Molecular and morphological systematics of Elysia Risso, 1818 (Heterobranchia: Sacoglossa) from the Caribbean region Author Krug, Patrick J. Author Vendetti, Jann E. Author Valdés, Ángel text Zootaxa 2016 4148 1 1 137 journal article 10.11646/zootaxa.4148.1.1 a757adc6-2763-41f7-ae26-b1cae841091c 1175-5326 256946 91353147-FDA8-45CC-A8F1-1DE801C835A6 Elysia christinae new species ( Figs. 56 E, 64–66) Elysia sp. 2 — Valdé s et al. 2006: 74–75. Elysia sp. 18 — Krug et al. 2015: 990-991, figs. 3B, 4 Type material. Bimini , Bahamas , July 2010 , ( Holotype LACM 3308 , Paratype LACM 3309 [ 2 in lot]), collected by PJK. Type locality. Bimimi , Bahamas Material examined. Bimini , Bahamas , July 2010, 10 specimens, ( Holotype LACM 3308 , Paratype LACM 3309 [ 2 in lot], isolate Echr_10Bim04-10). Live animal. Slugs resting on the algae held their parapodia open and flattened against the algal surface ( Fig. 64 A–B). When crawling, slugs typically elevated the parapodia along the anterior two-thirds of the body length, assuming a more typical slug-like shape, but the posterior third of the parapodia remained open and flattened, creating a widened, rounded end of the body ( Fig. 64 C–D). On crawling slugs, parapodial edges undulate. The head can be used to grip the substrate, and the front two-thirds of the body can rear up like a snake. External anatomy. Four largest specimens ranged from 8–11 mm , remaining six slugs were 3–6 mm in length. Overall coloration dark green due to digestive diverticula, which ramify throughout body, head, and parapodia ( Fig. 64 A–D). Epidermis of head and body covered by network of brownish-rust colored lines and patches. Body elongate when parapodia contracted over dorsum. Head green and brown; prominent white patch just anterior to eyes and posterior to rhinophores on some specimens ( Fig. 64 B, F). Faint blue and white flecks dot sides of head. Underside of head green-brown with scattered blue dots, and brown line on lower “lip” of mouth ( Fig. 64 D). Rhinophores short, rolled, with flat tips. Proximal half of rhinophores green with brown specks, distal half mostly white due to dense covering of white speckles; white patches form transverse white line at beginning of distal half of each rhinophore. Penis extends from relaxed animals from under right rhinophore; white, tapering to blunt end. Foot pale green. Transverse groove separates underside of head from foot, connecting to genital groove ( Fig. 64 D). Digestive diverticula present in foot but concentrated on either side, missing entirely from medial pale strip giving appearance of pale stripe down center of foot. Edges of foot not delineated from parapodia. Body not wider than head, narrowing towards posterior end. Some specimens with parapodia fusing at posterior end with no visible tail ( Fig. 64 A); other specimens with short, pointed tail ( Fig. 64 D). Live animal sometimes having pear-shaped appearance due to widening parapodia at posterior end of body ( Fig. 64 A, D). Exterior of parapodia almost uniformly green with scattered white spots irregular in size and shape, more dense near outer parapodial margin, and fewer small blue spots. Interior of parapodia densely penetrated by digestive diverticula; pigmented by brown flecks (and larger black spots on some specimens) ( Fig. 64 B, E–F). Parapodial margin scalloped, with thin brown-black marginal line, but margin otherwise pale relative to rest of parapodia due to absence of digestive diverticula. Row of white spots runs along margin, with one spot under each pointed tip of scalloped margin ( Fig. 64 A–B). On largest specimen, front third of parapodia was twice as high as posterior portion of parapodia, forming wing-flaps on both sides just behind head. Anterior parapodial flaps held flat against substrate in resting animal; anterior region of parapodia sometimes folded over dorsum on living animal when crawling actively. Pericardium small and rounded, pale or white in center, brown around periphery, dotted with a few white spots ( Fig. 64 E–F). Anterior face of pericardium flattened where it merges with head, and penetrated by digestive diverticula. Anal papilla emerges from right side of pericardium, just posterior and distal to a dark circle, tapering to a rounded tip. Female genital aperture a white, eye-shaped opening at anterior edge of right parapodium near point of fusion with body; some specimens with small papilla directly above this opening. FIGURE 64. Elysia christinae n. sp. , external morphology and egg mass. A–B, Live specimens resting on host alga Rhipilia tomentosa , showing characteristic flattening of parapodia. Images taken within 2 weeks of field collection. Length of slugs = 11 mm (A), 8 mm (B). C, Crawling specimen showing tendency of posterior parapodia to bell out and remain flattened, giving the body a pear-shaped appearance. Length = 10 mm. D, Crawling specimen with body fully elongated and parapodia folded over dorsum; length = 9 mm. E–F, Relaxed specimens showing elongated renopericardial extension and dorsal vessel network. Images taken after specimens were held in aquaria for three months, at which point algal food was depleted and color faded to brownish-yellow. Length = 6 mm (E), 10 mm (F). G, Egg mass showing orange ECY ribbon and gastrula-stage embryos. Actual diameter = 2 mm. FIGURE 65. Elysia christinae n. sp. , drawing of renopericardial complex and dorsal vessel network traced from photographs of live animals from Bimini, Bahamas (A: 10 mm long; B: 6 mm long). Grey areas represent sperm-storage vesicles. Renopericardium runs length of body, red-brown in outline and with red-brown dots across its length ( Fig. 64 F). Renal extension makes immediate S-turn upon exiting pericardium. Thick dorsal vessels (>500 µm wide) are either unbranched, or bifurcate about halfway to parapodial margin ( Fig. 64 E–F, 65). Some branches anastomose near point of bifurcation, about midway along parapodial surface; vessels do not anastomose at parapodial margin, terminating in blunt ends at point where digestive diverticula cease filling parapodium. Either one or no vessel emerges from pericardium on each side; remaining vessels emerge from renopericardial extension at irregular intervals along whole body length. Most vessels not paired with a vessel on opposing side of body. On slugs 8–10 mm in length, 6–8 vessels emerged from renopericardial extension on each side of body; largest specimen ( 11 mm long) had at least twelve vessels per side. Vessels densely spotted with white. Sperm storage vesicles apparent on both sides of body as white rounded protrusions inside parapodia, 3–5 per side on four largest specimens, dotting posterior ⅔ of body ( Fig. 64 E–F, 65). Vesicles irregular in size but typically about width of rhinophores; occur at irregular intervals along body, and not mirrored on opposite side. Anteriormost vesicle on each side closest to parapodial margin, with successive vesicles more proximal to midline, creating a “V”-shape towards posterior end of dorsum. Internal anatomy. Radula with 10 teeth (LACM 3309), 5 teeth in ascending limb and 5 in descending limb ( Fig. 66 A). Leading tooth elongate with a serrated, curved cusp, bearing 20 sharp denticles ( Fig. 66 B). Housing depression for interlocking teeth “V”-shaped and extending approximately ½ of total tooth length. Base of tooth approximately ½ of total tooth length. Ascus lost during radular preparation. Penis small, narrow, and elongate ( Fig. 56 E), with rigid musculature that did not deform after drying, tapering into a conical apex lacking armature ( Fig. 66 C). Deferent duct short, thin, and loosely convoluted. FIGURE 66. Elysia christinae n. sp. , SEM of the radula and penis (LACM 3309). A, Complete radula. B, Leading tooth. C, Penis. Reproduction and development. Slugs held in aquaria from Jul–Oct 2010 produced six egg masses. The egg strand formed a tight spiral, with one egg per capsule. Within the strand, capsules alternated on either side of a continuous ribbon of bright orange ECY ( Fig. 64 G). The ECY ribbon twisted around on the upper surface of the egg strand, under the outer covering of the egg mass. The ribbon was not perfectly flat, but rather formed raised peaks or folds in between capsules. Mean clutch size was 523.8 eggs (± 254.8 SD, n = 5; range: 239–820). Mean diameter of uncleaved ova was 61.2 µm (± 1.8 SD, n = 14) for one clutch and 58.7 µm (± 1.7, n = 25) for a second clutch; grand mean egg size was thus 60.0 µm ± 1.8 SD. For one egg mass, the long axis of egg capsules measured roughly 870 µm, while capsules in a second clutch were about 950 µm wide. Clutches held at ~25°C hatched relatively synchronously after 12.3 d (± 1.0 SD, n = 4; range: 11–13), releasing veliger larvae that swam actively. Mean larval shell length per clutch ranged from 102.0 µm (± 3.8 SD, n = 25) to 115.3 µm (± 10.0 SD, n = 25); grand mean shell length for five clutches was 107.8 µm ± 5.0 SD. Larvae were not cultured to competence, but their small size, short encapsulated period, and absence of eyespots or a developed propodium all indicated larvae were planktotrophic. Host ecology. About 15 specimens of E. christinae n. sp. were recovered from a sample of the udotacean alga Rhipilia tomentosa . The alga was growing at ~ 6 m depth, in a sandy patch within a large seagrass bed. Slugs were mostly juveniles < 5 mm long. Slugs were maintained in aquaria on R. tomentosa and observed for three months. All specimens preferentially associated with R. tomentosa and not other related algae growing in the aquaria ( Udotea , Caulerpa , Halimeda ). Slugs were observed feeding only on R. tomentosa in the laboratory. White spots on a green background color make live animals highly cryptic on their host alga, due to the numerous white calcareous structures made by fouling organisms on the surface of Rhipilia ( Fig. 64 A–B). Phylogenetic relationships. Elysia christinae n. sp. belongs to subclade 1, a group of Caribbean species. Within this lineage, no sister species was recovered with significant support. Subclade 1 includes lineages that have radiated onto multiple genera of udotacean algae, and are physically associated with their hosts — Penicillus ( E. papillosa , E. taino n. sp. ), Udotea ( E. zuleicae , E. buonoi n. sp. ), Rhipilia ( E . christinae ), and Halimeda ( E. patina ). These lineages thus remain ecologically partitioned at a fine spatial grain due to host specificity, and being co-distributed but differentiated by host use, may represent an adaptive radiation driven by ecological speciation. Range . Bimini, Bahamas (present study); Cozumel, Mexico (Valdés et al. 2006) Etymology . Named in honor and fondest memory of Christine Marie Donnelly Lee and her daughter Christine Marie Lee, loving grandmother and aunt of PJK. Remarks. No other Caribbean elysiid resembles E. christinae n. sp. in morphology, development or host use, and the species is genetically distinct from all sampled species. The characteristically flattened, open parapodia of E. christinae n. sp. are distinctive, although a similar flattening behavior is sometimes expressed by specimens of E. zuleicae resting on Udotea . Like other members of subclade 1, E. christinae n. sp. shares the feature of sperm storage vesicles that form after mating. However, only in E. christinae n. sp. do multiple vesicles form along both sides of the body, rather than one pair as in other subclade members. No specimens of E. christinae n. sp. were observed to swim, which is a characteristic of all related species. Elysia christinae n. sp. is the only Caribbean elysiid currently known to have both planktotrophic development and orange ECY; other taxa with orange ECY ( E. velutinus , E. patina , E. subornata , E. pratensis , E. pawliki n. sp. ) have lecithotrophic development, although the larval type of E. hamanni (which also has orange ECY) remains undetermined. In terms of host ecology, E. christinae n. sp. is the only Elysia sp. known to feed on Rhipilia , a tropical genus with 11 species; at least one undescribed Indo-Pacific species ( Elysia sp. 11 in Krug et al. 2015) feeds on the related alga Tydemania (Lam & Zeckman 2006) . Rhipilia is typically a deep-water alga in the Caribbean, and is often misidentified as Avrainvillea , factors that may have previously impeded collection and obscured the host-association of E. christinae .