A cryptic radiation of Caribbean sea slugs revealed by integrative analysis: Cyerce ‘ antillensis’ (Sacoglossa: Caliphyllidae) is six distinct species Author Moreno, Karina Department of Biological Sciences, California State Polytechnic University Pomona, Pomona, CA 91768, USA Author Rico, Diane M. Department of Biological Sciences, California State University, Los Angeles, CA 90032 - 8201, USA Author Middlebrooks, Michael Department of Biology, University of Tampa, Tampa, FL 33609, USA Author Medrano, Sabrina Department of Biological Sciences, California State Polytechnic University Pomona, Pomona, CA 91768, USA Author Valdés, Ángel A. Department of Biological Sciences, California State Polytechnic University Pomona, Pomona, CA 91768, USA Author Krug, Patrick J. Department of Biological Sciences, California State University, Los Angeles, CA 90032 - 8201, USA pkrug@calstatela.edu text Zoological Journal of the Linnean Society 2024 2023-10-12 200 4 940 979 http://dx.doi.org/10.1093/zoolinnean/zlad111 journal article 10.1093/zoolinnean/zlad111 0024-4082 PMC10983082 38566915 11240925 E8CC81A3-E625-4C48-B783-29AA9BFC83C3C Cyerce browneveorum sp.nov. ( Figs 1–3 , 5 , 8E , 18 , 19 ) ZooBank registration: lsid:zoobank.org:act: 226D89BA- 1F0F-47E8-A4AE-D304A6C3DF3F Cyerce antillensis – Clark and Defreese 1987: 259–279 ; Clark 1994: 906 ; Jensen 1993a: 137–140 , fig. 1a; Christa et al. 2015 : fig. 1b. Type material Holotype : Geiger Beach , Florida Keys , USA , April 2018 ( LACM 3840 ; isolate 18Gei01). Additional material examined Geiger Beach , Florida Keys , USA , October 2009 , one specimen preserved, isolate 09Gei01, 4 mm length alive; Geiger Beach, Florida Keys, USA , July 2010 , one specimen preserved, isolate 10Gei01, 5 mm length alive; Geiger Beach, Florida Keys, USA , April 2018 , two specimens , isolates 18Gei02 and 18Gei03 . Range Summerland Key and Geiger Beach, Florida Keys ( Christa et al. 2015 ; present study). Description External morphology: Body colour pale olive green. Notum with white border, white patches and burgundy to brown speckling ( Fig. 18 ). Head white, flecked with burgundy or brown speckles. Large burgundy Y-shaped patch between eyes, extending up to the base of each rhinophore. Rhinophores and tentacles smooth, translucent, mottled with patches of white and burgundy. Rhinophores elongated, half body length on live animal; oral tentacles less than half the length of rhinophores. Transverse groove separating head from foot on ventral surface. Pericardium round, with few white papillae; white ground colour, with patches of brown to burgundy speckles ( Fig. 18 ). Anal papilla white with brown speckling, round, anterior and to the right of pericardium. Dorsal vessels clear, with one pair of posterior vessels running length of notum and one lateral vessel emerging immediately posterior to anal papilla. Both vessels branching, then fusing with a long vessel running parallel to margin of notum, from which one short vessel connects to base of each ceras; separate, elongated branch of posterior vessel running towards pointed tail. Cerata flat, narrow, translucent, containing clumps of embedded white granules. Burgundy oval-shaped patch at base of ceras; second dark burgundy patch surrounded by indigo ring near ceratal tip, and lighter burgundy speckles and irregular patches scattered across surface of cerata ( Fig. 18 ). Ceratal margin a thickened crenulate white rim of rounded, finger-like protrusions. Internal morphology: Radula of 5 mm specimen (10Gei01) with six teeth on ascending limb and eight teeth on descending limb ( Fig. 19A ). Teeth moderately angled, tapering to a rounded tip. Leading tooth 165 µm long, ~17 µm deep at bend, with a row of 10 rectangular denticles along each cutting edge. Denticles 4–5 μm wide but becoming longer and narrower towards tip of tooth, and angled out from cutting edge ( Fig. 19B ). Ascus with ≥ 10 pre-radular teeth ( Fig. 19C ). Penis with straight, cylindrical stylet, 180 µm long, 8–10 µm wide; embedded in tissue for most of length except for pointed triangular tip ( Fig. 8E ). Ecology Live specimens were collected from Pe. dumetosus in 2008 and 2010 and from R. phoenix in 2018. Based on COI sequences, conspecific specimens were also collected independently in the Florida Keys and included in a DNA barcoding analysis of the gut contents of diverse sacoglossans ( Christa et al. 2015 ). Sequences of the rbcL marker were amplified from undigested chloroplasts within C. browneveorum and matched to reference algal sequences to determine what slugs had ingested recently; slugs were reported to contain plastids from a range of udotacean algae, including Halimeda , Udotea , Bryopsis , Avrainvillea and Pseudochlorodesmis , but no live specimens were obtained from those hosts (see Remarks). Reproduction Clark and Jensen (1981) reported lecithotrophic development for nominal C. antillensis non-Engel, 1927 collected from Penicillus in the Florida Keys, with an egg size of 112 μm. We attribute these records to the Florida Keys endemic C. browneveorum , because both other Florida species ( C. antillensis and C. piercei ) are planktotrophic, and of those, only C. antillensis has been sampled in the Keys. Etymology Named in honour of Dr Tegan Eve and Kimberly Brown Eve, long-term friends of P.J.K., who have allowed their home and many vacation rentals to be turned into makeshift field stations for sea slug research over 20 years, facilitating extended study of Florida Keys sacoglossans. Remarks Both external and internal anatomical features differentiate C. browneveorum from other complex members. In particular, three species that are superficially similar could be distinguished externally by markings on the head. In C. browneveorum , there is a burgundy Y-shape on an otherwise solid white head, separating the eyes and extending up to the rhinophores but not carrying forwards onto the front of the head. In C. piercei , there is a similar but darker purple triangular wedge, more V- than Y-shaped, that separates the eyes but carries forwards onto the dark pigmented front of the head; as a result, the eyes appear to sit in white patches in C. piercei , as opposed to a white head bearing a Y-shaped mark in C. browneveorum . Finally, in C. nicholasi there is a dark patch between the eyes that extends forwards as a thickened line anterior to the eyes up to each rhinophore, with a faint dusting of pigment on the front of the head of specimens. Likewise, while all C. antillensis complex members had translucent cerata, with a white crenulate margin, bearing a dark brown or burgundy spot at the base and another near the margin, there were subtle distinctions between species. For instance, the dark oval patch near the ceratal margin was surrounded by a thin yellow ring in C. nicholasi , whereas the corresponding dark oval patch in C. browneveorum had an indigo border. The ceratal margin of both C. nicholasi and C. browneveorum is solid white, with thickened finger-like projections, whereas that of C. piercei is speckled white, giving a lace-like appearance. Figure 17. Cyerce willetteorum , radular scanning electron micrographs of LACM 3844 (isolate 07Stir01). A, complete radula, with ascus. B, close-up view of tissue-free teeth from descending limb, showing irregularly shaped denticles. C, close-up view of ascus. Internally, the radular teeth of C. browneveorum are distinct from the teeth of other complex members. Teeth of C. nicholasi and C. ellingsonorum bear rectangular denticles oriented straight down in a plane with the cutting edge of the tooth. In C. piercei , denticles are not rectangular, tilt outwards from the plane of the cutting edge, and angle forwards or backwards. In C. browneveorum , rectangular denticles tilt outwards from the plane of the cutting edge without angling forwards or backwards and are longer and narrower towards the tooth tip. Figure 18. Cyerce browneveorum , living specimen from Geiger Beach, Florida Keys (isolate 10Gei01); actual length, 5 mm. Although we did not obtain reproductive data for Florida Keys specimens, it is likely that C. browneveorum comprises the lecithotrophic ‘ C. antillensis ’ of Clark and Jensen (1981) sampled in the Florida Keys. We show here that C. antillensis Engel, 1927 , which also occurs in Florida , is planktotrophic. The other C. antillensis complex member from Florida , C. piercei , is planktotrophic and has not been sampled in the Florida Keys. Clark and Jensen (1981) also reported a diet of Pe. dumetosus for their ‘ C. antillensis ’ from Florida , matching the host of C. browneveorum but not C. antillensis . The other lecithotrophic species specializing on Pe. dumetosus , C. nicholasi , does not occur in the Florida Keys. It is thus most probable that reports of lecithotrophy for ‘ C. antillensis ’ correspond to C. browneveorum . Strikingly, sampled host use in C. browneveorum was the same as that for C. nicholasi , with most specimens collected from Pe. dumetosus but multiple specimens being sampled from R. phoenix in both species. No prior work reported Rhipocephalus as a host of Western Atlantic Cyerce . Given their phylogenetic affinity, it is not surprising that Rhipocephalus might be a suitable alternative host for sacoglossans that otherwise specialize on Penicillus , such as E. papillosa ( Krug et al. 2016 ) . Different conclusions were reached by Christa et al. (2015) using molecular analyses of chloroplasts in slug digestive glands to infer the diet of specimens they termed ‘ C. antillensis ’; we confirm that their specimens were C. browneveorum based on the published COI barcode sequence. Sequences of the rbcL gene were interpreted as indicating that the diet of C. browneveorum included diverse algae from at least six udotacean genera, but notably not Penicillus or Rhipocephalus , the hosts from which we recovered live specimens ( Christa et al. 2015 ). However, several potential concerns with the interpretation of these data should be noted. First, phylogenetic analyses of recovered rbcL sequences from Cyerce specimens and reference algal sequences returned a polytomy, in which species of Penicillus , Rhipocephalus , Udotea and Chlorodesmis were intermingled (Supporting Information, Fig. S 1 in Christa et al. 2015 ). Without monophyletic groups of reference sequences, it remains uncertain from which of these genera C. browneveorum might have contained plastids. Second, biased amplification of rbcL could lead to over-representation of some algal species in this approach, which sequenced a limited number of clones instead of the next-generation sequencing used in most metabarcoding studies to achieve deep coverage of gut contents. Third, slugs might feed briefly on algae that are not ecologically relevant hosts and contain their chloroplasts, yet have no ecologically meaningful association with those algae. Notably, no specimens of Cyerce were obtained even from extensive collections of Udotea , Bryopsis and Avrainvillea in Florida , making the ecological relevance of the barcoding work unclear. However, it remains possible that the real niche breadth for C. browneveorum is comparable to that of C. piercei , encompassing numerous host genera; laboratory feeding experiments could test this in the future. Taxon inquirendum Based on the original description, the following species does not conform to the generic diagnosis for Cyerce , as noted by prior authorities. We exclude this name from recognized western Atlantic species of Cyerce for the reasons given below.