Brooding in Cocculiniform Limpets (Gastropoda) and Familial Distinctiveness of the Nucellicolidae (Copepoda): Misconceptions Reviewed from a Chitonophilid Perspective

Biological Journal of the Linnean Society, 2002, 75, 187-217. With 16 figures

Brooding in cocculiniform limpets (Gastropoda) and familial distinctiveness of the Nucellicolidae (Copepoda): misconceptions reviewed from a chitonophilid perspective

RONY HUYS FLS1*, PABLO J. LÓPEZ-GONZÁLEZ2, ELISA ROLDÁN3 a n d ÁNGEL A. LUQUE3

1Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK 2Departamento de Fisiología y Zoología, Facultad de Biología, Universidad de Sevilla, Reina Mercedes, 6,41012 Sevilla, Spain 3Laboratorio de Biología Marina, Departamento de Biología, Universidad Autónoma, 28049 Madrid, Spain

Received 21 January 2001; accepted for publication 16 August 2001

Nauplii, copepodids and adults of a new mesoparasitic genus and species of Chitonophilidae,Lepetellicola brescia nii, are described from the palliai cavity of a deepwater cocculiniform limpet,Lepetella sierrai, collected in the Bay of Biscay and Gulf of Cádiz. Re-examination of the type material of the recently established Nucellicolidae revealed several important observational errors in the original description, such as the oversight of the rootlet system in the adult female and misinterpretations of the tagmosis and antennulary segmentation in the late copepodid. Lamb et al.’s (1996) criteria used to justify the familial distinctiveness of the Nucellicolidae are all invalid. The family is relegated to a junior synonym of the Chitonophilidae on the basis of overwhelming support provided by copepodid and adult morphology. The impact of heterochrony on the body plan of adults and developmental stages is discussed. Phylogenetic analysis supports a basal dichotomy dividing the Chitonophilidae into a mesoparasitic clade, utilizing exclusively polyplacophoran hosts, and a sisterclade grouping genera associated with chitons, prosobranch gas tropods and cocculiniform limpets. The presence of maxillipeds and postmaxillipedal apodemes in the adult males of the latter clade is considered as apomorphic rather than plesiomorphic, being the result of incomplete moulting and correlated with the ventral position of the genital apertures.Nucellicola is identified as the sistergroup of the only other endoparasitic genus,Tesonesma, found in the body cavity of chitons. The inferred relationships indicate that host switching has occurred twice in the evolution of the Chitonophilidae. Examination of the antennulary segmentation and setation patterns of copepodidsLepetellicola in andNucellicola unequivocally refutes both the current placement of the Chitonophilidae in the Poecilostomatoida and its alterna tive assignment to the Siphonostomatoida. Exclusion from the Poecilostomatoida is reinforced by the absence of a coxo-basis in the antenna. The family is placed with the cyclopoids, providing further evidence for the crown-group status of the Poecilostomatoida within the currently paraphyletic Cyclopoida. A critical review of the published reports of brooding in cocculiniform limpets demonstrated that there is, as yet, no tangible evidence for this phe nomenon in either the Lepetelloidea or Cocculinoidea. © 2002 The Linnean Society of London,Biological Journal of the Linnean Society, 2002, 75, 187—217.

ADDITIONAL KEYWORDS: Chitonophilidae - heterochrony -Lepetella - Lepetellicola gen. nov. - molluscan hosts - phylogeny.

““Corresponding author. E-mail:[email protected]

INTRODUCTION Vaigamidae and Amazonicopeidae (Thatcher& Robertson, 1984; Thatcher, 1986). These families were Copepods, together with nematodes and insects, are proposed for highly modified fish parasitic copepods the most abundant multicellular organisms on Earth. but in reality are merely specialized lineages of the Current evidence suggests that copepods originated Ergasilidae (Abdelhalimet al., 1993; Amado et al., from the marine hyperbenthic habitat (Huys & 1995). The uncritical acceptance of such familial iden Boxshall, 1991) and subsequently spread out in tity, without considering the possibility of the taxon sediments across the entire salinity spectrum. Their being at a terminal node in a larger encompassing current position of world predominance, however, can clade, is potentially naive and unscientific, particu be attributed to two principal, recurrent, radiation larly when no attempt has been made to identify the events, i.e. their major habitat shift into the marine hypothetical outgroup. plankton, and the evolution of parasitism. Given their We have used mollusc-infesting copepods as an moderately high host specificity in conjunction with example to demonstrate these misconceptions. The the dazzling spectrum of potential marine hosts, it discovery of a new genus of Chitonophilidae prompted is highly conceivable that parasitic copepods sig us to challenge the widely assumed phenomenon of nificantly outnumber their free-living counterparts brooding in cocculiniform limpets, an anatomically in species diversity. It would not be extravagant to and biologically very diverse group currently classified assume that every other kind of marine macro as an order or suborder of the streptoneurous Gas invertebrate on earth has at least one copepod species tropoda (Haszprunar, 1998). It has also provided new acting as its own personalized parasite. This suc morphological information of high phylogenetic signif cessful colonization or utilization of virtually every icance, causing serious implications for the taxonomic metazoan phylum has generated a great diversity in status of the Nucellicolidae, a recently proposed family copepod body morphology, which is arguably unparal of highly transformed endoparasitic copepods based leled among the Crustacea. Regrettably, this variety solely on autapomorphies. in body plan has also given rise to unfortunate conse quences for phylogenetic analysis. More specifically, MATERIAL AND METHODS the highly transformed forms frequently lie at the root of two important misconceptions. Museum collections of Lepetella sierrai D antart & Failure to recognize the extreme modification of Luque (1994) (Mollusca, Cocculiniformia, Lepetel certain copepods has repeatedly resulted in them loidea), deposited in the Naturhistoriska Riksmuseet being misinterpreted as part of the host. Garstang in Stockholm (NRS) and the Museo Nacional de (1890) recorded ‘pieces of spawn’ attached to the den- Ciencias Naturales in Madrid (MNCN), were exam dronotid nudibranchLomanotus genei (Verany, 1846) ined for parasitic copepods. This material was col but these were later identified by Scott & Scott (1895) lected in the Iberian-Moroccan Gulf (BALGIM as egg-sacs of the splanchnotrophid copepodLoman expedition, 1984, coordinated by the Muséum oticola insolens Scott & Scott (1895), the urosome of National d’Histoire Naturelle, Paris) and in the Bay which is typically protruding through the host integu of Biscay (Fauna Ibérica II expedition, coordinated ment. Another example of such observational error is by MNCN). Host material was obtained from empty shown by Muñozet al. (1996) who figured an unusual or occasionally inhabited, corneous tubes of the penis with ‘... a row of hooks on each side’ in their sedentary polychaete Hyalinoecia tubicola (O.F. redescription of the goniodoridid nudibranchOkenia Müller, 1776), on which Lepetella sierrai lives and luna (Millen et al., 1994). The offset lobate anterior feeds. Copepods were cleared and dissected in lactic end, the traces of segmentation in the posterior part, acid; the dissected parts were mounted on slides the number of lateral processes and the presence of an in lactophenol mounting medium. Preparations were unpaired median outgrowth leave no doubt that the sealed with Glyceel (BDH Laboratory Supplies, supposed penis is an adult female ofIsmaila Bergh, Poole, UK) or transparent nail varnish. All drawings 1867, a genus of endoparasitic copepods (Splanch have been prepared using a camera lucida on an notrophidae) typically associated with the kidney, Olympus BH (Hamburg, Germany) or a Leica DMR pericardium or the digestive gland of nudibranch and (Wetzlar, Germany) differential interference contrast sacoglossan opisthobranchs (Huys, 2001). microscope. Both host and parasite specimens were Secondly, failure to place strange copepods in examined with a Philips XL 30 scanning electron existing classifications, often compounded by imper microscope (Eindhoven, The Netherlands). Specimens fect dissection and observation, has recently led to the were prepared by dehydration through graded proposal of a number of monogeneric or monotypic acetone, critical point dried, mounted on stubs and families. An example of such undesirable inflation sputter-coated with gold or palladium. of higher categories is illustrated by the families The descriptive terminology is adopted from Huys&

© 2002 The Linnean Society of London,Biological Journal of the Linnean Society, 2002, 75, 187-217 F igure 1. Lepetellicola brescianii gen. et sp. nov. A, paratype ? attached to host, dorsal view showing attached dwarf male ($), eggs and spermatophores [e.s., remaining egg string of hatched egg; sp., spermatophore; ov., ovary]; B, same, ventral view showing two dwarf males (

Figure 2. Lepetellicola brescianii gen. et sp. nov. Adult $. A, habitus, dorsal view; B, same, lateral view; C, same, ventral view.

Boxshall (1991). Abbreviations used in the text are: ae, RESULTS aesthetasc; P1-P4, first to fourth thoracopod. Scale bars in Figures 1-13 are in pm. M orphological observations Type material examined in the present paper has Family Chitonophilidae Avdeev & Sirenko, 1991 been deposited in the MNCN, NRS and the Natural Syn. Nucellicolidae Lambet al., 1996. History Museum in London (NHM). In order to study the effect of the copepod parasite History on the host, 51 specimens of L. sierrai (ranging All genera currently allocated to the Chitonophilidae between 0.6 and 2.0 mm), 26 of which were infested, have been discovered in the last 15 years. Jones & were examined for gonad development and maturaMarshall (1986) described ovigerous females of Coc tion. Material for this histological study comes from culinika myzorama from the wood-associated coc- the Iberian-Moroccan Gulf (BALGIM expedition). culinid limpet Coccopigya hispida Marshall, 1986 in Specimens were preserved in 70% ethanol and their New Zealand bathyal waters. They were unable to external anatomy was examined using a stereomicro- place the species in any existing family or order and scope. Material for histological studies was dehy ranked it incertae sedis in the Copepoda. Franz & drated, embedded in paraplast, serially sectioned atBullock (1990) described both sexes ofIschnochitonika 7 pm and stained with Mayer’s haematoxylin-eosin. lasalliana from the branchial cavity of two polypla- The phylogenetic software package PAUP 3.1.1, cophoran hosts, Ischnochiton striolatus (Gray, 1828) written by David Swofford of the Laboratory of Mole andStenoplax boogi (Haddon, 1886), collected in the cular Systematics, Smithsonian Institution (Swofford, Caribbean. A comparison was made with the highly 1993), was used to analyse phylogenetic relationships modified Herpyllobiidae and a number of enigmatic within the Chitonophilidae. mollusc-infesting genera such as Teredoika Stock,

F igure 3. Lepetellicola brescianii gen. et sp. nov. Nauplius. A, antennule; B, antenna; C, mandible;D, habitus, dorsal view.

1959, Axinophilus Bresciani & Ockelmann, 1966 and added by Nagasawaet al. (1991) from Ischnochiton Pectenophilus Nagasawa, Bresciani & Lützen, 1988, (.Ischnoradsia ) hakodadensis (Pilsbry, 1893) in the Sea but Franz & Bullock (1990) were unsuccessful in of Japan but, despite the first description of the nau assigning Ischnochitonika to any of the parasitic plius, and SEM observations, their discovery shed no copepod orders. A second species,I. japonica was new light on affinities.

Figure 4. Lepetellicola brescianii gen. et sp. nov. Copepodid. A, habitus, lateral view; B, same, dorsal view; C, antenna (minute claw arrowed); D, maxilliped and oral tube, lateral view.

A-E

F igure 5. Lepetellicola brescianii gen. et sp. nov. Copepodid. A, antennule; B, leg 1 and intercoxal sclerite, anterior; C, leg 2 and intercoxal sclerite, anterior; D, leg 3 and intercoxal sclerite, anterior; E, leg 4 and intercoxal sclerite, ante rior; F, urosome, lateral view.

© 2002 The Linnean Society of London,Biological Journal of the Linnean Society, 2002, 75, 187-217 F igure 6. Lepetellicola brescianii gen. et sp. nov. (SEM micrographs). A, ovigerous female attached to dorsal side of cephalic region of Lepetella sierrai (shell removed); B, same, dorsal view showing paired egg-sacs; C, ovigerous female largely removed from host, dorsal, showing neck (arrowed) and egg-strings of previous batches; D, close-up of neck pene trating host integument. LIMPET BROODING AND NUCELLICOLID IDENTITY 195

F igure 7. Lepetellicola brescianii gen. et sp. nov. (SEM micrographs). A, female with two attached males in palliai cavity of Lepetella sierrai (position arrowed); B, close-up showing males (arrowed); C, ovigerous female with paired egg masses; D, group of three late copepodids found along with nauplii in egg mass. Scale bars: 500 |im (A), 150 |im (B), 250|im (C), 100|im (D).

© 2002 The Linnean Society of London,Biological Journal of the Linnean Society, 2002, 75, 187-217 F igure 8. Lepetellicola brescianii gen. et sp. nov. (SEM micrographs). Late copepodid. A, body, lateral; B, body, ventral; C, rostrum, frontal; D, céphalothorax (posterior part), P2-bearing somite (condylar articulation arrowed) and pedigerous double-somite, lateral. F igure 9. Lepetellicola brescianii gen. et sp. nov. (SEM micrographs). Late copepodid. A, céphalothorax, ventral view showing maxillipeds, oral eone and bases of antennae (coxa arrowed); B, oral eone (pore on maxillipedal basis arrowed); C, antennule (segment 3 arrowed); D, P3 outer basal seta. 198 R. HUYS ET AL.

□ DA SI : □ SI

□ Dà A A O A...... A"

£ 3

° 2 □ □ a □

□ O A □ without copepod a with copepod -•A... A..... A A 0,6 0,8 1,0 1,2 1,4 1,8 2,0 Shell length (mm)

Figure 10. Lepetella sierrai Dantart & Luque, 1994. Scatterplot of the level of gonad maturity against shell length (mm). Different levels of gonad maturity: 0, no gonad; 1, spermatids, no ovary; 2, spermatids, previtellogenic oocytes; 3, mature sperm, no ovary; 4, mature sperm, previtellogenic oocytes; 5, mature sperm, vitellogenic oocytes.

These three publications were probably overlooked Diagnosis by Avdeev & Sirenko (1991) when they proposed the family Chitonophilidae for two new genera of highly Adult female highly transformed, lacking appendages transformed copepods from shallow water polypla- and external traces of segmentation. Body comprising cophorans in the north-western Pacific.Chitonophilus well developed rootlet system, serving as holdfast and laminosus Avdeev & Sirenko, 1991 was described from absorptive system inside the host, and sac-like trunk, Tonicella submarmorea (von Middendorfi, 1848), containing reproductive system and digestive tract. whereas the host of Leptochitonicola latus Avdeev & Trunk either external (ectosoma) in mesoparasites or Sirenko, 1991 was Leptochiton assimilis (Thiele, entirely internal in endoparasites; in mesoparasites 1909). Little or no justification was provided for the often with lateral lobes anteriorly and/or posteriorly. proposal of the new family and no ordinal placement Genital system paired; position of gonopores variable. was suggested. In 1994 Avdeev & Sirenko accepted Eggs forming grape-like masses in mesoparasitic Cocculinika andIschnochitonika as valid members of members, often attached to the genital opening by the Chitonophilidae and established two new genera individual egg-strings; in endoparasites laying free in Tesonesma and Cookoides found in chiton hosts of membranous cyst or tube, communicating with exte the genus Stenosemus von Middendorfi, 1847. rior via initial point of entry. Unlike other Chitonophilidae, Tesonesma reniformis Adult male highly transformed, distinctly smaller Avdeev & Sirenko, 1994 was found to be entirely than female, lacking segmentation; often with lateral endoparasitic. or frontal lobes; with either antennae or maxillipeds Lamb et al. (1996) recognized the Chitonophilidae as only appendages. Typically attached near the as a poecilostomatoid family but gave no justification genital apertures of the female. Genital system paired; for this assignment. They proposed a new family vasa deferentia each terminating in specialized sper- Nucellicolidae for Nucellicola holmanae Lamb, matophore sac; genital apertures either posterior to Boxshall, Mill & Grahame, 1996, an endoparasite of maxillipeds (when present) or dorsal. Spermatophores the dogwhelk Nucella lapillus (Linné, 1758) in the elongate-pyriform, attached in clusters to female British Isles. The same copepod had previously been genital apertures. described as Nucellicola kilrymontis in an unpub Nauplius lecithotrophic; antennule 1- or 2-seg- lished Ph.D. thesis (Fitches, 1966). Our results below mented; antennae and mandibles without gnatho- provide strong evidence for the inclusion ofNucellicola basal elements. Caudal rami represented by one seta in the Chitonophilidae and consequently the Nucelli each. Nauplii developing in palliai cavity of host in colidae is relegated here to a junior synonym of the mesoparasitic species or inside egg-tube (or cyst) latter. in endoparasites.

F.L. L.L.

F igure 11. Lepetellicola brescianii gen. et sp. nov. A, copepodid, ventral view of cephalic appendages [Ai, basal segment of antennule; A2, base of antenna].Nucellicola holmanae Lamb, Boxshall, Mill & Grahame, 1996. B, late male copepodid, last two urosomites and caudal rami, dorsal view; C, adult$ enclosed in membranous vesicle together with exuvium of preceding copepodid, ventral view (F.L., frontal lobe; L.L., lateral lobe; S., spermatophore sac); D, adult$, lateral view; E, leg 1, anterior view. Asterisk in C-D denotes maxillipeds beneath frontal lobe. pmxp.ap.

pmxp.ap. g.m.

200

2mm

1mm

Figure 12. Nucellicola holmanae Lamb, Boxshall, Mill & Grahame, 1996. A, adult ?, habitus (arrow indicates rootlet system); B, rootlet system of adult ? shown in (A); arrows marking constriction between cephalic lobe and rootlet system; C, adult $, dorsal view (d.s., discharged spermatophore; s., spermatophore; both attached to female); D, adult$, maxil lipeds, ventral view (g.m., genital muscle; pmxp. ap., postmaxillipedal apódeme; atrophied intrinsic musculature indicated by dotted contours); E, adult$, maxilliped, lateral view. Lepetellicola brescianii gen. et sp. nov. F, adult $, maxil lipeds, ventral view (m., mouth opening; pmxp. ap., postmaxillipedal apódeme). LIMPET BROODING AND NUCELLICOLID IDENTITY 201

Figure 13. Nucellicola holmanae Lamb, Boxshall, Mill & Grahame, 1996. Late male copepodid. A, habitus, lateral view (arrow marking ventral slit in exuvium); B, habitus, dorsal view.

Late copepodid cyclopiform with 3-segmented with distinct postmaxillipedal apódeme. Oral eone prosome, comprising céphalothorax, leg 2-bearing small, without sclerotized mouth-ring. Genital system somite and pedigerous double-somite bearing legs completely paired, comprising testes, vasa deferentia 3-4; urosome 3-segmented. Céphalothorax with 5- and spermatophore sacs associated with ventral segmented antennules, uniramous antennae bearinggenital apertures located posterior to maxillipeds. three elements at tip, and subchelate maxillipeds; oral Typically attached midventrally to posterior region of eone short, without trace of mandibles. Swimming legs female. 1-4 biramous; rami 1-segmented with exopodal setae Nauplius with 1-segmented antennules; antennae arranged around distal and inner margins. Caudaland mandibles both with 1-segmented bisetose rami with one dorsal and three terminal elements. endopod and 3-segmented exopod bearing one seta on Additional metanaupliar and copepodid stageseach segment. sometimes present (Nucellicola). Infection of host Late copepodid as for the family; segment 2 of anten at copepodid stage. nule with three setae plus aesthetasc; anterior Adults mesoparasitic in palliai groove or endopara element on segment 4 spiniform. Leg 1 without outer sitic in viscera of Polyplacophora, Cocculiniformia and seta on basis. Caudal rami with smooth terminal N eogastropoda. setae. Note: Avdeev & Sirenko (1991, 1994) regarded Mesoparasitic in palliai cavity of deepwater coc the fleshy swellings on either side of the mouthcone in culiniform limpets. the males of Chitonophilus laminosus andTesonesma reniformis as highly transformed mandibles. This is Type species highly unlikely as no mandibles are found in the late Lepetellicola brescianii gen. et sp. nov. copepodids ofNucellicola holmanae andLepetellicola brescianii. Etymology The generic name is derived from the generic name Type genus of the host, Lepetella, and the Latincolere, meaning Chitonophilus Avdeev & Sirenko, 1991. inhabiting. Gender: masculine.

Other genera Cocculinika Jones & Marshall, 1986; Ischnochitonika L epetellicola b r e s c i a n i igen. et sp. nov. Franz & Bullock, 1990; Leptochitonicola Avdeev & Type locality Sirenko, 1991; Cookoides Avdeev & Sirenko, 1994; Vizcaya, Bay of Biscay, Fauna Ibérica II Stn 168 A Tesonesma Avdeev & Sirenko, 1994; Nucellicola (43°45.13'-43°46.53'N, 8°10.09'-8°9.59'W), 116-120m Lamb, Boxshall, Mill & Grahame, 1996; Lepetellicola depth. Host: Lepetella sierrai, paratypes MNCN reg. gen. nov. no. 15.05/5252, coli. 07 June 1991.

Material examined L epetellicola g e n . n o v. Following specimens were found on L.sierrai from: Diagnosis (1) Type locality: holotype ovigerous Î (MNCN reg. Chitonophilidae. Female body comprising ectosoma no. 20.04/5259a); paratypes preserved in alcohol are connected to branching rootlet system (endosoma) two ovigerous and two juvenile îî (NHM reg. nos. inside host via distinct cylindrical neck. Ectosoma 2000. 1782-1785), two juvenile ÎÎ and one nauplius typically heart-shaped, with large lateral lobes con (MNCN reg. no. 20.04/5259b), and one ovigerous Î taining ovaries and elongate paired posterior lobes (MNCN reg. no. 20.04/5259c); paratypes mounted on originating subventrally from genital apertures. Neck slides are four nauplii (on three slides; NHM reg. nos. arising midventrally from anterior part of ectosoma. 2000. 1790-1793), one copepodid (on two slides; NHM Genital system completely paired, comprising ovaries, reg. no. 2000. 1794), one copepodid and one egg (on one oviducts and posterodorsal genital apertures located slide; MNCN reg. no. 20.04/5259d), one copepodid (on in depressions at base of posterior lobes. Egg masses one slide; MNCN reg. no. 20.04/5259e), one egg mass shaped like bunch of grapes; eggs attached individu with developing nauplii (on 1 slide; NHM reg. no. ally by long strings. 2000. 1797), and 1 Î with attached1 (on one slide; Adult male smaller than female, elongate; compris NHM reg. no. 2000. 1798); four ovigerous ÎÎ mounted ing trilobate cephalic region and sac-like posterior on SEM stubs (not registered); trunk, separated by bilateral constriction; lateral lobes (2) Off Gijon, Fauna Ibérica II Stn 185 A (43°42.59'- weakly developed. Antennae and antennary projec43°43.48'-N, 5°55.40'-5°56.40'W), 116-112 m depth; tions absent. Subchelate maxillipeds only appendages, coli. 4 July 1991; on paratypes of L. sierrai (MNCN

© 2002 The Linnean Society of London,Biological Journal of the Linnean Society, 2002, 75, 187-217 LIMPET BROODING AND NUCELLICOLID IDENTITY 203 reg. no. 15.05/27875): paratype $ on slide (MNCN reg. Infested hosts usually contained a single female but no. 20.04/5260); in some cases up to three females were observed (e.g. (3) Cabo Mayor (N. Cabo Mayor), Fauna Ibérica II females illustrated in Figures 1A-B,F-G were found Stn 128 A(43°35.92'-43°35.63'-N, 3°47.53'-3°49.21'W), on the same gastropod individual). 141-142 m depth; coli. 19 June 1991; on paratypes of L. sierrai (MNCN reg. no. 15.05/27877): one ovigerous Young females Î with attached $, mounted on SEM stub; Body (Fig. IF—G) about 260pm in length and 390pm (4) BALGIM stn. CP21: Gulf of Cádiz (36°36'-N, wide. Posterior lobes weakly developed and widely 07°24'-W), 478-491 m depth; coli. 31 May 1984; divergent. Reproductive system already fully devel deposited in NRS: paratypes are eight ovigerous oped. Young female illustrated in Figure 1F-G with females in alcohol (reg. nos MNHN-Cp 1900-1907) two males attached (not shown) but no sper and one young Î with twoS S mounted on slide (reg. matophores or traces of egg batches (egg-strings) no. MNHN-Cp 1908). discernible. Descriptions based on (1) and (2); SEM observations based on (1) and (3). A dult male Body (Figs 2A-C and 7B) highly transformed, without Adult female any traces of segmentation or clear tagmosis; dis Body (Figs 1A-E, 6A-C and 7B) highly transformed, tinctly smaller than Î; length 250-275pm (mean: consisting of sac-like ectosoma connected via distinct 265 pm), width 105-130pm (mean: 120pm, N = 5); neck to internal rootlet system. Ectosoma with large comprising trilobate cephalic region and sac-like rounded anterolateral lobes and widely separatedposterior trunk, separated by bilateral constriction. elongate posterior lobes extending subventrally; Frontal and posterolateral expansions of cephalic length 400-590 pm (mean: 510 pm, N = 7) including region lobate, rounded and directed ventrally. Medial posterior lobes; width 475-630pm (mean: 570 pm, mouth eone very small, located just anterior to maxil N= 7. Posterior lobes longer and heart shape of ante lipeds (Fig. 12F). rior part of ectosoma more pronounced in larger spec Maxillipeds only appendages present; located imens (Fig. 1A). Cylindrical neck arising midventrally midventrally on raised area at level of bilateral con from anterior part of ectosoma (arrowed in Fig. 6C), striction (Fig. 2B-C); subchelate (Fig. 12F), com with longitudinal furrows (Fig. 6D); in smaller speci prising robust proximal segment (representing basis) mens shorter and originating slightly more anteriorly; and strong endopodal claw; no traces of syncoxa integument reinforced around site of penetration. discernible; palmar margin of basis with medially Posterior lobes arising from ventral margin of genital directed spinous process. Maxillipeds internally apertures; in bigger specimens often obscured by supported by a symmetrically convoluted post numerous spermatophores and remains of egg-strings maxillipedal apódeme running across entire width (Figs 1A,D,E and 6C). Genital apertures paired, of cephalic region. No trace of muscles in basis of posterodorsal; concealed in depressions between maxilliped. hind margin of ectosoma and posterior lobes. Reproductive system completely paired (Fig. 2C). Genital system clearly visible with paired ovaries, oc Testes large, extending into frontal lobe and passing cupying lateral lobes, and long convoluted oviducts dorsally to posterior end of trunk (Fig. 2B); vasa (Fig. 1A-B). Radiating branches of digestive system deferentia making characteristic loop with ascending discernible ventrally, connected via neck with rootlet and descending branches in mid-region of trunk; system (Figs 1B,D). Endosoma represented by delicate distal parts of vasa deferentia dilated, forming small rootlet system branching inside host; branching spermatophore sac opening to exterior via paired pattern irregular and only partly revealed after pro gonopores located on either side of mid-ventral longed preservation in lactic acid. protuberance. Spermatophores elongate-pyriform, Egg masses in the shape of a bunch of grapes attached in clusters to female genital apertures (Figs 6A-B and 7C). Eggs typically spherical (Fig. 1C, (Figs 1A,E). e.) but becoming more ovoid later in development Adult male attached medially to posteroventral area (Fig. 1C, nj; 110-120pm in diameter, attached indi of female (Fig. ID); up to six males can be attached to vidually by thin egg string to dorsal surface of poste a single female, often with one of them located mid rior lobes (Fig. 1A); larger females can have up to 40 ventrally between both posterior lobes and any addi eggs attached. Egg strings often remain attached to tional males positioned submedially (Fig. IB; arrowed the female following eclosión of the nauplii (Fig. 6C). in Fig. 7B). In one case the site of a dislodged male Muscles associated with developing naupliar limbs was revealed by the maxillipeds left behind on the visible through egg membrane. female’s ventral surface (arrowed in Fig. ID).

Nauplius margin of céphalothorax, forming condylar articula Lecithotrophic. Body 120-125 um long and 75-80 um tions (Fig. 4A; arrowed in Fig. 8D); with transverse broad, based on five specimens in lactophenol. Newly internal ribs (anterior to middorsal pore; stippled in released nauplii (Fig. 3D) elongate-ovoid; maximum Fig. 4B) dorsally, representing attachment sites of well width measured in anterior half. Exoskeleton developed intersomitic membranes (Fig. 4A); posterior extremely thin and fragile. A pigmented nauplius eye width smaller than that of céphalothorax. appears to be absent. No labrum, functional mouth or Pedigerous double-somite with six pairs of sensillae; anal slit present. Caudal region with pair of naked free lateral margin of pleurotergite with short incision setae. marking original segmentation of P3- and P4-bearing Antennule (Fig. 3A) 1-segmented; basal portion somites; posterolateral angles backwardly produced. corresponding to pedestal, incompletely separated by Urosome (Fig. 5F) very slender and narrow; ante suture from large paddle-like distal segment. Arma rior margin of first urosomite covered by membranous ture consisting of plumose lateral seta and two ring; middle urosomite longest; anal somite shortest. pinnate setae plus aethetasc apically. Caudal rami rectangular, longer than wide; each with Antenna (Fig. 3B) biramous; protopod consisting of short dorsal seta, two long terminal setae and one unarmed coxa and basis, both without gnathobasal stubby spine at outer distal corner; all elements projections or setae. Exopod 3-segmented, middle smooth. and distal segments minute; each segment with one Antennule (Figs 5A and 9C) 5-segmented; arm ature pinnate seta. Endopod distinctly wider than exopod;formula 1-[1], 2-[3 + ae], 3-[l], 4-[l + 1 spine + ae], 1-segmented; with two apical pinnate setae. 5-[5 + ae]; segment 3 (arrowed in Fig. 9C) much nar Mandible (Fig. 3C) biramous, more slender than rower than proximal and distal segments, anterio- antenna but with similar segmentation and without proximal part often telescoped into distal portion of gnathobasal structures on protopodal segments. segment 2. Aesthetasc on segment 2 much longer than Exopod 3-segmented, segments 2-3 minute; each others and typically constricted halfway its length. segment with one pinnate seta. Endopod narrower Antenna (Fig. 4C) 4-segmented, comprising coxa, than exopod, dilating distally; inner distal corner probasis and 2-segmented endopod; exopod absent. Coxa duced into pointed process; with two apical pinnate with small sclerite around the base (Fig. 9A, arrowed; setae. Fig. 11A). Coxa, basis and proximal endopod segment Several nauplii were observed still attached with unarmed. Proximal endopod segment elongate; distal their caudal end to the female’s body via the proximal segment very short, with two equally long claws api part of the egg membrane and its connecting string cally and one minute element (arrowed in Fig. 4C) at (Fig. 1C: n2). Late nauplii with yolk clearly partitioned outer distal corner. have been observed, possibly indicating metamery of Mandible, maxillule and maxilla absent. Oral eone developing copepodid. All nauplii belonging to same short (Figs 4D, 9A-B and 11A), positioned on midven- stage; no evidence for metanaupliar instars. tral crest between maxillipeds; with two apical open ings separated by medial septum. Late copepodid Maxilliped (Fig. 4D) subchelate; syncoxa not Body length 160—170pm (N = 3), greatest width expressed, basis articulating directly with ventral (90 um ) measured mid-way céphalothorax; integument surface of céphalothorax; 2-segmented, comprising with irregular pattern of surface pits (Fig. 8D). Body basis and endopod. Basis robust and swollen; palmar cyclopiform (Figs 4A-B, 7D and 8A-B), comprising margin produced into spinous process bearing apical 3-segmented prosome and 3-segmented urosome. First pore (arrowed in Fig. 9B). Endopod represented by pedigerous somite incorporated in cephalosome, strong curved claw. forming céphalothorax (Fig. 8D). Prosome comprising Swimming legs 1-4 (Fig. 5B-E) with well developed céphalothorax, P2-bearing somite and pedigerous intercoxal sclerites (distinctly smaller in leg 4), 2-seg- double-somite bearing P3-P4. Céphalothorax broadly mented protopods and 1-segmented rami. Coxae ovoid; posterolateral corners produced into lobate unarmed. Bases with outer seta on legs 2 and 3; basal extension; with well developed sensillar pattern and seta with distinct longitudinal ridges (Fig. 9D). accessory pores as indicated in Figure 4A; separated Exopods distinctly longer than endopods; without dorsally from P2-bearing somite by membranous zone outer spines or setae but outer distal corner produced (Fig. 8D). Rostrum represented by ventrally directed, into minute spinous projection; with two apical and tapering protuberance bearing one median slit-like two (legs 1 and 4) or three (legs 2 and 3) inner ele pore and two lateral sensillae (Fig. 8C). ments. Endopods with two apical setae. All elements P2-bearing somite with three pairs of sensillae and plumose except for outer distal element of exopod middorsal pore; anterior margin with lateral rounded being serrate along outer margin and inner distal protuberances articulating in sockets of posterior element tri pinnate.

Etymology uration of the female gonads and, in extreme cases, The species is named after our colleague Dr José causes complete cessation of development and proba Bresciani, in recognition of his valuable contributions bly parasitic castration as reported by Jensen (1987) to the biology and anatomy of parasitic copepods. for splanchnotrophid copepods in shell-less opisthobranchs. Among the infested individuals studied, only three mature simultaneous hermaphroditic specimens Affinities were found; these were considerably larger (1.4, 1.8 Lepetellicola brescianii is most closely related to and 1.9 mm) than fully mature, noninfested specimens Cocculinika myzorama, the only other chitonophilid (Fig. 10). known to be associated with a cocculiniform limpet. Prevalence expressed as the number of host indi Both species occupy the same niche on the host, i.e. viduals infested vs. the number of host individuals above the head region inside the palliai cavity. Adult examined was 0.39 (169/425) in station CP21, 0.233 females are morphologically similar in the possession (14/60) in station 185 A, 0.72 (8/11) in station 128 A of lateral expansions but differ in the position of the and 0.48 (36/75) in station 168 A). The intensity (based paired genital apertures. InCocculinika the gonopores on the number of female parasites only) was never are located far anteriorly, near the transition between higher than one parasite per infected host. the ectosoma and the short neck region, whereas in Lepetellicola they are concealed in depressions between the hind margin of the ectosoma and the pos New observations on N ucellicola Lamb, terior lobes. L. brescianii has very well developed pos Boxshall, M ill & Grahame, 1996 terior lobes that are only incipient inC. myzorama. Diagnosis (Modified from Lamb et al. (1996)) The former is also readily distinguishable by the long Chitonophilidae. Female body cylindrical, comprising cylindrical neck. In view of the uniformity observed in small cephalic lobe, bearing branching rootlet system, the genus Ischnochitonika (Franz & Bullock, 1990; and large vermiform trunk containing highly convo Nagasawa et al., 1991) we regard these differences as luted gonads. Reproductive system completely paired, sufficiently distinct to warrant separate generic comprising ovaries, oviducts and posteriorly located status. genital apertures. Female enclosed within membra nous tube extending through host viscera to point of Host-parasite relationships entry of parasite; tube containing eggs and developing All families of the Cocculiniformia, except for the nauplii, not arranged in egg-sacs. Choristellidae, have been considered simultaneous Adult male dwarfed, located in membranous vesicle hermaphrodites displaying internal fertilization and at posterior end of female; comprising trilobate having distinct vesicles acting as testis and ovary cephalic region and sac-like posterior trunk, separated (Haszprunar, 1988b, 1998). Both testis and ovary are by strong bilateral constriction; lateral lobes of mature in fully grown noninfested adultsLepetella of cephalic region strongly developed. Antennae and sierrai. However, the former always appears, and antennary projections absent. Subchelate maxillipeds reaches maturity, before the latter. Mature testes were only appendages, with distinct postmaxillipedal observed in noninfested individuals as small as apódeme. Oral eone small, overlain by frontal lobe, 0.8-1.0mm in shell length (Fig. 10). At this size the without sclerotized mouth-ring. Genital system com ovary consists solely of previtellogenic oocytes, indi pletely paired, comprising testes, vasa deferentia and cating th at L. sierrai may only function as a male at spermatophore sacs associated with ventral genital this size. Testis and ovary both reach maturity in spec apertures located posterior to maxillipeds. imens larger than 1.0 mm, which therefore can be Nauplius with 1-segmented antennules; antennae considered as simultaneous hermaphrodites. These and mandibles with 1-segmented bisetose endopod observations indicate that L. sierrai is a functional and 3-segmented exopod bearing one seta on each protandric hermaphrodite likeAddisonia excentrica segment. Metanauplius with maxillipeds and two Tiberi, 1857, another lepetelloidean belonging to the pairs of swimming legs. Early copepodid with 4-seg- family Addisoniidae (Roldán& Luque, 1999). In mented prosome and 2-segmented urosome; anten infested individuals the development and maturation nary exopod and mandible vestigial; with four pairs of of the ovary is strongly affected by the presence of the swimming legs. parasites. In general, the testis matures without Late copepodid as for the family; segment 2 of anten development of the ovary, which is a condition (Fig. 10, nule with four setae plus aesthetasc; anterior element stage 3) never observed in noninfested specimens on segment 4 setiform. Leg 1 with outer seta on basis. where the ovary always contains previtellogenic Caudal rami with sparsely plumose terminal setae. oocytes at stages 2 and 4. Infection of the host by Endoparasitic in visceral whorl ofNucella lapillus chitonophilids often results in severe delay in the mat (Gastropoda: Muricidae).

Type species Late male copepodid Nucellicola holmanae Lamb, Boxshall, Miii & Description based on exuvium dissected out of mem Grahame, 1996 [by monotypy]. branous sac containing adult male (Fig. 11C). Body length 190 pm (N = 1), greatest width (100 pm) mea N u c e l l ic o l a h o l m a n a e Lam b, sured at posterior third of céphalothorax. Body cyclop- Boxshall, M ill & Grahame, 1996 iform (Fig. 13A-B), comprising 3-segmented prosome and 3-segmented urosome. First pedigerous somite Material examined incorporated in cephalosome forming céphalothorax. Holotype Î with adult allotype î attached (NHM reg. Prosome comprising céphalothorax, P2-bearing somite nos. 1995.664^665); two paratype îî each with $ and pedigerous double-somite bearing P3-P4. attached (NHM reg. nos. 1995.666-667); endoparasitic Céphalothorax broadly ovoid, abruptly tapering in Nucella lapillus-, intertidal zone of rocky shore in posteriorly; posterolateral corners not produced into Robin Hood’s Bay, North Yorkshire, England. lobate extension; with well developed sensillar pattern and accessory pores as indicated in Figure 13A-B; sep Adult female arated dorsally from P2-bearing somite by membra As described by Lambet al. (1996) except for presence nous zone. Rostral area as inLepetellicola. Ventral of well developed rootlet system (Fig. 12A-B) sepa surface with large slit through which the adult male rated from the elongated trunk by a distinct constric emerged; corresponding to position of maxillipeds and tion (arrowed in Fig. 12B). postmaxillipedal apódeme in copepodid Lepetelli of cola (compare Fig. 4A). A dult male P2-bearing somite with two pairs of sensillae and Enclosed along with exuvium of late male copepodid middorsal pore; with condylar articulations and trans in membranous vesicle (Fig. 11C); attached in between verse internal ribs as inLepetellicola-, widening poste genital apertures of female. Body highly transformed, riorly so that width at hind margin is greater than 550pm long and 390pm wide; comprising trilobate posterior width of céphalothorax. cephalic region and heart-shaped sac-like trunk, Pedigerous double-somite with nonfunctional artic separated by strong bilateral constriction. Cephalic ulation middorsally, fading out in transverse surface region with very large lateral lobes, extending dor suture dorsolaterally and laterally (Fig. 13B); free sally; rounded frontal lobe directed ventrally, over- lateral margin of pleurotergite without trace of origi lying small oral eone and maxillipeds (Fig. 11C-D). nal segmentation of P3- and P4-bearing somites; with Trunk with lateral lobate extensions in anterior six pairs of sensillae; posterolateral angles backwardly half. produced; posterior part with sigmoid chitinous ribs Medial mouth eone very small, located just anterior dorsally. to maxillipeds. Maxillipeds (indicated by asterisk in Urosome (Figs 11B and 13A-B) less slender than Fig. 11C-D) located in deep depression formed by in Lepetellicola but somites of similar proportions frontal lobe and anterior midventral face of trunk (foreshortened in Fig. 13B). Caudal rami rectangular, (Fig. 12E) at level of bilateral constriction; subchelate longer than wide; each with short dorsal seta, two (Fig. 12D), comprising robust proximal segment (rep long-terminal setae and one stubby spine at outer resenting basis) and strong endopodal claw; no tracesdistal corner; terminal setae sparsely plumose. of syncoxa discernible; palmar margin of basis with Antennule (Fig. 13A-B) 5-segmented; armature medially directed spinous process. Maxillipeds inter formula 1-[1], 2-[4 + ae], 3-[l], 4-[2 + ae], 5-[5 + ae]; nally supported by a symmetrically postmaxillipedal segmental proportions and relative lengths of aes- apódeme (Fig. 12D-E). Intrinsic muscles in basis atro thetascs as in L. brescianii. phied. Two intermaxillipedal processes present Antenna (Fig. 13A) 4-segmented, comprising coxa, (Fig. 12D-E). basis and 2-segmented endopod; distal segment very Reproductive system completely paired, as short, with two equally long claws apically and one described by Lambet al. (1996). Distal part of each vas minute element subapically. deferens dilated, forming distinct spermatophore sac Maxillipeds and mouth area missing. (Fig. 11C: S), opening to exterior via paired gonopores Swimming legs 1-4 with well developed intercoxal located midventrally, posterior to postmaxillipedal sclerites, 2-segmented protopods and 1-segmented apódeme; genital muscles opening gonopores inserting rami. Coxae unarmed, bases with outer seta on legs medially on postmaxillipedal apódeme (Fig. 12D-E). 1-3. Rami with setal formula as in L. brescianii. All Elongate-pyriform spermatophores attached in clus elements plumose except for outer distal element of ters to genital apertures of Î (Fig. 12C); both full and exopod being pinnate along outer margin and inner discharged spermatophores present. distal element tripinnate; setae shorter than in L.

© 2002 The Linnean Society of London,Biological Journal of the Linnean Society, 2002, 75, 187-217 LIMPET BROODING AND NUCELLICOLID IDENTITY 207 brescianii. Outer margin of exopod denticulate, that has completely enclosed the copepodid, resulting in of endopod with long setules (Fig. 11E). the shedding of the old cuticle whilst maintaining a grip on the female with the maxillipeds. This explains Remarks why no trace of these appendages can be found in the Lamb et al. (1996) described the prosome of the late exuvium and why they are of exactly the same size in male copepodid stage as 5-segmented, comprising a both the infective copepodid and adult, despite their cephalosome and four free pedigerous somites. This large disparity in body size (Fig. 11C). As space within is based on a double observational error as the first the vesicle is limited, the male is almost immobilized pedigerous somite is in fact incorporated in the and no longer requires attachment devices. Conse cephalosome forming a céphalothorax (as in the early quently, the maxillipeds become redundant, which is copepodid; Lambet al., 1998), and the last two somites reflected in the atrophy of the basal and extrinsic mus are fused, forming a pedigerous double-somite. Exces culature (Fig. 12D). The genital apertures are located sive squashing of the céphalothorax could have caused close to the maxillipeds and are intimately associated the misinterpretation of the narrow posterior part as with the postmaxillipedal apodemes. Two pairs of the first pedigerous somite, whereas the dorsal surface muscles originate on the common median extension suture on the double-somite was mistakenly drawn of the apodemes, one inserting directly on the as a fully functional articulation separating the last lateral wings of the apodemes, and the other inserting two pedigerous somites. Additional oversights in on the body wall posterior to the genital apertures Lamb et al.’s (1996) illustrations include the accessory (Fig. 12D-E). These muscles (g.m.) are responsible for element on the antennary endopod, the outer basal opening the genital apertures and are presumably spine on legs 1-3, the dorsal seta on the caudal ramus, opposed by the elasticity of the thickened cuticle in the setal ornamentation on the swimming legs and this area. The maxillipeds and postmaxillipedal caudal rami, and the sensillar pattern, which was apodemes present in the infective copepodid are the incompletely illustrated for the pedigerous somites only juvenile exoskeletal elements transferred to the and not shown at all on the céphalothorax. The anten adult male during metamorphosis. This developmen nules are clearly 5-segmented instead of 4-segmented tal mechanism maintains grasping efficiency during and the proximal aesthetasc in the specimen exam the initial stages of attachment of the male and more ined is about twice as long as figured by Lambet al. importantly ensures continuity in the build-up of the (1996). genital apparatus. Our observations of the infective Lamb et al. (1996) described the late copepodid copepodid (Fig. 4A) and male (Figs 2B-C and 12F) of based on the exuvium enclosed along with the adult L. brescianii suggest a similar mechanism of incom male inside the vesicle (Fig. 11C). They emphasized plete moulting in all genera that have retained max the lack of maxillipeds in this copepodid and postu illipeds in the adult malesNucellicola, ( Lepetellicola, lated that their sudden appearance in the adult male Tesonesma and possiblyCocculinika). may have been the result of extreme developmental The similarity between the copepodids ofN. hol delay as found in some other poecilostomatoids such manae and L.brescianii is, to say the least, remark as the Ergasilidae. In a later report, Lambet al. (1998) able. Only a few differences can be observed, all of claimed that they had overlooked the maxillipeds in minor significance: (1) antennule setation pattern the copepodid exuvium because its presence had been (segment 2 with additional seta inNucellicola-, ante demonstrated in preceding stages, including therior element of segment 4 spiniform inLepetellicola) metanauplius and the early copepodid, as well as the (2) outer basal seta on leg 1 (absent inLepetellicola) infective copepodid that they obtained in culture. This (3) degree of fusion of pedigerous somites 3-4 (4) statement is clearly incorrect as the exuvium invari proportional length of urosomites (5) length and orna ably shows a longitudinal slit on the ventral surface mentation of swimming leg setae, and (6) terminal of the céphalothorax, coinciding with the position of setae on caudal rami (plumose inNucellicola). the maxillipeds (arrowed in Fig. 13A). The infective male copepodid attaches to the posterior end of the Familial distinctiveness of Nucellicolidae female, near the genital apertures, using its maxil The Nucellicolidae and Chitonophilidae are unique in lipeds. The strong muscles in the bases of the maxil having both sexes highly transformed. Although this lipeds (as found inL. brescianii) ensure that the similarity was noted by Lambet al. (1996), they dis copepodid is kept in position during formation of tinguished the latter from the former by the presence the vesicular wall around it. Although the origin of the of a highly developed rootlet system in females, and vesicle is unknown it is likely to be derived from an by the possession of paired antennae and a common extension of the female body wall. Metamorphosis into median genital aperture in males. These discrepan the adult male presumably initiates once the vesicle cies, in conjunction with the structure of the swim-

© 2002 The Linnean Society of London,Biological Journal of the Linnean Society, 2002, 75, 187-217 208 R. HUYS ET AL. ming legs in the late male copepodid stage, were con the presence of two claws plus one accessory element sidered as sufficient justification to attribute separate on the antenna and the caudal ramus setation pattern. family status to Nucellicola. Our observations have Concordance is found even in small details such as the shown that none of these differences is real and that integumental sensillar pattern and the specialized the resemblance between both families extends far condylar articulations between the céphalothorax and beyond the shared transformation of both sexes. A sug the leg 2-bearing somite. gested relationship between the Nucellicolidae and Finally, the adult males ofLepetellicola andNucel Mytilicolidae on the basis of mandibular loss (Lamb licola, despite differences in gross body morphology, et al. 1998) could not be corroborated. are remarkably similar in the configuration of the A branching rootlet system is clearly developed in reproductive system, the position of the genital aper N. holmanae (Fig. 12A-B) and we suspect that its tures, the possession of a pair of almost identical max reported absence in the original description is due to illipeds (with spinous process on the basis but without imperfect dissection of the parasite from the host’s syncoxa) and the presence of a characteristic post viscera and the egg-tube enclosing it. Examination of maxillipedal apódeme. These characters, the presence the anterior cephalic lobe in the paratypes ofN. hol of a rootlet system in the adult female in conjunction manae showed it to be incomplete, the irregularly with several lines of evidence based on copepodid mor shaped fracture plane coinciding with the bilateral phology decisively reject the claim that Nucellicola constriction between the swollen cephalic region and deserves distinct familial status. Its phylogenetic posi the much larger rootlet system (arrowed in Fig. 12B). tion within the Chitonophilidae is discussed below. The diagnostic significance of the presence of paired antennae in male chitonophilids is not to be taken as Phylogeny of Chitonophilidae absolute as this character has thus far only been The monophyly of the Chitonophilidae is supported reported in the males ofChitonophilus andLeptochi by the shared presence of a branching rootlet system tonicola (Avdeev & Sirenko 1991). Antennae are embedded in the host’s tissues, serving both as an entirely absent in the generaCookoides, Ischnochi absorptive system and attachment mechanism. The tonika, Tesonesma and Lepetellicola, all of which rootlet system transports nutrients to the digestive resemble Nucellicola in this feature. The males ofCoc tract in the ectosoma. Members of the highly trans culinika are as yet unknown. formed family Herpyllobiidae have a similar holdfast, Lamb et al.’s (1996) claim of a median genital aper the endosoma, containing the entire digestive system ture in male Chitonophilidae is based on a misinter of the copepod (Lützen, 1964, 1966). However, in this pretation of Avdeev & Sirenko’s (1991) illustrations of family, digestion takes place in the endosoma, indi C. laminosus and L. latus. In both species the male cating that the herpyllobiid holdfast is not homo genital system is paired and each vas deferens termi logous to the chitonophilid rootlet system. A close nates in a spermatophore sac with associated genital relationship between both families is also ruled out on aperture, as in Nucellicola. This configuration is also the basis of copepodid antennule morphology (see found inLepetellicola, Ischnochitonika, Tesonesma below). and possibly also Cookoides although Avdeev & Assessing the phylogenetic relationships within the Sirenko’s (1994) illustrations of the latter are not Chitonophilidae is difficult as the adults of both sexes conclusive in this respect. Lamb et al.'s (1996) ob are highly transformed and the more morphologically servational error is in all probability based on a informative developmental stages are not known for misinterpretation of the conspicuous midventral oral all the genera. Naupliar stages have been described disc illustrated by Avdeev & Sirenko (1991). for Ischnochitonika, Cookoides, Lepetellicola and The late copepodid stage provides overwhelming Nucellicola, while information on copepodids is only evidence for a close relationship betweenNucellicola available for the latter two genera. The present analy and the chitonophilid genera. Lambet al. (1996) had sis is based on nine naupliar and ten adult characters already emphasized the uniqueness of the swimming (Table 1) and their character states are summarized legs in N. holmanae, having elongate 1-segmented in matrix format in Table 2. All characters were set rami, and with exopodal setae arranged exclusively irreversible, which suppresses reversals at the along the distal and inner margins. The discovery of expense of introducing extra convergences and the only copepodid stage ofLepetellicola revealed a vir thereby increasing tree length. A ‘Branch and Bound tually identical swimming leg morphology, differing search’ was run, which guarantees finding all most from Nucellicola only in the absence of the outer basal parsimonious trees, and the ‘Acctran’ optimization seta on leg 1 and minor deviations in setal length andused. ornamentation. Copepodids of both genera also share Despite the inclusion of several underdetermined the same tagmosis (including the pedigerous double taxa, analysis of the data matrix of 19 characters somite), the form and segmentation of the antennules, (Tables 1,2) resulted in one fully resolved, most parsi

© 2002 The Linnean Society of London,Biological Journal of the Linnean Society, 2002, 75, 187-217 LIMPET BROODING AND NUCELLICOLID IDENTITY 209 monious tree (MPT) with a length of 21 steps, consis affected the size of the antennary projections in this tency index 0.905 and retention index 0.931 (Fig. 14). genus (Nagasawa et al., 1991). Omission of naupliar characters 1-9 from the data The monophyletic group in apposition to the matrix also resulted in one MPT with identical topol Chitonophilus lineage shows a basal split, with a ogy but obviously with a shorter tree-length of 12 steps mesoparasitic clade, includingCocculinika andLep- and consistency index 0.833. The topology supports a basal dichotomy dividing the family into a mesopara sitic clade utilizing only polypi acophoran hosts Table 1. Morphological characters used in the phyloge (■Chitonophilus, Ischnochitonika, Cookoides andLepto netic analysis. Apomorphic states are referred to in square chitonicola) and a clade grouping genera associated brackets with cocculiniform limpets (Cocculinika, Lepetelli 1 Antennule nauplius 2-segmented [1-segmented] cola), chitons (Tesonesma) and prosobranch gastropods 2 Antennary endopod nauplius 2-segmented (Nucellicola). [1-segmented] The Chitonophilus clade is supported by two 3 Antennary endopod nauplius with lateral seta synapomorphies (17, 19), both relating to the mor [absent] phology of the adult male. Males of Chitonophilus, 4 Antennary exopod nauplius 5-segmented Leptochitonicola andIschnochitonika all have a raised [3-segmented] mouth eone characterized by a strongly sclerotized 5 Antennary exopod nauplius with 4 lateral setae [2 ring circumscribing the mouth opening. Nagasawa lateral setae] et al. (1991) showed that this eone has both an ante 6 Mandibular endopod nauplius 2-segmented rior and a posterior opening. Avdeev& Sirenko (1994) [1-segmented] were unable to observe the mouth opening in the 7 Mandibular endopod nauplius with lateral seta strongly reduced male ofCookoides cordatus, possibly [absent] resulting from the strong dorsoventral flexure of 8 Mandibular exopod nauplius 4-segmented the anterior part of the body. In males of the [3-segmented] Chitonophilus clade the spermatophore sacs and asso 9 Mandibular exopod nauplius with 3 lateral setae ciated genital apertures assume a dorsal position. This [2 lateral setae] condition is considered as apomorphic due to sec 10 Females mesoparasitic, with egg-sacs or masses ondary displacement as in most copepods the male [endoparasitic, without egg-sacs] genital apertures are located ventrally (Huys & 11 Adult Í without lateral lobes [present] Boxshall, 1991). This ancestral position is retained 12 Adult Í without posterior lobes [present; incipient in Nucellicola, Lepetellicola and Tesonesma-, it is in Cocculinika] 13 Genital apertures of Í lateral [dorsal] unknown inCocculinika as the male has yet to be dis 14 Céphalothorax S without distinct lobes [trilobate] covered. The genus Chitonophilus is identified as the 15 Antenna present in adultS [absent] sistergroup of a 3-taxon clade, which is characterized 16 Adult S without paired antennary projections by the possession of paired antennary projections [present] in the male (character 16). These projections are well 17 Adult S without sclerotized mouth-ring [present] developed inLeptochitonicola andIschnochitonika but 18 Maxilliped absent in adultS [present] are secondarily reduced inCookoides due to the loss of 19 Spermatophore sac and genital apertures ofS the antennary claws. The loss of these claws (charac located ventrally [dorsally displaced] ter 15) is shared with Ischnochitonika but has yet not

Table 2. Character data matrix [0 = ancestral (plesiomorphic) state, 1 = derived (apomorphic) state, ? = missing data]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Chitonophilus ? ? ? ? ? ? ? ? ? 0 0 0 0 0 0 0 1 0 1 Cocculinika ? ? ? ? ? ? ? ? ? 0 1 1 1 ? ? ? ? ? ? Cookoides 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 ? 0 ? Ischnochitonika 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 1 0 1 Lepetellicola 1 1 1 1 1 1 1 1 1 0 1 1 1 0 1 0 0 1 0 Leptochitonicola ? ? ? ? ? ? ? ? ? 0 0 0 0 0 0 1 1 0 1 Nucellicola 1 1 1 1 1 1 1 1 1 1 ? 0 ? 1 1 0 0 1 0 Tesonesma ? ? ? ? ? ? ? ? ? 1 1 0 ? 1 1 0 0 1 0

Chitonophilus

17, 19 Leptochitonicola Polyplacophora Cookoides

Ischnochitonika

Cocculinika 12-13 Cocculiniformia5 Lepetellicola 1-9, 11, 15, 18

Tesonesma Polyplacophora T.L. = 21 10, 14 C.I. = 0.905 R.I. = 0.931 Nucellicola Prosobranchia

Figure 14. Phylogenetic tree depicting relationships between genera of the Chitonophilidae. Host groups indicated in bold. For explanation of apomorphic states see Tables 1,2 and text.

etellicola, and an endoparasitic clade, encompassing(or their absences) are mutually exclusive in Tesonesma andNucellicola. It is supported by a suite the two clades. Such a disjunct distribution of major of nine naupliar characters, the loss of the antennae character states (presence/absence) simultaneously in the adult male and the presence of lateral lobes in expressed in two primary appendages is extremely the adult female. Hypermorphosis along the antero unusual for a taxon that is highly advanced in all posterior axis inNucellicola prevented us from scoring other respects, particularly when they determine the the latter character confidently and consequently we basal dichotomy of the group. This is a typical example have treated it as a missing entry (Table 2: character demonstrating that what appear to be reversals in 11). An additional apomorphy of major importance is copepod evolution should not be taken unquestionably the presence of maxillipeds in the malesoí Nucellicola, at face value. It is noteworthy that the postmaxil Lepetellicola andTesonesma (the male is unknown in lipedal apódeme is only present in those genera that Cocculinika). These appendages were misinterpreted have retained ventral genital apertures, showing once as the antennae by Avdeev & Sirenko (1994) in their again the close functional correlation between both description ofT. reniformis, presumably because they structures. had orientated the male incorrectly, the ventral view The relationship between Cocculinika andLepetel being the dorsal one, and vice versa. The maxillipeds licola is supported by two female characters, the are not acquired as a result of normal moulting but dorsal displacement of the genital apertures and the represent structures retained from the previous devel formation of paired posterior lobes. These lobes are opmental stage (see above). It is postulated here that incipient inCocculinika (Jones & Marshall, 1986) and all chitonophilid adults (both sexes) typically acquire strikingly resemble the condition found in young their highly transformed body shape through extreme females of Lepetellicola (Fig. 1G). metamorphosis at the final moult, leaving no trace of A key innovation in the evolution of the Chitonophil similarity with the preceding cyclopiform copepodid idae is the adoption of a completely endoparasitic life and resulting in the loss of virtually all appendages. style by the female and the associated loss of egg-sac This is a common phenomenon observed in many par development. This transition conceivably permitted asitic copepods that are intimately associated with the major adaptive shift from deepwater hosts to those their hosts. The absence of maxillipeds in males of living in the upper and middle eulittoral zones of rocky the Chitonophilus lineage is therefore regarded as the shores such as the predatory gastropodNucella lapil plesiomorpic state (Tables 1,2: character 18). Their lus. The completion of the naupliar phase inside the presence in the other genera is not the result of hete host allows for the offspring to be released at an rochrony but simply reflects incomplete moulting advanced state of development (metanauplius), possi and is interpreted as the apomorphic state. This ex bly in a controlled manner depending on whether tidal plains why the presence of antennae and maxillipedsconditions are favourable for dispersal. The position of

Nucellicola females inside the dogwhelk is typically may be modified vetigastropods whereas the Cocculi near the tip of the visceral whorl, but occasionally they noidea (utilized byCocculinika) are possibly of neriti- can be found elsewhere in the host nearer to their morph affinities. point of entry in the mantle (Lambet al., 1996). Each female is contained within a highly convoluted tube that extends through the host viscera to the presumed DISCUSSION point of entry. The tube is filled with eggs, nauplii and metanauplii, the latter being closest to the opening Brooding in cocculiniform limpets? through which they are released to the exterior. Brooding in Lepetellidae was first reported by Warén Avdeev & Sirenko (1994) emphasized that Tesonesma (1972) in Lepetella laterocompressa (De Rayneval & reniformis, unlike other chitonophilids, occupies the Ponzi, 1854) from the Swedish west coast. Warén body cavity of its chiton host. They observed large observed in the right part of the m antle cavity a heart- numbers of eggs at various stages of development as shaped egg cluster of about 25 eggs at different stages well as nauplii, lying freely in a semitransparent cyst of development, attached by a narrow string where surrounding the female. The cyst wall is of a complex the anus should be situated. This report, undoub nature, consisting of a ramifying system of membra tedly being based on an infection by chitonophilid nous partitions that separate the linear, loosely copepods, has been persistently cited in the literature arranged egg-clusters. It is penetrated by the long, as evidence for brood protection in lepetelloidean Coc simplified rootlet system of the parasite and presum culiniformia (e.g. Haszprunar, 1987a, 1988b, 1998; ably has a second opening leading to the exterior as in Haszprunar & McLean, 1996). Subsequent records of N. holmanae. It is highly conceivable that the cyst of eggs in the mantle cavity of other families of the Lep Tesonesma and the egg-tube ofNucellicola are homo etelloidea fed the conjecture that brooding was wide logous structures. Similarity is also noted in the spread in the Cocculiniformia (e.g. Haszprunar distinctly coiled paired ovaries and oviducts T.of reni 1988b). For example, Moskalev (1978) reported about formis that are reminiscent of the highly convoluted 100 eggs of irregular triangular shape (about reproductive system of N. holmanae (Lamb et al. 56 X 140 pm), situated under the mantle ofBathy 1996). Although Avdeev & Sirenko (1994) did not give phytophilus caribaeus Moskalev, 1978 (Bathyphyto any information on the exact position of the sexes philidae) from the abyssal Caribbean. A similar case relative to each other, the resemblance in male mor of presumed brooding was recently reported in the phology provides additional evidence in support congenerB. diegensis Haszprunar & McLean, 1996 of a Nucellicola-Tesonesma sistergroup relationship. from the San Diego Trough. Haszprunar & McLean Males of both genera differ essentially in the degree of (1996) photograph of a brooding limpet shows eggs at development of the lateral lobes and the overall shape the left side of the mantle roof and subpallial cavity, of the posterior trunk. Haszprunar (1987a) briefly near the head region. Haszprunar (1988a) reported described (but did not figure) an endoparasite ofthe presence of many ripe eggs in the palliai and sub unknown affinities, living in the haemocoel of the coc- pallial cavities of two specimens of Notocrater ponderi culinid Coccopigya hispida and maintaining contact Marshall, 1986 that he regarded as clear evidence for with the exterior via a duct opening at the pedal sole, brood protection in the family Pseudococculinidae. In however, it is unclear whether this animal is a the second superfamily of the Cocculiniformia, the chitonophilid. Cocculinoidea, supposed brooding has been observed The outgroup of the Chitonophilidae and hence its by Haszprunar (1987a) who found ripe eggs in the host affiliation are as yet unknown. However, as no palliai cavity of a single specimen of Coccopigya other copepods have been recorded from chitons thus hispida. far, we postulate that the Polyplacophora is the ances D antart & Luque (1994) pointed out that many spec tral host group of the Chitonophilidae. This implies imens of L. sierrai, collected in the Bay of Biscay, had that host switching has occurred on two occasions, the palliai cavity filled with a mass of up to 70 white once in the ancestor of theCocculinika-Lepetellicola spherical eggs, measuring about 100 pm in diameter. clade (cocculiniforms) and once in theNucellicola In accordance with previous reports of brooding in coc lineage (prosobranchs) (Fig. 14). It is interesting that culiniform limpets they regarded these as developing both cocculiniform parasites cluster together as the embryos of the gastropod. However, during a recent proposed monophyly of the host group (Haszprunar study of the Lepetellidae from the Iberian-Moroccan 1988b) has been the subject of recent debate. Ponder Gulf and North Spain it was concluded that these & Lindberg’s (1996, 1997) phylogenetic analyses indi ‘embryos’, typically found as a cluster dorsal of the cated that the Cocculiniformia is polyphyletic, cephalic tentacles (Dantart & Luque, 1994: fig. 52; comprising two divergent superfamilies of deepwater fig. 6A herein), are in reality the egg-masses of limpets. The Lepetelloidea (utilized byLepetellicola) mesoparasitic copepods. Warén (pers. comm.) con

© 2002 The Linnean Society of London,Biological Journal of the Linnean Society, 2002, 75, 187-217 212 R. HUYS ETAL. firmed the presence of similar ovigerous parasites though more than 500 individuals of the Atlantic- in the palliai cavity of Caymanabyssia vandoverae Mediterranean species A. excentrica have been McLean, 1991, collected from sunken driftwood at a examined until now. The scanty data available for the depth of 2750m off the coast of Oregon (44°45'47'-N, Pseudococculinidae and Bathyphytophilidae are based 125°31'14'-W). on very few specimens and leave considerable scope The published records for both superfamilies of the for re-interpretation, whereas the single specimen Cocculiniformia made H aszprunar (1988b, 1998) pos record for the Cocculinidae (Haszprunar, 1987a) is tulate that brood protection was a common phenome almost certainly based on an ovigerous female of non and possibly typical for the whole group, but all C. myzorama. Hence, the function of the supposed these records should now be re-interpreted in the light ‘brood pouch’ formed by the hypobranchial gland in of the present results. The position ofL. brescianii on the Cocculinidae (Haszprunar, 1998) requires recon L. sierrai (Figs 6A and 7A) is similar to that described sideration. In conclusion, there is at present no sound for Cocculinika myzorama by Jones & Marshall (1986) evidence for brooding in cocculiniform limpets. who found a single ovigerous female attached above and behind the head, near the anus of each infested specimen of the cocculinid hostCoccopigya hispida. H eterochrony in C hitonophilidae The latter is the only known host ofC. myzorama and The development inNucellicola holmanae and other it is therefore highly conceivable that Haszprunar’s chitonophilids is a remarkable example of dissociated (1987a) report of brooding in this species is based on heterochrony, whereby different appendages or growth an observational error. The similar position of the ‘ripe fields can undergo different heterochronic changes. In eggs’ described inNotocrater ponderi by Haszprunar the male developmental sequence for example (1988a) and inLepetella laterocompressa by Warén (Fig. 15) predisplacement and postdisplacement are (1972) leave little doubt that they were dealing combined in a single stage. Lambet al. (1998) identi with gastropods infested by chitonophilid copepods. fied the early copepodid ofN. holmanae as the equiv Although Haszprunar & McLean’s (1996) illustration alent of copepodid II observed in copepods passing of Bathyphytophilus diegensis lacks sufficient focus to through the complete series of six postnaupliar stages. be conclusive, the position and size of the egg-masses This identification was based on body segmentation, raise the strong suspicion that they also belong to a comprising a céphalothorax and five free somites, chitonophilid. which serves as a useful reference background against Haszprunar (1988b, 1998) assumed th at the which the timing of appearance of the various reduced ciliated gili leaflets displayed by many appendages can be assessed. The mandible and anten- families of the Lepetelloidea are no longer primarily nary exopod were shown by Lambet al. (1998) as respiratory in function but are instead involved in shrivelled appendages. The illustration of the former creating water currents in the palliai cavity, supply is not convincing, however, the rudimentary anten- ing the brooded eggs with fresh, oxygenated water. A nary exopod is clearly reminiscent of the condition similar inference of brood protection based on ana found in copepodid I of many Cyclopoida (e.g. Dudley, tomical features was made specifically for the Lepet 1966) and Poecilostomatoida (e.g. Izawa, 1986). The ellidae. Haszprunar (1988b) stated that this family is persistence of this vestige in copepodid II ofN. hol exceptional in possessing a deeper mantle cavity that manae is the result of postdisplacement, i.e. the late serves as a ‘brood pouch’ for the lecithotrophic eggs, initiation of the reduction of the antennary exopod. On and he considered this a synapomorphy for the Lep the contrary, the presence of the fourth pair of swim etellidae. However, detailed examination of more than ming legs in this stage contradicts the generalized 500 specimens ofL. sierrai and otherLepetella species pattern of leg development proposed by Ferrari (1988). from different localities and depths in the Atlantic As this condition is typical for copepodid III, the early failed to reveal any evidence of brooding. These ob onset of the development of this character is due to servations have demonstrated unequivocally that at predisplacement. Lamb et al. (1998) attributed the least in the Lepetellidae the reported phenomenon of mosaic morphology of the copepodid II to the combi brooding is fallacious. Conversely, members of the nation of retarded (antenna, mandible) and acceler lepetelloidean family Addisoniidae possess a well deated development (leg 4). This is somewhat misleading veloped gili, and the development of the nonrespira- as these heterochronic changes are rate concepts, com tory epithelial zones of the gili, for which Haszprunar monly known as neoteny (slower rate) and accelera (1987b) suggested a possible function in protecting tion (increased rate), and describe perturbations in the eggs or embryos, is significantly related to the grade rate of developmental events. Predisplacement and of gonad maturity (Roldán& Luque, 1999). Neverthe postdisplacement are time concepts describing the rel less, no specimens of any species ofAddisonia Dali, ative timing of onset (initiation) and offset (cessation) 1882 have been found to brood eggs thus far even of ontogenetic processes as expressed, for example, in

naupliar copepodid adult d"

i lí 1 lí || iii 1

1 : 1 1 Mxp 2-scgmcntcd ☆ 1 1

1...... j . ± ] 1 Legs 1-2 1 -segmented ik

Hfl 1 A 2 cxp/Md vestigial ik HI i | BHH Leg 4 ★ 1

Somatic 1 1 growth ik 1

Figure 15. Heterochronic events in development of maleNucellicola holmanae (indicated by asterisk) compared with normal development. Arrows indicate predisplacement, postdisplacement or hypermorphosis.

the early copepodid ofN. holmanae. The metanauplius They also placed the Nucellicolidae in this order on the of the latter is a unique case of predisplacement, basis of the structure of the antenna in the copepodid demonstrated by the presence of a 2-segmented stage, and the presence of maxillipeds in males com maxilliped and two pairs of 1-segmented swimming bined with their apparent absence in females. One of legs, both of which are characters normally expressed the antennary characters used by Lambet al. was the at copepodid I (Fig. 15). Lamb et al. (1998) regarded alleged presence of a coxo-basis, which is a diagnostic this as an adaptation to enhance the locomotory autapomorphy of the Poecilostomatoida (Huys & capability of the metanauplius. Boxshall, 1991). This claim is however, contradicted by The abbreviation of the life cycle in Nucellicola is their own illustration (Lambet al., 1996: Fig. 2C and extreme, comprising only two naupliar and two cope our observations (Fig. 13A-B), showing that there is podid stages plus the adult. There is virtually no size no evidence for such compound segment and that the increase between the nauplius and the late male cope coxa is discrete from the basis as in Lepetellicola podid, suggesting a rapid moulting sequence. The (Figs 4C, 9A and 11A). This antennary segmentation simultaneous presence of eggs, first nauplii and late pattern precludes placement of the Chitonophilidae in copepodids still maintaining close contact to the adult the Poecilostomatoida. female in L. brescianii (Fig. 1C) suggests that rapid The segmentation pattern of the male copepodid moulting may also be common in other Chitonophili antennules is identical Nucellicolain andLepetellicola dae. The adults of both sexes ofN. holmanae not only (Figs 5A, 9C and 13B). Lamb et al. (1996) erroneously undergo extreme transformation at the final moult but described the antennule as 4-segmented, discounting also gross size increase as a result of hypermorphosis. the small third segment as a true segment. However, The extent of hypermorphosis expressed in the adult this segment is crucial as it permits unequivocal male is demonstrated by comparison with the exuvium homologization of the four segment boundaries size of the late copepodid (III) (Fig. 11C). In the adult (Fig. 16). The presence of the posterior seta on this female the effect is an order of magnitude larger and segment unambiguously identifies it as ancestral is correlated with extreme elongation along the segment XXIV as no other posterior setae are found antero-posterior axis as a result of allometric growth. on more proximally located segments. The fourth segment, carrying a posterior seta and an anterodistal seta with associated aesthetasc, is identified as O r d i n a l p o s i t i o n o f C hitonophilidae segment XXV. The distal compound segment corre Ordinal placement of the Chitonophilidae was not sponds to fused ancestral segments XXVI-XXVIII. discussed by Avdeev & Sirenko (1991, 1994) but Proximal to segment XXIV is another compound Lamb et al. (1996) ranked the family among the segment bearing a large aesthetasc and threeLep ( Poecilostomatoida without giving any explicit reason. etellicola) or four setae (Nucellicola). The aesthetasc

Asterocheres Clausidiidae Erebonasteridae Archinotodelphys profundus Nucellicola adult copepodid III adult copepodid III last copepodid (= III)

SIPHONOSTOMATOIDA POECILOSTOMATOIDA CYCLOPOIDA

Figure 16. Segmentation and setation pattern of antennule part distal to XX-XXI articulation (equivalent to neocopepo- dan geniculation) for various taxa. Based on personal observations{Archinotodelphys profundus, Nucellicola) or data derived from Boxshall & Huys (1998) CAsterocheres), Kim & Ho (1992) (.Hemicyclops) and Huys & Boxshall (1990) (Erebonasteridae). See text for explanation.

originates from ancestral segment XXI, inferring that cilostomatoida is expressed as a distinct segment, the second segment represents the fused ancestral carrying two setae and an aesthetasc. Although this segments XXI—XXIII. The boundary between the first signature can be regarded as an ordinal character two segments is homologous to the articulation istic, it is by no means exclusively diagnostic as many separating ancestral segments XX and XXI, indi cyclopoids, both free-living (e.g. Gurney, 1933) and cating that no separation has occurred proximal to associated (e.g. Dudley, 1966), show a similar pattern this boundary (i.e. segments I-XX). This articulation (Fig. 16). Consequently, it does not provide conclusive is highly conserved, both in ontogeny and phylogeny, evidence justifying placement of the Chitonophilidae and is already expressed at copepodid I in most copein the Poecilostomatoida, however, it unequivocally pods. Examination of a range of calanoid developmen excludes the family from the Siphonostomatoida in tal stages showed that it is the first articulation to be which the double segment XXIV-XXV remains undi expressed in ontogeny, often from nauplius I onwards vided (Fig. 16) or is part of a larger compound segment (Huys & Krsinic, unpublished data). incorporating additional proximal and/or distal seg The male copepodid antennule in Chitonophilidae ments (Boxshall & Huys, 1998). Particularly relevant reveals three characters of particular interest: seg is segment XXIV, which is distinct in the Chitono mentation, aesthetasc pattern and armature on the philidae (arrowed in Fig. 9C). Within the Poecilo compound segment XXI-XXIII. stomatoida, this segment is only expressed in the Boxshall & Huys (1998) pointed out that the penul Erebonasteridae (e.g. Martinez Arbizu, 1996/97; Huys timate segment (XXV) of virtually every copepodid & Boxshall, 1990), that is generally accepted as the stage of almost every described member of the Poe most primitive lineage in the order (Huys & Boxshall,

1991). The presence of this feature in the highly mod atoida, an ordinal position already refuted on the ified Chitonophilidae would imply a basal position in basis of antennary segmentation. Placement in the the poecilostomatoid phylogenetic tree for this family, Cyclopoida is supported by antennulary segmentation a scenario that is not, however, reinforced by addi and aesthetasc pattern, both of which preclude assign tional morphological evidence. ment to the Siphonostomatoida. This position is, Copepodid stages of poecilostomatoids typically however, admittedly based on the principle of elimi have three aesthetascs on the distal part of the anten- nation rather than on common ancestry. The ple- nule. These aesthetascs originate from ancestral seg siomorphic condition of the three antennulary ments XXI, XXV and XXVIII (Boxshall & Huys, 1998) characters provides further evidence for the paraphyly and are found on the last three segments in the major of the order Cyclopoida, a status already indicated by ity of poecilostomatoids. In the Erebonasteridae and the recent discovery of the Fratiidae. Hoet al. (1998) Chitonophilidae, four segments are expressed distal to placed this family in the Cyclopoida but its mosaic the XX-XXI articulation but the origin and number of morphology, combining gnathostome and poecilostome aesthetascs on them is similar. The poecilostomatoid features, clearly reflects the phylogenetic continuum aesthetasc pattern is highly conservative during post- between both orders. naupliar development, being expressed from copepo The Chitonophilidae is the third family in the did I onwards, and differs from the siphonostomatoidCyclopoida utilizing molluscan hosts. The three arrangement by the presence of an aesthetasc on genera of the Mantridae are all associated with segment XXV. This dissimilarity demonstrates that bivalves of the families Chamidae and Mytilidae the current placement in the Siphonostomatoida (Huys, 1990; Ohtsuka et al., 2000). The only known (Bowman & Abele, 1982; Huys & Boxshall, 1991) representative of the Ozmanidae inhabits the hae- of some highly transformed families such as the Her mocoel of the freshwater mesogastropod snail pyllobiidae is unjustified as the infective copepodid Pomacea maculata Perry, 1810 in the Brazilian stage exhibits the poecilostomatoid aesthetasc pattern. Amazon (Ho & Thatcher, 1989). However, it does not unequivocally substantiate assignment of the Herpyllobiidae and Chitonophilidae to the Poecilostomatoida as many cyclopoids, such as ACKNOWLEDGEMENTS members of the Fratiidae (Hoet al., 1998) and Notodel phyidae (Dudley, 1966), display the same pattern. We thank Dr Anders Warén (Naturhistoriska In poecilostomatoids, the anterior seta on segment Riksmuseet Stockholm) for arranging the loan of the XXIV appears at copepodid II but those on segments BALGIM material and for additional information on XXI and XXII never appear due to the early offset of Lepetella. Dr Bruce A. Marshall (National Museum of setal development on these segments (local progene New Zealand) is acknowledged for the initial sugges sis). This results in the presence of only three anterior tion about the copepod. This work was supported by a and one posterior setae on the compound segment research grant of the Dirección General de Investi XXI'-XXIV in most families and only two anterior gación Científica y Técnica within the framework of setae on the compound segment XXI-XXIII in the the Fauna Ibérica II project (PB-89-0081), and a F.P.I. Erebonasteridae (Fig. 16). The report of six setae on Fellowship from the Comunidad Autónoma de Madrid antennulary segment 5 (= XXI-XXIV) inClausidium to E. Roldán. PJL-G acknowledges financial support vancouverense (Haddon, 1912) by Huys& Boxshall for a visit to the Natural History Museum (London) (1991) is based on an observational error, the tiny seta under the EC-funded TMR Bioresource Large-Scale being a reduced aesthetasc (originating from XXI) and Facility Programme. We also acknowledge the assis the long seta arising from a socle near the proximal tance of Esperanza Salvador, Luis Ángel Garrido and articulation being the anterodistal seta of segment José Luis Poderoso (SIDI, U.A.M) in preparing the XX. In Nucellicola (Fig. 13B) the second compound SEM micrographs, and Zara Frenkiel (NHM) for help segment possesses the full complement of four setae in translating Russian literature. and one aesthetasc, representing the ancestral setation of segments XXI (two anterior setae + aesthetasc), XXII and XXIII (each with one anterior seta). Either REFERENCES the anteroproximal seta on segment XXI or the ante Abdelhalim AI, Lewis JW, Boxshall GA. 1993. The exter rior seta on segment XXII failed to develop in the cope nal morphology of adult female ergasilid copepods (Cope podid ofLepetellicola (Fig. 5A) as only three elements poda: Poecilostomatoida): a comparison betweenErgasilus plus an aesthetasc are present on segment 2. The andNeoergasilus. Systematic Parasitology 24:45-52. retention of the full setation on these segments in Amado MA, Pinto da Motta, Ho J-S, da Rocha CEF. 1995. 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