taxonID	type	description	language	source
745B522BFFEAFFD5FEFC599BFB84FE3D.taxon	materials_examined	Type species: Vetulina stalactites Schmidt, 1879 (by monotypy). Emended diagnosis: Polymorphic lithistid Demospongiae: encrusting to hemispherical, lamellate or vase shaped; acrepid polyaxial desmas (sphaeroclone to astroclone) as megascleres; microscleres if present are styles, (sub) tylostyles and strongyles (modified from Pisera et al., 2017). Remarks: Only lamellate or vase-shaped growth forms are published in the diagnosis of the genus (Pisera & Lévi, 2002 b; Pisera et al., 2017). However, in the present study we describe two new species, the first with a thick encrusting, spreading morphology, and the second forming a thick hemispherical encrustation with a spherical outline, requiring amendment to the diagnosis of the genus. In addition, (sub) tylostyles and strongyles of various sizes (75 – 125 µm) are discovered and considered here as microstyles, despite their size, following the same argument as Pisera et al. (2017), who considered the location / function of these styles in the skeleton as being microscleres.	en	Schuster, Astrid, Pisera, Andrzej, Kelly, Michelle, Bell, Lori J., Pomponi, Shirley A., Wörheide, Gert, Erpenbeck, Dirk (2018): New species and a molecular dating analysis of Vetulina Schmidt, 1879 (Porifera: Demospongiae: Sphaerocladina) reveal an ancient relict fauna with Tethys origin. Zoological Journal of the Linnean Society 184: 585-604
745B522BFFEAFFD5FEFC599BFB84FE3D.taxon	materials_examined	Material examined: HBOM 003: 01011 (HBOI 21 - V- 00 - 1 - 004): Johnson Sea-Link II (JSL-II) dive 3226, South Central Coast, off Piscadera Bay, Curaçao, 12 ° 6 ′ 38.77 ″ N, 68 ° 58 ′ 43.25 ″ W, 212 m, May 2000. HBOI 11 - V- 00 - 3 - 006: JSL-II dive 3210, South Central Coast, Seamount off Porto Mari Bay, Curaçao, 12 ° 12 ′ 51.19 ″ N, 69 ° 5 ′ 50.21 ″ W, 180 m, May 2000. HBOI 11 - V- 00 - 3 - 004: JSL-II dive 3210, South Central Coast, Seamount off Porto Mari Bay, Curaçao, 12 ° 12 ′ 51.19 ″ N, 69 ° 5 ′ 50.21 ″ W, 219 m, May 2000. HBOI 31 - VIII- 93 - 4 - 004: JSL-I dive 3601, North Coast, 4 Nautical miles (NM) SE of Galina Port, Jamaica, 18.355 ° N, 76.833 ° W, 350 m, August 1993. HBOI 14 - V- 00 - 1 - 007: JSL-II dive 3214, West Coast, Wecua Port, Bonaire, 12 ° 13 ′ 20.35 ″ N, 68 ° 25 ′ 2.93 ″ W, 256 m, May 2000. HBOI 13 - XI- 98 - 3 - 003: JSL-I dive 4098, Andros Island, East of Stafford Creek, Bahamas, 24 ° 54 ′ 49.79 ″ N, 77 ° 52 ′ 12.61 ″ W, 498 m, June 1998. Ty p e l o c a l i t y: E a s t e r n C a r i b b e a n, B a r b a d o s (13 ° 10 ′ 0 ″ N, 59 ° 40 ′ 0 ″ W), 183 – 329 m, RV Hassler Expedition (Fig. 1 A). Distribution: Barbados (13 ° 10 ′ 0 ″ N, 59 ° 40 ′ 0 ″ W), 135 – 601 m (Schmidt, 1879); Curaçao, 12 ° 6 ′ – 12 ° 12 ′ N, 68 ° 58 ′ – 69 ° 05 ′ W, 137 – 219 m (present study); Jamaica, 18 ° 21 ′ 18.07 ″ N, 76 ° 49 ′ 59.41 ″ W, 350 m (present study); Turks and Caicos, 21 ° 31 ′ – 21 ° 52 ′ N, 71 ° 8 ′ – 72 ° 20 ′ W, 520 – 550 m (present study); Bahamas, 23 ° 38 ′ – 24 ° 54 ′ N, 74 ° 56 ′ – 77 ° 52 ′ W, 498 – 569 m (present study); St Vincent Island, 13 ° 9 ′ 38.99 ″ N, 61 ° 16 ′ 31.19 ″ W, 247 m (present study); Bonaire, 12 ° 13 ′ 20.35 ″ N, 68 ° 25 ′ 2.93 ″ W, 256 m (present study); Martinique 14 ° 30 ′ 18 ″ N, 61 ° 6 ′ 12 ″ W, 220 m (Pomponi et al., 2001). Description: The gross morphology is irregularly lamellate to undulating and foliose, occasionally with tubular outgrowth (Fig. 2 A – E) or cup to vase shaped (Fig. 2 F); the base is attached to hard substratum. Dimensions of the cups are ~ 5 – 20 cm in diameter and ~ 7 – 26 cm high, with walls of 0.6 – 1 cm thickness and round, flattened margins. Outer surfaces contain well-developed, regularly concentric growth lines (Fig. 2 A – F) and irregularly distributed pores of ~ 0.1 mm in diameter (Fig. 3 A, B), while the inner surface is rather smooth and finely porous (Fig. 2 E). Texture stony. Colour in life is pale yellow, yellowish brown to tan in ethanol. The skeleton is composed of a dense and regular network of sphaeroclone desmas (Fig. 3 D – F) stacked upon one another (Fig. 3 D). Megascleres are sphaeroclone desmas, 100 – 350 µm in diameter, with an irregularly globular centrum, 0.03 – 0.06 mm in diameter, with spinose root-like outgrowths emanating from the top of each desma (Fig. 3 E). Immature desmas have a hollow centre (hole: ~ 20 – 25 µm in diameter; Fig. 3 E, F). Three to eight sparsely spinose rays branch from the lower part of the centre (Fig. 3 E). Zygomes of the branching rays attach the upper centre part of adjacent desmas to build up the skeleton. The globose centre aligns to the surface, with rays branching and extending towards the choanosome (Fig. 3 A – C). Microscleres are microstyles ~ 50 – 150 µm long and 4 µm thick, with mucronate pointed tips (Fig. 3 C). Microstyles exclusively protrude from the ectosome surface and are sparsely distributed around some ostia (Fig. 3 C). DNA barcodes: In the present study, we sequenced partial cox 1 (‘ Folmer’ fragment) of specimens: HBOI 19 - XI- 94 - 1 - 012, 2 - VII- 89 - 2 - 012, 21 - V- 00 - 1 - 004, 31 - VIII- 4 - 004, 12 - XI- 94 - 3 - 005, 14 - V- 00 - 1 - 007, 14 - XI- 02 - 3 - 001, 13 - XI- 98 - 3 - 003, 11 - V- 003 - 004, 11 - V- 00 - 3 - 008, 11 - V- 00 - 3 - 006, 31 - III- 89 - 1 - 003 and 14 - V- 00 - 1 - 009. All 13 sequences are identical in all specimens; partial 28 S fragment C 1 – D 2 for HBOI 31 - VIII- 93 - 4 - 004, 13 - XI- 98 - 3 - 003, 11 - V- 00 - 3 - 006, 21 - V- 00 - 1 - 004 and 14 - V- 00 - 1 - 007, all of which are identical; ITS for HBOI 11 - V- 00 - 3 - 006, 21 - V- 00 - 1 - 004, 31 - VIII- 93 - 4 - 004, 13 - XI- 98 - 3 - 003 and 11 - V- 00 - 3 - 004, all of which are identical; and partial 18 S for HBOI 14 - XI- 02 - 3 - 006, 13 - X- 03 - 3 - 003 and 13 - XI- 98 - 3 - 003, all of which show identical sequences. Additionally, GenBank accession no. KC 901963 (Redmond et al., 2013) and AJ 224648 (McInerney, Adams & Kelly, 1999) (18 S) are added, whereupon sequence AJ 224648 has ambiguous characters towards the 3 ′ end. Remarks: Before this study, V. stalactites was reported only from the type locality of Barbados, between 153 and 601 m (Schmidt, 1879; Van Soest & Stentoft, 1988) and Martinique (229 m; Pomponi et al. 2001). Here, we expand the distribution of this species in the Tropical Western Atlantic to the Bahamas, Turks and Caicos Islands, Jamaica, Curaçao, St Vincent and Bonaire, all collected between depths of 137 and 569 m (see Fig. 1). Our re-examination of V. stalactites, in particular the detailed observation of the ectosomal membrane, clearly produces evidence of the presence of microscleres (styles) throughout the species (Fig. 3 C), which were already noticed by Sollas (1888), but considered by Pisera & Lévi (2002 b) to be contamination. Finally, spinose microxeas, as observed from the inner surface of the syntype MCZ 6640 by Pisera & Lévi (2002 b), could not be detected in any of our examined specimens.	en	Schuster, Astrid, Pisera, Andrzej, Kelly, Michelle, Bell, Lori J., Pomponi, Shirley A., Wörheide, Gert, Erpenbeck, Dirk (2018): New species and a molecular dating analysis of Vetulina Schmidt, 1879 (Porifera: Demospongiae: Sphaerocladina) reveal an ancient relict fauna with Tethys origin. Zoological Journal of the Linnean Society 184: 585-604
745B522BFFE5FFD7FC505CD7FD3FF923.taxon	description	(FIGS 2 G – J, 4 A – F, 5 A – C) Diagnosis: Dome-shaped Vetulina with a thick, steepsided body and circular to oval base and an osculum opening (1 – 4 mm in diameter) on the apex of the sponge. Microscleres are styles and (sub) tylostyles. Holotype: HBOI 30 - X- 96 - 2 - 003: JSL-I dive 2799, SW Grand Bahama Island, lithoherm (deep-water c a r b o n a t e m o u n d), B a h a m a s, 2 6 ° 3 7 ′ 2 7.2 3 ″ N, 78 ° 58 ′ 48.83 ″ W, 428 m, October 1996. Paratypes: HBOI 11 - XI- 02 - 3 - 011: JSL-I dive 4500, Eleuthera, Weymyss Bight, Bahamas, 24 ° 45 ′ 53.46 ″ N, 76 ° 19 ′ 21.58 ″ W, 427 m, November 2002. 19 - XI- 98 - 1 - 005: JSL-I dive 4109, Rum Cay, South Coast of Port Nelson, Bahamas, 23 ° 37 ′ 24.42 ″ N, 74 ° 50 ′ 4.74 ″ W, 440 m, November 1998. 13 - XI- 02 - 1 - 009: JSL-I dive 4503, Crooked Island, NW Tip, Bahamas, 22 ° 40 ′ 0.66 ″ N, 74 ° 21 ′ 7.13 ″ W, 367 m, November 2002. Type locality: Northern Caribbean, Bahamas. SW Grand Bahama Island lithoherm (26 ° 37 ′ 27.23 ″ N, 78 ° 58 ′ 48.83 ″ W), 428 m (Fig. 1 A). D i s t r i b u t i o n: B a h a m a s (2 6 ° 3 7 ′ 2 7. 2 3 ″ N, 78 ° 58 ′ 48.83 ″ W), 428 m; Bahamas, 22 ° 49 ′ – 26 ° 37 ′ N, 74 ° 21 ′ – 78 ° 58 ′ W, 367 – 440 m. Description: Dome-shaped sponge with a thick, steepsided body and circular to oval base (Fig. 2 G – J). Surface smooth or with several oval depressions (Fig. 2 G – J). The entire plain base of the sponge is attached to the hard substratum (Fig. 2 G – J). Dimensions of the spherical – bulbous sponge are 0.7 cm × 2.5 cm. Circular osculum, flush with the surface, 1 – 4 mm in diameter, located apically or laterally on the sponge (Fig. 2 G – J). Outer surface smooth; pores not visible. Colour in life is pale yellow, and yellowish brown (Fig. 2 G, I) to dark brown in ethanol. Choanosomal skeleton consists of very dense sphaeroclone desmas (150 – 300 µm in diameter). Megascleres are sphaeroclones; the centre from the outer choanosomal part has spinose root-like outgrowth (Fig. 4 E, F), whereas the inner part shows lower tubercles with low, smooth tubercled rays. Three to five main rays branch from the centre and connect with the upper centre part of adjacent desmas, building up the skeleton network (Fig. 4 A – D). Rarely, immature desmas are visible, showing a typical hollow of 20 – 25 µm in diameter instead of a globular centre (Fig. 4 E). Microscleres are styles with mucronate tips (Fig. 5 A), subtylostyles with a slight swelling on the upper part of the shaft (= polytylote; Fig. 5 B) and tylostyles. Both style types are sparsely distributed (perpendicular) in the ectosomal part and concentrated around the osculum openings (not shown). Etymology: From Greek tholos, meaning a beehive tomb or domed tombs, which describes the oval dome-shaped gross morphology of the sponge. surface. Wide and tiny vein-like channels radiate from the osculum. Megascleres are sphaeroclonar desmas. Microscleres are styles and diverse subtylostyles. Holotype: NIWA 109682, BSPG 8020, 0 CDN 3443 - A, USNM 1470701: Davao, north side of Talikud Island. Deep inside crevices. Local name: ‘ Angels Cove’, Philippines, 6 ° 56 ′ 43 ″ N, 125 ° 40 ′ 56 ″ E, 7 – 12 m. Collected by Dr Patrick L. Colin, Coral Reef Research Foundation, Republic of Palau, March 1996. DNA barcodes: In the present study, we sequenced partial cox 1 (‘ Folmer’ fragment), ITS and partial 28 S (C 1 – D 2 fragment) of the following specimens: HBOI 30 - X- 96 - 2 - 003, 19 - XI- 98 - 1 - 005, 13 - XI- 02 - 1 - 009 and 11 - XI- 02 - 3 - 011. Sequences from each of the gene fragments were identical; partial 18 S for HBOI 19 - XI- 98 - 1 - 005, 13 - XI- 02 - 1 - 009 and 11 - XI- 02 - 3 - 011. Remarks: This new species differs from V. stalactites, V. rugosa and V. indica, which form foliose vases and cups (Pisera et al. 2017), by having a dome-shaped external growth form instead of being flabellate vase to cup shaped (Fig. 2). The choanosomal sphaeroclones are very similar to V. rugosa, V. indica and V. stalactites, and differ only in minor details, such as tuberculation and arborescent outgrowth density of the desma centre. In terms of microsclere composition, the new species differs from V. rugosa, V. indica and V. stalactites in that rare stubtylostyles and tylostyles were detected aside from styles. Vetulina tholiformis sp. nov. differs from the new species V. incrustans sp. nov. (for description, see next subsection) by being thinly (vs. thickly) encrusting.	en	Schuster, Astrid, Pisera, Andrzej, Kelly, Michelle, Bell, Lori J., Pomponi, Shirley A., Wörheide, Gert, Erpenbeck, Dirk (2018): New species and a molecular dating analysis of Vetulina Schmidt, 1879 (Porifera: Demospongiae: Sphaerocladina) reveal an ancient relict fauna with Tethys origin. Zoological Journal of the Linnean Society 184: 585-604
745B522BFFE7FFD1FEEE5BA8FCD5F8C4.taxon	description	(FIGS 6 A – F, 7 A – I) Diagnosis: Thick encrusting, biscuit-shaped Vetulina, with remarkable bright yellow coloration and several large oscula openings (1 mm in diameter) on the Type locality: Talikud Island, Davao, Philippines, 7 – 12 m (Figs 1, 6 A). Distribution: Davao, Philippines. Description: Thick encrusting, biscuit-shaped sponge, ~ 1 cm thick and up to 20 cm in greatest dimension, with broad hemispherical lobes or domes in the surface (Fig. 6 A). Oscules are ~ 1 mm in diameter, scattered irregularly on the apex of the surface domes, and flush with the surface (Fig. 6 B). Aquiferous canals radiate towards the oscules (Fig. 6 B). Ostia, 0.1 – 0.3 mm in diameter, are evenly dispersed over the entire surface (Fig. 6 B), forming little pits. Texture is stony but breakable, slightly velvety to the touch owing to a fine plush of microstyle brushes projecting from the surface. Colour in life is a remarkable bright yellow to light orange that extends to ~ 1 mm deep; the choanosome is beige (Fig. 6 A). Beige in ethanol. Skeleton is composed of sphaeroclone desmas (Fig. 6 C), the ectosome being differentiated by the presence of brushes of microstyles projecting from the surface between the ostial pits (Fig. 6 C). Immature desmas are obvious in the surface skeleton. Megascleres are sphaeroclones, with a centrum from which arises an elongated, spined apex, and below which emanate four to five spined rays (Fig. 6 E, F) that are joined to other desmas in zygosis. The apex is ornamented with ragged (heavily acanthose) spires (Fig. 6 E, F); the branches are more simply spined. Immature desmas are characterized by a hole in the centre (Fig. 6 D). In addition, hastate, slightly curved oxea megascleres of various sizes (75 – 217 µm in length and 3 – 10 µm thick, N = 15; Fig. 7 A – E) were detected but considered here as artefacts of haplosclerid origin. Microscleres are abundant slender, slightly curved, occasionally polytylote microstyles (Fig. 7 F – I). Substrate and ecology: Sponges were found deep inside crevices on vertical limestone reef faces with small caves, between 7 and 12 m. Etymology: Named after its unusual encrusting growth form (incrustans = encrusting, Latin). DNA barcodes: In the present study, we sequenced partial cox 1 (‘ Folmer’ fragment) and 28 S (C 1 – D 2 fragment). Remarks: The encrusting domed morphology of V. incrustans sp. nov. is highly distinctive, clearly separating it from all species of Vetulina, particularly V. indica and V. rugosa from Western Australia, which are lamellate. The sphaeroclones are of a similar general structure and diameter, but the ornamentation is characteristic of the new species; the apical spine is covered with ragged spires. Hastate oxeas (Fig. 7 A – E), reminiscent of haplosclerid forms, were found to be moderately common in places in the ectosome and shallow choanosome. They represent a variety of sizes and shapes, ranging in length from 75 to 270 µm, and 2 – 10 µm thick (Fig. 7 A – E), suggesting that they are artefacts. We consider the diverse polytylote subtylostyle microscleres to belong to the species owing to their position in the skeleton. and V. rugosa from Western Australia is dated to ~ 22.5 Mya (Early Miocene, Aquitanian), whereas the split of V. tholiformis and V. stalactites is dated earlier, to ~ 16.1 Mya (Early Miocene, Burdigalian). Furthermore, our relaxed molecular clock approach indicates a possible split of freshwater sponges (Spongillida) and marine Sphaerocladina at ~ 247.5 Mya (Early Triassic). MOLECULAR SYSTEMATICS AND RELAXED MOLECULAR CLOCK APPROACH The present study comprises the largest dataset of Vetulina sequences to date, encompassing cox 1, 28 S, ITS and 18 S sequences of all known extant species except for V. incrustans sp. nov., where no ITS and 18 S could be amplified (Figs 8, 9). Sequence variation among different Vetulina species is found to be low for ITS (Fig. 8 A). Nevertheless, the three Indo-Pacific Vetulina species (V. rugosa, V. indica and V. incrustans sp. nov.) group separately from the Tropical Western Atlantic species (V. tholiformis sp. nov. and V. stalactites; Figs 8, 9). In addition, there is no genetic variation observed between V. rugosa and V. indica from the Indian Ocean. Pairwise sequence differences of these two species to V. incrustans sp. nov. from the Philippines is low in cox 1 (0.5 %) and 28 S (1.6 %). There is no variation between V. stalactites and V. tholiformis sp. nov. within cox 1, 18 S and 28 S (C 1 – D 2), but within ITS we observed pairwise sequence difference of 0.8 %). GenBank sequence AJ 224648 has several ambiguous sites towards the 3 ′ end of the sequence, which causes the long branch in the 18 S gene tree. Our dated phylogeny based on datasets of cox 1 and 28 S (C 1 – D 2) calculated in a relaxed molecular clock framework is illustrated in Fig. 10. The Asian Vetulina species form a monophylum, which is sister to the Tropical Western Atlantic Vetulina clade. The origin of all extant Vetulina species is dated to ~ 43.3 Mya (Eocene, Bartonian), with a credibility interval of 21.1 and 84 Mya, thus before the closure of the Tethyan Seaway, which was dated to 13 – 11 Mya. The split of V. incrustans sp. nov. from the Philippines and V. indica	en	Schuster, Astrid, Pisera, Andrzej, Kelly, Michelle, Bell, Lori J., Pomponi, Shirley A., Wörheide, Gert, Erpenbeck, Dirk (2018): New species and a molecular dating analysis of Vetulina Schmidt, 1879 (Porifera: Demospongiae: Sphaerocladina) reveal an ancient relict fauna with Tethys origin. Zoological Journal of the Linnean Society 184: 585-604
