Muricy et al., 1996a). Plakina jani and P. trilopha are sibling species, but sympatric populations of the two species have a genetic identity (Nei s 1) of 0.83, as measured by allozyme electrophoresis (Muricy et al., 1996a). In addition, two diagnostic loci differentiate the two populations. This is considered strong evidence for divergence at the species level (Thorpe, 1982, 1983; Nei, 1987). Plakina endoumensis showed greater differences in morphological characters, in agreement with the lower genetic identities to both P. jani and P. trilopha (Nei s 1=0.49 and 0.54, respectively Muricy et al., 1996a). It shares the small discoidal habit and simple internal anatomy with P. monolopha and P. crypta, from which it differs by the simultaneous presence of mono-, di-, tri-, and tetralophose calthrops. Since Schulze (1880), the taxonomy of the genus Plakina has been based mostly on the possession of particular combinations of spicule types by different species. Spicule types have been defined by the number of actines (diods, triods, and calthrops), and by the number of ramified (lophose) actines in calthrops (mono-, di-, tri-, and tetralophose calthrops). Usually no reference is made to the precise shape of the actines or to their ramification patterns. Analysis of these characters in light microscopy and particularly in SEM micrographs may greatly add to the combination of spicule types to aid in discrimination of sibling species of Plakina. Also, most species diagnoses seem to point to a linear evolution of mono-, di-, tri-, and tetralophose calthrops replacing each other in the spicule complement of different species (as in the series P. monolopha P. dilopha P. trilopha P. tetralopha). Our study shows instead that many different forms of mono-, di-, tri-, and tetralophose calthrops exist, not simply replacing each other in a linear series, but co-occurring extensively in the same individuals. We have obtained relevant taxonomic information from the following parameters