First record of the semi-slug Omalonyx unguis ( d ’ Orbigny , 1837 ) ( Gastropoda , Succineidae ) in the Misiones Province , Argentina

Omalonyx unguis (d’Orbigny, 1837) is a semi-slug inhabiting the Paraná river basin. This species belongs to Succineidae, a family comprising a few representatives in South America. In this work, we provide the first record for the species from Misiones Province, Argentina. Previous records available for Omalonyx in Misiones were identified to the genus level. We examined morphological characteristics of the reproductive system and used DNA sequences from cytochrome oxidase subunit I (COI) gene for species-specific identification. These new distributional data contribute to consolidate the knowledge of the molluscan fauna in northeastern Argentina.

According to potential distribution models for Omalonyx species in South America, the Argentine province of Misiones represents a suitable area for the occurrence of O. unguis and O. convexus (Heynemann, 1868) (Coscarelli et al. 2018).Both species occur in the Paraná river basin and are typical inhabitants of hygrophilous environments (Coscarelli andVidigal 2011, Coscarelli et al. 2018).Although the genus has been recorded in the Misiones Province, these records have not been identified at a specific level (Gutiérrez Gregoric et al. 2013).To date, specific-identity of the Omalonyx species in Misiones Province is unknown.Recently, the first molecular data for Omalonyx in South America became available, which comprises DNA sequences for the mitochondrial COI gene, and the nuclear internal transcribed spacer 2 (ITS2) region (Vidigal et al. 2018).Nonetheless, DNA sequences for Omalonyx from Argentina are still scarce, and are only available for O. convexus.
In this study, we report new records for the genus Omalonyx in Argentina, with that finding also constituting the first anatomically and molecularly confirmed records for O. unguis in the Misiones Province.Moreover, DNA sequences obtained here constitute the first genetic data generated for this species in Argentina.

Methods
Living specimens were manually collected in the margins of the High Paraná River and 4 tributary streams between 2017 and early 2018.Individuals were captured during the sunset while they were involved in full activity on the leaves and flowers of Eichornia crassipes (Pontederiaceae) and on riparian vegetation (Figs 1-4).The animals were photographed using a Sony DSC Hx400v camera.Geographic coordinates were recorded with GPS Garmin eTrex Legend®.Living specimens were relaxed in water with menthol crystals for 4-10 h, subsequently immersed in hot water (80 °C), and finally fixed in 96% ethanol.Shells were photographed in dorsal, ventral, lateral and protoconch views following Arruda et al. (2016).Voucher material was deposited in the malacological collection at the Instituto de Biología Subtropical (IBS-Ma), UNaM-CONICET, Misiones Province, Argentina.
We examined the genital anatomy of 8 ethanolpreserved adult specimens (IBS-Ma 042, 073/1, 074/1, 280, 281/1, 282/3, 283, 284/1) following Tillier (1981), Arruda et al. (2006), Arruda (2011), andCoscarelli andVidigal (2011).Specimens were dissected using a Labomed Luxeo 4D stereomicroscope.A 0.1% (w/v) neutral-red solution was used to increase the contrast of tissues as in Vogler et al. (2014).DNA was isolated from muscle tissue of the same 8 anatomically dissected specimens using a CTAB protocol (Beltramino et al. 2018).Partial sequences of the mitochondrial COI marker were amplified by polymerase chain reaction (PCR) through the use of the universal primers LCO1490 and HCO2198 (Folmer et al. 1994).The PCR reaction was performed in a total volume of 50 μl containing 30-50 ng of template DNA, each primer at 0.1 μM, 1× reaction buffer, 2.5 mM MgCl 2 , 50 μM dNTPs and 1.2 U Taq Pegasus DNA polymerase (Productos Bio-Lógicos, Argentina).Amplification was performed on a T18 thermocycler (Ivema Desarrollos) as follows: an initial denaturing for 3 min at 94 °C; 5 cycles of 30 sec at 94 °C, 40 sec at 45 °C, 1 min at 72 °C; 35 cycles of 30 sec at 94 °C, 40 sec at 51 °C, 1 min at 72 °C; followed by a final extension at 72 °C for 10 min.Success of PCR reactions was verified by agarose gel electrophoresis.The PCR products were purified by means of an AccuPrep PCR Purification Kit (Bioneer, Korea).In some cases, owing to the co-amplification of nonspecific fragments, PCR products were purified from 1.5% (w/v) agarose gel with an ADN PuriPrep-GP Kit (Inbio Highway, Argentina).Both DNA strands were directly cycle sequenced (Macrogen Inc., Seoul, Korea), and the resulting sequences were trimmed to remove the primers.The consensus sequences of the individuals between the forward and reverse sequencings were assembled using the BIOEDIT 7.0.5 software (Hall 1999).In order to confirm the species-specific identification, the consensus sequences were compared with reference sequences in GenBank using the BLASTN algorithm (Altschul et al. 1990).1, Fig. 5).

New
Identification.The semi-slugs were firstly identified as belonging to Omalonyx by their typically reduced, flat, fingernail-shaped shells (Fig. 6), as well as by the animal bodies twice than shell lengths (Arruda and Thomé 2008a).Species-specific identification was achieved based on morphological features of the reproductive system, and on partial COI sequence data using the BLASTN algorithm.Anatomically, the specimens matched the morphological characters defined for O. unguis as described by Arruda (2011), and in particular exhibited the presence of a serpent-like fold on the outer surface of the epiphallus, a diagnostic character for the species (Figs 7-9).In addition, the following distinctive characters were observed: ovotestis spherical with hermaphroditic duct contorted in distal portion.Penial retractor muscle attached to distal region of vas deferens.Epiphallus shorter than penis, with epiphallic sphincter in proximal region.Penis uniform in diameter with inner papillae similar to those described by Tillier (1981) and Arruda (2011); diameter of proximal penis similar to epiphallus.Spermatheca spherical with duct of spermatheca thin, inserted at junction of free oviduct and vagina.Vagina cylindrical with elliptic papillae on inner surface.The complete reproductive system is shown in Fig. 9.The total lengths of the mitochondrial sequences obtained were 655 bp for all individuals.The BLASTN search results with the COI sequences here obtained confirmed their specific identities as O. unguis, as they showed 100% coverage and top-ranking scores between 100-96% similarity with the COI sequences available for the species in the GenBank database.The specimens reported herein were collected in calm-water environments with abundant vegetation, at sites subject to high anthropogenic disturbance, as the Itá stream.This is an urban stream flowing through Posadas city, which is affected by the disposal of anthropogenic wastes (Secretaría de Planificación Estratégica y Territorial 2012).In addition, all the streams in the area were also affected by the filling stages of the Yacyretá Reservoir (Argentina-Paraguay) resulting in a slower surface-water flow velocity near their confluence with the High Paraná River, conditions that favor the growth of Eichornia crassipes (Flores et al. 2009, Fulco 2012).In agreement with observations by Poi de Neiff et al. (1977), the specimens collected in March 2018 during the flowering period of Eichornia crassipes showed a preference for feeding on the flowers of this plant.
Recently, Coscarelli et al. (2018) made predictions on the potential distribution area of 5 Omalonyx species, where Misiones Province is indicated as a suitable area for the occurrence of O. unguis and O. convexus.Nonetheless, although more than 150 years have passed since both species were described, these species continue to be poorly represented for Misiones Province and occurrence data are almost lacking in the literature for the area.To our knowledge, only Gutiérrez Gregoric et al. (2013) reported the presence of the genus in Misiones Province based on fieldwork conducted between 2009 and 2010; however, species-level identification was not achieved.
Specific morphological identification of Omalonyx species rely on characters of the genital system (Tillier 1981, Arruda et al. 2006, 2008a, b, Coscarelli and Vidigal 2011).All the individuals analyzed in this study presented Our BLASTN searches of the COI sequences revealed that samples of O. unguis from Itá, Zaimán, and Garupá streams were identical (100% query coverage, 100% maximum identity) to Foz do Iguaçu, Brazil samples, whereas our sample from Mártires stream was identical (100% query coverage, 100% maximum identity) to sequences from a tributary stream of the Paraguay River, as well as with High Paraná River samples from locations situated before its confluence with the Paraguay River.On the other hand, DNA sequences from the remaining samples differed from the previously characterized by Vidigal et al. (2018), thus representing new haplotypes.Further research is required to gain insights into the genetic background of the O. unguis populations in Misiones Province, as the molecular data presented here represent the first DNA sequences for the species in Argentina.
By last, considering the previous records of O. unguis in the bordering countries of Brazil and Paraguay where similar environmental conditions exist, the lack of previous reports in Misiones Province might be attributable  to low-sampling efforts for this molluscan fauna, and further fieldwork is required to comprehensively assess the distribution pattern of this semi-slug species in the region.

Discussion
This work documents for the first time the occurrence of the semi-slug O. unguis in the Misiones Province, Argentina from 7 localities near Posadas city.Omalonyx unguis is the type species of the genus and its type locality was emended from Corrientes, Argentina to Asunción, Paraguay(Arruda and Thomé 2008a, b).In Argentina, the known distribution of this species-based on literature and museum data-includes the provinces of Buenos Aires, Catamarca, Chaco, Corrientes, Entre Ríos, Formosa, Santa Fe, and Tucumán(Fernández 1973, Cazzaniga 1985, Coscarelli et al. 2018 and references  therein).However, due to misconceptions and contradictions in Omalonyx systematics, those previous records should be interpreted with caution as they may include what today is recognized as O. convexus(Arruda and Thomé 2008a).Despite this, the records reported herein constitute the first anatomically and molecularly confirmed occurrences of O. unguis in Misiones and increase

Figure 5 .
Figure 5. Distribution map of Omalonyx unguis in the Paraná river basin.Triangles indicate molecularly-confirmed occurrences according to Vidigal et al. (2018).Circles indicate the new localities where the specimens of O. unguis were found in Misiones Province, Argentina.The localities numbers correspond to those presented in Table1.
the serpent-like fold on the outer surface of the epiphallus, the most robust diagnostic taxonomic character for diagnosing O. unguis, exhibiting different extent of undulation among specimens.Omalonyx unguis differs from O. matheroni, O. pattersonae, and O. brasiliensis (Simroth, 1896), in having the insertion of the penial retractor muscle on the distal region of the vas deferens, and differs from O. matheroni, O. pattersonae, O. brasiliensis, and O. convexus in having the diameter of proximal penis similar to that of epiphallus.Omalonyx unguis differ from O. geayi Tillier, 1980 and O. pattersonae in lacking longitudinal folds on the inner surface of the free oviduct.In addition, O. unguis differs from O. geayi, and the whole genus, in the shape and distribution of the inner penis surface papillae.Further information on the anatomical differences amongst Omalonyx species can be found in Arruda (2011) and Vidigal et al. (2018).

Table 1 .
Reports of Omalonyx unguis obtained here and literature records for which COI sequences are available.IBS-Ma: Malacological collection at Instituto de Biología Subtropical, Universidad Nacional de Misiones-CONICET.LMSM: Malacological collection of the Laboratório de Malacologia e Sistemática Molecular, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais.