Phenotypic features of Helicina variabilis ( Gastropoda : Neritimorpha ) from Minas Gerais , Brazil

Helicina variabilis Wagner, 1827 (Neritimorpha, Helicinidae) is redescribed based on a sample collected in Nanuque, northern Minas Gerais, Brazil. The species description, previously based only on the shell, is expanded to the phenotypic features. The study revealed absorption of the internal shell whorls; a diaphragm muscle connected to the floor of the pallial cavity; a monoaulic pallial oviduct, with the female genital aperture inside the anal aperture, and the lack of a seminal receptacle and provaginal sac; and the pleural ganglia of the nerve ring connected with each other. The significance of these findings is discussed in the light of current taxonomic and phylogenetic knowledge. Key-Words. Atlantic Rainforest; Helicinidae; Anatomy; Morphology; Phenotype.


INTRODUCTION
With the main goal of filling a gap in knowledge of neritimorph phenotypic features, a more complete anatomical description of Helicina variabilis Wagner, 1827 is provided herein.Specimens of this species were collected during an expedition by the naturalist and conchologist José Coltro Jr. and his team to the region of Nanuque in northern Minas Gerais state, which recovered many land snails, most of them reported by Simone & Salvador (2016).This is another example of how much remains to be discovered in a region of Brazil that is historically the most explored and exploited, and in which the natural areas, mainly remnants of the Atlantic Rainforest, are highly threatened, lacking information that allows their preservation.
Knowledge of the helicinids as a whole is slight, although it is a relatively speciose group (up to 750 species; Richling & Glaubrecht, 2008), particularly in the Americas (Richling, 2004(Richling, , 2014)).Anatomical features in particular have been very useful in comparative analyses, such as taxonomy and phylogeny, at all levels, from closely related species (e.g., Richling, 2009;Richling & Bouchet, 2013) to wider systematic studies, specifically in helicinids (Richling, 2004) and in gastropods in general (Simone, 2011).However, anatomical features have been barely used in neritimorphs.As part of a larger project on phenotypic features and phylogeny of the Neritimorpha (= Neritopsina), Helicina variabilis was selected as one of the terrestrial representatives of the group, as it appears to be a typical member of the family.Eventually, the estimated systematic relationships based on comparative anatomical information will be compared to those inferred using molecular approaches (e.g., Uribe et al., 2016).
The Helicinidae constitute a terrestrial branch of the Neritimorpha (Richling & Glaubrecht, 2008), a taxon with ~2,000 species in four superfamilies, Neritopsoidea, Hydrocenoidea, Helicinoidea (which includes the family Helicinidae), and Neritoidea (Uribe et al., 2016), most of the species in the last two taxa.The helicinids are easily distinguished from the more common Pulmonata snails in having a well-developed, normally calcareous operculum, and the eyes placed at the base of a single pair of cephalic tentacles.Their shells are discoid to globose, with a suture plane, sometimes carinate at the periphery, and an umbilicus absent.These features easily distinguish them from the other operculate land snails belonging to Cyclophoroidea, which is basal to the Caenogastropoda (Simone, 2011).
Focusing on the Caenogastropoda, Simone (2011) included only a few neritimorph species as outgroups; none of these were terrestrial, a gap filled by the present study.As in most branches of Mollusca, the anatomical features of helicinids have been considered uniform and of limited use in comparative analyses (e.g., Bourne, 1911;Boss & Jacobson, 1974).For this reason, most studies of helicinid systematics have focused on the shell and radula (e.g., Wagner, 1907Wagner, -1911;;Baker, 1922Baker, , 1923)).Only more recently have some anatomical characters, primarily from the reproductive system, been used as an additional tool to resolve the systematics of the group (e.g., Richling, 2004Richling, , 2009)), mainly focused on the reproductive system.
The helicinids share some important neritimorph features, such as the absorption of the inner whorls of the shell, producing a sac-like, coil-less visceral mass (Bourne, 1911).The type of radula, the shape of the columellar muscles, and the calcareous operculum are additional synapomorphies (Simone, 2011).On the other hand, using the study of Helicina variabilis as an example, together with literature data, it is possible to suggest some features that distinguish the helicinids at least from the marine Neritidae, as discussed below.

MATERIAL AND METHODS
The list of material examined follows the species description.The specimens studied were collected in the vicinity of the city of Nanuque (~17°51'S 40°23'W, ~120 m elevation) in northern Minas Gerais state, very close to the border with the states of Bahia and Espírito Santo.The locality is a tiny fragment of Atlantic Rainforest close to the Mucuri River, surrounded by tomato fields and thus highly subject to anthropogenic impacts and degradation.See Simone & Salvador (2016) for more details.

Central nervous system (Figs. 32, 33):
Nerve ring located in ventral region of snout base, occupying ~1/20 of haemocoel volume (Fig. 24: nr), surrounding oral tube close to its posterior region (Fig. 27: nr).Pair of cerebral ganglia (ce), each ganglion occupying ~20% of nervering volume, relatively spherical; cerebral commissure slightly longer than each ganglion length.Pair of pedal ganglia (gp) about as large as cerebral ganglia, very closely connected with each other; each ganglion subspherical, tapering to large anterior pedal nerve.Statocyst (cy) ~1/5 pedal ganglion volume, bearing several statoconia; located on ventral side of middle region of each pedal ganglion.Pair of pleural ganglia (pl) located close to pedal ganglia, ~1/2 of their size; both widely connected on median side.Cerebro-pleural and cerebro-pedal connectives relatively symmetrical, about as long as cerebral commissure; right pair somewhat more widely separated from each other than left pair.

DISCUSSION
The previously known distribution of H. variabilis includes the Brazilian states of Paraíba, Bahia, Espírito Santo, and Rio de Janeiro (Wagner, 1827;Simone, 2006).The present record slightly extends the species distribution to NE Minas Gerais state.
As the epithet suggests, this species is known for its wide variability in the shell color pattern, from completely white to almost completely brown (Figs.5-7), and commonly shows a banded pattern (Figs.1-4), easily seen in the numerous specimens in sample MZSP 128202.Despite this variability, H. variabilis can be distinguished Simone, L.R.L.: Phenotypy of Helicina variabilis from Brazil Pap.Avulsos Zool., 2018; v.58: e20185832 4/9 from its Brazilian congeners (Simone, 2006) in having a relatively tall shell, a rather weak peripheral carina, the peristome slightly displaced downwards, and the presence of spiral striae with irregular interspaces.
Helicina variabilis shows the normal anatomical pattern of the family (Bourne, 1911;Richling, 2004), with the single main monoaulic pallial oviduct characteristic of the genus Helicina, in which the single aperture is anterior.Also, although it apparently is a common helicinid feature, the female genital aperture inside the anal aperture is noteworthy, but not exclusive, as Alcadia hollandi (C.B.Adams, 1849) also has this feature (Bourne, 1911: fig. 25).Another remarkable character is the connection of the pleural ganglia with each other.Normally this pair  has been reported at least in A. hollandi (Bourne, 1911: fig. 44) as well as in the neritid Neritina zebra (Bruguière, 1792) (Barroso et al., 2012: fig. 30).This feature may be a neritimorph synapomorphy.In the genital system, the main exclusive character of H. variabilis is the lack of a seminal receptacle (re-   4B), as well as the lack of a provaginal sac and duct (e.g., Richling & Boucher, 2013: fig. 20), and only a well-developed bursa copulatrix is present.The bursa with branched profile appears to be a common feature of the helicinids (Baker, 1926).The characteristics of the branches can be used as a taxonomic feature.The bursa of H. variabilis has three main branches, each branch subdividing distally (Fig. 34: bc), which appears to be an exclusive character.
The kidney of H. variabilis is relatively simple for a terrestrial invertebrate.It is mostly hollow, possessing a small renal lobe (Fig. 25: kl).The auricle, on the other hand, is huge (Fig. 25: au), and is triangular with vertices at the right connection with the kidney, the connection with the remnant of the ctenidial vein (cv), and with the ventricle (ve).Another noteworthy character is the diaphragm muscle (Figs. 25,26: dm), which may help in breathing movements, as it is absent in the aquatic forms.A similar muscle is present in the non-related  Ampullariidae (basal Caenogastropoda) (Simone, 2004, as gastric muscle), another air-breathing group.
With respect to the odontophore, H. variabilis, as well as other helicinids (Bourne, 1911: figs. 5-7), has some similarity with that of the neritid N. zebra (Barroso et al., 2012: figs. 25-29), with an inner arrangement in two pairs of cartilages, one anterior and one posterior.However, the helicinids so far known have the horizontal muscle (m6) single, not divided into two components (Figs. 30,31).The pair of odontophore protractors (m10) is inserted far posteriorly (Fig. 29) in both species, separating them from related taxa (Simone, 2011); however, that of H. variabilis is longer and slender.The pair of retractor odontophore muscles (Figs. 29, 30: m2) is simple in H. variabilis, but is highly branched in N. zebra ("ml" Barroso et al., 2012).Details of the pairs m3 are different from those of N. zebra, but these m3 muscles, as well as the jugal muscles m1, normally are the most variable, even in closely related species.The stomach of H. variabilis has a pair of ducts to the digestive gland, located close to each other (Fig. 26: dd); the other known helicinids usually have these ducts on opposite sides of the stomach (e.g., Bourne, 1911: figs. 10-15), but the remaining digestive features appear similar, including the well-developed pair of esophageal pouches (Fig. 24: ep).
In the phylogenetic scheme of Simone (2011), H. variabilis would certainly be placed in the "node U -Neritimorpha" (fig.20), in having the main neritimorph synapomorphies, such as the absorption of the inner shell whorls, the pair of columellar muscles inserting directly on the columellar side of the aperture, and the calcareous operculum.The presence of well-developed pallial gonoducts, indicating internal fertilization and production of egg capsules, as shown in Figs. 23,34 and 35, are one of the synapomorphies of the Adenogonogastropoda Simone, 2011, a taxon uniting Neritimorpha with the Apogastropoda.Other main characters are the eyes with a lens, loss of the right pallial structures (gill, osphradium, and hypobranchial gland), and loss of the right kidney.All features were confirmed in the species studied here.
The monotocardian condition of the Helicinidae, i.e., a single auricle in the heart, has been an interesting enigma (Little, 1972), as this character is shared with the higher gastropods.The basal neritimorphs, despite having lost the right pallial structures, still retain the left auricle (Estabrooks et al., 1999;Barroso et al., 2012).Taking into consideration the information provided by Simone (2011), the monotocardian condition of the helicinids is a remarkable convergence with the apogastropods (sensu Simone, 2011), also known as Monotocardia.However, a different situation was found by Uribe et al. (2016), using a molecular approach, in that the marine diotocardian neritids appeared as a more derivative taxon inside Neritimorpha, preceded by the representatives of the three terrestrial superfamilies Neritopsoidea, Hydrocenoidea, and Helicinoidea in their phylogram (Uribe et al., 2016: fig. 2).From a morphological point of view, this arrangement is not the most parsimonious, as the most derived taxon (Neritidae) supposedly would have reverted to the marine environment and recovered several morphological plesiomorphies, such as the bipectinate gill, the osphradium, and the right auricle.Possibly to resolve this dilemma, Uribe et al. (2016) suggested a non-parsimonious explanation, in which the three most basal superfamilies evolved independently from different marine ancestors.