Molecular and morphological systematics of Elysia Risso, 1818 (Heterobranchia: Sacoglossa) from the Caribbean region Krug, Patrick J. Vendetti, Jann E. Valdés, Ángel Zootaxa 2016 4148 1 1 137 6F5PC [151,321,727,753] Gastropoda Plakobranchidae Elysia Animalia Sacoglossa 98 99 Mollusca species pawliki sp. nov.    Elysia papillosa[non Verrill, 1901] —Thompson 1977: figs. 26, 27; Espinosa & Ortea 2001: 44; Espinosa et al.2005: 56; Ortea et al.2005: 498–502 (part), figs. 1–2, pl. 1, fig. A; Redfern 2001: 162, figs. 672A–F; Collin et al.2005: 690; Valdés et al.2006: 65.   Elysiasp. A —Redfern 2013: 286–287, figs. 793A–B.   Elysiacf. tomentosasp.2 —Krug et al.2013: 1109-1113, figs. 2C, 4; Krug et al.2015: 990, fig. 3B.     Typematerial. Sweetings Cay, Bahamas, 2003, ( Holotype  LACM3303), collected by PJK;  AbacoIslands, Bahamas, 2003, ( Paratype  LACM3304), collected by Colin Redfern.    Typelocality. Sweetings Cay, Bahamas    Materialexamined. Bahamas: AbacoIslands, 2003, 2 specimens( Paratype  LACM3304,  LACM178674);  Sweetings Cay, 2003, 1 specimen, 35 mmlong alive (isolate Epaw_03Swe01, Fig. 58A);  July 2007, 1specimen, 25 mmlong alive ( Holotype  LACM3303, isolate Epaw_07Swe01, Fig. 58B–E).  Live animal.Both Sweetings Cay specimens were collected four years apart in the same tidal channel, which opens to a mangrove lagoon. Slugs were collected off a large clump of  Caulerpa racemosa. Resting slugs held their parapodia apart, forming a series of siphonal openings ( Fig. 58A–B). One specimen was observed to associate, and potentially attempt to mate with, a large  E. subornatawhen the two were held together in a container. Slugs often rested with their head tucked inside their expansive parapodial flaps. Both specimens from Sweetings Cay, Bahamaswere observed in the laboratory for 3–4 weeks after collection; neither slug was ever observed to swim or flap its parapodia when disturbed. When stressed, slug released a cloud of iridescent blue-white mucus from the parapodial margin, and contorted its parapodia, but did not swim.   External anatomy.Overall coloration yellow-green with patches of brown. Body turning dark green after feeding, due to digestive diverticula ramifying throughout body, head and parapodia, visible through epidermis at low density. Overall body shape dominated by large parapodia with series of three laterally extended side-flaps folding away from body ( Fig. 58A–B). When parapodia close over dorsum, flaps create three siphonal openings, the middle being the largest. Anterior-most opening small, representing little more than a fold over pericardium. When held open, middle parapodial flaps extending out roughly as wide, tip to tip, as body length from pericardium to tail. Elongated middle flaps forming largest opening when parapodia are closed, giving live animal a cruciform appearance. Third, posterior-most pair of parapodial flaps intermediate in size. Larger specimens with 2–3 additional siphonal openings present along posterior half of body, including a posterior pair of laterally extended parapodial flaps; examples include isolate Epaw_03Swe01 ( Fig. 58A), and a specimen from Jamaicain the BMHN labeled “  E. papillosa” by T. E. Thompson, 30 mmlong. Exterior surface of parapodia covered by elongate, white papillae rising from tan-brown patches of pigment ( Fig. 58C). Upper portion of parapodia greywhite inside and out, with occasional blotches of plum color; one large plum-color patch appearing on anteriormost parapodial flap near margin. Brown band running along inner and outer edge of parapodial margin. Row of papillae extending straight up from edge, running to end of parapodia; marginal papillae grey-brown tipped with white. Interior of parapodia featuring white patches, pink at center, scattered about. Outer surface of body heavily papillose. Irregular, white patches like lichen covering head and upper portion of parapodia; long, conical papillae rising out of these white patches, spotted with irregular small patches of pink. Glands appearing as scattered brown spots cover sides of parapodia, head and rhinophores; black-edged openings slightly elevated above surface of epidermis, with brown spherical inclusion (gland) lying beneath, discharging heavy mucus secretion when animal is alarmed.   FIGURE 58.  Elysia pawliki  n. sp., external morphology and egg mass of specimens from Sweetings Cay, Bahamas. A,Live specimen with six siphonal openings formed by parapodial flaps (LACM 3303; length = 35 mm). B–E,Live specimen viewed from above (B), right side (C), underneath (D), or with parapodia spread open (E) to show renopericardial complex and dorsal vessels (isolate Epaw_07Swe01; length = 25 mm). Purple egg masses of parasitic copepod visible. F,Egg mass (deposited by specimen LACM 3303), showing orange ECY ribbon and early-stage embryos. Field of view = 3 mm.   FIGURE 59.  Elysia pawliki  n. sp., drawing of the renopericardial complex and dorsal vessel network traced from a photograph of a live animal (isolate Epaw_07Swe01; 2.5 cm long). Grey shapes are egg masses of parasitic copepod. Front of head covered by large patches of tan, white and pink, over yellow-green background color. Eyes tiny, located in patch of background coloration posterior to base of rhinophores. Upper lip split into two curved sections; lower lip flattened, broader. Diverticula extend into both top and bottom lips. Upper lip with moustache of brown spots. Rhinophores short relative to body length ( 3 mmat maximum extension on 25 mmanimal); rolled, bluntended, with long white papillae. Surface of rhinophores white-brown, dotted with dark brown spots (glands) and pink spots of equal size, and occasional irregular white patches. Faint, longitudinal white stripes run tip to base. At base, inner surface of rhinophores penetrated by green digestive diverticula, but outer surface white and devoid of diverticula. Foot with same yellow-green or brown color as rest of body, with rows of minute white papillae but no brown glands or white patches ( Fig. 58C–D). Transverse groove separating underside of head from foot, opening into wider genital groove on right side of head, with a white genital aperture at the top of the groove. Endof foot wide, blunt, not narrowing to a tip; no extended tail. Pericardium large, rounded, white-pink patch on top, dotted with brown glands. Thick renopericardial extension runs to halfway point of body, between second and third parapodial flaps ( Fig. 58B, E). Dorsal vessels asymmetric; typespecimen with four vessels emerging on left side, five on right side of pericardial complex ( Fig. 59). Vessels branch and anastomose forming complex network running up to inner parapodial margin. Main branch of elongated posterior vessel running to tail on each side; posterior vessel otherwise notably asymmetric in placement and branching pattern. Vessels run under, or terminate in, papillae that dot inner parapodial surface.  Internal anatomy.Radula with ~24 teeth (LACM 3303, LACM 3304), 6 teeth in ascending limb and ~ 18 indescending limb ( Fig. 60A). Leading tooth wide and flat, with fine, blunt denticles on cusp, with slightly rounded apex ( Fig. 60B, E). Housing depression for interlocking teeth “V”-shaped and extending ⅔ of tooth length. Base of tooth approximately ¼ total tooth length. Ascus of small teeth in a single row with some jumbled teeth at the end ( Fig. 60F). Penis elongate and curved ( Fig. 60C) with rigid musculature resistant to desiccation and tapering distally into a conical apex bearing a resistant, hollow tip ( Fig. 60D). Penial stylet is not a scoop or barb; hardened penial tip visible by SEM, but not light microscopy. Deferent duct long, narrow, and highly convoluted.  Reproduction and development.One egg mass was produced by specimen 03Swe01 ( Fig. 58F). The egg strand was wound in a typical elysiid spiral on the surface of the bubble-like “grapes” of the host alga  C. racemosa. Egg capsules alternated around a continuous, thick ribbon of bright orange ECY on the upper surface of the egg strand, under the outer covering of the egg mass. The ribbon was molded around each individual capsule. The clutch released swimming, lecithotrophic veliger larvae with eyespots; neither egg nor larval shell size was obtained, however.   Host ecology.Both live specimens of  E. pawliki  n. sp.were recovered from  Caulerpa racemosa, on which both specimens fed readily in the laboratory. Slugs became green upon feeding, but reverted to a brown color after a few days without food. Specimens collected at different times from the Bahamaseach had one pair of vivid purple egg masses of a parasitic copepod poking out of the dorsal surface near the pericardium; ( Fig. 58B, E). Although copepods parasitize a wide range of opisthobranchs, the purple eggs of this unknown copepod species were only observed on the related  E. zemi  n. sp.and not on any other sacoglossan over the past 12 years, suggesting a specialized relationship.  Phylogenetic relationships.  Elysia pawliki  n. sp.belongs to the  E. tomentosaspecies complex, together with at least five distinct species from the Indo-Pacific, and three sympatric Caribbean species:  E. subornata, E. pratensisand  E. zemi  n. sp.( Fig. 4). All species in this clade feed on  Caulerpaspp., except  E. pratensis. We recovered as sister to  E. pawliki  n. sp.an undescribed, morphologically similar species collected from  Caulerpa cupressoidesin Australiaand an unknown  Caulerpasp. in Thailand(  Elysiacf. tomentosasp. 5; Krug et al.2013). Both species are brown, highly papillose, and share a characteristic cruciform body shape due to the wide lateral extension of the parapodia. Molecular data were not available for the morphologically similar species  E. manriqueiOrtea & Moro, 2009from the Canary Islands.   Range.Bahamas (Redfern 2001, 2013; present study), Costa Rica (Espinosa & Ortea 2001), Venezuela (Valdés et al.2006)   Etymology.Named in honor of colleague and “evil twin” Joseph R. Pawlik, in recognition of his landmark achievements studying Caribbean reef ecosystems, and the larval and chemical ecology of sea slugs. Without the opportunity to participate in four research cruises on which Dr. Pawlik was Chief Scientist, the present study would not have been possible, and the holotypespecimen would not have been collected.   Remarks.Both Thompson (1977) and Ortea et al.(2005) described material most closely matching  E. pawliki  n. sp.(but potentially also  E. zemi  n. sp.) as  E. papillosa. However,  E. pawliki  n. sp.has many features that are incompatible with the details provided by Verrill (1901) in his description of  E. papillosa. The only similar feature shared by both species is a highly papillose body surface, but the papillae of  E. pawliki  n. sp.are notably different from the “small conical papillae” described and figured by Verrill (1901) for  E. papillosa. Ortea et al.(2005) claim that Verrill’s illustration shows branching, digitiform papillae, but they misinterpreted Verrill’s drawing (Verrill, 1901: pl. 4, fig 3), which shows the specimen of  E. papillosasitting on a branching stipe of the alga  Halimeda incrassatta; the alga was interpreted as papillae on the slug by Ortea et al.(2005). Further, although Verrill’s typematerial from the Bermudaexpedition was lost, we found among Verrill’s surviving material a preserved specimen of  E. pawliki  n. sp.marked “  Elysiasp.”; thus, Verrill considered  E. pawliki  n. sp.to be distinct from his  E. papillosa. Moreover,  E. pawliki  n. sp.has never been reported from the typelocality of  E. papillosa( Bermuda).  A majorbehavioral difference further confirms that  E. pawliki  n. sp.is distinct from  E. papillosaVerrill 1901, which was described as swimming freely using its parapodia. Two live specimens of  E. pawliki  n. sp.were observed over a period of weeks, but never swam no matter the degree to which they were disturbed; thus,  E. pawliki  n. sp.cannot be  E. papillosaVerrill 1901. At least five Caribbean species swim when disturbed by flattening and rapidly undulating their thin parapodia; all five belong to subclade 1, and are phylogenetically distinct from  E. pawliki  n. sp. Morphologically, no named Caribbean species is similar in gross anatomy to  E. pawliki  n. sp.In  E. pawliki  n. sp., as in most species belonging to subclade 4, the renopericardial extension runs about halfway down the body, whereas the renopericardial extension of related species  E. subornataand  E. pratensisruns the whole body length. The most similar Atlantic species is  E. manriqueiOrtea & Moro 2009from the Canary Islandsoff WestAfrica. In addition to its east Atlantic typelocality,  E. manriqueidiffers from  E. pawliki  n. sp.in its external morphology in having symmetrical and non-anastomosing dorsal vessels, large black spots dotting the entire body and head, shorter rhinophores, and more vertically exaggerated siphonal openings. The radular teeth are also markedly different. The tooth of  E. manriqueihas a flat, serrated cutting edge, and a curving top edge; in  E. pawliki  n. sp., both the cutting and non-cutting surfaces taper together towards a rounded tooth tip. The radular teeth of  E. pawliki  n. sp.are most similar to those of  E. zemi  n. sp.in overall morphology, both bearing fine denticles and possessing a sharp tooth tip on a rounded apex, and downward-facing angle at ⅔ the length of the tooth. The teeth of  E. pawliki  n. sp.are distinct in having straighter tooth cusps and less club-like tooth apices. Like in  E. ellenaeand  E. zemi  n. sp.the resistant penial tip in  E. pawliki  n. sp.is not scoop or barblike, but based on Gascoigne’s (1974) description we refer to it as a stylet. Developmentally, all members of the  E. tomentosaclade studied to date have orange ECY ribbons. However, the lecithotrophic larvae of  E. pawliki  n. sp.swim upon hatching; thus, development mode (pelagic lecithotrophy) differentiates  E. pawliki  n. sp.from  E. subornataand  E. pratensis(non-pelagic lecithotrophy with encapsulated metamorphosis), and from its Pacific sister species  E.cf. tomentosasp. 5, which is planktotrophic (Krug et al.2013). In terms of host ecology,  E. pawliki  n. sp.feeds on  Caulerpa racemosa, which is also consumed by most related species. The co-occurring  E. subornatafeeds on several  Caulerpaspp. including  C. racemosa, but is much more common throughout the Caribbean. 1291049052 [151,1246,1023,1048] LACM Bahamas Sweetings Cay 98 99 LACM 3303 2 holotype 1291049055 LACM Colin Redfern Bahamas Abaco 98 99 LACM 3304 1 Abaco paratype 1291049058 [199,646,1095,1120] Bahamas Sweetings Cay 98 99 1 holotype 1291049060 [199,1432,1132,1157] LACM Bahamas Material 98 99 LACM 3304, LACM 178674 2 Abaco paratype 1291048866 2007-07-01 LACM Bahamas Sweetings Cay 98 99 LACM 3303 1 Abaco holotype