Halisarca dujardini

Ereskovsky, Alexander V., Lavrov, Dennis V., Boury-Esnault, Nicole & Vacelet, Jean, 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, pp. 5-31 : 10-25

Redescription of Halisarca dujardini type species

( Figs. 2–23 )

Previous reviews. Lévi 1956; Bergquist 1996; Bergquist & Cook 2002.

Halisarca dujardini Johnston, 1842

Synonymy. Halisarca dujardini Johnston, 1842: 192 –193; Lendenfeld 1889: 729–730; de Laubenfels 1948: 175–176; Lévi 1956: 184; Chen 1976: 113–139; Ereskovsky 1993: 8; Bergquist 1996: 24–27; Bergquist & Cook 2002: 1090–1091. Halisarca franz-schulzei Merejkowsky 1878: 27–36, Pl. I, fig. 1–6; Pl. II, fig. 9–15. Hymeniacidon dujardini Bowerbank 1866: 224 . Halisarca sputum Topsent 1893: 38 –39.

Original description. Halisarca dujardini, Johnston, 1842 , p. 192.

Type material. Holotype: presumably lost. Neotype: BMNH 1960.1.7.1—Black Rock, Brighton, English Channel (epizootic on algae, collected close to the type locality) ( Bergquist & Cook 2002).

Comparative material examined. Sponge specimens used in the revision of Halisarca dujardini were collected using SCUBA, in White Sea (Kandalaksha Bay, Chupa Inlet (66°17'32"N – 33°39'39"E), 3–5 m depth, from algae Fucus ramosus and Laminaria saccharina , July 2000–2004; in Barents Sea, Kola Peninsula, Dalnije Zelentsi (69°07'19"N – 36°03'21"E) 0.3–0.5 m depth, July, August 2003; in Bering Sea, Avacha Bay (52°46'32"N – 158°36'16"E) 10-11 m depth, October 18 2008; in Japan Sea the Gulf of Peter the Great near of the Marine Biological station “Vostok” (Marine Biological Institute of Russian Academy of Sciences) (42°54'N – 132°38'W) from rocks and boulders at a depth 0.4–4 m; in North Sea, Norway, Bergen (60°23'29"N – 5°13'46"E), June 2009, 6 m depth; in North Sea, Netherlands, Zeeland, Goese Sas (51°38'52"N – 3°48'0"E) 18 m depth, July, 2009; NE Atlantic, North Wales (53°14'6"N – 4°7'48"W) July 1988, 3 m. We also investigated histologically a piece of the neotype and a piece of Halisarca franz-schulzei Merejkovsky, 1879 from Zoological Institute RAN, St-Petersburg, Russia.

Description. External morphology. Predominantly incrusting sponge, 0.3–12 mm thick, forming smooth crusts on shallow water hard surfaces, or globular, 0.5–2 cm in diameter. Frequently a fouling organism (on red and brown algae, ascidians, shells of bivalves and brachiopods, tubes of sedentary polychaetes, hydroid stalks) ( Figs. 2, 3 ). Incrusting forms have multiple oscula 0.3–1 mm in diameter, scattered regularly; globular sponges have one osculum or, occasionally, two oscula with raised rims, approximately 1–2 mm in diameter. Pores are dispersed. A significant reduction in the aquiferous system is characteristic for the latest phases of reproduction (embryo and larvae development). During these periods oscula and pores are rarely observed. Colour is yellow-beige to pale brown ( Fig. 2 ); during last phases of reproduction the specimens have ivory or whitish colour due to the presence of embryos and larvae ( Fig. 3 ). Surface is smooth and slimy. Consistency is soft, gelatinous and slightly elastic.

Anatomy. Soft tissues. An ectosomal region up to 27 µm thick, consists of three layers: (1) The upper layer composed of a mucous acellular cuticle 0.1–0.2 µm thick ( Figs. 4 , 6 ), underlying external parts of T-shaped exopinacocytes and a diffuse collagen region 0.9–3 µm thick beneath it; (2) the middle layer ~ 5–10 µm thick containing interlaced collagen fibrils organized into firm tracts. This layer can include rare spherulous cells; and (3) the inner layer, up to 3–7 µm thick, consisting of condensed collagen fibrils and the cell bodies of T-shape exopinacocytes. The choanosome makes up the greatest volume of the sponge body and is composed of choanocyte chambers, canals and mesohyl ( Fig. 4 ). The organization of the aquiferous system is reminiscent of a syconoid system described earlier ( Lévi 1956; Vacelet et al. 1989). The long, branching, tubular, meandering choanocyte chambers are arranged radially around a large exhalant canal ( Fig. 4 ). Choanocyte chambers could vary in abundance and size depending on life cycle. During active growth period choanocyte chambers are 120–600 µm long and 24–90 µm in diameter. Abundance and length of choanocyte chambers decrease during reproduction ( Fig. 5 ).

Cytology. Choanocytes ( Figs. 7, 8 ) are cylindrical or trapeziform, with long and numerous basal pseudopods ( Vacelet et al. 1989; Boury-Esnault et al. 1990; Gonobobleva & Maldonado 2009). The cells are 6–9.5 μm long and 3.5–5 μm wide, with a smooth flagellum and a collar composed of 30–40 microvilli. Some but not all cells have a periflagellar asymmetrical cytoplasmic sleeve. Choanocytes within a choanocyte chamber are connected with each other at the basal-lateral surfaces with numerous interdigitations. Nuclei are pyriform (2.5 x 4.8 μm) with apical thinning and often with one to three nucleoli. Cytoplasm contains phagosomes, electron transparent vacuoles, perinuclear Golgi complexes, and mitochondria.

Exopinacocytes ( Figs. 9–11 ) are T-shaped. External flat cytoplasmic parts connect with each other by interdigitations. Cell bodies, including the nucleus and the main cytoplasm volume, are situated beneath the ectosome. Cell bodies are connected with flat cytoplasmic parts by thin and long cytoplasmic bridges. Nuclei are nucleolated.

Endopinacocytes ( Figs. 12, 13 ) are flat fusiform or polygonal in shape. A nucleolated nucleus is oval, 1.6– 2 x 3–3.8 μm in size, and is located in the center of the cell. The cytoplasm includes well-developed Golgi complexes, small electron dense spherical inclusions and abundant electron-transparent vacuoles.

Basopinacocytes ( Figs. 14, 15 ) are flattened and polygonal, about 8–14 μm in length and about 2 μm in width. The cytoplasm includes a well-developed Golgi complex, small electron dense spherical inclusions and some phagosomes.

Mesohylar cells. Lévi (1956) was the first to describe the free cells in the mesohyl of H. dujardini at light microscopy level. He distinguished three cells populations (types): (1) amoeboid or star-shaped cells with spherical central nucleus and clear cytoplasm containing small granules, called “amoebocytes I and II”, or amoebocytes and collencytes; (2) the cells with the cytoplasm filled with acidophilic granules, called “amoebocytes III”, or fuchsinophil cells, or granular cells; and (3) cells with large peripheral nucleus and the cytoplasm filled with big spherical inclusions – spherulous cells. In subsequent works ( Bergquist 1996; Bergquist & Cook 2002) three types of mesohylar cells were also mentioned: (1) lophocytes; (2) spherulous cells, common in the zone between ectosome and choanosome and along canals; and (3) fuchsinophilic cells scattered throughout the choanosome. However, the use of electron microscopy led to recognition of six mesohylar cells types in H. dujardini : (1) amoebocytes, (2) collencytes or lophocytes, (3) spherulous cells, (4) granular cells, (5) microgranular cells, and (6) vacuolar cells ( Volkova & Zolotareva 1981; Korotkova & Ermolina 1986; Sukhodolskaya & Krasukevitch, 1984), which are described below.

(1) Archaeocytes (“amoebocytes I”) ( Figs. 16, 17 ) are abundant cells in H. dujardini mesohyl. These cells have an irregular amoeboid shape and often produce pseudopodia. A large spherical nucleolated nucleus (2.5–3.5 µm in diameter) is located in the center of the cell. Cytoplasm is characterized by the absence of any special or monotypical inclusions, although it can contain heterophagosomes and various osmiophilic or electron-transparent inclusions. There are well developed Golgi complex and endoplasmic reticulum. Archaeocytes are distributed throughout the mesohyl.

(2) Lophocytes ( Figs. 18, 19 ) have a star-like, clavate, or oval form, often with a tuft of secreted collagen fibrils attached to the lateral and posterior ends. The cytoplasm contains some phagosomes and small osmiophilic inclusions. An oval nucleolated nucleus is located in the anterior part of the cell.

(3) Spherulous cells ( Fig. 20 ) are oval or round in appearance (8–12 µm in diameter) and are characterized by the presence in the cytoplasm of 2–7 large trapeziform, spherical, or oval homogenous osmiophilic inclusions 2–8 µm in diameter. These inclusions have a microgranular structure. The cytosol is reduced to a thin layer between these inclusions. The cytoplasm sometimes contains large heterogenous inclusions and small electron transparent vacuoles. The Golgi complex is well developed. A nucleus without nucleoli could be in the cell’s center or at the periphery. Spherulous cells can be distributed anywhere in the mesohyl, but are more common in external parts of the choanosome. They often occupy the collagen-reinforced layer in the sponge ectosome.

(4) Granular cells (amoebocytes III or fuchsinophilic cells) ( Fig. 20 ) form one of the more numerous cell types in the mesohyl. The shape of these cells is highly variable; usually cells are irregular, ovoid or elongate with a mean diameter about 8 µm. The cytoplasm is filed with round or oval electron-dense homogenous granules (0.5– 2 µm in diameter). The nucleus has a peripheral, or, rarely, central location in the cell. The cells are distributed throughout the choanosome.

(5) Microgranular cells ( Fig. 20 ). We propose to distinguish this cell type in H. dujardini from granular cells. Microgranular cells are characterized by irregular, amoeboid or, rarely, oval shape and have large lobopodia. The cytoplasm is filed with abundant small (0.2–0.5 µm in diameter) electron-dense spherical or oval inclusions. These inclusions can be of oval shape or rod-shaped. The Golgi complex is well developed. The nucleus can be nucleolated or without nucleoli, located in the cell’s center or at the periphery. Microgranular cells do not display any special localization in the choanosome.

(6) Vacuolar cells ( Fig. 20 ) are rare in the mesohyl of H. dujardini but do not represent a stage in the ontogenesis of spherulous cell, as thought by Lévi (1956). Cells are irregular to oval in shape, 5x 7–9x12 µm in size. The cytoplasm contains one to 12 vacuoles of oval or elongated shape 0.5–7.2 µm in diameter. Vacuoles are electrontransparent or could contain very loose mucus-like inclusions, and in sometimes intact symbiotic bacteria. Vacuolar cells are predominantly distributed in the middle part of the choanosome.

Additional transient mesohylar cell types could differentiate at various stages in the life cycle. They can be nutritive cells, macrophages, and special granular cells containing cationic peptides and proteins.

Nutritive cells ( Fig. 21 ) are a mixed heterogeneous cell population with predominance of dedifferentiated choanocytes. Choanocytes filled with phagosomes leave choanocyte chambers and enter the mesohyl during oocytes vitellogenesis ( Korotkova & Apalkova 1975; Korotkova & Aisenshtadt 1976; Aisenshtadt & Korotkova 1976). Among the nutritive cells there are also amoebocytes I and spherulous cells. Nutritive cells are phagocyted by growing oocytes for yolk granules formation.

Macrophages ( Fig. 22 ) are large cells that appear at the end of sexual reproduction when begins the postreproductive restoration phase of the life cycle (Ereskovsky 2000), or during the regeneration when it undergoes destructive processes in the mesohyl ( Korotkova & Movchan 1973; Volkova & Zolotareva 1981; Korotkova & Ermolina 1986b; Sukhodolskaya & Krasukevitch 1984). Cell dimensions can reach 30–35 µm, the cytoplasm is filled with large heterogeneous phagosomes. A large spherical nucleus is nucleolated.

Special eosinophilic granular cells ( Fig. 23 ) contain cationic peptides and proteins ( Krylova et al. 2004). These cells are amoeboid or oval in shape (about 8–12 µm in diameter); the nucleus is oval and voluminous (about 3.2 µm in diameter) with condensed heterochromatin or a small nucleolus. The cytoplasm contains several specific inclusions: (1) vacuoles that include one to ten round or oval electron-dense granules with peripheral thinly-fibrillar material; (2) vacuoles with electron-dense fine-granular inclusions; (3) electron-dense thinly-fibrillar inclusions located in the cytoplasm. Cells differentiate at the beginning of vitellogenesis of oocytes. During the embryonic development they concentrate in the mesohyl surrounding embryos. These eosinophilic granular cells (or eosinophilic amoebocytes) of maternal sponge penetrate developing embryos and remain there until the beginning of metamorphosis ( Korotkova & Ermolina 1982; Ereskovsky & Gonobobleva 2000; Gonobobleva & Ereskovsky 2004a, b).

Symbiotic bacteria. Symbiotic bacteria of H. dujardini are represented by a single morphotype found both in the mesohyl of all investigated adults ( Fig. 20 ) and during all stages of embryonic development of the White Sea specimens ( Ereskovsky et al. 2005). Bacteria are almost evenly distributed in the mesohyl and do not form accumulations. Sometimes bacteria are located in vacuoles of archaeocytes. Bacteria (likely gram-positive) have a curved spiral form, characteristic of spirills ( Fig. 20 ). Their length is about 0.45 µm, thickness 0.18 µm, the cell wall is 0.05 µm thick. No flagella or piles are found at the surface of bacterial wall. The cytoplasm is heterogeneous. Its peripheral part has a medium electronic density, while the central part is electron dense and possibly corresponds to the nucleoid. In-between, a thin electron-transparent layer is visible.

Reproduction. The sponge is dioecious (however some rare individuals could be hermaphrodites) with short seasonal period of reproduction ( Table 2 ). Spermatogenesis occurs only during the cold part of the year (Ereskovsky 2000). Larvae are sub-spherical to oval dispherulae, 100–200 µm in diameter, completely ciliated but only sparsely so at the posterior pole ( Ereskovsky & Gonobobleva 2000; Gonobobleva & Ereskovsky 2004a, b).

Habitat. Halisarca dujardini is usually found at depths from 1 to 20 m but also from upper littoral (Barents Sea) to 300 m (Skagerrak), mainly on the algae Fucus vesiculosus , F. serratus, Ascophyllum nodosum and Laminaria saccharina , under and on stones and boulders, in empty shells of lamellibranchs, on carapaces of crabs, at the base of gorgonians, and on the ascidian Styela rustica .

Distribution. Halisarca dujardini species complex is considered to be cosmopolitan ( Bergquist & Cook 2002). In fact, however, its distribution is limited to temperate waters: in the North Atlantic—along the European costs from English Channel to White Sea and in Atlantic coast of USA (Massachusetts) and in the North-West of Mediterranean Sea; in North Pacific—Bering Sea, Avacha Bay and in Russian coast of Japan Sea; in South Pacific—the New Zealand coasts ( Fig. 24 ). See Annex 1 for the detailed distribution.

Material examined. Holotype: collected in “Grotte à Corail” Maire Island, 16 m depth, 43°12'38.04"N – 5°19'56.80"E, Muséum National d'Histoire Naturelle (Paris, France) MNHN DJV 128. Paratype: collected in “Grotte à Corail” Maire Island, 16 m depth, 43°12'38.04"N – 5°19'56.80"E, Zoological Institute RAS (Saint-Petersburg, Russia) № 11141.

Description. External morphology. Thinly incrusting sponge, 0.17–0.19 mm thick, spreading on the surface of the living bryozoan Smittina cervicornis ( Fig. 25, 26 ). The sponge covers the host’s colony from the base to the tips of the branches with a continuous layer. Most often, the sponge coating is very thin and transparent, and thus hardly visible. The sponge may become relatively thick and then appears as an opaque sheath. The surface is skinlike, lustreless and smooth, even ( Fig. 26 ). Oscula have a cone shape (height: 0.1–0.5 µm and diameter about 0.5 mm) and are located exclusively along the bryozoan branch edges that are devoid of zooid orifices. Texture is soft, very delicate and easily torn. The colour is pale-yellow.

Internal organization. Soft tissue organization. There is a thin ectosomal region, 4–14 µm deep, consisting of the flat part of exopinacocytes and of an underlying dense collagen layer, about 1.7–2.6 µm deep ( Fig. 27 ). This layer contains symbiotic bacteria and some secretory cells ( Fig. 27 ). Cell bodies of T-shape exopinacocytes and rare spherulous cells occur beneath the collagen layer.

The choanosome makes up most of the volume of the sponge body and is composed of choanocyte chambers and mesohyl ( Figs. 27, 28 ). The individual choanocyte chambers are tubular in shape, sometimes branched, measuring from 76 up to 175 μm in length and are 9.3–14 μm wide. There are mobile cells present in the mesohyl.

The basal part ( Fig. 29 ), in contact with the bryozoan surface, includes a dense, thin (0.17 µm) cuticle secreted by the basopinacocytes. Collagen cuticle includes spiral symbiotic bacteria and other bacteria, which are not present in other sponge parts.

Cytology. Exopinacocytes ( Fig. 30 ) are T-shaped with a thin, flattened external cytoplasmic part, and an internal cell body. The cell body is irregular or triangular in shape, about 4.9x5.3 μm. The two parts of the cell are connected by a thin cytoplasmic bridge. Nucleus is lightly ovoid (2.2–2.4 µm in diameter), often without nucleolus.

Choanocytes ( Fig. 28 , 31 ) are elongate or roughly pyramidal, 2.3–5.3 µm wide at the base and 1.9–4.2 µm wide at the nucleus area, the height is 4–5.9 µm. Choanocyte collar arises at approximately 4/5 of the height of the cell and is composed of about 18 microvilli. Nucleus is central or apical, spherical about 1.9 µm in diameter, occasionally with a nucleolus, 0.5 µm in diameter. The apical part of choanocytes encompasses many small vacuoles. The central and basal cytoplasm usually contains 2–4 phagosomes, 0.8–1.5 µm in diameter with heterogeneous inclusions. The choanocytes also contain mitochondria, osmiophilic granules (0.3–0.6 µm), electron transparent vacuoles (0.1–0.8 µm) and other inclusions. The choanocytes contact each other only at the base. Long fine pseudopodia frequently arise from the central and basal parts of the choanocytes; the latter anchor choanocytes in the mesohyl. The free apical surface of the choanocytes and the flagella are covered by a thin (1 µm) glycocalyx layer.

Endopinacocytes ( Fig. 32 ) are flat cells with very thin, long, and flat cytoplasmic outgrowths (about 0.2–0.3 µm in thickness), non-flagellated, 9–13.7 µm wide by 2.1–2.5 µm high in the nucleus area. The cytoplasmic outgrowths can make branching diverticules. Endopinacocytes are anchored in the mesohyl by short, thin basal pseudopodia, which develop from the central part of the cell. Nucleus is ovoid (2 x 2.3 µm), often without nucleolus. The cytoplasm usually contains 1–3 phagosomes 0.4–0.6 µm in diameter, transparent vacuoles, and osmiophilic inclusions.

Basopinacocytes ( Fig. 33 ). These cells constitute the loose layer underlying the base of the sponge. They are oval, flattened in basal part, about 5.7 μm in length and about 1.8 μm in width in the nucleus region. The nucleus is oval 0.9 x 2.8 μm in size. The cytoplasm includes a well-developed Golgi complex, small electron dense spherical inclusions and abundant mitochondria.

Mesohylar cells. Archaeocytes ( Figs. 29 , 34 ) are amoeboid cells, 5– 7 x 4–2.9 µm in size with a round, nucleolate nucleus (1.9–2.2 µm in diameter, nucleolus: 0.5 µm). The cytoplasm contains phagosomes (0.5–2 µm in diameter) with heterogeneous inclusions, round electron dense inclusions (0.2–0.7 µm in diameter) and, sometime, lipid droplets 0.5–0.7 µm in diameter. Archaeocytes are not abundant and are dispersed in all parts of the sponge mesohyl.

Lophocytes ( Fig. 35 ). Clavate, drop-like or oval cells (6.2–8.3 x 1.3–2.4 µm), often with a tuft of secreted collagen fibrils attached to the posterior end. The nucleus, located in the anterior part of cell, is oval (about 1.4 x 2.7 µm) with a nucleolus 0.5 µm in diameter. Cytoplasm contains mitochondria, some digestive vacuoles, and small osmiophilic inclusions.

Spherulous cells ( Figs. 28 , 36 ). Oval, round, or irregular in shape cells, 7.9–11.2 µm in diameter. Nucleus is round (about 3.4 µm in diameter) or oval, commonly without the nucleolus. The cytoplasm contains large, trapeziform or, more rarely, spherical (0.6–2.9 µm in diameter) osmiophilic inclusions. The Golgi complex is well developed. Spherulous cells are more abundant in the exterior part of the choanosome and in the ectosome, but they are also present in the choanosome with a preferential distribution under the choanocyte layer.

Vacuolar cells ( Fig. 29 , 37 ) with big electron transparent vacuoles are often situated in the basal part of sponge choanosome. Cell form is irregular, sometimes dumbbell-like, 5.4–11.7 µm in length and 3.5–4.6 µm in width, with an oval, anucleolate nucleus, in the central part 2x 2.4 µm in dimension. The cytoplasm is reduced to a fine layer surrounding the vacuoles and the nucleus. The vacuoles are oval or irregular, from 2.5x4.2 to 4 to 6.8 µm in size.

Microgranular cells ( Fig. 38 ) are large amoeboid cells, 5.7 x 7.4 µm. The nucleus is round (2.7 µm in diameter) with a nucleolus 0.4 µm. The cytoplasm is filled by oval (length 0.5–1.15 µm; diameter 0.15–0.45 µm) or round (0.2–0.4 µm in diameter) electron-dense granules, surrounded by a membrane and by abundant small irregular, electron transparent vacuoles.

Granular cells ( Fig. 39 ) are oval or irregular cells with thin cytoplasmic outgrowths. Cell length is 4.1–5.6 µm and width is 2.7–3.6 µm. The anucleolate nucleus is round or ovoid, about 1.7 µm in diameter. The cytoplasm is filled by oval or round electron-dense homogenous granules 0.2–1.5 µm in diameter. The granules are surrounded by a thin electron transparent space.

Rhabdiferous cells ( Fig. 40 ) are amoeboid in shape (6.2– 11 x 2.7–6.1 µm) with long pseudopodia. The nucleus is oval, about 2 µm in diameter with a nucleolus 0.4 µm. The cytoplasm is filled with rhabdite-like or oval osmiophilic inclusions 0.5–2.3 µm in length and 0.2–0.9 µm in diameter.

Symbiotic bacteria. Two morphotypes of symbiotic bacteria, B1 and B2, occur in the mesohyl. Both are extracellular. The type B1 ( Figs. 41, 42 ) is found in the mesohyl as well as in the surface layer of the sponge. These bacterial cells are spiral-shaped, slightly curved, with flagella 0.24–0.31 µm in diameter and 2–4.5 µm in length. The cytoplasm is electron-dense. Thick nucleoid filaments form a regular, thin central structure. The cell wall is surrounded by a thin wavy extracellular layer. An electron-dense thickening of the cell wall forms a spiral around the cell surface. The type B2 ( Fig. 43 ) is located only in the basal collagen layer lining the border with the bryozoan host. These bacteria are oval, 0.8–1.2 µm in length and 0.4–0.5 µm in diameter. The cytoplasm is homogeneous and of medium electron density. The nucleoid zone contains a filamentous, irregular network which appears characteristic of these bacteria. Most remarkable are numerous thin projections, up to 0.6 µm long, that extend from the surface of the bacteria.

Mitochondrial genome. Mitochondrial genome of H. harmelini is identical in both the gene content and the gene arrangement to those of H. dujardini and Chondrilla nucula (Demospongiae, Chondrosida ) (Fig. 44). Most of the protein genes and the structural RNA genes are identical or similar in size between the two Halisarca genomes ( Table 3 ), but the total length of intergenic regions is larger by ~ 1,300 bp (6.8% of the genome size) in H. harmelini . Comparison of coding sequences among H. harmelini , H. dujardini , and C. nucula revealed that genetic distances between the two Halisarca species (on average ~ 0.12 for all sites, 0.53 for 4-fold degenerate sites, see Table 3 for individual proteins) are similar to genetic distances between either of them and C. nucula ( Fig. 45 ). The extent of sequence divergence between the two species greatly exceeds the amount of differences found between two specimens of H. dujardini and within closely related C. nucula ( Fig. 45 ), sampled across a large geographic range and various habitats ( Duran & Rützler 2006). Given that the rate of sequence evolution is very low in sponges ( Lavrov 2010), these large genetic distances indicate an ancient divergence between the two Halisarca species. Nevertheless, they still form a strongly supported clade in demosponge phylogeny based on mitochondrial DNA data ( Fig. 46 ).

FIGURE 44. Genomic maps of Halisarca harmelini and Halisarca dujardini mtDNA. Protein (blue) and ribosomal genes (green) are atp6, atp8–9: subunits 6, 8 and 9 of F0 adenosine triphosphatase (ATP) synthase; cox1–3: cytochrome c oxidase subunits 1–3; cob: apocytochrome b; nad1–6 and nad4L: NADH dehydrogenase subunits 1–6 and 4L; rns and rnl: small and large subunit rRNAs. tRNA genes (black) are identified by the one-letter code for their corresponding amino acids. Two arginine, isoleucine, leucine, and serine tRNA genes are differentiated by subscripts with trnR(ucg) marked as R1, trnR(ucu) —as R2, trnI(gau) —as I1, trnI(cau) —as I2, trnL(uag) —as L1, trnL(uaa) as L2, trnS(ucu) —as S1, and trnS(uga) —as S2. Genes for inferred initiator and elongator tRNA(Met) are marked as Mf and Me, respectively. Overlaps between adjacent genes are shown in yellow. All genes are transcribed clockwise.

# nucleotides ab Rates of sequence evolution between HH and HD Predicted initiation and termi-

nation codons in HH and HD c a Data for Halisarca dujardini are from ( Wang & Lavrov 2008). b Bold numbers indicate sequences of different lengths comparing to their homologues in Halisarca harmelini . Asterisks denote internal indels. c Identical initiation/termination codons in H. harmelini and H. dujardini are shown only once.

Ecology and distribution. Specimens of Halisarca harmelini sp. nov. occur only as thin sheets on the rigid erect adeoniform ascophoran bryozoan Smittina cervicornis ( Figs. 25, 26 ). The species has been found in all investigated sites along the Northwestern Mediterranean coast (from 43°46'26"N – 7°30'56"E to 42°28'46"N – 3° 9'10"E) ( Fig. 1 ) 14–65 m depth, on coralligenous rocks and detritical sand. It has not been observed on all other large rigidly erect bryozoans living in the same microhabitats.

Etymology. The species name refers to its first collector, Dr. Jean-George Harmelin, Centre d’Océanologie de Marseille, Station marine d’Endoume, Marseille, France.

Figures

FIGURE 1. a—Geographical distribution of Halisarca harmelini sp. nov. in Mediterranean Sea (red dots); b—Map of the Northwest Mediterranean coast showing the site of collection of holotype, paratype and specimens of Halisarca harmelini sp. nov. for ultrastructural and the total DNA extraction.

FIGURE 6. Scheme of the ectosomal region (modified from Bergquist 1996). 1 — the upper layer; 2 — the middle layer; 3 — the inner layer. Abbreviation: ex—exopinacocytes.

FIGURE 20. Halisarca dujardini. Comparative TEM micrographs of mesohylar cells with inclusions and symbiotic bacteria of Halisarca dujardini from different seas. Abbreviations: G—granules; N—nucleus; Ph—phagosomes; S—spherulous inclusions; V—vacuoles.

FIGURE 24. Geographical records of Halisarca dujardini in World Ocean (red drops).

FIGURE 45. Cox 1 divergence between Halisarca harmelini and Halisarca dujardini in comparison to cox 1 polymorphism within Chondrilla nucula. Neighbor-joining tree was computed using uncorrected “ p ” distances for nucleotide data.

FIGURE 46. Phylogenetic position of Halisarca harmelini in demosponge phylogeny. Posterior majority-rule consensus tree obtained from the analysis of 3411 aligned amino acid positions from 33 taxa under the CAT + GTR + Γ model is shown. Phylogenetic analysis was conducted with the PhyloBayes 2.3 d program with four independent chains running in parallel until convergence (maxdiff = 0.06). The numbers at each node are Bayesian posterior probabilities. Fungi, Homoscleromorpha, and major clades of demosponges are collapsed for clarity. The two species of Halisarca are in bold. The complete tree is shown as supplementary data.

FIGURES 2 – 5. Halisarca dujardini. In situ and in vivo photos of non reproductive sponge from Bering Sea (Fig. 2) and reproductive sponge (arrows indicated embryos) from White Sea (Fig. 3) sponges. Histological section of non-reproductive sponge (Fig. 4) and reproductive (Fig. 5) sponge. Abbreviations: Bc—blastocoel; CC—choanocyte chambers; CE—cleaving embryos; Ex—exopinacoderm; M—mesohyle. Scale bars: 2 – 10 mm; 3 – 5 mm; 4 – 650 µm; 5 – 150 µm.

FIGURES 7 – 15. Halisarca dujardini. Ultrastructure of epithelial cells. TEM of choanocyte chamber of sponge from Barents Sea (Fig. 7); SEM of choanocytes of sponge from White Sea (Fig. 8); TEM (Fig. 9) and SEM (Figs. 10, 11) of exopinacocytes of sponges from White Sea; TEM (Fig. 12) and SEM (Fig. 13) of endopinacocytes of sponges from Japan (12) and White (13) Seas; TEM (Fig. 14) and SEM (Fig. 15) of basopinacocytes of sponges from Barents (14) and White (15) Seas. Abbreviations: B—symbiotic bacteria; Bp—basopinacocytes; C—external cuticle; CB—cytoplasmic bridge; CS—periflagellar cytoplasmic sleeve; EC—external cytoplasmic part; Enp—endopinacocyte; Exp—exopinacocytes; F—flagellum; Mv—microvilli; Nnucleus; Ph—phagosomes. Scale bars: 7 — 7 µm; 8 — 2 µm; 9 — 3 µm; 10 — 2 µm; 11 — 7 µm; 12 — 3 µm; 13 — 7 µm; 14 — 1.5 µm; 15 — 10 µm.

FIGURES 16 – 19. Halisarca dujardini. Ultrastructure of mesohylar cells. TEM of nucleolated amoebocytes (archaeocytes) from Barents (16) and Japan (17) Seas; TEM of lophocytes from Barents Sea (18) and SEM of lophocyte from White Sea (19). Abbreviations: B—symbiotic bacteria; L—lophocyte; N—nucleus; Nu—nucleolus; Ph—phagosomes. Ps—pseudopodia. Scale bars: 16 — 2 µm; 17 — 2 µm; 18 — 1.5 µm; 19 — 7 µm.

FIGURES 21 – 23. Halisarca dujardini. TEM of additional temporary mesohylar cell. Nutritive cells (21); macrophages (22); special eosinophilic granular cells (23). Abbreviations: G—granules; N—nucleus; Nu—nucleolus; Ph—phagosomes. V—vacuoles. Scale bars: 21 — 2.5 µm; 22 — 15 µm; 23 — 3 µm.

FIGURES 25, 26. Halisarca harmelini sp. nov. in situ and in vivo (arrowheads) (photos J. Harmelin). Abbreviations: Ooscula; Z—bryozoan zooids. Scale bars: 25 — 10 mm; 26 — 3 mm.

FIGURE 27 – 29. Anatomy of Halisarca harmelini sp. nov. 27 — General view (semi-thin section); 28 – choanocyte chamber (TEM); 29 — basal part (TEM). Abbreviations: Ar, archaeocyte; B 2, bacteria type 2; C, cuticle; CC, choanocyte chambers; Ch, choanocytes; SC, spherulous cell; VC, vacuolar cell. Scale bars: 27 — 100 µm; 28 — 10 µm; 29 — 5 µm.

FIGURES 30 – 35. TEM micrographs of the cells of Halisarca harmelini sp. nov. 30 — exopinacocyte; 31 — choanocytes; 32 — endopinacocyte; 33 — basopinacocyte; 34 — nucleolate amoebocyte; 35 — lophocyte. Abbreviations: B 1 — bacteria type 1; Ccuticle; CB—cytoplasmic bridge; EC—external cytoplasmic part; F—flagellum; Mv—microvilli; N—nucleus; T—cell’s “ tail ”. Scale bars: 30 – 2.5 µm; 31 — 2 µm; 32 — 2.5 µm; 33 — 2 µm; 34 — 1 µm; 35 — 2 µm.

FIGURES 36 – 43. Micrographs of secretive cells (36 – 40) and symbiotic bacteria (41 – 43) of Halisarca harmelini sp. nov. 36 — Spherulous cell; 37 — Vacuolar cell; 38 — Microgranular cell; 39 — Granular cell; 40 — Rhabdiferous cell; 41, 42 — Symbiotic bacteria Type B 1; 43 — Symbiotic bacteria Type B 2. Abbreviations: B 1 — symbiotic bacteria type 1; G—granules; GC—Golgi complex; N—nucleus; R—rhabdite-like inclusions; S—spherule; V—vacuole. Scale bars: 36 — 3 µm; 37 — 7 µm; 38 — 2 µm; 39 — 1. 5 µm; 40 — 2 µm; 41 — 0. 5 µm; 42 — 1 µm; 43 — 0. 5 µm.

Tables

TABLE 2. Period of reproduction (embryonic development and larvae release) in Halisarca dujardini from different geographical regions.

Region White Sea	Months End of June-July	Ref Ereskovsky 2000
Barents Sea North Sea (Bergen) English Channel (Roscoff) SW Pacific (New Zealand)	July-August June-July June-September October-November	Korotkova & Apalkova 1975 Personal observations Lévi 1956 Bergquist 1996
Halisarca harmelini sp. nov. (Figs. 25–46).

TABLE 3. Comparison of mitochondrial coding and rRNA sequences in Halisarca harmelini sp. nov. (HH) and Halisarca dujardini (HD).

Gene	HH	HD	All positions dN	dS	dN/dS	Initiation codons	Stop Codons
atp6	735	735	0.0905 0.0124	0.5593	0.0327	ATG	TAG
atp8	222	225*	0.1391 0.0587	0.7465	0.0221	ATG	TAG/TAA
atp9	237	237	0.0834 0.0005	0.4871	0.0787	ATG	TAG
cob	1146	1146	0.1349 0.0388	0.9105	0.001	ATG	TAG
cox1	1563	1563	0.1236 0.0144	1.1801	0.0426	ATG	TAA/TAG
cox2	771	771	0.1066 0.0147	0.7751	0.0122	ATG	TAG
cox3	786	786	0.1035 0.0168	0.6306	0.019	ATG	TAA
nad1	975	975	0.1418 0.0171	1.0451	0.0267	ATG	TAA
nad2	1425	1428*	0.1581 0.0523	1.0367	0.0164	ATG	TAG
nad3	360	360	0.1386 0.051	1.0314	0.0504	ATG	TAA/TAG
nad4	1458	1470*	0.1337 0.0008	0.7713	0.0495	ATG	TAA
nad4L	300	300	0.0993 0.0219	1.0811	0.001	ATG	TAA/TAG
nad5	1827	1827	0.1221 0.0375	0.8358	0.0203	ATG	TAA/TAG
nad6	576	591*	0.1096 0.0315	1.0601	0.0449	GTG	TAG/TAA