A molecular contribution to the controversial taxonomical status of some freshwater snails (Caenogastropoda: Rissooidea, Cochliopidae) from the Central Andes desert to Patagonia

For over 40 years malacologists have been discussing the taxonomical status of Heleobia species, an enigmatic genus from Cochliopidae family (Caenogastropoda: Rissooidea). As with other rissooidean families, the considerable character convergence and the paucity of anatomical synapomorphies has proved to be a problem in resolving cochliopid phylogenetic relations and establishing the validity of several nominal cochliopid species. Here we present a molecular contribution to solve the taxonomical status of one of the most abundant Southern South America cochliopid genera which has many endemic species. We report molecular evidence that supports three of the four Heleobia groups described for this region, the “australis”, “parchappii” and “piscium” groups. The fourth, the “hatcheri” group, belongs not to Heleobia but to a different genus which itself should not be considered as part of the family Cochliopidae but closely related to genus Potamolithus Pilsbry & Rush, 1896.

Cochliopidae is a family of rissooidean snails composed of more than 30 genera and more than 260 species that mainly inhabit freshwaters in tropical and températe regions of América and several regions of Eurasia (Hershler & Thompson, 1992). The status of this enigmatie family remained unstable during many years, until Wilke et al. (2001), using molecular tools, confirmed that Coehliopidae is a family distinet from Hydrobiidae as it is aeeepted by Bouchet & Rocroi (2005). The monophyly of the family, the eonsisteney of molecular and anatomieal eharaeters (mainly elosed spermatheeal duet and oviduet jointed direetly to the albumen gland), and its phylogenetie relationships have been assessed and diseussed by Liu et al., 2001 (as Coehliopinae) and Wilke et al. (2001) who mainly utilized DNA sequenees of mitoehondrial genes. Previous attempts to resolve systematie and/or phylogeny of Hydrobiids based only on morphologieal data (e.g. Kabat & Hershler, 1993;Falniowsky & Szarowska, 2000) poorly resolved the uneertainty due to considerable character convergence and the scarcity of anatomical synapomorphies.
Cochliopidae are abundant snails worldwide. However, sequenced taxa from South America are underrepresented, as was pointed out by Liu et al. (2001) diseussing biogeography. Reeent molecular eharaeterization of endemie gastropod fauna from Titieaea Lake (Kroll et al., 2012) and Northern of Chile (Collado et al., 2013) appear to be the only available eoehliopid information for this subcontinent.
The Argentinian Coehliopidae were studied originally by M. C. Gaillard (unpublished data) and Gaillard & Castellanos (1976) both as family Hydrobiidae and genus Littoridina. They listed 18 nominal speeies, gathered in 4 groups ("australis'": South Atlantie littoral waters; "piscium"'. subtropical freshwaters; "parchappei": Pampean región, North Patagonia and Centre-West of Argentina; "hatcheri": Patagonian eontinental región) using morphologieal eharaeters of the shell, opereulum, radula and penis. A eompilation of gastropod snails from the freshwater of Argentina (Rumi etal., 2008) reported 16 speeies of Coehliopidae (all belonging to the genus Heleobia and 10 of them endemies). A review of the taxonomy of Heleobia with emphasis on Argentina was reported by Cazzaniga (2011); none of these previous studies for the Argentinian cochliopidae species included molecular data with the exeeption of Kroll et al. (2012), where two sequences of Heleobia are mentioned, both eolleeted from Laguna Mar Chiquita by C. G. de Francesco and identified by R. Hershler (Museum records) as H. australis and H. parchappii (both preserved in the Smithsonian Natural History Museum).
The Centre-West of Argentina, extending between 28° and 37°S and 65° and 71°W, lies within the dominión of the South American Arid Diagonal, which is considered to have been climatically sensitive to the latitudinal shift of the Pacific and Atlantic anticyclone centers during the late Pleistocene and the Holocene (Abraham de Vázquez et al., 2000). The dominant climate is semiarid with a mean annual rainfall of 250 mm in the eastern foothills of the Andes (Capitanelli, 2005). With respect to freshwater gastropods, this región constitutes the 'Cuyo Malacological Province' (CMP) (Núñez et al., 2010) and covers approximately 280,000 km2. The gastropod diversity of this región is one of the lowest in the country and comprises only 18 known species spread over the families Ampullariidae (1 species), Cochliopidae (5), Physidae (4), Planorbidae (2), Lymnaeidae (2), and Chilinidae (4) (Núñez et al., 2010;Gutiérrez Gregoric et al, 2014). Despite this low biodiversity, the cochliopid species from the CMP are interesting issue due to their confused taxonomical status. The five Cochliopidae species originally described for the CMP belong to the genus Heleobia; one included in the "hatcheri group" H. hatcheri (Pilsbry, 1911) and four in the "parchappii group": H. parchappii (d'Orbigny, 1835), H. kuesteri (Strobel, 1874), H. occidentalis (Doering, 1885), and H. vianai (Parodiz, 1960). According to Cazzaniga (1980), this last ñame is a synonym of H. occidentalis (as Littoridina occidentalis). Gaillard & Castellanos (1976) proposed H. occidentalis as a geographical variation of H. parchappii for saline waters, while Cazzaniga (1980) considered H. occidentalis a valid species after examining penis morphology. De Francesco (2007) suggested H. occidentalis as synonym of H. parchappii based on conchological characters, criteria adopted in recent ecological studies in saline areas from the CMP by Ciocco & Scheibler (2008) and De Francesco & Hassan (2009). Heleobia kuesteri also remain enigmatic. Based on geographic distribution and the original conchological description of Strobel, M. C. Gaillard (unpublished data) considered this species within the "parchappii group". Cazzaniga (1981) proposed H. kuesteri as species inquirenda and Ciocco (2011) suggested that it could be a valid taxon related to the "parchappii group".
Heleobia hatcheri, abundant in Patagonian waters, differs from the other Heleobia species from the CMP in, among other characters, the presence of a so called nuchal papilla in all females studied (pseudohermaphroditism or natural imposex, Martín, 2002), the only reported sex in CMP populations where sex ratios have been studied (Uspallata River;Martín, 2002;Ciocco, 2011). This organ was previously mistakenly interpreted as a reduced and functional penis from hypothetical H. hatcheri males (Gaillard & Castellanos, 1976;Cazzaniga, 1981), to the point that a new genus was proposed (Strobeliella;Cazzaniga, 1981).
Additionally, a new morphotype with similar shell features to H. hatcheri (ovate-conic shell), but discontinuous peristome, was recently found in several localities of the CMP (Heleobia sp.; Ciocco & Koch, unpublished data).
The goal of this study is to develop a molecular approach to solve the taxonomical status of Heleobia species from the Centre-West of Argentina as a first step towards an integrated phylogenetic study of the Southern South América cochliopids. Considering: i) the proteincoding mitochondrial citocrome oxidase I (COI) gene does not show insertions or deletions in the superfamily Rissooidea; ii) the vast information on COI gene sequence available in NCBI GeneBank for the Gastropoda in general and iii) that this sequence shows good phylogenetic signáis from population to family levéis (Wilke et al, 2001), we analyze COI sequences from 7 taxa (5 cochliopid and 2 non cochliopid ones) without previous data in GenBank, in an attempt to provide new considerations tending to solve a long term controversial issue.

MATERIALS AND METHODS
Specimens. Individuals from CMP were eolleeted in Aguas Negras (30°18'6.72"S, 68°43'46.62"W, San Juan Province), Uspallata stream (32°40'11.1"S, 69°21'52.8"W, Mendoza Province), and Laguna Bebedero (33°39'S, 66°34'W, San Luis Province) during expeditions to the CMP between 2011 and 2013. Heleobiapiscium (d'Orbigny, 1835) and Potamolithus spp. specimens were eolleeted in 2014 from Martin Garcia Island, upper portion from de la Plata River basin (34°11'S, 58°15'W, Buenos Aires Province). In all cases, animals were alcohol preserved following previous menthol relaxation. Voucher specimens for all studied taxa were deposited in the Museo de La Plata collection under voucher numbers: MLP MA 13806 to 13812. DNA isolation, PCR and sequencing. The total DNA was extracted from the foot of dissected snails. Tissues were rinsed in TE buffer (lOmM Tris lmM EDTA, pH 8) and digested overnight in CTAB (Cetyl trimethylammonium bromide) buffer containing proteinase K (0.14 mg at 60°C) and 2-Mercaptoethanol. DNA was purified by a threefold extraction with chloroform-isoamyl alcohol (24:1) foiiowéd by precipitation with ethanol. The DNA was then resuspended in DNAse/RNAse free distilled water. A 655bp fragment of the COI gene was amplified by means of the primers of Folmer et al. (1994). Amplification by the polymerase chain reaction (PCR) was performed in a final volume of 50 pl containing: 50-100 ng of template DNA, 0.1 pM of each primer, IX PCR buffer, 50 pM dNTPs, 2.5 mM MgCl2, and 1 U Taq polymerase (Invitrogen, Brazil). The thermocycling sequence was conducted at 94°C for 3 min; with 5 eyeles at 94°C for 30 s, 42°C for 30 s, and 72°C for 1 min 30 s; followed by 34 eyeles at 94°C for 30 s, 45°C for 30 s, and 72°C for 1 min 30 s; with a final chain extensión at 72°C for 5 min. 5 gl of each PCR product was tested on a 1% (w/v) agarose gel electrophoresis. The remainders (45gL) of reactions with the expected PCR product were purified with AccuPrep® PCR purification Kit (Bioneer Corporation, Korea), then sequenced in both directions (Instituto de Biotecnología, Unidad de Genómica, INTA Castelar, Argentina). The resulting sequences were analyzed with BioEdit (Hall, 1999) to obtain consensus sequences for each individual.
Sequence alignment. The COI sequences were unambiguously aligned in MEGA6 (Tamura et al., 2013) and trimmed to a total length of 638bp. Phylogenetic analysis was undertaken comparing gene sequences from this study and related sequences in GenBank (Tab. I). A phylogenetic tree was constructed using the Máximum Likelihood method based on the Tamura-Nei model (Tamura & Nei, 1993). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% of the bootstrap replicates are collapsed.
The initial tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Máximum Composite Likelihood (MCL) approach, and then selecting the topology with the best log likelihood valué. The analysis involved 38 nucleotide sequences. All codon positions were included. All positions containing gaps and missing data were eliminated. There were a total of 636 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).

RESULTS
The bootstrap consensus tree yielded from the ML analysis comprises the outgroup taxon Pomatiopsis lapidaria (Say, 1817), 4 dominant clades and several subclades (from top to bottom; Fig 1.): 1. Clade Cochliopidae was represented by 4 genera (Heleobia, Semisalsa, Heleobops and Tryonia), 3 subclades and 4 lineages: Tab. I. Taxon, collection locality data, reference and GenBank accession numbers for specimens analyzed in this study. Three of the Heleobia spp. from Subelade 1.3 eorrespond to taxa reeorded from the CMP: H. parchappii, H. occidentalis and H. kuesteri. The topology of this subelade showed that two first species are very close, reinforcing that H. occidentalis is synonym of H. parchappii. Also, analysis of this subclade confirmed that Heleobia kuesteri belongs to "parchappii group" and suggested that it should be aeeepted as a valid speeies. Heleobia limariensis, from Huaseo River Basin from northern Chile and the three CMP speeies of this subelade, share an arid Andean environment.
Heleobia hatcheri (and the very similar morphotype Heleobia sp.) from CMP, resolved outside Coehliopidae. Both were integrated in the well-defined Tateidae subclade 3.2 eomposed of 3 Potamolithus speeies: P agapetus (Pilsbry, 1911) and P buschii (Fraunfeld, 1865), sympatrie taxa from de la Plata River, and P ribeirensis (Pilsbry, 1911, sensu Davis & Pons da Silva, 1984, from Iporanga River, Southern Brazil, part of the Paraná and La Plata River drainage systems. "Heleobia hatcheri" and "Heleobia sp." were elosely-related to the three Potamolithus speeies studied in this work and these three taxa are more closely linked to Tateidae family than to Lythoglyphidae, as was pointed out by Wilke et al. (2013) for P ribeirensis.

DISCUSSION
Our results suggest that only two Coehliopidae speeies of the "parchappii group" should be reeognized in the Centre-West of Argentina: H. parchappii and H. kuesteri. Heleobia parchappii is a elongate-eonie shell species abundant in oligohaline waters from the Pampean Región, able to develop populations in estuaries (De Francesco & Isla 2004) or in hard eontinental waters sueh as those of Desaguadero, Llaneanelo and Bebebero saline's from the CMP (Ciocco & Scheibler, 2008;De Francesco & Hassan, 2009, and this work). Heleobia vianai (cited from only one CMP locality, M. C. Gaillard, unpublished data) and H. occidentalis from Bebedero and Llaneanelo saline areas, should be considered as synonymus of H. parchappii as was suggested by Cazzaniga (1980) and De Francesco (2007), respeetively. We were unable to obtain COI sequences for preserved H. vianai material studied (Lote MLP 9224 Invertebrates Collection of Museo de La Plata, Argentina), or eolleet any H. vianai speeimen from the same area as the type loeality where saline waters are predominant. Cazzaniga (1980), based in penial morphology, did not detect significant differences between H. vianai and H. occidentalis, and proposed that the former is a synonym of H. occidentalis whieh, aeeording to the COI sequenees studied in this work, should itself be eonsidered as a synonym of H. parchappii.
Heleobia kuesteri, meanwhile, is an elongate-eonie shell endemie speeies from the Centre-West of Argentina abundant in relatively soft waters of the subandean foothills of the CMP. Despite this speeies needs to be redeseribed ineluding soft parts. The shell features of the searee available material in malacological collections identified as H. kuesteri (Lote 20997/1 Invertebrates Colleetion of Museo Argentino de Ciencias Naturales Bernardino Rivadavia, Buenos Aires, Argentina) appear identieal to the numerous speeimens we eolleeted in the Centre and North of the CMP. Although shell morphology of this speeies is different from typieal H. parchappii, De Francesco & Hassan (2009) eited for the South of the CMP another abundant bateh of individuáis, with identieal eonehologieal features to those we found in the Centre and North of the CMP, as Heleobia aff. parchappii based on similarity of penial eomplex. However, these authors reeognized difficulties in identifying this material and considered the possibility that it eould be an undeseribed speeies or H. kuesteri.
Preliminary studies using seanning eleetron mieroseopy (SEM) performed on shell, penis and radula of many Coehliopidae from Argentina indieated that i) the radulae of H. parchappii and H. kuesteri are similar, ii) although both speeies penial eomplexes are similar, there are small differenees in the shape and the porosity porous of the papillae, and distal end shape, iii) the whorls of H. kuesteri are less eonvex than those of H. parchappii, supporting the possibility that they are different speeies. The COI sequenees analyzed here seemed to reinforee this possibility. However, more detailed anatomieal studies, ineluding female genitalia and use of other molecular markers are necessary to solve the question definitively.
Our results suggest that Heleobia hatcheri and the morphologically similar Heleobia sp. should not be included among the family Cochliopidae, and that they would be closely-related to the three studied Potamolithus species. The latter has two novel and significant implications: i) the conspicuous group "hatcheri". traditional component of the Cochhopidae from Chile (Biese, 1944) and Argentina (Gaillard & Castellanos, 1976), would disappear as part of this family; ii) as was recently suggested by Wilke et al. (2013), the Potamolithus genus endemic from South America would not be Lithoglyphidae as was proposed originally by Davis & Pons Da Silva (1984).
Several other morphoiogicai features of H. hatcheri in addition to its ovate-conic and small shells have previously suggested that this taxon departs from typical cochliopid characters. These include the absence of a penis and the parthenogenetic (and pseudohermaphroditic) condition of the species shown by Martín (2002). This reproductive mode is infrequent in Cochliopidae. Moreover, unlike other parthenogenetic rissooideans such as Potamopyrgus, H. hatcheri is oviparous (Cazzaniga, 2011). Although mistaken, the original interpretation of the species nuchal papillae as a reduced penis, also suggested that H. hatcheri was different from the remaining Cochliopidae, to the point that a new genus (Strobeliella) was proposed for H. hatcheri (Cazzaniga, 1981). In the same sense, the two or three cusps present in the basis of rachidean teeth of H. hatcheri radula are absent in all other Cochliopidae from the southern end of South American (Cazzaniga, 2011). Although Hershler & Thompson (1992) maintained the synonymy of Strobeliella with Heleobia, assuming that the new proposed genus was based on gerontic specimens having a degenerate penis, our molecular data reinstate the requirement for a distinct generic name for H. hatcheri.
Intéréstingiy, there is no fossil record of H. hatcheri from the Centre-West of Argentina although the Holocene aquatic malacofauna of the región is, with the exception of this species and the exotic Physa acuta Draparnaud, 1805, identical to the current gastropod and bivalve assemblages (De Francesco & Hassan, 2009). These observations suggest that this enigmatic taxon may have colonized the Centre-West of Argentina in the last ca. 11,000 years.
Heleobia sp., as previously mentioned, must undoubtedly be considered as very close to H. hatcheri, a taxon with which it shares in sympatry the relatively soft waters of the CMP. Morphological studies indicate that the species have an identical radula, similar pigmentation in the prosboscis and tentacles together with nuchal papillae and absence of males in all the examined populations. The only notable difference from H. hatcheri is that the Heleobia sp. shell has a discontinuous peristome, a character that could be interpreted as an intraspecific variation, as also appears to be the case in H. kuesteri. Nevertheless, a detailed morphological description of this morphotype and the eventual incorporation of other molecular markers should be taken into account before considering it as a new species or a H. hatcheri variation.
With respect to the phylogenetic proximity of H. hatcheri to the South American genus Potamolithus and the suggestion that the latter belong not to Lithoglyphidae but to Tateidae (Wilke et al., 2013), these authors indicated that "We do not know of any unique characters defining this group". Nevertheless, the diagnosis of the Paleartic-Neartic Lithoglyphidae is made by the closed ventral wall of the female capsule gland and the blade-like penis lacking large appendages and specialized glands, remarking finally that the genus Potamolithus was resolved as a member of the Tateidae Clade in all their molecular analysis (Wilke et al., 2013).
The Potamolithus species incorporated in this study, P. buschii and P. agapetus, are sympatric in the La Plata River basin. Potamolithus agapetus presents a marked secondary sexual dimorphism on shell shape and size (López Armengol, 1996). Females of both taxa show a nuchal node on the right side of the neck as was described by Davis & Pons Da Silva (1984) for P ribeirensis. This fleshy protuberance is situated where the base of the simple, and without appendages, penis is located (Davis & Pons Da Silva, 1984;López Armengol, 1996) in the three mentioned Potamolithus species, also coinciding with the position of the nuchal papilla of the parthenogenetic H. hatcheri females (Martín, 2002). Unfortunately, the female genitalia of P buschii and P agapetus have not been described.
The only description of female genitalia available for the genus corresponds to that of P ribeirensis (Davis & Pons Da Silva, 1984). While it has served as the basis to define the "typical" idealized anatomical ground plan of the Lithoglyphidae (Wilke et al., 2001), it is not incompatible with the characterization of the Tateidae female genitalia as "simple, usually with one distal seminal receptacle and a bursa copulatrix; ventral channel occasionally separated to form a vestibule", (Wilke et al., 2013). In H. hatcheri the spermathecal tube seems not be separated from the albumen gland, which would distinguish it from the Cochliopidae. However a deeper anatomical study of H. hatcheri, with emphasis on the female genitalia, and the incorporation of other mitochondrial markers is necessary to determine the genus and, more importantly, the family to which H. hatcheri belongs.