Taeniogyrus albulus Yamana, Hirabayashi, Tanaka, and Ogawa, 2022

Yamana, Yusuke, Tanaka, Hayato, Hirabayashi, Isao, Dan, Ichinosuke, Hirai, Atsushi & Ogawa, Akito, 2022, Three New Infaunal Species of Taeniogyrus (Echinodermata: Holothuroidea: Apodida: Chiridotidae: Taeniogyrinae) from Southern Coast of Wakayama, Japan, Species Diversity 27 (1), pp. 139-158 : 141-152

Subfamily Taeniogyrinae Smirnov, 1998 Genus Taeniogyrus Semper, 1867 [Japanese name: Kuruma-kagi-namako-zoku] Taeniogyrus albulus Yamana, Hirabayashi, Tanaka, and Ogawa , sp. nov. [New Japanese name: Kushimoto-kuruma-kagi-namako] ( Figs 2 , 3A , 4 ; Tables 1, 2)

Material examined. Holotype: WMNH-INV-2017-100 (length 134 mm, width 10 mm, collected from Kushimoto on 12 July 2017; INSD accession number LC655787 ,

433 bps). Paratypes: 7 specimens, WMNH-INV-2017-101 (length 81 mm, width 11 mm, collected from Kushimoto on 3 September 2017); WMNH-INV-2018-21 (length 69 mm, width 7 mm, collected from Kushimoto on 24 April 2018); WMNH-INV-2018-22 (length 76 mm, width 9 mm, collected from Kushimoto on 24 April 2018); WMNH- INV-2018-23 (length 151 mm, width 13 mm, collected from Kushimoto on 22 May 2018); WMNH-INV-2019-300 (length 55 mm, width 4 mm, collected from Kushimoto on 22 May 2019); WMNH-INV-2020-10 (length 55 mm, width 9 mm, collected from Kushimoto on 26 June 2019; LC655788 , 628 bps); WMNH-INV-2020-11 (length 121 mm, width 13 mm, collected from Kushimoto on 30 December 2019) .

Diagnosis. Body large, up to 150 mm. Living body color semi-transparent pink, anterior tip and tentacles pale brown. Fixed body color yellowish white, anterior tip and tentacles ocher yellow. Tentacles ten, with thick, palmate, bumpy skin, with up to 12 digits. Single Polian vesicle in RI. Body wall containing wheel, curved rod, sigmoid-hook ossicles. Sigmoid-hook ossicles large and outer bend edge with paired rows of spinelets, each row 3–5 spinelets, between 2 rows forming depression. Curved rod ossicles, with spinous ends, centrally with sparse teeth, sometimes teeth along entire length, rods only found sparsely from the anterior body surface. A band of ciliated funnels situated near right side of midventral longitudinal muscle. Funnels long (about 0.3–0.5 mm), curved like French horn, wide opening (about 0.2 mm).

Holotype description. Body cylindrical, large; length of holotype specimen (WMNH-INV-2017-100) 134 mm; width 10 mm ( Fig. 2A ; Table 1). Living body color semitransparent pink, anterior tip and tentacles pale brown ( Fig. 2B ); preserved body color yellowish white, anterior tip and tentacles ochre yellow ( Fig. 2A ). Mouth anterior; anus posterior; oral disc strongly inclined toward ventral side. Dorsal skin smooth, ventral skin rough.

Ten tentacles, non-retractile; thick, palmate, bumpy skin; each tentacle with maximum 12 digits ( Table 1); sensory cups absent ( Fig. 2C ). Tentacle digitations more frequent on ventral side than dorsal side ( Table 1).

Single Polian vesicle in RI, vesicle shaped fusiform (in 2.9 mm, width about 1/3 of length) ( Fig. 2H ). Stone canal single, thin, coiled and short (about 0.9 mm), madreporite small, worm-like, twisted (about 0.7 mm in length), situated left side of the anterior end of dorsal mesentery ( Fig. 2H ). A band of ciliated funnels situated near right side of medioventral longitudinal muscle RI, not clustered, lacking in anterior or posterior ends ( Fig. 2D ). Funnels long (approximately 0.4 mm), strongly curved (> about 90°), like a French horn, laterally depressed narrow opening (approximately 0.7 mm in width; 0.2 mm in height), slit along proximal side to the funnel base, and with short stalk ( Fig. 3A ). Intestine with no loop, situated along IR5. Hermaphrodite; having two ovaries and two testes; ovary in two clusters in anterior body, one each side of dorsal mesentery, tubules well-branched; testis in 2 tubules in middle body, both adhered to abdominal canal, one in mid-dorsal region of left side of dorsal mesentery, one on midventral side, tubules unbranched.

Calcareous ring with 5 thick radial, 5 thick inter-radial plates; distally flattened ends ( Fig. 4A ); all with deep posterior depression and shallow anterior depression, RI with wide anterior notch, others without notch or perforation.

Body wall thin (less than 0.5 mm in thickness), containing wheel, curved rod, sigmoid-hook ossicles ( Figs 2E, F , 4B–D ; Table 2), wheel ossicles scattered sparsely in anteri- or to middle body surface, occasionally lacking posteriorly ( Table 2). Wheel ossicle aggregations (wheel-papillae) absent; wheel ossicles scattered sparsely in anterior to middle body surface, occasionally lacking posteriorly ( Table 2). Wheel ossicle diameter 33–82 µm (means 59–68 µm in different tissues) ( Table 2), rounded-hexagonal, 6 spokes ( Fig. 4B ). Inner margin of rim parallel to outer margin. Teeth triangular-shaped, tip gradually blunted, 8–19 per inter-spokes area (means 13–15 in different tissues) ( Table 2); often reduced in number and size at corners, but no discontinuities in tooth series. Spokes broad, breadth 15–25% of wheel ossicle diameter (means 19–22%) ( Table 2).

Curved rod ossicles, length 107–120 µm ( Table 2), very rarely present, spinous ends, centrally with sparse teeth, sometimes teeth along entire length ( Fig. 4C ), rods only found from the anterior body surface ( Table 2).

Sigmoid-hook ossicles scattered densely throughout body surface ( Fig. 2E, F ), scattered even on longitudinal muscles, not in clear rows or circles. Sigmoid-hook ossicles thick, large, outer bend edge with paired rows of spinelets, each row with 3–5 spinelets, internal space between 2 rows forming depression ( Fig. 4D ). Open-loop end bending strongly, few teeth in shank tip; length 144–185 µm (means 165– 173 µm in four different tissues) ( Table 2).

Tentacles containing small rods ( Fig. 4E ; Table 2), spinous, curved, mostly branching distally, weakly expanding centrally. Rod ossicles length 37–77 µm, breadth 3–10 µm ( Table 2).

Variation including paratypes. Length of largest paratype specimen (WMNH-INV-2018-23) 151 mm; width 13 mm ( Fig. 2A ; Table 1). Tentacle digitations more frequent in medium-sized specimens than large- and small-sized specimens ( Table 1). In all eight type specimens with single Polian vesicle, vesicle shaped fusiform . Wheel teeth triangular-shaped, tip gradually blunted, 7–19 per radiant (means 12–16 in measured five type specimens) ( Table 2). Spokes breadth 15–27% of wheel ossicle diameter (individual means 19–25% in measured five type specimens) ( Table 2). Occasionally spokes bifurcate at close to rim and forming Y-shaped spokes observed at very low frequencies. Wheel diameter 33–86µm (means 53–72 µm in different tissues and in measured five type specimens) ( Table 2), differed significantly among individuals in all body parts (Kruskal– Wallis test, P s<0.01) and differed significantly among parts of the body in three specimens (WMNH-INV-2017-101, 2018-21, and 2018-23) among the present measured five type specimens ( Kruskal – Wallis test, P <0.05) . Curved rod length 88–120 µm ( Table 2), rods only found from the anterior body surface ( Table 2). Occasionally, rods with 1 short central branch. Sigmoid-hook length 137–208 µm (means 162–189 µm in four different tissues and in measured five type specimens) ( Table 2), length of posterior body parts significantly larger than anterior parts in two of five measured specimens (WMNH-INV-2018-21 and 2018-23). Tentacles rod length 28–106 µm (means 49–68 µm in measured five type specimens) ( Table 2).

Remarks. Taeniogyrus albulus sp. nov. has curved rod ossicles in the skin of the anterior body. Among all the taeniogyrid genera, although this feature has been reported in two Rowedota species [ R. mira (Cherbonnier, 1988) and R. chippiru Yamana, Tanaka, and Nakachi, 2017 ], T. albulus sp. nov. is easily distinguishable from these two species based on the shape of the wheel ossicle. However, the presence of body wall rods has not been reported in the genus Taeniogyrus at present. Therefore, T. albulus sp. nov. is also easily distinguishable from other nominal Taeniogyrus species.

Although a detailed description of the position of reproductive organs has not been made in most studies of Taeniogyrus congeners, there have been limited studies regarding hermaphroditism (e.g., Heding 1928). Among the Taeniogyrus congeners in Japanese waters, T. mijim has been reported as hermaphroditic, having two long tubules of ovaries and testis, which are unbranched and isolated from the intestine (Yamana et al. 2017). On the other hand, T. albulus sp. nov. is also hermaphroditic. However, the position of its reproductive organs is unique in T. albulus sp. nov.: the ovary is in two clusters of well-branched tubules in the anterior body, one on each side of the dorsal mesentery; the testis is in two tubules in the middle body, adhered to the intestine, one in the of the mitochondrial CO1 gene, 433 bps and 628 bps were obtained from the holotype (WMNH-INV-2017-100: INSD accession number LC655787 , base frequency A=19.9%, C=14.6%, G=20.1%, T =45.4%) and a paratype specimen (WMNH-INV-2020-10: LC 655788 , base frequency A= 19.1%, C=14.8%, G=21.8%, T =44.3%), respectively . In BLAST searches, the closest hits to the partial CO1 sequence are from Leptosynapta clarki Heding, 1928 ( HM 542244 – HM542248 and HM542251 – HM 542253 , with 83.8–84.0% similarity in 98–100% coverage) .

Taeniogyrus flavus Yamana and Tanaka , sp. nov. [New Japanese name: Shirahama-kuruma-kagi-namako] ( Figs 3B , 5 , 6 ; Tables 3, 4)

mid-dorsal of the left side of the dorsal mesentery and one in the midventral side. There is a possibility that T. albulus sp. nov. will also be distinguishable from the other congeners by the position of its reproductive organs, but which must be studied in more Taeniogyrus species.

Distribution. Known only from the type locality, in shallow water (9–13 m deep) at Kushimoto, Wakayama, southern end of the mainland of Japan.

Etymology. The specific name is derived from the Latin adjective albulus (meaning whitish), based on its body color.

DNA barcode sequences. Two specimens of sequence

Material examined. Holotype: WMNH-INV-2016-135 (length 17.3 mm, width 2.0 mm, collected from Shirahama on 23 June 2016). Paratypes: 5 specimens, WMNH- INV-2016-136 (length 16.8 mm, width 1.9 mm, collected from Shirahama on 23 June 2016); WMNH-INV-2016-137 (length 14.1 mm, width 1.6 mm, collected from Shirahama on 23 June 2016; INSD accession number LC637701 , 819 bps); WMNH-INV-2016-138 (length 13.1 mm, width 1.8 mm, collected from Shirahama on 24 June 2016); WMNH-INV-2016-139 (length 3.8 mm, width 0.9 mm, collected from Shirahama on 24 June 2016); WMNH- INV-2016-140 (length 8.3 mm, width 0.9 mm, collected from Shirahama on 24 June 2016). Other materials: 64 specimens, WMNH-INV-2016-141–204 (length 1.2–16.1 mm breadth 0.8–2.1 mm, collected from Shirahama on 24 June 2016).

Diagnosis. Body color translucent pale yellow, with whitish spots of wheel-papillae along three dorsal interradii. Tentacles ten, with 3–5 pairs of digits. Polian vesicles 1–3 in RI. Ciliated funnels solitary arranged along with inter-radius of near right of medioventral longitudinal muscle RI, and near left side of left lateral longitudinal muscle RII. Funnels curved at middle about 90 degrees, with bugle horn like opening. Outer bend edge of sigmoid hook ossicles frequently having very minute spinelets.

Holotype description. Body small, anaesthetized length of largest specimen (holotype, WMNH-INV-2016-135) 17.3mm (after preservation), cylindrical, straight, or weakly bend downward, slightly tapered toward posterior end ( Fig. 5A ; Table 3). Body color (of living and preserved specimens) translucent pale yellow. Mouth anterior; anus posterior. Oral disc inclined toward ventral side.

Tentacles ten, non-retractile, slender, with smooth or rarely bumpy skin. Each tentacle with 3–5 pairs of digits; sensory cups absent ( Fig. 5C ). Color of tentacles (of living and preserved specimens) pale yellow.

Polian vesicles two in RI (1.3 mm and 1.7 mm in length), branched in basal part ( Table 3). Stone canal single, thin, coiled and short (about 0.2 mm), with a small coffee beanlike madreporite (about 0.15 mm in diameter) just behind of IR5 calcareous plate ( Fig. 5C ). One ambiguous row of ciliated funnels situated in inter-radius of near right of medioventral longitudinal muscle RI ( Fig. 5F ), and another ambiguous row situated interradial zone IR3, near left side of left lateral longitudinal muscle RII, both running through anterior to posterior body cavity, dense in anterior to middle. Each funnel moderately curved (= about 90°), with deep notch at upper ends of opening, and slit with granular layer along proximal side to the funnel base ( Fig. 3B ). Funnels separately standing, small (about 0.05–0.2mm), with short stalk. Intestine with no loop, situated along mediodorsal with mesentery IR5. Each 2–4 tubules of ovaries or testis present in both sides of dorsal mesentery, branched in basal part, rarely branched in other part.

Calcareous ring composed of five radial and five interradial plates, these all being thick and broad, all with low anterior projection and shallow posterior depression ( Fig. 6A ), with distally spread and flattened. Low trapezoid anterior projection in RI ( Fig. 6F ), and low triangle anterior projection in IR5 ( Fig. 6A ), both symmetrical designs.

Body-wall thin (less than 0.5 mm in thickness), containing wheel and sigmoid-hook ossicles ( Figs 4 , 5 ; Table 4). Wheel ossicles only present in wheel-papillae ( Fig. 5F ), which scattered only in three dorsal inter-radial body-wall, ranging from tentacle base to body posterior end. Wheel ossicles rounded-hexagonal with six spokes. Inner margin of rim approximately parallel to outer margin. Teeth sharp, with 9–16 per inter-spokes area. Spokes narrow, their breadth amounting to 20–26% of wheel ossicle diameter ( Fig. 6C ; Table 4). Wheel ossicle diameter 46–75 µm in anterior dorsal side and 43–74 µm in posterior dorsal side.

Sigmoid-hook ossicles in body-wall scattered in interradii. Along both sides of longitudinal muscles, with point- ed end facing toward inter-radius, and open-loop end facing toward longitudinal muscles ( Fig. 5E ). Outer bend edge frequently having minute teeth ( Fig. 6D ), but mostly blunt and not easy to detect. Breadth of shank almost constant, sometimes broadest at bend. Length 91–104 µm in anterior dorsal side, 84–111 µm in posterior dorsal side, 85–96 µm in anterior ventral side, and 74–94 µmin posterior ventral side.

Tentacles containing rod ossicles ( Fig. 6B ; Table 4), these mostly bifurcate distally, rarely bifurcate twice or more and with another branch at centrally. Distal ends spinous. Rod ossicle length 45–69 µm, breadth 4–9µm.

Variation including paratypes. Two Polian vesicles in the holotype, but 1–3 Polian vesicles in five paratype specimens ( Table 3). In individuals with single Polian vesicle (width about 1/2 of length), vesicle shaped fusiform, and in individuals with 2 or 3 Polian vesicles, vesicle shaped globular. Calcareous ring plate with low trapezoid anterior projection in RI, and low triangle anterior projection in IR5, in small specimen (WMNH-INV-2016-140) ( Fig. 6E ), each projection slightly higher than that of large specimens (WMNH-INV-2016-135 and 2016-137) ( Fig. 6F, G ).

Wheel diameter means of 53–62 µm in anterior dorsal side and means of 62–67 µm in posterior dorsal side, differing significantly among specimens in anterior part (Kruskal– Wallis test, P <0.05), but not in posterior part (Kruskal– Wallis test, P>0.05). Wheel diameters in four specimens (WMNH-INV-2016-136–139) significantly different between anterior and posterior parts ( Kruskal – Wallis test, P s<0.05), but not in the largest specimen (WMNH- INV-2016-135) . Sigmoid-hook length 77–114 µm (individual means of 90–107 µm in measured five type specimens) in anterior dorsal side, 78–111 µm (individual means 83–95 µm in measured five type specimens) in posterior dorsal side, 76–112 µm (individual means 80–97 µm in measured five type specimens) in anterior ventral side, and 65–102 µm (individual means 78–90 µm in measured five type specimens) in posterior ventral side. Length significantly different among specimens in all four body parts (Kruskal– Wallis test, P s<0.05), but not significantly different between anterior and posterior parts of five specimens ( Kruskal – Wallis test, P s>0.05) . Length of sigmoid-hooks also significantly different among four body parts in all five specimens (Kruskal– Wallis test, P s<0.05), and in four specimens (WMNH-INV-2016-135, 137–139) hook in dorsal parts significantly larger than that of ventral parts ( Kruskal – Wallis test, P s<0.05) . Tentacles rod length 43–77 µm, means of 57–63 µm in different five specimens, significantly different among them ( Kruskal – Wallis test, P <0.05) .

Remarks. Up to date, seven species of Taeniogyrus with wheel-papillae have been reported ( Table 5): 1) T. bamberi O’Loughlin, 2015 ; 2) T. cidaridis ; 3) T. clavus Heding, 1928 ; 4) T. dayi Cherbonnier, 1952 ; 5) T. heterosigmus Heding, 1931 ; 6) T. prydzi O’Loughlin and VandenSpiegel, 2010 ; and 7) T. yvonnae . Since T. flavus sp. nov. has 1–3 Polian vesicles, and ciliated funnels along with medioventral and left dorsal, and such features are not reported in these seven species and most of the other congeners ( Table 5), but arrangement of ciliated funnels of T. bamberi have not been reported. Although ciliated funnels have not been reported, sigmoid hooks of this species apparently larger (means 247 µm in anterior and 273 µm in posterior) than that of T. flavus sp. nov. (ranged in 77–114 µm) ( Table 5), and body color of this species (off-white to grey with faint orange to purple tinge in preserving status) also different from that of T. flavus sp. nov. (translucent pale yellow) ( Table 5). Thus, T. flavus sp. nov. is distinguishable from most of the nominated species, by the number of Polian vesicles, the arrangements of two rows of the ciliated funnels, size range of sigmoid hooks, and its body color. Additionally, another new species described below T. rubrus sp. nov. also possess wheel-papillae, but body color of T. rubrus sp. nov. (red or reddish brown in living and preserving status) apparently different from this new species ( Table 5).

Although the body size of T. australianus ( Stimpson, 1855) has been reported as at least twice as large as that of T. flavus sp. nov. ( Table 5), ossicle shapes and body appearances of T. australianus previously reported ( O’Loughlin and VandenSpiegel 2010) are similar to those of T. flavus sp. nov. However, according to Heding (1928) and O’Loughlin and VandenSpiegel (2010), the sigmoid-hook ossicles are not scattered but gathered in small papillae in T. australianus , and this feature is quite different from those of T. flavus sp. nov. The measurements of the ossicles of T. australianus are reported to be larger than those of T. flavus sp. nov., with the length of the sigmoid-hook ossicles of T. australianus ranging within 120–136 µm ( O’Loughlin and VandenSpiegel 2010). The original evaluation of T. australianus was quite simple and without figures ( Stimpson 1855); but, detailed descriptions and figures have been reported by Heding (1928) and O’Loughlin and VandenSpiegel (2010). According to these reports, there are five pairs of digits on the tentacles ( Heding 1928) or up to seven pairs ( O’Loughlin and VandenSpiegel 2010). There are two-series of ciliated funnels in an inter-radius of near right of the mid-ventral longitudinal muscle RI and near left of the left lateral longitudinal muscle RII ( O’Loughlin and VandenSpiegel 2010). These features conform well to T. flavus sp. nov. Furthermore, in the photograph provided by O’Loughlin and VandenSpiegel (2010), the wheel-papillae are confined to only three dorsal inter-radial zones, identical to T. flavus sp. nov. On the other hand, a single Polian vesicle occurs in the descriptions of T. australianus provided by both Heding’s (1928) and O’Loughlin and VandenSpiegel’s (2010), which differs from T. flavus sp. nov. having mostly two (or sometimes more) Polian vesicles ( Table 5).

Distribution. Known only from the type locality at Shirahama, in sandy-gravel shore, Wakayama, southern end of the mainland of Japan.

Etymology. The specific name is derived from the Latin adjective flavus (meaning yellow), based on its body color.

DNA barcode sequence. A 819 bps sequence of the mitochondrial CO1 gene was obtained from a paratype specimen (WMNH-INV-2016-137: INSD accession number LC637701 ) . Base frequency was A=21.2%, C=13.1%, G=22.8%, T=42.9%. In BLAST searches, the closest hit to the partial COI sequence is from Taeniogyrus verruculosus Yamana and Tanaka, 2017 ( LC203483 , with 83.0% similarity in 100% coverage) . The results of BLAST search are correspond to the morphological identification that the present species belongs to Taeniogyrus , but it is shown that present genus includes great genetic diversity at the same time.

Figures

Fig. 2. External and internal morphologies of Taeniogyrus albulus sp. nov.A–G: holotype (WMNH-INV-2017-100); H: paratype (WMNH- INV-2018-23). A, B, Lateral views of preserved state and anaesthetized state, respectively; C, tentacles of left side (viewed from left side, dorsal upper); D, ciliated funnels along with lm RI (anterior left); E–G, microphotograph of anterior dorsal skin containing ossicles (E), anterior ventral skin containing ossicles (F), and ciliated funnels (G); H, left internal view of preserved states (anterior left). Abbreviations: ad, anterior dorsal; av, anterior ventral; CR, calcareous ring; fun: ciliated funnels; lm: longitudinal muscle; M, madreporite; pd, posterior dorsal; POL, Polian vesicle; pv, posterior ventral; SC, stone canal; sh, sigmoid-hook ossicle; te, tentacle; wh, wheel ossicle.

Fig. 3. SEM images of ciliated funnels of holotypes of three new Taeniogyrus species. A, T. albulus sp. nov. (WMNH-INV- 2017-100); B, T. flavus sp. nov. (WMNH-INV-2016-135); C, T. rubrus sp. nov. (WMNH-INV-2019-299).

Fig. 4. SEM images of calcareous ring parts and body ossicles of Taeniogyrus albulus sp. nov. A–E: holotype (WMNH-INV-2017-100); F: paratype (WMNH-INV-2017-101). A, Right parts of calcareous ring (viewed from right side, dorsal upper); B–D, wheel ossicles from a wheel papilla of anterior dorsal (B), curved rod ossicles (C) and sigmoid-hook ossicles (D) from anterior dorsal skin; E, rod ossicles from a lateral tentacle; F, calcareous plate IR5 of a paratype.

Fig. 5. External and internal morphologies of Taeniogyrus flavus sp. nov. A, D, G–I: holotype (WMNH-INV-2016-135); B, E: paratype (WMNH-INV-2016-138); C, F: paratype (WMNH-INV-2016-137). A, B, Lateral views of preserved states of the holotype and paratype, respectively; C, F, Right internal views of preserved states of a paratype (WMNH-INV-2016-137); D, E, tentacles of left side (viewed from oral disc) of the holotype and paratype, respectively; G, H, microphotograph of anterior dorsal skin of the holotype containing ossicles; I, microphotograph of ciliated funnels of the holotype. Abbreviations: ad, anterior dorsal; av, anterior ventral; CR, calcareous ring; fun: ciliated funnels; lm: longitudinal muscle; M, madreporite; pd, posterior dorsal; POL, Polian vesicle; pv, posterior ventral; SC, stone canal; te, tentacle.

Fig. 6. SEM image of calcareous ring parts and body ossicles of Taeniogyrus flavus sp. nov. A–D, F: holotype (WMNH-INV-2016-135); E: small-sized paratype (WMNH-INV-2016-140); G: middle sized paratype (WMNH-INV-2016-137). A, Right parts of calcareous ring (viewed from right side, dorsal upper); B–D, rod ossicles from a lateral tentacle (B), wheel ossicles from a wheel papilla of anterior dorsal (C), and sigmoid-hook ossicles from anterior dorsal skin (D); E–G, calcareous plate RI and IR5 of a small-sized paratype (E), RI of holotype (F), and IR5 of a middle-sized paratype (G) (anterior upper). Abbreviations: ro, rod ossicles; sh, sigmoid-hook ossicles; wh, wheel ossicles; wh’, internal side of wheel ossicles.