Supraspecific taxonomy in the Vertiginidae (Gastropoda: Stylommatophora)

The genus VertigoMüller, 1774 consists of c. 100 species of terrestrial microsnails c. 1.5–3 mm in length with a rounded aperture and 0–6 (sometimes more) apertural lamellae at maturity. As currently defined, the genus is largely Holarctic in distribution with only a few Neotropical species being known (Pilsbry, 1948; Nekola & Rosenberg, 2013). Consensus does not exist concerning supraspecific taxonomy of the genus. Pilsbry (1919, 1927, 1948) placed Vertigo, Columella Westerlund, 1878 and Truncatellina Lowe, 1852 in the family Pupillidae, subfamily Vertigininae. Based largely on anatomy, he differentiated these from the subfamily Nesopupinae, into which he placed 11 mostly tropical genera: Bothriopupa Pilsbry, 1898, Campolaemus Pilsbry, 1892, Costigo Boettger, 1891, Cylindrovertilla Boettger, 1880, Lyropupa Pilsbry, 1900, Nesopupa Pilsbry, 1900, Pronesopupa Iredale, 1913, Ptychalaea Boettger, 1889, Pupisoma Stoliczka, 1873, Staurodon Lowe, 1854 and Sterkia Pilsbry, 1898. Bouchet & Rocroi (2005) assigned Vertigo to the family Vertiginidae, which in their scheme comprised three subfamilies: Vertigininae, Nesopupinae (both largely as designated by Pilsbry, 1927, but with Pupisoma being moved to the Valloniidae) and Gastrocoptinae (including Gastrocopta Wollaston, 1878 and 10 related genera). The Gastrocoptinae have subsequently been assigned to the Chondrinidae (Pokryszko et al., 2009). Pilsbry (1919, 1948) recognized four subgenera within Vertigo: Vertigo s.s. and the monotypic Angustula Sterki, 1888, Vertilla Moquin-Tandon, 1855 and Vertillaria Pilsbry, 1919. He further divided Vertigo s.s. into several informal sections and groups. This treatment was followed until Turgeon et al. (1998) and Sysoev & Schileyko (2009), respectively, considered the group Nearctula Sterki, 1892 and the subgenus Vertilla to be of generic rank. Similar intrageneric division has been prominent within Nesopupa; e.g. Gittenberger & van Bruggen (2013) recognized nine genera originally proposed as sections by Pilsbry & Cooke (Pilsbry, 1919): Afripupa, Cocopupa, Helenopupa, Indopupa, Infranesopupa, Insulipupa, Nesodagys,Nesopupa andNesopupilla. To address these issues empirically, we have assembled and phylogenetically analysed DNA sequence data from the nuclear 28S ribosomal RNA (28S) and mitochondrial 16S ribosomal RNA (16S) genes. The species selected for study included the full conchological, biogeographic and ecological range of Vertigo, plus representatives of Columella, Gastrocopta, Nearctula and Truncatellina, and a variety of putative nesopupillids and other genera within the Pupillidae (sensu Pilsbry, 1927). We also included a representative sampling across the infraorder Orthurethra as designated by Wade, Mordan & Clarke (2001). Among the analysed taxa, the following represent the type species of their respective genera: Acanthinula aculeata (Müller, 1774), Chondrina avenacea (Bruguière, 1792), Cochlicopa lubrica (Müller, 1774), Columella edentula (Draparnaud, 1805), Helix pomatia Linné, 1758, Lauria cylindracea (da Costa, 1778), Leiostyla anglica (Wood, 1828), Nearctula californica (Rowell, 1862), Planogyra asteriscus (Morse, 1857), Pupilla muscorum (Linné, 1758) Pyramidula rupestris (Draparnaud, 1801), Solatopupa similis (Bruguière, 1792), Sterkia calamitosa (Pilsbry, 1889), Strobilops labyrinthica (Say, 1817), Vallonia costata (Müller, 1774), Vertigo pusilla (Müller, 1774), Vertilla angustior (Jeffreys, 1830) and Zoogenetes harpa (Say, 1824). Carychium tridentatum, Helix pomatia and Cornu aspersum were used as comparative outgroups. Archived GenBank sequences were used for 24 specimens, including data first reported by Armbruster et al. (2005) [AY546471], Dinapoli, Zinssmeister & Klussmann-Kolb (2010) [GU331954], Gaitan-Espitia, Nespolo & Opazo (2013) [JQ417194], Ketmaier et al. (2010) [jGU046389], Nekola & Rosenberg (2013) [KF214500, KF214496], Nekola et al. (2012) [JN941017, JN941032, JN941041, JN941044], Nekola, Coles & Bergthorsson (2009) [GQ921543], Wade, Mordan & Naggs (2006) [AY841284, AY841285, AY841286, AY841333], Wade et al. (2001) [AY014019, AY014020, AY014022, AY014023, AY014024, AY014025, AY014027, AY014028, AY014028, AY014030, AY014032, AY014033, AY014040, AY014148] and Weigand et al. (2013) [KC206171]. Sequences for the remaining 35 specimens (Table 1) were newly obtained. DNA extraction, purification, PCR amplification and sequencing were performed using previously published methods and primers (see Wade & Mordan, 2000; Nekola & Rosenberg, 2013). The amplified 28S region (Fig. 1) encompasses ITS-2, a region that cannot be aligned because of its intergeneric hypervariability. As a result, all sequence more than 284 bp upstream of the LSU2 primer of Wade &Mordan (2000) was excluded from analysis. The resultant analysed 28S amplicon ranged in length from 809 bp (Acanthinula aculeta) to 827 bp (Columella edentula). The entire 16S amplicon was used for analysis and ranged in length from 403 bp (Cornu aspersum) to 594 bp (Truncatellina cylindrica). Sequences were aligned using ClustalX, with adjustment by eye. Mega v. 5.0 was used to conduct neighbour-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses separately for the 28S and 16S sequences. NJ analysis was based on maximum composite distance including transitions