Vansoestia caribensis gen. nov., sp. nov.: first report of the family Ianthellidae (Verongida, Demospongiae) in the Caribbean Author Díaz, Maria C. Author Thacker, Robert W. Author Redmond, Niamh E. Author Perez, Thierry Author Collins, Allen G. text Zootaxa 2015 3956 3 403 412 journal article 10.11646/zootaxa.3956.3.5 fd562f8c-5541-478c-8827-6464cc13f053 1175-5326 240004 25F9FA82-28BA-4C94-8EF5-D0EE88B0B48E Vansoestia , gen. nov. Diagnosis. Ianthellidae represented by individuals with a thin ( 1–4 mm ) encrusting growth form that presents a cellularized cortex of variable thickness (10–200 µm) composed of a collagenous matrix and various types of cells with inclusions (i.e. granular, spherulous, and/or vacuolar). Abundant subdermal canals and lacunae are found immediately underneath the cortex. The choanosome consists of numerous aquiferous canals, a mesohyl where two or three types of cells with inclusions can be distinguished, and oval eurypylous choanocyte chambers 20–50 µm, all supported with abundant fibrillar collagen. Type species: Vansoestia caribensis gen. nov. sp. nov. Material examined. All specimens studied were found in coral reefs from Bocas del Toro, Panama , between 10–20 m in depth. This species grows over dead coral and other organisms, in open and cryptic reef habitats. Holotype : USNM 1133773, Buoy 19 (N9°18.11, W 82°17.66), Bocas del Toro, Panama (P67), 10 m deep, coll: M.C. Díaz and R.W. Thacker; 7/18/2009 Paratypes : USNM 1133782, Buoy 19 (N9°18.11, W 82°17.66), Bocas del Toro, Panama (P76), 15 m deep, coll: M.C. Díaz and R.W. Thacker; 7/18/2009 ; USNM 1204851 (P 12x 342), Punta Caracol (N9°22.66, W 82°18.19), 15 m deep, coll: M.C. Díaz and S.A. Pomponi, 8/8/2012 . Additional material. P 12x 402, and P 12x 403 were consumed in histological, or TEM preparations. Etymology. The species is named in honor of Dr. Rob van Soest, an essential contributor to the understanding of Demospongiae, including Verongida , in the Caribbean and worldwide. Description. External morphology : The sponge is soft, thinly encrusting ( 1–4 mm thick), orange to yellow in color internally and externally. Color turns slowly brown to purple in ethanol. The sponge grows over dead coral or over other organisms, particularly other sponges ( Figure 1 ). Its surface is dominated by thin reticulated subdermal canals ( 3–8 mm wide) that branch and decrease in width away from the oscules ( 0.5–1 cm wide). Small ostia ( 0.4– 0.8 mm wide) are dispersed between subdermal canals. The soft and fragile consistency of this sponge reflects the lack of a reticulate fiber skeleton. Internal morphology ( Figures 2–4 ). There is a clear separation between ectosome and choanosome ( Figure 2 A). The ectosome is a collagenous cortex of variable thickness (10–200 µm), with collagen in the form of an amorphous matrix containing loose cells with inclusions ( Figure 2 A, B), and an external cuticle ( Figure 3 A). Large channels and lacunae (subdermal spaces) are observed immediately underneath the ectosome ( Figure 2 A). The choanosome consists of densely arranged oval-shaped choanocyte chambers (20–40 µm in diameter) ( Figure 2 A, C), and a mesohyl reinforced by collagen fibrils arranged in strands ( Figure 3 B, C), packed with various types of cells with inclusions, and with abundant aquiferous canals. The chambers are densely and homogeneously distributed in some areas ( Figure 2 A, C), and rare in others that are instead densely packed by conglomerates of cells ( Figure 3 D). Choanocytes (4–6 µm in diameter) appear oval to triangular in shape ( Figure 2 D). Larger cells with inclusions of various shapes and sizes (4–12 µm in diameter) can be observed in both the mesohyl and the ectosome ( Figures 2–3 ). The largest cells with inclusions (Ci1) are widespread in the mesohyl, in the cortex, and lining canals ( Figure 3 B and 3D). There are some canals lined by these large cells with inclusions ( Figure 3 D), and others without them ( Figure 3 C). FIGURE 1. Vansoestia caribensis gen. nov. sp. nov. growing on the edge of a dead plate coral. A. General habit showing its overall growth form, with algae and polychaete calcareous tubes mingled within the body. Scale bar = 2 cm. B. Close-up view of a more relaxed specimen showing the subdermal canals (Sc), 3–8 mm wide, and the profusely abundant pores. Scale bar = 1.2 cm TEM sections allowed further distinction of the various types of cells with inclusions, and the observation of archaeocytes, choanocytes, and collagen fibrils. Spherulous cells 1 ( Figure 4 A, B and D) are ovoid to roundish slightly deformed (6–10 µm in diameter) with spherules (0.7–2 µm wide), containing microgranular inclusions that are dense but grey or black in color (0.06–0.15 µm in diameter), with a clear round nucleus (1.3–2 µm in diameter). By the size and distribution of spherules, these cells probably correspond to the cells with inclusions 1 (Sc1) observed in the histological cross sections ( Figures 2–3 ). Spherulous cells 2 ( Figure 4 B) are similar in size and shape of Sc1, but their spherules are larger and ellipsoid in shape (0.1–0.3 µm in diameter), and electron dense dark. These cells might represent a different stage of the spherulous cell 1. A third type , which seems like vacuolar cells ( Figure 4 C), occurs with variable shapes, smaller size (5–8 µm in diameter), and containing a single round, dark dense granule (0.4–0.8 µm in diameter) within each vacuole (1–1.5 µm in diameter). By the shape and size of the granules, these cells likely correspond to the cell with inclusion 2 observed in the histological cross sections. Finally, a microgranular cell with membrane-bound inclusions with striated content, was observed once ( Figure 4 D). A thick layer of collagen fibrils (Co) with loose bacteria surrounds the choanocyte chambers and long cytoplasmic projections could be seen in some choanocytes ( Figure 4 E). One choanocyte with a basal nucleolated nucleus was observed ( Figure 4 F). Remarks. The delicate and thin specimens of Vansoestia caribensis have been found in Bocas del Toro, Panama , since 2006; however, only the slow oxidation observed when specimens were placed into alcohol suggested an affinity to verongids. Due to the lack of a fiber skeleton, we initially affiliated this sponge to the genus Hexadella . Histological observations corroborated its ianthellid nature (large, sac-shaped choanocyte chambers), but its distinct identity and closest affinity to the known sequences of fiber-bearing ianthellid genera from the Western Tropical Pacific appeared after analyzing 18S and 28S ribosomal gene sequences (Redmond et al. , 2013; Thacker et al. , 2013). Its genetic distance from the only previously known fiber-less verongid genus, Hexadella , prompted the need to erect a new genus to classify this species. This biological record constitutes the first member of family Ianthellidae ever encountered in the Caribbean Sea. FIGURE 2. Photomicrographs of Vansoestia caribensis gen. nov. sp. nov. histological cross sections. A. Upper body region showing a thin ectosome (Ec) followed by wide subdermal canals (Sc) and the choanoderm with densely arranged choanocyte chambers (Cc). Scale = 110 µm. B. Detail of an area of the ectosome showing a dense “amorphous matrix” (Am) of collagen with dispersed dark stained cells with inclusions (Ci), Scale = 30 µm; C. Basal region where the sponge is attached to the substrate. Note the corallites (Co) of the dead coral where the sponge was growing, the choanocyte chambers (Cc) and canals (Ca), Scale = 200 µm. D. Detail of choanocyte chamber with more than 20 choanocytes (oval to triangular in shape). Notice at least two types of cells with inclusions (Ci) and collagen strands (Cs) in between the chambers and the cells, Scale = 15 µm. There is a striking similarity between V. caribensis gen nov. sp. nov. and the external appearance of Hexadella pruvoti Topsent, 1896 ( Reveillaud et al. , 2012 ) , particularly with respect to the abundant subdermal canals converging in a wide oscule, and its yellowish coloration. However, 18S and 28S gene sequence analyses confirmed the very distinct nature of these two species. A cellularized cortex of variable thickness (10–200 µm thick) composed of amorphous collagen and various types of cells is present in V. cari bensis gen. nov. sp. nov. and forms an important morphological difference between these two genera, since Hexadella possesses a thinner cortex with a 1 µm thick cuticle. A well-developed cellularized cortex might represent a potential synapomorphy between Vansoestia gen. nov. , Ianthella , and Anomoianthella ( Bergquist, 1980 , 1995; Bergquist and Kelly-Borges, 1995 ; Bergquist and Cook, 2002; present study). Indeed, Bergquist, (1995) and Bergquist and Kelly-Borges (1995) described a collagenous ectosome (70 to 300 µm thick) containing cells for all Ianthella species and a cellularized cuticle for Anomoianthella species ( Bergquist, 1980 ). Together with the presence of a collagen-reinforced cortex combined with a cuticle, the high degree of reinforcement of the choanosome with collagen is also a characteristic shared by Vansoestia gen. nov. , Ianthella and Anomoianthella . Some similarities among the spherulous cells of V. caribensis gen. nov. sp. nov. , and other verongids were also noticed ( Vacelet, 1967 ). The cells with striated membrane-bound bodies are very similar to cells observed in Aplysina aerophoba by Maldonado (2009) . The dark granules found in cells of Vansoestia gen. nov. might represent glycogen granules ( Maldonado, 2009 ), or they might contain brominated compounds (Thompson, 1986) like has been found for microgranular and spherulous cells of two different Aplysina species. The large diversity of cells with inclusions, and their similarities with species of Hexadella and other verongid genera invites continued research to understand the morphology and physiological functions of these cells and their products. FIGURE 3. Detail of the internal morphology of Vansoestia caribensis gen. nov. sp. nov. at 400–600 X. A. Ectosomal cellularized cortex, where a 1 µm cuticle (Cu) lining the outer surface of the sponge is evidenced, Scale = 13 µm. B. Various cells with inclusions (Ci1, Ci2) and smaller choanocytes (Ch) embedded in a mesohyl with abundant strands of collagen (Cs), Scale = 16 µm. C. Portion of the sponge body in contact with the substrate (Co), showing abundance of collagen strands (Cs), and cells. Notice an aquiferous canal (Ac) that is not lined by larger cells with inclusions, Scale = 25 µm. D. Detail of the mesohyl showing an aquiferous canal that is lined by cells with inclusions type 1, and “cell conglomerates” and other types of cells, Scale = 32 µm. FIGURE 4. TEM sections of Vansoestia caribensis gen. nov. sp. nov. A. Roundish to ovoid Spherulous cells type 1 (Sc1) with nucleus (Nu) bearing round spherules containing light and dark inclusions, and abundant collagen fibrils (Co), Scale = 2.5 µm. B. Sperulous cell type 2 with electron dense dark granules, and bacteria within it, close to an aquiferous channel (Ac), Scale = 2 µm. Bacteria can be seen in the interior of the channel. C. Vacuolar cells (Sc 3) seen in the mesohyl close to an aquiferous channel, Scale = 2 µm. D. A microgranular cell (Mc) with striated membrane-bound bodies, among spherulous cells 1, and 2. Note the presence of abundant collagen fibrils (Co) between the cells, Scale = 1.3 µm. E. Portion of a choanocyte chamber where main body of the choanocytes can be seen, some with cytoplasmatic projections (Cy). A collagen fibril layer (Co) is found surrounding all choanocyte chambers, Scale = 1.6 µm. F. A triangular shaped choanocyte (Ch) with a basal nucleolated nucleus, contiguous to a layer of fibrillar collagen (Co) containing bacteria (Ba), Scale = 1.3 µm. FIGURE 5. Schematic representation of the phylogenetic relationships of the genera of Verongida , summarizing four previously published phylogenies (Erwin & Thacker 2007 [ITS2+28S]; Díaz et al. , 2013 [cox1]; Redmond et al. , 2013 [18S]; Thacker et al. , 2013 [28S];). Black circles indicate nodes with 100% maximum likelihood bootstrap support in at least 2 studies; gray circles indicate nodes with 100% maximum likelihood bootstrap support in 1 study, and none under 85%; white circles indicate poorly resolved nodes, or phylogenies incongruent between studies. Proposed common characters are indicated with numbers: 1. Verongida with bundles of interstitial collagen fibrils either in the cortex, mesohyl, or within fibers; 2. Family Chondrosiidae ( sensu Redmond et al. , 2013) with a thick, acellular collagenous cortex; 3. Clade represented by the rest of the Verongida , all containing spherulous cells and brominated secondary metabolites; 4. Clade represented by Hexadella , with eurypylous choanocyte chambers, a thin cuticle, and lacking fibers; 5. Poorly supported clade (62%) containing all the non - Hexadella classical genera of Verongida ; 6. Moderately supported clade (85%) containing all eurypylous verongids with a cellularized collagenous cortex. 7. Verongids with diplodal choanocyte chambers; 8. Verongids with an isotropic, welldeveloped fiber reticle, with oval to hexagonal meshes, and fibers with a distinct organic pith and a bark (concentrically layered) throughout the body. 9. Verongids with a variably developed reticulation of fibers, sometimes absent, with thick fibers with or without apparent bark; a morphological synapomorphy is yet to be discovered. Phylogenetic remarks . 18S and 28S sequences demonstrate that this fiber-less member of Ianthellidae is a sister group to the clade that includes Ianthella and Anomoianthella . (Redmond et al. , 2013; Thacker et al. , 2013). Figure 5 summarizes the current phylogenetic interpretation of the relationships among verongid genera based on the latest molecular, and morphologic studies of the group ( Erwin and Thacker, 2007 ; Erpenbeck et al. , 2012 ; Díaz et al. , 2013; Redmond et al. , 2013; Thacker et al. , 2013). The two sister clades make up the subclass Myxospongiae, one containing the amended Verongida (after Redmond et al. , 2013) and the other containing Chondrilla and Halisarca (Order Chondrillida sensu Redmond et al. , 2013). All verongids are grouped in a strongly supported monophyletic clade ( Figure 5 , clade 1), which shares the presence of collagen fibrils forming bundles either in the cortex, in the mesohyl, or within fibers. Chondrosia (family Chondrosiidae ) forms a wellsupported sister clade to all other verongids ( Figure 5 , clade 2), and is represented by sponges with extremely high collagen content, a thick (> 1 mm thick) “collagenous cortex”, and a fiber-less body. The rest of the verongids form a highly supported clade ( Figure 5 , clade 3), containing all taxa that produce brominated secondary metabolites and have abundant spherulous cells. Basal within this group, a highly supported clade ( Figure 5 , clade 4) contains all Hexadella species which share eurypylous choanocyte chambers, a thin cuticle (1–2 µm) and a very thin ectosome. The next clade ( Figure 5 , clade 5) is a moderately supported clade containing all non- Hexadella verongids. Within clade 5, two clades are distinguished: a moderately (85%) supported clade ( Figure 5 , clade 6) containing Vansoestia gen. nov. (skeleton less) and the fiber bearing Pacific taxa of genera Ianthella and Anamoianthella , and a moderately to highly supported clade ( Figure 5 , clade 7) including all the taxa possessing diplodal choanocyte chambers. Within clade 7 ( Figure 5 ), a clearly monophyletic group of sponges (clade 8) contains all Aplysina species, whereas a poorly supported clade (clade 9) contains at least six genera ( Aiolochroia , Aplysinella , Porphyria , Pseudoceratina , Suberea , Verongula ) and an undescribed genus from Moorea (Díaz et al. , 2013). The Verongida taxa with eurypylous, sac-shaped choanocyte chambers are placed in two distinct clades ( Figure 5 , clades 4 and 6), demonstrating that this feature is paraphyletic within Verongida . Current studies suggest the classification of Verongida comprises four main clades: (1) a clear monophyletic group containing all Aplysina species studied, and representatives of all Aplysinellidae , and Pseudoceratiniidae genera; (2) a clade that contains Ianthella , Anomoianthella , and Vansoestia gen. nov. , but excludes Hexadella , which should be defined by an amended family Ianthellidae ; (3) a clade that presently contains only Hexadella and that should be erected as a new family; and (4) a clade containing the family Chondrosiidae sensu Redmond et al. (2013) . We demonstrate here that the suprageneric classification of Verongida must be further revised in order to reflect our current knowledge of phylogenetic relationships among the genera of Myxospongiae. Considering the diagnostic characters that we have in hand, a thorough comparative review of the histological and ultrastructural features of representatives of each genus is definitely needed to improve the taxonomic diagnoses of these taxa.