A new cave-dwelling species of Plakina (Porifera: Homoscleromorpha) from Crete, Greece (South Aegean Sea) Author Lage, Anaíra Author Araujo, Hannah Paola Mota Author Gerovasileiou, Vasilis Author Muricy, Guilherme text Zootaxa 2018 2018-08-31 4466 1 39 48 journal article 29372 10.11646/zootaxa.4466.1.5 1980a3df-0542-48ec-a594-e6bc4dc8f3aa 1175-5326 1442100 2FACA737-6533-4EFE-B2E8-17DCF3D62FEC Plakina strongylata sp. nov. ( Figs. 1–3 ) Diagnosis. Plakina thinly encrusting, with a highly folded surface and white color. Spicules are diods, triods and calthrops with rounded extremities plus mono- and dilophose diods; mono-, di- and trilophose triods; and mono-, di-, tri- and tetralophose calthrops. Most spicules are very irregular, and the ramification pattern of lophose spicules often varies in different actines of a single spicule. Material examined. Hοlοtype MNRJ 21242 , Blue Cave , Crete , 15 m depth, 11.vii.2017 , coll. V. Gerovasileiou and G . Muricy. Paratype MNRJ 21215 , same data as holotype . Material examined for comparison. Plakina trilοpha Schulze, 1880 : Neοtype. MNHN.D.NBE.1468, 7.vii.1995 , 3PP Cave, La Ciotat, France, 43°13’N, 5°35’E, 15 m depth, coll. N. Boury-Esnault (Muricy et al. 1998) . MNRJ 18370, Fara Cave, Fara Bay Islet, Lesvos Island, Greece (38°58’11.64”N, 26°28’39.54”E), 18 m depth, 17.vii.2010 , coll. V. Gerovasileiou (Lage et al. 2018) . Plakina anisοactina Lage, Gerovasileiou, Voultsiadou & Muricy, 2018 : Hοlοtype MNRJ 18372, Agios Vasilios Cave, Agios Vasilios Islet, Lesvos Island, Greece (38°58’13.25”N, 26°32’30.46”E), 23–40 m depth, 25.vi.2010 , coll. V. Gerovasileiou. Plakina bοwerbanki (Sarà, 1960), Lesvos Island, Greece: MNRJ 18365, 04.vi.2010 ; MNRJ 19634, MNRJ 19635, 18.v.2015 ; Fara Cave, Fara Bay Islet, 18 m depth, coll. V. Gerovasileiou (Lage et al. 2018) . Plakina anοmala Lage, Gerovasileiou, Voultsiadou & Muricy, 2018 : Hοlοtype MNRJ 18374-A, Paratype MNRJ 18374-B, both from Fara Cave, Fara Bay Islet, Lesvos Island, Greece, 18 m depth, 01.vi.2010 , coll. V. Gerovasileiou. Plakina hellenica Lage, Gerovasileiou, Voultsiadou & Muricy, 2018 : Hοlοtype MNRJ 19637, Fara Cave, Fara Bay Islet, Lesvos Island, Greece, 15–18 m depth, 18.v.2015 , coll. V. Gerovasileiou. Description. Sponge thinly encrusting, with irregular outline, up to 8 x 8 cm wide by 2–3 mm thick ( Figs. 1A– B ). Color white. Surface strongly folded, with many irregularly cylindrical canals, 3–5 mm in diameter, pressed against each other and forming ridges in the whole surface ( Figs. 1A–B ). The canals converge to circular oscules, 1–2 mm in diameter, with a thin transparent rim. The surface is punctuated by many small inhalant openings. Marginal canal absent. Consistency very soft, fragile. Skeletοn . Choanosomal skeleton forms a dense, irregular to alveolar reticulation with meshes 30–50 µm in diameter surrounding choanocyte chambers ( Fig. 1C ). Basal cavities well developed, approximately 100–150 µm high ( Fig. 1C ). Lophose calthrops are concentrated in the ectosome, often with lophose actines pointing outwards ( Fig. 1D ). Subectosomal cavities absent. Spicules ( Figs. 1–3 , Table 1 ). Diods common, irregular in shape; straight, curved or centrally bent. Central swelling conspicuous, ‘S-bent’, centrotylote, or button-shaped. Extremities rounded, strongyloid ( Fig. 1E–F ). Full size: 14–36–62 / 1.1–2.5–5.1 µm. Triods abundant, irregular in shape, uni- or bi-planar, Y- to T-shaped. Actine extremities rounded or, more rarely, acerate ( Fig. 1G ). Actine size: 8–19–34 / 1.1–2.3–4.9 µm. Calthrops rare, variable in shape, with straight or irregular actines and rounded extremities. One actine is sometimes reduced to a small button ( Fig. 2A ). Actine size: 6–21–40 / 1.0–2.0–4.2 µm. Monolophose diods common, irregular in shape. Non-lophose actine straight or curved, with blunt end; center very irregular, often with button-shaped protuberances similar to reduced actines. Lophose actine ramifies in 2–4 short rays with rounded ends (ramification pattern ‘1m’) or with a second round of ramification and acerate or blunt ends (pattern ‘ 1m , 2d’) ( Fig. 2B ). Full size: 19–35–47 / 1.4–2.4–4.3 µm. Dilophose diods very rare, small, irregular, with a large button-shaped center and straight or sinuous actines, which ramify in 2 rays that may ramify again in 2 secondary rays with strongyloid or bifurcated ends ( Fig. 2C ). Full size: 22–33–40 / 1.6–2.2–3.4 µm. Monolophose triods common, irregular, variable in shape; non-lophose actines even or annulated, with blunt or slightly bifurcated ends. Central irregularity conspicuous. Lophose actine ramifies once in 2–4 rays, which rarely ramify again distally in 2–3 secondary rays with rounded or acerate ends ( Fig. 2D ). Full size: 17–28–44 / 1.3–2.5– 4.3 µm. Dilophose triods uncommon, asymmetrical, general shape similar to monolophose triods. Non-lophose actine even or slightly annulated, with blunt ends. Lophose actines ramify once at medial or distal position in 2–4 rays, which may be stout with rounded ends or slender, ramifying again at distal position and with blunt ends ( Fig. 2E ). Full size: 17–31–40 / 1.4–2.5–4.4 µm. TABLE 1. Spicule types and size of specimens of Plakina strongylata sp. nov. compared to its most similar congeners from the Aegean Sea. Measurements of triods and calthrops are actine sizes, and of all other spicules are full (maximum) spicule sizes.

Species / Specimens	Diods	Triods	Calthrops	Monolophose diods	Dilophose diods	Monolophose triods	Dilophose triods	Trilophose triods	Monolophose Dilophose calthrops calthrops		Trilophose calthrops	Tetralophose calthrops	Irregular spicules
P. strongylata sp. nov.
MNRJ 21215 (P)	14–38–62 / 1.3–2.2–4.3 (36)	8–14–21 / 1.2–2.1–2.8 (36)	6–19–40 / 1.0–2.0–2.9 (30)	20–34–47 / 1.4–2.4–4.3 (28)	30–37–40 / 2.1–2.2–3.4 (3)	17–28–41 / 1.5–2.8–3.9 (32)	19–32–39 / 1.6–2.6–4.4 (27)	15–21–32 / 2.2–2.4–3.0 (4)	14–23–43 / 1.2–2.1–3.6 (24)	18–31–49 / 1.7–2.2–3.4 (27)	21–27–35 / 1.5–2.5–4.2 (18)	22 / 1.9 (1)	19–35 (2)
MNRJ 21242 (H)	21–35–59 / 1.1–2.7–5.1 (33)	13–23–34 / 1.1–2.7–4.9 (34)	7–23–36 / 1.1–2.1–4.2 (30)	19–35–46 / 1.9–2.5–3.3 (16)	22 / 1.6 (1)	18–27–44 / 1.3–2.4–4.3 (31)	17–30–40 / 1.4–2.5–3.5 (23)	20–23–25 / 1.8–2.0–2.2 (2)	12–27–34 / 1.4–2.3–2.9 (14)	19–26–34 / 1.4–2.4–4.3 (27)	16–23–35 / 1.5–2.1–3.6 (12)	21–21.5–22 / 2.2–2.4–2.6 (2)	23–27 (3)
P. bowerbanki *	–	–	10–14–19**	–	–	–	–	17–23–35	18–24–33	17–25–37	16–24–31	3 distinct shapes	15–29
P. hellenica *	37–59–80	13–24–35	10–18–33	–	16–26–45	14–26–54	16–25–40	15–21–27	15–25–35	15–25–40	7–12–21	11–18–30	17–26
P. anisoactina *	40–59–78 / 2–3	13–24–30 / 2–4	12–21–30 / 3–4	–	–	25–38–55	20–29–38	–	28–34–45	20–34–51	25–32–41	–	–
P. trilopha *	37–56–75 / 1–2	15–22–29 / 1.0–1.5	11–18–27 / 1.5–2.0	–	–	–	–	–	20–24–29	18–22–26	17–21–24	15–19–23	–

H, holotype. P, paratype. –, absent. Number of measurements in parentheses. Measurements are minimum–average–maximum length / width or full spicule size in micrometers (μm). *, measurements from Lage et al. (2018) ; Lesvos Island , Greece . **, calthrops with large spines. FIGURE 1. Plakina strongylata sp. nov. A–B, living specimens in situ (os, oscules). C, transverse section of the whole sponge body (bc, basal cavities; ch, choanosome; em, embryos; s, surface). D, ectosome in transverse section (ls, lophose spicules). E, diods. F, detail of the extremities of diods. G, triods (E–G, SEM). Trilophose triods rare, irregular, variable in shape ( Fig. 2F ). Center thick, irregular; actines straight, ramifying in 2–4 rays that may have round tips or ramify again in 2–3 short secondary rays with rounded or bifurcated ends. Each actine often has a different ramification pattern. Full size: 15–23–32 / 1.8–2.3–3.0 µm. Monolophose calthrops rare, variable in shape, sometimes extremely irregular. Non-lophose actines with blunt or rounded ends, sometimes reduced to irregular buttons. Lophose actines ramify distally in 3–4 short rays that may ramify again at distal position in 2–3 short secondary rays with blunt ends ( Fig. 3A ). Full size: 12–25–43 / 1.2–2.2– 3.6 µm. FIGURE 2. Spicules of Plakina strongylata sp. nov. (SEM). A, calthrops. B, monolophose diods. C, dilophose diods. D, monolophose triods. E, dilophose triods. F, trilophose triods. Dilophose calthrops abundant, irregular, highly variable in shape. Non-lophose actines with blunt ends. Lophose actines ramify at medio-distal position in 2–4 short rays that may bifurcate distally, with terminal spines or blunt ends ( Fig. 3B ). Full size: 18–29–49 / 1.4–2.3–4.3 µm. Trilophose calthrops common, shape and ramification pattern similar to dilophose calthrops ( Fig. 3C ). Each actine often has a different ramification pattern. Full size: 16–27–35 / 1.5–2.4–4.2 µm. Tetralophose calthrops very rare, relatively regular, with straight actines that ramify distally in 2–3 rays with rounded ends ( Fig. 3D ). Full size: 21–21.5–22 / 1.9–2.2–2.6 µm. Irregular spicules are common, variable in shape, and difficult to ascribe to any traditional category. They have mainly 3–4 contorted, ill developed or cylindrical actines, usually with high variation in thickness and with thin terminal ramifications or large spine-like projections ( Fig. 3E ): Full size 19–24.5–35 µm. Reproduction. Cinctoblastula larvae and embryos 50–150 µm in diameter were found in the two specimens examined, collected in July 2017 ( Fig. 1C ). FIGURE 3. Spicules of Plakina strongylata sp. nov. (SEM). A, monolophose calthrops. B, dilophose calthrops. C, trilophose calthrops. D, tetralophose calthrops. E, irregular spicules. Habitat. This species was found in a large semi-submerged cave, exclusively on vertical walls in a narrow semicircular siphon-like passage at a depth range of 14– 17 m . The tunnel was approximately 15 m long and no more than 2–3 m wide, leading to a chamber with small openings to the cave exterior. The population of the new species was aggregated in few small patches apparently restricted to the darker middle zone of the tunnel. Cave walls were dominated by doughnut-like bioconstructions and biostalactites built by Prοtula serpulids, similar to those described from the dark zones of other Aegean caves ( Sanfilippo et al. 2017 ) and some typical sponge species of Mediterranean dark and semi-dark caves ( Gerovasileiou & Voultsiadou 2012 ), e.g. Dendrοxea and Merlia crusts, Myrmekiοderma spelaeum ( Pulitzer-Finali, 1983 ), and small discolored specimens of Petrοsia (Petrοsia) ficifοrmis ( Poiret, 1789 ). Distribution. Known only from the Blue Cave in Agia Pelagia, North coast of Crete Island, Greece ( South Aegean Sea). Nevertheless, more caves with narrow dark passages exist in the same area and further explorations could possibly reveal a broader distribution of this species. Etymology. The species name strοngylata refers to the strongyloid shape of the diods, due to their rounded extremities. From Greek στρογγύλος (strοngylοs) – rounded. Taxonomic remarks. Only one species of Plakina was previously reported from Crete Island: P. weinbergi (see Ereskovsky et al. 2009b ). It greatly differs from the new species by a massive drop-shaped habit pending from the cave ceiling, the rarity of diods, triods and calthrops, the spicule shape with simple ramification pattern and acerate or blunt extremities, and the absence of monolophose diods and of mono- and dilophose triods ( Muricy et al. 1998 ). Increasing molecular evidence supports the paraphyly of the genus Plakina , with recognition of at least two separate clades usually called B3 and B4 (Gazave et al. 2010, 2012; Cruz-Brraza et al . 2014; Ruiz et al . 2015, 2017). Although DNA sequences are not available for the new species, it fits morphologically in Clade B3 (= PhyloCode clade Tetralοphοsa Gazave et al . 2012) due to the synapomorphic presence of tetralophose calthrops. This clade also includes P. trilοpha , P. jani Muricy, Boury-Esnault, Bézac & Vacelet, 1998 , P. jamaicensis Ereskovsky, Lavrov & Willenz, 2014 and P. kanaky Ruiz & Pérez, 2015 (Gazave et al . 2010; Ruiz et al . 2015, 2017). Plakina strοngylata sp. nov. shares with P. jani , P. jamaicensis and P. trilοpha the presence of mono-, di-, tri- and tetralophose calthrops and a folded surface ( Muricy et al. 1998 ; Lage et al. 2018 ). Plakina trilοpha is white in color as the new species, but P. jani and P. jamaicensis are yellow ( Muricy et al. 1998 ). In P. jamaicensis the spicules are regular, uniform and acerate, with a simple ramification pattern ( Ereskovsky et al . 2014 ). In P. kanaky the only spicules are mono-, tri- and tetralophose calthrops, while non-lophose diods, triods and calthrops are absent (Ruiz et al . 2015). Plakina strοngylata sp. nov. further differs from all these species by its very fragile consistency and especially by the exclusive presence of abundant monolophose diods, monolophose triods, and dilophose triods. Plakina hellenica Lage, Gerovasileiou, Voultsiadou & Muricy, 2018 shares many spicule categories with the new species, including diods, triods, calthrops, dilophose diods, mono-, di- and trilophose triods, mono-, di-, tri- and tetralophose calthrops, and irregular spicules ( Lage et al. 2018 ). It differs however by the absence of monolophose diods, the peculiar shape of its sagittal trilophose triods, the more symmetrical shape of all lophose spicules ( vs . typically asymmetrical in the new species), and the acerate ends of most spicules ( Lage et al . 2018 ). They also differ greatly in shape, consitency and colour. Some spicules of P. anοmala Lage, Gerovasileiou, Voultsiadou & Muricy, 2018 from the North Aegean Sea also have rounded extremities (lophose spicules type 6 in Lage et al . 2018 ), but their shape is very different from those of P. strοngylata sp. nov. : their actines are stout, annulated, cylindrical, and ramified terminally in short cylindrical rays with blunt or spined ends. They form a distinct category of lophose spicules, and non-lophose diods, triods and calthrops are absent in P. anοmala . In contrast, in the new species most non-lophose actines of all spicule types have rounded ends, and the actines are generally thinner and more irregular than in the lophose spicules type 6 of P. anοmala . The two species also differ greatly by the unique cavity-filling habit of P. anοmala ( Lage et al. 2018 ) The spicule complement of the new species is most similar to that of P. anisοactina Lage, Gerovasileiou, Voultsiadou & Muricy, 2018 . However, these species differ by the microtuberculate surface and the absence of monolophose diods in P. anisοactina , and especially by the shape of the extremities of the spicules: in P. anisοactina they are acerate or terminally spined and often different in the same spicule, while in the new species most spicules have markedly rounded ends, which make the diods look like irregular strongyles. Also, the lophose spicules of the new species vary from slender actines with 1–2 rounds of ramification and blunt ends to unusually stout, cylindrical actines with a single ramification and rounded extremities. The highly folded surface, the fragile consistency, the types of lophose spicules (especially the unusual monolophose diods and the asymmetrical mono- and dilophose triods and calthrops), and the markedly rounded extremities of most diods distinguish this species from all other species of Plakina .