Halisarca magellanica Topsent, 1901

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, Zootaxa 4208 (6), pp. 501-533 : 503-515

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 , Antártica 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 , Quellón, 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 ), Reñihué 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 , Quellón, 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 ), Reñihué 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 µm in morphotypes 1 & 3 and reaches 900 µm 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 µm thick, whereas its inward side consists of interlaced collagen fibrils forming dense tracts 2 to 25 µm, 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 µm to 30 µm 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 µm long x 10 to 40 µm 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 µm thick ectosome ( Fig 3 D). In some sections, choanocyte chambers are located radially around several thin, probably inhalant, canals 4 to 16 µm 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 µm 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 µm x 4.0–4.5 µm), 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 µm in size, occur infrequently throughout the mesohyl and have a classical amoeboid shape with pseudopodia. The nucleolated nucleus (3.2 to 3.9 µm 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 µm) 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 µm in diameter) packed with large dense osmiophilic homogeneous membrane bound inclusions of variable diameter (1.1 to 4.8 µm) 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 µm in diameter) with large inclusions of variable diameter (1.3 to 4.2 µm) 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 µm) than the two previous types and contain heterogeneous inclusions (1.1–2.1 x 2.2–3.3 µm) 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 µm) 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 µm 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 µm), 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 µm) 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 µm) with short pseudopodia. The nucleolated nucleus is relatively large and spherical (2.1–3.6 µm in diameter). The cytoplasm is filled with osmiophilic elliptical membrane bound inclusions (0.7–1.7 x 0.4–0.7 µm), 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 µm) 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 µm). 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 µm 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 µm long and 1.2 µm 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.

Figures

FIGURE 2. Halisarca magellanica. A, B & C, morphotype 1 with stretched filamentous outgrowths (specimen RBINS-POR. 10827 in situ); D, type specimen, re-photographed as published by Topsent (1901), corresponding to morphotype 1; E, morphotype 2 thinly encrusting (specimen RBINS-POR. 10753 in situ); F & G, morphotype 3 bushy with assembled digitations (specimen RBINS-POR. 12921 in situ). (O = osculum). Arrows indicate ostia.

FIGURE 3. Halisarca magellanica. Histological sections perpendicular to the surface of all three morphotypes stained with a combination of methylene blue / azure II and basic fuchsine. Cytoplasm stains light blue and nuclei darker blue, while collagen and mesohyl stain pink to purplish-red. Ectosome is thinner in morphotypes 1 and 3 than in morphotype 2. A, morphotype 1 (holotype RBINS-POR. 034) with structures still clearly recognizable after preservation for more than a hundred years in ethanol. B to C, morphotype 1 (RBINS-POR. 10827); D & E, morphotype 2 (RBINS-POR. 10753); F to I, morphotype 3 (F, RBINS-POR. 12918; G to I, RBINS-POR. 12921). (IC = inhalant canal; CC = choanocyte chamber; CL = complex collagen layer; EC = exhalant canal; In = invagination of the basal region of the collagenous layer; SC = spherulous cell). White doubleheaded arrows indicate ectosome; black double-headed arrows indicate choanosome; arrowheads indicate choanocyte chambers radially grouped around aquiferous canals.

FIGURE 4. Halisarca magellanica. Ectosome in TEM. Sections perpendicular to the surface. A to C, exopinacocyte bodies mixed among spherulous cells Type 1 are located under a complex collagenous layer and reach the surface through thin processes ending as superficial flat extensions (arrowheads); D, limit between the unorganized collagenous structure (UC) and collagen fibrils (CF) of the complex collagen layer; E, narrow projection emerging from an exopinacocyte cell body (right), ending as a thin superficial extension (left); F, junction between superficial extensions of two exopinacocytes (arrow) with glycocalyx at the surface of the barely visible external dermal membrane. A, RBINS-POR. 10827; B to D, RBINS-POR. 12921; E & F, RBINS-POR. 12922. (B = bacterium; CL = complex collagenous layer; Ex = exopinacocyte; G = glycocalyx; In = invagination of the basal region of the complex collagen layer; N = Nucleus; n = nucleolus; Out = outside of the sponge; SC 1 = spherulous cell Type 1). Black and white double-headed arrows indicate respectively outward (unorganized collagenous structure) and inward portions (tracts of collagen fibrils) of the complex collagenous layer.

FIGURE 5. Halisarca magellanica. Choanocyte chambers and choanocytes in TEM and SEM (A to C & E, RBINS-POR. 12921; D, RBINS-POR. 8798). (C = choanocyte; F = flagellum; M = mesohyl; Mi = microvilli; N = nucleus; n = nucleolus; PS = periflagellar sleeve). Arrows indicate basal pseudopodial extensions of choanocytes.

FIGURE 6. Halisarca magellanica. Endopinacocytes in TEM and SEM. A, inhalant canal lined with one exopinacocyte (RBINS-POR. 12920); B, exhalant canal padded with several endopinacocytes (RBINS-POR. 10753); C, fragment of exhalant canal (RBINS-POR. 12922). (IC = inhalant canal; En = endopinacocyte; EC = exhalant canal; N = nucleus).

FIGURE 7. Halisarca magellanica. Mesohylar cells in TEM. A, archaeocyte (RBINS-POR. 8798); B, lophocyte with anteriorly located nucleus (RBINS-POR. 10753); C & D, lophocyte with posteriorly located nucleus (RBINS-POR. 12922); E, spherulous cell Type 1 (RBINS-POR. 12918); F, spherulous cell Type 2 (RBINS-POR. 8888); G, spherulous cell Type 3 (RBINS-POR. 12920); H, granular cell (RBINS-POR. 8888). (CF = collagen fibrils; ChI = chestnut inclusion; Gr = electron dense granule; HI = homogeneous osmiophilic (electron dense) inclusion; M = mesohyl; N = nucleus; n = nucleolus; P = pseudopod; Ph = phagosome; RI = rough inclusion). Arrows indicate small electron-dense vacuoles.

FIGURE 8. Halisarca magellanica. Mesohylar cells in TEM. A, microgranular cell (RBINS-POR. 12920); B, rhabdiferous cell in upper part of the ectosome, under its complex collagen layer (RBINS-POR. 10827); C, enlargement of B; D, rhabdiferous cell enclosed in a choanocyte chamber (RBINS-POR. 10827); E, enlargement of D. (CL = complex collagenous layer; HI = homogeneous osmiophilic (electron dense) inclusion; P = pseudopod; Ph = phagosomes; R = rhabdite).

FIGURE 9. Halisarca magellanica. Pocket cells in TEM and SEM. A & B, early stage of mesohyl engulfment through extension of pseudopods (respectively RBINS-POR. 12918 & 12921); C, mesohyl within pocket (RBINS-POR. 12921); D, similar view in SEM (RBINS-POR. 10753); E & F, later stages with pockets of variable size and number containing mesohyl material and associated bacteria (respectively RBINS-POR. 12920 & 10753); G, ultimate stage with numerous pockets containing bacteria and mesohylar content (RBINS-POR. 12921). (B = bacterium; Gr = electron dense granules of the mesohyl; M = mesohyl; N = nucleus; n = nucleolus; P = pseudopod; Po = pocket).

FIGURE 10. Halisarca magellanica. Bacteria in TEM. A & B, bacteriocyte containing bacteria Type B 1 (RBINS-POR. 10827); C, bacteria Type B 1 (RBINS-POR. 12918); D, bacteria Type B 2 in the mesohyl (RBINS-POR. 12922); E, bacterium Type B 2 (RBINS-POR. 12921); F, bacterium Type 3 (RBINS-POR. 12920); G, detail of electron dense granules of the mesohyl (RBINS-POR. 12921). (F = fimbria; Gr = electron dense granules of the mesohyl; N = nucleus; P = pseudopodium). Arrowheads indicate nucleus.

FIGURE 19. Phylogenetic positions of Halisarca magellanica and H. desqueyrouxae sp. nov. Shown is a posterior majorityrule consensus tree obtained from the Bayesian analysis of cox 1 sequences under the “ mixed ” model of nucleotide substitutions with 4 gamma categories in Mr. Bayes. The tree was drawn in FigTree (http: // tree. bio. ed. ac. uk / software / figtree /). The number at each node represents the Bayesian posterior probability. Sequences from five specimens of H. magellanica, seven specimens of H. desqueyrouxae sp. nov. and 13 specimens of Chondrilla aff. nucula are shown as “ collapsed ” subtrees (triangles). The height of a triangle reflects molecular divergence among sequences it contains. No differences were found among sequences of H. magellanica or among sequences of H. desqueyrouxae sp. nov. Sequences of Chondrilla aff. nucula are 0 – 1.2 % different.