Comparative anatomy of the fasciolariids Pustulatirus ogum and Hemipolygona beckyae from Brazil ( Gastropoda : Buccinoidea : Peristerniinae )

The Brazilian species Pustulatirus ogum and Hemipolygona beckyae were examined, and certain morphological characters were described. Both species were originally assigned to the genus Latirus, considered as a heterogeneous complex. The radulae of both species are like that which characterizes Latirus, in which the innermost cusp of the rachidian tooth is well developed but always smaller than the other cusps. This feature differs from Leucozonia, in which this cusp is reduced or absent. The penis tapers terminally, and the tapered part may be long (more than half the total penis length), as in H. beckyae, or very short (less than half the total penis length), as in P. ogum. The anatomical data observed in both species are discussed under the framework of fasciolariid systematics and they appear to be widespread among other fasciolariid species. For this reason, to date, the soft-part features here provided and those known from previously studied species of Latirus are not useful for delineating precise generic diagnoses.

Pustulatirus ogum and Hemipolygona beckyae, which occur in Brazilian waters, were treated in the most recent bibliographic records as subgenera of Latirus (Rios 1994(Rios , 2009)).The former species occurs from Espírito Santo to Bahia state, and the latter only in Espírito Santo; P. ogum inhabits tide pools, while H. beckyae occurs at depths of about 30 m. Kosyan et al. (2009) studied the anatomy of some species of Fasciolariidae, including Turrilatirus turritus (Gmelin, 1791), Pustulatirus mediamericanus (Hertlein and Strong, 1951) and Latirus polygonus (Gmelin, 1791), all of which were previously regarded as members of Latirus.In Brazil, only the anatomy of species belonging to Leucozonia has been studied: L. nassa (Gmelin, 1791) by Marcus and Marcus (1962); L. nassa (Gmelin, 1791), L. ocellata (Gmelin, 1791) and L. ponderosa (Vermeij and Snyder 1998) by Couto and Pimenta (2012); and Teralatirus roboreus by Simone et al. (2013).Fraussen et al. (2007) reported that a combination of traits is diagnostic for Fasciolariidae: multicuspidate lateral teeth and straight rachidian teeth, proboscis retractor muscle as a single or paired tuft of fibres, ducts of the salivary glands embedded in the esophagus wall, and a stomach without a posterior mixing area.Kosyan et al. (2009) studied the anatomy of eight fasciolariid species belonging to seven genera.These authors distinguished fasciolariids from buccinids studied by them and by Kosyan and Kantor (2009), based on the stomach morphology: low folds with transverse striations, absence of differentiation of the gastric chamber, absence of a posterior mixing area (Kantor 2003), and proboscis retractor muscles as a single muscle or paired (Fraussen et al. 2007).The orange-red colour of the foot and head-foot mass is typical for fasciolariids.
Morphological characters may prove useful in validating phylogenetic relationships and may help to resolve internal clades (Strong 2003, Simone 2011).However, no formal anatomical characterization within Latirus and related species exists.They are presently distinguished solely on shell features (Vermeij andSnyder 2006, Lyons andSnyder 2013), and hence prone to hypotheses of polymorphism and convergence.The present contribution provides morphological descriptions and comparisons of Pustulatirus ogum and Hemipolygona beckyae.

MATERIALS AND METHODS
The material used for this study is deposited in the Museu Nacional / Universidade Federal do Rio de Ja-neiro (MNRJ) and Museu de Zoologia / Universidade de São Paulo (MZSP).
The specimens collected were fixed in 70% ethanol.Shells were measured with a caliper, and photographs of individuals were taken with a digital camera.The anatomical dissections were done under a stereomicroscope.All drawings were made using a camera lucida.Radulae were manually extracted and prepared by immersion in KOH, followed by ultrasonic cleaning and subsequent immersion in distilled water for scanning electron microscope photography.
Head-foot (Fig. 2A-B).colour cream in fixed species.Head prominent, of medium size (width ~1/3 of adjacent width of head-foot), cephalic tentacles blunt and short (length ~1/2 of anterior width of head), situated very close to each other; bases lying side by side.Eyes dark, small, rounded, situated in middle region of outer edge of tentacles.Foot short, rounded, anterior region bifid.Pedal gland as shallow median anterior slit, with anterior furrow extending along entire anterior edge.Columellar muscle thick, with ~1.25 whorls in length.
Circulatory and excretory systems (Fig. 4G).Pericardium spanning ~1/5 of total renal cavity area.Auricle pyriform, wall thin, translucent; ventricle large and rounded (~larger than auricle), triangular in shape, with thick walls.Aorta bifurcate immediately after leaving ventricle; posterior aorta following visceral mass close to stomach; anterior aorta crossing diaphragmatic sep- tum anteriorly.Anterior aorta running anteriorly along whole length of posterior esophagus, crossing gland of Leiblein in mid-esophagus, with branches forming sinus surrounding nerve ring.Anterior aorta bifurcating anteriorly to nerve ring; one branch following anteriorly to pedal ganglia as pedal aorta, another branch accompanying anterior esophagus, following anteriorly to buccal mass and odontophore.Kidney bearing ventral and dorsal lamellar lobes similar in shape; nephridial gland situated on dorsal side of membrane between renal cavity and pericardium; renal aperture as slit in membrane between pallial and renal cavities, flanked on its right side by transversal folds, longitudinal to roof of pallial cavity.Part of intestine running longitudinally on inner side of kidney, ventrally adhered to its membrane  Digestive system (Figs 3A-E, 4A-E).Rhynchostome as small longitudinal slit, located between and below cephalic tentacles.Proboscis straight, of moderate length (~2/3 of haemocoel length), with thick muscular walls bearing 2 lateral grooves.Pair of proboscis retractor muscles originating in ventral posterior wall of proboscis; series of short lateral muscle fibres connected to inner walls of haemocoel.Mouth small, circular.Odontophore long, slender (~same length as proboscis), pair of odontophore cartilages dorsally concave, fused anteriorly at ~1/4 of total cartilage length.Series of transversal muscle fibres connecting odontophore tube with anterior esophagus; superficial circular muscles (m3) enveloping entirely odontophore, except for most posterior end; horizontal muscle (m6), originating on ventral surface of odontophore Male genital system (Fig. 4F).Testis brownish, occupying all whorls of visceral mass, except for last one; surrounding apically entire length of digestive gland.Visceral vas deferens running from testis.Seminal vesicle coiled, located on mid-ventral region of last whorl of visceral mass; vas deferens narrow, simple, running along ventral wall of kidney.Prostate thin and long, tubular, located along right side of roof of pallial cavity, next to rectum and ~equal in width.Penis long, close to head-foot, ~circular in transverse section; penis becoming narrower at middle of its length, terminating in extension of ~1/2 of total penis length; duct of penis linear, simple.Female genital system (Fig. 2B).Ovaries brownish, with same texture and length as testis.Female cement gland opening at ~1/2 from anterior edge of foot, forming somewhat elongated and deep sac of ~same depth as foot thickness, recurved anteriorly.Pallial oviduct not observed.
Nervous system (Fig. 5A-B).Nerve ring highly concentrated, occupying ~1/6 of total haemocoel area, surrounding mid-esophagus posteriorly.All commissures internal.Cerebral ganglia bean-shaped, occupying ~1/2 of total nerve ring volume, right ganglion slightly larger and more dorsal than left ganglion, its posterior halves broadly connected with each other; pair of lateral tentacular nerves following anteriorly to pedal aorta.Pleural ganglia as pair of bulges ventral to cerebral ganglia, strongly attached to these; left pleural ganglia emitting thick nerve accompanying proboscis anteriorly.Pedal ganglia anterior, elongated, ~1/2 of total nerve ring volume; bearing anterior nerves; right pedal ganglion slightly larger and dorsal than left pedal ganglion.Buccal ganglia circular, small, ~1/5 of cerebral ganglia and dorsal to these, emitting pair of cerebro-buccal nerves, following anteriorly to anterior aorta.Supra-esophageal ganglion posterior to cerebropleural ganglia complex, slightly larger than buccal ganglia, emitting thick osphradial nerve.Subesophageal ganglion as ventral bulge in left cerebro-pleural ganglia complex emitting thick pallial-siphon nerve.Pair of vitreous statocysts with one anterior and associated with right pedal ganglion; and one posterior, associated with left pedal ganglion.
Genus Hemipolygona Rovereto, 1899 Diagnosis.Shell extremely nodulose with blunt to sharp nodes where axial ribs cross spiral cords, especially on shoulder angulation and central cord, but also on base of shell and subsutural ramp; columella bearing up to 3 weak to strong folds medially; outer lip crenulated, marked internally by several beaded lirae; siphonal fasciole and pseudoumbilicus usually present.(Snyder, 2000) (Figs 6-9) Distribution.Espírito Santo to São Paulo states, southeast coast of Brazil.
Head-foot (Fig. 7A-B).Colour cream in fixed species, Head prominent, small (width ~1/4 of adjacent width of head-foot), cephalic tentacles blunt and of medium size (length ~same as anterior width of head), situated very close to each other, bases lying side by side.Eyes dark, small, rounded, situated in middle region of outer edge of tentacles.Foot short, rounded, its anterior region bifid.Pedal gland as shallow median anterior slit, with anterior furrows extending along entire anterior edge.
Circulatory and excretory systems.Not analysed.7C-D, 8, 9A-C).Rhynchostome as transversal slit, located slightly below right cephalic tentacle.Proboscis straight, of moderate length (~2/3 of haemocoel length), with thick muscular walls; strong proboscis retractor muscles originating in right ventral posterior wall of proboscis; laterally to proboscis, series of short muscle fibres connect to inner walls of haemocoel.Mouth small, circular.Odontophore long, very slender (~1/2 total length of proboscis).Pair of odontophore cartilages dorsally concave, fused anteriorly at ~1/5 of total cartilage length; series of transversal muscle fibres connect odontophore tube with anterior esophagus, and series of thin muscle fibres, superficial circular muscles (m3) entirely envelope odontophore, except for most posterior end.Horizontal muscle (m6), on ventral surface of odontophore cartilages, except for most posterior region (~1/6 of total odontophore length).Pair of odontophore retractor muscles (m2) originating from posterior end of odontophore cartilages, near to radular sac, inserted in inner wall of proboscis.Pair of accessory odontophore retractor muscles (m2a), originating from inner surface of proboscis, near origin of m2, running adjacent to esophagus, insertion enveloping radular sac; pair of secondary, long branches of m2a accompanying anterior aorta reaching up to posterior level of nerve ring.Pair of dorsal tensor muscles of radula (m4) originating from posterior dorsal end of odontophore, covering its dorsal surface, inserting m2a.Pair of auxiliary dorsal tensor muscles of radula (m5) originating from posterior end of odontophore, covering its ventral    oral tube.Accessory salivary glands absent.Middle esophagus short; duct of gland of Leiblein short, situated after nerve ring.Gland of Leiblein brownish, long, of ~same length as posterior esophagus, posterior end acute.Posterior and esophagus of ~same width.Inner wall of anterior esophagus with thin dorsal longitudinal folds, salivary ducts immersed in marked lateral folds.Stomach as wide sac with thin walls bearing many internal folds.Digestive gland dark brown, occupying all whorls of visceral mass, from apex to kidney/pericardium area, surrounding stomach and emitting two narrow ducts that discharge into stomach near esophagus and intestine apertures.Pallial portion of digestive system not analysed.

Digestive system (Figs
Male genital system (Fig. 9D).Testis brownish.Visceral and pallial portion of male genital system not observed.Penis long and thin, close to head-foot mass, ~circular in transverse section; at ~2/3 of its length penis becomes narrower (~1/4 diameter), terminating in short, blunt extension; duct of penis linear.
Nervous system (Fig. 9E-F).Nerve ring highly concentrated, occupying ~1/4 of total hemocoel area, surrounding mid-esophagus posteriorly.All commissures and internal.Cerebral ganglia bean-shaped, occupying ~1/3 of total nerve ring volume, of about same size, posterior halves broadly connected; pair of lateral tentacular nerves follow pedal aorta anteriorly.Pleural ganglia as pair of bulges ventral to cerebral ganglia, strongly attached to these; left pleural ganglia emits thick nerve that accompanies proboscis anteriorly.Pedal ganglia anterior, elongated, ~1/2 of total nerve ring volume, emitting anterior zigzag nerves; right pedal ganglion slightly larger and dorsal than left.Buccal ganglia subcircular, ~1/3 of cerebral ganglia and dorsal to these, emitting pair of nerves that form cerebro-buccal nerves, that follow anterior aorta anteriorly.Supra-esophageal ganglion posterior to cerebro-pleural ganglia complex, elongated, ~same volume as cerebral ganglion, emitting thick osphradial nerve.Subesophageal ganglion as ventral bulge in left cerebro-pleural ganglia complex that emits thick branching pallialsiphon.Pair of vitreous statocysts with one anterior and associated with right pedal ganglion, and one posterior, associated with left pedal ganglion.Ponder (1973) pointed out the anatomical similarity among members of the Buccinoidea, concluding that there are no consistent differences among the families; hence they could be treated as subfamilies (e.g.Buccininae, Fasciolariinae).However, later taxonomic studies (e.g.Bouchet and Rocroi 2005) recognized family entities within the superfamily Buccinoidea.The morphological results obtained in this study are in agreement with the diagnostic characteristics established by Fraussen et al. (2007) for Fasciolariidae.These are the multicuspidate lateral teeth, the straight shape of the rachidian teeth of the radula, the proboscis retractor muscle as a single or paired tuft of fibres, ducts of the salivary glands embedded in the esophagus wall, and the stomach without a posterior mixing area.

DISCUSSION
The taxonomy of fasciolariids is based on the shell and radula (e.g.Tryon 1880, Thiele 1929-1935, Vermeij and Snyder 2002, 2006), and taxonomic approaches based on soft-part anatomy are few.Anatomical data for the buccinoideans, particularly the stomach (e.g.Kosyan and Kantor 2013, Kantor 1996, Strong 2003), the anterior digestive system including the radula (e.g.Kosyan et al. 2009, Simone 1996) and the reproductive system (Fraussen et al. 2007), suggest that they are highly advanced Neogastropoda that lack accessory salivary glands and anal glands.
The accessory salivary glands and anal glands are synapomorphic to neogastropods (Ponder and Lindberg 1997, Harasewych 1998, Strong 2003, Simone 2011), although these organs are lacking in buccinoideans.Kantor and Fedosov (2009) asserted the dual appearance of the valve of Leiblein in Buccinoidea; therefore, this clade shares none of the previously hypothesized autapomorphies with other neogastropods; and in this case, Neogastropoda is a paraphyletic group.
Historically, the taxonomy of the subfamily Peristerniinae, especially that of Latirus, has been confused, because the genus was used indiscriminately to include several species, some of them doubtfully related.Latirus was initially considered to have a worldwide distribution.However, Vermeij and Snyder (2006) considered the known geographic range of the genus to be restricted to the western Indo-Pacific, and consequently raised several taxa previously considered as subgenera to genus rank (e.g.Hemipolygona) and proposed new genera (e.g.Pustulatirus, Turrilatirus).Vermeij and Snyder (2003) transferred several species to the genus Benimakia Habe, 1958, including Benimakia ogum, originally described in Latirus.These authors characterized Benimakia as high-spired fasciolariids with prominent axial ribs and a labral tooth at the end of the central cord of the outer lip.Benimakia ogum differs from other species of the genus in having a discontinuous beaded lira on the inner side of the outer lip (Fig. 1A, D), in this respect resembling Latirus (Vermeij and Snyder 2003) and Pustulatirus (Vermeij and Snyder 2006).Species included in Benimakia by Habe (1958) and Vermeij and Snyder (2003) occur in the western Pacific, with the exception of B. ogum, which putatively differs from other members of Peristerniinae related to Latirus in having a small labral tooth at the end of the basal cord.However, the presence of this tooth is questionable.A labral tooth is not mentioned in the original description by Petuch (1979), nor was it found in the present study (Fig. 1A-F).A pseudoumbilicus is also present, differentiating it from Benimakia, although it occurs in Pustulatirus.Therefore B. ogum clearly belongs to the genus Pustulatirus, in agreement with Landau and Vermeij (2012) and Lyons and Snyder (2013).
Hemipolygona beckyae was originally included in Latirus by Petuch (1979), and was later allocated to Hemipolygona by Snyder (2003), as agreed to by Vermeij and Snyder (2006), due to the highly nodulose shell with a deep slit-like pseudoumbilicus and whitish spiral cords (Fig. 6A-G).
The morphology of the two species is similar and in accordance with other descriptions of fasciolariids (Fraussen et al. 2007, Kosyan et al. 2009, Couto and Pimenta 2012), with the main differences occurring in the anterior digestive and male reproductive systems.Details of the anatomy, histology and ultrastructure of the anterior digestive system (including the radula) have been noted as useful traits for phylogenetic analyses (Ponder and Lindberg 1997), and the anterior structures of the foregut are generally used to distinguish neogastropod families (Fraussen et al. 2007).A recent phylogenetic analysis based on comparative morphology (Simone 2011) consistently recovered all the major caenogastropod clades.
The rhynchostome occurs as a lip-like slit bearing longitudinal lamellar folds, which may be longitudinal to the adjacent head-foot mass as in P. ogum (Fig. 3A) or transverse, although located slightly to the right side of the animal, not between its cephalic tentacles as in H. beckyae (Fig. 7C).Golding et al. (2009a) studied the snout and proboscis morphology in species belonging to 33 caenogastropod families, among them a buccinoidean (Columbellidae), but included no fasciolariid.In their study they reported the ventro-lateral insertion of the proboscis retractor muscles as occurring in all Neogastropoda, and the presence of aortic muscles that flank the aorta in the anterior esophagus; both characters are confirmed for members of Fasciolariidae so far studied.On the other hand, Goulding et al. (2009b) studied the anatomy of odontophoral cartilages in Caenogastropoda through the use of micro-CT scanning, although none of the species studied were buccinoideans.This method allows observation of the cartilages in their natural orientation, without anatomical dissections that would otherwise cut or displace structures.Despite the methodological differences, Pustulatirus ogum and H. beckyae showed a close resemblance to the muricoidean studied by Golding et al. (2009b) in having greatly elongated anterior cartilages and lacking subradular cartilages.As noted by these authors, the Neogastropoda possess the most dramatic modifications of the plesiomorphic odontophoral cartilage morphology.Also, the morphology of the odontophoral cartilages may be conserved within families and superfamilies: hence the resemblance of these structures among the Fasciolariidae (Couto and Pimenta 2012) and to other buccinoideans (Simone 1996(Simone , 2011) ) The lateral teeth of the radula of the Peristerniinae observed in this study and in Leucozonia (Couto and Pimenta 2012) have the innermost cusp (defined as a 'denticle' by Bullock, 1974) as a small projection at its base, next to the rachidian tooth.This projection may vary considerably in size and shape.In Leucozonia, it is reduced or even absent (Couto and Pimenta 2012: 1Q, 5G and 9O), while in P. ogum (Fig. 1A-J) and Hemipolygona beckyae (Fig. 6J-K) it is developed, although smaller than the outer cusps, and recurved outward.All species of Peristerniinae studied by Bullock (1974) and Bandel (1984) have this same conformation, and Bullock (1974) also noted that this feature distinguishes Latirus and related species from Leucozonia.
Within the Fasciolariidae, members of Peristerniinae possess fewer cusps of the lateral teeth than members of other subfamilies (Bandel 1984, Taylor and Lewis 1995, Snyder and Bouchet 2006).However, recent findings from moderate/deep-sea regions of the Indo-West Pacific led to the description of several species and genera that deviate from this pattern (e.g.Amiantofusus, Fraussen et al. 2007;Chryseofusus, Hadorn et al. 2008;and Angulofusus, Fedosov and Kantor 2012).All aforementioned genera have the radula closer to Peristerniinae than to Fusininae.Table 1 lists relevant radular features of P. ogum and H. beckyae, as well as those of other members of Peristerniinae compiled from the literature.
According to Fraussen et al. (2007), the ducts of the salivary glands embedded in the esophagus wall is diagnostic for the family; this feature was reported for Latirus polygonus, but not for Pustulatirus mediamericanus, Turrilatirus turritus, Peristernia nassatula, P. ustulata, Opeatostoma pseudodon and Tarantinae lignaria studied by Kosyan et al. (2009), and therefore a reinvestigation is needed in these species.In the species  studied here and those reported by other authors (e.g.Marcus and Marcus 1962, Couto and Pimenta 2012, Fedosov and Kantor 2012), this feature also occurs.
Hemipolygona beckyae has a single powerful proboscis retractor muscle, which emerges posteriorly and ventrally from the proboscis (Fig. 8A).Pustulatirus ogum has a pair of muscles (Fig. 3B).In the species studied by Kosyan et al. (2009), all fasciolariids but Latirus polygonus and Fusinus tenerifensis have a single muscle, while in the buccinids multiple fibres occur posteriorly to the proboscis.Golding et al. (2009a) distinguished different proboscis types among caenogastropods, although they studied only one species of Buccinoidea, the columbellid Euplica scripta, which possesses two ventro-lateral proboscis retractors, resembling those of H. beckyae.Both fasciolariids have the proboscis retractor passing outside the nerve ring and originating in the posterior hemocoel floor, near the diaphragm septum.Kantor (2003) distinguished species of Fasciolariidae from other buccinoideans by the low relief of the folds on the inner stomach wall; presence of transverse striations on the low longitudinal fold; absence of clear differentiation of the gastric chamber into dorsal and ventral parts; absence of a posterior mixing area; and a shallow lateral sulcus.Despite this thorough examination of representatives of the three subfamilies (Fasciolariinae: Fasciolaria lilium, F. filamentosa; Fusininae: Fusinus nicobaricus and Peristerniinae: Leucozonia nassa), Kantor (2003) noted the difficulties of examination and the necessity of specially preserved specimens for stomach analysis, although the differences observed are likely due to phylogenetic relationships.While both P. ogum and H. beckyae have stomach morphology similar to the fasciolariids cited by Kantor (2003), species-level differentiation is unlikely.
Both species, as well as Leucozonia (Marcus andMarcus 1962, Couto andPimenta 2012), have penises with terminal tapering.In Leucozonia (Couto and Pimenta 2012: Figs 4E and 8F) and H. beckyae (Fig. 9D) the terminal extension extends for more than half of the total penis length, while in P. ogum it extends less than half of its length (Fig. 4F).
Several morphological characters occur in both species and also occur diffused among other fasciolariids (Fraussen et al. 2007, Kosyan et al. 2009, Couto and Pimenta 2012).These include the outline of the gill lamellae, the length and anterior fusion of the odontophore cartilages, and the extension of the anus to the edge of the pallial cavity.For this reason, the soft-part traits of Latirus and related species studied so far do not allow a precise anatomical diagnosis.Table 2 lists the main differentiating characteristics.
A, head-foot mass in dorsal view; B, longitudinal section of head-foot mass, female; C, roof of pallial cavity in ventral view, male; D, transverse section of roof of pallial cavity.

Table 1 .
-Main comparative radular features of the Peristerniinae based on our data and those of 1 Couto and Pimenta (2012), 2Kosyan et al.  (2009),3 Bandel (1984)and4 Snyder and Bouchet (2006).Table2.-Comparison between major anatomical features among Peristerniinae species based on this study and literature.Data was extracted, when available, from 1