Pulmonata Stylommatophora Helicidae)
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Biodiversity Journal , 2015, 6 (1): 401–411 The genus Erctella Monterosato, 1894: new molecular evid- ence (Pulmonata Stylommatophora Helicidae) Maria Stella Colomba 1* , Armando Gregorini 1, Fabio Liberto 2, Agatino Reitano 3, Salvatore Giglio 4 & Ignazio Sparacio 5 1Università di Urbino, Dipartimento di Scienze Biomolecolari (DiSB), via Maggetti 22, loc. Sasso, 61029 Urbino, Pesaro-Urbino, Italy; e-mail: [email protected], [email protected] 2Strada Provinciale Cefalù-Gibilmanna n° 93, 90015 Cefalù, Palermo, Italy; email: [email protected] 3Via Gravina 77, 95030 Tremestieri Etneo, Catania, Italy; email: [email protected] 4Contrada Settefrati, 90015 Cefalù, Palermo, Italy; email: [email protected] 5Via E. Notarbartolo 54 int. 13, 90145 Palermo, Italy; email: [email protected] *Corresponding author, email: [email protected] ABSTRACT In this paper we report on new molecular data (COI sequences) of different and represent- ative populations of Erctella mazzullii (De Cristofori et Jan, 1832), E. cephalaeditana Gian - nuzzi-Savelli, Oliva et Sparacio, 2012 and E. insolida (Monterosato, 1892) (Pulmonata, Stylommatophora, Helicidae). Present results are compared with those from recent literature and the current knowledge on phylogenetic relationships among Helicidae pulmonate gastropods is reviewed. Obtained results suggest that: i) Cornu Born, 1778 and Cantareus Risso, 1826 are separate and well distinct from Helix Linnaeus, 1758 ; ii) Erctella Monterosato, 1894 is a valid and independent genus rather than a subgenus of Cornu ; iii) Cornu aspersum (O.F. Müller, 1774) is a group of species (i.e. " aspersum " group) whose taxonomic status needs to be defin further studies; iv) Cornu , Cantareus and Erctella might belong to the same tribe that, still, remains to be defined. KEY WORDS Erctella; Helicidae; mitochondrial markers; phylogenetic reconstruction. Received 11.02.2015; accepted 18.03.2015; printed 30.03.2015 Proceedings of the 2nd International Congress “Speciation and Taxonomy”, May 16th-18th 2014, Cefalù-Castelbuono (Italy) INTRODUCTION and nearby mountains, E. mazzullii s. str., (ii) the endemic population of Cefalù, La Rocca, E. ceph- Colomba et al. (2011) reported on a multidiscip- alaeditana Giannuzzi-Savelli, Oliva et Sparacio, linary study based on genital morphology, DNA 2012, and (iii) the populations living in the analysis, distribution, ecology and fossil records of mountains of Trapani surroundings, E. insolida Cornu mazzullii (De Cristofori et Jan, 1832), a (Monterosato, 1892). species endemic to North-Western Sicily. Obtained Based on the phylogenetic reconstruction results supported the hypothesis that C. mazzul- obtained by the multigenic analysis of nuclear lii should be attributed to the genus Erctella (ITS2) and mitochondrial (16S rDNA, 12S rDNA) Monterosato, 1894 and that this genus was probably molecular markers, Colomba et al. (2011) strongly structured in three discrete clades (i.e., the mazzullii suggested that the genus Erctella should be kept group) recognized as species including: (i) the distinct from the closely related genera Cornu populations living in Monte Pellegrino (Palermo) Born, 1778 and Cantareus Risso, 1826. In the 402 M.S. C OLOMBA ET ALII same paper, this hypothesis was also corroborated nomic frame showing Helix distinct from Canta- by the analysis of several 16S rDNA partial reus and Cornu, the latter two considered the same sequences downloaded from GenBank for other genus (Manganelli et al., 2005; Koene & Schu- genera representatives of Western Palaearctic lenburg, 2005; Wade et al., 2006, 2007). Helicidae taxa; noteworthy, the phylogenetic tree Nevertheless, despite all these anatomical and topology clearly showed Cornu and Cantareus molecular evidence, recently Welter-Schultes et al. distinct from Helix Linnaeus, 1758 (see Colomba (2011) and Welter-Schultes & Audibert (2012) con- et al., 2011, fig. 42). sidered Cornu and Cantareus to belong to the genus Cornu Born, 1778 (type species: Cornu copiae Helix. Bank (2012) argued that such a systematic Born, 1778) was reintroduced as distinct genus by position is wrong, and, above all, it does not take Waldén (1976) with Cryptomphalus De Charpen- into account a number of studies (cited above) tier, 1837 (type species: Cryptomphalus aspersum suggesting a taxonomic choice closer to the real af- O.F. Müller, 1774) as junior synonim; it was some- finities among these taxa. Welter-Schultes et al. times considered as subgenus of Helix Linnaeus, (2012), however, reaffirmed their beliefs and, 1758 (type species: Helix pomatia Linnaeus, 1758) besides, Welter-Schultes (2012) reported Erctella and sometimes as a distinct genus. The description as synonym of Helix. of Cornu copiae was based on a teratological Nordsieck (2013), reviewing the papers, pub- specimen of “Helix” aspersa; due to different lished in the last decades, dealing with anatomical interpretations of the Article 1.3.2 of the Code, a and molecular data, concluded, in summary, that: request for conservation of the name Cornu is still “According to genital morphology and DNA ana- pending a ruling of the International Commission lysis, “Helix“ aspersa and relatives are not more on Zoological Nomenclature. related to Helix than Eobania and other genera of Cantareus Risso, 1826 (type species: Cantareus the Helicinae […] These species must therefore be apertus Born, 1778) was sometimes considered as generically separated from Helix. The shell and the subgenus of Helix and sometimes as a distinct genital differences, especially those of the penis genus. (Giusti et al. 1995, Neubert & Bank 2006, Schileyko (1978) was the first one who de- Colomba et al. 2011), are sufficient for the generic scribed the internal structure of male sexual organs separation of Cantareus and Cornu (or Cryptom- of "Helix" aspersa characterized by a penial papilla phalus, if the name Cornu is not valid because of and a prominent semicircular fold in the distal part Art. 1.3.2 ICZN, cf. Giusti et al. 1995: 491). of the penis (see also Nordsieck, 2013). Because of Erctella is regarded as a subgenus of Cornu instead these anatomical differences, the Author attributed of a genus, because it is more closely related to this species to the genus Cryptomphalus. Cornu than to Cantareus”. Giusti et al. (1995) showed a close similarity More recently, detailed molecular genetics between genitalia of "Helix" aperta and "Helix" studies (Korábek et al., 2014; 2015; Razkin et al., aspersa and, therefore, attributed these two 2015) confirmed Cornu and Cantareus as two species to the same genus, Cantareus, morpholo- distinct genera forming a group with no sign of a gically well distinct from Helix. Moreover, they close relationship with Helix. In addition, Erctella reported that Helix has a real penial papilla inside DNA sequences, when included in such analysis the penis and, distally, an accessory penial papilla, (see Korábek et al., 2015), confirmed this item, in whereas Cantareus shows a system of a real penial line with Colomba et al. (2011). papilla, a false penial papilla and, distally, an At present there seems to be broad agreement in "annular pad". considering Cornu and Cantareus distinct genera, Neubert & Bank (2006) mainly confirmed these while on the position of Erctella opinions are still morphological differences and concluded in con- diverging. In order to be able to further test the sidering Cornu and Cantareus as related but distinct “genus hypothesis” (Erctella as a distinct genus, genera. One year later, similar observations were Colomba et al., 2011) versus the “subgenus hypo- reported by Alonso & Ibáñez (2007). thesis” (Erctella as a Cornu subgenus, Nordsieck, At the same time, findings of scientific studies 2013), we performed an additional molecular ana- based on molecular data were in line with the taxo- lysis to characterize and define even better, from a The genus Erctella Monterosato, 1894: new molecular evidence (Pulmonata Stylommatophora Helicidae) 403 molecular standpoint, the identity and reliability of (Folmer et al., 1994). PCR cycles were as follows: Erctella . 95°C for 5 min; 95°C for 1 min, 42°C for 1 min, In particular, phylogenetic relationships among 72°C for 1 min (35 cycles); 72°C for 5 min. To taxa under study were analysed by comparing par - remove primers and unincorporated nucleotides, tial sequences of the gene encoding for the cyto - amplified products were purified with the Wizard chrome oxidase subunit I (COI) - which is one of SV gel and PCR Clean-up kit (Promega). Sequen - the most commonly used mitochondrial markers in cing of purified PCR products was carried out using molecular evolution and molecular phylogeny. automated DNA sequencers at Eurofins MWG Besides, to provide a little contribute in sheding Operon (Germany). All COI sequences generated some more light on Helicidae systematics, the ana - in this study were uploaded in GenBank (accession lysis was extended to hundreds of specimens of the numbers: KR921883-KR921914). family Helicidae whose COI sequences were down- loaded from GenBank database. A similar analysis Phylogenetic analyses was carried out including 16S rDNA partial sequences of the same taxa. Molecular analyses The analysis was conducted on two partial gene have been performed either with single (16S or sequences: COI and 16S rDNA, integrating our data COI) or combined (16S+COI) molecular datasets. with those obtained from GenBank database. In particular, in addition to the sequences obtained from specimens