Leucosolenia variabilis Haeckel, 1870

Lavrov, Andrey, Ekimova, Irina, Schepetov, Dimitry, Koinova, Alexandra & Ereskovsky, Alexander, 2024, The complex case of the calcareous sponge Leucosolenia complicata % Porifera: Calcarea): hidden diversity in Boreal and Arctic regions with description of a new species, Zoological Journal of the Linnean Society 200, pp. 876-914 : 893-899

Leucosolenia variabilis Haeckel, 1870

( Figs 11–16 ; Table 6)

Type material: Syntype BMNH-1910.1.1.421. Other type material is not known.

Type locality: Norway * Bergen .

Material studied: Forty specimens. Molecular data— 40 specimens * external morphology— 40 specimens * skeleton organization— three specimens %WS11643* WS11708* WS11735)* spicules %light microscopy* SEM)— seven specimens %WS11707* WS11714* WS11731* WS11732* WS14637* WS14671* WS14681)* cytology %TEM)—three specimens %WS11643* WS11644* WS11645) %Supporting Information* Table S1).

External morphology: Length of cormus up to 5 cm. Cormus massive* often spherical* otherwise formed by basal reticulation of tubes. Cormus built as reticulation around one or several largest central tubes. Outline of cormus formed by numerous* short diverticula. Largest tubes of cormus always end with oscula. Main oscular tubes large* prominent* erect* bearing many small diverticula* spreadingtotwo-thirdsoftubes’length. Osculartubegraduallynarrows to oscular rim* possessing short spicular crown % Fig. 11A * B). In addition to main oscula on largest tubes of cormus* smaller oscular tubes usually scattered all over the cormus. Surface minutely hispid. Coloration of living specimens greyish white. Coloration of preserved specimens from greyish white to ochre % Fig. 11A ).

Spicules: Diactines % Figs 12A * 13* 16A). Two populations: %i) curved* smooth* lanceolate diactines % Fig. 12A )* mean length 306.7 µm* mean width 9.8 µm* % Table 6)* slightly curved* smooth* with lanceolate outer tip* variable in length; %ii) trichoxeas % Fig. 13 )* thin %mean width 0.9 µm) % Table 6)* with numerous* irregularly distributed spines % Fig. 13C )* long* but usually represented by fragments of variable length %up to 362.4 µm long) % Table 6).

Triactines % Figs 12B * C* 16A). Predominantly T-shaped* sagittal %mean angle 138.5°)* unpaired actines* variable in length: most frequently equal to paired actines* commonly shorter or rarely longer than paired %mean length: 122.3 µm—unpaired* 127.9 µm—paired) % Table 6). Abnormal triactines with one of paired actines undulated also common % Fig. 12C ). Actines equal in width %mean width: 8.1 µm—unpaired* 8.5 µm—paired) % Table 6).

Tetractines % Fig. 12D ). Predominantly T-shaped* sagittal %mean angle 142.2°)* unpaired actines variable in size: equal to* shorter* or longer than paired actines %mean length: 147.6 µm—unpaired* 142.0 µm—paired* 22.8 µm—apical) % Table 6). Unpaired actines usually slightly slender than paired %mean width: 8.5 µm—unpaired* 9.1 µm—paired) % Table 6). Apical actines curved* smooth* and slender %mean width 5.9 µm) % Table 6).

Skeleton: Skeleton of both oscular and cormus tubes formed by dense net of tetractines and triactines % Fig. 11C * D). In oscular tubes* spicules constitute organized array with their unpaired actines directed toward cormus and oriented more or less in parallel to proximo-distal axis of oscular tube % Fig. 11C ). In cormus tubes* spicule array completely disordered % Fig. 11D ). Diactines form small oscular crown up to 100 µm % Fig. 11B ) and cover tubes’ surface* orienting in different directions and extending outside by lance-shaped tip.

Cytology: Body wall* 9–13.8 µm thick* three layers: exopinacoderm* loose mesohyl* and choanoderm % Fig. 14A * B; Supporting Information* Table S2). Flat endopinacocytes located only in distal part of oscular tube %oscular ring) replacing choanocytes. Inhalant pores scattered throughout exopinacoderm* except the oscular ring area.

Exopinacocytes non-flagellated* T-shaped* rarely flat % Fig. 14C ). External surface covered by glycocalyx. Cell body %height 6.3 µm* width 3.7 µm)* containing spherical to oval nucleus %diameter 2.7 µm)* submersed in mesohyl. Cytoplasm with specific spherical electron-dense inclusions %0.2–0.35 µm diameter) % Fig. 14C ).

Endopinacocytes non-flagellated* flat cells* size 16 µm × 2.8 µm. External surface covered by glycocalyx. Nucleus %3.2 µm × 2.3 µm) spherical to oval with nucleolus. Cytoplasm without specific inclusions % Fig. 14F ).

Choanocytes flagellated trapeziform or prismatic %height 10.7 µm* width 4.1 µm) % Fig. 14D ). Flagellum surrounded by collar of microvilli. Characteristic pyriform nucleus %diameter 2.5 µm) in apical position. Cytoplasm with phagosomes and small vacuoles % Fig. 14D ).

Porocytes tubular cylindrical %height 2.5–4.7 µm* width 4.3–5 µm)* connecting external milieu with choanocyte tube % Fig. 14E ). Nucleus spherical %diameter 2.7 µm)* containing nucleolus. Cytoplasm with spherical electron-dense inclusions* identical with inclusions of exopinacocytes % Fig. 14E ).

Sclerocytes amoeboid* size 6 µm × 3.1 µm % Fig. 15G ). Nucleus usually oval or pear-shaped %diameter 2.2 µm)* containing a single nucleolus. Well-developed Golgi apparatus and rough endoplasmic reticulum. Cytoplasm usually with phagosomes and/or lysosomes % Fig. 15G ).

Amoebocytes of different shape %from oval to amoeboid) without special inclusions* size 3 µm × 4–7.5 µm % Fig. 15A ). Nucleus spherical %diameter 2.7 µm)* sometimes with nucleolus.

Large amoeboid cells of different shape %from elongate to amoeboid)* size 20 µm × 4.2 µm % Fig. 15B ). Rare cells located under choanoderm. Nucleus oval %size 4.8 µm × 1.7 µm). Cytoplasm with numerous* large heterophagosomes %diameter 1.1–3.2 µm)* well-developed Golgi apparatus % Fig. 15B ).

Granular cells small oval* size 4 µm × 3.3 µm % Fig. 15C ). Rare cell type * located under the choanoderm. Nucleus in peripheral position* spherical %diameter 1.7 µm) with large amounts of heterochromatin* associated with nucleus membrane. Cytoplasm with electron-dense oval inclusions %size 0.7–6 µm × 0.4–1.1 µm) and rare* spherical* electron-transparent vacuoles %diameter 1.2 µm) % Fig. 15C ).

Spherulous cells with irregular shape from amoeboid to crescent* size 2.7–9.2 µm × 4.7–5.3 µm % Figure 15E * F). Regularly distributed numerous cells* usually located under choanocytes. Distance between cells 2–9 µm % Fig. 15F ). Nucleus deformed %size 2.4 µm × 1.7 µm). Cytoplasm mostly occupied by large crescent or irregular electron-dense homogenous inclusions %diameter 1.8–4.5 µm) and less electron-dense fine-granular inclusions %diameter 0.7–2.6 µm). Granular or foamy material fills cytoplasm spaces between inclusions % Figure 15E ).

Myocytes rare fusiform cells* size 18 µm × 2.7 µm* located in mesohyl % Fig. 15D ). Nucleus oval %3.5 µm × 2.7 µm)* with nucleolus. Cytoplasm with mitochondria* ribosomes* small vesicles* and cytoplasmic myofilaments. Myofilaments grouped in bundles %diameter 0.07-0.2 µm) located along long axis of myocyte % Fig. 15D ).

Three morphotypes of bacterial symbionts in mesohyl % Fig. 15 H-J). Morphotype 1 numerous % Fig. 15H ). Bacteria large* spiral-shaped* diameter 0.2 µm* length 2.5–3.9 µm. Spiral turns regular and compact. Single-membrane cell wall* cytoplasm granular* nucleoid region tubular % Fig. 15H ).

Morphotype 2 rare % Fig. 15I ). Bacteria small* spiral-shaped* diameter 0.3 µm* length 1.5–1.8 µm. Spiral turns irregular and sparse. Cytoplasm transparent* nucleoid region tubular % Fig. 15I ).

Morphotype 3 rare % Fig. 15J ). Bacteria small* rod-shaped bacteria* diameter 0.23 µm* length 0.8 µm. Double-membrane cell wall* cytoplasm with dark filamentous materials* no distinction between cytoplasm and nucleoid region % Fig. 15J ).

Distribution: Boreal-Arctic species* described from Norway. Molecular identity confirmed for the White Sea and Greenland % Alvizu et al. 2018). In the White Sea occurs in low intertidal and subtidal zones up to 40–45 m depth* on rocks and kelps.

Reproduction: No data about reproduction time for this species.

Remarks: We studied three type specimens %slides with spicules) of Leucosolenia variabilis from the British Museum of Natural History % BMNH):BMNH-1910.1.1.421*BMNH-1906.12.1.40* and BMNH-1906.12.1.50. Spicules are similar morphologically across these specimens % Fig. 16B–D )* which supports the idea that they belong to the same species. At the same time* their type status should be reconsidered due to the data represented in the revision by Minchin %1904). Slide labels contain specific information %exact page and number)* allowing an unambiguous comparison with the collection data of these samples given in Minchin %1904). Accordingly* BMNH-1906.12.1.50 was collected from Bantry Bay* Ireland * by C. Norman and identified by him as Leucosolenia botryoides ; this label was endorsed by Haeckel ‘ Ascandra variabilis ’ %slide no. 1; Minchin 1904: 385). BMNH-1906.12.1.40 was received by Haeckel for re-examination from Bowerbank and collected from Guernsey %slide no. 4; Minchin 1904: 385). Finally* BMNH-1910.1.1.421 was collected by Haeckel in Bergen* Norway * the type locality of this species* and contained a printed label ‘ Ascandra variabilis H’ %slide no. 3; Minchin 1904: 385). Therefore* the slide BMNH-1910.1.1.421 could be designated as a syntype.

The analysis of L. variabilis syntype BMNH-1910.1.1.421 indicated two diactine types %lanceolate diactines and trichoxeas)* and V- and T-shaped tri- and tetractines with shorter unpaired actines % Fig. 16D ). Although Haeckel’s description lacks long trichoxeas* it should be mentioned that such spicules are easily broken during preparation. It may also be suggested that the second type of diactine without lanceolate tips described by Haeckel %1872) is in fact broken* long trichoxeas. Direct comparison of spicule slides of specimens from the White Sea with L. variabilis syntype BMNH-1910.1.1.421 shows strong correspondence between them.

Leucosolenia variabilis has a large* massive* sometimes spherical cormus* which could be a good distinctive trait* since all other sympatrically living species % Leucosolenia complicata * L. corallorrhiza * and Leucosolenia sp. A ) are represented by basal reticulation of tubes with extended oscular tubes. In spicular characters* L. variabilis differs from L. somesii by the presence of lanceolate spined diactines; and from L. complicata and Leucosolenia sp. A by the presence of extremely long and highly spined trichoxeas. Leucosolenia variabilis also has the highest diversity of mesohyl cells and symbiotic bacteria among the studied Leucosolenia species %Supporting Information* Table S2). In addition to the usual amoebocytes* L. variabilis also has rare large amoebocytes and small granular cells* as well as numerous unusual spherulous cells of different shapes regularly distributed in the body wall. The composition of symbiotic bacteria of L. variabilis includes three morphotypes: one typical rod-shaped and two unusual spiral-shaped.

Figures

Figure 11. Leucosolenia variabilis Haeckel* 1870 external morphology and skeleton.A* general morphology %Wº11731); B* skeleton of oscular rim %Wº11731); C* skeleton of oscular tube; D* skeleton of cormus %Wº11643). Abbreviations: c* cormus; d* diverticulum; o* osculum; or* oscular rim; ot* oscular tube.

Figure 12. Leucosolenia variabilis Haeckel* 1870 spicule types* scanning electron microscopy.A* curved smooth lanceolate diactines; B* triactines; C* abnormal triactines; D* tetractine.

Figure 13. Leucosolenia variabilis Haeckel* 1870 trichoxeas. A* B* general view of trichoxea fragments; C* enlarged view of trichoxea* white arrowhead marks spines. Abbreviations: tx* trichoxeas.

Figure 14. Leucosolenia variabilis Haeckel* 1870 body wall structure and cell types of bordering tissues.A* B* semi-thin sections of body wall of sponge; C* exopinacocytes; D* choanocytes; E* porocyte; F* endopinacocyte. Scale bars: A* 50 µm; B* 20 µm; C* D* 5 µm; E* F* 2 µm. Abbreviations:am* amoeboid cell; ch* choanocytes; chd* choanoderm; ex* exopinacocyte; exp* exopinacoderm; f* flagellum; m* mesohyl; mv* microvilli; n* nucleus; po* porocyte; sb* symbiotic bacteria; sc* spherulous cells.

Figure 15. Leucosolenia variabilis Haeckel* 1870 mesohyl cell types and symbiotic bacteria.A* amoebocytes; B* large amoeboid cell; C* granular cell; D* myocyte* inset—bundles of myofilaments; E* spherulous cell; F* spherulous cells in the body wall; G* sclerocyte; H* symbiotic bacteria* morphotype 1; I* symbiotic bacteria* morphotype 2; J* symbiotic bacteria* morphotype 3. Scale bars: A* 2 µm; B* 5 µm; C–E* 2 µm; F* 50 µm; G* 2 µm; H–J* 1 µm. Abbreviations: am* amoebocyte; bam* large amoeboid cell; ch* choanocytes; gc* granular cell; gr* granule; m* mesohyl; mf* myofibrils; n* nucleus; sc* spherulous cells; sp* spicule.

Figure 16. Leucosolenia variabilis Haeckel* 1870 spicule types in the White ºea specimen %A) and in the specimens from the British Natural History Museum collection %B–D).A* spicules from Wº11731; B* spicules from BMNH 1906.12.1.40; C* spicules from BMNH 1906.12.1.50; D* spicules from the syntype BMNH 1910.1.1.421.a. Abbreviations: d* diactines; tx* trichoxeas.