Description and molecular phylogeny of Tethya leysae sp. nov. (Porifera, Demospongiae, Hadromerida) from the Canadian Northeast Pacific with remarks on the use of microtomography in sponge taxonomy Author Heim, Isabel Author Nickel, Michael text Zootaxa 2010 2422 1 21 journal article 10.5281/zenodo.194503 93c97456-1abb-49bf-aee5-40d6917e2b3b 1175-5326 194503 Tethya leysae sp. nov. Holotype : NHM 2009.5.1.1, Leg. Sally P. Leys, 25.06.2003 . Paratype : PMJ Porif 287, Leg. Sally P. Leys, 27.09.2006 Type locality: Rocky hard bottom substrate in the shallow Infralittoral ( 10 - 25 m depth) near Ohiat Islet, Northeast Pacific, Barkley Sound, Bamfield, Vancouver Island, British Columbia , Canada ( Fig. 1 ), coordinates 48°51’3.00’’ N , 51’3’’ 125°11’60.00’’ W . Diagnosis. Tethya leysae sp. nov. is the only Tethya species in the NE Pacifc possessing a massive uniform cortex with few lacunae and densely packed megasters (oxyspherasters 41-115 µm; R/C 0.41), which are larger and display shorter rays compared to T. californiana . It also lacks the alveolar exocortex and the bilayered megastrer distribution typical for T. californiana ; Two categories of oxeas/strongyloxeas, both slightly larger/thicker than in T. californiana : main (1580-2540 µm x 18-53 µm) and auxiliary (490-1490 µm x 7–30 ), lacking tylostrongyles. Etymology. We have chosen the name in honor of Prof. Dr. Sally P. Leys, Edmonton, BC, Canada , who collected and kindly provided the type specimens and is an inspiring colleague and friend. Description. General body morphology. The body is spherical, with a diameter of 5 x 4 cm ( holotype ; Fig 2A ). Sections show an unambiguous division into a cortex region and a choanosomal core ( Fig. 3 A). The colour in life is orange-yellow to light red ( Fig. 2 C). The colour in alcohol is white, with a greyish core ( Fig. 2A ). In living specimens, the body is slightly contractile. However, the overal body consistency is incompressible. The verrucose surface is frequently loaded lightly with sediment. The surface lacks tubercles, filaments and stalked buds (at least they have not been found in the examined material). The cortex is dense and compact, with 3-6 mm in thickness. It lacks lacunae, but is packed with megasters ( Figs. 3 and 4 ). FIGURE 1. Type locality of T. leysae sp. nov. in the Northeast Pacific, around Ohiat Islet, Barkley Sound, near Bamfield, Vancouver Island, British Columbia, Canada, North America. FIGURE 2. A– B. True to scale comparison between the habitus of T. leysae sp. nov. (A, paratype) and T. californiana (B; figure modified from Sarà & Corriero, 1993). C. Tethya leysae sp. nov in situ in Barkley Sound. Asterisks indicate stalkless buds. The image is a scan of a diapositive; neither the used film material nor the scanner was color-calibrated; therefore, the colors might deviate from natural colors (image courtesy of S. Leys, Edmonton). Skeletal morphology. The dense radiate bundles of main megascleres (oxeas and strongyloxeas) display diameters between 400–600 µm (see sections, Fig. 3 C, F; and microtomographic reconstructions, Fig. 4 A, B). The bundles terminate in compact cortical fans which are formed by the main and auxiliary megascleres and make up broad cortical tubercles ( Figs. 3 B) which contribute to the external verrucose appearance. Groups of auxiliary megascleres are also present interstitially in the choanosome between the main bundles ( Fig. 3 C). The megasters (spherasters/oxyspherasters) are evenly and densely scattered throughout the whole cortex ( Figs. 3 B, 3F, 4), but almost lacking in the peripheral 200–500 µm of the cortex ( Fig. 3 D) and in some basal parts of the cortex near the cortical-choanosomal boundary ( Fig. 3 E). Micrasters form a discrete layer allocated in the exopinacoderm surface ( Fig. 3 D) and are most dense in the peripheral cortex. In addition, micrasters are evenly but sparsely distributed throughout the cortex and the choanosome. Spicules. The main megascleres are constituted by oxeas, anisostrongyles and strongyloxeas ( Fig. 5 ), 1580–2540 µm (2049 ± 259 µm; n=40) in length, 18–53 µm (34 ± 7 µm; n=40) in diameter. Auxiliary megascleres are constituted by oxeas, anisostrongyles and strongyloxeas, 490–1490 µm (1055 ± 215 µm; n=128) in length, 7–30 µm (19 ± 5 µm; n=128) in diameter. Main and auxiliary megascleres form two significantly different length categories (independent t-test, p<0.001; Fig. 6 B), both of normal distribution (Kolmogorov-Smirnov test). The megasters are represented by spherasters to oxyspherasters of varying size and morphology, as evidenced by SEM and microtomography reconstructions ( Figs. 5 B, C, 6A and 7C, Tab. 1 ). Cortical megasters ( Fig. 5 B) display 8–20 rays and are 41–115 µm (84 ± 12 µm; n=40) in diameter with R/Cs of 0.34– 0.69 (0.46 ± 0.07; n=227). Choanosomal megasters ( Fig. 5 C) display 12–18 rays and are 24–81 µm in diameter, with R/Cs of 0.25–0.81 (0.41 ± 0.1; n=85). The form of the rays varies in both regions from slender to stout ( Fig. 5 B, C). A Kolmogorov-Smirnov test (independent t-test, p<0.001) suggests that choanosomal oxyspherasters size is significantly smaller than in cortical megasters. FIGURE 3. Skeletal and overall anatomy of T. leysae sp. nov. (resin slice preparation of the holotype). A. Cross section through cortex (Co) and choanosome (Ch); radial megasclere bundles (rMSB) fan out slightly in the peripheral cortex region. B. – C. Details of the cortex (B) and choanosome (C). The cortex appears solid with almost no subdermal lacunae; it is densely filled with megasters, in contrast to the very low megaster density of the choanosome. Auxiliary megascleres (aMS) are present in the cortical megasclere bundle fans and separately or grouped in the choanosome. D. – G. Asters in the cortex and the choanoderm; peripheral micrasters (ma) are associated with the exopinacoderm (D); megasters (MA) dominate the cortex; the average distance between megasters is lower than one megaster diameter (D & E, see Fig. 4); A peripheral cortical layer 200 – 400 µm thick is almost completely free of megasters (D), subcortical lacunae are present near the inner cortical boundary, thus appearing partly free of megascleres (E); the choanoderm is largely free of megasters (E–F) or they show up in clouds (G), with a much lower density compared to the cortex. Micrasters ( Fig. 5 D) fall into four categories: acanthoxyspherasters ( Fig 5 D, top left), 10–19 µm in diameter with 10–12 rays (the main category); acanthostrongylasters ( Fig. 5 D, top middle) 8–18 µm in diameter, with 8–12 rays; a few acanthotylasters with only slight terminal knobs ( Fig. 5 D, top right), 6–8 µm in diameter, with 10–14 slightly spinulated rays; and small oxyspherasters ( Fig. 5 D, bottom), 4–10 µm in diameter, with 10–15 slender rays. Molecular characters. The nucleotide sequences of the cytochrome oxidase subunit I (Folmer fragment) are accessible in Genbank ( holotype : GQ292532 ; paratype : GQ292533 ) and at www.spongebarcoding.org (record no. 222). The base pair exchanges in the COI fragment and the deduced amino acid sequences (Tab. 2) clearly distinguish T. leysae sp. nov. from T. californiana (4 nt/2 aa), T. minuta Sarà, Sarà, Nickel & Brümmer, 2001 (22 nt/3 aa) and T. actinia (18 nt/3 aa). FIGURE 4. Virtual 3D reconstructions of the cortical skeleton of T. leysae sp. nov. (holotype) imaged using synchrotron radiation-based x-ray micro computer tomography (SR µCT). The massive megasclere bundles reach diameters of up to 500 µm; there is no free space between asters and megasclere bundles as sometimes seen in other Tethya species; patchy aster-free regions are occupied by canals (tissue not visible in spicule-optimized SR µCT, see Nickel et al. 2006a, b). A. – B. Block diagrams of cortex preparations from the holotype (A) and the paratype (B). C. Detail cropped from the paratype (coordinate system in mm). Additional 3D-renderings of the holotype as well as the paratype are available upon request. FIGURE 5. Spicule types of T. leysae sp. nov. (A–D; SEM micrographs) in comparison to T. californiana (E–F; drawings modified from Sarà & Corriero 1993, re-evaluated by own light microscopy of spicule preparations from the specimen BMNH 29.8.22.15.). A. Main and auxiliary megascleres. B. The highly variable cortical megasters. C. Choanodermal megasters. D. Micrasters. E. Megasters. F. Micrasters. Reproduction. Asexual reproduction by bud formation near the sponge surface is indicated ( Fig. 2 C; asterisks). No data exist to date on the sexual reproduction of T. leysae sp. nov. Ecology. The type habitat at Barkley Sound is infralittoral hard bottom influenced by strong tidal changes such as regular periods of strong currents. Usually, T. leysae is found in aggregates of several specimens (presumably due to asexual reproduction by budding). Larger specimens of up to 8 cm diameter sometimes cluster in sheltered small canyons of wave exposed areas. Tethya leysae sp. nov. is most abundant in depths between 15–20 m with moderate water flow but no direct wave exposure or current. It usually lives in lighted conditions and on shaded rocks, but avoids dark habitats. Specimens of T. leysae sp. nov. are frequently found to be covered by debris which might be particulate organic matter, but also algae and other small epibionts like foraminifers. In some areas of Barkley Sound, it is the most obvious subtidal sponge. Other common sponges are Neopetrosia vanilla (de Laubenfels,1930) and Cliona sp. (for a species list compare Austin et al. 1999 –2007). Distribution. At present, T. leysae sp. nov. has only been reported for its type locality Barkley Sound, near Bamfield, British Columbia , Canada . It is likely to occur more widely along the North American Pacific coast but its biogeographical limits are presently not known. Related species. Comparative morphology (general anatomy, skeleton structure, megasclere and microsclere sizes, forms and distribution) suggests that T. leysae sp. nov. might be closely related to T. aurantium , T. robusta ( Bowerbank, 1873 ) and T. californiana . At present, T. californiana seems to be the only species which eventually occurs sympatrically. However, T. leysae sp. nov. can be clearly distinguished from T. californiana by the lack of an alveolar cortex and the extremely high density of megasters in the cortex. Another striking difference is the megaster morphology. Their R/C values differ significantly between T. californiana and T. leysae sp. nov. ( Tab. 1 ) and the oxyspherasters of the latter rarely display bent rays. In addition, spherules have not been found among the micrasters of T. leysae sp. nov. However, this character can only be accessed by extensive and very careful study of spicule preparations. In addition to the morphological differences between T. californiana and T. leysae sp. nov. , extensive nucleotide (4 nt) and amino acid (2 aa) exchanges are present within the molecular marker COI (Tab. 2). Tethya leysae sp. nov. also differs from T. aurantium in respect to cortex architecture, which is more massive and much more densely packed with megasters in the new species. In comparison to T. aurantium , the variance in relative ray length of the megasters is higher: while T. aurantium displays spherasters, T. leysae sp. nov. displays a range from spherasters to oxysherasters (compare data in Sarà & Melone 1965 ; Sarà et al. 1992 ). In addition to the morphological differences between T. aurantium and T. leysae sp. nov. , extensive nucleotide (52 nt) and amino acid (4 aa) exchanges are present within the molecular marker COI (Tab. 2). Tethya robusta seems to be the species with the most similar cortical architecture ( Bowerbank 1873 ; Sarà & Sarà 2004 ), since in both species, megasters are so closely packed “that the rays of each pass between those of the adjoining ones, and the whole become, as it were, cemented into a solid mass” ( Bowerbank 1873 ). However, the megasters in T. robusta display a much higher number of rays (24–32) than those of T. leysae sp. nov. Both species also differ in micraster types : T. leysae sp. nov. lacks the fine rayed oxyasters of T. robusta which in turn lacks the stout acanthoxyspherasters of T. leysae sp. nov.