Abstract

The sewage snail Physa acuta, native to North America, is an effective invasive species around the world. In Chile, it was first reported in 2014 in the north central area of the country. So far, the species has not been recorded in southern Chile. Sampling performed in 2015 in three localities from Llanquihue Lake, Chilean Patagonia, only provided native freshwater snails. However, new collections performed in February 2018 in the same three sites were successful for physid specimens suggesting a biological invasion entailing a large southward range expansion of these snails. Here we performed morphological, microstructural and phylogenetic analyses to investigate whether the new samples belong to Physa acuta. The shell morphology, male copulatory complex and radula microstructure of the new material agree with those of the sewage snail. The molecular phylogenetic analyses using the cytochrome c oxidase subunit 1 (COI) gene confirmed morphological identification. We suggest to take prompt measures to prevent the expansion of Physa acuta in Llanquihue Lake or nearby aquatic ecosystems.

Key words
Chile; invasive species; molecular identification; Physa; South America; taxonomy

INTRODUCTION

The planet is currently undergoing two processes of global environmental variation, climate change and the globalization of trade, which have impacted the biosphere generating favorable opportunities for an increase in bioinvasions (Lockwood et al. 2007LOCKWOOD J, HOOPES M & MARCHETTI M. 2007. Invasion ecology. Blackwell, Oxford.). The introduction of invasive species can generate a series of effects in native ecosystems such as economic loss, damage to human health, landscape alterations and threats to biodiversity (Gordon 1998GORDON DR. 1998. Effects of invasive, non-indigenous plant species on ecosystem processes: lessons from Florida. Ecol Appl 8(4): 975-989., Perrings 2001PERRINGS C. 2001. The economics of biological invasions. Land Use and Water Resources Research 1(3): 1-9., Bax et al. 2003BAX N, WILLIAMSON A, AGUERO M, GONZALEZ E & GEEVES W. 2003. Marine invasive alien species: a threat to global biodiversity. Mar Policy 27: 313-323., Juliano & Lounibos 2005JULIANO SA & LOUNIBOS LP. 2005. Ecology of invasive mosquitoes: effects on resident species and on human health. Ecol Lett 8(5): 558-574., Lovell et al. 2006LOVELL S, STONE S & FERNANDEZ L. 2006. The economic impacts of aquatic invasive species: a review of the literature. Agric Resour Econ Rev 35(1): 195-208.). Among a variety of taxa, mollusks are an important source of invasive species and it is not strange that have appeared published in “the list of 100 of the world’s worst invasive alien species” (Lowe et al. 2000LOWE S, BROWNE M & BOUDLEJAS S. 2000. 100 of the world’s worst invasive alien species. Invasive Species Specialist Group, Auckland, p. 1-11.) six species of this phylum, the bivalves Dreissena polymorpha (Pallas, 1771), Mytilus galloprovincialis (Lamarck 1819) and Potamocorbula amurensis (Schrenck, 1861), and the gastropods Achatina fulica (Bowdich, 1822), Euglandina rosea (Férussac, 1821) and Pomacea canaliculata (Lamarck, 1828). Among the invasive mollusks, some fall into the category of “ecosystem engineers” (Jones et al. 1994JONES CG, LAWTON JH & SHACHAK M. 1994. Organisms as ecosystem engineers. Oikos 69: 373-386.), as is the case of the aggressive invasion of the golden mussel Limnoperna fortunei (Dunker, 1857) in South America (Darrigran & Damborenea 2011DARRIGRAN G & DAMBORENEA C. 2011. Ecosystem engineering impacts of Limnoperna fortunei in South America. Zoolog Sci 28: 1-7.).

Physidae are ubiquitous pulmonate gastropods that inhabit a variety of aquatic ecosystems around the world (Appleton 2003APPLETON CC. 2003. Alien and invasive fresh water Gastropoda in South Africa. Afr J Aquat Sci 28: 69-81., Dillon et al. 2002DILLON RT JR, WETHINGTON A R, RHETT J & SMITH T. 2002. Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra. Invertebr Biol 121: 226-234., Taylor 2003TAYLOR DW. 2003. Introduction to Physidae (Gastropoda: Hygrophila), biogeography, classification, morphology. Rev Biol Trop 51: 1-263.). One of them is the freshwater snail Physa acuta Draparnaud, 1805, native to North America (Dillon et al. 2002DILLON RT JR, WETHINGTON A R, RHETT J & SMITH T. 2002. Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra. Invertebr Biol 121: 226-234., 2005DILLON RT, ROBINSON JD, SMITH TP & WETHINGTON AR. 2005. No reproductive isolation between freshwater pulmonate snails Physa virgata and P. acuta. Southwest Nat 50(4): 415e22., Lydeard et al. 2016LYDEARD C, CAMPBELL D & GOLZ M. 2016. Physa acuta Draparnaud, 1805 should be treated as a native of North America, not Europe. Malacologia 59(2): 347-350.), which has profusely invaded all continents, except Antarctic (e.g. Madsen & Frandsen 1989MADSEN H & FRANDSEN F. 1989. The spread of freshwater snails including those of medical and veterinary importance. Acta Trop 46: 139-146., Ali 1993ALI JH. 1993. The distribution of Physa acuta Draparnaud (Gastropoda: Physidae) in Malaysia and its suitability as test material for insecticide toxicity studies. J Med Appl Malacol 5: 129-134., Brackenbury & Appleton 1993, Dillon et al. 2002, Zukowski & Walker 2009ZUKOWSKI S & WALKER KF. 2009. Freshwater snails in competition: alien Physa acuta (Physidae) and native Glyptophysa gibbosa (Planorbidae) in the River Murray, South Australia. Mar Freshwater Res 60(10): 999-1005., Núñez 2010NÚÑEZ V. 2010. Differences on allocation of available resources, in growth, reproduction, and survival, in an exotic gastropod of Physidae compared to an endemic one. Iheringia Sér Zool 100(3): 275-279., Vinarski 2017VINARSKI MV. 2017. The history of an invasion: phases of the explosive spread of the physid snail Physella acuta through Europe, Transcaucasia and Central Asia. Biol Invasions 19(4): 1299-1314.). The species has been commonly called the sewage snail (Aditya & Raut 2002ADITYA G & RAUT SK. 2002. Potential of the leech Glossiphonia weberi (Blanchard) in controlling the sewage snails Physa acuta Draparnaud. Curr Sci 83(11): 1317-1319., Appleton 2003APPLETON CC. 2003. Alien and invasive fresh water Gastropoda in South Africa. Afr J Aquat Sci 28: 69-81., de Kock & Wolmarans 2007DE KOCK KN & WOLMARANS CT. 2007. Distribution and habitats of the alien invader freshwater snail Physa acuta in South Africa. Water SA 33: 717-722., Guo & He 2014GUO Y & HE H. 2014. Identification and characterization of a goose-type lysozyme from sewage snail Physa acuta. Fish Shellfish Immunol 39(2): 321-325.) because it is frequently found in polluted habitats which are plentiful in human waste (Brown 1994BROWN DS. 1994. Freshwater snails of Africa and their medical importance, 2nd edn. Taylor and Francis, London, 609 p.). Physa acuta has also been considered of medical importance since it can potentially acts as an intermediate host of human trematodes (Kanev 1994KANEV I. 1994. Life-cycle, delimitation and description of Echinostoma revolutum (Froelich, 1802) (Trematoda: Echinostomatidae). Syst Parasitol 28: 125-144., Dreyfuss et al. 2002DREYFUSS G, VIGNOLES P, ABROUS M & RONDELAUD D. 2002. Unusual snail species involved in the transmission of Fasciola hepatica in watercress beds in central France. Parasite 9: 113-120.). In South America, Physa acuta was identified for the first time by Paraense & Pointier (2003)PARAENSE WL & POINTIER JP. 2003. Physa acuta Draparnaud, 1805 (Gastropoda: Physidae): a study of topotypic specimens. Mem Inst Oswaldo Cruz 98(4): 513e7. who reported the synonymy of Physa cubensis Pfeiffer, 1839 with Physa acuta but it is apparent that the species was present from before in the subcontinent (Miquel 1985MIQUEL SE. 1985. Presencia del género Physella Haldeman, 1842 en la República Argentina (Mollusca Pulmonada Physidae). Neotrópica 31: 38., Paraense 1987PARAENSE WL. 1987. Physa cubensis Pfeiffer, 1839 (Pulmonata: Physidae). Mem Inst Oswaldo Cruz 82: 15-20., Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578., Darrigran et al. 2020DARRIGRAN G ET AL. 2020. Non-native mollusks throughout South America: emergent patterns in an understudied continent. Biol Invasions 22(3): 853-871.).

Despite there is not agreement among the authors, in Chile there has been reported five native physid species: Physa peruviana Gray, 1828, Physa chilensis Claessin, 1886, Physa rivalis Sowerby, 1874, Physa porteri Germain, 1913 and Physa nodulosa Biese, 1948BIESE WA. 1948. Revisión de los moluscos terrestres y de agua dulce provistos de concha de Chile. Familia Ancylidae y Physidae. Bol Mus Nac Hist Nat 24: 217-239. (Stuardo 1961STUARDO J. 1961. Contribución a un catálogo de los moluscos chilenos de agua dulce. Con una clave adicional de géneros. Gayana 1: 7-32., Valdovinos 1999VALDOVINOS C. 1999. Biodiversidad de moluscos chilenos: Base de datos taxonómica y distribucional. Gayana 63: 111-164., 2006VALDOVINOS C. 2006. Estado de conocimiento de los gastrópodos dulceacuícolas de Chile. Gayana 70: 88-95., Sielfeld 2001SIELFELD W. 2001. Phylum Mollusca. Guías de identificación y biodiversidad fauna chilena. Apuntes de Zoología. Universidad Arturo Prat, Iquique, 15 p.). In 2014, the alien Physa acuta was added to this list, being recorded in the north central area of the country. The first identification was made from the Elqui River, Región de Coquimbo, and Mataquito River, Región del Maule, both rivers separated by about 560 km (Bousset et al. 2014BOUSSET L, POINTIER JP, DAVID P & JARNE P. 2014. Neither variation loss, nor change in selfing rate is associated with the worldwide invasion of Physa acuta from its native North America. Biol Invasions 16: 1769-1783.). More recently, several other populations were reported and collected from different basins in the country, partially filling the gap in the invasive range of the species (Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.):

• Región de Coquimbo: Choapa River, Illapel River and Consuelo Stream in the Choapa River basin.

• Región de Valparaíso: Santa Juana near Concón in the Aconcagua River basin.

• Región Metropolitana: Parque O’Higgins Spring in the Maipo River basin.

• Región de Valparaíso: El Salto in the Marga Marga River basin.

New collections performed in Llanquihue Lake, Región de Los Lagos, Chilean Patagonia, provided physid snails from Puerto Chico, a beach east from Puerto Varas City, Llanquihue City and Maullín River, the effluent of the lake. As these three places were sampled in 2015 by the first author together with assistants, being positive only for native snails (unpublished data), we hypothesized a biological invasion of this species into the lake, including a large dispersion and increase in the range of expansion in the country. This is supported by the fact that physid snails have not been reported for the area in any of the checklist of Chilean mollusk species or other works related to native snails of the lake (Marshall 1933MARSHALL WB. 1933. New fresh-water gastropod mollusks of the genus Chilina of South America. Proceedings U.S. National Museum 82: 1-6., Biese 1944BIESE WA. 1944. Revisión de los moluscos terrestres y de agua dulce provistos de concha de Chile. Parte I, Familia Amnicolidae. Bol Mus Nac Hist Nat 22: 169-190., Stuardo 1961STUARDO J. 1961. Contribución a un catálogo de los moluscos chilenos de agua dulce. Con una clave adicional de géneros. Gayana 1: 7-32., Valdovinos 1999VALDOVINOS C. 1999. Biodiversidad de moluscos chilenos: Base de datos taxonómica y distribucional. Gayana 63: 111-164., 2006, Sielfeld 2001SIELFELD W. 2001. Phylum Mollusca. Guías de identificación y biodiversidad fauna chilena. Apuntes de Zoología. Universidad Arturo Prat, Iquique, 15 p.).

Taxonomic identification of physid species has been historically difficult since species are morphologically similar due to overlapping shell characters due to genetic and environmental influences (Dewitt et al. 2000DEWITT TJ, ROBINSON BW & WILSON DS. 2000. Functional diversity among predators of a freshwater snail imposes an adaptive trade-off for shell morphology. Evol Ecol Res 2: 129-148., Gustafson et al. 2014GUSTAFSON KD, KENSINGER BJ, BOLEK MG & LUTTBEG B. 2014. Distinct snail (Physa) morphotypes from different habitats converge in shell shape and size under common garden conditions. Evol Ecol Res 16: 77-89.). The aim of the present study is to assess whether the recently sampled populations of physid snails from Llanquihue Lake correspond to the invasive Physa acuta or a closely related species. For this, we performed morphological, microstructural and phylogenetic analyses.

MATERIALS AND METHODS

Llanquihue Lake is the second largest of the lakes of Chile after Lake General Carrera. It is located between 40°58’ and 41° 20’ S and between 72°31’ and 73°00 W in the Llanquihue Province, comprising an area of about 585 km², with an altitude of 52 m and a depth of 350 m (Solano Asta-Buruaga & Cienfuegos 1899, Chile365 2018LOCKWOOD J, HOOPES M & MARCHETTI M. 2007. Invasion ecology. Blackwell, Oxford.). It drains on its Southwest side to the Pacific Ocean nine kilometers to the NW of Puerto Varas giving origin to the Maullín River. Sampling was performed by hand using a sieve from 11 sites along the shoreline of the Llanquihue Lake in February 2018, including streams, irrigation canals and small lagoons, as well as the Maullín River (Fig. 1). The snails were examined and photographed using a Motic SMZ-168 Stereomicroscope adapted with a Moticam 2000 digital camera. The radula was cleaned in a diluted sodium hypochlorite solution and observed using a Hitachi SU3500 scanning electron microscope (SEM). Initial snail identification was based on the original description and additional literature (Paraense 1987PARAENSE WL. 1987. Physa cubensis Pfeiffer, 1839 (Pulmonata: Physidae). Mem Inst Oswaldo Cruz 82: 15-20., Paraense & Pointier 2003PARAENSE WL & POINTIER JP. 2003. Physa acuta Draparnaud, 1805 (Gastropoda: Physidae): a study of topotypic specimens. Mem Inst Oswaldo Cruz 98(4): 513e7., Núñez 2011NÚÑEZ V. 2011. Revisión de dos especies de Physidae. Rev Mex Biodivers 82: 93-108., Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.). Voucher specimens were deposited in the Museo de Ciencias Naturales y Arqueología Profesor Pedro Ramírez Fuentes (MCNPPRF-CC 148-01 to MCNPPRF-CC 148-05), Chillán, Chile. Partial sequences of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene were obtained following Collado (2017)COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578., edited in BioEdit (Hall 1999HALL TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analyses. Nucl Acids Symp Ser 41: 95-98.) and aligned in Clustal X (Thompson et al. 1997THOMPSON JD, GIBSON TJ, PLEWNIA KF, JEANMOUGIN F & HIGGINS DG. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acid Res 25: 4876-4882.). Haplotype diversity was estimated using DnaSP 4.0 (Rozas et al. 2003ROZAS J, SÁNCHEZ-DELBARRIO JC, MESSEGUER X & ROZAS R. 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19: 2496-2497.). Maximum parsimony (MP) and Bayesian inference (BI) analyses were implemented in PAUP* (Swofford 2003SWOFFORD DL. 2003. PAUP*: Phylogenetic analysis using parsimony (*and other methods). Ver. 4. Sinauer Associates, Sunderland.) and MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003RONQUIST F & HUELSENBECK JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.), respectively, following Collado (2017)COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.. For the BI analysis we set the best model of sequence evolution obtained in jModelTest 0.1.1 (Posada 2008POSADA D. 2008. jModelTest: phylogenetic model averaging. Mol Biol Evol 25: 1253-1256.) based on the Bayesian information criterion (TPM2uf+I+G). The support nodes were evaluated using 100 bootstrap (bt) pseudoreplicates (Felsenstein 1985FELSENSTEIN J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.) in the MP analysis while in BI analysis were obtained as posterior probability values (p.p). We generated a matrix of 75 COI sequences including those obtained in the present study plus others of Physa acuta and related species retrieved from GenBank (Wethington & Lydeard 2007WETHINGTON AR & LYDEARD C. 2007. A molecular phylogeny of Physidae (Gastropoda: Basommatophora) based on mitochondrial DNA sequences. J Molluscan Stud 73: 241-257., Pip & Frank 2008PIP E & FRANK JPC. 2008. Molecular phylogenetics of central Canadian Physidae (Pulmonata: Basommatophora). Can J Zool 86: 10-16., Wethington et al. 2009WETHINGTON AR, WISE J & DILLON RT. 2009. Genetic and morphological characterization of the Physidae of South Carolina (Pulmonata: Basommatophora), with description of a new species. Nautilus 123: 282-292., Albrecht et al. 2009ALBRECHT C, KROLL O, TERRAZAS EM & WILKE T. 2009. Invasion of ancient Lake Titicaca by the globally invasive Physa acuta (Gastropoda: Pulmonata: Hygrophila). Biol Invasions 11: 1821-1826., 2014ALBRECHT C, FÖLLER K, HAUFFE T, CLEWING C & WILKE T. 2014. Invaders versus endemics: alien gastropod species in ancient Lake Ohrid. Hydrobiologia 739: 163-174., Gates et al. 2013GATES KK, KERANS BL, KEEBAUGH J, KALINOWSKI S & VU N. 2013. Taxonomic identity of the endangered Snake River physa, Physa natricina (Pulmonata: Physidae) combining traditional and molecular techniques. Conserv Genet 14: 159-169., R. Aguilar, M.B. Ogburn and A.H. Hines, unpublished data, D.S. Park and H.W. Oh, unpublished data). A sequence of the species Lymnaea stagnalis (Linnaeus, 1758) was used as outgroup (Remigio 2002REMIGIO EA. 2002. Molecular phylogenetic relationships in the aquatic snail genus Lymnaea, the intermediate host of the causative agent of fascioliasis: insights from broader taxon sampling. Parasitol Res 88(7): 687-696.).

Figure 1
Sampling localities in Llanquihue Lake. Physa acuta was found in three places (red triangles). Negative sampling sites for the species are shown in green circles. Principal cities are depicted by black squares.

RESULTS

Sampling performed in Llanquihue Lake allowed to collect eight physid specimens in Llanquihue City (41°15’15.00 S; 73°00’06.50’’ W), forty three in Puerto Chico (41°19’37.06” S; 72°57’26.64” W) and one in Maullín River (41°15’41.07” S; 73°00’09.14” W), which were identified as Physa acuta (Fig. 2 a-r). Shell measurements are shown in Table I Eight additional sampling sites only provided native specimens of the genus Potamolithus Pilsbry, 1896 and/or Chilina Gray, 1828.

Figure 2
a-f. Specimen shells of Physa acuta from Llanquihue Lake under stereomicroscope. a, b. Specimen from Maullín River (MCNPPRF-CC 148-01). c, d. Specimen from Puerto Chico (Puerto Varas) (MCNPPRF-CC 148-02). e, f. Specimen from Llanquihue City (MCNPPRF-CC 148-03). g, h. The same specimen of a and b imaged using SEM. i-m. Male copulatory complex of a specimen from Puerto Chico (i, j) (MCNPPRF-CC 148-02) and Llanquihue City (k, l, m) (MCNPPRF-CC 148-03), j,l, left side, k, right side (p: penis, pg: preputial gland, pp: prepuce, ps: penis sheath, psg: penis sheath gland, vd: vas deferens). n. Anterior section of the radular ribbon of a specimen from Llanquihue City (MCNPPRF-CC 148-04). o. Zoom of the central area of n showing the radular teeth augmented (1: central tooth, 2: first pair of lateral teeth, 3: second pair of lateral teeth, 4: marginal teeth). p-r. Radular teeth of a specimen from Puerto Chico (the numbers of the teeth correspond to those denoted in o). Scale bar: a-h= 3 mm, i= 300 µm, j-l= 1 mm, m= 300 µm, n= 50 µm, o= 30 µm, p-r= 5 µm.

The shell shape of specimens examined in the present study is elongate-ovate, thin, sinisterly coiled, light brown and translucent (Fig. 2a-h). The mantle is black with white spots, foot gray. The distal portion of the male copulatory complex includes prepuce, penis sheath gland, penis sheath and vas deferens (Fig. 2i-m). The prepuce is a large structure which includes a lenticular preputial gland near its posterior end. The penis sheath encloses an elongated conical penis. The radula is V-shaped (Fig. 2 n-o). It consists of a central tooth composed by three major cusps and other minor ones (Fig. 2o), followed by two pairs of lateral teeth and a series of marginal teeth (Fig. 2p-r).

We yielded 11 COI sequences of Physa acuta from Puerto Chico (6) and Llanquihue City (5), which were submitted to GenBank (accession numbers: MK024393-MK024403). The sequence analysis allowed to identify five haplotypes, two from Puerto Chico, two from Llanquihue City and one shared by seven specimens of both localities. Both, the MP (tree not shown) and BI (Fig. 3) analyses recovered the snails from Llanquihue Lake in the Physa acuta clade with high node supports (1.00 p.p, 100% bt). Within this clade, the five haplotypes clustered in three different subclades.

Figure 3
Bayesian phylogenetic tree obtained using COI sequences. Snails sequenced in the present study are depicted in bold. Numbers above the nodes indicate posterior probability values obtained in the BI analysis (only values equal to or greater than 0.95 are given) followed by the bootstrap values obtained in the MP analysis (only values above 50% are given).

DISCUSSION

The shell shape, male copulatory complex and radular microstructure of specimens from Llanquihue Lake were congruent with the characteristics previously reported in Physa acuta (e.g. Draparnaud 1805DRAPARNAUD JJ. 1805. Histoire naturelle des mollusques terrestres et fluviatiles de la France. Ouvrage posthume. Chez Luis Colas, Paris, p. 55., Paraense 1987PARAENSE WL. 1987. Physa cubensis Pfeiffer, 1839 (Pulmonata: Physidae). Mem Inst Oswaldo Cruz 82: 15-20., 2003PARAENSE WL. 2003. Planorbidae, Lymnaeidae and Physidae of Peru (Mollusca: Basommatophora). Mem Inst Oswaldo Cruz 98: 767-771., Appleton 2003APPLETON CC. 2003. Alien and invasive fresh water Gastropoda in South Africa. Afr J Aquat Sci 28: 69-81., Paraense & Pointier 2003PARAENSE WL & POINTIER JP. 2003. Physa acuta Draparnaud, 1805 (Gastropoda: Physidae): a study of topotypic specimens. Mem Inst Oswaldo Cruz 98(4): 513e7., Núñez 2011NÚÑEZ V. 2011. Revisión de dos especies de Physidae. Rev Mex Biodivers 82: 93-108., Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.). Among all the morphological features, the position and characteristic of the preputial gland, lenticular in shape, has been considered as relevant to recognize this species (Paraense 1987PARAENSE WL. 1987. Physa cubensis Pfeiffer, 1839 (Pulmonata: Physidae). Mem Inst Oswaldo Cruz 82: 15-20., 2003, Paraense & Pointier 2003PARAENSE WL & POINTIER JP. 2003. Physa acuta Draparnaud, 1805 (Gastropoda: Physidae): a study of topotypic specimens. Mem Inst Oswaldo Cruz 98(4): 513e7.), although this need to be verified with caution since it is present in other members of the genus (Wethington & Lydeard 2007WETHINGTON AR & LYDEARD C. 2007. A molecular phylogeny of Physidae (Gastropoda: Basommatophora) based on mitochondrial DNA sequences. J Molluscan Stud 73: 241-257.). The BLASTn search and phylogenetic analysis based on COI sequences also allowed to recognize the specimens from Llanquihue Lake as belonging to Physa acuta. The shared haplotype showed 100% identity to other two sequences of Physa acuta, one from Anne Arundel County, Maryland, USA (MH087660) (R. Aguilar, M.B. Ogburn and A.H. Hines, unpublished data) and another from South Korea (KF966541) (D.S. Park and H.W. Oh, unpublished data). Another haplotype recovered in Llanquihue City showed 100% identity to the sequence EU038367 obtained from Santiago de Cuba (Wethington & Lydeard 2007WETHINGTON AR & LYDEARD C. 2007. A molecular phylogeny of Physidae (Gastropoda: Basommatophora) based on mitochondrial DNA sequences. J Molluscan Stud 73: 241-257.) and several snails from northern and central Chile (Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.). A sequence of Puerto Chico differs in one substitution of sequences from El Salto (KX108847) in central Chile (Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.) and Lake Titicaca (FJ373016) (Albrecht et al. 2009ALBRECHT C, KROLL O, TERRAZAS EM & WILKE T. 2009. Invasion of ancient Lake Titicaca by the globally invasive Physa acuta (Gastropoda: Pulmonata: Hygrophila). Biol Invasions 11: 1821-1826.).

The presence of Physa acuta in Chile has put in doubt the identity of some native species of the genus. For instance, the native Physa chilensis was described in 1886 with samples from Chile as type locality and later collected from El Salto River in the Región de Valparaíso (Sielfeld 2001SIELFELD W. 2001. Phylum Mollusca. Guías de identificación y biodiversidad fauna chilena. Apuntes de Zoología. Universidad Arturo Prat, Iquique, 15 p.) but samples from this locality were unambiguously identified as Physa acuta (Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.). The same occur in the case of Physa nodulosa, originally described in 1948 from the Choapa River, Salamanca and other places in the Región de Coquimbo, where the presence of Physa acuta was also verified (Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.).

To become a pest, an invasive species must possess life history traits that confer ability to survive, reproduce and growth in the new ecosystem and then spread to additional places. Among the traits that have allowed Physa acuta to overcome the growth of ecologically analogous native species are a higher salinity and temperature tolerances, faster growth rate, higher fecundity, shorter incubation period and continuous reproduction during the year (Brackenbury & Appleton 1991BRACKENBURY TD & APPLETON CC. 1991. Effect of controlled temperatures on gametogenesis in the gastropods Physa acuta (Physidae) and Bulinus tropicus (Planorbidae). J Molluscan Stud 57(4): 461-469., Zukowski & Walker 2009ZUKOWSKI S & WALKER KF. 2009. Freshwater snails in competition: alien Physa acuta (Physidae) and native Glyptophysa gibbosa (Planorbidae) in the River Murray, South Australia. Mar Freshwater Res 60(10): 999-1005., Ng et al. 2015NG TH, TAN SK & YEO DCJ. 2015. Clarifying the identity of the long-established, globally-invasive Physa acuta Draparnaud, 1805 (Gastropoda: Physidae) in Singapore. BioInvasions Rec 4(3): 189-194.). The success of Physa acuta as an invader has also been shown by its great resilience capacity to repeated flooding, overcoming the population growth and expansion capability of native species (Brackenbury & Appleton 1991BRACKENBURY TD & APPLETON CC. 1991. Effect of controlled temperatures on gametogenesis in the gastropods Physa acuta (Physidae) and Bulinus tropicus (Planorbidae). J Molluscan Stud 57(4): 461-469.). In some invaded habitats like the Umsindusi River, South Africa, Physa acuta reaches densities of up to 4561 individuals/m² (Brackenbury & Appleton 1991BRACKENBURY TD & APPLETON CC. 1991. Effect of controlled temperatures on gametogenesis in the gastropods Physa acuta (Physidae) and Bulinus tropicus (Planorbidae). J Molluscan Stud 57(4): 461-469.) while the net reproductive rate has been recorded to be 116.1 in India (Saha et al. 2017SAHA C, PARVEEN S, CHAKRABORTY J, PRAMANIK S & ADITYA G. 2017. Life table estimates of the invasive snail Physa acuta Draparnaud, 1805, occurring in India. Ekológia 36(1): 60-68.). Several authors have shown that this species has replaced populations of endemic species of different genera (de Kock & Walmarans 2007, Dobson 2004DOBSON M. 2004. Replacement of native freshwater snails by exotic Physa acuta (Gastropoda: Physidae) in southern Mozambique, a possible control mechanism for schistosomiasis. Ann Trop Med Parasitol 98: 543-548., Gashaw et al. 2008GASHAW F, ERKO B, TEKLEHAYMANOT T & HABTESELLASIE R. 2008. Assessment of the potential of competitor snails and African catfish (Clarias gariepinus) as biocontrol agents snail hosts transmitting schistosomiasis. Trans R Soc Trop Med Hyg 102: 774-779.). At present, Physa acuta in Llanquihue Lake coexist with two ecologically analogous native freshwater snails, Potamolithus australis Biese, 1944 and Chilina llanquihuensis Marshall, 1933, with which competitive interactions can be expected as a consequence of niche overlap.

With the data reported in this study, the range of the invasion in Chile is extended several kilometers to the south than previously known (Bousset et al. 2014BOUSSET L, POINTIER JP, DAVID P & JARNE P. 2014. Neither variation loss, nor change in selfing rate is associated with the worldwide invasion of Physa acuta from its native North America. Biol Invasions 16: 1769-1783., Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.), being the southernmost record for the country. Given the high invasiveness of Physa acuta, and the absence of previous records in southern Chile, it is possible that climate change may have favored the invasion of the species and subsequent propagation in the lake. Alternatively, it is also possible that the contamination by sewage waters, which produced several problems to inhabitants of Puerto Varas in 2017 (e.g. https://www.elciudadano.cl/chile/contaminacion-lago-llanquihue, http://www.terram.cl/2017/12/salud-confirma-puntos-de-alta-contaminacion-en-lago-llanquihue/) could have also favored the spreading of this alien species. In this context, it is worth highlighting an increasingly important variant of dispersal, such as the Jump dispersal model of non-native species, which is associated with humans (MacIssac et al. 2001). This dispersal mode is determined by the probability of movement of propagules transported through human activities from a source of origin to a recipient ecosystems (for example, a tourist center). This type of dispersal is much less dependent on time or on the distance between source and recipient site. Once the non-native species reaches this place, it can spread according to its natural capacity or be transported again by humans through Jump dispersal to a new ecosystem. The positive about establishing the model by which non-native species propagate, lies in, for example, focusing attention on the probabilities of invasion from that receptor site now known to another site, in order to prevent it.

Physids can be spread via different active and/or passive dispersal vectors (Dillon & Wethington 1995DILLON RT JR & WETHINGTON AR. 1995. The biogeography of sea islands: clues from the population genetics of the freshwater snail, Physa heterostropha. Syst Biol 44: 400-408., de Kock & Wolmarans 2007DE KOCK KN & WOLMARANS CT. 2007. Distribution and habitats of the alien invader freshwater snail Physa acuta in South Africa. Water SA 33: 717-722., van Leeuwen et al. 2013VAN LEEUWEN C, HUIG N, VAN DER VELD G, VAN ALEN TA, WAGEMAKER C, SHERMAN C, KLAASSEN M & FIGUEROLA J. 2013. How did this snail get here? Several dispersal vectors inferred for an aquatic invasive species. Freshwater Biol 58: 88-99.). The date and mechanism of introduction of Physa acuta in Chile is unknown (Collado 2017COLLADO GA. 2017. Unraveling cryptic invasion of a freshwater snail in Chile based on molecular and morphological data. Biodivers Conserv 26: 567-578.). The introduction route to Llanquihue Lake is also unknown, but after the summer of 2015, considering that the three places in which the species was found in this study were exhaustively surveyed that year. It is reasonable that there may have been more than one introduction event in the lake, judging from the different haplotypes found, including the arrival of animals from South Korea or Maryland. However, considering that four of the five haplotypes were already identified in northern and central Chile, we think the most parsimonious interpretation would be that a single introduction of several genetically variable snails occurred from one of these northernmost regions of the country, or both. However, regardless of the causes that entry mechanism of the species, as the invasion is recent, it is advisable to take prompt measures to prevent the expansion of this invasive mollusk in Llanquihue Lake or nearby aquatic ecosystems.

ACKNOWLEGMENTS

To the staff of the Museo de Ciencias Naturales y Arqueología Profesor Pedro Ramírez Fuentes, Chillán, Chile and Cristian Suarez for technical support. GAC, MAV and CT-D are grateful to Grupo de Investigación Biodiversidad y Cambio Global (GI 170509/EF), Universidad del Bío-Bío. We also thank the CONICYT-FONDEQUIP Program (No. EQM-140088) for acquisition of the Hitachi Scanning Electron Microscope. This work was partially supported by the CONICYT-FONDECYT under Grant 11130697 (GAC).

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