Amer. Malac. Bull. 31(2): 245–256 (2013) 245 First records of four exotic slugs in Argentina Diego E. Gutiérrez Gregoric1,2, Ariel A. Beltramino1,3, Roberto E. Vogler1,2, María G. Cuezzo2,4, Verónica Núñez1,2, Suzete R. Gomes5, Marisol Virgillito6, and Sergio E. Miquel2,6 1División Zoología Invertebrados, Museo de La Plata, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n, B1900WFA, La Plata, Argentina 2Consejo Nacional de Investigaciones Científi cas y Técnicas (CONICET) 3Agencia Nacional de Promoción Científi ca y Tecnológica (ANPCyT) 4Instituto de Biodiversidad Neotropical, Facultad de Ciencias Naturales, Universidad Nacional de Tucumán, Miguel Lillo 205, CP 4000, Tucumán, Argentina 5National Malacology Laboratory, USDA APHIS PPQ NIS, Academy of Natural Sciences, 1900 Benjamin Franklin Parkway, Philadelphia, PA, U.S.A., 19103-1101 6División Invertebrados, Museo Argentino de Ciencias Naturales Bernardino Rivadavia, Av. Ángel Gallardo 470, (1405), Buenos Aires, Argentina Correspondence, Diego E. Gutiérrez Gregoric: dieguty@fcnym.unlp.edu.ar Abstract: This paper reports for the fi rst time the occurrence of four exotic terrestrial slug species in Argentina: Lehmannia valentiana (Férussac, 1823) (Limacidae), Deroceras invadens Reise et al. 2011 (Agriolimacidae), Arion intermedius Normand, 1852 (Arionidae) and Meghimatium pictum (Stolyczka, 1873) (Philomycidae). The study is based on specimens deposited in museums in Argentina. Both the morphologic characteristics and the mitochondrial cytochrome oxidase I gene sequences were used to identify the exotic species. Phylogenetic analyses were also carried out in order to explore the location of their origins. Lehmannia valentiana had the oldest records and has been widely distributed in Argentina. Deroceras invadens and A. intermedius were found to be restricted to the southern portion of the country. Meghimatium pictum was recorded in the northwest and northeast Argentina, and the DNA sequences analyzed from this species were more closely related to specimens from the west of the Strait of Taiwan. A determination of the origin of the other species was impossible because either the sequences analyzed grouped with samples from different geographical origins or only few sequences were available for comparison. In view of the invasive potential of these slug species, the present work provides new and potentially useful DNA sequence data obtained from morphologically-confi rmed specimens. Information provided from these analyses should assist in making a rapid identifi cation of these exotic slugs by nonspecialists and governmental authorities who are responsible for managing and controlling the presence of exotic species. Key words: Lehmannia valentiana, Deroceras invadens, Arion intermedius, Meghimatium pictum, molecular phylogenetic analyses Biologic invasions, together with climate change and hab- itat fragmentation, constitute one of the most serious threats affecting the maintenance of global biodiversity (Vitousek et al. 1996, Nentwig 2007). Bioinvasions have mostly caused disrup- tive effects on native species, alteration of ecological processes, changes in natural systems, and economic and human-health problems (Mooney et al. 2005). In South America, for exam- ple, certain invasive mollusk species – e.g., the continental bivalve Limnoperna fortunei (Dunker, 1857), the marine gas- tropod Rapana venosa (Valenciennes, 1846) and the terrestrial snail Achatina (Lissachatina) fulica Bowdich, 1822 – have been reported to reduce biodiversity (Darrigran and Damborenea 2006, Giberto et al. 2006, Thiengo et al. 2007). Some introduc- tions have been deliberate (e.g., A. fulica), while others were unintentional (e.g., L. fortunei), with the latter being the most common case in point (Cowie and Robinson 2003). Twenty-three exotic terrestrial gastropod species have been reported in Argentina (Parent and Miquel 1999, Miquel et al. 2007, Rumi et al. 2010, Gutiérrez Gregoric et al. 2011). Six of these species are slugs: Limacus fl avus Linnaeus, 1758, Limax maximus Linnaeus, 1758, Deroceras agreste (Linnaeus, 1758), Deroceras laeve (Müller, 1774), Deroceras reticulatum (Müller, 1774), and Milax gagates (Drapaurnaud, 1801) (Rumi et al. 2010). Because of the high intraspecifi c morpho- logic variability in slugs, molecular studies have proven to be extremely useful for species identifi cation (McDonnell et al. 2008, 2011). Phylogenetic studies conducted on several spe- cies have, moreover, enabled a more complete understanding of the history of the invasions and an establishment of the origins of the invaders along with the pathways of their intro- duction (e.g., Facon et al. 2003). These molecular studies, combined with anatomical taxonomic identifi cation and an understanding of those pathways, constitute crucial informa- tion for enabling governmental entities to adjust their strate- gies for controlling invading species (Cowie and Robinson 2003, Lizarralde et al. 2008). The aim of this report is to document for the fi rst time in Argentina the presence of four alien terrestrial slugs from 246 AMERICAN MALACOLOGICAL BULLETIN 31 · 2 · 2013 the families Limacidae, Agriolimacidae, Arionidae and Philo- mycidae, on the basis of morphologic and molecular-genetic identifi cations and, wherever possible, to make inferences about the origins of those species and the possible conse- quences of their introductions. MATERIALS AND METHODS Specimen collections and morphology-based identifi cations Malacological collections from the Museo de La Plata (MLP), the Museo Argentino de Ciencias Naturales (MACN-In), the Instituto Fundación Miguel Lillo (IFML), and the United States Department of Agriculture (USDA) were surveyed (Table 1). The specimens chosen (Limacidae N = 43; Agrio limacidae N = 62; Arionidae N = 14; Philomycidae N = 12) were dis- sected under a stereomicroscope and identifi ed mainly on the basis of the reproductive system and/or the external mor- phology. Adult specimens, properly relaxed, were measured to establish a length range. DNA extraction, polymerase chain reaction (PCR) amplifi - cation, and genetic sequencing Total DNA was extracted from 2 mm3 samples from the foot of each dissected slug. The tissue was rinsed in distilled Table 1. Material examined from slugs from Argentina. IFML, Instituto Fundación Miguel Lillo; MACN-In, Museo Argentino de Ciencias Naturales; MLP, Museo de La Plata; N, number of specimens; USDA, United States Department of Agriculture. Family Province Site Date Collection N Coordinates Limacidae Tucumán San Miquel 1962 IFML 604 28 26°49′S, 65°12′W Chubut Lago Puelo Nat. Park 2003 IFML 14431 4 42º05′S, 71°36′W Buenos Aires Sierras Bayas 1924 MACN-In 14602 5 36°56′S, 60°09′W La Plata 2012 MLP 13636 8 34°55′S, 57°55′W Río Negro El Bolsón 2004 MACN-In 36158 1 51°58′S, 71°32′W Neuquén Neuquén 2004 MACN-In 36162 1 38°56′S, 68°03′W Agriolimacidae Río Negro El Bolsón 2004 MACN-In 36157 MACN-In 36158/1 2 51°58′S, 71°32′W Dina Huapi 2004 MACN-In 36167 MACN-In 36168/1 4 41°05′S, 71°10′W San Carlos de Bariloche 2004 MACN-In 36173 MACN-In 36179–80 MACN-In 36182–83 MACN-In 36186/2 MACN-In 36188–92 33 41°08′S, 71°18′W Cerro Otto 2004 MACN-In 36177 4 41°08′S, 71°19′W Nahuel Malal 2004 MACN-In 36176 2 41°06′S, 71°26′W Manantial 2004 MACN-In 36181/1 2 40°40′S, 71°37′W Neuquén Neuquén 2004 MACN-In 36162 1 38°56′S, 68°03′W Isla Victoria 2004 MACN-In 36164 12 40°58′S, 71°31′W Villa La Angostura 2004 MACN-In 36169 4 40°45′S, 71°38′W Arionidae Chubut Los Alerces Nat. Park 2010 MLP 13405 IFML 15569 12 42°36′S, 71°38′W Los Alerces Nat. Park 2003 MLP 13404 IFML 15463 5 42°44′S, 71°44′W Lago Puelo Nat. Park 2003 IFML 15449 IFML 15566 13 42°05′S, 71°36′W Lago Puelo Nat. Park 2004 MACN-In 36184 2 42°05′S, 71°37′W Río Negro Guillelmo lake 2010 IFML 15568 A 3 41°21′S, 71°29′W San Carlos de Bariloche 2004 MACN-In 36170 MACN-In 36186/1 4 41°05′S, 71°27′W Philomycidae Misiones Iguazú Nat. Park 2009 MLP 13402 3 25°41′S, 54°27′W Tabay Fall 2009 MLP 13403 5 27°00′S, 55°11′W Puerto Iguazú 2011 IFMLP 15570 A 1 25°35′S, 54°34′W Tucumán San Miguel 1999 IFML 15571 A 1 26°48′S, 65°17′W Brazil Foz do Iguaçu 2011 USDA 110442 2 25°34′S, 54°34′W EXOTIC SLUGS FROM ARGENTINA 247 water, ground in 100 mM EDTA and 20 mM Tris, and digested overnight in CTAB buffer containing proteinase K. DNA was purifi ed by a threefold extraction with chloroform-isoamyl alcohol (24:1) followed by precipitation with isopropanol. The DNA was then resuspended in TE buffer. A 655 bp frag- ment of the gene encoding the mitochondrial cytochrome c oxidase subunit I (COI) was amplifi ed by means of the primers of Folmer et al. (1994). Amplifi cation was performed in a fi nal volume of 50 μl containing: 50–100 ng of template DNA, 0.1 μM of each primer, 1X PCR buffer, 50 μM dNTPs, 2.5 mM MgCl 2 , and 1.2 U Platinum Taq polymerase (Invitrogen, Brazil). The thermocycling sequence consisted of 3 min at 94 °C; 5 cycles of 30 s at 94 °C; 40 s at 45 °C; 1 min at 72 °C; followed by 35 cycles of 30 s at 94 °C; 40 s at 51 °C; 1 min at 72 °C; with a fi nal extension for 10 min at 72 °C. After purifi cation of the PCR products by electrophoresis in 1.5% (w/v) agarose gels through the use of a Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Orange, California), both DNA strands were sequenced (Macrogen Inc., Seoul, Korea). The resulting sequences were trimmed to remove the primers, and the con- sensus sequences of the individuals were compared to refer- ence sequences in GenBank through the use of the BLASTN algorithm (Altschul et al. 1990) to identify similarities. Phylogenetic analyses Phylogenetic analyses were conducted in order to con- fi rm the morphology-based identifi cation of the specimens found in Argentina and to make inferences on the source location whenever that identifi cation was possible. For the Agriolimacidae we used the COI sequences of almost all the species represented in Reise et al. (2011) (Table 2). For the Arionidae, we analyzed the COI sequences available in Gen- Bank mainly for the Arion Férussac, 1819 species, those being reported in the literature as either invasive or potentially in- vasive (McDonnell et al. 2009, Thomas et al. 2010) (Table 3). For the Philomycidae, we conducted the phylogenetic analy- ses with the COI sequences for only Meghimatium pictum (Stolyczka, 1873) since we counted on a large number of available sequences for that species from various locations Table 2. Information on the specimens used in the phylogenetic reconstruction of Deroceras species. *GenBank unpublished sequences: the sequence author and submission year are indicated. Taxon GenBank # Country Reference Outgroup Arion distinctus Mabille, 1868 EF128218 Taiwan Tsai and Wu 2008 Arion rufus (Linnaeus, 1758) FJ481178 - Tsai 2008* Pallifera dorsalis (A. Binney, 1842) FJ896618 U.S.A. Tsai et al. 2011 Philomycus carolinianus (Bosc, 1802) EF128221 U.S.A. Tsai et al. 2011 Megapallifera ragsdalei (Webb, 1950) EF128220 U.S.A. Tsai and Wu 2008 Ingroup Deroceras golcheri (Altena, 1962) JN248291–293 Malta Reise et al. 2011 Deroceras invadens FJ358222 South Africa Reise et al. 2011 JN248295 Germany Reise et al. 2011 JN248296 United Kingdom Reise et al. 2011 JN248297 Canada Reise et al. 2011 JN248298–300 Italy Reise et al. 2011 JN248301–302 Germany Reise et al. 2011 JN248303 United Kingdom Reise et al. 2011 JN248314 France Reise et al. 2011 JN248315 U.S.A. Reise et al. 2011 JQ743070 Argentina This work Deroceras laeve AF239733 U.S.A. Reise et al. 2011 EF128217 Taiwan Tsai and Wu 2008 HM584699 - Reise et al. 2011 Deroceras panormitanum JN248304–306 Italy Reise et al. 2011 JN248307–311 Malta Reise et al. 2011 JN248312–313 Italy Reise et al. 2011 Deroceras reticulatum AF239734 U.S.A. Reise et al. 2011 AM259702–703 United Kingdom Reise et al. 2011 FJ481179 - Reise et al. 2011 248 AMERICAN MALACOLOGICAL BULLETIN 31 · 2 · 2013 Table 3. Information on the specimens used in the phylogenetic reconstruction of Arion species. *GenBank unpublished sequences: the sequence author and submission year are indicated. Taxon GenBank # Country Reference Outgroup Deroceras leave EF128217 Taiwan Tsai and Wu 2008 Deroceras reticulatum FJ481179 - Reise et al. 2011 Limacus fl avus FJ481181 - Tsai 2008* Megapallifera ragsdalei EF128220 U.S.A. Tsai and Wu 2008 Philomycus carolinianus EF128221 U.S.A. Tsai and Wu 2008 Ingroup Arion circumscriptus Johnston, 1828 AY094600 Lithuania Soroka 2002* AY987872 Ireland Davison et al. 2009 AY987873 Belgium Davison et al. 2009 DQ647392 Lithuania Soroka 2006* Arion distinctus AY094599 Poland Soroka and Skujienė 2011 AY987874 Belgium Pinceel et al. 2005* AY987876 Belgium Pinceel et al. 2005* DQ647393 Poland Soroka and Skujienė 2011 Arion fasciatus (Nilsson, 1823) AF239735 U.S.A. Remigio and Hebert 2003 AY094598 Lithuania Soroka 2002* AY987877 Germany Davison et al. 2009 AY987878 Austria Davison et al. 2009 Arion fuscus (Müller, 1774) AY094597 Lithuania Soroka and Skujienė 2011 AY987885 Poland McDonnell et al. 2011 AY987886 Bulgaria McDonnell et al. 2011 DQ647391 Lithuania Soroka 2006* Arion hortensis Férussac, 1819 AY423670 - Davison et al. 2009 AY423688 - Dodd et al. 2003* AY987889 United Kingdom Davison et al. 2009 EU382742 U.S.A. McDonnell et al. 2008 Arion intermedius AM259724 United Kingdom McClymont 2006* AY987891 Belgium Davison et al. 2009 EU382756 U.S.A. McDonnell et al. 2008 JQ743069 Argentina This work Arion lusitanicus Mabille, 1868 AY987894 Belgium Davison et al. 2009 EF520640 Poland Soroka et al. 2009 EF535149 Poland Soroka et al. 2009 EU734828 Belgium Soroka and Kałuski 2011 Arion rufus EF520644– 647 Poland Soroka et al. 2009 Arion owenii Davies, 1979 AY423702 - Davison et al. 2009 AY423703 - Dodd et al. 2003* AY987897 United Kingdom Pinceel et al. 2005* AY987898 United Kingdom Davison et al. 2009 Arion silvaticus Lohmander, 1937 AF513018 Lithuania Góbbeler and Klussmann- Kolb 2010 AY987917– 918 Belgium Davison et al. 2009 Arion subfuscus (Draparnaud, 1805) AY987905 Belgium McDonnell et al. 2011 AY987914 France McDonnell et al. 2011 AY987916 France McDonnell et al. 2011 GU249583 U.S.A. McDonnell et al. 2011 EXOTIC SLUGS FROM ARGENTINA 249 cited in Gomes et al. (2011) and Tsai et al. (2011) (Table 4). Finally, phylogenetic analyses were not possible for the Lima- cidae because few sequences were available in GenBank, with the molecular identifi cation of this species based only on the BLASTN algorithm. In all instances, the phylogenetic analyses were carried out as follows: the sequence alignment was performed with the Clustal X 2.0.12 software (Larkin et al. 2007), optimized by visual inspection, and edited with a word processor. The total lengths of the matrices analyzed were 534 bp for the Limacidae, 552 bp for the Arionidae, and 654 bp for the Philomycidae. The data were subjected to four different phy- logenetic analyses by the methods of Neighbor-Joining (NJ), maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI). The NJ analysis was conducted with MEGA 5.05 software (Tamura et al. 2011) through the use of the maximum-composite-likelihood option for computing evolutionary distances (Tamura et al. 2004). The MP analysis was carried out with the PAUP*4.0b10 software (Swofford 2002), through the use of heuristic search, characters equally Table 4. Information on the specimens used in the phylogenetic reconstruction of Meghimatium pictum. CMS-DPE, Superintendência de Controle de Endemias do Estado de São Paulo, Brazil; ESRI-MOL, Endemic Species Research Institute, Taiwan; MLP, Museo de La Plata, Argentina; MZSP, Museu de Zoologia da Universidade de São Paulo, Brazil; USDA, United States Department of Agriculture, U.S.A. *GenBank unpublished sequences: the sequence author and submission year are indicated. Codifi cation GenBank # Country Location Reference Outgroup Arion distinctus EF128218 Taiwan Wuling farm, Taichung Tsai and Wu 2008 Arion rufus FJ481178 - - Tsai 2008* Deroceras laeve EF128217 Taiwan Guansi, Hsinchu Tsai and Wu 2008 Deroceras reticulatum FJ481179 - - Reise et al. 2011 Limacus fl avus FJ481181 - - Tsai 2008* Megapallifera ragsdalei EF128220 U.S.A. Seatey, Alaska Tsai and Wu 2008 Pallifera dorsalis FJ896618 U.S.A. - Tsai et al. 2011 Philomycus carolinianus EF128221 U.S.A. Carter, Tennesee Tsai and Wu 2008 Ingroup Meghimatium pictum ARP1 (MLP 13403/1) JQ712575 Argentina Tabay Fall, Misiones This work ARP2 (MLP 13403/2) JQ712574 Argentina Tabay Fall, Misiones This work BRP1 (CMS-DPE-95) HM233929 Brazil Ribeirão Pires, São Paulo Gomes et al. 2011 BRP2 (MZSP 93842) HM233928 Brazil Curitiba, Paraná Gomes et al. 2011 BRP3 (MZSP 93836) HM233930 Brazil Palhoça, Santa Catarina Gomes et al. 2011 BRP4 (USDA 110442) JQ712572 Brazil Foz do Iguaçu, Paraná This work BRP5 (USDA 110442) JQ712573 Brazil Foz do Iguaçu, Paraná This work CNP1 (ESRI-MOL-07-0123) FJ896666 China Linan, Zhejiang Tsai et al. 2011 CNP2 (MZSP 93847) HM233931 China Zhongcun, Guangzhou Gomes et al. 2011 TLP1 (ESRI-MOL-05-0024) FJ896667 Thailand Bangkok Tsai et al. 2011 TWP1 (ESRI-MOL-08-0565) FJ896651 Taiwan Wushikeng, Taichung Tsai et al. 2011 TWP2 (ESRI-MOL-07-0117) FJ896652 Taiwan Yousheng river, Taichung Tsai et al. 2011 TWP3 (ESRI-MOL-08-0264) FJ896653 Taiwan Lugu, Nantou Tsai et al. 2011 TWP4 (ESRI-MOL-08-0566) FJ896654 Taiwan Wanluan, Pingtung Tsai et al. 2011 TWP5 (ESRI-MOL-08-0138) FJ896655 Taiwan Litao, Taitung Tsai et al. 2011 TWP6 (ESRI-MOL-06-0295) FJ896656 Taiwan Lanyu, Taitung Tsai et al. 2011 TWP7 (ESRI-MOL-99-0001) FJ896657 Taiwan Ruisui, Hualien Tsai et al. 2011 TWP8 (ESRI-MOL-02-0003) FJ896658 Taiwan Meifeng farm, Nantou Tsai et al. 2011 TWP9 (ESRI-MOL-08-0567) FJ896659 Taiwan Jiufen, Rueifang, Taipei Tsai et al. 2011 TWP10 (ESRI-MOL-01-0002) FJ896660 Taiwan Taman, Wulai, Taipei Tsai et al. 2011 TWP11 (ESRI-MOL-07-0121) FJ896661 Taiwan Kinhu, Kinmen Tsai et al. 2011 TWP12 (ESRI-MOL-07-0122) FJ896662 Taiwan Lieyu Tsai et al. 2011 TWP13 (ESRI-MOL-07-0125) FJ896663 Taiwan Beigan, Matsu Tsai et al. 2011 TWP14 (ESRI-MOL-06-0296) FJ896664 Taiwan Nangan, Matsu Tsai et al. 2011 TWP15 (ESRI-MOL-07-0124) FJ896665 Taiwan Dongyin, Matsu Tsai et al. 2011 250 AMERICAN MALACOLOGICAL BULLETIN 31 · 2 · 2013 weighted, tree bisection and reconnection branch-swapping and 10 random stepwise additions. The ML inference was performed by means of the PhyML program (Guindon and Gascuel 2003) available at the public Phylemon2 webserver (http://phylemon.bioinfo.cipf.es; Sánchez et al. 2011). The optimal model of nucleotide substitution was evaluated by the likelihood-ratio test and selected by means of the cor- rected Akaike Information Criterion with the Jmodeltest 0.1.1 software (Posada 2008). The HKY+I+G (for Limacidae), the TIM2+I+G (for Arionidae), and the GTR+I+G (for Phylomicidae) substitution models were used as evolution- ary paradigms. The statistical support for the resulting phy- logenies were assessed by bootstrapping with either 1,000 (NJ, MP) or 100 (ML) replicates (Felsenstein 1985). The BI was carried out with the Mr. Bayes 3.1.2 software (Ronquist and Huelsenbeck 2003). Two runs were performed simulta- neously with 4 Markov chains that went for 1,000,000 gen- erations, sampling every 100 generations. The fi rst 10,000 generations of each run were discarded as burnin, and the remaining 18,000 trees were used to estimate posterior prob- abilities. All the trees were edited with the TreeGraph 2 soft- ware (Stöver and Müller 2010). RESULTS Family LIMACIDAE Lamarck, 1801 The adult specimens had a mean total length of 27.21 mm (N = 13; SD = 5.24; range = 20.7–39.5 mm), with an ill- defi ned keel at the hind end. The external morphology and reproductive system of the dissected adult specimens were consistent with descriptions by Castillejos and Garrido (1996) for Lehmannia valentiana (Férussac, 1823). Specimens from IFML 14431 were identifi ed only by external morphology. Body color of slugs varied from chestnut or auburn to yellow- ish gray, with two or three black longitudinal bands on the mantle shield and only two on the rest of the body, fi nalizing in the posterior end. The central band on the mantle shield was diffuse in some cases. Pneumostome was on the right posterior part of mantle. The specimens had been found in urban and protected zones (e.g., Lago Puelo National Park). Lehmannia valentiana has a widespread distribution in Argentina (Fig. 1), and specimens were recorded together with other native and exotic gastropods (e.g., Deroceras re- ticulatum and Limacus fl avus in La Plata city). The molecu- lar data confi rmed the morphology-based identifi cation. The BLASTN search results, with the obtained partial COI sequence as the query sequence (Gen Bank JX117876, 655 bp), showed top-ranking scores and a 99% sequence iden- tity with the only two 554 bp reference COI sequences avail- able in the GenBank nucleotide database (AM259710 and AM259711). Family AGRIOLIMACIDAE Wagner, 1935 The length of the adult specimens was in general around 12.74 mm (N = 51; SD = 1.38; range = 9.5–16.4 mm). The exter- nal morphology and reproductive system of the dissected speci- mens (N = 62) were consistent with descriptions by Reise et al. (2011) for Deroceras invadens Reise et al., 2011. This species has been recorded in both urban and protected areas (e.g., Nahuel Huapi and Arrayanes National Parks) (Fig. 1). The identifi cation of the Agriolimacidae specimen analyzed from Argentina based on molecular analysis confi rmed its specifi c identity as D. in- vadens. The trees obtained by different methods indicated a similar topology that was in agreement with Reise et al. (2011; Fig. 2 cf. the MP and BI trees). In all instances, this specimen from Argentina belonged to the D. invadens clade, which assign- ment was highly supported (NJ = 100, MP = 98, ML = 100, BI = 1), and was furthermore classifi ed in a subgroup inside this clade (NJ = 87, MP = 93, ML = 84, BI = 0.85) one that included se- quences from several parts of the world (i.e., Canada, England, France, Germany, South Africa and U.S.A). Figure 1. Distribution of new exotic slugs recorded in Argentina. ▲: Lehmannia valentiana; ●: Deroceras invadens; ■: Arion intermedius; *: Meghimatium pictum. Provinces: BA, Buenos Aires; C, Chubut; M, Misiones; N, Neuquén; RN, Río Negro; T, Tucumán. EXOTIC SLUGS FROM ARGENTINA 251 Family ARIONIDAE Gray, 1840 The specimens had a mean of 10.07 mm of total length (N = 3; SD = 0.74; range = 9.5–10.9 mm). The external mor- phology and reproductive system of the dissected adult speci- mens were consistent with the Barker’s descriptions (1999) for Arion intermedius Normand, 1852. Specimens from IFML 15449, 15566, 15568 (Table 1) were identifi ed by external morphology. General coloration was golden yellow in most of the collected specimens, without lateral or central bands; ommatophores were dark brown; the mantle was oval with pneumostome on the right side in the antemedial portion; the posterior body section was rounded without keel. The slugs were collected under woodpiles or sheltering under rocks, in synanthropically disturbed locations, such as the service areas of Lago Puelo National Park, and the camping areas in Los Alerces National Park. The identifi cation of the Arionidae specimen (MLP 13405) analyzed by molecular- genetic sequencing confi rmed its specifi c identity as A. inter- medius. Different phylogenetic analyses gave very similar topological organization for the NJ, MP, and ML trees and minor differences in the BI-tree organization (Fig. 3 cf. the MP and BI trees). The specimen from Argentina was placed within the highly supported A. intermedius group (NJ = 100, MP = 100, ML = 100, BI = 1). Family PHILOMYCIDAE Gray, 1847 Only the specimen from Puerto Iguazú was sexually mature (total length = 50 mm). The morphology of its reproductive system and external morphology coincided with descriptions by Tsai et al. (2005) and Gomes et al. (2011) for Meghimatium pictum (Stolyczka, 1873). Specimens from IFML 15570 A and MLP 13402 (Table 1) were identifi ed by external morpholo- gy, which had an opaque beige background color of mantle, with two dark brown to black lateral stripes, and one medial stripe, often lighter than the lateral ones. This species was found in sites ranging from highly anthropically disturbed (in the Misiones and Tucumán provinces) to undisturbed areas (Iguazú National Park; Fig. 1, Table 1). The identifi cation of Philomycidae specimens by DNA-data analysis confi rmed their specifi c identity as M. pictum (MLP 13403 and USDA 110442; Table 1). The sequences obtained were identical and larger (655 bp) than previously reported for the species in South America (Gomes et al. 2011). All four phylogenetic approaches provided similar results and indicated a topology Figure 2. Phylogenetic trees of several Deroceras species based on 534 nucleotides of the partial COI gene. A, The most parsimonious tree (179 parsimony-informative characters, total length = 519, CI = 0.5934, RI = 0.8252 and, RC = 0.4897). B, Bayesian consensus tree. The support values: bootstrap values (MP) and posterior probabilities (BI) are shown on the branches. References to the sequences are given in Table 2. 252 AMERICAN MALACOLOGICAL BULLETIN 31 · 2 · 2013 in agreement with Tsai et al. (2011; Fig. 4, cf. the MP and BI trees). In all instances, three groups were identifi ed for M. pictum: Group 1 (NJ = 81 , MP = 86, ML = 88, BI = 0.55) was geographically restricted to Taiwan Island (TWP1, TWP2, TWP3, TWP5, TWP6, TWP7, TWP8, TWP9 and TWP 10); Group 2 (NJ = 100, MP = 100, ML = 100, BI = 1) comprised M. pictum from Kinmen (TWP11) and Lieyu (TWP12) Is- lands as well as the South-American specimens (BRP1, BRP2, BRP3, BRP4, BRP5, ARP1 and ARP2) for which only a single haplotype was found. One sequence from southern Taiwan Island (TWP4) and a sequence from continental China (CNP2) were also found within Group 2. Finally, Group 3 (NJ = 99, MP = 91, ML = 100, BI = 0.81) included sequences from continental China (CNP1), Thailand (TLP1) and Matsu Island (i.e., TWP13, TWP14, TWP15). DISCUSSION The present study revealed the existence of established populations of four exotic slugs in Argentina. The taxonomic identifi cation of the alien species reported here is based on anatomical studies in addition to sequences of COI gene and constitutes the fi rst report of the presence of these species, all four of which have already shown an invasive behavior in several South American countries. Deroceras panormitanum (Lessona and Pollonera, 1882) and Lehmannia valentiana— both native of Europe—had been previously recorded for Chile and Colombia, while L. valentiana had also been reported in Brazil and Peru (Rumi et al. 2010). Based on anatomical and molecular evidence Reise et al. (2011) had split D. panormita- num into several species, including D. panormitanum s.s., as well as Deroceras invadens, that latter as the single successful invader worldwide. According to this new taxonomic ar- rangement, the classifi cation of D. panormitanum remains restricted to only the area where was originally described (i.e., Malta and Sicily). Arion intermedius, a typical European Ari- onidae, has already been recorded in Chile and Colombia (Hausdorf 2002, Letellier et al. 2003, Cádiz and Gallardo 2007). Meghimatium pictum, a native from eastern and southern Asia, had recently been reported as an alien species in southern Brazil, with that record being the fi rst one of the genus in South America (Gomes et al. 2011). Lehmannia valentiana is the species with the oldest oc- currence records in Argentina in comparison to the other three reported here, with data as early as 1924 (Buenos Aires Figure 3. Phylogenetic trees of several invasive or potentially invasive Arion species in South America based on 552 nucleotides of the partial COI gene. A, One of the two most parsimonious trees (229 parsimony-informative characters, total length = 1074, CI = 0.4181, RI = 0.8085 and, RC = 0.3380). B, Bayesian consensus tree. The support values: bootstrap values (MP) and posterior probabilities (BI) are shown on the branches. References to the sequences are given in Table 3. EXOTIC SLUGS FROM ARGENTINA 253 province, MACN-In 14602) and 1962 (Tucumán province, IFML 604). To determine when and how this species was in- troduced into the country, however, is diffi cult because the molecular information at hand was not enough to shed any light on the area of origin of the species. Lehmannia valentiana could be considered as an established species, with an incipient invasive behavior since specimens have been recorded in remote sites (in both urban and protected areas), feeding on ornamental plants. In other countries, this species is consid- ered to be a greenhouse pest, damaging orchid fl owers and ornamental plants (Chichester and Getz 1969, South 1992, Ester et al. 2003). Deroceras invadens was recorded in southern Argentina; but inferences about the site of the species’ introduction were not possible because the sequence from Argentina grouped into a clade that included samples from diverse geographical origins (e.g., Canada, U.S.A., Germany, South Africa), thus indicating that the species had already been widely distrib- uted worldwide. The specimens examined were from several towns of southern Argentina, a distribution that suggested the species had acquired an invasive behavior. The probable pathway for the introduction of D. invadens into Argentina could be related to commerce, as has been reported in the United States (Meissner et al. 2009). Deroceras invadens feeds on a great variety of plants and organic material in de- composition and is known to be a pest in pastures, gardens and agricultural fi elds (Barker 1999). Moreover, this slug spe- cies is identifi ed as an intermediate host for the nematode Gallegostrongylus australis Spratt, Haycock and Walter, 2001 (Spratt et al. 2001), which species parasitizes rodent lungs, causing mild pathologic changes. We could neither clarify the source location nor assign a precise date of introduction for the specimens of Arion inter- medius studied. Cádiz and Gallardo (2007) had reported this species for the fi rst time in neighboring Chile, but the date cited could be considered controversial because Letelier et al. (2003) had recorded many specimens of A. intermedius de- posited into National Museum of Natural History of Chile (but with no collection number) from southern Chile. We could nevertheless, conclude that populations of A. interme- dius had been living in Argentina since at least 2003, the year when they were fi rst collected (Table 1). This species can be considered to be established in Argentina since live speci- mens had also been found at the same locations seven years later, during the summer 2010 (Table 1). The pathway of in- troduction of the species into southern Argentina, however, remains unclear. Cádiz and Gallardo (2007) could not estab- lish the introduction pathway into Chile, although they con- cluded that human accidental transport was the probable cause since species of that genus were being recovered from U.S. sea- ports on limestone, machinery and rocks (Meissner et al. 2009). Arion intermedius exhibits polyphagous or phytophagous Figure 4. Phylogenetic reconstruction of Meghimatium pictum, based on 654 nucleotides of the partial COI gene. A, One of the six most par- simonious trees (245 parsimony-informative characters, total length = 987, CI = 0.464, RI = 0.6399 and, RC = 0.2969). B, Bayesian consensus tree. The support values: bootstrap values (MP) and posterior probabilities (BI) are shown on the branches. References to the sequences are given in Table 4. 254 AMERICAN MALACOLOGICAL BULLETIN 31 · 2 · 2013 feeding habits and a predominant autofecundation, a strategy that increases its invasive capabilities (Wiktor et al. 2000, Reise et al. 2001). They are known to have a high capacity for pen- etrating undisturbed native forest, more so than any other type of exotic slug species (Hausdorf 2002, Cadiz and Gal- lardo 2007). In Argentina, occurrences of the species were recorded within national park areas, far away from urban en- vironments. Within the Arionidae, the genus Arion Férussac, 1819 is recognized by international organizations concerned with pest control, such as the Eastern Region Cooperative Agricultural Pest Survey, as potentially damaging for agricul- ture (Cádiz and Gallardo 2007). Specimens could also act as intermediate hosts for nematodes such as Filaroides martis (Werner, 1783), a parasite that affects the respiratory tract of mammals (Grewal et al. 2003). In both the most parsimoni- ous tree and the Bayesian consensus tree (Fig. 3), the Arion species analyzed grouped in a single clade, suggesting that phylogenetic analysis is as reliable a tool as the morphology in corroborating the identity of the different invasive members of the Arion genus. The introduction of Meghimatium pictum into South America, and particularly into Brazil, was suggested to have been accidental through agricultural products in the 1990s, coinciding with the beginning of trade between China and Brazil in order to boost the production of mushrooms in the latter country (Gomes et al. 2011). On the basis of position of the South-American samples in all the topologies obtained in our phylogenetic reconstructions, we suggest that the origin of the South-American invasive lineage might be located in a region west of the Strait of Taiwan, either along the coast of China or within the islands close to that coast such as the Tai- wanese Kinmen and Lieyu Islands. Currently, M. pictum can be found in several states of Brazil (Paraná, Santa Catarina and Río Grande do Sul) bordering northeast Argentina and also in São Paulo State (Gomes et al. 2011). Occurrences of this species in the Misiones province of Argentina suggest that the introduction pathway into Argentina could possibly be linked either to an active dispersion of the slug from Brazil and/or to the commerce of fl ora between those aforemen- tioned Brazilian areas and Argentina. That we recorded one specimen of M. pictum in a garden with orchids collected from the Misiones province plus other plants acquired from nurseries in the Tucumán province supports our hypothesis about the commercial trade as being the principal vector of introduction. In Brazil, Meghimatium pictum was associated with the attack of plants in private gardens in Santa Catarina (Gomes et al. 2011) and has affected grape vines in Rio Grande do Sul (Baronio et al. 2011). In Argentina, further studies are re- quired to know if incipient grape vines in Misiones province, covering about 50 hectares, are already affected; though, the extent of the invasion could be even worse if the species has reached the western provinces (Mendoza, San Juan, La Rioja and Catamarca) where the 97% of the domestic production of wine is concentrated. Such a development could have seri- ous implications on the country’s national and international wine trade. In the Argentine areas where M. pictum is present, several other gastropods species (both natives and exotics) that can act as intermediate hosts for the zoonotic parasitic nematode Angiostrongylus costaricensis Moreira and Céspedes, 1971 can be found, with the potential risk that M. pictum could likewise begin to act as a new vector. Meghimatium bi- lineatum (Benson, 1842), a very close species to M. pictum, has furthermore been reported to be a host of Angiostrongylus cantonensis (Cheng, 1935) (Li et al. 2006), another zoonotic parasitic nematode. We provide here the fi rst report of four new exotic slugs in Argentine territories. Because of the invasive potential of these slug species, the present work provides new COI se- quences obtained from morphologically confi rmed speci- mens. 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Submitted: 26 December 2012; accepted: 6 April 2013; fi nal revisions received: 14 June 2013