Molecular and phylogenetic analyses

The taxonomic sampling scheme used herein included 23 individuals tentatively identified as Clea helena, six specimens of Nassodonta dorri, and a reduced subset of 27 taxa used in the comprehensive phylogenetic analysis of the Nassariidae of Galindo et al. (2016). Six representatives from other families among the Buccinoidea were also included and the tree was rooted with Cancellaria cooperii Gabb, 1865 (Cancellariidae, Cancellarioidea).

Molecular sequencing was carried out at the Service de Systématique Moléculaire (MNHN-Paris VI UMR 7138 UMS 2700). Partial COI, 16S, 12S, 28S (partial C1 and D2 domains) and H3 genes were amplified, using the primers of Galindo et al. (2016), Palumbi et al. (1991), Simon, Franke & Martin (1991), Chisholm et al. (2001) and Colgan et al. (1998), respectively. PCR reactions were performed in 20 μl volumes, containing between 1 and 2 μl of genomic DNA, 1× reaction buffer, 2.5 mM MgCl₂, 0.26 mM dNTP, 0.3 μM of each primer, 5% DMSO and 5% BSA (10 mg/l) and 1.5 units of Q-Bio Taq (QBiogene, Carlsbad, CA, USA). Annealing temperature was 54 °C for 40 s for COI, 53 °C×35 s for 16S, 63 °C × 40 s for 12S, 58 °C × 40 s for 28S and 55 °C × 35 s for H3. Bidirectional sequencing was carried out by the Centre National de Séquençage (Genoscope, Essonne, France). Chromatograms were visually inspected and edited as necessary in CodonCode Aligner 4.0.4 (CodonCode Corporation, Dedham, MA, USA), and aligned with Clustal W (Larkin et al., 2007) as implemented in MEGA5 (Tamura et al., 2011). COI was translated into amino acids to check for stop codons and frameshift mutations. All newly generated sequences have been deposited in GenBank (Table 1).

PartitionFinder 1.0 (Lanfear et al., 2012) was used to select the best-fit partitioning schemes and models for phylogenetic analysis, which favored two partitions (first COI codon position, 12S, 16S vs second and third COI codon positions, 28S, and H3) and the GTR+I+G substitution model for both partitions. In the absence of significant incongruence, Bayesian phylogenies for three datasets (mitochondrial, nuclear and concatenated datasets) were each inferred using MrBayes 3.1.6 (Ronquist & Huelsenbeck, 2003) as implemented on the CIPRES Science Gateway (Miller, Pfeiffer & Schwartz, 2010). Bayesian analyses, consisting of two independent replicates with eight chains each were run for 60,000,000 Markov chain Monte Carlo (MCMC) generations with a sampling frequency of one tree every 100 generations. MCMC convergence, likelihood curves and standard deviation of split frequencies were assessed using Tracer 1.6 (Rambaut et al., 2014). The first 25% trees were discarded as burn-in and a 50% majority rule consensus tree constructed. Vouchers from the molecular and anatomical investigations have been deposited in the collections of the Muséum national d’Histoire naturelle in Paris (MNHN) and the National Museum of Natural History in Washington, D.C. (USNM) (see Table 1).

Anatomy

• Anentome sp. A

Material examined: Specimens purchased through aquarium trade, Washington DC, (USNM 1405293); freshwater pond, Phuket, Thailand, July 2010 (MNHN uncataloged).

External Anatomy: Mature animal comprising roughly 3.5 whorls in preserved specimens. Animal light yellow, with dense pattern of irregular black blotches covering external surface of headfoot; black blotches fewer and more dispersed on foot sole. Mantle margin, internal surface of siphon and external surface of siphon along seam marked with small, whitish granules (Fig. 5). Head small with long, slender cephalic tentacles (Fig. 5: t), roughly half of siphon in length in preserved specimens, with eyes slightly elevated on prominent ocular peduncles at tentacle outer bases. Foot narrowly oval with small attachment disc to operculum (Fig. 5: op). Prominent propodium separated from mesopodium by conspicuous constriction, and with very deep propodial pedal gland along anterior edge, lined with two histologically distinct subepithelial gland cells. Short, deep transverse ovipositor short distance behind front edge of foot. Metapodium lacking posterior tentacles. Operculum large, spanning nearly entire aperture, elongate oval, concave, with basal nucleus slightly turned to left, and thickened, pointed, elevated process behind (Figs. 6A, ​,7A7A and ​and7B).7B). Mantle cavity long, roughly one whorl in length, posteriorly bounded by reno-pericardial complex (Fig. 6). Mantle cavity markedly asymmetrical, deeper along left side in front of pericardium. Columellar muscle thick, simple, not subdivided, spanning roughly one whorl from attachment at rear of mantle cavity to operculum. Ctenidium (Fig. 6: ct) extending along left side of mantle cavity from near mantle edge to narrow, posterior base of mantle cavity at left; anterior tip of ctenidium slightly curving to end adjacent to more anterior siphonal flap. Long siphon (Fig. 6: si) emerging from behind left anterior mantle edge in front of gill, to left of head. Short, narrow osphradium lying to left of anterior ctenidium, at base of siphon, slightly posterior to curved anterior tip of ctenidium. Osphradium bipectinate, length somewhat variable in preserved specimens, roughly one-third of ctenidium in length, nearly symmetrical, with lamellae along ctenidial axis slightly wider. Lamellae not numerous, rather narrow, roughly 35 in total. Pallial oviduct and rectum bordering mantle cavity at right. Anus opening well back from mantle edge, near anterior third of mantle cavity. Female gonopore opening short distance in front of anus. Hypobranchial gland (Fig. 6: hg) forming thick, elongate pad with sharp, well defined borders in mantle roof and partially overlying rectum, supported by white, calcium-bearing connective tissue; extending anterior to anus and female reproductive pore.

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Figure 5

Living animal of Anentome sp. A. Apertural and abapertural views.

Aquarium-trade specimen. e, eye; f, foot; op, operculum; si, siphon; t, cephalic tentacle. Scale bar: 5 mm.

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Figure 6

External anatomy of Anentome sp. A.

(A) Apertural view. (B) Abapertural view. au, auricle; c, caecum; ct, ctenidium; dg, digestive gland; ebv, efferent branchial vein; f, foot; hg, hypobranchial gland; int, intestine; kd, kidney; oes, esophagus; op, operculum; os, osphradium; p, propodium; r, rectum; rs, rectal sinus; si, siphon; t, cephalic tentacle; te, testis. Scale bar: 1 mm.

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Figure 7

Operculum and radula morphology of Anentome sp. A.

(A) External, and (B) internal views of operculae. Scale bars: (A, B) 1 mm, (C) 100 μm.

Radula (Fig. 7C): Radula with three teeth per row. Rachidian with arched basal plate and six main cusps on posterior margin. Lateral cusps slightly shorter than central cusp. Occasionally, very short denticles present on one or both sides, external to lateralmost cusps. Lateral teeth tricuspid, with central cusp much narrower and situated much closer to inner cusp.

Foregut (Figs. 8A and ​and8B):8B): Proboscis in contracted state not long, slightly shorter than aperture, with smooth walls, pigmented with irregular black blotches. Mouth forming triangular slit. Length/diameter ca. 4.5 in retracted proboscis and 6.5 in protracted one. Powerful paired proboscis retractors attached to posterior outer rhynchodaeum (Figs. 8A and ​and8B:8B: prr) in retracted proboscis and posteriormost proboscis walls in protracted proboscis and fuse with columellar muscle at posterior limit of hemocoel. Seven or eight smaller retractors attached to lateral walls of rhynchodaeum (Fig. 8A: lpr) and to lateral walls of hemocoel. Esophagus forming short loop upon leaving proboscis before passing through large nerve ring. Valve of Leiblein situated immediately anterior to nerve ring, poorly defined and very slightly broader than anterior esophagus, lacking ciliary cone. Esophagus expanding slightly in diameter after passing through nerve ring, paralleled by anterior aorta of slightly lesser diameter. Gland of Leiblein absent. Salivary glands acinous, fused, forming half-ring embracing posterior rhynchodaeum latero-ventrally. Salivary ducts free along anterior esophagus, entering its walls at half proboscis length, then continuing in walls of esophagus beneath lateral folds. Odontophore rather long, spanning roughly two-thirds of protracted proboscis in length, nearly equal in length to retracted proboscis. Radular sac equally long as odontophore. Odontophoral retractors fusing with proboscis walls.

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Figure 8

Anatomy of the digestive system of Anentome sp. A.

(A, B) Foregut. (A) Right view of the foregut, rhynchodaeum partially removed to expose the proboscis. (B) Protracted proboscis opened along right-dorsal line. (C, D) Stomach. (C) Stomach opened along dorsal line and outer wall reflected. Only short portion of posterior mixing area is shown. (D) Enlarged area of opening of the posterior esophagus into stomach. Outer stomach wall removed, esophagus opened along mid-line. c, caecum; ddg, ducts of digestive gland; ig, intestinal groove; lf, longitudinal fold on the inner stomach wall; lpr, lateral proboscis retractors; ls, lateral sulcus; mnt, minor typhlosole; mt, major typhlosole; nr, nerve ring; od, odontophore; oeo, esophageal opening into gastric chamber; oes, esophagus; pr, proboscis; prr, proboscis retractors; rd, radula; rr, radular retractor; sd, salivary duct; sg, fused salivary glands; vL, valve of Leiblein. Scale bars: 1 mm.

Midgut (Figs. 8C and ​and8D):8D): Stomach small, partially covered by digestive glands. Posterior mixing area very long, forming “caecum” spanning nearly three-quarters of whorl, equally narrow along its length (Fig. 8C). Caecum oval in transverse section, dorsoventrally compressed, lined with nine prominent, tall, longitudinal folds, several continuous with folds of posterior esophagus (Fig. 8C: oeo). Epithelial cells of folds mostly ciliated, with sporadic glandular, non-ciliated cells. Gastric chamber comparatively short. Small, paired, closely spaced, oval openings of digestive gland ducts adjacent to opening of esophagus. Each duct opening situated in rather deep depression. Longitudinal fold (Fig. 8C: lf) rather short, oblique, bordering posterior part of distinct, albeit narrow, lateral sulcus (Fig. 8C: ls). Lateral sulcus present on outer stomach wall, although shallower. Typhlosoles well-developed, bordering deep intestinal groove. Major typhlosole narrower and more prominent, while minor typhlosole broader but short, becoming obsolete at level of posterior kidney border. Inner wall of dorsal channel of style sac lined with transverse folds, replaced with inconspicuous longitudinal folds anteriorly.

Style sac region crossing below floor of posterior kidney, from posterior left to anterior right side, ventrally embedded in digestive gland. At base of mantle cavity, major typhlosole becoming inconspicuous, intestine turning and emerging into mantle roof. Intestine expanding somewhat into broad, flattened rectum, surrounded by voluminous rectal sinus. Rectum terminating anteriorly in small, simple, non-papillate anus.

Reproductive anatomy (Figs. 9 and ​and10):10): Gonad overlying digestive gland at right in apical whorls, anteriorly bordering caecum at right to level of esophageal opening, extending to tip of apical whorls in mature specimens. Vas deferens emerging from testes ventrally, continuing anteriorly along ventral aspect of visceral whorls, forming thickened, coiled seminal vesicle (Fig. 9A: vs) behind base of mantle cavity on ventral surface of visceral whorl, dorsally embedded in digestive gland below and slightly behind kidney (Fig. 9A). Vas deferens straightening and narrowing before penetrating base of mantle cavity at right. In mantle roof, vas deferens continuing anteriorly as thickened glandular prostate (Fig. 9A: prs). Prostate long, slender, thin-walled, lying below rectum, crossing to neck at level of anus. Prostate narrowing anteriorly to pallial vas deferens with thick muscular walls. Pallial vas deferens completing two half loops within cephalic hemocoel, first looping through tight half turn posteriorly, then reversing course through broad half loop anteriorly, before continuing as ejaculatory duct down center of penis lying on right side of neck behind cephalic tentacle. Penis (Fig. 9A: p) long, slender, simple, without external glands or elaborations, tapering to simple, narrowly rounded tip with apical pore.

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Figure 9

Anatomy of the reproductive system of Anentome sp. A.

(A) Male reproductive anatomy. Visceral mass and posterior part of the mantle, uncoiled, above; right view of the head-foot with prostate and anterior muscular seminal duct seen by transparency, below. (B) Female reproductive anatomy. Left lateral view, above, anterior is at left; right lateral view, below, anterior is at right. Hashed line indicates boundaries of lumen. ag, albumen gland; bu, copulatory bursa; c, caecum; cg, capsule gland; dg, digestive gland; fp, female pore; hg, hypobranchial gland; int, intestine; kd, kidney; op, operculum; p, penis; per, pericardium; prs, prostate; r, rectum; te, testis; v, vagina; vc, ventral channel; vd, pallial vas deferens; ve, vestibule; vs, seminal vesicle. Scale bars: 1 mm.

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Figure 10

Histology of the pallial oviduct of Anentome sp. A.

(A) Posterior albumen gland. (B) Anterior albumen gland. (C) Posterior capsule gland. (D) Anterior capsule gland. (E) Posterior copulatory bursa and non-glandular extension of ventral channel. (F) Middle copulatory bursa, vestibule and vaginal opening. (G) Anterior copulatory bursa and vestibule near entrance to ventral channel extension. aa, anterior aorta; bu, copulatory bursa; fp, female pore; kd, kidney; mc, mantle cavity; oes, esophagus; ovi, renal oviduct; r, rectum; v, vagina; vc, ventral channel; ve, vestibule; vg, visceral ganglion. Scale bar: 500 μm.

Oviduct emerging ventrally from ovary, continuing anteriorly along ventral aspect of visceral whorls. Gonopericardial duct lacking (Fig. 9B). Renal oviduct penetrating base of mantle cavity to enter pallial oviduct. Albumen gland (Figs. 9B and ​and10:10: ag) short, broad, with flattened lumen, comprising roughly one-third of glandular pallial oviduct. Albumen gland lumen narrowing anteriorly at junction with more distal capsule gland. Capsule gland (Figs. 9B and ​and10:10: cg) roughly twice albumen gland in length, also with flattened lumen. Capsule gland regionated into at least five externally and histologically differentiable sets of glands (Figs. 9B and ​and10).10). Precise pattern of folds somewhat variable between individuals, but symmetrical on right and left sides of lumen. Capsule gland continuous anteriorly with narrow, flattened, longitudinally ridged, non-glandular continuation of ventral channel, or vestibule. Vestibule (Figs. 9B and 10E–10G: ve) opening dorsally at anterior end to cylindrical, posteriorly directed copulatory bursa (Figs. 9B and 10E–10G: bu) containing unorientated sperm. Just in front of opening to bursa, vestibule expanding dorsally into small, muscular, bulbous vagina (Figs. 9B and 10G: v) terminating ventrally in elongate female pore. Right wall of vagina conspicuously thicker and deeply longitudinally ridged, with bifid fold extending into female pore. Entrance to vestibule just above female pore, between branches of bifid fold.

Remarks: We here follow the terminology of Fretter (1941) who termed the non-glandular continuation of the ventral channel anterior to the capsule gland, the vestibule, and the region between the female pore and the vestibule, the vagina. Kantor (2003) studied the stomach of Anentome sp. A (as “Clea helenae”), while Coelho, Dinis & Reis (2013) described the life history traits of “Clea helena” obtained through the aquarium trade, which is likely also conspecific with Anentome sp. A, judging from the rather poor figure of a single adult shell in abapertural view. In Anentome sp. B (MNHN IM-2013-52176), the odontophore is roughly one-half the length of the retracted proboscis.

• Nassodonta dorri (Wattebled, 1886)

Material examined: Vietnam: Bình Thuận Province, Tuy Phong, Phan Ri River, Sông Lũy, 11°10.57′N, 108°33.70′E (MNHN uncataloged).

External anatomy (Fig. 11): Mature animal comprising 2.25 whorls in preserved specimens. Head small, broad, with very short, thick cephalic tentacles (Fig. 11: t), with eyes slightly elevated on prominent ocular peduncles at tentacle outer bases. Foot broad, fleshy, overlapping sides of operculum in preserved specimens. Propodium narrow with posterior extent marked by indistinct notch, poorly demarcated from mesopodium. Shallow propodial pedal gland (Fig. 11: ppg) along anterior edge, with two histologically distinct subepithelial gland cells. Ovipositor forming deep, simple pore surrounded by weakly developed subepithelial glands. Metapodium lacking posterior tentacles. Operculum (Fig. 11: op) thin, elongate, oval with basal nucleus. Mantle cavity short, less than one-half whorl in length, posteriorly bounded by reno-pericardial complex. Mantle cavity slightly asymmetrical, slightly deeper at left side in front of pericardium. Columellar muscle long, extending just past posterior end of kidney. Ctenidium (Fig. 11: ct) extending along left side of mantle cavity from near mantle edge to pericardium behind left base of mantle cavity. Short siphon (Fig. 11: si) emerging from behind mantle edge at left anterior mantle cavity in front of gill, to left of head. Long osphradium lying to left of central two-thirds of ctenidium. Osphradium bipectinate, asymmetrical, with leaflets along ctenidial axis much larger than those along mantle floor. Osphradium separated from efferent branchial vein by deep cleft. Pallial oviduct and rectum bordering mantle cavity at right. Papillate anus opening well back from mantle edge, near anterior third of mantle cavity. Female gonopore opening just behind anus. Hypobranchial gland (Fig. 11: hg) forming elongate, thick pad with sharp borders in mantle roof and partially overlying rectum, thinning and extending short distance anterior to anus and female reproductive pore.

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Figure 11

External anatomy of Nassodonta dorri.

(A) Apertural view. Hashed line at anterior end of nephridial gland indicates nephropore, seen by transparency. (B) Abapertural view. ag, albumen gland; au, auricle; c, caecum; cg, capsule gland; cm, columellar muscle; ct, ctenidium; dg, digestive gland; ebv, efferent branchial vein; f, foot; hg, hypobranchial gland; kd, kidney; ngl, nephridial gland; op, operculum; os, osphradium; ov, ovary; p, propodium; ppg, propodial pedal gland; r, rectum; si, siphon; t, cephalic tentacle; v, ventricle. Scale bar: 1 mm.

Radula: (after Kantor & Kilburn, 2001) Radula comprising roughly 75 rows. Lateral teeth with four to six cusps, with number of cusps varying even on adjacent rows. Outermost cusps ~2.5 longer than innermost cusps. Intermediate cusps sharply pointed or bifurcating at their tips. Innermost cusp with seven to eight denticles on inner, lateral side. Rachidian with 11–12 cusps, central cusp serrated, with number of cusps varying even on adjacent rows. Basal plate evenly and deeply notched along anterior edge.

Foregut (Figs. 12A–12C): Proboscis very short and coniform, deeply retracted into rhynchodaeum, spanning only about half of rhynchodaeum length. Mouth forming narrow ventral slit at anterior tip of proboscis (Fig. 12C). In retracted proboscis, odontophore and radular sac significantly protruding from rear of proboscis (Fig. 12A: od). Buccal tube rather long, spanning nearly half of proboscis length (Fig. 12B: bt). Proboscis retractors arranged in bundles, attached to antero-lateral side of rhynchodaeum (Fig. 12A: lpr). Esophagus forming long loop, as long as rhynchodaeum, upon emerging from proboscis, before passing through large nerve ring. Valve of Leiblein pyriform, well defined, situated immediately anterior to nerve ring. Esophagus significantly expanding in diameter after passing through nerve ring, forming thin walled, spoon-shaped “crop,” embracing posterior part of protruded odontophore (Fig. 12A: cr). Gland of Leiblein absent. Salivary glands acinous, separate, large. Right salivary gland nearly completely covering nerve ring laterally (displaced in Fig. 12A to show nerve ring and valve of Leiblein). Salivary ducts thick, paralleling anterior esophagus. Odontophore large, muscular, nearly as long as proboscis. Large unpaired odontophoral retractor attached to ventral side of rhynchodaeum (Fig. 12B: odr). Subradular cartilages fused anteriorly along third of odontophore length. Radular sac equally long as odontophore.

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Figure 12

Anatomy of the digestive system of Nassodonta dorri.

(A–C) Foregut. (A) Right view of the foregut, right salivary gland displaced to show the nerve ring, dashed line indicates proboscis retracted within rhynchodaeum. (B) Proboscis opened along left-dorsal line. (C) Proboscis tip, ventral view. (D–F) Midgut. (D–E) Views of visceral mass showing layout of posterior digestive system. (F) Gastric chamber opened along right side. adg, anterior duct of digestive gland; bt, buccal tube; c, caecum; cr, crop; dg, digestive gland; gc, gastric chamber of stomach; gs, gastric shield; lf, longitudinal fold on outer stomach wall; lpr, lateral proboscis retractors; ls, lateral sulcus; mo, mouth; nr, nerve ring; od, odontophore; odr, odontophoral retractor; oeo, esophageal opening into gastric chamber; oes, esophagus; ov, ovary; pdg, posterior duct of digestive gland; pr, proboscis; rd, radula; sd, salivary duct; sg, salivary gland; st, statocyst; vL, valve of Leiblein. Scale bars: 1 mm.

Midgut (Figs. 12D–12F): Stomach very long, spanning approximately one whorl from posterior kidney border, roughly three-quarters of length comprising very long caecum, situated along right aspect of visceral whorls, parallel to longitudinal axis (Figs. 12D and 12E. Gastric chamber and style sac situated obliquely to longitudinal axis, across visceral whorls. Relief of stomach epithelium very low. Very broad, oval posterior opening of digestive gland duct (Fig. 12F: pdg) adjacent to opening of esophagus (Fig. 12F: oeo). Anterior duct smaller (Fig. 12F: adg), situated in depression in lateral sulcus. Longitudinal folds of posterior esophagus continuous into caecum, with folds also orientated parallel to main stomach axis. Longitudinal fold separating ducts and esophageal opening from dorsal chamber of stomach broad, poorly pronounced. At esophageal opening in ventro-dorsal wall, distinct cuticularized gastric shield present (Fig. 12F: gs). Broad but low longitudinal fold on outer wall continues from esophageal opening into caecum, bordered by transverse folds in narrow zone immediately adjacent to fold. Caecum otherwise bearing very low, narrow, inconspicuous longitudinal folds. Typhlosoles less pronounced than intestinal groove. Inner wall of dorsal channel of style sac lined with weak transverse folds. Stomach integuments in area of style sac pigmented.

Reproductive anatomy (Figs. 13 and ​and14):14): Gonad overlying digestive gland at right in apical whorls, posteriorly bordering caecum, extending to tip of apical whorl in mature specimens. Vas deferens very thin, emerging from testis ventrally, continuing anteriorly along ventral aspect of visceral whorls. Seminal vesicle not defined. Vas deferens broadening very slightly before penetrating base of mantle cavity at right, continuing anteriorly below rectum, crossing to base of poorly glandular prostate near posterior end of mantle cavity. Prostate medium short, with thin walls, narrowing anteriorly to pallial vas deferens. Pallial vas deferens coiling slightly before continuing as ejaculatory duct down center of penis lying on right side of neck behind cephalic tentacle. Penis long, slender, simple, without external glands or elaborations, tapering to very short and narrow papilla with apical pore.

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Figure 13

Female reproductive anatomy of Nassodonta dorri.

Left lateral view, above, anterior is at left; right lateral view, below, anterior is at right. Hashed line indicates boundaries of lumen. Note small, separate opening of vestibule to mantle cavity below anterior end of female pore. ag, albumen gland; al, anterior lobe; bu, copulatory bursa; cg, capsule gland; fp, female pore; gd, gonopericardial duct; ml, middle lobe; pl, posterior lobe; v, vagina; vc, ventral channel; ve, vestibule. Scale bar: 1 mm.

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Figure 14

Histology of female reproductive anatomy of Nassodonta dorri.

(A) Longitudinal section of ovipositor. (B) Longitudinal section of glandular pallial oviduct. ag, albumen gland; al, anterior lobe; bu, copulatory bursa; cg, capsule gland; cm, columellar muscle; fs, foot sole; ml, middle lobe; ovp, ovipositor; pl, posterior lobe; r, rectum; rs, rectal sinus; v, vagina; ve, vestibule. Scale bars: (A) 100 μm, (B) 500 μm.

Oviduct emerging ventrally from ovary, continuing anteriorly along ventral aspect of visceral whorls. Near base of mantle cavity, renal oviduct turning posteriorly. Gonopericardial duct present (Fig. 13: gd). Renal oviduct entering pallial oviduct well behind base of mantle cavity, along right side of kidney. Glandular pallial oviduct externally divisible into three distinct lobes (Figs. 13 and 14B). Posterior lobe (Figs. 13 and 14B: pl) long, weakly u-shaped, comprising roughly two-thirds of glandular pallial oviduct, with broad flattened lumen bordered by albumen glands (Figs. 13 and 14B: ag). Lumen turning slightly to right at anterior end of lobe and narrowing, before turning sharply left to enter small, distinct central lobe (Figs. 13 and 14B: ml). Within central lobe, lumen of oviduct oriented transversely across longitudinal axis, bordered posteriorly by albumen gland and anteriorly by capsule gland. Lumen turning sharply again at left side of oviduct, to continue anteriorly in anterior lobe (Figs. 13 and 14B: al), bordered by distinctly regionated capsule glands. Lumen in anterior lobe again oriented parallel to longitudinal axis. Glands of capsule gland (Figs. 13 and 14B: cg) darkly pigmented with dispersed black granules at anterior end. Capsule gland continuous anteriorly with short, narrow, flattened, longitudinally ridged, non-glandular vestibule. Vestibule (Figs. 13 and 14B: ve) opening dorsally near anterior end to vestigial copulatory bursa, empty in histological sections. Vestibule opening to mantle cavity via small, separate, slit-like pore below anterior end of large separate entrance to rounded, muscular vagina (Figs. 13 and 14B: v).

Remarks: Several specimens used in the anatomical investigations of this species, including those used in midgut dissections and for histological sectioning, had been previously dried and rehydrated. This has introduced some artefacts, and may have influenced some of the observations, including the low relief of the stomach epithelium and the size of the openings of the digestive gland ducts, the posterior of which was observed to be significantly smaller in a second specimen. In addition, only a single male (SL 12.4 mm) was available for study, and while being adult, was obviously between reproductive seasons. Nassodonta is a genus with only two species currently recognized (Nassodonta dorri, Nassodonta insignis Adams, 1867), traditionally placed in the Nassariidae (Smith, 1895b). Nassodonta species live in turbid brackish waters from India to Vietnam, with an unconfirmed report from China (Smith, 1895b), and are capable of withstanding a wide range of salinities. Kantor & Kilburn (2001) reported on the rediscovery of Nassodonta dorri, described the shell, radula and provided some preliminary anatomical observations based on a single, poorly preserved female, and discussed the family placement of the genus. Simone (2007) provided a description of the external anatomy, proboscis musculature and radula, based on “semi-mummified” specimens.

Diversification of the Anentominae

Results of the molecular analyses support the recognition of at least four lineages of Anentome “helena.” All but one are restricted to a single site, with Anentome sp. C recovered from two sites in peninsular Malaysia and one in Sumatra. Despite the allopatric distribution of these populations, the modern distribution of this species reflects paleo-drainage connectivity across the Malacca Straits river system, which drained large portions of the west coast of peninsular Malaysia and the east coast of northern Sumatra, northwest to the Andaman Sea during the last glacial maximum (de Bruyn et al., 2013). Connectivity between these populations would have been maintained at least until ~13,000 years bp when extensive land bridges still existed in this area (Voris, 2000; Sathiamurthy & Voris, 2006). Faunal links across the Straits are well documented, especially among freshwater fishes, and have formed the basis for recognition of the Northern Central Sumatra—Western Malaysia ecoregion (Abell et al., 2008).

While all four lineages received high support (Figs. 3A and ​and4;4; PP ≥ 0.97) in the analyses of the mitochondrial and concatenated datasets, specimens identified here as species B from a single site in northern Thailand displayed unusually high Kimura-corrected average pairwise distances for COI and 16S (COI: 0.080; 16S: 0.026–0.045). Compared to the distances within species A, C and D (COI: 0.000–0.012; 16S: 0.000–0.016), this suggests that there may be three lineages represented among the three specimens available for study of species B. Comparison of the shells, however, reveals them to be almost identical, and it is difficult to imagine that these represent three species, particularly as they were all sampled from the same locality. We conservatively interpret this pattern to be indicative of the pronounced mitochondrial structuring that can be found within some terrestrial (Greve et al., 2010; Kotsakiozi et al., 2012) and freshwater (Whelan & Strong, 2016) gastropod populations and which, in the latter, may be maintained through balancing selection (Whelan & Strong, 2016). Additional population-level sampling is necessary to fully characterize the magnitude and geographic scale of mitochondrial structuring among these isolated freshwater populations and to assess the number of discrete lineages.

Regardless of the precise number of molecular lineages, they are all referable to the current broad concept (Brandt, 1974) of Anentome helena, formerly Clea helena, with its fusiform, prominently ribbed and spirally banded shell. However, none of the populations analyzed here are from Java, so determining which, if any, of these lineages represents true Anentome helena is problematic. As stated, all but one of the lineages are restricted to single sites, with one lineage having a relictual distribution reflecting paleo-drainage connectivity across the Malacca Straits in what is today recognized as the Northern Central Sumatra—Western Malaysia ecoregion. Indeed, all other sampled lineages represent distinct paleo-drainages and distinct freshwater ecoregions: Anentome sp. A is found in the Lower Salween ecoregion that would have drained west to the Andaman Sea; Anentome sp. B is found in the Mekong Delta ecoregion, representing the Mekong paleo-drainage that drained south to the South China Sea; and Anentome sp. D is found in the Southern Annam ecoregion which would have drained east to the South China Sea. Java, southeast Sumatra and western Borneo comprise several ecoregions today, but large portions of these islands would have been drained by the East Sunda paleo-drainage, a large river system that flowed south to the Java Sea (Voris, 2000; de Bruyn et al., 2013). Thus, the type specimens of Anentome helena would correspond to both a modern ecoregion and a paleo-drainage distinct from any of those represented by the populations included here. Phylogenies of freshwater fishes from Southeast Asia have been found to display a high degree of concordance with paleo-drainage patterns (de Bruyn et al., 2013). Consequently, given the geographic scale of this species complex, the conchological distinctiveness of the lineages, their modern distributions and inferred historical connectivity, it is likely that none of the molecular lineages revealed here represents true Anentome helena. This is especially true of the aquarium trade specimens conspecific with species A from Thailand and which bear little conchological resemblance to the types (Fig. 1).

While our analysis has revealed a previously unrecognized radiation within what was formerly recognized as a single species, comprehensively revising the systematics remains a significant challenge. Establishing the identity of Melania helena requires topotypic material, but the types are poorly localized simply to “Java.” With the benefit of the insights obtained from the molecular analyses presented herein, it is possible to conclude that the type material likely represents at least two conchologically distinct species given the range of morphological variation (see Fig. 1). Consequently, the prospect that additional members of the Anentome helena complex are present on Java is high, or at least historically were present. A further complicating factor is that the freshwaters of Java, as of so many other areas in SE Asia, have been devastated by human-mediated impacts, including those from agriculture, mining, pollution, impoundment and habitat loss (Köhler & Glaubrecht, 2005). A number of freshwater species, including snails and shrimp, are believed to already have gone extinct (Köhler & Glaubrecht, 2005; Marwoto & Isnaningsih, 2012; De Grave et al., 2015). In addition, we require topotypic or near-topotypic material for the other eight nominal species currently in the synonymy of Anentome “helena,” with type localities stretching from Thailand and Cambodia to Java, a number of which are similarly poorly localized. Consequently, revising the taxonomy of this assemblage would be premature. For the present, we prefer to keep these lineages in open nomenclature pending a thorough systematic revision including broad geographic and population-level sampling. It is probable that the actual diversity of the genus is higher than presently appreciated, although the validity of the other currently recognized species remains to be tested.

The majority of specimens included in the present study were obtained during expeditions organized by the MNHN and Pro-Natura International as part of the Our Planet Reviewed program, and by the MNHN and the Institut de Recherche pour le Développement as part of the Tropical Deep-Sea Benthos program. Funding was provided by the Total Foundation, Prince Albert II of Monaco Foundation, Stavros Niarchos Foundation, Richard Lounsbery Foundation, the French Ministry of Foreign Affairs, and the Philippines Bureau of Fisheries and Aquatic Resources. In addition, samples provided by Ivan Bolotov (INEP—Institute of Environmental Problems of the North, Russian Academy of Sciences), Manuel Caballer (formerly of Observatorio Ambiental Granadilla, Santa Cruz de Tenerife), Pongrat Dumrongrojwattana (Burapha University), Alexander Fedosov (Russian Academy of Sciences), the late R.N. Kilburn (Natal Museum), Guido Pastorino (Museo Argentino de Ciencias Naturales), Jean-Pierre Pointier (University of Perpignan), L.R.L. Simone and D. Cavallari (University of São Paulo, Brazil), Tan Tu Vuong and Do Van Tu (Vietnam Academy of Science and Technology), and Manuel Malaquias (University of Bergen) helped complete our dataset. We are grateful to Richard Preece (University Museum of Zoology, Cambridge) and Fred Naggs (The Natural History Museum, London) for sharing photos of the syntype of Clea annesleyi; to Manuel Caballer (E-RECOLNAT ANR-11-INBS-0004, MNHN) for photographing the types; to Delphine Gey (MNHN) for her efforts in optimizing sequencing protocols for freshwater Nassariidae; to Nicolas Puillandre (MNHN) for informative discussions and on the molecular aspects of the study; and to Philippe Bouchet (MNHN) who made this study possible and provided thoughtful advice and constructive feedback, especially on aspects related to nomenclature and classification.