Re-characterization of the Red-lip Megalobulimus ( Gastropoda : Strophocheilidae ) from Peru with description of a new species

Megalobulimus K. Miller, 1878 is a genus of land snails that includes the largest living snails in the Neotropics. The main goal of this paper was to review all species of Megalobulimus that have a red lip, and which are distributed in Peru. We carried out a detailed description of their shells and soft parts, and conducted a multivariate analysis on their shells and geographic distribution. There are two species reported from Peru, Megalobulimus capillaceus (Pfeiffer, 1855) and Megalobulimus separabilis (Fulton, 1903). Megalobulimus capillaceus is known to occur in three regions - San Martin, Huanuco and Cusco - but the Cusco population is undoubtedly different from all remaining populations, and is recognized herein as a new species, Megalobulimus florezi sp. nov. This species has a more elongated shell, penis club-shaped, epiphallus longer, and free oviduct longer than M. capillaceus. By contrast, the male genitalia of M. separabilis is filiform and does not present an external diverticulum in the free oviduct.

Description.Living animals: Color: orange-brown; head and foot equal in color.Two lobulated buccal palps.Gonopore posterior to right omatophore.Shell (Figs 2-4, Table I): conic, small (up to 66 mm tall).Wider region above middle shell height.First and second whorls flattened.Third and fourth whorls with thinner riblets, from suture to suture.Small granulations with spiral disposition between each riblet, appearing from second whorl and reaching corporal whorl.Spire about 1/7 height of shell.Sutures deep.Periostracum between orange and brown.Aperture elliptic, red peristome.Umbilicus covered by outer lip.Digestive system : Buccal mass elliptic, highly muscular, with posterior region slightly later- Middle segment (crop) expanded with thicker ridges, except in middle zone, which is smooth.Posterior segment with many thin ridges and expansion seeming slightly cropped.Posterior segment receiving duct of anterior digestive gland.Esophagic typhlosole arising from duct of anterior digestive gland, reaching middle of stomach, increasing in thickness, with blunt end.Thin ridge arising from same duct, running parallel to esophagic typhlosole.Both end up in middle region (gastric shield) of stomach.Stomach reniform, muscular.Posterior digestive gland connected to stomach through duct terminating   middle of septum.Triangular kidney, similar length to septum (6/7).Ureters absent.Nephrostome on anterior third of right side of kidney.Posterior end of kidney does not reach posterior end of ureteric groove, which is flanked by septum and rectum.Heart covered by pericardium, sited anterior to kidney, which is half its length.Reproductive system : Ovotestis attached to posterior digestive gland; with three to five conducts ending on hermaphroditic duct.Central portion of hermaphroditic duct dilated, stowed.Distal portion ending at talon.Talon attached to annex glandular sac, with thick wall; two chambers internally, one connected to hermaphroditic duct; chambers converging to common duct into which albumin gland and annex glandular sac deposit their secretions.Annex glandular sac piriform, attached to concave region of albumin gland; inner surface rough with small concavities.Albumin gland reniform, variable in size, creamy color.Spermoviduct cylindrical, muscular, slightly curved.Prostatic gland extending over dorsal surface of spermoviduct, pink in color with transversal folds.Ventral surface of spermoviduct slightly translucent.Internally, with two parallel regions; one with surface with protrusions arranged like irregular plates, other with small transversal folds.Both regions have creamy color.Internally, prostatic gland with many small ducts that end independently on collector channel that runs along the gland and continues with deferens duct, which is hidden at the beginning, then appears externally on free oviduct.Free oviduct muscular with external bulb-like diverticulum located in middle of its length and opposite to spermatolitic bursa duct.Inner surface of oviduct with many wide folds, some of which reach diverticulum.Vagina longer than free oviduct.Outer surface of vagina similar to free oviduct.On inner surface, proximal region with thicker folds than those of free oviduct, but thickness of these folds decreases as they arrive at genital atrium.Spermatolitic bursa piriform, variable in size.Deferens duct thin, ending suddenly on epiphallus, which lies on penis.Epiphallus with two short flagella of similar or different lengths.Inner surface of epiphallus with small granulations; with thick fold (pilaster) that originates at its proximal end and then divides in two at end of first fifth of epiphallus length.Two new folds have homogenous surfaces, their thickness decreases as they approach distal end.In some cases, these folds converge in a single fold close to distal end.Another fold that begins from the middle of epiphallus, terminates at penis.This fold wider but low, almost at the same level of the epiphallus inner surface.Penis muscular, cylindrical, threefold epiphallus length.Retractor muscle with unique insertion at variable position on penis, apical or subapical.Its length is one third of penis length.Internally, penial folds anastomosed to finish at proximal third; these folds have network aspect, their thickness decreases to a low relief.Genital atrium almost indistinguishable.Pedal gland (after BORDA et al. 2010;Fig. 11): Triangular, borders undulated, depressed, length (23.45 mm) spans visceral cavity in contracted specimen.Anterior third with width constant.Dorsal side of anterior third covered by thin connective tissue, then free to posterior end; posterior third thin, blunt, attached to foot by few connective bundles arising from lateral borders.Gland capsule semi-transparent.Lumen reduced, spanning entire gland length.Glandular body prominent with cream color, smooth surface; it dangles from capsule roof by projections retained to dorsal surface; medium longitudinal groove spans its entire length.Groove depth shallower in posterior region.Inside the glandular body, glandular acinous separated from each other by capsule roof projections.Capsule floor smooth with small lifting along middle line of anterior portion coinciding with gland body groove.
Distribution (Fig. 21): Distributed in the San Martín and Huánuco regions, Peru.In San Martín, it is present throughout almost the entire region, whereas in Huánuco, it is distributed only in the northern part of the region, which is humid.It is important to note that SMITH (1894) reported this species for Yurimaguas in the Loreto region.Yurimaguas is only a few kilometers from the San Martín region; for this reason it is likely that the distribution of M. capillaceus may reach Loreto.
Habitat.Megalobulimus capillaceus inhabits dry forest and premontane humid forests, being also common in gardens and backyards next to the forest.Individuals of this species are very conspicuous during the rainy season, whereas in the dry season they remain buried.
Remarks.The thickness of the peristome differs between the material examined and the types of M. capillaceus from the NHMUK (NHMUK 1855 -20130002) (Fig. 22).This feature is variable and depends on the age of the individuals.Otherwise, the proportions, shape, and sculpture of the shell are the same.Megalobulimus capillaceus has often been confused with M. oblongus (Müller, 1774) and M. musculus, both from Argentina, and with M. separabilis.It is also similar to M. intertextus (Pilsbry, 1895), known from Brazil and Bolivia, and which can be found in dry habitats, contrasting with the humid habitats of M. capillaceus.Furthermore, M. capillaceus differs from M. intertextus, M. oblongus, and M. musculus in the shape of the shell.In the latter three species, the shell is more conical and has slender spires when compared with the shell of M. capillaceus.BEQUAERT (1948) reported this species from Peru, Bolivia, Brazil and Paraguay.The proportions of the specimen described from Paraguay and which is deposited at the FLMNH (UF 166264) are similar to the proportions of the typical M. capillaceus from Moyobamba, but the external shell features of that specimen are more similar to M. musculus.We therefore assign that specimen to M. musculus.The same holds true for the material from the FMNH (#25925) and FLMNH (UF 022143) from Brazil.The material from the FLMNH (UF 022143) has been described from "Marigua" (sic FLMNH; Maringá), a locality in the state of Paraná, Brazil, which is next to Paraguay.The material previously attributed to M. capillaceus reported from Bolivia and deposited at the FLMNH and FMNH, is actually similar to M. intertextus and M. florezi sp.nov., and is herein assigned to M. intertextus.The morphological differences between M. capillaceus and M. separabilis are provided below, under M. separabilis.From the above, we conclude that M. capillaceus Megalobulimus florezi sp.nov.Diagnosis.Shell medium-sized, conic, with riblets; clubshaped penis, internal penial folds divided at the proximal third.Free oviduct longer than vagina.Kidney with triangular shape and is 2/3rds of pulmonary septum length.
Description.Living animals: Head and foot with brown color.With pair of bucal palps with lobulated border.Gonopore posterior to right omatophore.
Shell (Figs 5-7, Table I): Conic and small (up to 70 mm tall): Wider region in middle shell height.First and second whorls flattened.Third and fourth with thinner riblets, from suture to suture.Between riblets, small granulations with spiral disposition, appearing from the 2.6 whorls and reaching corporal whorl.Spire about 1/5 shell height.Sutures deep.Periostracum between orange and brown.Umbilicus totally covered by outer lip.Digestive system (Figs 27 and 28): Buccal mass elliptic, highly muscular with posterior region slightly laterally expanded, radular pouch ventral.Retractor muscle inserted ventrally to buccal mass, anteriorly to radular pouch.Esophagus with three segments of same size: anterior, middle and posterior.Anterior segment with taller ridges and attached salivary glands.Ducts of salivary glands inserting dorso-posterior to buccal mass.Middle segment (crop) expanded, with thicker ridges, except in middle zone, smooth.Posterior segment with many thinner ridges and expansion seeming slightly cropped.Posterior segment receiving duct of anterior digestive gland.Esophagic typhlosole arising from duct of anterior digestive gland, reaching middle of stomach, increasing in thickness, with blunt end.Thin ridge arising from same duct, running beside esophagic typhlosole.Both end up in gastric shield.Stomach reniform, muscular.Posterior digestive gland connected to stomach through duct that ends next to gastric shield.Near intestine, internal surface with wavy ridges.These ridges are thinner than those in M. capillaceus.Stomach with central surface smooth in which both esophagic and intestinal typhlosole converge.Thicker walls near esophagus and intestine.Intestine with three segments.First segment extending between stomach and pre-rectal valve.Externally, prerectal valve appears as two bulges with lobed edges.Intestinal typhlosole with folded surface, blunt end, arising from duct of posterior digestive gland, ending close to pre-rectal valve.Many ridges parallel to intestinal typhlosole, two thicker and near typhlosole, one ending close to terminus of typhlosole, other running to pre-rectal valve.Middle segment extending from prerectal valve, with oblique ridges that converge in a longitudinal ridge in V-disposition.Distal region (rectum) is the longest; with ridges in V-disposition, opening in the anus beside pneumostome in mantle collar.Pallial complex (Fig. 26): Septum continuous with anterior end of kidney, covered by network of vessels (rete mirabile-like) that expands to both sides of septum, partially covers kidney and pericardium (heart).Pulmonary network with two principal and longitudinal vessels.Vessel nearest to septum with major caliber, more ramified than other longitudinal vessels, 1/4th septum length.Second nearest originates at anterior end of cavity, ends on anterior end of kidney.Triangular kidney, 2/3rds septum length.Nephrostome on anterior third of right side of kidney.Posterior end of kidney does not reach the posterior end of ureteric groove, which is flanked by septum and rect.Heart covered by pericardium, sited anterior to kid- Gonad attached to posterior digestive gland, with three to five ducts that lead to hermaphroditic duct.Central portion of hermaphroditic duct dilated, stowed.Talon attached to annex glandular sac, receiving distal end of duct.Internally, with two chambers, of which one connects with hermaphroditic duct.Both chambers converge on a common duct through which albumin gland and annex glandular sac deposit their secretions.Annex glandular sac piriform, attached to concave region of albumin gland, inner surface rough with small concavities.Al-bumin gland reniform, variable in size, creamy color.Spermoviduct cylindrical, muscular, slightly curved.Prostatic gland extending over dorsal surface of spermoviduct, pink, with transversal folds.Ventral surface of spermoviduct slightly translucent.Internally, with two parallel regions; one with protrusions arranged like irregular plates that change their appearance to folds at the distal third; the other with small transversal folds.Both regions creamy in color.Internally, prostatic gland with many small ducts ending independently in collector channel that runs along gland, continues with deferens duct hidden at Free oviduct with external bulb-like diverticulum located at mid-length, opposite to spermatolitic bursa duct.Inner surface of oviduct with many wide folds, some of which continue in diverticulum.Free oviduct longer than vagina.Vagina outer surface similar to that of free oviduct.Proximal region of its inner surface with thicker folds than those of free oviduct, reducing their thickness as they extent toward genital atrium.Spermatolitic bursa piriform, variable in size.Deferent duct ending suddenly in epiphallus, which lies over penis.Epiphallus with two short flagella, the same length or not.Epiphallus inner surface with small granulations; with thick fold (pilaster) on proximal region and then divided in two at the end of first fifth of epiphallus length.The two new folds with homogenous surfaces decrease in thickness as they approach distal end.These folds may converge to a single fold close to distal end or reach distal end independently.Another fold from middle of epiphallus to its end, wider but low, almost at the same level of epiphallus surface.Penis muscular, club-shaped, two times length of epiphallus.Retractor muscle half length of penis, with unique insertion to penis at variable position, which may be apical or subapical.Internally, penis distinguished by abrupt increase in fold number and rough surface.Some penial folds anastomosed to finish at proximal third; these folds have network aspect; also, on the distal half, penial folds increasing in thickness but then decreasing to low relief.Genital atrium almost indistinguishable.Pedal gland (Fig. 25): Depressed, triangular form, undulated borders, length (23.45 mm) spans visceral cavity in contracted specimen.Anterior third constant in width, covered by thin connective tissue, and then free.Posterior third thin, blunt, attached to foot by few connective bundles from lateral borders of gland.Gland capsule semi-transparent.Habitat.Humid tropical forest but also in gardens and backyards.
Etymology.This species is dedicated to Dr. Ángel Flórez (UNSAAC, Cusco, Peru) for his contribution to Peruvian malacology.
Diagnosis.Body mass with yellowish-orange coloration, shell small-sized, conic with gray and brown coloration; fili- a ridge arising from this duct, reaching middle of stomach (gastric shield); at the same time, typhlosole increases its thickness and has blunt end.Stomach reniform, muscular.Posterior digestive gland connected to stomach through a duct that ends next to gastric shield.Internal surface near intestine with wavy ridges.Center of stomach with smooth surface where both the esophagic and intestinal typhlosole converge.Thicker stomach walls near esophagus and intestine.Intestine with three segments, three times esophagus length.First segment extends between stomach and pre-rectal valve.Externally, pre-rectal valve appears as two bulges with lobed edge.Internal surface of this segment with intestinal typhlosole with folded surface, with blunt end, arising from the duct of posterior digestive gland and ending close to pre-rectal valve.This typhlosole is thicker than in M. capillaceus and M. florezi sp.nov.Many ridges parallel intestinal typhlosole, two of which are thicker; one with the same size of typhlosole; other running to pre-rectal valve.Middle segment extends from pre-rectal valve, with oblique ridges that converge in a longitudinal ridge in V-disposition.Rectum longest, has ridges with V-disposition, opens in anus beside pneumostome in the mantle collar.Pallial complex (Fig. 35): Septum continuous to anterior end of kidney, covered by network of vessels (rete mirabile-like) that expands to both sides of septum and covers kidney and pericardium (heart).Major expansion of this network in pulmonary region.Pulmonary network with two principal and longitudinal vessels.Vessel nearest to septum with major caliber, more ramified than other, 1/4th septum length.Second nearest originates at second third of septum and extends to kidney.Triangular kidney, 2/3rd septum length.Nephrostome sited on anterior third of right side of kidney.Posterior end of kidney does not reach posterior end of ureteric groove which is flanked by septum and rectum.Heart covered by pericardium, sited anterior to kidney.Reproductive system : Gonad attached to posterior digestive gland, with three to five ducts that end on hermaphroditic duct.Central portion of hermaphroditic duct dilated, stowed.Talon spherical attached to annex glandular sac, receives distal end of duct.Comprised of two chambers, one of them with connection with hermaphroditic duct.Both chambers converge on a common duct through which albumin gland and annex glandular sac deposit their secretions.Annex glandular sac piriform, attached to albumin gland, inner surface rough with small concavities.Albumin gland reniform with variable size, creamy color.Spermoviduct cylindrical, slightly curved.Prostatic gland with transversal folds, pink coloration.Ventral surface of spermoviduct slightly translucent.Internally, with two parallel regions of creamy color, one with surface folded, other having smooth surface.Internally, prostatic gland with many small ducts which end independently on collector channel that continues with the deferens duct, which is hidden at its origin.Muscular free oviduct without external diverticulum.Oviduct inner surface with wide folds (10 to 14).Thicker folds in proximal half that reduce lumen; at this level, with internal diver-ticulum, and folds continue inside it.Free oviduct longer than vagina.Outer surface of vagina similar to free oviduct.Proximal region of inner surface presents thinner folds than those of free oviduct, but these folds reduce their thickness as they arrive at genital atrium.Spermatolitic bursa piriform, variable in size.Externally, no clear demarcation between deferens duct and epiphallus; instead, there is slight expansion at end of deferens duct that continues to penis, folded over the latter.Internally, deferens duct with thin folds of oblique disposition; likewise, there are longitudinal folds that extend to the first half of the distal expansion (epiphallus).Epiphallus differentiated by presence of two longitudinal folds that increase their thickness at their middle and and run parallel up to the epiphallus-penis limit; surface with small granulations.Epiphallus almost 1/6th penis length.Epiphallus diameter constant, without flagellum.Penis bottle-shaped.Retractor muscle 1/3rd penis length, attached to penis only by a bundle of variable position, apical or subapical.Penis inner surface distinguished by abrupt increase of folds and rough surface.Penial folds divided, finishing at proximal third; these folds have network aspect, also their thickness decreases to a low relief.Genital atrium almost indistinguishable.Pedal gland (Fig. 34): Triangular form, undulated borders, depressed, length (23.6 mm) spans visceral cavity in the contracted specimen.Anterior third with constant width, covered by thin connective tissue, then free to its posterior end.Its posterior third thin, blunt, retained to foot by few connective bundles from its lateral borders.Gland capsule not transparent.Lumen reduced, its width almost half that of gland, spans entire length of gland.Glandular body with cream color, smooth surface, obliterates lumen, dangles on cap- Distribution (Fig. 21).This species is found only in the Ambo province in the upper Huallaga basin, in two localities, Ambo and Tomayquichua.BEQUAERT (1948) did not find differences between them, and we agree with him.The specimens examined by us have the same characteristics of the syntype of M. separabilis (NHMUK 1903.11.20.39) (Fig. 24).Megalobulimus separabilis is clearly different from the other redlipped Megalobulimus from Peru studied herein.Differences are in the shell (slender and gray coloration), epiphallus (not externally conspicuous) and free oviduct with an internal diverticulum.Megalobulimus separabilis is also considered endemic.The shell of M. separabilis is very different from those of other Megalobulimus, but some features of the genital system of this species are shared with other species of the genus.The shape of the penis of M. separabilis is similar to that of M. parafragilior, but M. parafragilior has a prominent epiphallus and an external diverticulum on the free oviduct (LEME & INDRUSIAK 1990).The internal diverticulum is a character that has not been described before for any species of Megalobulimus.Likewise, the pedal gland of M. separabilis is different from the other red-lipped Megalobulimus described herein because the glandular body dangles on capsule on its lateral-dorsal surface; for this reason, the width of the lumen is reduced.

Multivariate analysis
The shells of the three red-lipped Megalobulimus species from Peru were differentiated by PCA using seven variables.The first component accounted for 60.67% of the variance and the variables that best explain this variation are lateral width (0.964) and shell width (0.918).The second component accounted for 29.53% of the variance with shell height (0.977) and position of major diameter (0.737) best explaining this variation (Table II).The scatter plot of the two first principal components shows that M. separabilis occupies a different morphospace than the other species.This morphospace is wider than that of M. separabilis.Megalobulimus florezi sp.nov.has a morphospace very close to M. capillaceus but is better discriminated by its slender shell with a larger spire (Fig. 44).The populations of M. capillaceus from the two regions of Peru (Huánuco and San Martin) overlap.Individuals from Huánuco are inside the morphospace of the San Martín population and have great similarity with the largest individuals from San Martín.

DISCUSSION
The main characters that provide information for differentiation among species are shell characters, the internal surface of the epiphallus, the internal folds of the penis, the free oviduct and the distribution of veins in the pulmonary cavity.Several authors have questioned the validity of shell information owing to the plasticity of conchological characters (KEMP & BERTNESS 1984, KOOL 1993, EMBERTON 1995).However, the PCA analysis allowed us to discriminate among the three species studied herein, based on shell characteristics.BORDA et al. (2010) described the pedal gland of several species of Megalobulimus, which can be clustered in two groups based on its capsule.Species of the first group, such as M. capillaceus, have a transparent capsule.We found that M. florezi sp.nov.belongs to this group because it displays a transparent capsule.This character is also shared with M. lorentzianus (HYLTON-SCOTT 1939).In the second group, the pedal gland has a thicker capsule.One example of a species in this group is M. lichtensteini; M. separabilis has this characteristic too.The diverticulum of the free oviduct described herein for both M. capillaceus and M. florezi sp.nov.was also described for other species of Megalobulimus, although by three different names; BAKER (1926)  referred to it as the "short diverticulum" in M. oblongus, HYLTON-SCOTT (1939), as the "bursa hastae" in M. lorentzianus, and SIMONE & LEME (1998) as the "free oviduct appendix" in M. riopretensis and M. mogianensis.Although this structure was described on each of these species, its function remains unknown (BAKER 1926, HYLTON-SCOTT 1939, LEME 1973, THOMÉ et al. 1994)

Figure 21 .
Figure 21.Distribution Map of red lip Megalobulimus from Peru.

Figure 44 .
Figure 44.Discrimination of Peruvian red lip Megalobulimus from the principal component analysis (PCA) of 7 quantitative shell characters.The percentage of variation accounting for each principal component (PC) is given in parentheses.Two populations of M. capillaceus are distinguished by letters.M. capillaceus_H from Huánuco and M. capillaceus_SM from San Martín.

Table I
. Measurements of specimens of Megalobulimus capillaceus, M. florezi, and M. separabilis.Variables M. capillaceus (n = 80) M. florezi sp.nov.(n = 18) M. separabilis (n = 40) Restricted to northern Cusco, Peru.Was reported only in the humid rain forest of La Convención and Calca province.In La Convención it is found from Huayopata to Quellouno, near the Vilcanota River, which is part of the Urubamba basin.In Calca, it is found only in Lares Valley, near La Convención.

Table II .
Component Matrix for the variables evaluated in the ACP for the red lip Megalobulimus.
(RAMÍREZ et al. 2012)internal diverticulum in the free oviduct of M. separabilis is considered a new character state, but it is not clear whether this state is ancestral or derived.The shape of the two folds of the epiphallus of both M. capillaceus and M. florezi sp.nov. is very similar to the state found in other species of Megalobulimus, as in M. oblongus, another red-lipped species; by contrast M. separabilis has very different folds, both in shape and disposition.In a molecular phylogeny of Megalobulimus from Peru(RAMÍREZ et al. 2012), M. capillaceus formed a monophyletic group with M. oblongus, but not with M. separabilis, showing that shell lip color is a homoplastic character.