BioInvasions Records (2021) Volume 10, Issue 1: 10–20 CORRECTED PROOF Rapid Communication Occurrence in Mexico of two European invasive slug species: Arion vulgaris Moquin-Tandon, 1855 and Arion intermedius (Norman, 1852) Victoria Araiza-Gómez1,*, Edna Naranjo-García2 and Gerardo Zúñiga1 1Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Zoología, Prolongación de Carpio y Plan de Ayala s/n, Col. Casco de Santo Tomás, 11340 Mexico City, Mexico 2Instituto de Biología, Departamento de Zoología, Universidad Nacional Autónoma de México, Avenida Universidad 3000, Apartado Postal 70-153, 04510 Mexico City, Mexico Author e-mails: [email protected] (VAG), [email protected] (ENG), [email protected] (GZ) *Corresponding author Citation: Araiza-Gómez V, Naranjo- García E, Zúñiga G (2021) Occurrence in Abstract Mexico of two European invasive slug species: Arion vulgaris Moquin-Tandon, Knowledge of invasive terrestrial gastropods species in Mexico, and their potential 1855 and Arion intermedius (Norman, impacts, is practically unknown. We document in this study, the occurrence of the 1852). BioInvasions Records 10(1): 10–20, large invasive slug Arion vulgaris Moquin-Tandon, 1855 for the first time in https://doi.org/10.3391/bir.2021.10.1.02 Mexico and only the third time in North America. In addition, we also report Arion Received: 30 December 2019 (Kobeltia) intermedius Normand, 1852 from seven localities demonstrating that the Accepted: 2 August 2020 species is already well established in Mexico. To identify both species, a Maximum- Published: 14 December 2020 Likelihood phylogenetic analysis was performed using partial sequences, alone or concatenated, of nicotinamide adenine dinucleotide dehydrogenase 1 (ND1) and Handling editor: Norine Yeung cytochrome C oxidase 1 (COI) genes. In addition, we used the morphological characters Thematic editor: Stelios Katsanevakis reported in other studies of these species to confirm their taxonomic identity. The Copyright: © Araiza-Gómez et al. phylogenetic analysis performed with the concatenated ND1 and COI genes, showed This is an open access article distributed under terms of the Creative Commons Attribution License that the Mexican sequences of A. vulgaris were grouped with the corresponding (Attribution 4.0 International - CC BY 4.0). sequences of this species from Canada and European countries. In addition, attributes such as the shape and position of the ligula were useful to confirm the taxonomic OPEN ACCESS. identity of this species. On the other hand, the phylogenetic analysis using ND1 sequences alone clustered Mexican specimens of A. intermedius with sequences of this species from Spain and UK. Characters such as the body color pattern, prickles on the dorsum, as well as characters of the reproductive system of A. (K.) intermedius corroborated the taxonomic identity of Mexican specimens of this species. Key words: Arionidae, invasive slugs, molecular identification, North America Introduction The settlement of invasive species in areas outside their natural distribution can cause damage to biodiversity and ecosystems, and also produce negative effects on the human, animal and plant health. The environmental, economic and social impact of invasive slugs in Mexico is unknown, because they are very poorly studied species. The genus Arion Férussac 1819, the most diverse within the family Arionidae, is divided into four subgenera: Arion Férussac, 1819, Kobeltia Seibert, 1873, Carinarion Hesse, 1926 and Mesarion Hesse, 1926 (Breugelmans et al. 2013). The actual number of species worldwide is Araiza-Gómez et al. (2021), BioInvasions Records 10(1): 10–20, https://doi.org/10.3391/bir.2021.10.1.02 10 Arion in Mexico unknown due to their wide polymorphism, which makes it difficult to identify many species (Quinteiro et al. 2005). About 25–50 nominate species exist (Barker 1999; Pinceel et al. 2005), suggesting that the taxonomic status of some of them should be confirmed combining molecular and morphological evidence such as in other groups of slugs (Nitz et al. 2009; Reise et al. 2011; Vendetti et al. 2019). Several species of Arion have expanded their distribution range within the past century. For example, at least seven species have invaded other regions into Europe: A. distinctus J. Mabille, 1868; A. fasciatus (Nilsson, 1823); A. flagellus Collinge, 1893; A. hortensis A. Férussac, 1819; A. rufus (Linnaeus, 1758); A. silvaticus Lohmander, 1937 and A. vulgaris Moquin- Tandon, 1855 (Hulme 2009). Other Arion species have reached other continents as a consequence mainly of globalization, and where the unintentional introduction of eggs, immature individuals or adults have provided for a steady barrage of slugs via shipments of mushrooms, cut flowers, flower bulbs, horticultural plants and vegetables (Barr et al. 2009). European species have been recorded both in synanthropic and disturbed environments in North America. For example, A. rufus and A. subfuscus (Draparnaud, 1805) were recorded as pests in the USA and Canada (Robinson 1999; Forsyth 2004), Arion (Carinarion) circumscriptus Johnston, 1828 was reported in Mexico City, Mexico, and Arion (Kobeltia) intermedius Normand, 1852 recorded from the border with the USA (Naranjo-García and Castillo-Rodríguez 2017). The distribution of A. vulgaris includes Spain, Portugal, and the Azores (Zajac et al. 2017), and recently was reported in USA and Canada (Zemanova et al. 2018); although the native range is unknown. This species is commonly confused with A. lusitanicus, however, Quinteiro et al. (2005) used molecular markers to demonstrate that this species is restricted to the north-west region of the Iberian Peninsula. A. vulgaris sensu stricto is considered to be an invasive species which is widely distributed across Europe, which may have a negative impact on native slugs (Hulme 2009; Zemanova et al. 2017). In addition, this species inflicts damage to domestic gardens, vegetables, strawberry cultures, and cereal fields (Davies 1987; von Proschwitz 1997); and it is an intermediate host for several lungworm species that cause respiratory or systemic disease in canids and felids (Penagos-Tabares et al. 2020). Arion intermedius is naturally distributed in North-Western Europe, and it has been introduced recently in Austria (Duda et al. 2019), North and South Africa (Connolly 1912; Cowie 1997), North and South America (USA, Chile and Colombia) (Hausdorf 2002; Cádiz and Gallardo 2007; Mc Donnell et al. 2008), Australia and New Zealand (Barker 1999). The presence of this species worldwide can be explained by its adaptability to different environmental conditions, its mixed breeding strategy that allows the production of viable offspring by single specimens (Jordaens et al. 2013), Araiza-Gómez et al. (2021), BioInvasions Records 10(1): 10–20, https://doi.org/10.3391/bir.2021.10.1.02 11 Arion in Mexico Table 1. Species, sampling sites, habitats and catalogue number/specimens deposited (CNMO). Species Sampling sites Habitat CNMO/N Arion intermedius Hidalgo, Pueblo Nuevo. 20°08′43.6″; 98°41′05.5″W (HPN) Pine-oak forest 8043 / 3 Tlaxcala, Yauhquemecan, Cascadas de Atlihuetzia. Under rocks, surrounding A. intermedius 8044 / 13 19°21′53″N; 98°10′57″W (TLX) habitat buildings (hotel) A. intermedius CDMX, Bosque de Tlalpan. 19°17′38.8″N; 99°12′15.9″W (BT) Oak forest-xerophytic scrub 8045 / 7 Puebla, Teopancingo, Protected Natural Area (Zona Protectora A. intermedius Forestal Vedada, Cuenca Hidrográfica del Río Necaxa). Pine-oak forest 8046 / 11 20°06′96.6″N; 98°04′49″W (TE) A. intermedius Puebla, Venta Grande. 20°07′51.68″N; 98°05′21.63″W (VG) Pine-oak and Abies forest 8047 / 9 CDMX, Parque Nacional Desierto de los Leones. A. intermedius Coniferous forest 3470/ 12 19°19′32.96″N; 99°18′30″W (DLIB) CDMX, Parque Nacional Desierto de los Leones, lago de los A. intermedius Coniferous forest 8048/ 2 patos. 19°18′41.8″N; 99°18′34.33″W (DL) A. vulgaris CDMX, Parque Nacional Fuentes Brotantes. (Mxd, Mx81, Mx82) Secondary vegetation 8049-50/ 3 and the intensive commercial exchange between countries. No effect of A. intermedius has been documented on native populations, and it has been regarded as a pest only rarely. Although Barker (2002) reports effects on plant productivity in pastures of New Zealand and Cádiz and Gallardo (2007) suggested that this species can have negative effects on biodiversity by displacing native species from their habitats. Introduced species act as vectors for parasites like rat lungworm, flukes, and nematodes that may impact native species, crops, pets, livestock and human health. In addition, given these species are potential pests, and a threat for native species; their populations should be monitored and controlled frequently. Hence, we report the presence of Arion vulgaris Moquin-Tandon, 1855 (Arion lusitanicus auct. non-Mabille) in Mexico for the first time, along with new records from A. intermedius, which was previously known from one report. Materials and methods Between 2014–2018 we surveyed 20 locations in Mexico, Arion species were collected in eight of these sites. Specimens were collected by hand after visual inspection under logs, rocks, stones, on vegetation, in cracks and between rocks. These animals were drowned and fixed with 70% ethanol. In addition, some samples were obtained from the Colección Nacional de Moluscos (CNMO) Instituto de Biología, Mexico City, Mexico (Table 1). The taxonomic identification was performed based on Maximun- likelihood phylogenetic analysis using two molecular