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Halisarca desqueyrouxae sp. nov. (Figs 11–18, Table 1) Material examined. Holotype: RBINS-IG 32236- POR. 10803 (Fragments: MNRJ 10803, MHNG 90139), Reñihue Fjord, Palena Province, Chile (42°31'55.02"S –72°35'30.72"'W), 3 m depth, coll. Ph. Willenz & E. Hajdu, 22.v. 2007. Paratypes: RBINS-IG 3 2235- POR. 10754 (Fragments: MNRJ 10754, MHNG 90094), Liliguapi Island, Comau Fjord, Palena Province, Chile (42°09’43.32”S – 72°35’54.90”W), 14 m depth, coll. Ph. Willenz & E. Hajdu, 07.v.2007. RBINS-IG 3 2236- POR. 10757 (Fragments: MNRJ 10757, MHNG 90097), Liliguapi Island, Comau Fjord, Palena Province, Chile (42°09’43.32”S – 72°35’54.90”W), 13 to 16 m depth, coll. Ph. Willenz & E. Hajdu, 07.v.2007. RBINS-IG 3 2238- POR. 12917 (Fragments: MNRJ 12917, MHNG 90251), Liliguapi Island, Comau Fjord, Palena Province, Chile (42°09'43.32"S – 72°35'54.90"W), 26 m depth, coll. Ph. Willenz & J. Biro, 01.ii.2009. RBINS-IG 32238- POR. 1 2919 (Fragments: MNRJ 12919, MHNG 90253), Liliguapi Island, Comau Fjord, Palena Province, Chile (42°09'43.32"S – 72°35'54.90"W), 26 m depth, coll. Ph. Willenz & J. Biro, 01.ii.2009. RBINS-IG 32238- POR. 12924 (Fragments: MNRJ 12924, MHNG 90258), Liliguapi Island, Comau Fjord, Palena Province, Chile (42°09'43.32"S – 72°35'54.90"W), 26 m depth, coll. Ph. Willenz & J. Biro, 01.ii.2009. Additional paratypes not observed in Electron Microscopy. RBINS-IG 32 232- POR. 8795 (Fragment: MNRJ 8795) Liliguapi Island, Comau Fjord, Palena Province, Chile (42°09’44.23”S – 72°35’42.99”W), 20 m depth, coll. Ph. Willenz & E. Hajdu, 24.ii.2005. RBINS-IG 32236- POR. 9193 (Fragment: MNRJ 9193), Estero Farquhar, Bernardo Fjord, Capitán Prat Province, Chile (48˚29'18,70"S–74˚12'25,70"W), 4 m depth, coll. V. Häusssermann & G. Försterra, 29.iii.2005. RBINS-IG 32236- POR. 9214 (Fragments: MNRJ 9214, Estero Farquhar, Bernardo Fjord, Capitán Prat Province, Chile (48˚29'18,70"S–74˚12'25,70"W), 5 m depth, coll. V. Häusssermann & G. Försterra, 29.iii.2005. RBINS-IG 32236- POR. 9215 (Fragments: MNRJ 9215, Tempano Fjord, Capitán Prat Province, Chile (48°42'59.70"S – 74°00'18.80"W), 8 m depth, coll. V. Häusssermann & G. Försterra, 26.iii.2005. RBINS-IG 32233- POR. 9961 (Fragments: MNRJ 9961, MHNG 90662), Isla Lavinia, Última Esperanza Province, Chile (49°00'48.10"S – 74°58'37.50"W), 4 m depth, coll. Ph. Willenz & L. Atwood, 13.iii.2006. RBINS-IG 32 238- POR. 12923 (Fragments: MNRJ 12923, Liliguapi Island, Comau Fjord, Palena Province, Chile (42°09’44.23”S – 72°35’42.99”W), 11 m depth, coll. Ph. Willenz & J. Biro, 1.ii.2009. External morphology. Polymorphic sponge. The first morphotype corresponding to the holotype is the most frequently encountered. It consists of an encrusting wrinkled mat, up to 2 cm thick, composed of ridges and short digitations with well-defined oscula (morphotype 1, Figs 11 A–11D). The surface is slimy with clear and evenly spread ostia. The consistency is resistant to tearing. The second morphotype is represented by individuals of tubular form varying in length, with a single and sometimes stretched out osculum (morphotype 2, Figs 11 E–11H). Colour. Both morphotypes are light brown when exposed to the light and beige when hidden under overhangs. Anatomy. The ectosome varies from 80 to 250 µm in thickness (Figs 12 A, 12B, 12D, 12E & 12G–12H). On its outside, it is delimited by a superficial layer made of plane extensions of exopinacocytes connecting through thin processes with their cell bodies, which are located under a thick complex collagenous layer (Figs 13 B & 13C). No glycocalyx covers the external membrane of the exopinacocytes in any of the observed samples. A complex collagenous layer contains two distinct regions: its outward side has a loose and diffuse unorganized structure, 1 to 14 µm thick; its inward side, 3 to 5 µm thick, is made of collagen fibrils arranged in dense tracts, which are in contact with the underlying choanosome (Figs 13 A, 13C & 13E). The global thickness of the collagenous layer is not consistent and ranges between 6 to 19 µm among the samples, but without any particular widening. Spherulous cells are scattered in the collagenous layer and are abundantly concentrated in the ectosome. They are more dispersed in the choanosome (Figs 12 A– 12I). Symbiotic bacteria are also present from the outermost collagenous layer to the deepest regions of the mesohyl. The choanosome includes typical elongate and convoluted choanocyte chambers (50 to 260 µm long x 20 to 60 µm wide) surrounded by numerous aquiferous canals of various diameters (Figs 12 A– 12I). The mesohyl is very lax and remarkably clear, lacking dense granules (Figs 14–17), but contains collagen tracts (Figs 12 D–12F) and different wandering cell types at low density. Cytology. Exopinacocytes (Figs 13 A–13E) are "T-shaped" in transverse section. Their cell bodies (2.8 to 6.7 µm in diameter), with basal nucleolated nuclei, are deeply rooted under the complex collagenous layer, and connect with thin flake-like superficial extensions, revealed in SEM, through outstretched projections (Figs 13 A– 13C). Inter-cellular junctions between superficial extensions connect exopinacocytes to each other (Fig. 13 D). Choanocytes (Figs 14 A–14D) are stretched and cylindrical with a roughly pyramidal base (6.9 to 10.5 µm long x 2.1 to 8.3 µm wide) provided with basal pseudopods extending toward the mesohyl. The cytoplasm comprises electron dense inclusions and phagosomes, mainly concentrated on the basal side of the cell, and an apical nucleus. A periflagellar sleeve surrounds the base of the flagellum. Endopinacocytes (Figs 15 A & 15B) are flat with a prominent oval nucleus and constitute the walls of the canals. Basopinacocytes were not observed. Archaeocytes (Fig 16 A) are few and have a conventional amoeboid shape (6.2–8.0 µm) with a spherical nucleolated nucleus (3.7 to 4.3 µm in diameter). The cytoplasm is dense with phagosomes and vacuoles of different densities varying from 270 to 670 nm in diameter. Lophocytes (Figs 16 B & 16C) are elongated (ranging 2.3 x 9.6 µm) with tufts of collagen fibrils emerging from their posterior side or surrounding them. The nucleus is located in the anterior part of the cell. The cytoplasm is dense, contains phagosomes and small osmiophilic inclusions (330 x 730 nm). Spherulous cells of two different types are distinguished in TEM (Figs 13 A & 13C). Spherulous cells Type I (Figs 16 D & 17C) are oval cells (7.8–10.9 x 11.5–12.0 µm in diameter) with short pseudopodial extensions. Large dense osmiophilic homogeneous membrane-bound inclusions of variable diameter (1.0 to 3.8 µm) squeeze the central nucleolated nucleus, leaving exiguous space for a cytoplasm containing phagosomes and small electron translucent vesicles (100 to 200 nm). They are the most abundant cells in the mesohyl. Spherulous cells Type III (Figs 13 A, 16E–16G & 17F) are large cells (5.0–7.4 x 8.8–10.6 µm) with irregular shape. Large inclusions (0.8 to 2.0 µm in diameter) of porous texture with a denser periphery occupy most volume of the cytoplasm that contains large phagosomes, except for the cells occurring near the exopinacocytes (Fig. 16 G). These cells are common and mainly located in the upper part of the ectosome and occur less often in the choanosome; they are not found in all specimens examined (Table 1). Granular cells (Fig. 16 H) are round cells (4.5–7.0 x 6.7–7.7 µm in diameter) with homogeneous spherical inclusions of variable size and density (0.6–1.0 x 1.6–3.6 µm in diameter) and large phagosomes. These cells are found in the choanosome but do not occur in all specimens (Table 1). Rhabdiferous cells (Figs 17 A & 17B) are irregularly shaped cells (7.9–4.0 x 6.3–1.7 µm) with short pseudopodia. The nucleus is spherical and anucleolated (1.7–2.6 µm in diameter). The cytoplasm contains abundant osmiophilic elliptical membrane bound rhabdites (up to 1.3 µm in length and 0.8 µm in diameter), which include a less dense spherical sub-inclusion. Rhabdiferous cells occur in the ectosome and in the choanosome, where they are either free in the mesohyl or occasionally enclosed within choanocyte chambers and even larvae. Pocket cells (Figs 17 C–17H) are amoeboid cells (6.3–13.3 x 3.9–7.2 µm) with long pseudopods extending actively around the cell to phagocytize components of the mesohyl, including cells from other types and bacteria, forming large "pockets". The nucleolated nucleus is round to ovoid (2.9 to 4.0 x 2.6 to 3.5 µm). These cells occur everywhere in the sponge, from the choanosome to the ectosome where they appear to be more developed. Their membrane is folded in many thin and puffed layers, forming small electron translucent microvesicles located at the periphery of the cell. Some pocket cells with numerous pockets and smaller electron translucent vacuoles are crossing the collagen layer to be rejected from the sponge as they are found at the surface of the exopinacocytes. Extracellular symbiotic bacteria (Figs 18 A–18E) of three different morphotypes occur in the mesohyl. The nomenclature used here corresponds to the one used above for H. magellanica. Type B1 is absent. Type B2 is rod shaped and reaches 1.9 µm in length and 0.3 µm in diameter with an electron dense thick wall and an electron translucent cytoplasmic matrix. The electron translucent nucleoid presents a central rod surrounded by filamentous material. Type B2 occurs only in H. desqueyrouxae sp. nov. morphotype 2 (Table 1). Type B3 is spiral-shaped and thick walled, its width reaches 0.4 to 0.5 µm. The cytoplasmic matrix is clear and the nucleoid is electron dense. Type B3 occurs in the choanosome of all specimens of both morphotypes where it is evenly distributed. Type B4 is larger in diameter than Type B2 (1.1 to 1.4 x 0.5 µm), and has a more ovoid shape and a wrinkled capsule. No central rod appears in the clear nucleoid. This type was found in only one specimen of each morphotype (Table 1). Types B2 and B4 are exclusively found in the collagenous layer of the ectosome where they are frequent. Reproduction. One of the three specimens collected in early February 2009 contained embryos and prelarvae. Larvae appeared in two out of the three specimens collected in May 2007 (Figs 12 A, 12C & 12H). Distribution. Halisarca desqueyrouxae sp. nov. is presently known from Chilean Patagonia (42°S). Habitat. Halisarca desqueyrouxae sp. nov. is commonly found at depths ranging from 3 to 26 m, on mussel beds at shallow depths or covering rocks exposed to the light and under overhangs below 10 m. Etymology. The specific epithet is a tribute to Dr. Ruth Desqueyroux-Faúndez who inspired our first sponge collections in Chilean Patagonia and actively participated in several expeditions in that region. Phylogenetic analysis. Partial cox1 sequences were determined for seven specimens of H. desqueyrouxae sp. nov. (RBINS 10803, 10757, 12917, 12919, 12924 and 12923 = morphotype 1; RBINS 10754 = morphotype 2) and were found to be identical. Phylogenetic analysis placed H. desqueyrouxae sp. nov. as a sister group to H. dujardini, but revealed 2.3% difference between their cox1 sequences (Fig. 19).

Genus Halisarca Johnston, 1842 Diagnosis. Chondrillida in which the choanocyte chambers are tubular, branched and wide mouthed. Larvae are incubated disphaerulae (a larval form found only in Halisarca) with simple undifferentiated histology and cilia of uniform length. Skeleton is composed of fibrillar collagen only, there are no fibrous or mineral elements present; ectosomal and subectosomal collagen is highly organised and structurally diversified (Ereskovsky et al. 2011)., Published as part of Willenz, Philippe, Ereskovsky, Alexander V. & Lavrov, Dennis V., 2016, Integrative taxonomic re-description of Halisarca magellanica and description of a new species of Halisarca (Porifera, Demospongiae) from Chilean Patagonia, pp. 501-533 in Zootaxa 4208 (6) on page 503, DOI: 10.11646/zootaxa.4208.6.1, http://zenodo.org/record/214957, {"references":["Johnston, G. (1842) A History of British Sponges and Lithophytes. In. Lizars, W. H., Edinburgh, pp. i - xii, 1 - 264, pls I - XXV. http: // dx. doi. org / 10.5962 / bhl. title. 51495","Ereskovsky, A. V., Lavrov, D. V., Boury-Esnault, N. & Vacelet, J. (2011) Molecular and morphological description of a new species of Halisarca (Demospongiae: Halisarcida) from Mediterranean Sea and a redescription of the type species Halisarca dujardini. Zootaxa, 2768, 5 - 31."]}

Re-description of Halisarca magellanica Topsent, 1901 type species (Figs 2���10, Table 1) Synonymy. Halisarca dujardini var. magellanica, Topsent, 1901; Thiele, 1905; Burton, 1929, 1932, 1940. Original description. Halisarca dujardini var. magellanica, Topsent, 1901, p. 44���45. Type material. Holotype: RBINS-POR.034���4 fragments (alcohol), Antarctic Expedition of the Belgica 1897���99, sample n��47, Torrent Bay, Londonderry Island, Ant��rtica Chilena Province, Chile (55��03'S ��� 69��23'W), intertidal, coll. M. Racovitza, 18.xii.1897. Material examined. RBINS-IG 32232- POR. 8798 (Fragment: MNRJ 8798), Liliguapi Island, Comau Fjord, Palena Province, Chile (42��09���44.23���S ��� 72��35���42.99���W), 20 m depth, coll. Ph. Willenz & E. Hajdu, 24.ii.2005. RBINS-IG 3 2232 - POR. 8888 (Fragments: MNRJ 8888, MHNG 89996), Piedra Lile, Quell��n, Chiloe Province, Chile (43��10���58.60���S ��� 73��38���27.20���W), 16 m depth, coll. E. Hajdu & Ph. Willenz, 04.iii.2005. RBINS-IG 32235 - POR. 10753 (Fragments: MNRJ 10753, MHNG 90093), Liliguapi Island, Comau Fjord, Palena Province, Chile (42��09���44.23���S ��� 72��35���42.99���W), 13 m depth, coll. Ph. Willenz & E. Hajdu, 07.v.2007. RBINS-IG 32 235- POR. 10827 (Fragments: MNRJ 10827, MHNG 90161), Re��ihu�� Fjord, Palena Province, Chile (42��30'48.06"S ��� 72��37'49.74"W), 13 m depth, coll. Ph. Willenz & E. Hajdu, 23.v.2007. RBINS-IG 32238- POR. 12918 (Fragments: MNRJ 12918, MHNG 90252), Liliguapi Island, Comau Fjord, Palena Province, Chile (42��09'43.32"S ���72��35' 54.90W), 26 m depth, coll. Ph. Willenz & J. Biro, 01.ii.2009. RBINS-IG 32238- POR. 12920 (Fragments: MNRJ 12920, MHNG 90254), Liliguapi Island, Comau Fjord, Palena Province, Chile (42��09'43.32"S ���72��35' 54.90W), 26 m depth, coll. Ph. Willenz & J. Biro, 01.ii.2009. RBINS-IG 32238- POR. 12921 (Fragments: MNRJ 12921, MHNG 90255), Liliguapi Island, Comau Fjord, Palena Province, Chile (42��09���44.23���S ��� 72��35���42.99���W), 10 m depth, coll. Ph. Willenz, 01.ii.2009. RBINS-IG 32238- POR.12922 (Fragments: MNRJ 12922, MHNG 90256), Liliguapi Island, Comau Fjord, Palena Province, Chile (42��09���44.23���S ��� 72��35���42.99���W), 10 m depth, coll. Ph. Willenz, 01.ii.2009. Additional material not observed in Electron Microscopy. MNRJ 8797 (Fragment: RBINS-IG 32232- POR. 8797), Liliguapi Island, Comau Fjord, Palena Province, Chile (42��09���44.23���S ��� 72��35���42.99���W), 20 m depth, coll. Ph. Willenz & E. Hajdu, 24.ii.2005. MNRJ 8892 (Fragment: RBINS-IG 32232- POR. 8892), Piedra Lile, Quell��n, Chiloe Province, Chile (43��10���58.60���S ��� 73��38���27.20���W), 16 m depth, coll. E. Hajdu & Ph. Willenz, 04.iii.2005. RBINS-IG 32238 - POR. 12933 (Fragment: MNRJ 12933), Re��ihu�� Fjord, Palena Province, Chile (42��30���20.70���S ��� 72��46���29.70���W), 30 m depth, coll. Ph. Willenz & J. Biro, 08.ii.2009. External morphology. Polymorphic sponge. The first morphotype, 0.2 to 10 mm thick, corresponding to the type specimen (Topsent 1901), is encrusting and massive with a slimy surface, slippery to the touch (morphotype 1, Figs 2 A���2D). The consistency is gelatinous and elastic. When growing on mussel banks or under overhangs, it forms stretched filamentous outgrowths of different lengths and shapes "dripping" downward, resulting in propagules bearing a discrete osculum at their end (Figs 2 A���2C). Ostia are microscopic and barely visible. The second morphotype is thinly encrusting, without propagules but with the same viscous surface and consistency (morphotype 2, Fig. 2 E). Both morphotypes are found on vertical to horizontal hard substrates or under overhangs. The third morphotype is "bushy", 1 to 5 cm high, made of grouped digitations, each with obvious pores (ostia) and always ending with a clearly visible osculum. The surface is velvety and the consistency is more brittle and less elastic than morphotypes 1 & 2. It is found growing mainly on vertical substrates (morphotype 3, Figs 2 F & 2G). Colour. All three morphotypes are bright pink when exposed to the light and usually ivory or whitish when hidden under overhangs. Anatomy. The ectosome has a thickness varying from 80 to 280 ��m in morphotypes 1 & 3 and reaches 900 ��m in morphotype 2 (Figs 3 A��� 3I). The structure of the ectosome is similar in all three morphotypes and is composed of a superficial layer made of flat extensions of exopinacocyte cell bodies, which themselves are located under an intermediary complex collagenous layer and connect to the surface of the sponge by thin processes (Figs 4 A���4C, 4E & 4F). Exopinacocyte cell bodies are distant from each other and are only tightly joined at the level of their superficial extensions. The external membrane of the exopinacocytes is covered by a thin glycocalyx varying from 30 to 150 nm (Fig. 4 F). The collagenous layer shows two regions: its outward side consists of a loose and diffuse unorganized structure 1 to 6 ��m thick, whereas its inward side consists of interlaced collagen fibrils forming dense tracts 2 to 25 ��m, which are in contact with the rest of the ectoderm lying underneath (Figs 4 C &4D). The thickness of the collagenous layer ranges from 4 ��m to 30 ��m with varying internal composition. In the arborescent morphotype, regularly spaced invaginations of the basal region of the collagenous layer extend into the mesohyl (Figs 3 I & 4B). Both regions of the collagenous layer include few scattered spherulous cells and bacteria (Figs 3 C & 3I, 4A & 4B). The choanosome is typical of all Halisarca species previously described in the literature, with irregularly shaped, elongated and curved choanocyte chambers (30 to 180 ��m long x 10 to 40 ��m wide), aquiferous canals and a mesohyl containing a variety of cell types, bacteria and collagen bundles (Figs 3 A���3C, 3E��� 3I & 5A). No difference in abundance or size of choanocyte chambers is observed between morphotypes 1 & 3 (Figs 3 B, 3C & 3F���3H). Chambers are less abundant in morphotype 2 (MNRJ 10753) and occur deeper in the sponge, under a 900 ��m thick ectosome (Fig 3 D). In some sections, choanocyte chambers are located radially around several thin, probably inhalant, canals 4 to 16 ��m in diameter (Fig 3 F). Larger canals, most likely exhalant, occur less frequently in the choanosome (Figs 3 A & 3D). In semi-thin sections stained with methylene blue/azure II and basic fuchsine, the mesohyl stains pink to purplish-red, indicating the abundance of collagen. TEM sections reveal the granular structure of the mesohyl; abundant electron dense granules 19 to 93 nm in diameter amalgamate in a fuzzy loose matrix crossed by collagen fibrils and bundles (Figs 7 D, 9A, 10D & 10G). Cytology. Exopinacocytes (Figs 4 A���4C, 4E & 4F) are "T-shaped" in transverse sections with an extremely thin superficial extension situated at the tip of a narrow projection emerging from the cell body and connecting cells to each other through inter-cellular junctions (Figs 4 E & 4F). Cell bodies (2.5 to 8.6 ��m in diameter), with a basal nucleolated nucleus, are anchored in the mesohyl below the complex collagen layer of the ectosome. Choanocytes (Figs 5 A���5E) are long and cylindrical (10.5���14.5 ��m x 4.0���4.5 ��m), with abundant basal pseudopodial extensions. The cytoplasm contains many electron translucent vacuoles, abundant larger electron dense basal inclusions and an apical nucleolated nucleus. An irregular periflagellar sleeve, sometimes longer than the microvilli of the collar, surrounds the base of the flagellum. Endopinacocytes (Figs 6 A���6C) are flat cells lining the canals of the aquiferous system. Their shape is thin and flat with a central oval nucleolated nucleus. Basopinacocytes were not observed. Archaeocytes (Fig. 7 A) are around 7.0 to 15.0 ��m in size, occur infrequently throughout the mesohyl and have a classical amoeboid shape with pseudopodia. The nucleolated nucleus (3.2 to 3.9 ��m in diameter) is spherical and located centrally. The cytoplasm is dense with phagosomes and electron-dense vacuoles of variable sizes ranging from 150 to 470 nm. Lophocytes (Figs 7 B���7D) are abundant elongated cells (around 1.5 x 14.0 ��m) with a tuft of collagen fibrils emerging from their posterior end. The nucleus is either located in the anterior (Fig. 7 B) or the posterior (Fig. 7 C) side of the cell. The cytoplasm is dense, contains phagosomes and small electron-dense inclusions (140 to 960 nm). Spherulous cells are of three types, two of which were never previously described in Halisarca. Spherulous cells Type I (Fig. 7 E) are globular oval cells (6.5���8.2 x 9.0���10.6 ��m in diameter) packed with large dense osmiophilic homogeneous membrane bound inclusions of variable diameter (1.1 to 4.8 ��m) squeezing a central nucleolated nucleus, and a relatively small remaining cytoplasmic volume. They are the most abundant mesohylar cells in the ectosome and the choanosome of all investigated specimens. Spherulous cells Type II (Fig. 7 F) are large, bulbous and spherical (7.8��� 8.9 x 7.8���11.6 ��m in diameter) with large inclusions of variable diameter (1.3 to 4.2 ��m) composed of two distinct parts: a large crescent-shaped osmiophilic portion encompasses a smaller elliptical less osmiophilic region, giving a "chestnut aspect" to these inclusions. The cytoplasm volume is also minimized and the central nucleus is nucleolated. These cells do not occur in all specimens but, when present, are common in the choanosome (Table 1). Spherulous cells Type III (Fig. 7 G) are more spherical (5.8���9.4 x 7.1���11.7 ��m) than the two previous types and contain heterogeneous inclusions (1.1���2.1 x 2.2���3.3 ��m) characterized by a rough internal texture with a more osmiophilic periphery. The cytoplasm volume is larger than in Type I and Type II, with a spherical nucleolated nucleus. These cells are rare, found only in the choanosome and are not observed in all specimens (Table 1). Granular cells (Fig. 7 H) are oval shaped cells (5.5��� 63 x 7.1���11.3 ��m) with a nucleolated nucleus and spherical homogeneous osmiophilic inclusions of variable size, but smaller than in spherulous cells Type 1 (0.6 x 1.6���2.3 ��m in diameter). These cells are infrequent, occur only in the choanosome and are not present in all specimens (Table 1). Microgranular cells (Fig. 8 A) are amoeboid cells (7.5���10.6 x 4.2���7.5 ��m), distinct from granular cells by their anucleolated nucleus and the abundance of electron dense spherical or elliptical inclusions of smaller size (0.5���0.8 x 0.2 ��m) contained in the cytoplasm. Small phagosomes and a developed endoplasmic reticulum are also present. Microgranular cells occur in all specimens examined but are not abundant, and are found randomly in the choanosome. Rhabdiferous cells (Figs 8 B���8E) are amoeboid in shape (4.5���10.7 x 3.3���7.6 ��m) with short pseudopodia. The nucleolated nucleus is relatively large and spherical (2.1���3.6 ��m in diameter). The cytoplasm is filled with osmiophilic elliptical membrane bound inclusions (0.7���1.7 x 0.4���0.7 ��m), which include one or two less dense spherical sub-inclusions (rhabdites). Rhabdiferous cells are numerous in all specimens examined and occur equally in the ectosome and the choanosome where they are even occasionally found within choanocyte chambers (Figs 8 D & 8E). Pocket cells (Figs 9 A���9G) are amoeboid in shape, of variable size, (6.3���8.8 x 8.9���16.9 ��m) characterized by one or several large "pockets" formed by junction of long and thin pseudopodial extensions engulfing portions of the mesohyl that often include bacteria. As the size and the number of pockets progressively increase, the volume of the cytoplasm decreases until it becomes a thin layer surrounding a nucleolated nucleus (1.7���2.1 x 2.2���3.3 ��m). The final content of the pockets has the same density as the mesohyl. The cytoplasm contains phagosomes and abundant electron dense and electron translucent small inclusions. Bacteriocytes (Figs 10 A���10C) are roundish massive cells (10.0���18.3 ��m in diameter) with pseudopodial extensions and packed with large bacteria of a single morphotype (Type B1). The cytoplasm is dense and the anucleolated nucleus is squeezed at the periphery of the cell. Bacteria are rod-shaped up to 4.5 ��m long and 1.2 ��m wide and are isolated from the cytoplasm by a membrane. All present a thick electron dense cytoplasm with a thin central electron translucent filamentous nucleoid. Bacteriocytes do not occur in all specimens, are infrequent and appear randomly in the choanosome. Extracellular symbiotic bacteria (Figs 10 D���10F). Two other morphotypes of bacteria are found extracellularly, evenly distributed in the mesohyl, spanning from the outward collagenous layer of the ectosome to the entire choanosome (Types B2 and B3). Type B2 is abundant, rod-shaped and reaches 910 nm in length and 380 nm in diameter. The cytoplasmic matrix is homogeneous and electron dense. The nucleoid is more electron translucent, with a central rod surrounded by filamentous material. The cell wall is thick and covered with discrete fimbriae. Type B3 is spiral-shaped and has a thick cell wall. The length is undetermined in TEM since an entire longitudinal section is unlikely obtained. The width ranges 200 to 470 nm. The cytoplasmic matrix is clear and the nucleoid is homogeneous and electron dense. Type B3 is much less frequent, occurs in a minority of specimens and is found exclusively in the choanosome (Table 1). Reproduction. One of both specimens collected in March 2005 and two out of four collected in February 2009 contained oocytes in different stages of development. Distribution. Halisarca magellanica is known from austral Chile (56�����54��S), Argentina and Uruguay (56����� 34��S) and Malvinas Islands (Desqueyroux & Moyano 1987). Habitat. Halisarca magellanica is commonly found at depths ranging from 1 to 26 m, on mussels beds at shallow depths or covering rocks below 10 m. Phylogenetic analysis. Partial cox1 sequences were determined for five specimens of H. magellanica (RBINS 10827 = morphotype 1; RBINS 10753 = morphotype 2; RBINS 12918, 12920 and 12921 = morphotype 3) and were found to be identical. Phylogenetic analysis of these data along with previously published sequences of H. dujardini, H. caerulea, H. harmelini, Halisarca sp. GW3298 from the Red Sea and selected outgroups showed H. magellanica forming a sister group to all other analysed Halisarca sequences except for H. harmelini (Fig. 19). The cox1 sequence of H. magellanica was 9.1���9.7% different from the other Halisarca species., Published as part of Willenz, Philippe, Ereskovsky, Alexander V. & Lavrov, Dennis V., 2016, Integrative taxonomic re-description of Halisarca magellanica and description of a new species of Halisarca (Porifera, Demospongiae) from Chilean Patagonia, pp. 501-533 in Zootaxa 4208 (6) on pages 503-515, DOI: 10.11646/zootaxa.4208.6.1, http://zenodo.org/record/214957, {"references":["Topsent, E. (1901) Spongiaires. Resultats du voyage du S. Y. Belgica en 1897 - 99 sous le commandement de A. de Gerlache de Gomery. Expedition antarctique belge. Zoologie, 4, 1 - 54.","Thiele, J. (1905) Die Kiesel- und Hornschwamme der Sammlung Plate. Supplement 6 (Fauna Chiliensis III). Zoologische Jahrbucher, 1905, 407 - 496, pls 427 - 433.","Burton, M. (1929) Porifera. Part II. Antarctic sponges. British Antarctic (\" Terra Nova \") Expedition, 1910. Natural History Report. London, British Museum (Natural History). Zoology, 6, 393 - 458, pls I - V.","Burton, M. (1932) Sponges. Discovery Reports, pp. 237 - 392, pls 248 - 257. http: // dx. doi. org / 10.5962 / bhl. part. 24379","Desqueyroux, R. & Moyano, H. (1987) Zoogeografia de Demospongias Chilenas-Zoogeography of Chilean Demospongiae. Boletin de la Sociedad de Biologia de Concepcion, 58, 39 - 66."]}

International audience; The germline definition in metazoans was first based on few bilaterian models. As a result, gene function interpretations were often based on phenotypes observed in those models and led to the definition of a set of genes, considered as specific of the germline, named the " germline core ". However, some of these genes were shown to also be involved in somatic stem cells, thus leading to the notion of germline multipotency program (GMP). Because Porifera and Ctenophora are currently the best candidates to be the sister-group to all other animals, the comparative analysis of gene contents and functions between these phyla, Cnidaria and Bilateria is expected to provide clues on early animal evolution and on the links between somatic and germ lineages. Our present bioinformatic analyses at the metazoan scale show that a set of 18 GMP genes was already present in the last common ancestor of metazoans and indicate more precisely the evolution of some of them in the animal lineage. The expression patterns and levels of 11 of these genes in the homoscleromorph sponge Oscarella lobularis show that they are expressed throughout their life cycle, in pluri/multipotent progenitors, during gametogenesis, embryogenesis and during wound healing. This new study in a nonbilaterian species reinforces the hypothesis of an ancestral multipotency program.

Background Wnt proteins are secreted signalling molecules found in all animal phyla. In bilaterian animals, including humans, Wnt proteins play key roles in development, maintenance of homeostasis and regeneration. While Wnt gene repertoires and roles are strongly conserved between cnidarians and bilaterians, Wnt genes from basal metazoans (sponges, ctenophores, placozoans) are difficult or impossible to assign to the bilaterian + cnidarian orthologous groups. Moreover, dramatic differences in Wnt numbers among basal metazoan exist, with only three present in the genome of Amphimedon queenslandica, a demosponge, and 21 in the genome of Sycon ciliatum, a calcisponge. To gain insight into the ancestral Wnt repertoire and function, we have chosen to investigate Wnt genes in Halisarca dujardini, a demosponge with relatively well described development and regeneration, and a very distant phylogenetic relationship to Amphimedon. Results Here we describe generation of a eukaryotic contamination-free transcriptome of Halisarca dujardini, and analysis of Wnt genes repertoire and expression in this species. We have identified ten Wnt genes, with only one orthologous to Amphimedon Wnt, and six appearing to be a result of a lineage specific expansion. Expression analysis carried out by in situ hybridization of adults and larvae revealed that two Halisarca Wnts are expressed in nested domains in the posterior half of the larvae, and six along the adult body axis, with two specific to the osculum. Strikingly, expression of one of the Wnt genes was elevated in the region undergoing regeneration. Conclusions Our results demonstrated that the three Poriferan lineages (Demospongiae, Calcarea and Homoloscleromorpha) are characterized by highly diverse Wnt gene repertoires which do not display higher similarity to each other than they do to the non-sponge (i.e. ctenophore, cnidarian and bilaterian) repertoires. This is in striking contrast to the uniform Wnt repertoires in Cnidarians and Bilaterians, suggesting that the Wnt family composition became “fixed” only in the last common ancestor of Cnidarians and Bilaterians. In contrast, expression of Wnt genes in the apical region of sponge adults and the posterior region of sponge larvae suggests conservation of the Wnt role in axial patterning across the animal kingdom. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0700-6) contains supplementary material, which is available to authorized users.

International audience; Matrotrophy, the continuous extra-vitelline supply of nutrients from the parent to the progeny during gestation, is one of the masterpieces of nature, contributing to offspring fitness and often correlated with evolutionary diversification. The most elaborate form of matrotrophy—placentotrophy—is well known for its broad occurrence among vertebrates, but the comparative distribution and structural diversity of matrotrophic expression among invertebrates is wanting. In the first comprehensive analysis of matrotrophy across the animal kingdom, we report that regardless of the degree of expression, it is established or inferred in at least 21 of 34 animal phyla, significantly exceeding previous accounts and changing the old paradigm that these phenomena are infrequent among invertebrates. In 10 phyla, matrotrophy is represented by only one or a few species, whereas in 11 it is either not uncommon or widespread and even pervasive. Among invertebrate phyla, Platyhelminthes, Arthropoda and Bryozoa dominate, with 162, 83 and 53 partly or wholly matrotrophic families, respectively. In comparison, Chordata has more than 220 families that include or consist entirely of matrotrophic species. We analysed the distribution of reproductive patterns among and within invertebrate phyla using recently published molecular phylogenies: matrotrophy has seemingly evolved at least 140 times in all major superclades: Parazoa and Eumetazoa, Radiata and Bilateria, Protostomia and Deuterostomia, Lophotrochozoa and Ecdysozoa. In Cycliophora and some Digenea, it may have evolved twice in the same life cycle. The provisioning of developing young is associated with almost all known types of incubation chambers, with matrotrophic viviparity more widespread (20 phyla) than brooding (10 phyla). In nine phyla, both matrotrophic incubation types are present. Matrotrophy is expressed in five nutritive modes, of which histotrophy and placentotrophy are most prevalent. Oophagy, embryophagy and histophagy are rarer, plausibly evolving through heterochronous development of the embryonic mouthparts and digestive system. During gestation, matrotrophic modes can shift, intergrade, and be performed simultaneously. Invertebrate matrotrophic adaptations are less complex structurally than in chordates, but they are more diverse, being formed either by a parent, embryo, or both. In a broad and still preliminary sense, there are indications of trends or grades of evolutionarily increasing complexity of nutritive structures: formation of (i) local zones of enhanced nutritional transport (placental analogues), including specialized parent–offspring cell complexes and various appendages increasing the entire secreting and absorbing surfaces as well as the contact surface between embryo and parent, (ii) compartmentalization of the common incubatory space into more compact and 'isolated' chambers with presumably more effective nutritional relationships, and (iii) internal secretory ('milk') glands. Some placental analogues in onychophorans and arthropods mimic the simplest placental variants in vertebrates, comprising striking examples of convergent evolution acting at all levels—positional, structural and physiological.

A series of recent expeditions in fjords and canals of Southern Chilean Patagonia allowed the re-collection of Halisarca magellanica Topsent, 1901 and the discovery of a new species, Halisarca desqueyrouxae sp. nov. The material studied was collected at depths ranging from 3 to 30 m at latitudes comprised between 42° and 49°S. Both species share the same habitat and show a morphological plasticity, but differ in their colour. Halisarca magellanica is bright pink to whitish with three morphs whereas H. desqueyrouxae sp. nov. is light brown to beige with two morphs. An extensive investigation in TEM and SEM reveals several differences among cell types with inclusions between both species. Three distinct spherulous cells occur. Type 1 is shared by both species, Type 2 is occasional in H. magellanica but absent from H. desqueyrouxae sp. nov. Type 3 is rare in H. magellanica and occurs abundantly in half of the specimens of H. desqueyrouxae sp. nov. Granular cells are shared by both species but do not occur in all specimens. Microgranular cells are characteristic of H. magellanica. Both species also clearly differ by their endobiotic bacteria. Phylogenetic analysis of cox1 sequences places H. magellanica as a sister group to all other previously published Halisarca species sequences (9.1–9.7% difference) except H. harmelini , while H. desqueyrouxae sp. nov. is placed as a sister group to H. dujardini (2.3% difference).

International audience; The cod, Gadus morhua, is a common and abundant demer-sal fish in the White Sea coastal zone. The published data on the maximum age of White Sea cod still requires additional documentation, because some authors have noted difficulties in age determination by otoliths of large specimens. To obtain accurate data on the longevity of this species in the White Sea, an image analysis of thin-sections of otoliths was made for age estimation. Research surveys in the Chupa Inlet and adjacent waters of the Kandalaksha Bay were con-ducted in June to August from 2007 to 2013. The five largest specimens of a total of 3564 captured fish were selected for age determination. Age of these largest individuals ranged between 7 and 12 years, total length and weight varied from 60.2 to 77.0 cm and from 2.4 to 6.1 kg, respectively. Accord-ing to the data, maximal White Sea cod longevity is greater than in previously published data, and demonstrates similar longevity to conspecifics from the Baltic and North seas, whereas its life cycle is much shorter than cod from the Northeast Arctic, Iceland, Greenland, Newfoundland and Labrador stocks.

International audience; Diatoms are considered unicellular eukaryotic organisms exclusively depositing biogenic silica. Heretofore there has been no report of calcification by these algae. Here it is shown that calcium carbonate within the stalks of Didymosphenia geminata, a nuisance species that has prolifically colonized streams and rivers globally, is biogenic in origin and occurs as a network of calcite nanofibers. The nanofibrous framework in the mineralized polysaccharide matrix imparts mechanical support to the stalks, providing stability in variable flow conditions. The results demonstrate that D. geminata possesses cellular and periplasmic carbonic‐anhydrases that contribute to carbon fixation and biomineralization, respectively. The activity of external carbonic‐anhydrase was more than 50% of the total activity, which points to its role in anchoring this bioeroding diatom on hard surfaces. The first evidence of multiphase biomineralization by diatoms that deposit both biogenic silica and crystalline biogenic calcite which are imparting distinct functional advantage to the organism is provided.

Borisenko, I. and Ereskovsky, A.V. 2011. Tentacular apparatus ultrastructure in the larva of Bolinopsis infundibulum (Lobata: Ctenophora). —Acta Zoologica (Stockholm) 00: 1–10. Most ctenophores have a tentacular apparatus, which plays some role in their feeding. Tentacle structure has been described in adults of only three ctenophore species, but the larval tentacles have remained completely unstudied. We made a light and electron microscopic study of the tentacular apparatus in the larvae of Bolinopsis infundibulum from the White Sea. The tentacular apparatus of B. infundibulum larvae consists of the tentacle proper and the tentacle root. The former contains terminally differentiated cells, while the latter contains stem cells and cells undergoing differentiation. The core of the tentacle is formed by myocytes, and its epidermis contains colloblasts (hunting cells), wall cells, degenerating cask cells, refractive vesicles, and ciliated sensory cells. Stem cells, colloblasts, and cask cells at various stages of differentiation and putative myocytes progenitors were revealed in the tentacle root. Two different populations of the stem cells in the tentacle root give rise to epidermal (colloblasts and cask cells) and mesogleal (myocytes) cell lines. Nervous elements, glandular cells, and basal lamina were not found. Step-by-step differentiation of colloblasts and cask cells is described.

International audience; Over the past few years, there has been growing interest among the sponge community in the phylogenetic position of the Homoscleromorpha (i.e. within or outside the class Demospongiae). Recent molecular analyses clearly show that the Homoscler-omorpha forms a distinct clade separated from the Demospongiae and is composed of two families, Oscarellidae and Plakinidae. Within the currently more widely accepted hypothesis of a monophyletic Porifera, we formally propose here to raise Homos-cleromorpha to the class rank (the fourth one). We, therefore, provide a definition and a formal diagnosis. In the supplementary materials, we also present an alternative classification of the Homoscleromorpha, following the PhyloCode.

Lysophospholipids (LPLs) are recognized as important signaling molecules in metazoan cells. LPLs seem to be widely distributed among marine invertebrates, but their physiological role remains poorly known. Marine sponges produce original phospholipids and LPLs whose isolation and structural elucidation rarely have been reported. Two LPLs were isolated for the first time from the Mediterranean Homoscleromorph sponge Oscarella tuberculata: a bioactive lyso-PAF already identified in some other sponge species; and the new lysophosphatidylethanolamine C20:2 (LPE 1). The expression of LPL metabolites was investigated over time to determine their baseline variations and to relate them to the sponge reproduction pattern in order to better understand their putative role in the sponge life cycle. Expression levels of both compounds appeared to be highly correlated displaying significant seasonal fluctuations with maximal values in summer and minimal in winter. A significant higher LPL content was detected in reproductive sponges and especially in females, with a peak occurring during embryogenesis and larval development. The results suggest that LPLs could play a role of mediators in sponge embryogenesis and morphogenesis.

A new homoscleromorph sponge species was observed for the first time in 2002 along the coast of Provence (France) and since then, several investigations have been implemented. The new homoscleromorph species is devoid of skeleton and it is thus related to the Oscarella genus. Oscarella balibaloi sp. nov. has been found in several littoral submarine caves of the Western Mediterranean Sea (France and Spain). After several years of monitoring, we show that this sponge is becoming abundant in several locations. The species has a peculiar habit, often overgrowing massive sponges, gorgonians or some erected bryozoans. Oscarella balibaloi sp. nov. is thinly encrusting, with an irregular morphology, lobate surface, and soft and very slimy consistency. Its color is white to orange. At the cytological level, this new species is characterized by three distinct types of cells with inclusions in its mesohyl: one with vacuoles and the others with paracrystalline inclusions. The reproduction pattern is very similar to other Oscarella species. We also present a specific metabolic fingerprint and compare it to that of other Mediterranean Oscarella species.

The work was aimed at performing long-term cultivation of primmorphs in vitro from freshwater sponge Lubomirskia baikalensis (Pallas 1776), collected from Lake Baikal, obtaining its long-term primmorph culture in both natural (NBW) and artificial (ABW) Baikal water and at identifying the impact of different environmental factors on formation and growth of primmorphs. The first fine aggregates of L. baikalensis are formed in vitro 10-15 min after dissociation of sponge cells. Epithelization of aggregates begins 4 h later after the dissociation. Young primmorphs are formed 1 or 2 days later. The surface of primmorphs is covered with a layer of exopinacocytes. The primmorphs remain viable for more than 10 months at 3-6 °C. Over 50% of primmorphs in NBW and 25% in ABW are attached to the substrate and grow like adult sponges. Thus, the long-term primmorph cultivation in vitro allows the creation of a controlled live model system under experimental conditions. The results of this work will allow the creation of a cell culture collection of Baikal freshwater sponges for studying morphogenesis of primmorphs during cultivation at different stages and transdifferentiation of their cells, physiological functions of sponge cells, processes of spiculogenesis, identification of proteins involved in biomineralization process, decoding of their genes, as well as a spectrum of secondary metabolites.

In order to evaluate the biomedical potential of three-dimensional chitinous scaffolds of poriferan origin, chondrocyte culturing experiments were performed. It was shown for the first time that freshly isolated chondrocytes attached well to the chitin scaffold and synthesized an extracellular matrix similar to that found in other cartilage tissue engineering constructs. Chitin scaffolds also supported deposition of a proteoglycan-rich extracellular matrix of chondrocytes seeded bioconstructs in an in vivo environment. We suggest that chitin sponge scaffolds, apart from the demonstrated biomedical applications, are highly optimized structures for use as filtering systems, templates for biomineralization as well as metallization in order to produce catalysts.

Marine invertebrate organisms including sponges (Porifera) not only provide an abundant source of biologically active secondary metabolites but also inspire investigations to develop biomimetic composites, scaffolds and templates for practical use in materials science, biomedicine and tissue engineering. Here, we presented a detailed study of the structural and physico-chemical properties of three-dimensional skeletal scaffolds of the marine sponges Aiolochroia crassa, Aplysina aerophoba, A. cauliformis, A. cavernicola, and A. fulva (Verongida: Demospongiae). We show that these fibrous scaffolds have a multilayered design and are made of chitin. (13)C solid-state NMR spectroscopy, NEXAFS, and IR spectroscopy as well as chitinase digestion and test were applied in order to unequivocally prove the existence of alpha-chitin in all investigated species.

Sponges can filter large volumes of seawater and accumulate highly diverse and abundant microbial communities within their tissue. Culture-independent techniques such as fluorescent in situ hybridization (FISH), 16S small subunit (SSU) rRNA gene analyses, and transmission electron microscopy (TEM) were applied to characterize the presence and distribution of microbes within sponges abundant on south Florida reefs. This study found that coral disease-associated bacteria (CDAB) are harbored within Agelas tubulata and Amphimedon compressa. FISH probes detected several potential bacterial pathogens such as Aurantimonas coralicida, Cytophaga sp., Desulfovibrio spp, Serratia marcescans, and Vibrio mediterranei within A. compressa and A. tubulata host sponges. Spatial differences in the distribution of targeted bacteria were seen within sponge hosts. Transmission electron microscopy of A. compressa indicated there was a higher concentration of bacteria in the choanosome compared to the ectosome. These observed spatial distributions support the presence of internal sponge niches, which could play a role in the location of the CDAB within the sponges.

Biological materials are a rewarding area of modern materials science, yielding both evolutionary insights and inspiration for biomimetic research. In particular, biocomposite structures are valuable sources of novel structures with unusual chemical properties, and they are very informative for the mechanisms of biomineralization. Here we describe a unique biocomposite of amorphous silica, crystalline aragonite, and chitin from species of the order Verongida, a group of marine sponges. The structures have been analyzed with a diverse suite of techniques, revealing a chitinous template for siliceous overgrowth containing aragonite-based crystal aggregates. Sponge chitin is an example of a specific template where two minerals in amorphous and crystalline forms are formed together with an organic molecule.

Morphological investigations of morphogenesis accompanying the metamorphosis of the cinctoblastula larva of poriferan Plakina trilopha (Homoscleromorpha) have been made. The larva possesses a distinct columnar epithelium which subdivides into three cellular areas: antero-lateral, postero-lateral, and posterior one. Characteristic morphological features of the cells in each area can be used as natural markers when tracing the fate of larval cells during metamorphosis. The ciliated epithelium of the larva is transformed directly into choanoderm and pinacoderm, without losing its organization. This transformation is a peculiar feature of the metamorphosis in Homoscleromorpha. Metamorphosis in P. trilopha is based on epithelial morphogenesis and includes the mechanisms of flattening of the exopinacoderm, evagination and invagination of larval epithelium in the course of the development of the rhagon aquiferous system. The flattening of larval cells during exopinacoderm formation in metamorphosing P. trilopha is a common change of cell shape during epithelial morphogenesis of this species. The separation of proximal fragments of cells has been observed here. This phenomenon, that we have called “cytoplasmic shedding”, appears to play an important role in the change of epithelial cell shape in P. trilopha. Mechanism of epithelial–mesenchymal transition, i.e., ingression of epithelial ciliated cells into the cavity of the metamorphosing larva of P. trilopha participates in mesohylar cell origin.

The capacity of all cells to respond to stimuli implies the conduction of information at least over short distances. In multicellular organisms, more complex systems of integration and coordination of activities are necessary. In most animals, the processing of information is performed by a nervous system. Among the most basal taxa, sponges are nerveless so that it is traditionally assumed that the integrated neuro-sensory system originated only once in Eumetazoa, a hypothesis not in agreement with some recent phylogenomic studies. The aim of this review is to show that recent data on sponges might provide clues for understanding the origin of this complex system. First, sponges are able to react to external stimuli, and some of them display spontaneous movement activities. These coordinated behaviors involve nervous system-like mechanisms, such as action potentials and/or neurotransmitters. Second, genomic analyses show that sponges possess genes orthologous to those involved in the patterning or functioning of the neuro-sensory system in Eumetazoa. Finally, some of these genes are expressed in specific cells (flask cells, choanocytes). Together with ultrastructural data, this gives rise to challenging hypotheses concerning cell types that might play neuro-sensory-like roles in sponges.

Symbiotic bacteria from six Oscarella species (adults and embryos) collected in the Mediterranean Sea (O. lobularis, O. tuberculata, O. imperialis, O. microlobata, O. viridis) and the Sea of Japan (O. malakhovi) were investigated by scanning electron microscopy and transmission electron microscopy. In most cases, symbionts are rather numerous. Each sponge species has a definite set of bacterial morphological types. All bacteria are extracellular. Symbionts occupy the mesohyl of adult sponges or intercellular space in embryos and are often in contact with mesohylar filaments or cells. Bacteria of some morphotypes have characteristic blebs. Most symbionts are gram-negative, and two types of bacteria have traits of Archaea and one type of bacteria is similar to Planctomycetes. Data on morphology of bacterial symbionts can be a good additional character for identification of Oscarella species, which have no skeleton.

Sponges branch basally in the metazoan phylogenetic tree and are believed to be composed of four distinct lineages with still uncertain relationships. Indeed, some molecular studies propose that Homoscleromorpha may be a fourth Sponge lineage, distinct from Demospongiae in which they were traditionally classified. They harbour many features that distinguish them from other sponges and are more evocative of those of the eumetazoans. They are notably the only sponges to possess a basement membrane with collagen IV and specialized cell-junctions, thus possessing true epithelia. Among Homoscleromorphs, we have chosen Oscarella lobularis as a model species. This common and easily accessible sponge is characterized by relatively simple histology and cell composition, absence of skeleton, and strongly pronounced epithelial structure. In this review, we explore the specific features that make O. lobularis a promising homoscleromorph sponge model for evolutionary and developmental researches.

Larval development and follicle structure of a representative of the Calcinea (Calcispongiae) Guancha arnesenae from the White Sea have been studied for the first time at the ultrastructural level. The follicle in G. arnesenae has an unusual structure: it consists of trapezoid cells rich in phagosomes and a surrounding dense collagen layer. Follicular cells differentiate from choanocytes. Cleavage results in formation of a hollow, equal, non-polarized coeloblastula. Larval morphogenesis occurs by means of direct hollow blastula formation without any individual cell or cell layer movements. The coeloblastula (calciblastula) larva of G. arnesenae is completely ciliated. The larva also contains rare non-ciliated cells: vacuolar cells, bottle-shaped cells and free cells in a central cavity. The basal ciliary apparatus of larval cells includes the basal body, an accessory centriole oriented perpendicularly to it, the basal foot, and two cross-striated rootlets. A bundle of microtubules emerges from the side of the basal body, opposite to the basal foot, running parallel to the outer surface. All bundles of cells are parallel to each other and oriented towards the posterior larval pole, forming a transverse cytoskeletal system. Specialized intercellular junctions in the apical regions of all ciliated cells are revealed for the first time in a Calcispongiae larva. The central larval cavity contains symbiotic bacteria, which are included inside the embryo at the blastula stage.

Esperiopsis koltuni, a new species of the sponge family Esperiopsidae, is described from deep water of the Sea of Okhotsk. The new species has a unique growth form: there is a basal plate with radiating cylindrical branches, whose oval flat distal parts bear filament-like outgrowths. Megascleres are arranged differently in the main part of branches and in their distal flat parts. The dermal membrane contains isochelae with protruding alae and abundant sigmancistras. Microscleres are represented by large anchorate spatuliferous isochelae, small isochelae, sigmas and sigmancistras. Esperiopsis koltuni sp. nov. is hermaphroditic, with parenchymella larva. The larval skeleton consists only of sigmas and sigmancistras. Esperiopsis koltuni sp. nov. can be easily distinguished from other Esperiopsis species by dimensions and combination of spicule types. In particular, it is the only species in the genus with three different size categories of styles. The new species lacks the aquiferous system, has a characteristic body plan with symmetrical lateral expansions, an unusual arrangement of microscleres in the dermal membrane and true sigmancistras. Moreover, prey capture by a filament-like outgrowth of the flat distal part was observed. All of these characteristics indicate that Esperiopsis koltuni sp. nov. is a carnivorous sponge.

The Mediterranean Sea is a hotspot for marine biodiversity. Past studies of Mediterranean marine caves have revealed the unique biocoenotic and ecological characteristics of these habitats, which are protected by European Union legislation. The Maltese Islands have an abundance of partially and fully submerged marine caves with di fferent geomorphological characteristics, yet there have been no systematic studies on these habitats and their associated species. This study is a firrst synthesis of existing information on the biotic assemblages and physical characteristics of Maltese marine caves. The work combines a review of the available information with a preliminary survey of some marine caves in Gozo, during which several species were recorded for the first time for the Maltese Islands. Characteristic species recorded from local marine caves are highlighted, including several species of red and brown algae, sessile invertebrates including bryozoans, ascidians and sponges, and mobile forms including crustaceans and fi sh. A marked zonation from the cave entrance to the inside of the caves was identifi ed: photophilic algae at the mouth of the cave are progressively replaced by more sciaphilic species, followed by a middle section dominated by sessile invertebrates, and then a completely dark inner section that is mostly devoid of sessile organisms. Several species protected by national and international legislation were found to occur., peer-reviewed

International audience; Sponges are known to possess remarkable reconstitutive and regenerative abilities ranging from common wounding or body part regeneration to more impressive re-building of a functional body from dissociated cells. Among the four sponge classes, Homoscleromorpha is notably the only sponge group to possess morphologically distinct basement membrane and specialized cell-junctions, and is therefore considered to possess true epithelia. The consequence of this peculiar organization is the predominance of epithelial morphogenesis during ontogenesis of these sponges. In this work we reveal the underlying cellular mechanisms used during morphogenesis accompanying ectosome regeneration in the homoscleromorph sponge model: Oscarella lobularis. We identified three main sources of novel exopinacoderm during the processes of its regeneration and the restoration of functional peripheral parts of the aquiferous system in O. lobularis: (1) intact exopinacoderm surrounding the wound surface, (2) the endopinacoderm from peripheral exhalant and inhalant canals, and (3) the intact choanoderm found on the wound surface. The basic morphogenetic processes during regeneration are the spreading and fusion of epithelial sheets that merge into one continuous epithelium. Transdifferentiation of choanocytes into exopinacocytes is also present. Epithelial-mesenchymal transition is absent during regeneration. Moreover, we cannot reveal any other morphologically distinct pluripotent cells. In Oscarella, neither blastema formation nor local dedifferentiation and proliferation have been detected, which is probably due to the high morphogenetic plasticity of the tissue. Regeneration in O. lobularis goes through cell transdifferentiation and through the processes, when lost body parts are replaced by the remodeling of the remaining tissue. Morphogenesis during ectosome regeneration in O. lobularis is correlated with its true epithelial organization. Knowledge of the morphological basis of morphogenesis during Oscarella regeneration could have important implications for our understanding of the diversity and evolution of regeneration mechanisms in metazoans, and is a strong basis for future investigations with molecular-biological approaches.

International audience; Secondary metabolites play a crucial role in marine invertebrate chemical ecology. Thus, it is of great importance to understand factors regulating their production and sources of variability. This work aimed to study the variability of the bromotyrosine derivatives in the Mediterranean sponge Aplysina cavernicola, and also to better understand how biotic (reproductive state) and abiotic factors (seawater temperature) could partly explain this variability. Results showed that the A. cavernicola reproductive cycle has little effect on the variability of the sponges’ secondary metabolism, whereas water temperature has a significant influence on the production level of secondary metabolites. Temporal variability analysis of the sponge methanolic extracts showed that bioactivity variability was related to the presence of the minor secondary metabolite dienone, which accounted for 50 % of the bioactivity observed. Further bioassays coupled to HPLC extract fractionation confirmed that dienone was the only compound from Aplysina alkaloids to display a strong bioactivity. Both dienone production and bioactivity showed a notable increase in October 2008, after a late-summer warming episode, indicating that A. cavernicola might be able to induce chemical changes to cope with environmental stressors.

International audience; The ability to regenerate is widespread in the animal kingdom, but the regenerative capacities and mechanisms vary widely. To understand the evolutionary history of the diverse regeneration mechanisms, the regeneration processes must be studied in early-evolved metazoans in addition to the traditional bilaterian and cnidarian models. For this purpose, we have combined several microscopy techniques to study mechanisms of regeneration in the demosponge Halisarca dujardini. The objectives of this work are to detect the cells and morphogenetic processes involved in Halisarca regeneration. We show that in Halisarca there are three main sources of the new exopinacoderm during regeneration: choanocytes, archaeocytes and (rarely) endopinacocytes. Here we show that epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) occur during Halisarca regeneration. EMT is the principal mechanism during the first stages of regeneration, soon after the injury. Epithelial cells from damaged and adjacent intact choanocyte chambers and aquiferous canals assume mesenchymal phenotype and migrate into the mesohyl. Together with archaeocytes, these cells form an undifferentiated cell mass beneath of wound, which we refer to as a blastema. After the blastema is formed, MET becomes the principal mechanism of regeneration. Altogether, we demonstrate that regeneration in demosponges involves a variety of processes utilized during regeneration in other animals (e.g., cell migration, dedifferentiation, blastema formation) and points to the particular importance of transdifferentiation in this process. Further studies will be needed to uncover the molecular mechanisms governing regeneration in sponges. Subjects Developmental Biology

Asexual reproduction by external budding in Homoscleromorpha is reported for the first time. Two Mediterranean sponge species were studied, Oscarella lobularis and O. tuberculata. Buds are formed in the marginal basal part of sponge. Budding takes from 1 to 4 days and is defined in three budding stages. First, small irregular protuberances, consisting of external parental tissue, are formed. Second, they elongate and acquire more regular, nipple-like shape. These protuberances are tube like, their internal cavity derived from parental exhalant canal. The wall consists of three layers: (a) external layer is flagellated exopinacoderm, (b) internal one is flagellated endopinacoderm and (c) intermediate one is a thin layer of mesohyl. Third, a spherical bud with a large central cavity is formed. During budding, we did not observe cell proliferation or transdifferentiation either in budding zone or in any special mitotically active region. The bud attached to the substrate is similar to the rhagon developing after larva metamorphosis, it has a syconoid organization. Morphogenetically, budding in Oscarella differes from that in other sponges. Occurring by epithelial morphogenesis, it is similar to morphallaxis during regeneration. The presence in Homoscleromorpha of an epithelial morphogenesis is unique among sponges. This feature is shared by Homoscleromorpha and Eumetazoa.

Nowadays the formation of germ layers (endoderm and mesoderm) is associated with gastrulation. The question of whether the cell movements during early embryonic development in sponges (Porifera) are gastrulation as in eumetazoans remains in dispute. Recent data on the histological organization, digestion and embryonic morphogenesis in sponges are analyzed here in an attempt to answer this question. Unique features of these basal Metazoa are the lack of intestinal epithelium, digestive parenchyma or any cell population specialized in digestion. Food particles are captured by cells of almost all types. These data show that sponges have no embryonic layers such as ectoderm or endoderm, characteristic to eumetazoans, and, consequently, no gastrulation. We make an assumption that the formation of germ layers cannot be considered as a recapitulation of events that took place in the common ancestor of Porifera and Eumetazoa. The unity of Metazoa is expressed not in the presence of gastrulation processes per se, but in the universal nature of cell movement mechanisms ensuring various types of morphogenesis, including those underlying gastrulation. It is concluded that metazoan mechanisms of morphogenetic movements must have emerged in the course of evolution prior to the separation of the germ layers like endoderm and ectoderm.

Having descended from the first multicellular animals on earth, sponges are a key group in which to seek innovations that form the basis of the metazoan body plan, but sponges themselves have a body plan that is extremely difficult to reconcile with that of other animals. Adult sponges lack overt anterior–posterior polarity and sensory organs, and whether they possess true tissues is even debated. Nevertheless, sexual reproduction occurs as in other metazoans, with the development of embryos through a structured series of cellular divisions and organized rearrangements of cellular material, using both mesenchymal and epithelial movements to form a multicellular embryo. In most cases, the embryo undergoes morphogenesis into a spatially organized larva that has several cell layers, anterior–posterior polarity, and sensory capabilities. Here we review original data on the mode of cleavage, timing of cellular differentiation, and the mechanisms involved in the organization of differentiated cells to form the highly structured sponge larva. Our ultimate goal is to develop interpretations of the phylogenetic importance of these data within the Porifera and among basal Metazoa.

Embryonic development from coeloblastula to fully developed larva was investigated in 8 Mediterranean homoscleromorph species: Oscarella lobularis, O. tuberculata, O. microlobata, O. imperialis, Plakina trilopha, P. jani, Corticium candelabrum, and Pseudocorticium jarrei. Morphogenesis of the larva is similar in all these species; however, cell proliferation is more active in species of Oscarella than in Plakina and C. candelabrum. The result of cell division is a wrinkled, flagellated larva, called a cinctoblastula. It is composed of a columnar epithelium of polarized, monoflagellated cells among which are scattered a few non-flagellated ovoid cells. The central cavity always contains symbiotic bacteria. Maternal cells are also present in O. lobularis, O. imperialis, and P. jarrei. In the fully developed larva, cell shape and dimensions are constant for each species. The cells of the anterior pole have large vacuoles with heterogeneous material; those of the postero-lateral zone have an intranuclear paracrystalline inclusion; and the flagellated cells of the posterior pole have large osmiophilic inclusions. Intercellular junctions join the apical parts of the cells, beneath which are other specialized cell junctions. A basement membrane underlying the flagellated cells lines the larval cavity. This is the first observation of a basement membrane in a poriferan larva. The basal apparatus of flagellated cells is characterized by an accessory centriole located exactly beneath the basal body. The single basal rootlet is cross striated. The presence of a basement membrane and a true epithelium in the larva of Homoscleromorpha—unique among poriferan clades and shared with Eumetazoa—suggests that Demospongiae could be paraphyletic.

All stages of vertical transmission of symbiotic bacteria, from the penetration into oocytes to the formation of rhagon, were investigated in the White Sea (Arctic) representatives of Halisarca dujardini Johnston (Demospongiae). Small populations of free-living specific symbiotic bacteria inhabit the mesohyl of H. dujardini. They are represented by a single morphotype of small spiral gram-positive bacteria. Vertical transmission of symbiotic bacteria between generations in sponges may occur in different ways. In the case of H. dujardini the bacteria penetrate into growing oocytes by endocytosis. A part of the bacteria plays a trophic role for oocytes and the other part remains undigested in membrane-bound vacuoles within the cytoplasm. In cleaving embryos bacteria are situated between the blastomeres or in the vacuoles. In the blastula all bacteria are disposed in the blastocoel. The symbionts are situated in intercellular spaces in free-swimming larvae and during metamorphosis. Symbiotic bacteria do not play any trophic role in the period of embryonic and postembryonic development of H. dujardini. No signs of destruction and digestion of bacteria were revealed at any stage of development.

Summary The fine structure of the free-swimming larvae of the sponge Ircinia oros (Dictyoceratida, Demospongiae) from the northern Mediterranean was studied using light and electron microscopy. Larvae are of the parenchymella type, oval. The larva is extensively ciliated. A band of long cilia and pigment-filled protrusions separates the posterior region from the antero-lateral region. The parenchymella is three-layered, with a ciliated epithelium, an intermediate layer and an internal zone. The surface of the larva is made of pear-shaped monociliated cells, which form a pseudo-stratified columnar epithelium. Zonula adhaerens, which join the apical parts of the ciliated cells, can be observed. The single basal ciliary rootlet is fibrillar and not associated with nucleus. A network of microtubules extends from the side of the basal body and forms electron-dense bundles. These bundles are opposite to the basal foot, run parallel to apical part of the plasmalemma and are oriented towards the posterior pole of...

The cleavage and the first stages of development of larvae in five Oscarella species, from the north-west Mediterranean, O. lobularis, O. tuberculata, O. imperialis, O. microlobata and Oscarella sp., are investigated. Eggs are isolecithal, and rich in yolk inclusions. The cleavage is holoblastic, equal and asynchronous. During cleavage, there is no central cavity (blastocoel). The result of cleavage is the formation of a solid morula constituted by equal blastomeres. The polarity of the blastomeres is not expressed prior to the beginning of larva differentiation, i.e. approximately up to 64-cell stage. The superficial membrane of the blastomeres shows numerous filopodia which form blastomere contact. Symbiotic bacteria and cells with inclusions of the maternal mesohyl are present in the intercellular spaces of the embryo from the beginning of cleavage. Whereas maternal symbiotic bacteria are present in the embryo of the five species studied, maternal cells with inclusions are absent from two species (O. t...

Matrotrophy, the continuous extra‐vitelline supply of nutrients from the parent to the progeny during gestation, is one of the masterpieces of nature, contributing to offspring fitness and often correlated with evolutionary diversification. The most elaborate form of matrotrophy—placentotrophy—is well known for its broad occurrence among vertebrates, but the comparative distribution and structural diversity of matrotrophic expression among invertebrates is wanting. In the first comprehensive analysis of matrotrophy across the animal kingdom, we report that regardless of the degree of expression, it is established or inferred in at least 21 of 34 animal phyla, significantly exceeding previous accounts and changing the old paradigm that these phenomena are infrequent among invertebrates. In 10 phyla, matrotrophy is represented by only one or a few species, whereas in 11 it is either not uncommon or widespread and even pervasive. Among invertebrate phyla, Platyhelminthes, Arthropoda and Bryozoa dominate, with 162, 83 and 53 partly or wholly matrotrophic families, respectively. In comparison, Chordata has more than 220 families that include or consist entirely of matrotrophic species. We analysed the distribution of reproductive patterns among and within invertebrate phyla using recently published molecular phylogenies: matrotrophy has seemingly evolved at least 140 times in all major superclades: Parazoa and Eumetazoa, Radiata and Bilateria, Protostomia and Deuterostomia, Lophotrochozoa and Ecdysozoa. In Cycliophora and some Digenea, it may have evolved twice in the same life cycle. The provisioning of developing young is associated with almost all known types of incubation chambers, with matrotrophic viviparity more widespread (20 phyla) than brooding (10 phyla). In nine phyla, both matrotrophic incubation types are present. Matrotrophy is expressed in five nutritive modes, of which histotrophy and placentotrophy are most prevalent. Oophagy, embryophagy and histophagy are rarer, plausibly evolving through heterochronous development of the embryonic mouthparts and digestive system. During gestation, matrotrophic modes can shift, intergrade, and be performed simultaneously. Invertebrate matrotrophic adaptations are less complex structurally than in chordates, but they are more diverse, being formed either by a parent, embryo, or both. In a broad and still preliminary sense, there are indications of trends or grades of evolutionarily increasing complexity of nutritive structures: formation of (i) local zones of enhanced nutritional transport (placental analogues), including specialized parent–offspring cell complexes and various appendages increasing the entire secreting and absorbing surfaces as well as the contact surface between embryo and parent, (ii) compartmentalization of the common incubatory space into more compact and ‘isolated’ chambers with presumably more effective nutritional relationships, and (iii) internal secretory (‘milk’) glands. Some placental analogues in onychophorans and arthropods mimic the simplest placental variants in vertebrates, comprising striking examples of convergent evolution acting at all levels—positional, structural and physiological.

Five new species of Homoscleromorpha (Porifera) of four genera, Oscarella, Plakortis, Plakina and Corticium, are described from vertical walls of reef caves at depths ranging from 23 to 28 m in the Caribbean Sea. Oscarella nathaliae sp. nov. has a leaf-like thinly encrusting, flat body, loosely attached to the substrate and a perforated, not lobate surface. Oscarella nathaliae sp. nov. contains two bacterial morphotypes and is characterized by two mesohylar cell types with inclusions. Plakortis myrae sp. nov. has diods of two categories: abundant large ones (83–119 µm long) and rare small ones (67–71 µm long) with sinuous, S-bent centres; triods Y- or T-shaped (18–5 µm long), and abundant microrhabds (5–12 µm long). Plakortis edwardsi sp. nov. has diods of one category with thick, sinuous, S-bent centres (110 to 128 µm long); triods T-shaped (actines 28–59 µm long). It is the only species of this genus showing small diods (22–31 µm long). Plakortis dariae sp. nov. has diods of two categories: large ones (67–112 µm long) and small, rare, irregular ones, slightly curved, often deformed with one end blunt (30–59 µm long); triods rare and regular (actines 20–44 µm long). Corticium diamantense sp. nov. has oscula situated near its border, regular non-lophose calthrops of one size-class, very rare tetralophose calthrops and candelabra with the fourth actine ramified basally in 4–5 microspined rays. In addition, a re-description of Plakina jamaicensis is based on newly collected material and the type specimen. Plakortis jamaicensis has a convoluted brain-like surface; well developed sub-ectosomal cavities; irregular sinuous diods, triods, calthrops, rare monolophose calthrops, rare dilophose calthrops, rare trilophose calthrops and common tetralophose calthrops. Molecular 'barcoding' sequences for mitochondrial cob are given for Plakortis edwardsi sp. nov., P. dariae sp. nov., Plakina jamaicensis and Corticium diamantense sp. nov. An identification key for all western Atlantic Homoscleromorpha is provided.