Description of the First Caribbean Oscarellidae (Porifera: Homoscleromorpha)

Zootaxa 4369 (4): 501–514 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2018 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4369.4.3 http://zoobank.org/urn:lsid:zoobank.org:pub:A216E1AB-8A63-4E42-A525-5B161F8B5D00 Description of the first Caribbean Oscarellidae (Porifera: Homoscleromorpha)

THIERRY PEREZ1,2 & CESAR RUIZ1 1Institut Méditerranéen de Biodiversité et d’Ecologie marine et continentale, CNRS, Aix Marseille Univ, IRD, Avignon Univ. Station Marine d’Endoume, chemin de la Batterie des Lions, 13007 Marseille, France 2E-mail: [email protected]

Abstract

The sponge class Homoscleromorpha has a challenging taxonomy and its systematics is still a matter of debate. A significant effort has recently been deployed to better evaluate the diversity of these sponges, and each new exploration of cryptic habitats reveals new species. Although several undescribed or wrongly determined Oscarella-like sponges have been reported by different authors, the Oscarellidae family still lacks description of its true Caribbean representatives. The exploration of various submarine caves in the Lesser Antilles has allowed us to find and to formally describe the first two Oscarellidae of the Caribbean Sea, Oscarella filipoi sp. nov. and Oscarella zoranja sp. nov. Both new species are quite common in semi-dark habitats throughout the Lesser Antilles. Moreover, O. filipoi sp. nov. is the largest Oscarellidae ever observed world-wide. Both species harbor a sylleibid aquiferous system and a high density of ovoid to spherical choanocyte chambers. The molecular taxonomy clearly shows their affiliation to the Oscarella clade containing the type species of the Oscarellidae family. In addition to their growth forms, these two species differ in the shape of their mesohylar cells, and in the degree of development of their basal region. This latter trait is particularly remarkable in large specimens of O. filipoi which can have a very thick ectosome.

Key words: Sponges, Homosclerophorida, Oscarella, Lesser Antilles, Submarine caves

Introduction

Sponge diversity is underestimated in a great number of the islands of the Lesser Antilles. A recent inventory performed in Martinique has begun to complete this knowledge gap, highlighting a reservoir of hidden sponge diversity dwelling in cryptic habitats such as crevices, overhangs, tunnels and submarine caves (Pérez et al. 2017). The knowledge gain was evident for Homoscleromorpha, with 11 species added after this inventory, including several new species. This sponge group was recently elevated to the rank of class (Gazave et al. 2012), and in spite of its challenging taxonomy, it presents one of the highest rates of new taxa descriptions (Cárdenas et al. 2012). The Caribbean Sea today counts 18 Homoscleromorpha species, but they currently belong to the Plakinidae family (Van Soest et al. 2017; Ruiz et al. 2017). Some of these species have been described recently, such as Oscarella nathaliae, which was the only Caribbean representative of the Oscarellidae family for three years (Ereskovsky, Lavrov & Willenz 2014; Ruiz et al. 2017). Indeed, one of the main motivations of Ereskovsky et al. (2014) in attempting to assign this new species to the genus Oscarella was its lack of a skeleton. However, later molecular evidence and the re-examination of certain morphological traits demonstrated that this species actually belongs to Plakina (Ruiz et al. 2017). The Plakinidae definition has been further overturned following the description of the new aspiculate sponge Aspiculophora madinina by Ruiz, Muricy, Lage, Domingos, Chenesseau & Pérez (2017). Consequently, the lack of a skeleton is no longer a specific characteristic of the Oscarellidae, and this family remains with no properly described Caribbean representatives. All former reports of Oscarella specimens referred to undescribed specimens or to wrongly named O. lobularis (Schmidt, 1862) (e.g. Vacelet 1998; Diaz & Van Soest 1994) which has long been thought to be cosmopolitan. Several research projects are currently underway to improve our knowledge of Caribbean sponge fauna, with particular attention paid to underwater cave communities similar to those in other seas where Homoscleromorpha

Accepted by M. Klautau: 22 Nov. 2017; published: 8 Jan. 2018 501 were known to be an abundant and diverse taxonomic group (Ereskovsky et al. 2009; Gerovasileiou & Voultsiadou 2012). Every recently explored cave in the Lesser Antilles has revealed a huge diversity of this sponge Class (Fauvelot 2015; Pérez 2015). Descriptions have used an integrative taxonomy approach on the most common representatives (see Ruiz et al. 2017). Hereafter, we describe two new sympatric Oscarella species which were originally collected in Martinique Island and later in various islands of the Lesser Antilles. They are among the most common Homoscleromorpha species and were found in every semi-dark community investigated. One of these is the largest Oscarella ever recorded world-wide.

Material and methods

Sampling. Specimens were collected by SCUBA diving between June 2011 and April 2016 in the South of Martinique Island and during the PACOTILLES cruise across the Lesser Antilles, from Anguilla to Mayreau Island (May–June 2015) (Fig. 1). Some testimonies accompanied by underwater pictures were also used to delineate the distribution of the new species, but these records were not confirmed by samples.

FIGURE 1. Sampling sites, current distribution of the two new Caribbean Oscarellidae ; black stars : Oscarella filipoi sp. nov. ; grey stars: Oscarella zoranja sp. nov.

The two new species live in sympatry. All specimens were found on vertical walls, under horizontal rocky surfaces or on the ceilings of submarine caves varying in depth from very shallow (3 m) to 50 m depth.. Underwater photographs and general information, such as the size, shape, color and consistency, were registered for each individual. Each sample was fixed in 95% ethanol and in 2.5% glutaraldehyde for molecular and morphological analyses, respectively. Prior to further analysis, the absence of spicules was verified for each specimen.

502 · Zootaxa 4369 (4) © 2018 Magnolia Press PEREZ & RUIZ Cytology. A small fragment of each specimen was fixed in 2.5% glutaraldehyde in 2 M phosphate buffer and filtered sea water (1 vol: 4 vol: 5 vol), then post-fixed in 2% OsO4 in sea water (Boury-Esnault et al.1984). For semi-thin and ultra-thin sections, each fragment was embedded in AralditeTM. Semi-thin sections were stained with toluidine blue and observed under a Leica DMBL light microscope (LM). Ultra-thin sections were made using a RCMC ultramicrotome PTXL. The cuts were placed on a copper grid (3.05 mm in diameter, 300 mesh) and stained with 2% uranyl acetate for 15 min. Observations were carried out with a JEOL JEM-1400 transmission electron microscope (TEM). The cytological and prokaryotic compositions of each sample were analyzed, taking into account cell morphology, dimensions and cytoplasm characteristics.

TABLE 1. NCBI data base accession numbers of specimens used for the molecular taxonomy. Specie Sample code Accession number Aspiculophora madinina KU674367.1 Plakinastrella onkodes EU237487 Plakortis halichondroides HQ269359.1 Plakortis angulospiculatus Plakina jani NC_014860.1 Corticium candelabrum HQ269363.1 Oscarella zoranja 131203MT3CR2 MG009495 Oscarella zoranja GR27HOM16 MG009496 Oscarella zoranja 150516MT8CR4 MG009497 Oscarella zoranja GR26HOM4 MG009498 Oscarella zoranja GR34HOM29 MG009499 Oscarella zoranja GR30HOM25 MG009500 Oscarella zoranja 110611MT4TP12 MG009501 Oscarella zoranja 110611MT3TP5 KX348266 Oscarella zoranja 150516MT4CR13 MG009502 Oscarella zoranja 150516MT1TP7 KX348267 Oscarella zoranja 150516MT4CR8 MG009503 Oscarella zoranja GR26HOM1 MG009504 Oscarella zoranja GR27HOM20 MG009506 Oscarella filipoi 131203MT3CR1 MG009507 Oscarella filipoi 120325MT1TP14 KX348268 Oscarella filipoi 120325MT4TP13 MG009508 Oscarella filipoi 120325MT1TP12 MG009509 Oscarella tuberculata 3170974238 Oscarella lobularis 317134132 Oscarella balibaloi Pseudocorticium jarrei HQ269357.1 Xestospongia muta EU237490.1

DNA analyses. For the molecular taxonomy study, we chose CO1 markers which had proved reliable in differentiating closely related species among Homoscleromorpha in previous works (see for instance Pérez et al. 2011; Boury-Esnault et al. 2013, Ruiz et al. 2015, 2017). DNA extractions were performed from small sponge fragments (2 cm3) using QIAamp DNA Mini Kit (QIAGEN). Universal primers C1-Npor2760 and C1-J2165 were used to amplify a 507-bp fragment (I3-M11) of the CO1 mitochondrial gene (Misof et al. 2000; Erpenbeck et al. 2006). Amplifications were done in a 40 µl total reaction volume with: 4 µl of each primer (10 µM), 6.4 µl dNTPs

(10 mM), 8 µl polymerase buffer, 5 µl MgCl2 (25 mM), 0.2 µl Taq polymerase (5 U*µl-1), 2.4 µl extracted DNA

DESCRIPTION OF THE FIRST CARIBBEAN OSCARELLIDAE Zootaxa 4369 (4) © 2018 Magnolia Press · 503 and 14 10 µl of ultrapure (Milli-Q) water. PCR were performed using a Mastercycler gradient PCR-S Eppendorf thermocycler with an initial step of 5 min at 94° C followed by 40 amplification cycles (denaturation at 94° C for 1 min; annealing at 42° C for 1 min; and extension at 72° C for 1 min), and a final extension step at 72° C for 5 min. PCR products were directly sequenced in each primer direction by the Eurofins laboratory (Ebersberg, Germany). To construct the phylogenetic tree, another four sequences were taken from our previous works and others were downloaded from GenBank (Table 1). Sequences were aligned using BIOEDIT 7.0.5.3 (Hall 1999). Phylogenetic trees were constructed using both the neighbor-joining (NJ) method (1000 bootstrap replicates) with CLUSTAL X 2.0 (Larkin et al. 2007) and the maximum likelihood method (ML), under the GTR + I + G model (previously tested using Jmodel test software; Darriba et al. 2012) with a nonparametric bootstrap resampling of 100 replicates using PhyML algorithms (Guindon et al. 2010). The Demospongiae Xestospongia muta (Schmidt, 1870) was used as an out-group.

Results

Class Homoscleromorpha Bergquist, 1978

Order Homosclerophorida Dendy, 1905

Family Oscarellidae Lendenfeld, 1887

Genus Oscarella Vosmaer, 1884

Type species: Halisarca lobularis Schmidt, 1862 (by monotypy). (Oscaria) Vosmaer, 1881: 163 (preocc. by Oscaria Gray, 1873—Reptilia); Oscarella Vosmaer, 1884: pl. 8 (explanation); 1887: 326 (nom. nov. for Oscaria Vosmaer). Octavella Tuzet and Paris, 1963: 88. Diagnosis: Homoscleromorphorida without skeleton, with a variable development of the ectosome. The aquiferous system has a sylleibid-like organization, with spherical to ovoid eurypylous or diplodal choanocyte chambers arranged around large exhalant canals (Muricy & Diaz 2002).

Oscarella filipoi sp. nov.

Holotype: Muséum National d’Histoire Naturelle de Paris, MNHN—DJV193: Martinique, Le Diamant, Caribbean Sea. Site «Tunnel du Diamant » (14°26.556' N, 61°2.408' W), 10 m depth, on a vertical wall of the tunnel, date 11/ 06/2011. Sample code: 120325-MT1TP14 collector T. Pérez (Fig. 2). GenBank accession number : KX3482268. Paratype 1: Muséum National d’Histoire Naturelle de Paris, MNHN—DJV194: Martinique, Le Diamant, Caribbean Sea. Site « Grotte Fer à Cheval » (14°27.882' N, 61°01.162 W), 17 m depth, overgrowing an octocoral in the cave entrance, date 25/03/2012. Sample code: 120325-MT4bTP01, collector T. Pérez. Paratype 2: Muséum National d’Histoire Naturelle de Paris, MNHN—DJV195: Saint Martin, Caribbean Sea. Site « Rocher Créole » (18°07.038' N, '063°03.419' W), 10 m, under an overhang, date 26/05/2015. Sample code: 150526-SN3CR08 collector C. Ruiz. Other material examined: Martinique, 131203-MT3CR1, Grotte Couleur at 8 m depth, Anses d'Arlet, collector C. Ruiz ; 120325-MT1TP12, 120325-MT1TP13, 120325-MT1TP14, Tunnel du Diamant at 15 m depth, collector T. Pérez. This material is kept in the collection of the Station Marine d’Endoume (Marseille—France). Etymology: the new species is dedicated to Philippe Thélamon, a resourceful diver of Anses d’Arlet. Known by his nickname “Filipo”, he is deeply involved in nature conservation and the promotion of Martinique’s marine biodiversity. Filipo was the first to take us to the type locality of the new species. Diagnosis. The largest Oscarella recorded world-wide, covering up to 1m², inhabiting shaded habitats. Color from purple to yellow and light green, with the largest specimens always purple, thick, with a pale-green base. Large specimens are often associated to tiny hydroids. Thick ectosome, especially in the basal part, is mostly devoid of cells. Ovoid to spherical eurypylous choanocyte chambers. Only one type of vacuolar cell is abundant and is grouped in clusters in the mesohyl.

504 · Zootaxa 4369 (4) © 2018 Magnolia Press PEREZ & RUIZ Description. External morphology: An encrusting sponge with irregular clathrate surface and small lobes. Size up to 1 m² and 2–3 cm thick. Different color morphotypes have been observed: the most common is deep purple, but light green and light orange were also observed in natural conditions (Fig. 2). The surface is mostly colored while the basal part is pale or green in large specimens, turning green in all cases when put in alcohol. The consistency is soft, fragile and easy to tear. The surface presents small lobes that converge to oscula of about 1–4 cm diameter. Large and heavy specimens tend to partially detach from the substrate, often hanging from the ceiling of semi-dark caves and tunnels.

FIGURE 2. In situ pictures of Oscarella filipoi. A) O. filipoi is the biggest Oscarella species ever recorded, surface covered reaching 1m² in certain cases. B) Green chromotype, very rare. C) Orange chromotype, rare. D) Detailed view of a common tiny cerianthid associated with O. filipoi.

Soft tissue organization: No spicule or fiber skeleton. The ectosome is 20 to 50 µm in the apical zone, but can be considerably thicker than 100 µm in the basal part (Fig. 3A). Inhalant canals, 13 to 22 µm wide, often run perpendicular to the surface. The exhalant canals run towards a well-developed system of irregular basal cavities from 2 to 150 µm in diameter before finally reaching the oscula of the sponge. Eurypylous choanocyte chambers are ovoid to spherical, from 30 to 72 µm in diameter (Fig. 3). Apopylar cells are located around the apopyle of the choanocyte chambers. Basopinacocytes delimit the basal part from the substrate (Fig. 3). The ectosome is mostly devoid of cells, with only a few vacuolar cells distributed randomly underneath the pinacocytes (Fig. 3). Cytology: Choanocytes have a rather pyramidal shape, 3 to 7 µm wide at the central part and 3.5 to 7 µm high. (Fig.4). Their collar is 3 µm wide, composed of about 30 microvilli. Their nucleus is basal, centrally positioned, and 1.5 to 3 µm in diameter. Their cytoplasm usually contains 1 to 3 phagosomes of 0.8 to 1.7 µm in diameter, osmiophilic inclusions and mitochondria. Apopylar cells also have a pyramidal shape, and are about the same size

DESCRIPTION OF THE FIRST CARIBBEAN OSCARELLIDAE Zootaxa 4369 (4) © 2018 Magnolia Press · 505 as the choanocytes. Pinacocytes are flat and flagellated, 7 to 12 µm wide and 1 to 3 µm high. Their nucleus is ovoid, up to 2 µm in diameter. Several osmiophilic inclusions between 0.4 and 0.7 µm were observed only in the cytoplasm of endopinacocytes (Fig. 4). No archaeocytes were observed in the mesohyl. One type of vacuolar cell, with an ovoid to irregular shape, 8 to 16 µm in diameter, is quite abundant and is grouped in clusters in the mesohyl (Fig. 4). Its cytoplasm contains between 2 and 5 vacuoles, 3.5 to 10 µm in diameter, and their content is always clear. No bacteriocytes were observed. A single morphotype of symbiotic prokaryote is distributed in the mesohyl. It has a bacillus morphology, 1 to 1.5 µm long and 0.5 µm in diameter, with a cell wall consisting of many layers of dense filaments (Fig. 4). Each bacteria is surrounded by a surface devoid of collagen fibers (Fig. 4). No reproductive elements were observed.

FIGURE 3. Histological sections of Oscarella filipoi sp. nov. A. General soft tissue organization. B. Detailed view of the mesohyl showing the aquiferous system and the ectosome layer. C. Detailed view of the basal part of O. filipoi. cc: Choanocyte chamber; ect: Ectosome; bas: basopinacocyte; vc: Vacuolar cell.

Ecology. Oscarella filipoi sp. nov. was found in rather shallow waters, from 3 to 20 m depth. This new species prefers half-light conditions, and can thus be found on vertical walls of semi-dark marine caves, under overhangs, underneath rocks or inside crevices. The only sign of epibiosis is an undetermined tiny hydroids that grows into the lobes of the sponge, especially on large specimens. No sign of predation was observed. Taxonomic remarks. The new species cannot be confused with the previous aspiculate Homoscleromorpha described in the Caribbean, as it has very classic Oscarella external morphology: it is clathrate with a very soft consistency, whereas A. madinina, for instance, has a cushion or globulous shape with a gelatinous consistency. On the other hand, the aspiculate P. nathaliae is a leaf-like, thinly encrusting sponge, which is a rather classic external morphology for Plakina. According to the in situ photos available in previous reports of Oscarella in the Caribbean

506 · Zootaxa 4369 (4) © 2018 Magnolia Press PEREZ & RUIZ Sea, the new species may well have formerly been called O. lobularis (e.g. Vacelet 1998). The external morphology (color, consistency and aspect of the surface) of the smallest specimens of O. filipoi sp. nov. is indeed very similar to the Mediterranean species, but the largest specimens do not correspond to any previous reports of Oscarella. The largest specimens tend to detach from the substratum, allowing a part of the body to hang like a curtain. The mesohylar content of the new species is very different from that of O. lobularis, as it includes abundant clusters of vacuolar cells, a trait shared with O. tuberculata (Boury-Esnault et al. 1992).

FIGURE 4. TEM micrographs of Oscarella filipoi sp. nov. A) Detailed view of choanocytes. B) Detailed view of endopinacocytes. C) Detailed view of vacuolar cell. D) Detailed view of some of the few prokaryote morphotypes found in O. filipoi. Cho: Choanocyte; end: Endopinacocyte; vc: part of a vacuolar cell.

Oscarella zoranja sp. nov.

Holotype: Muséum National d’Histoire Naturelle de Paris, MNHN—DJV196: Martinique, Anses d’Arlet, Caribbean Sea. Site « Grotte Couleur » (14°29.752´N, 61°05.407´W), 7 m depth, under an overhang, date 11/06/ 2011. Sample code: 110611-MT3TP5, collector T. Pérez (Fig.5). GenBank accession number : KX3482266. Paratype 1: Muséum National d’Histoire Naturelle de Paris, MNHN—DJV197: Guadeloupe, Caribbean Sea. Site “Grotte aux Barracudas”, (16°27.301'N, 61° 32.253'W), 19 m depth, under an overhang, date 15/02/2012. Sample code: GR26HOM1, collector A. Ereskovsky. Paratype 2: Muséum National d’Histoire Naturelle de Paris, MNHN—DJV198: Martinique, Le Diamant, Caribbean Sea. Site « Grotte Zeb » (14°27.832' N, 61°01.065' W), 19 m depth, on a vertical wall at the entrance of the cave, date 15/05/2016. Sample code: 150516-MT4CR13, collector C. Ruiz. Other material examined: Martinique, 131203-MT3CR2, Anses d'Arlet, Grotte Couleur at 8 m depth. collector C. Ruiz ; 150516-MT4CR4, 150516-MT4CR13, 150516-MT4CR8 Le Diamant, Grotte Zeb at 19 m depth, collector C. Ruiz ; 110611-MT4TP12, 110611-MT4TP5, Le Diamant, Grotte Zeb at 19 m depth, collector T. Pérez; 110613-MT1TP7 Le Diamant, tunnel du Diamant at 15 m depth, collector T, Pérez . Guadeloupe, 150516- MT8CR4, GR26HOM9, GR27HOM16, GR26HOM4, Grotte aux Barracudas at 19 m depth, collector A. Ereskovsky; GR34HOM29, GR30HOM29, GR26HOM1, GR26HOM8, GR27HOM20 Grotte la Cathédrale at 18 m depth, collector A. Ereskovsky. This material is kept in the collection of the Station Marine d’Endoume (Marseille—France).

DESCRIPTION OF THE FIRST CARIBBEAN OSCARELLIDAE Zootaxa 4369 (4) © 2018 Magnolia Press · 507 Etymology: From the creole zoranj (orange), the species name thus refers to the color of the new species. Diagnosis: Oscarella inhabiting shaded habitats. Orange color. Smooth, regularly lobate surface and rather cartilaginous consistency. Ovoid to spherical eurypylous choanocyte chambers. A single type of vacuolar cell, containing up to 10 vacuoles, and one type of spherulous cell.

FIGURE 5. In situ picture of Oscarella zoranja sp. nov.. A) The orange chromotype is the most common color of this species. B) Pale yellow specimen mostly found in semi-dark habitats.

Description. External morphology: A sponge with regular lobate surface which can be up to 15 to 20 cm large and 1 to 3 cm thick. The consistency is soft, and the sponge is easily removed from the substrate. The color in vivo is always a nuance of orange. The sponge does not exude any substance in contact with air, nor does its color change in alcohol. Its surface is smooth, and it presents between 1 and 3 oscula of about 12 mm in diameter. The lobes are small, rounded and regular, and only a few of them have inhalant ostia with a flared-out margin. Soft tissue organization: No spicule or fibers. The ectosome is thick, from 30 to 83 µm. The inhalant canals (13 to 19 µm) run perpendicular to the surface. Exhalant canals run towards a well-developed system of irregular basal cavities leading to the oscula. The eurypylous choanocyte chambers are ovoid to spherical, with diameters between 30 and 72 µm (Fig. 6 and 7). The apopyle is surrounded by apopylar cells.

FIGURE 6. General soft tissue organization of Oscarella zoranja sp. nov.. cc: Choanocyte chamber; pi: pinacoderm.

Cytology: Choanocytes are ovoid to pyramidal, 2.2 to 5 µm large and 4 to 6 µm high. (Fig.7B). Their collar averages 3 µm wide and is composed of about 30 microvilli. Their nucleus, 1.5 to 2.5 µm in diameter, is in central to basal position. The cytoplasm usually contains 1 to 3 phagosomes, 0.6 to 1.7 µm in diameter, osmiophilic inclusions and mitochondria. Apopylar cells are ovoid to pyramidal in shape and of the same size as the choanocytes (Fig. 7). Their nucleus is centrally positioned and 1.5 to 2.5 µm in diameter. Small vacuoles up to 2 µm wide are present in their cytoplasm. Pinacocytes are flat and flagellated, 7 to 12 µm large and 1 to 3 µm high. Their ovoid nucleus is up to 2 µm in diameter. Several osmiophilic inclusions between 0.4 and 0.7 µm were observed only in the cytoplasm of endopinacocytes. No archaeocyte was observed, although two types of cells with

508 · Zootaxa 4369 (4) © 2018 Magnolia Press PEREZ & RUIZ vacuoles were recorded in the mesohyl and in the ectosome. Both types are ovoid to irregular, from 8 to 16 µm in diameter, with a cytoplasm containing between 2 and 10 vacuoles of 3.5–10 µm in diameter (Fig.7). The only difference between the two types lies in the content of the vacuoles: the first type can be defined as a vacuolar cell, with almost empty vacuoles (Fig. 7), whereas the second type is a spherulous cell with vacuoles occupied by electron- dense materials. Prokaryotic cells are quite abundant in the mesohyl. The main morphotype is a bacillus- like shape, 1 to 1.5 µm long and 0.5 µm in diameter (Fig. 7). The cell wall consists of many layers of dense filaments, and the cytoplasm can be dense to electrons. Each bacterium is surrounded by a layer devoid of collagen fibrils. No reproductive element was observed.

FIGURE 7. TEM micrographs of Oscarella zoranja sp. nov. A) Detailed view of a choanocyte chamber. B) Detailed view of choanocytes. C) Flagellated endopinacocyte. D, E) Detailed views of vacuolar cell type 1. F) Detailed view of the spherulous cell of O. zoranja. G) General view of the prokaryotes in the mesohyl. apo: Apopylar cells; cc: Choanocyte chamber.

Ecology. Oscarella zoranja sp. nov. can be found in the same habitats as O. filipoi, on vertical walls of semi- dark caves, tunnels, overhangs and small crevices. This species was found from shallow waters down to 50 m depth in a cave off the Rocher du Diamant in Martinique. This species is never subject to epibiosis and no sign of predation was observed. Taxonomic remarks. Oscarella zoranja sp. nov. has very classic Oscarella external morphology, lobate and with a rather cartilaginous consistency compared to O. filipoi sp. nov., again removing any possibility for confusion with the previous aspiculate Homoscleromorpha described in the Caribbean Sea (Ruiz et al. 2017). This species has the same external morphology and consistency as O. tuberculata, but the new species differs in color and in its mesohylar cells. Unlike both O. tuberculata and O. filipoi sp. nov., this new species does not present clusters of vacuolar cells. DNA analysis. The positioning of the two new Oscarellidae within the Homoscleromorpha phylogeny was achieved after sequencing all examined individuals and downloading some GenBank sequences (Fig. 8). The two families of Homoscleromorpha (Gazave et al. 2010) are well supported in the phylogenetic reconstruction. Oscarellidae is composed of two main clades, and both new species clearly belong to the clade containing the type

DESCRIPTION OF THE FIRST CARIBBEAN OSCARELLIDAE Zootaxa 4369 (4) © 2018 Magnolia Press · 509 species of the family Oscarella lobularis. Oscarella filipoi sp. nov. is the closest to the type species, its sequences of the I3-M11 CO1 fragment differing by 25 and 26 base-pairs from O. lobularis and O. tuberculata, respectively. Oscarella zoranja sp. nov. is not included in the O. filipoi sp. nov. clade and the type species of the family, because its sequences differ by 32 to 35 base-pairs on average from the previous group of species. Oscarella zoranja sp. nov. presents the highest genetic variability recorded among the Homoscleromorpha with this molecular marker, with up to 8 different base pairs between the sequenced individuals, compared with the single base-pair that differs between Oscarella tuberculata (Schmidt, 1868) and O. lobularis. However, our analyses of the morphology of a great number of individuals of O. zoranja sp. nov. failed to reveal any trait which might support this variability.

FIGURE 8. Phylogenetic reconstruction positioning the two new Oscarellidae among Homoscleromorpha on the basis of the I3-M11 fragment of the CO1 mitochondrial gene using neighbor-joining analysis, and indicating maximum-likelihood. Bootstrap values are given for both analyses, with ML values in parenthesis.

Discussion

Among Homoscleromorpha, the Oscarellidae family is currently composed of two genera, Oscarella and Pseudocorticium (Boury-Esnault, Muricy, Gallissian & Vacelet 1995), defined by the absence of a skeleton. This peculiarity is now observed in two genera of the Plakinidae family, Aspiculophora and Plakina (Ruiz et al. 2017), extending previous observations in Demospongiae belonging to various orders, such as Hexadella (Topsent, 1896 (Verongida)), Thymosiopsis (Vacelet & Pérez, 1998 (Chondrosida)), or Myceliospongia (Vacelet & Pérez, 1998 (Demospongiae incertae sedis)). Oscarella is widely distributed, with 18 valid species listed in the World Data Base (Van Soest et al. 2017). Even if both new species are thus far the only valid representatives of this genus in the Caribbean Sea, several species remain to be described (unpublished data).

510 · Zootaxa 4369 (4) © 2018 Magnolia Press PEREZ & RUIZ Identifying or describing Oscarella is always challenging because of the lack of a skeleton, which is the basis of sponge systematics. In this case, only histology and cytology provide subtle diagnostic morphological traits. For example, Oscarella species are usually characterized by a poorly developed ectosome, essentially composed of a single cell layer of pinacoderm, a sylleibid organization of the aquiferous system, and a ratio lower than 1 between the volumes occupied by the mesohyl and the choanocyte chambers (Muricy & Diaz 2002). However, regarding these morphological traits, the new Oscarella species are unusual as they both present a much better developed ectosome than all previously reported Oscarellas. This trait is particularly remarkable in large specimens of O. filipoi which can harbor a thick ectosome in the basal part, defined as “hypophare” by Topsent in his report of O. lobularis in the “Etude Monographique des Spongaires de France” (Topsent 1895). When it is well developed, this basal ectosome is composed principally of collagen with few widespread cells and large canals or lacunae. Most Oscarella species are represented by various chromotypes, generally with a dominant chromotype, for example, yellow in O. tuberculata or orange in Oscarella balibaloi (Pérez, Ivanisevic, Dubois, Pedel, Thomas, Tokina & Ereskovsky 2011). This is also the case for O. filipoi sp. nov., but with a lower variability observed so far. This species was generally recorded as being purple, with the green differentiation of its basal part corresponding to the well-developed ectosome of large specimens. Few small pale-yellow specimens were recorded across the Lesser Antilles. On the other hand, the color of O. zoranja sp. nov. varies only across several shades of orange. Consistency and surface aspect are also important morphological traits (Boury-Esnault et al. 1992; Muricy et al. 1996; Muricy & Diaz 2002). Regarding these traits, O. filipoi sp. nov. shares both its very soft consistency and its main color, with O. lobularis, which might explain the use of this species name for the first reports of Oscarella in the Caribbean Sea (Vacelet 1998). On the other hand, O. zoranja sp. nov. shares the rather cartilaginous consistency of O. tuberculata. Today, an integrative taxonomy often takes into account the cell composition of the mesohyl as well as detailed analysis of the morphology of each encountered cell type (for a review see Cárdenas et al. 2012 or Boury-Esnault et al. 2013). Cells with vacuoles and inclusions of the mesohyl were proposed as discriminating traits at the species level. Our two new species do not present particular vacuolar or spherulous cells which could be used with confidence to distinguish them from most of the other Oscarella species. Only the abundant clusters of vacuolar cells of O. filipoi sp. nov. are noteworthy, albeit very similar to those of O. tuberculata. The two new species can also be differentiated by the spherulous cell recorded in O. zoranja sp. nov.. In this genus, the most useful trait is indeed the presence of a spherulous cell type which harbors paracrystalline inclusions in a well-defined clade containing Pseudocorticium jarrei (Boury-Esnault, Muricy, Gallissian & Vacelet 1995), Oscarella microlobata (Muricy, Boury-Esnault, Bézac & Vacelet 1996) , Oscarella imperialis (Muricy, Boury-Esnault, Bézac & Vacelet 1996) , Oscarella kamchatkensis (Ereskovsky, Sanamyan & Vishnyakov 2009b), and O. balibaloi (Boury-Esnault et al. 1995; Muricy et al. 1996; Ereskovsky et al. 2009a; Pérez et al. 2011), and might call for the reassignment of some Oscarella to Pseudocorticium. If so, this should be undertaken when the next new Oscarellidae with paracrystalline inclusions enters this clade. In our case, the convergence of the molecular and cytological results solidly supports the affiliation of these two new species to the Oscarella clade including the type species. The role of these vacuolar or spherulous cells is still highly debated. A number of Demospongiae have been shown to produce, or at least store, specialized biomolecules (e.g. Turon et al. 2000), which may then be released into the sea in a process recently named spherulization (Ternon et al. 2016). Accordingly, the appearance of these vacuoles may be related to their chemical content. No significant predation on Oscarella species is known, leading to the assumption that their ecological success in restrictive environmental conditions such as submarine caves and their ability to sometimes overgrow invertebrates, may be explained by their ability to produce efficient allelochemicals (Pérez et al. 2011). At least one of the new species, O. filipoi, does cover large surfaces, and thus seems to be a strong competitor for space. However, some preliminary metabolomic analyses of both new species have revealed rather low chemical diversities compared to what is known from Mediterranean Oscarella species (Ivanišević et al. 2011) or from Plakinidae. Further investigation is needed to identify this chemical diversity which, although low, may well involve specialized metabolites explaining the particular behavior of O. filipoi, the biggest Homoscleromorpha ever recorded.

This work was performed in the framework of the French-Brazilian Associated International Laboratory “LIA MARRIO” and the French-Colombian cooperation project ECOS-NORD. It was funded by the CNRS and the Total Foundation. The authors are grateful to Sandrine Chenesseau (CNRS/IMBE), Joël Courageot and Alexandre Altié (Aix Marseille University / Plateforme de microscopie) for help with light and electron microscopy. The authors would like to thank the crew of the N/O Antea for their involvement in the PACOTILLES campaign in the Lesser Antilles, Julien Chalifour in Saint Martin and to great friends “Filipo” and Jean-Claude Erin for hosting us in Martinique. The authors are also grateful to Virginie Baldy and Olivier P. Thomas who were present for the first sampling and preserving of both new species in Martinique Island, to Alexander (Sacha) Ereskovsky and Pierre Chevaldonné for the fruitful and friendly discussions, and finally to Nicole Boury-Esnault and Jean Vacelet for the great source of inspiration they represent, and for their ever positive criticism regarding our work.

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