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 piercei sp.nov. ( Figs 1–3 , 5 , 8B , 14 , 15 ) ZooBank registration: lsid:zoobank.org:act: 751FB893-3D0C- 4A3B-AE35-1BBE9343D248 Type material Holotype : Stirrup Cay , Bahamas , 2.5 mm preserved length ( LACM 3843 ; isolate 07Stir02). Additional material examined Plana Cay , Bahamas , one specimen preserved, isolate 07 Pla 01. Sweetings Cay , Bahamas , one specimen preserved, isolate 07 Swe 03. Tarpon Springs , FL , 2018, one specimen preserved, isolate KM66 . Range Bahamas , Florida (present study). Description External morphology: Body colour mottled dark yellow; notum flecked with brown. Head white, with Y-shaped burgundy patch extending between eyes up onto front of head and base of rhinophores ( Fig. 14 ). Rhinophores and tentacles smooth, translucent white, with faint burgundy mottling. Head with burgundy patch extending behind eyes to area surrounding pericardium, and down sides of head; dark purple line limning edge of head. Pericardium round, with scattered small white papillae ( Fig. 14A ) or smooth ( Fig. 14B–D ); light burgundy ground colour with darker outline, sometimes with darker spots on top. Anal papilla light grey or dark grey, anterior and to the right of pericardium. Dorsal vessels shaded grey; one pair of lateral vessels and paired posterior vessels emerging from pericardial sac and forking to feed into base of cerata ( Fig. 14A–D ). Posterior vessels running length of notum to base of pointed tail after forking once, with short side branches emerging to connect to cerata along body margin ( Fig. 14D ). Cerata flat, rectangular; translucent, with small white specks across surface and embedded grey, spherical, glandular inclusions. Small burgundy or brown oval-shaped patch at base of ceras; larger brown oval patch near tip of ceras ( Fig. 14A, B ). Ceratal margin with ragged appearance like torn cloth; translucent ceras abruptly transitioning to opaque white or brown speckled band along margin covering uneven crenulated edge. Table 5. Trait comparisons among Western Atlantic species of Cyerce .

Trait	C. cristallina	C. antillensis	C. nicholasi	C. piercei	C.	C. willetteorum	C.
ellingsonorum	browneveorum
Radular traits
Number of teeth	4, 6 (B101)	11, 9	8, 7	5, 7	6, 10	6, 7 (07Stir01)	6, 8 (10Gei01)
in ascending,	4, 6 (B103)	(MM109A)	(07LSS02)	(07PLA01)	(06Jam09)
descending	5, 7	10, 11	4, 9	5, 7 (07Stir02)
limbs	(MM98)	(10Bim01)	(10Bim03)
Length of lead-	380	67	145	180 (07Stir02)	125	97	165
ing tooth (µm)	(MM109A)	(07LSS02)	290a
60 (10Bim01)	115
(10Bim03)
Number of dent-	17	11	10	14	10	9	10
icles
Shape of dent-	Triangular	Pointed,	Rectangular,	Angled forward	Rectangular, ≤	Irregular width,	Longer and nar-
icles	curved	3 μm wide	or backward	6 μm wide	curved edge	rower at tip
Angle of tooth	15	25 (07LSS02)	15 (10BIM03)	15 (07PLA01)	20	18	18 (10GEI01)
(°)	(MM98)	15 (07Stir02)	(06JAM09)
Penial stylet traits
Shape	Slightly	Cylindrical	Curved,	elongCurved, em-	Cylindrical,	Curved, wide	Straight, wide,
curved	ated	bedded	straight	embedded
Opening	Pointed tip,	Pointed tip,	Wide tip, oval	Oval	Pointed tip,	Wide tip, oval	Pointed tip,
trioval	oval	triangular	angular
Length (µm)	100	<50	175	75	65	120	180
Host alga	Penicillus	Halimeda	Penicillus spp.	Penicillus	Penicillus	Penicillus	Penicillus spp.
capitatus ?	opuntia	Rhipocephalus	lamourouxii	dumetosus	capitatus	Rhipocephalus
phoenix	Halimeda in-	phoenix
crassate
Udotea luna
Udotea looensis
Developmental traits
Larval type	Planktotrophic	Lecithotrophic	Planktotrophic	Lecithotrophicb
Egg diameter	62.0 ± 1.7	112b
(μm)	61.6 ± 2.1
(mean ± SEM)
Shell diameter	101.9	308.3	143.3 ± 4.4
(μm)	μm ± 1.3	μm ± 4.3
(mean ± SEM)

aThompson (1977). bClark and Jensen (1981) . Internal morphology: Buccal mass < 1 mm ; buccal bulb slightly larger than pharyngeal pouch. Radula of two specimens (07PLA01, 2.5 mm long; LACM 3843 (isolate 07Stir02), 2.2 mm long) both with five teeth on ascending limb and seven teeth on descending limb ( Fig. 15A ). Teeth laterally flattened, shallow, wide at base but tapering; tip pointed, with dorsal inward groove between denticles ( Fig. 15B ). Leading tooth 180 μm long on specimen LACM 3843 , with 14 denticles splayed out along either side of cutting edge ( Fig. 15B ). Denticles widening away from tooth, with notched margin; denticles angled out from tooth, more conical than square in shape, and increasing in size towards the tooth tip. Ascus with a dense jumble of ≥ 10 pre-radular teeth ( Fig. 15C ). Penis with curved stylet embedded in tip, ~75 µm in length, with oval opening ( Fig. 8B ). Ecology Specimens of C. piercei from shallow water at Tarpon Springs, FL fed on Pe. lamourouxii and H. incrassata , but not U. flabellum . Specimens from Tarpon Springs, FL were also previously reported on Pe. capitatus ( Barber et al. 2021 ) . At ~ 15 m depth offshore of St. Petersburg, FL, specimens were collected on U. luna and U. looensis . Specimens survived well on either Pe. lamourouxii or U. looensis in the laboratory regardless of their field host. Figure 14. Cyerce piercei , live specimens. A, B, dorsal views of specimen collected on Penicillus lamourouxii in Tarpon Springs, FL, USA; actual size, 9 mm. A, photographed with lighting from above. B, photographed with oblique lighting. C, D, dorsal view of specimen collected on Udotea looensis at 15 m depth from Mary’s Ledge reef, offshore of St. Petersburg, FL, USA; actual size, 7 mm. C, photographed with lighting from above. D, photographed with oblique lighting. Reproduction Several clutches were laid by specimens collected from Pe. lamourouxii in Tarpon Springs, FL in 2020. The egg masses lacked extracapsular yolk and had one embryo per capsule. Larvae appeared planktotrophic, lacking eyespots or a developed propodium, and the mean ± SE larval shell width across the aperture was 143.3.3 ± 4.4 µm ( N = 11) for one clutch at hatching, in the size range for planktotrophic sacoglossans ( Krug et al. 2015 ). Etymology Named in honour of Sidney K. Pierce, in recognition of his distinguished career, including seminal contributions to the study of kleptoplasty in sacoglossans, and for all his support of our work. Dr Pierce was a long-term researcher in the Tampa Bay area, served as a mentor to M.M. and gave P.J.K. his first specimen of this species> 15 years ago. Remarks Cyerce piercei was supported as distinct by all species delimitation analyses and was recovered as the sister clade to a lineage of four Western Atlantic species (all the complex members except C. antillensis ). In external morphology, C. piercei was differentiated from the similar C. nicholasi and C. browneveorum by having a triangular dark patch extending forwards from the eyes to the front of the head, dividing the medial plane between the eyes, and extending posteriorly around the pericardium. Cyerce nicholasi has a thin V-shaped brown to burgundy line across the head, but anterior to that line the head is white or pale in colour, and the eyes are not clearly separated by a dark patch; the pericardium is also white in ground colour. In C. browneveorum , a thicker burgundy line separates the eyes and extends up each rhinophore in a Y-shape, but the anterior of the head is white, and the pericardium is light in ground colour. Figure 15. Cyerce piercei , radular scanning electron micrographs of LACM 3843 (isolate 07Stir02). A, complete radula, with ascus attached to descending limb. B, leading tooth. C, close-up view of ascus, slightly damaged. Radular morphology is very similar between C. piercei and C. antillensis ,butthedenticlesaremoreelongatedandangledin C.piercei vs. shorter and more squared off in C. antillensis . The penial stylet of C. piercei was deeply embedded in tissue and thus difficult to view, but appeared longer and more curved than the embedded, straighter stylets of C. antillensis , C. ellingsonorum and C. browneveorum .