Vetulina incrustans, Schuster & Pisera & Kelly & Bell & Pomponi & Wörheide & Erpenbeck, 2018

VETULINA INCRUSTANS View in CoL SP. NOV.

( FIGS 6A–F, 7A–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, 6A) .

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. 6A). Oscules are ~ 1 mm in diameter, scattered irregularly on the apex of the surface domes, and flush with the surface ( Fig. 6B). Aquiferous canals radiate towards the oscules ( Fig. 6B). Ostia, 0.1–0.3 mm in diameter, are evenly dispersed over the entire surface ( Fig. 6B), 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. 6A). Beige in ethanol.

Skeleton is composed of sphaeroclone desmas ( Fig. 6C), the ectosome being differentiated by the presence of brushes of microstyles projecting from the surface between the ostial pits ( Fig. 6C). 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. 6E, F) that are joined to other desmas in zygosis. The apex is ornamented with ragged (heavily acanthose) spires ( Fig. 6E, F); the branches are more simply spined. Immature desmas are characterized by a hole in the centre ( Fig. 6D). In addition, hastate, slightly curved oxea megascleres of various sizes (75–217 µm in length and 3–10 µm thick, N = 15; Fig. 7A–E) were detected but considered here as artefacts of haplosclerid origin.

Microscleres are abundant slender, slightly curved, occasionally polytylote microstyles ( Fig. 7F–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 cox1 (‘Folmer’ fragment) and 28S (C1–D2 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. 7A–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. 7A–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, 28S, ITS and 18S sequences of all known extant species except for V. incrustans sp. nov., where no ITS and 18S could be amplified ( Figs 8, 9).

Sequence variation among different Vetulina species is found to be low for ITS ( Fig. 8A). 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 cox1 (0.5%) and 28S (1.6%). There is no variation between V. stalactites and V. tholiformis sp. nov. within cox 1, 18S and 28S (C1– D2), but within ITS we observed pairwise sequence difference of 0.8%). GenBank sequence AJ224648 View Materials has several ambiguous sites towards the 3′ end of the sequence, which causes the long branch in the 18S gene tree.

Our dated phylogeny based on datasets of cox1 and 28S (C1–D2) 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