Nudibranch molluscs of the genus Corambe differ from most other Doridoidea by having ventral rather than dorsal anus and gills. Because of these and other features, such as separate cerebral and pleural ganglia, corambids have been considered as an archaic or enigmatic group. The first tropical eastern Pacific Corambe species is described in morphological and some histological detail. Selected organs such as circulatory and central nervous features are reconstructed from serial semithin histological slides and visualized in three dimensions using Amira software. Anatomical findings include two separate ganglia on the visceral loop and an additional ganglion on the right side of the body that is connected to the pedal ganglion. Corambe mancorensis n. sp. is dorsoventrally depressed, has an oval, fleshy notum covered with a cuticle, and has a wide posterior medial notch that can be closed completely by unique lobules. Gills are arranged in an unusual horseshoe-like manner including both phanerobranch anal (=medial) gills and corambid lateroventral gill rows, and are connected to the atrium by a complex vessel system. The three medial gills arise from a posterodorsal gill cavity within the notal notch, similar to the case in Corambe evelinae Marcus, 1958. By scanning electron microscopy a vestigial gill cavity is also detectable in C. pacifica MacFarland & O'Donoghue, 1929, but here it is situated ventrally. Our new information on adult corambids is compared with new and published ontogenetic data on phanerobranch and cryptobranch dorids, to contribute to a novel interpretation of the ontogeny of dorid mantle and gill complexes. The progenetic evolution of corambids ‘recapitulates’ early juvenile dorid stages – turning Haeckel's Law upside down.
Corambid dorid nudibranchs have long been regarded as aberrant (Bergh, 1871, 1892; Fischer, 1891; Odhner in Franc, 1968). Unusual features include a depressed body shape, a fleshy notum that is covered with a shedding cuticle in most members, separate cerebral and pleural ganglia, and a peripherally lobed digestive gland that is separated by paired dorsoventral muscle bundles (Schrödl & Wägele, 2001). Differing from lower diversity estimations by Edmunds (2007), the latest analysis of corambid phylogeny recognizes 10 valid species, including the genera Loy (3 species) and Corambe (7 species), plus 4 further, still undescribed Corambe species (Martynov & Schrödl, in press). According to that study, members of Loy are characterized by inhabiting soft bottoms and having an oral veil that is partly fused with the hyponotum, while most features thought to be characteristic for corambids actually are apomorphic for Corambe species, such as serial ventral gills, a cuticle covering the notum and pairs of dorsoventral muscle bundles separating the digestive gland into peripheral lobes.
All corambids show a typical suctorian buccal apparatus and radula; this led Millen & Nybakken (1991) to conclude that the corambids are a basal offshoot of suctorian phanerobranch dorids. However, the ventral position of the anus and gills of all corambids known at that time was intriguing and, as an apparently ancestral condition, led to classification of the group as an order Corambida at the base of the dorids (Baranets & Minichev, 1994). Martynov (1994a) was the first to describe two northern Pacific deep-water corambid-like species with dorsal or subventral anus and gills, thus bridging the gap to ‘normal’ dorids with dorsal anus and circumanal gills. An evolutionary scenario of derivation of corambids from suctorial Onchidorididae was proposed by Martynov (1994b, 1995) and confirmed by a recent cladistic approach (Martynov & Schrödl, in press); this explained the ventral shift of the anus and gills by paedomorphosis, which could also account for the comparatively low level of cephalization in corambids. The nervous system of corambids has, however, not yet been studied in adequate histological detail. Even the presence, position and homology of the heterogeneous corambid gills have not been clarified so far. Loy meyeniMartynov, 1994a, b shows three nonretractile dorsal gills (and anus) in a posteromedial notal cavity, thus closely resembling the usual phanerobranch dorid condition. These three medial gills lie in a subventral position in Loy millenae (Martynov, 1994a, b) (as Proloy) and ventrally, apparently without any cavity, in Loy thompsoni (Millen & Nybakken, 1991) (as Corambe). All other corambids have ventrolateral rows or pairs of gills. Having special gill glands and a similar gill vessel system, Schrödl & Wägele (2001) showed that ventrolateral gills in Corambe lucea Marcus, 1959 correspond to primary dorid gills, in contrast to the secondary, lamellate lateral gills present in Phyllidiidae. The left and right lateroventral gill rows of C. lucea and several other corambids are, however, neither connected with each other nor in close association with the anus as is usual in other dorids. In contrast, a continuous row of gills around the rear part of the animal is present in Corambe pacifica MacFarland & O'Donoghue, 1929. In the rather poorly known Brazilian C. evelinae Marcus, 1958 a special condition has been described: in addition to the lateral gill rows there are 2–3 medial gills situated in a more dorsal position, obviously close to the anus and within a kind of notal cavity (Marcus, 1958: pl. 7, figs 51, 54). The significance of this observation has not so far been explored.
The present paper gives detailed anatomical information on a new Corambe species from northern Peru, which is the first corambid record from the tropical eastern Pacific. The histology of selected organs is described; the nervous system is reconstructed from serial semithin slides and visualized three-dimensionally. The unique gill arrangement is compared with that of other corambids; it is interpreted as showing a continuous assemblage of both phanerobranch medial gills in a vestigial cavity and multiplied corambid gills in lateroventral rows. New observations on early phanerobranch and cryptobranch juveniles lead to a novel hypothesis on the ontogeny of the dorid notum, anus and gills, and a comparison with the progenetic evolution of these organs in corambids (Martynov & Schrödl, in press).
MATERIAL AND METHODS
More than 20 specimens of Corambe mancorensis n. sp. were collected at the pier of the fishery harbour of Mancora, Tumbes province, northern Peru by two of us (Y.H. and M.S.). Specimens were found on macroalgae (Padina durvillea, Spatoglossum cf. congestum) that were covered with encrusting bryozoans (Membranipora), at 0–3 m depth. Specimens were observed alive and photographed. Seven specimens were fixed in 96% ethanol and further specimens were fixed in 70% ethanol; the latter were used for dissections and SEM of body surfaces (after critical-point drying) and radulae. For histological study eight specimens were relaxed in isotonic MgCl2 solution and then fixed in 4% glutaraldehyde buffered in 0.2 M sodium cacodylate (0.1 M NaCl and 0.35 M sucrose, pH 7.2). These specimens were rinsed several times in the buffer solution and postfixed in buffered 1% OsO4 for 1.5 h. The specimens were dehydrated through a graded acetone series and finally embedded in Spurr's low-viscosity epoxy resin (Spurr, 1969) for semithin sectioning. Following Henry (1977) and Ruthensteiner (2008), glass knives were used to prepare two complete ribbons of serial cross-sections (1.5 µm) with a microtome (Microm HM 360, Zeiss, Thornwood, NY, USA). Sections were stained with methylene blue-azure II (Richardson, Jarett & Finke, 1960). One individual (the holotype) was used for three-dimensional reconstruction of selected organ systems using the software AMIRA 3.0 (TGS Template Graphics Software) as described, for example, by Neusser et al. (2006) and discussed by DaCosta et al. (2007).
To study the ontogeny of the direct developer Cadlina laevis, specimens and egg masses were taken from the vicinity of the Marine Biological Station of Moscow State University, Kandalaksha Bay, White Sea. In vitro, egg and juvenile development was observed daily, until juveniles reached 1 mm length. At relevant ontogenetic stages, i.e. whenever new features of body shape, anus position or structures related to notal lobes, gills or gill cavities appeared, specimens were fixed for light microscopy and SEM (CamScan; Leo II), the latter after critical-point drying (ZMMU Op-23, ZMMU Op-26). Preserved early juvenile specimens of other dorid species were available for SEM from Corsica, Mediterranean Sea (Paradoris indecora, ZSM 20011861; Doris ocelligera, ZSM 20012397; Onchidoris neapolitana, ZSM 20012382). Adult corambids used for light microscopy and SEM were obtained from Sevastopol, Black Sea (Corambe obscura, ZMMU Op-7), British Columbia (Corambe pacifica, ZMMU Op-14; C. steinbergae, ZMMU Op-31) and Peter the Great Bay, Japan Sea (Loy meyeni, holotype ZMMU Lc-25699).
Abbreviations: ZMMU, Zoological Museum Moscow State University; ZSM, Zoologische Staatssammlung München.
Family Onchidorididae Gray, 1827
Genus Corambe Bergh, 1869
Corambe mancorensis new species
Holotype: ZSM 20080543 (series of histological semithin sections; Bavarian State Collection of Zoology), pier of fishery harbour of Mancora, Tumbes Province, northern Peru (4°6′36″S, 81°4′2″W), 0–3 m depth (Hooker & Schrödl col. 02/xii/2006). Paratypes: Seven specimens, ZSM 20080536–42 (resin blocks); 6 specimens, ZSM 20091918–23 (entire), collected with holotype. Additional material: Several specimens, used for dissection and SEM; collected with holotype.
Named after the type locality Mancora.
Length of holotype 3 mm; in preserved condition and embedded for semithin sectioning 2.6 mm. Length of eight measured living specimens 1.3–3.2 mm, width 1–3.2 mm. Consistency of living animals soft, i.e. no spicules stiffening the body. Body dorsoventrally depressed. Notum broad, almost circular, posteriorly with two broad, slightly asymmetrical lobes forming a deep notch in the middle (Fig. 1A, B). Anterior and anterolateral edge of notch of notal lobes bears 3 broad, triangular, leaf-like, inwardly directed lobules (one specimen showed only 2 lobules) (Fig. 2A, D), each protecting one of the 3 dorso-terminal gills. Living specimens capable of opening notal cavity widely by spreading the lobes and bending their edges and lobules upwards, revealing the gills (Fig. 1A). When disturbed, edges of the cavities close rapidly. Gills arranged in horseshoe pattern comprising dorso-terminal semicircle of 5 gills, including 3 dorsalmost medial gills within a notal cavity (Fig. 2B). This gill semicircle merges gradually into the remaining gills, placed in two lateroventral rows. Usually 7–9 gills on each side of hyponotum, decreasing in size anteriorly; 2–3 anteriormost gills smallest and hardly visible. A juvenile specimen (1.3 mm) with only 3 and 4 ventrolateral gills on each side of hyponotum. Largest gills are those next to gill cavity. Gill rows do not reach middle part of foot and are restricted to its posterior part. All gills unipinnate; largest ones of 4–7 leaflets; smallest do not possess recognizable leaflets (Fig. 2B). Ventral anus located just anterior to notal cavity entrance, between two largest gills (Fig. 2B). Rhinophores short, retracted into raised sheaths with smooth, soft, not indented edges (Fig. 2A); edges capable of considerable contraction in living specimens. Rhinophore with short anterior smooth stalk, wrapped within two pairs of folds, and bearing posterior unpaired fold, which is a continuation of rhinophoral stalk (Fig. 1D). Notum sparsely covered with low hemispherical tubercles (Figs 1D, 2C), which are more densely arranged on notal notch lobules (Fig. 2D). Oral veil small, trapezoid; in living specimens anterolateral corners form short narrow triangular tentacles (Fig. 1B). Foot is broad, anteriorly thickened, beneath mouth it has a deep incision forming lateral lobes; posteriorly slightly narrowed and rounded (Fig. 1B).
Colour: Living specimens semitransparent, with irregular network of opaque bright white and yellowish broken lines or spots (Fig. 1A, B); brownish and pale lilac spots scattered within network, but in a deeper integument layer. Hyponotum covered by similar network of lines (Fig. 1B). Rhinophores translucent with few small white dots. Gills translucent white; some gills, including those in notal cavity, covered dorsally with conspicuous brownish or lilac spots. Three brownish lobes of digestive gland visible through the foot.
Integument (Figs 1A, D, 3A–D): Thin (10 µm), smooth epidermis forms notal surface, with few wart-like bumps. SEM reveals evenly distributed small papillae on entire dorsal surface in one specimen. Semithin sections show cuticle (8 µm thick) covering only dorsal epidermis (Fig. 3D); slightly darker ‘pegs’ of cuticular material are located apically large nuclei of epidermal cells (Fig. 3C, D). Fleshy notum formed of thick layer of densely fibrous connective tissue without detectable cell borders (Fig. 3A, B, D). Spacious vacuoles form numerous spherical to slightly pear-shaped cavities (50 µm diameter) within entire notum, mostly without connection to dorsal epidermis. These cavities appear empty but are lined with thin, distinct layer 2 µm thick; if apical pore is present it appears to be sealed by this lining. Dark-staining bottle-shaped glands sparsely distributed, opening through epidermis by apical pore that may be associated with aforementioned papillae (Fig. 3C). Except for dorsal surface of notum, epidermis is ciliated (patchy inside entire notal cavity, denser on gills and rhinophores, continuous on foot sole).
Digestive system (Figs 1B, E, 2E–I, 3E): Anterior part of buccal bulb modified to form prominent sessile (i.e. without stalk) buccal pump; pump encircled by narrow peripheral muscle (Fig. 2E). Rounded labial disk lined with smooth cuticle. Radula formula (in 2 studied specimens 2.5–3 mm length) 26–31 × 22.214.171.124.4. Central tooth absent. First lateral tooth large with long, wide base and long, slightly curved, attenuated beak-shaped cusp (Fig. 2G–I); that bears 5–7 sharp, long denticles. Further lateral teeth are slightly elongated small plates without cusps, all similar in size and shape. Salivary glands short. Stomach cavity large, broad, without caecum. Digestive gland of three lobes (Fig. 1B) – anterior pair and single posterior one, the latter notched terminally; anterior to and between lobes are three pairs of dorsoventral muscles (Figs 1E, 3B) connecting notum and foot.
Circulatory system (Figs 3C, D, 4): Pericardial sac with broadly triangular posterior auricle (atrium) and elongate oval ventricle. Ventricle with slightly thicker wall than auricle and no separating valve. Aorta leaves anterior tip of heart. A pair of spacious haemolymph lacunae begins at posterior tip of kidney, continuing as large paired afferent vessels leading to gills. Afferent vessels form five branches: one on each side of body connecting to all the smaller, anterior serial gills; a second paired one to largest, posteriormost serial and outer medial gills; a third single one to central medial gill (Fig. 4). Similarly, five efferent vessels emerge from gills, those from serial ventrolateral gills fuse on each side of body and three vessels enter the atrium. A lacunary space around heart also has at least one paired lateral connection to atrium. Some spherical gill glands (Fig. 3C) located ventral to large afferent lacunae, but opening to outside posteriorly, close to bases of medial and large serial gills. Gill glands of several cells surrounding small central lumen filled with mucus; cells possess either large basal nuclei or smaller apical nuclei. Blood gland (Fig. 1F) posterior to central nervous system (CNS). A smaller lobe of tissue lies anterior to CNS; it is not directly connected to blood gland so its identity is unclear.
Excretory system (Fig. 4): Ciliated syrinx emerges right dorsolaterally from pericardium. Long renopericardial duct runs along ventral side of kidney within a fold, entering kidney anteriorly. Kidney large, anchor-shaped, with two posterolaterally projecting lobes ventral to heart. Wide kidney lumen surrounded by strongly vacuolated wall. Posteriorly, the short, ciliated nephroduct leads to ventroterminal nephropore, close to anal opening.
Central nervous system (Figs 1E–G, 2F, 3E, 5): Large, elongate cerebral ganglia almost fused medially; smaller pleural ganglia broadly connect posterolaterally, but are separated from cerebral ganglia by a constriction (Figs 1F, 5). At anteroventral tip of each cerebral ganglion, a bundle of three nerves emerges next to cerebrobuccal connective, presumably comprising oral and labiotentacular nerves. Spherical buccal ganglia anteroventral to cerebral ganglia, just posteroventral to most proximal oesophagus (Fig. 1F). Smaller gastroesophageal ganglia connect to each buccal ganglion dorsally. Elongated rhinophoral ganglia connected to cerebral ganglia laterally via thick connective; distally, ganglia run into thick rhinophoral nerve. Smaller spherical optical ganglia are located more posterodorsally; comparably long thin optic nerve connects to eyes (20 µm diameter) with spherical clear lens and small dark-pigmented cup. Statocysts (diameter 25 µm) on top of pedal ganglia just medially to cerebropedal connective; thin static nerve emerges from cerebral ganglia posterolaterally. An additional nerve exits right cerebral ganglion laterally, leading towards genital opening but also connecting by one branch to anteriormost nerve emerging from right pleural ganglion (Fig. 5). Cerebropedal connective short, broad. Including connectives, left cerebral ganglion bears nine nerves at least; right one bears 10.
Pedal ganglia are large, spherical, with double commissure; parapedal commissure thinner and longer than pedal one. Two nerves emerge from left pedal ganglion (one thick and branching proximally), four from right pedal ganglion. Small unpaired, additional ‘genital’ (or better called ‘penial’) ganglion (Fig. 3E) connects to right pedal ganglion posterodorsally; a thick nerve leads to penis, a thinner one runs posterodorsally.
Pleural ganglia spherical. Left one bears two nerves (one running laterally, the other posteriorly); right pleural ganglion bears one nerve running posteriorly and two laterally, of which anteriormost connects to right cerebral ganglion. Pleural ganglia interconnected by comparatively long (for nudibranchs) visceral loop, with two small, but distinct, elongated ganglia (Figs 1G, 5). Left ganglion (a tentative subintestinal ganglion) consists of only a few loosely aggregated nerve cells; larger right ganglion (visceral ganglion) is more distinct and bears a single nerve running posteriorly towards viscera.
Reproductive system (Fig. 6): Hermaphroditic gonad tissue fills anterior left body cavity and covers digestive gland. Anterior genital system triaulic. Ampulla oval, swollen, relatively short, filled by sperm in all four studied specimens. Postampullary gonoduct bifurcates into vas deferens and oviduct. Proximal part of vas deferens muscular, long, bent, attached to nidamental gland; forms conspicuous swollen chamber before passing into prostate (Fig. 6C, D). Tubular prostatic part of vas deferens weakly defined, short, of 2–3 slightly swollen loops, which do not encircle bursa copulatrix. Prostate transits into short, almost straight, muscular part of vas deferens, which rapidly widens and enters short penial sheath; the latter contains well-defined, short, conical penis (Fig. 6B). Vagina (Fig. 6A) very long, thin, narrow, forming swollen elongated chamber (a ‘vaginal bursa’) before leading into bursa copulatrix. Bursa copulatrix large, compressed, irregularly spherical. Uterine duct short, narrow, emerging at junction of bursa base and vagina, and connecting with proximal oviduct (Fig. 6C). There is no separate seminal receptacle detectable, but a bent, slightly swollen part of proximal oviduct can be regarded as a serially arranged receptacle (Fig. 6C). Oviduct enters female gland mass, the parts of which were not studied in detail.
This species inhabits shallow-water algae covered with encrusting bryozoans (Membranipora), on which it preys. Corambe mancorensis mimics Membranipora colonies by its flattened shape and special colour pattern. Curiously, on the same algae, two further Membranipora mimics were found: a rounded, depressed ascidian species and a flatworm (Fig. 1C) that can easily be mistaken for C. mancorensis at first glance.
Corambe mancorensis is presently known only from the type locality Mancora, northern Peru. It is the first corambid reported from tropical eastern Pacific waters.
Only four previously known species of the genus Corambe (sensuValdés & Bouchet, 1998) possess rows of lateroventral gills and a posterior notal notch, i.e. Corambe testudinaria H. Fisher, 1889, C. pacifica, C. lucea and C. evelinae. Each of these species possesses a peculiar gill pattern that clearly differs from that of the new species. Both C. testudinaria and C. lucea have lateroventral gill rows which are not connected posteriorly (García, Urgorri & López-González, 1990; Schrödl & Wägele, 2001). They do not possess special, medially placed gills and lack any trace of a gill cavity. Due to their position, ‘small central gills’ found in C. lucea may correspond to vestiges of outer medial gills as found in C. mancorensis. In contrast, C. pacifica, C. evelinae and C. mancorensis all possess a more or less small gill cavity containing one to three medially placed gills. The gill cavity of C. pacifica is vestigial (Fig. 7J), and bears a single gill that is located strictly ventrally, i.e. under the notum, in between the rows of lateroventral gills. The medial gills and the gill cavity of C. evelinae and C. mancorensis are situated dorso-terminally. While the medial gills appear to be separated from the lateroventral gills in C. evelinae, the three medial gills of C. mancorensis are not arranged separately from ventral ones, but form a continuous horseshoe-shaped row. Corambe mancorensis is well distinguished from C. evelinae by a number of additional characters: the first radular teeth have longer cusps with a larger number of denticles (5–7 in C. mancorensis cf. 3–5 in C. evelinae), a short swollen ampulla (vs. a very long tube), a considerably shorter uterine duct, and a tubular and flow-through seminal receptacle (pyriform and semi-serial in C. evelinae). Apart from the unique gill pattern, C. mancorensis is well distinguished from all other known corambid species by the possession of posterior notal lobes with lobules that protect the gills. There is thus no doubt that the species described herein is distinct and undescribed. It is the first corambid species known to inhabit the tropical eastern Pacific.
Morphology and anatomy
Integument: Epidermal and notal features of C. mancorensis resemble those described for C. lucea by Schrödl & Wägele (2001). The notal cuticle of the new species is, however, thinner, and no shedding has yet been observed. Furthermore, there are small tubercles scattered over the notum and concentrated on special lobules emerging from the border of the posterior notal notch. This notch, with the aid of the lobules, is fully closable, which seems to be unique among known Corambe species.
Dorsoventral muscles: Three pairs of dorsoventral muscle bundles cross the body cavity of C. mancorensis, separating the gonad and digestive gland peripherally into lobes. A similar situation was described and discussed in C. lucea by Schrödl & Wägele (2001). While this appears to be the normal condition among Corambe species, none of the three species assigned to the genus Loy by Valdés & Bouchet (1998) has such muscles; at least, we could not detect them when re-examining some more or less well-preserved specimens of all of them for comparison.
Radula: The radula of C. mancorensis is very similar to that of congeners and Adalaria jannae (e.g. Marcus, 1958; Millen, 1987; Schrödl & Wägele, 2001; Martynov et al., 2009). In corambids the central tooth is always reduced, except for tiny and irregular central tooth vestiges in Loy thompsoni (see Millen & Nybakken, 1991). The first lateral teeth have long, slightly curved cusps in C. mancorensis, C. lucea and C. pacifica, whereas in Loy meyeni and Corambe obscura the cusp is considerably curved apically (Martynov, 1994a; A.M., unpubl.). The basal part of the cusp is provided with several distinct denticles in all corambids. Lateral tooth patterns differ between two members of the genus Loy, i.e. L. meyeni and L. millenae, and Corambe: the former have remarkable second laterals which are forked apically and long, knife-shaped further laterals (Martynov, 1994a), whereas Corambe species have more or less uniformly shaped elongate-triangular folded laterals with a peculiarly excavated inner side. Loy thompsoni, however, does not possess fork-shaped second laterals, and further lateral teeth are more similar to those of Corambe than the other Loy species. The number of outer laterals varies from 4–6 in Corambe species to 6–7 in species of Loy.
Buccal pump: There are two main types of suctorial buccal pumps within corambids: at least two species of Loy (L. meyeni and L. millenae) show only a poorly defined, slightly protruding elevation in the anteriormost part of the pharynx (Martynov, 1994a; A.M., unpubl.). The second type, found in C. mancorensis (Fig. 2E) and all other species of Corambe, is essentially similar to the buccal pump of noncorambid Onchidorididae, e.g. in the genera Onchidoris and Adalaria (Martynov, 1994b; Martynov et al., 2009; A.M., unpubl.). Both Loy and Corambe species have buccal pumps that are entirely encircled in the middle part by the peripheral muscle; however, there are certain differences among species: C. pacifica has a strongly developed, ovoid, swollen, buccal pump, C. lucea instead has a compressed, elongated pump, when compared at the same state of contraction. The pump of C. mancorensis is somewhat intermediate between C. pacifica and C. lucea, whereas C. obscura has a relatively short spherical pump (A.M., unpubl.).
Central nervous system: Corambe mancorensis shows a remarkable CNS (Fig. 5). Compared to most other dorids, it is little cephalized. Pleural ganglia are separated from cerebral ones, but not so clearly as in most other corambids, e.g. C. lucea. The small numbers of cerebral nerves and connectives (9 on the left, 10 on the right) reported here are provisional until more detailed study; the asymmetry is caused by an additional nerve leading to the genital opening on the right side of the body. The visceral loop is relatively long for dorid nudibranchs, which usually possess just one or lack any free ganglia (Schmekel & Portmann, 1982; Wägele & Willan, 2000). At least in one corambid species, C. testudinaria, a distinct nonpaired visceral ganglion has been reported at the base of the visceral loop (Fischer, 1891), although subsequent study did not detect such a structure (García et al., 1990). Uniquely, C. mancorensis possesses two clearly separated ganglia on the visceral loop (Fig. 1G). The right one of these can be supposed to be (or to contain) the visceral ganglion, because of the strong nerve emerging from it that leads backwards into the viscera. The left, very small ganglion might be parietal and/or suboesophageal. Equally unusual for nudibranchs, a small (‘genital’ or better ‘penial’) ganglion connects the right pedal ganglion with the copulatory organ; since it is neither situated on the visceral loop nor connected to it, its homology with the true genital ganglion of more basal opisthobranchs is unlikely.
Gills and circulatory system: Corambe mancorensis shows a very unusual arrangement of gills. As in other Corambe species, there are multiple gills in a posterior ventrolateral position, forming rows of feather-like gills as in e.g. C. lucea. These ‘ventral gills’ (or ‘serial gills’) in C. lucea have been shown to possess the usual dorid gill glands, and are connected to the body cavity and atrium via a paired system of vessels. Gills and gill vessels of Corambe have been interpreted as modified and multiplied from the normal dorid condition with ‘anal gills’ surrounding the usually dorsal anus in a (semi)circle (Schrödl & Wägele, 2001). According to the description and illustration of a histological slide by Marcus (1958), the Brazilian C. evelinae shows serial ventral gills, plus 2–3 special medial gills in a more dorsal position, obviously within the notal notch and on top of notal tissue, i.e. within a notal cavity; only the medial gills showed gill glands. Although neither the types nor any other specimens of C. evelinae were available for re-examination, the existence of this special condition is supported when comparing it with the gill arrangement observed in C. mancorensis. Here, serial ventral gills are present in two lateral rows that converge posteriorly in a horseshoe-like pattern, with the three more dorsal central gills emerging from a notal cavity. Gill glands appear to open at the bases of these medial gills and of the innermost serial gills only. Serial gill sizes increase posteriorly, with the pair of innermost serial gills being largest. Only these, and the central gills, are visible from above when the notal notch is fully opened in living specimens. A similarly continuous, though entirely ventral, placement of anal and lateral gills is found in C. pacifica, where just one central gill is developed, emerging from a vestigial gill cavity (Fig. 7J). Unfortunately, there is no information on gills of living C. evelinae, or on its circulatory system.
Based on the 3D reconstruction (not shown) of the complex circulatory system of C. mancorensis, a more instructive scheme was prepared (Fig. 4). All ventral gills but the innermost ones are connected to the body cavity by a common afferent vessel, and to the atrium by a common efferent vessel, as in C. lucea (see Schrödl & Wägele, 2001). The innermost ventral gills, however, receive their own pairs of vessels, which are basal spinoffs from the common serial gill vessels. This might be a consequence of the importance of the innermost serial gills for respiration, owing to their large size and position within the notal notch. Outer medial gills are connected to the serial gills vessel system as well, while the single inner medial gill receives haemolymph from its own, unpaired, medial vessel, and blood flows to the heart through its own efferent vessel, which enters the atrium in a terminal posterior position.
Corambe mancorensis, C. pacifica and most likely C. evelinae thus show at least two (though not strictly delineated) types of gills: (1) a multitude of serial, ventral gills arranged in rows; the innermost pairs have a special size, function, haemolymph supply, and their own gill glands at least in C. mancorensis; and (2) one to three central gills that are situated within a notal cavity, anterodorsally of the posteroventral anus, and provided with gill glands and their own vessel system, at least in C. mancorensis and C. evelinae. Apart from the ventral anus, this reflects the usual gill situation in dorids, and there is little doubt about the direct homology of medial gills of Corambe and dorid anal gills. Only those corambids with multiple ventral gills possess a fleshy notum that is covered with a cuticle, hindering oxygen diffusion; thus the transformation, multiplication and translocation of dorid anal gills into corambid serial gills correlates with, and was probably induced by, necessity for increased respiratory area on the ventral side (Martynov & Schrödl, in press).
As in the case of other genera of the Onchidoridae possessing gill pockets (Martynov et al., 2009), there is little doubt about the homology of the corambid notal cavities associated with anal gills with other doridoidean gill cavities, regardless of their dorsal, subventral or even ventral position. In C. mancorensis the gills can contract considerably, the notal notch edges and lobules closing to seal the gap. This is a unique cryptobranch condition of a phanerobranch species, which is structurally different from – thus analogous to – the retractable gills and closable gill cavities of Cryptobranchia and the onchidoridid Onchimira cavifera Martynov et al., 2009 (Martynov et al., 2009).
The ontogeny of the dorid notum, anus and gills
The anus of adult dorids usually opens in a medial, posterior dorsal position, and is surrounded by a (semi)circle of gills, as in Cadlina laevis (Fig. 7A). The ontogeny of the anus and gill arrangement is, however, poorly known. The posterior part of the notum in early postlarvae (250–500 μm length, Fig. 7B, C) of both cryptobranch (Cadlina, Glossodoris) and phanerobranch (Adalaria) Doridoidea is not entire as in adults, but forms asymmetrical primary notal lobes that are separated by a notch; the right lobe is larger than the left in early postmetamorphic Cadlina laevis (Fig. 8A) and in other species with available data (Thompson, 1958, 1967; Usuki, 1967). The anus is already ventral at this stage (Fig. 7C). Apparently, the differential growth of the right lobe causes the anus to move from a (plesiomorphic) right lateral (e.g. Wägele & Willan, 2000; Martynov & Schrödl, 2008) to a posterior position. Cadlina laevis of c. 1 mm living length have been recorded to have the anus in a terminal, ventral position under the notum (Thompson, 1967), which is confirmed herein. Juveniles of 650 µm living length had a ventral anus (Fig. 7C), but had no clearly visible primary notal lobes and lacked any gills and gill pocket. Similarly, juveniles of Glossodoris sibogae (Bergh, 1905) of about 0.8 mm length showed no gills (Usuki, 1967). As in C. laevis, the postlarval (=primary) notal lobes had completely disappeared at this stage, and the posterior notum resembled the morphology of the adult, except for not showing dorsal anus and gills (Fig. 8B). Thus, during further ontogeny, which was not been directly observed for any dorid before, the presumably always functional anus must shift from the ventral to the dorsal side, and a pocket with gills must develop. It is herein proposed that by differential growth of notal tissue the anus first comes into a terminal, subventral position and then, in cryptobranchs, becomes surrounded by secondarily developed notal lobes. Evidence for the real existence of such an ontogenetic stage was obtained herein: Cadlina laevis of 600–1,000 µm living length developed clearly visible, secondary notal lobes (Fig. 7D), but still no signs of gills or gill cavity were detectable. Field-collected, somewhat larger early juveniles of the cryptobranch Paradoris indecora (Bergh, 1881) (1–1.2 mm preserved length) already showed three gills in a posteriormost position and that were surrounded by notal lobes, forming the drop-shaped anlage of the gill cavity (Fig. 7E). This observed stage (Fig. 8D) must obviously be preceded by a similar stage, but without gills differentiated yet (Fig. 8C). The secondary notal lobes may fuse under and posterior to the now dorsal anus, forming a dorsal cavity in which anal gills develop. In fact, a slight suture was visible as a trace of the fused posterior notal lobes in all cryptobranch dorid species with adequately sized early juvenile stages available to us, i.e. Paradoris indecora and Doris ocelligera (Bergh, 1881) (Fig. 7E, F). Finally, the suture must vanish and the drop-shaped gill cavity must transform into a normal one with rounded opening, as is characteristic for adult cryptobranch dorids (Figs 7A, 8E).
In contrast, the phanerobranch onchidoridid Adalaria proxima, which has a dorsal gill circle around the anus but lacks a gill cavity (Fig. 8EE), shows a shortened ontogeny (Martynov, 1994b, 1995): the anus is in a dorsal position and notal lobes have already disappeared at a postlarval stage of 250–300 μm length (Thompson, 1958; Fig. 8BB). Juveniles of the confamiliar Onchidoris neapolitana (Delle Chiaje, 1841) 800 μm long do not show any macroscopic cavity (Martynov et al., 2009); our SEM examination, however, revealed a very small but evident cavity around the anus and remnants of the suture between almost entirely fused notal lobes (Figs 7G, 8DD). This small and potentially vestigial cavity is considered homologous with the well-defined juvenile drop-shaped gill cavity of corresponding cryptobranch stages (Figs 7E, F, 8C, D). It is thus possible that Thompson (1958) just did not recognize this small cavity during light microscopic examination of his still earlier juveniles of Adalaria proxima, and that an ontogenetic stage with a vestigial gill cavity is present among other onchidoridids and phanerobranchs as well.
Integrating the ontogenetic data available on dorids, Figure 8 shows schematically all stages described above plus one necessary (though never directly observed) intermediary stage in Figure 8C. One lineage of ontogenetic transformation (Fig. 8A–E) refers to Cryptobranchia, of which several species of different families (i.e. Chromodorididae and Discodorididae) were examined herein and in previous studies, with congruent results. Whether or not this ontogenetic series is valid for all taxa that are currently classified as Cryptobranchia (e.g. Odhner in Franc, 1968; Schmekel & Portmann, 1982; Wägele & Willan, 2000; Valdés, 2002; Schrödl, 2003) may be tested in future studies. The assumption that the early postmetamorphic ontogeny described here for C. laevis, a direct developer, is the general type for cryptobranchs needs to be confirmed by future studies of species with planktonic larvae. The other, shorter ontogenetic series (Fig. 8A–EE) refers to the few members of the phanerobranch family Onchidorididae for which information is available, i.e. species of the genera Onchidoris and Adalaria.
The absence of an accepted hypothesis of basal doridoidean phylogeny is problematic. Following conventional dorid classification, with monophyletic cryptobranchs that have evolved from a phanerobranch level of organization or paraphyletic phanerobranchs (e.g. Wägele & Willan, 2000; Valdés, 2002, 2004), the ‘short onchidoridid ontogeny’ was ancestral, and cryptobranchs not only elaborated the gill pocket but prolonged its development and even evolved an additional ontogenetic stage with secondary mantle lobes. The alternative hypothesis is that phanerobranch onchidoridids shortened an ancestral cryptobranch ontogeny, reducing the gill cavity and losing an ontogenetic stage with secondary mantle lobes. There is some evidence for the latter: some adult ‘phanerobranchs’ such as the basal onchidoridid genera Onchimira and Calycidoris (Martynov et al., 2009; Martynov & Schrödl, in press) show well-developed gill cavities (Fig. 8E) that are closely similar and thus probably homologous to cryptobranch ones rather than convergent organs. Great variation is observed in more derived onchidoridid genera. Adults of Diaphorodoris, Loy and some Corambe such as C. mancorensis (Figs 1A, B, 2B, 7I) and C. evelinae (see Marcus, 1958) retain a more or less well-developed gill cavity; this is very small and obviously without protective function in C. pacifica (Fig. 7J), but all these cavities can plausibly be regarded as remnants. Most other onchidoridids as well as other phanerobranchs lack gill cavities completely (Fig. 8EE). All these different stages can easily be explained as reductions reflecting an ‘abbreviated phanerobranch type’ of ontogeny which, however, remains to be tested for non-onchidoridid phanerobranchs.
Ontogenetic observations thus support a phylogenetic hypothesis in which one or several phanerobranch lineages evolved from a cryptobranch level of organisation (Martynov et al., 2009), implying that the possession of a gill cavity is ancestral for cryptobranchs and (at least one) phanerobranch lineages such as Onchidorididae. But why should such a protective gill cavity be lost during evolution? Having developed a faster way of bringing the anus into a dorsal position, or generally speeding up the entire postlarval ontogeny, the ontogenetic programme responsible for the anlage of a drop-shaped cavity could have been abbreviated or simply skipped.
The corambid time warp and the evolution of dorid notum, anus and gills
Intriguingly, adult corambids show a spectacular variety of anus positions and gill arrangements, which may reflect corresponding stages of dorid ontogeny (Fig. 8). The phylogeny of corambids is well known, for the topology by Valdés & Bouchet (1998) has essentially been confirmed by an updated and comprehensive cladistic analysis (Martynov & Schrödl, in press). The basal corambid species Loy meyeni has an anus that is associated with three small gills in a dorsal notal cavity (Fig. 7H). Different from a ‘normal’ adult dorid, but similar to an early juvenile dorid condition, is the drop-shaped rather than rounded gill cavity (compare Figs 7E, F, H, also Fig. 8C, D, I), and the presence of asymmetrical posterior notal lobes, which are nearly completely fused but still show a terminal suture (compare Fig. 7D–F). In other Loy species (Fig. 8II), the asymmetrical notal lobes are not fused, thus forming a notch, and the anal gills and their gill cavity are situated medially within the notch; the anus opens on the hyponotum, i.e. subventrally. The new C. mancorensis shows a similar terminal gill cavity with three gills and a subventral anus, but the notal lobes are more symmetrical, the notch is very well developed, equipped with protective lobules, and can be actively closed over the gills (Figs 1A, B, D, 7I). The Brazilian C. evelinae and the tropical Peruvian C. mancorensis show both gill types associated with the anus (‘anal gills’, ‘dorid gills’) and, in addition, special ventrolateral gills (‘serial gills’) (Fig. 8III). Most other corambid species with a notch, e.g. the Chilean C. lucea, have the anus in a derived ventral position between notum and foot, lack a gill cavity and lack gills clearly associated with the anus; instead, there are pairs or rows of multiple ventrolateral gills (Schrödl & Wägele, 2001). Comparing dorid ontogeny with a simplified corambid phylogeny (Fig. 8I–IV) there is evidence for a partial reversal of dorid postlarval development during corambid evolution. Within the corambid lineage, adults show an evolutionary translocation of anus and anal gills within a pocket from the dorsal to the ventral side and a successive opening of a notal notch; the underlying evolutionary process is progenesis resulting in pseudoarchaic paedomorphic features (Martynov, 1994b, 1995; Martynov & Schrödl, in press).
Accepting the presence of a gill pocket (i.e. a ‘cryptobranch’ condition) as the plesiomorphic state for Doridoidea, with reduction or loss in most phanerobranch lineages (Martynov et al., 2009), heterochronic juvenilization also explains the re-establishment of gill pockets between notal lobes in basal corambid taxa such as Loy (Figs 7H, 8I, II). This adult condition corresponds in shape and position to the earlier stages of gill pocket formation in cryptobranch juveniles (present study, Figs 7A–F, 8C, D). The pocket is still well developed in corambids with subventral anus and gills, i.e. C. mancorensis (Figs 1A, 2B, 7I, 8III) and C. evelinae, present but vestigial in C. pacifica with ventral anus and gills (Fig. 7J), and absent in other Corambe species. Lacking any notal notch during their entire ontogeny, C. steinbergae and C. obscura (Figs 7K, L, 8IV) may be considered as the most progenetic corambids. They may either show a very early, hypothetical ontogenetic stage before development of primary notal lobes, or a genuine modification of the latter stage. In contrast to other dorids, the posterior-ward shift of the anus is due to muscle retractor contraction rather than differential growth (Bickell, Chia & Crawford, 1981; Perron & Turner, 1977), corresponding to the fast generation times. In summary, as derived progenetic members of the Onchidorididae lineage, adult corambids display almost all the different stages of our novel hypothesis on notum and gill development in dorids. Interestingly, corambids such as C. mancorensis reflect an ontogenetic dorid stage with ventral anus and well-developed notal lobes gills and gill cavity, of which a similar type is known so far only from cryptobranchs (Figs 1A, 7D, 8C). Adult Loy meyeni most resembles the cryptobranch stage D (Figs 7H, 8D) and a, much smaller sized, juvenile stage of the phanerobranch Onchidoris neapolitana (Fig. 7G), which is in its adult condition devoid of any gill cavity. This supports our preferred hypothesis that the slow cryptobranch ontogeny is ancestral and the Onchidoris/Adalaria onchidoridid pathway represents a derived, shortened modification. Mantle and gill development may be variable among phanerobranch taxa and subject to evolutionary change according to ecological needs. Heterochrony, i.e. progenesis, is assumed to have already speeded up the ontogeny of early onchidoridids and this trend was accelerated within corambids. This ‘recapitulation’ of early juvenile dorid stages turns Haeckel's law upside down.
We are especially grateful to Tanya Korshunova (Moscow, Institute of Higher Nervous Activity and Neurophysiology) for preparing illustrations. Our sincere thanks go to Sandra Millen (Vancouver) and Gerhard Haszprunar (ZSM) for collecting and making available specimens of Corambe pacifica, C. steinbergae, Loy thompsoni and several early juvenile dorids. Georgy N. Davidovich, the head of the SEM laboratory of Moscow State University, the chief engineer Anatoly G. Bogdanov, and the staff of the laboratory are thanked for providing excellent SEM facilities. Histological and SEM work at the ZSM was assisted by Eva Lodde and Enrico Schwabe. The unforgettable fieldwork in Peru was supported by many friendly helpers, including fishermen, bus and taxi drivers, and local gods. Diving equipment and funds were contributed by the GeoBioCenter (LMU) and the German Research Foundation (DFG SCHR 667/4 to M.S.); guest stays of AM at the ZSM were financed by DFG grants SCHR 667/6–1 and 667/10–1.