Molecular analysis confirms the invasion of Amanita muscaria in native Nothofagus forests from Patagonia, Argentina

Molecular analysis confirms the invasion of Amanita muscaria in native Nothofagus forests from Patagonia, Argentina | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular analysis confirms the invasion of Amanita muscaria in native Nothofagus forests from Patagonia, Argentina Paula Victoria Giles, María Eugenia Salgado Salomón, María Belén Pildain, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6523152/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Biological Invasions → Version 1 posted 5 You are reading this latest preprint version Abstract Amanita muscaria , an ectomycorrhizal fungus native to the Northern Hemisphere, has been presumably introduced to Patagonia via exotic pine plantations. Recent findings confirm its presence in pure Nothofagus forests within several National Parks. Morphological and molecular analyses verified its association with N. dombeyi and N. pumilio , showing its capacity to invade native tree roots. Although its distribution in Nothofagus forests is still limited to small patches, its expansion could threaten the biodiversity in protected areas and other native forests of high landscape, ecological and economic value in Argentine Patagonia. Early detection and management strategies, critical to mitigate its spread, are discussed. Invasive ectomycorrhiza biological invasions exotic Pinus plantations phylogeny ITS Figures Figure 1 Introduction Afforestation with exotic conifers in Patagonia has facilitated the introduction and spread of ectomycorrhizal (EM) fungi, altering native forest ecosystems (Barroetaveña et al. 2010; Salgado Salomón et al. 2013, 2018; Salgado Salomón and Barroetaveña 2022). Amanita muscaria (L.) Lam., widely reported from Patagonian conifer plantations (Barroetaveña et al. 2007; Lorenzo 2018), forms natural associations in boreal and temperate, mature, conifers, birches and oaks forests from the Northern Hemisphere (Lebel 2024). The first formal report of the species fruiting in Patagonia is dated in 2001, from a pine plantation in Sectional Pucará, Lanín National Park (Lorenzo 2018). Since then, the species has been recorded in different locations in that National Park and increasingly to the south, always associated with Pinus L. species, each year with more abundant fruiting (Lorenzo 2018). Meanwhile, since 2014, several National Parks rangers have reported A. muscaria basidiomata presence in pure native Nothofagus forests. In this sense, the jump of A. muscaria to Nothofagaceae Kuprian. forests in New Zealand, Tasmania and Australia (Dunk et al. 2012) and to Quercus humboldtii Bonpl. in Colombia (Vargas et al. 2019) have been already reported. Confirming the effective invasion of Patagonian Nothofagus spp. with A. muscaria could imply the displacement of native EMs, a disturbance that threatens native biodiversity and eventually ecosystem balance (Fuhrer 2005). As this phenomenon is of great concern regarding the conservation of subantarctic forests of Argentina, but it has not yet been sufficiently studied in the region, this study aimed to determine the effective EM association of A. muscaria with endemic Nothofagus spp. and analyze the phylogenetic relationship of Patagonian A. muscaria specimens with those reported worldwide. Materials and methods Field work To identify sampling sites, consultations and records of A. muscaria basidiomata findings in Nothofagus forests within Arrayanes, Nahuel Huapi, Lago Puelo and Los Alerces, National Parks were conducted. Own records of plantations with sporocarps of the species were used. Sampling sites were located in northwestern Patagonian provinces: Neuquén, Río Negro and Chubut (Argentina). Twenty-eight samples of A. muscaria basidiomata were collected in pure Nothofagus spp. forests, pure and mixed Pinus ponderosa Dougl. ex Laws. and P. radiata D. Don. plantations with presences of Picea Link, Pseudotsuga , Betula L. and Quercus L., during autumn of 2020 to 2024 (Supplementary Table 1). When A. muscaria basidiomata were detected in pure Nothofagus forests (patches without the presence of exotic EM tree species more than 5000 m or more than 1000 m when natural barriers, such as rivers, were present), soil with root tips were taken below the A. muscaria basidioma until 10 cm depth (Supplementary Table 1). Morphological study Basidiomes: Macroscopical descriptions and measurements, color and macrochemical KOH reactions (KOH 30%) were done on fresh basidiomata. Microscopical characteristics were checked on oven dried (45°C) specimens revived in 3% KOH + floxin, Melzer's reagent and cotton blue following Salgado Salomón et al. (2018), and documented with a Nikon camera D70 in combination with the computer program LASX (https://www.leica-microsystems.com) and ImageJ (https://imagej.nih.gov/). Basidiospores obtained from the veil tissue were measured in 3% KOH, expressing (min) mean ± standard deviation (max) of 150 spores for statistical evaluation (Salgado Salomón et al. 2018); its dextrinoid reaction was tested with Melzer’s reagent acting five minutes. All material was deposited in the HCFC Herbarium (Supplementary Table 1). Species nomenclature and taxonomic classification followed the Index Fungorum database (www.indexfungorum.org). DNA extraction, PCR amplification and sequencing DNA extraction was performed following the CTAB method as described by Salgado Salomón et al. (2017, 2018). For basidiomes, 2-3 uncontaminated lamellae per collection were used. For ectomycorrhizal morphotypes, DNA was individually extracted from five representative root tips per site. The ITS region (rDNA ITS1-5.8S-ITS2) was amplified using the primers ITS4 and ITS5 for basidiomes, and ITS1F and ITS4 for ectomycorrhizal morphotypes (White et al. 1990; Gardes and Bruns 1993). PCR reactions were carried out in the MyCycler™ thermocycler (BioRad) with the protocol from Salgado Salomón et al. (2017). Amplified PCR products were visualized under UV illumination by gel electrophoresis on 1% agarose (w/v) stained with Gel-Red ™ (Biotium, California, USA). Purification and sequencing were performed at CERELA (Tucumán, Argentina) and MACROGEN (Seoul, South Korea). Data analysis Raw sequences were edited in BioEdit v7.0.9.0 (Hall 1999), aligned using the L-INS-i algorithm in MAFFT v7.0 (Katoh and Standley 2013), and manually refined in MEGA vX (Kumar et al. 2018). Reference sequences were retrieved via BLASTn from GenBank (Benson et al. 2000), and alignment was based on published datasets (Oda et al. 2004; Geml et al. 2006, 2008; Dunk et al. 2012), using Amanita ceciliae (GenBank AB015694: LEM950069) as outgroup (Supplementary Table 2). The best-fit evolutionary model (GTR+G) was selected using AIC in jModelTest (http://darwin.uvigo.es). Phylogenetic analyses were conducted under Maximum Likelihood in RAxML v7.2.8 (Stamatakis 2014) with 1000 bootstrap replicates. Newly generated sequences were deposited in GenBank (accession numbers OQ324766, OQ324769–70, OQ324774–75, OQ324778–79, PP490741–63; Supplementary Table 1). Results Seven patches of A. muscaria basidiomes were found in 4 protected areas associated with native N. dombeyi (Mirb.) Oerst. and N. pumilio (Poepp. and Endl.) Krasser, including Hua Huam Trail (Arrayanes National Park), El Manso (Nahuel Huapi National Park), Los Hitos, Camping Gendarmería, and Cerro Cuevas (Lago Puelo National Park), Puerto Chucao Trail and Los Pozones (Los Alerces National Park), with no evidence of nearby exotic plant species. A white EM morphotype was consistently found on roots below the basidiomata matching the characteristics of A. muscaria -type mycorrhizae. Twenty-eight basidiomes and two morphotypes were BLASTs as A. muscaria. Of them eleven basidiomes and both morphotypes were associated with N. dombeyi forests, one basidioma with N. pumilio forest, and the remaining were associated with P. radiata D. Don and P. ponderosa Dougl. ex Laws. Morphologically, some specimens of A. muscaria associated with Nothofagus spp. matched with the description of A. muscaria var. formosa Pers., presenting an orange to yellowish pileus and yellowish to cream mottling, although the phylogenetic analysis showed that the formosa variety belongs to a different clade. On the other hand, the specimens associated with coniferous forests corresponded morphologically to A. muscaria var. muscaria , with red pileus and white mottling (Geml et al. 2006). Despite these morphological differences, molecular analysis confirmed that all samples belonged to clade II, with Eurasian and Alaskan vouchers and with samples obtained by Dunk et al. (2012) from invaded Lophozonia cunninghamii (Hook.) Oerst. (Nothofagaceae) from Australia (Fig. 1 ). Discussion Amanita muscaria is invading pure stands of N. dombeyi and N. pumilio in Patagonia Argentina, evidenced by the presence of basidiomes in sites without exotic plant species, and confirmed by the molecular determination of A. muscaria morphotypes in Nothofagus spp. root tips. This coincides with what has been reported in Oceania, where A. muscaria was found in mixed native Lophozonia - Eucalyptus forest (Dickie et al. 2008; Dunk et al. 2012), in Q. humboldtii in Colombia (Vargas et al. 2019) and in South Africa, where Narh Mensah et al. (2024) found that it has jumped from exotics Pinus spp. to native forests. The sequences of invasive A. muscaria collections obtained in this study and those obtained by Dunk et al. (2012) for Australia, by Vargas et al. (2019) for Colombia and by Narh Mensah et al. (2024) for South Africa coincide with clade II proposed by Geml et al. (2008). This clade is the most widely distributed, likely due to the importation of soil and plants from North America and Europe to southern hemisphere countries (Wu et al. 2007; Vargas et al. 2019). This could apply to the Nothofagus forests in Patagonia. Although, to know the origin of the inoculum that could have originated its invasion, it would be necessary to do haplotype network studies and population studies to understand the distribution pattern, as it was done for other introduced fungi such as Suillus luteus (L.: Fries) Gray (Pildain et al. 2021). All sampled N. dombeyi sites invaded with A. muscaria shared common characteristics were areas of transhumance, well-travelled by tourists and cattle, with constant soil removal, proximity to watercourses, and a history of forest fires with subsequent reconditioning tasks. Invasive EM fungi are frequently associated with trails, camping sites, and other areas where they are likely to come into contact with shoes, vehicles, and camping gear (Dickie et al. 2016). A. muscaria , in particular, has been associated with disturbed environments where the presence of dispersal vectors and soil movement were high (Dunk et al. 2012). Anyway, when A. muscaria was found in N. pumilio forest in Cerro Cuevas, the context was different. This site is a Strict Nature Reserve, free of exotic species, with very low disturbance, where only Rangers can enter. In this case, it is possible to think of dispersion through local fauna or bagual cattle, as Nuñez et al. (2013) proposed to explain invasive EM fungi dispersion in Patagonian forests. Amanita muscaria distribution in invaded sites from Patagonian protected areas is still limited to small patches in disturbed areas. Although, this situation could progress compromising local biodiversity by displacing native fungal species and generating maladaptation of the associated forest species (Kranabetter et al. 2015). Despite scientific recognition of the spread and impact of invasive EM fungi, there is little practical advice on their management and few eradication strategies. Dickie et al. (2016) discussed possible strategies for prevention, slowing the spread, eradication and long-term management of invasive fungi, including manual removal of sporocarps, application of fungicide, trenching and elimination of associated tree species. A first experience aiming to control the spread of A. muscaria in Lago Puelo National Park (APN) included the removal of potential native associated trees in a delimited invaded area, but these actions increased the dispersal of the fungus, probably by increasing disturbance at the site (D. Raymundi, pers. comms.). Given that the active colonization of root tip in neighboring-invaded area host trees was not analyzed, invaded native trees could have been left standing. Also, once the spores are dispersed, they could remain infective in the spore bank even if trees are removed (Dickie et al. 2016). Moreover, the removal of possible associated native plant species in protected areas, with high conservation value, is not recommended as its fungal and plant biodiversity and ecosystem services could be compromised. With these considerations, a low impact protocol that preserves soil integrity, generating the least possible disturbance, but controlling A. muscaria dispersal is needed, along with periodical monitoring to assure early detection, key factors to control the spread (Dickie et al 2016). Previous studies highlight the role of EM mutualism in plant invasions (Richardson et al. 2008), but EM fungi as invaders remain understudied. The concept of invasive EM species is relatively novel, and requires deeper studies in Nothofagus forests, both when fungi are co-invading with the associated plant, or not. Hebeloma hiemale Bres. and Wilcoxina sp. Chin S. Yang & Korf, generalist EM associated exclusively with exotic Pinaceae in Patagonia, were found in invaded N. antarctica (G.Forst.) Oerst. root tips (Salgado Salomón et al. 2018; Salgado Salomón and Barroetaveña 2022), indicating that invasive exotic EM could be found in other Nothofagus species. The fact that the consequences of Nothofagus forest invasion by exotic EM species are poorly elucidated, but already established in four National Parks in Patagonia, emphasizes the importance of continued monitoring of their spread, the need for further research to understand the impact and the implementation and evaluation of innovative control measurements. Declarations Acknowledgments. We thank the residents and the Administración de Parques Nacionales-Delegación Patagonia (APN Research Permit Nº 1699) for allowing us to carry out the field work. The staff of Lago Puelo and Los Alerces National Parks are warmly thanked for their help and good predisposition during sampling trips and management tasks. PVG is a fellow and MESS, MBP and CB are researchers for the National Research Council of Argentina (CONICET). This work was supported by PICT Grupo en Formación Nº PICT 2020-SERIE A-02235. Author MESS has received research support from Dirección Nacional del Fondo para la Investigación Científica y Tecnológica (FONCYT), Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación. The authors have no relevant financial or non-financial interests to disclose. Author contributions. PVG: Data curation, Formal analysis, Writing – original draft, Writing – review & editing. MESS: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Visualization, Validation, Writing – original draft, Writing – review & editing. MBP: Data curation, Writing – review & editing. CB: Conceptualization, Validation, Writing – original draft, Writing – review & editing. Statement about use of generative AI. Non used. Competing interests. The authors have declared that no competing interests exist. References Barroetaveña C, Cázares E, Rajchenberg M (2007) Ectomycorrhizal fungal species associated with ponderosa pine and Douglas fir: a comparison of species richness in native forests and Patagonian plantations. Mycorrhiza 17:355–373 https://doi.org/10.1007/s00572-007-0121-x Barroetaveña C, Pildain MB, Salgado Salomón ME, Eberhart JL (2010) Molecular identification of ectomycorrhizas associated with ponderosa pine seedlings in Patagonian nurseries (Argentina). Canadian Journal Forest Research 40(10):1940–1950 https://doi.org/10.1139/X10-135 Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Rapp BA, Wheeler DL (2000) GenBank. Nucleic acids research 28(1):15–18 https://doi.org/10.1093/nar/28.1.15 Dickie IA, Johnston P, Singers N, Toft R, Waipara N, Walbert K, Council NR (2008) Invasive fungi research priorities, with a focus on Amanita muscaria . Landcare Research, Lincoln. Landcare Research Contract Report: LC0809/027 Dickie IA, Nuñez MA, Pringle A, Lebel T, Tourtellot SG, Johnston PR (2016) Towards management of invasive ectomycorrhizal fungi. Biological Invasions, 18:3383-3395. https://doi.org/10.1007/s10530-016-1243-x Dunk CW, Lebel T, Keane PJ (2012) Characterization of ectomycorrhizal formation by the exotic fungus Amanita muscaria with Nothofagus cunninghamii in Victoria, Australia. Mycorrhiza, 22(2):135-147. https://doi.org/10.1007/s00572-011-0388-9 Fuhrer BA (2005) A field guide to Australian fungi. Melbourne: Bloomings Books. pp. 24. ISBN 1-876473-51-7 Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology 2:113–118 https://doi.org/10.1111/j.1365-294x.1993.tb00005.x Geml J, Laursen GA, O'Neill K, Nusbaum HC, Taylor DL (2006) Beringian origins and cryptic speciation events in the fly agaric ( Amanita muscaria ). Molecular Ecology 15(1):225–239 https://doi.org/10.1111/j.1365-294X.2005.02799.x Geml J, Tulloss RE, Laursen GA, Sazanova NA, Taylor DL (2008) Evidence for strong inter- and intracontinental phylogeographic structure in Amanita muscaria . Molecular Phylogenetics and Evolution, 48(2):694-701. https://doi.org/10.1016/j.ympev.2008.04.029 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids symposium series 41:95–98 Lorenzo LE (2018) El hongo de las caricaturas ( Amanita muscaria ) en la Patagonia. Desde la Patagonia Nº3 UNCOMA Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution 30(4):772–780. https://doi.org/10.1093/molbev/mst010 Kranabetter JM, Stoehr M, O'Neill GA (2015) Ectomycorrhizal fungal maladaptation and growth reductions associated with assisted migration of Douglas‐fir. New Phytol 206(3):1135–1144 https://doi.org/10.1111/nph.13287 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular biology and evolution 35(6):1547 https://doi.org/10.1093/molbev/msy096 Lebel T, May TW, Cooper JA, Catcheside D, Catcheside P, Haska J (2024) Confirming the presence of five exotic species of Amanita in Australia and New Zealand. Swainsona 38:1–44 Narh Mensah DL, Allen B, Barnes I, Bivins C, Brewer S, Bryan C, Buthelezi S, Clark J, Coetzee MPA, Corby L, Dewing C, Drott M, Duong T, Fuqua SR, Harris M, Hoeksema J, Johnson D, Kopotsa K, Lane F, Zallek T (2024) Phylogenomics and specialized metabolism potential of invasive Amanita muscaria in South Africa (ID 2064). Dissertation, 12th International Mycological Congress Nuñez MA, Hayward J, Horton TR, Amico GC, Dimarco RD, Barrios-Garcia MN, Simberloff D (2013) Exotic mammals disperse exotic fungi that promote invasion by exotic trees. PLoS one 8(6):e66832. https://doi.org/10.1371/journal.pone.0066832 Oda T, Tanaka C, Tsuda M (2004) Molecular phylogeny and biogeography of the widely distributed Amanita species, A. muscaria and A. pantherina . Mycological Research, 108(8):885–896 https://doi.org/10.1017/S0953756204000620 Pildain MB, Marchelli P, Azpilicueta MM, Starik C, Barroetaveña C (2021) Understanding introduction history: Genetic structure and diversity of the edible ectomycorrhizal fungus, Suillus luteus , in Patagonia (Argentina). Mycologia 113(4):715-724 https://doi.org/10.1080/00275514.2021.1909449 Richardson DM, van Wilgen BW, Núñez MA (2008) Alien conifer invasions in South America: short fuse burning? Biol Invasions 10:573–577 https://doi.org/10.1007/s10530-007-9140-y Salgado Salomón ME, Barroetaveña C (2022) Native and exotic woodland from Patagonian Andes: anthropic impacts and Mycorrhizas. Chapter XII. Mycorrhizal Fungi in South America: Biodiversity, Conservation and Sustainable food production. Lugo M, Pagano M (Eds.) pp 237–254 https://doi.org/10.1007/978-3-031-12994-0_12 Salgado Salomón ME, Barroetaveña C, Pildain MB, Kuhar F, Rajchenberg M (2017) Tomentella (Thelephorales, Basidiomycota) en bosques de Nothofagaceae de Patagonia, Argentina: micorrizas de nuevas especies. Boletín de la Sociedad Argentina de Botánica, 52(3), 423-434. Salgado Salomón ME, Barroetaveña C, Pildain MB, Williams EA, Rajchenberg M (2018) What happens to the mycorrhizal communities of native and exotic seedlings when Pseudotsuga menziesii invades Nothofagaceae forests in Patagonia, Argentina? Acta Oecol 91:108–119 https://doi.org/10.1016/j.actao.2018.07.003 Salgado Salomón ME, Barroetaveña C, Rajchenberg M (2013) Pseudotsuga menziesii invasion in native forests of Patagonia, Argentina: what about mycorrhizas? Acta Oecol 49:5–11 http://dx.doi.org/10.1016/j.actao.2013.01.018 Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9):1312–1313 https://doi.org/10.1093/bioinformatics/btu033 Vargas N, Gonçalves SC, Franco-Molano AE, Restrepo S, Pringle A (2019) Amanita muscaria expanding into native Quercus forests in Colombia. Mycologia, 111(5):758-771. https://doi.org/10.1080/00275514.2019.1636608 White TJ, Burns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications. Innis AM, Gelford DH, Sninsky JJ, White JF (Eds.). Academic Press, New York, USA. pp. 315–322. Wu HX, Eldridge KG, Matheson AC, Powell MB, McRae TA, Butcher TB, Johnson IG (2007) Achievements in forest tree improvement in Australia and New Zealand 8. Successful introduction and breeding of radiata pine in Australia. Australian forestry 70(4):215–225. https://doi.org/10.1080/00049158.2007.10675023 Supplementary Files SupplementaryInformationSIGilesetal2025.docx Cite Share Download PDF Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Biological Invasions → Version 1 posted Reviewers agreed at journal 11 May, 2025 Reviewers invited by journal 05 May, 2025 Editor invited by journal 04 May, 2025 Editor assigned by journal 26 Apr, 2025 First submitted to journal 24 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6523152","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451964738,"identity":"349e9912-70e6-4ff0-8db3-2f6c93894c42","order_by":0,"name":"Paula Victoria Giles","email":"","orcid":"","institution":"CIEFAP: Centro de Investigacion y Extension Forestal Andino Patagonico","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"Victoria","lastName":"Giles","suffix":""},{"id":451964739,"identity":"a1be226c-0fad-436c-9822-06281c8032c3","order_by":1,"name":"María Eugenia Salgado Salomón","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4261-4532","institution":"CIEFAP: Centro de Investigacion y Extension Forestal Andino Patagonico","correspondingAuthor":true,"prefix":"","firstName":"María","middleName":"Eugenia Salgado","lastName":"Salomón","suffix":""},{"id":451964740,"identity":"4daf6c30-654d-401f-905f-a2b5896d87c5","order_by":2,"name":"María Belén Pildain","email":"","orcid":"","institution":"CIEFAP: Centro de Investigacion y Extension Forestal Andino Patagonico","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Belén","lastName":"Pildain","suffix":""},{"id":451964741,"identity":"c8b1cb09-59c3-4a72-979e-64e7b52b1b9a","order_by":3,"name":"Carolina Barroetaveña","email":"","orcid":"","institution":"CIEFAP: Centro de Investigacion y Extension Forestal Andino Patagonico","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Barroetaveña","suffix":""}],"badges":[],"createdAt":"2025-04-24 18:37:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6523152/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6523152/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10530-025-03712-3","type":"published","date":"2025-11-18T15:58:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82255716,"identity":"8a0a53ed-aba8-4f6a-8e27-4e26f024ba74","added_by":"auto","created_at":"2025-05-08 11:06:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":158961,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic position of the Patagonian isolates analyzed and others belonging to reference strains based on the ITS dataset, using Maximum Likelihood. Bootstrap values lower than 70% are not indicated. Sequences from Patagonia generated for this work are in bold\u003c/p\u003e","description":"","filename":"Fig.1Note.png","url":"https://assets-eu.researchsquare.com/files/rs-6523152/v1/bf54edad6ff923a2291ae488.png"},{"id":96650895,"identity":"80e2cbc1-462e-4807-89bf-7abaf653c45e","added_by":"auto","created_at":"2025-11-24 16:12:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":516376,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6523152/v1/d343fc6b-6707-41f8-8d09-80baf9fc718b.pdf"},{"id":82255709,"identity":"b8fa10a7-95f0-4f00-b302-b46b785ccf9f","added_by":"auto","created_at":"2025-05-08 11:06:43","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":19493,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationSIGilesetal2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6523152/v1/a972dd65f9c82e2822c19e32.docx"}],"financialInterests":"","formattedTitle":"Molecular analysis confirms the invasion of Amanita muscaria in native Nothofagus forests from Patagonia, Argentina","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAfforestation with exotic conifers in Patagonia has facilitated the introduction and spread of ectomycorrhizal (EM) fungi, altering native forest ecosystems (Barroetave\u0026ntilde;a et al. 2010; Salgado Salom\u0026oacute;n et al. 2013, 2018; Salgado Salom\u0026oacute;n and Barroetave\u0026ntilde;a 2022). \u003cem\u003eAmanita muscaria\u003c/em\u003e (L.) Lam., widely reported from Patagonian conifer plantations (Barroetave\u0026ntilde;a et al. 2007; Lorenzo 2018), forms natural associations in boreal and temperate, mature, conifers, birches and oaks forests from the Northern Hemisphere (Lebel 2024). The first formal report of the species fruiting in Patagonia is dated in 2001, from a pine plantation in Sectional Pucar\u0026aacute;, Lan\u0026iacute;n National Park (Lorenzo 2018). Since then, the species has been recorded in different locations in that National Park and increasingly to the south, always associated with \u003cem\u003ePinus\u003c/em\u003e L. species, each year with more abundant fruiting (Lorenzo 2018). Meanwhile, since 2014, several National Parks rangers have reported \u003cem\u003eA. muscaria\u003c/em\u003e basidiomata presence in pure native \u003cem\u003eNothofagus\u003c/em\u003e forests. In this sense, the \u003cem\u003ejump\u003c/em\u003e of \u003cem\u003eA. muscaria\u003c/em\u003e to Nothofagaceae Kuprian. forests in New Zealand, Tasmania and Australia (Dunk et al. 2012) and to \u003cem\u003eQuercus humboldtii\u003c/em\u003e Bonpl. in Colombia (Vargas et al. 2019) have been already reported. Confirming the effective invasion of Patagonian \u003cem\u003eNothofagus\u003c/em\u003e spp. with \u003cem\u003eA. muscaria\u003c/em\u003e could imply the displacement of native EMs, a disturbance that threatens native biodiversity and eventually ecosystem balance (Fuhrer 2005).\u003c/p\u003e \u003cp\u003eAs this phenomenon is of great concern regarding the conservation of subantarctic forests of Argentina, but it has not yet been sufficiently studied in the region, this study aimed to determine the effective EM association of \u003cem\u003eA. muscaria\u003c/em\u003e with endemic \u003cem\u003eNothofagus\u003c/em\u003e spp. and analyze the phylogenetic relationship of Patagonian \u003cem\u003eA. muscaria\u003c/em\u003e specimens with those reported worldwide.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eField work\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo identify sampling sites, consultations and records of \u003cem\u003eA. muscaria\u003c/em\u003e basidiomata findings in \u003cem\u003eNothofagus\u003c/em\u003e forests within Arrayanes, Nahuel Huapi, Lago Puelo and Los Alerces, National Parks were conducted. Own records of plantations with sporocarps of the species were used. Sampling sites were located in northwestern Patagonian provinces: Neuqu\u0026eacute;n, R\u0026iacute;o Negro and Chubut (Argentina). Twenty-eight samples of \u003cem\u003eA. muscaria\u003c/em\u003e basidiomata were collected in pure \u003cem\u003eNothofagus\u003c/em\u003e spp. forests, pure and mixed \u003cem\u003ePinus ponderosa\u003c/em\u003e Dougl. ex Laws. and \u003cem\u003eP. radiata\u003c/em\u003e D. Don. plantations with presences of \u003cem\u003ePicea\u003c/em\u003e Link, \u003cem\u003ePseudotsuga\u003c/em\u003e, \u003cem\u003eBetula\u003c/em\u003e L. and \u003cem\u003eQuercus\u003c/em\u003e L., during autumn of 2020 to 2024 (Supplementary Table 1). When \u003cem\u003eA. muscaria\u003c/em\u003e basidiomata were detected in pure \u003cem\u003eNothofagus\u003c/em\u003e forests (patches without the presence of exotic EM tree species more than 5000 m or more than 1000 m when natural barriers, such as rivers, were present), soil with root tips were taken below the \u003cem\u003eA. muscaria\u003c/em\u003e basidioma until 10 cm depth (Supplementary Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMorphological study\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBasidiomes: Macroscopical descriptions and measurements, color and macrochemical KOH reactions (KOH 30%) were done on fresh basidiomata. Microscopical characteristics were checked on oven dried (45\u0026deg;C) specimens revived in 3% KOH + floxin, Melzer\u0026apos;s reagent and cotton blue following Salgado Salom\u0026oacute;n et al. (2018), and documented with a Nikon camera D70 in combination with the computer program LASX (https://www.leica-microsystems.com) and ImageJ (https://imagej.nih.gov/). Basidiospores obtained from the veil tissue were measured in 3% KOH, expressing (min) mean \u0026plusmn; standard deviation (max) of 150 spores for statistical evaluation (Salgado Salom\u0026oacute;n et al. 2018); its dextrinoid reaction was tested with Melzer\u0026rsquo;s reagent acting five minutes. All material was deposited in the HCFC Herbarium (Supplementary Table 1). Species nomenclature and taxonomic classification followed the Index Fungorum database (www.indexfungorum.org).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDNA extraction, PCR amplification and sequencing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDNA extraction was performed following the CTAB method as described by Salgado Salom\u0026oacute;n et al. (2017, 2018). For basidiomes, 2-3 uncontaminated lamellae per collection were used. For ectomycorrhizal morphotypes, DNA was individually extracted from five representative root tips per site. The ITS region (rDNA ITS1-5.8S-ITS2) was amplified using the primers ITS4 and ITS5 for basidiomes, and ITS1F and ITS4 for ectomycorrhizal morphotypes (White et al. 1990; Gardes and Bruns 1993). PCR reactions were carried out in the MyCycler\u0026trade; thermocycler (BioRad) with the protocol from Salgado Salom\u0026oacute;n et al. (2017). Amplified PCR products were visualized under UV illumination by gel electrophoresis on 1% agarose (w/v) stained with Gel-Red \u0026trade; (Biotium, California, USA). Purification and sequencing were performed at CERELA (Tucum\u0026aacute;n, Argentina) and MACROGEN (Seoul, South Korea).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRaw sequences were edited in BioEdit v7.0.9.0 (Hall 1999), aligned using the L-INS-i algorithm in MAFFT v7.0 (Katoh and Standley 2013), and manually refined in MEGA vX (Kumar et al. 2018). Reference sequences were retrieved via BLASTn from GenBank (Benson et al. 2000), and alignment was based on published datasets (Oda et al. 2004; Geml et al. 2006, 2008; Dunk et al. 2012), using \u003cem\u003eAmanita ceciliae\u003c/em\u003e (GenBank AB015694: LEM950069) as outgroup (Supplementary Table 2). The best-fit evolutionary model (GTR+G) was selected using AIC in jModelTest (http://darwin.uvigo.es). Phylogenetic analyses were conducted under Maximum Likelihood in RAxML v7.2.8 (Stamatakis 2014) with 1000 bootstrap replicates. Newly generated sequences were deposited in GenBank (accession numbers OQ324766, OQ324769\u0026ndash;70, OQ324774\u0026ndash;75, OQ324778\u0026ndash;79, PP490741\u0026ndash;63; Supplementary Table 1).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eSeven patches of \u003cem\u003eA. muscaria\u003c/em\u003e basidiomes were found in 4 protected areas associated with native \u003cem\u003eN. dombeyi\u003c/em\u003e (Mirb.) Oerst. and \u003cem\u003eN. pumilio\u003c/em\u003e (Poepp. and Endl.) Krasser, including Hua Huam Trail (Arrayanes National Park), El Manso (Nahuel Huapi National Park), Los Hitos, Camping Gendarmer\u0026iacute;a, and Cerro Cuevas (Lago Puelo National Park), Puerto Chucao Trail and Los Pozones (Los Alerces National Park), with no evidence of nearby exotic plant species. A white EM morphotype was consistently found on roots below the basidiomata matching the characteristics of \u003cem\u003eA. muscaria\u003c/em\u003e-type mycorrhizae.\u003c/p\u003e \u003cp\u003eTwenty-eight basidiomes and two morphotypes were BLASTs as \u003cem\u003eA. muscaria.\u003c/em\u003e Of them eleven basidiomes and both morphotypes were associated with \u003cem\u003eN. dombeyi\u003c/em\u003e forests, one basidioma with \u003cem\u003eN. pumilio\u003c/em\u003e forest, and the remaining were associated with \u003cem\u003eP. radiata\u003c/em\u003e D. Don and \u003cem\u003eP. ponderosa\u003c/em\u003e Dougl. ex Laws. Morphologically, some specimens of \u003cem\u003eA. muscaria\u003c/em\u003e associated with \u003cem\u003eNothofagus\u003c/em\u003e spp. matched with the description of \u003cem\u003eA. muscaria\u003c/em\u003e var. \u003cem\u003eformosa\u003c/em\u003e Pers., presenting an orange to yellowish pileus and yellowish to cream mottling, although the phylogenetic analysis showed that the \u003cem\u003eformosa\u003c/em\u003e variety belongs to a different clade. On the other hand, the specimens associated with coniferous forests corresponded morphologically to \u003cem\u003eA. muscaria\u003c/em\u003e var. \u003cem\u003emuscaria\u003c/em\u003e, with red pileus and white mottling (Geml et al. 2006). Despite these morphological differences, molecular analysis confirmed that all samples belonged to clade II, with Eurasian and Alaskan vouchers and with samples obtained by Dunk et al. (2012) from invaded \u003cem\u003eLophozonia cunninghamii\u003c/em\u003e (Hook.) Oerst. (Nothofagaceae) from Australia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eAmanita muscaria\u003c/em\u003e is invading pure stands of \u003cem\u003eN. dombeyi\u003c/em\u003e and \u003cem\u003eN. pumilio\u003c/em\u003e in Patagonia Argentina, evidenced by the presence of basidiomes in sites without exotic plant species, and confirmed by the molecular determination of \u003cem\u003eA. muscaria\u003c/em\u003e morphotypes in \u003cem\u003eNothofagus\u003c/em\u003e spp. root tips. This coincides with what has been reported in Oceania, where \u003cem\u003eA. muscaria\u003c/em\u003e was found in mixed native \u003cem\u003eLophozonia\u003c/em\u003e-\u003cem\u003eEucalyptus\u003c/em\u003e forest (Dickie et al. 2008; Dunk et al. 2012), in \u003cem\u003eQ. humboldtii\u003c/em\u003e in Colombia (Vargas et al. 2019) and in South Africa, where Narh Mensah et al. (2024) found that it has \u003cem\u003ejumped\u003c/em\u003e from exotics \u003cem\u003ePinus\u003c/em\u003e spp. to native forests.\u003c/p\u003e \u003cp\u003eThe sequences of invasive \u003cem\u003eA. muscaria\u003c/em\u003e collections obtained in this study and those obtained by Dunk et al. (2012) for Australia, by Vargas et al. (2019) for Colombia and by Narh Mensah et al. (2024) for South Africa coincide with clade II proposed by Geml et al. (2008). This clade is the most widely distributed, likely due to the importation of soil and plants from North America and Europe to southern hemisphere countries (Wu et al. 2007; Vargas et al. 2019). This could apply to the \u003cem\u003eNothofagus\u003c/em\u003e forests in Patagonia. Although, to know the origin of the inoculum that could have originated its invasion, it would be necessary to do haplotype network studies and population studies to understand the distribution pattern, as it was done for other introduced fungi such as \u003cem\u003eSuillus luteus\u003c/em\u003e (L.: Fries) Gray (Pildain et al. 2021).\u003c/p\u003e \u003cp\u003eAll sampled \u003cem\u003eN. dombeyi\u003c/em\u003e sites invaded with \u003cem\u003eA. muscaria\u003c/em\u003e shared common characteristics were areas of transhumance, well-travelled by tourists and cattle, with constant soil removal, proximity to watercourses, and a history of forest fires with subsequent reconditioning tasks. Invasive EM fungi are frequently associated with trails, camping sites, and other areas where they are likely to come into contact with shoes, vehicles, and camping gear (Dickie et al. 2016). \u003cem\u003eA. muscaria\u003c/em\u003e, in particular, has been associated with disturbed environments where the presence of dispersal vectors and soil movement were high (Dunk et al. 2012). Anyway, when \u003cem\u003eA. muscaria\u003c/em\u003e was found in \u003cem\u003eN. pumilio\u003c/em\u003e forest in Cerro Cuevas, the context was different. This site is a Strict Nature Reserve, free of exotic species, with very low disturbance, where only Rangers can enter. In this case, it is possible to think of dispersion through local fauna or \u003cem\u003ebagual\u003c/em\u003e cattle, as Nu\u0026ntilde;ez et al. (2013) proposed to explain invasive EM fungi dispersion in Patagonian forests.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmanita muscaria\u003c/em\u003e distribution in invaded sites from Patagonian protected areas is still limited to small patches in disturbed areas. Although, this situation could progress compromising local biodiversity by displacing native fungal species and generating maladaptation of the associated forest species (Kranabetter et al. 2015).\u003c/p\u003e \u003cp\u003eDespite scientific recognition of the spread and impact of invasive EM fungi, there is little practical advice on their management and few eradication strategies. Dickie et al. (2016) discussed possible strategies for prevention, slowing the spread, eradication and long-term management of invasive fungi, including manual removal of sporocarps, application of fungicide, trenching and elimination of associated tree species. A first experience aiming to control the spread of \u003cem\u003eA. muscaria\u003c/em\u003e in Lago Puelo National Park (APN) included the removal of potential native associated trees in a delimited invaded area, but these actions increased the dispersal of the fungus, probably by increasing disturbance at the site (D. Raymundi, pers. comms.). Given that the active colonization of root tip in neighboring-invaded area host trees was not analyzed, invaded native trees could have been left standing. Also, once the spores are dispersed, they could remain infective in the spore bank even if trees are removed (Dickie et al. 2016). Moreover, the removal of possible associated native plant species in protected areas, with high conservation value, is not recommended as its fungal and plant biodiversity and ecosystem services could be compromised. With these considerations, a low impact protocol that preserves soil integrity, generating the least possible disturbance, but controlling \u003cem\u003eA. muscaria\u003c/em\u003e dispersal is needed, along with periodical monitoring to assure early detection, key factors to control the spread (Dickie et al 2016).\u003c/p\u003e \u003cp\u003ePrevious studies highlight the role of EM mutualism in plant invasions (Richardson et al. 2008), but EM fungi as invaders remain understudied. The concept of invasive EM species is relatively novel, and requires deeper studies in \u003cem\u003eNothofagus\u003c/em\u003e forests, both when fungi are co-invading with the associated plant, or not. \u003cem\u003eHebeloma hiemale\u003c/em\u003e Bres. and \u003cem\u003eWilcoxina\u003c/em\u003e sp. Chin S. Yang \u0026amp; Korf, generalist EM associated exclusively with exotic Pinaceae in Patagonia, were found in invaded \u003cem\u003eN. antarctica\u003c/em\u003e (G.Forst.) Oerst. root tips (Salgado Salom\u0026oacute;n et al. 2018; Salgado Salom\u0026oacute;n and Barroetave\u0026ntilde;a 2022), indicating that invasive exotic EM could be found in other \u003cem\u003eNothofagus\u003c/em\u003e species. The fact that the consequences of \u003cem\u003eNothofagus\u003c/em\u003e forest invasion by exotic EM species are poorly elucidated, but already established in four National Parks in Patagonia, emphasizes the importance of continued monitoring of their spread, the need for further research to understand the impact and the implementation and evaluation of innovative control measurements.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments.\u0026nbsp;\u003c/strong\u003eWe thank the residents and the Administraci\u0026oacute;n de Parques Nacionales-Delegaci\u0026oacute;n Patagonia (APN Research Permit N\u0026ordm; 1699) for allowing us to carry out the field work. The staff of Lago Puelo and Los Alerces National Parks are warmly thanked for their help and good predisposition during sampling trips and management tasks. PVG is a fellow and MESS, MBP and CB are researchers for the National Research Council of Argentina (CONICET). This work was supported by PICT Grupo en Formaci\u0026oacute;n N\u0026ordm; PICT 2020-SERIE A-02235. Author MESS has received research support from Direcci\u0026oacute;n Nacional del Fondo para la Investigaci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (FONCYT), Agencia Nacional de Promoci\u0026oacute;n de la Investigaci\u0026oacute;n, el Desarrollo Tecnol\u0026oacute;gico y la Innovaci\u0026oacute;n.\u0026nbsp;The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions.\u0026nbsp;\u003c/strong\u003ePVG: Data curation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. MESS: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Visualization, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. MBP: Data curation, Writing \u0026ndash; review \u0026amp; editing. CB: Conceptualization, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement about use of generative AI.\u0026nbsp;\u003c/strong\u003eNon used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u0026nbsp;\u003c/strong\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBarroetave\u0026ntilde;a C, C\u0026aacute;zares E, Rajchenberg M (2007) Ectomycorrhizal fungal species associated with ponderosa pine and Douglas fir: a comparison of species richness in native forests and Patagonian plantations. Mycorrhiza 17:355\u0026ndash;373 https://doi.org/10.1007/s00572-007-0121-x\u003c/li\u003e\n\u003cli\u003eBarroetave\u0026ntilde;a C, Pildain MB, Salgado Salom\u0026oacute;n ME, Eberhart JL (2010) Molecular identification of ectomycorrhizas associated with ponderosa pine seedlings in Patagonian nurseries (Argentina). Canadian Journal Forest Research 40(10):1940\u0026ndash;1950 https://doi.org/10.1139/X10-135\u003c/li\u003e\n\u003cli\u003eBenson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Rapp BA, Wheeler DL (2000) GenBank. Nucleic acids research 28(1):15\u0026ndash;18 https://doi.org/10.1093/nar/28.1.15\u003c/li\u003e\n\u003cli\u003eDickie IA, Johnston P, Singers N, Toft R, Waipara N, Walbert K, Council NR (2008) Invasive fungi research priorities, with a focus on \u003cem\u003eAmanita muscaria\u003c/em\u003e. Landcare Research, Lincoln. Landcare Research Contract Report: LC0809/027\u003c/li\u003e\n\u003cli\u003eDickie IA, Nu\u0026ntilde;ez MA, Pringle A, Lebel T, Tourtellot SG, Johnston PR (2016) Towards management of invasive ectomycorrhizal fungi. Biological Invasions, 18:3383-3395. https://doi.org/10.1007/s10530-016-1243-x \u003c/li\u003e\n\u003cli\u003eDunk CW, Lebel T, Keane PJ (2012) Characterization of ectomycorrhizal formation by the exotic fungus \u003cem\u003eAmanita muscaria\u003c/em\u003e with \u003cem\u003eNothofagus cunninghamii\u003c/em\u003e in Victoria, Australia. Mycorrhiza, 22(2):135-147. https://doi.org/10.1007/s00572-011-0388-9\u003c/li\u003e\n\u003cli\u003eFuhrer BA (2005) A field guide to Australian fungi. Melbourne: Bloomings Books. pp. 24. ISBN 1-876473-51-7\u003c/li\u003e\n\u003cli\u003eGardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology 2:113\u0026ndash;118 https://doi.org/10.1111/j.1365-294x.1993.tb00005.x\u003c/li\u003e\n\u003cli\u003eGeml J, Laursen GA, O\u0026apos;Neill K, Nusbaum HC, Taylor DL (2006) Beringian origins and cryptic speciation events in the fly agaric (\u003cem\u003eAmanita muscaria\u003c/em\u003e). Molecular Ecology 15(1):225\u0026ndash;239 https://doi.org/10.1111/j.1365-294X.2005.02799.x\u003c/li\u003e\n\u003cli\u003eGeml J, Tulloss RE, Laursen GA, Sazanova NA, Taylor DL (2008) Evidence for strong inter- and intracontinental phylogeographic structure in\u003cem\u003e Amanita muscaria\u003c/em\u003e. Molecular Phylogenetics and Evolution, 48(2):694-701. https://doi.org/10.1016/j.ympev.2008.04.029\u003c/li\u003e\n\u003cli\u003eHall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids symposium series 41:95\u0026ndash;98\u003c/li\u003e\n\u003cli\u003eLorenzo LE (2018) El hongo de las caricaturas (\u003cem\u003eAmanita muscaria\u003c/em\u003e) en la Patagonia. Desde la Patagonia N\u0026ordm;3 UNCOMA\u003c/li\u003e\n\u003cli\u003eKatoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution 30(4):772\u0026ndash;780. https://doi.org/10.1093/molbev/mst010\u003c/li\u003e\n\u003cli\u003eKranabetter JM, Stoehr M, O\u0026apos;Neill GA (2015) Ectomycorrhizal fungal maladaptation and growth reductions associated with assisted migration of Douglas‐fir. New Phytol 206(3):1135\u0026ndash;1144 https://doi.org/10.1111/nph.13287 \u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular biology and evolution 35(6):1547 https://doi.org/10.1093/molbev/msy096\u003c/li\u003e\n\u003cli\u003eLebel T, May TW, Cooper JA, Catcheside D, Catcheside P, Haska J (2024) Confirming the presence of five exotic species of \u003cem\u003eAmanita \u003c/em\u003ein Australia and New Zealand. Swainsona 38:1\u0026ndash;44\u003c/li\u003e\n\u003cli\u003eNarh Mensah DL, Allen B, Barnes I, Bivins C, Brewer S, Bryan C, Buthelezi S, Clark J, Coetzee MPA, Corby L, Dewing C, Drott M, Duong T, Fuqua SR, Harris M, Hoeksema J, Johnson D, Kopotsa K, Lane F, Zallek T (2024) Phylogenomics and specialized metabolism potential of invasive \u003cem\u003eAmanita muscaria\u003c/em\u003e in South Africa (ID 2064). Dissertation, 12th International Mycological Congress\u003c/li\u003e\n\u003cli\u003eNu\u0026ntilde;ez MA, Hayward J, Horton TR, Amico GC, Dimarco RD, Barrios-Garcia MN, Simberloff D (2013) Exotic mammals disperse exotic fungi that promote invasion by exotic trees. PLoS one 8(6):e66832. https://doi.org/10.1371/journal.pone.0066832 \u003c/li\u003e\n\u003cli\u003eOda T, Tanaka C, Tsuda M (2004) Molecular phylogeny and biogeography of the widely distributed \u003cem\u003eAmanita \u003c/em\u003especies, \u003cem\u003eA. muscaria\u003c/em\u003e and \u003cem\u003eA. pantherina\u003c/em\u003e. Mycological Research, 108(8):885\u0026ndash;896 https://doi.org/10.1017/S0953756204000620 \u003c/li\u003e\n\u003cli\u003ePildain MB, Marchelli P, Azpilicueta MM, Starik C, Barroetave\u0026ntilde;a C (2021) Understanding introduction history: Genetic structure and diversity of the edible ectomycorrhizal fungus, \u003cem\u003eSuillus luteus\u003c/em\u003e, in Patagonia (Argentina). Mycologia 113(4):715-724 https://doi.org/10.1080/00275514.2021.1909449 \u003c/li\u003e\n\u003cli\u003eRichardson DM, van Wilgen BW, N\u0026uacute;\u0026ntilde;ez MA (2008) Alien conifer invasions in South America: short fuse burning? Biol Invasions 10:573\u0026ndash;577 https://doi.org/10.1007/s10530-007-9140-y\u003c/li\u003e\n\u003cli\u003eSalgado Salom\u0026oacute;n ME, Barroetave\u0026ntilde;a C (2022) Native and exotic woodland from Patagonian Andes: anthropic impacts and Mycorrhizas. Chapter XII. Mycorrhizal Fungi in South America: Biodiversity, Conservation and Sustainable food production. Lugo M, Pagano M (Eds.) pp 237\u0026ndash;254 https://doi.org/10.1007/978-3-031-12994-0_12 \u003c/li\u003e\n\u003cli\u003eSalgado Salom\u0026oacute;n ME, Barroetave\u0026ntilde;a C, Pildain MB, Kuhar F, Rajchenberg M (2017) \u003cem\u003eTomentella \u003c/em\u003e(Thelephorales, Basidiomycota) en bosques de Nothofagaceae de Patagonia, Argentina: micorrizas de nuevas especies. Bolet\u0026iacute;n de la Sociedad Argentina de Bot\u0026aacute;nica, 52(3), 423-434.\u003c/li\u003e\n\u003cli\u003eSalgado Salom\u0026oacute;n ME, Barroetave\u0026ntilde;a C, Pildain MB, Williams EA, Rajchenberg M (2018) What happens to the mycorrhizal communities of native and exotic seedlings when \u003cem\u003ePseudotsuga menziesii \u003c/em\u003einvades Nothofagaceae forests in Patagonia, Argentina? Acta Oecol 91:108\u0026ndash;119 https://doi.org/10.1016/j.actao.2018.07.003\u003c/li\u003e\n\u003cli\u003eSalgado Salom\u0026oacute;n ME, Barroetave\u0026ntilde;a C, Rajchenberg M (2013) \u003cem\u003ePseudotsuga menziesii\u003c/em\u003e invasion in native forests of Patagonia, Argentina: what about mycorrhizas? Acta Oecol 49:5\u0026ndash;11 http://dx.doi.org/10.1016/j.actao.2013.01.018\u003c/li\u003e\n\u003cli\u003eStamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9):1312\u0026ndash;1313 https://doi.org/10.1093/bioinformatics/btu033\u003c/li\u003e\n\u003cli\u003eVargas N, Gon\u0026ccedil;alves SC, Franco-Molano AE, Restrepo S, Pringle A (2019) \u003cem\u003eAmanita muscaria\u003c/em\u003e expanding into native \u003cem\u003eQuercus \u003c/em\u003eforests in Colombia. Mycologia, 111(5):758-771. https://doi.org/10.1080/00275514.2019.1636608\u003c/li\u003e\n\u003cli\u003eWhite TJ, Burns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications. Innis AM, Gelford DH, Sninsky JJ, White JF (Eds.). Academic Press, New York, USA. pp. 315\u0026ndash;322.\u003c/li\u003e\n\u003cli\u003eWu HX, Eldridge KG, Matheson AC, Powell MB, McRae TA, Butcher TB, Johnson IG (2007) Achievements in forest tree improvement in Australia and New Zealand 8. Successful introduction and breeding of radiata pine in Australia. Australian forestry 70(4):215\u0026ndash;225. https://doi.org/10.1080/00049158.2007.10675023\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Invasive ectomycorrhiza, biological invasions, exotic Pinus plantations, phylogeny, ITS","lastPublishedDoi":"10.21203/rs.3.rs-6523152/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6523152/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eAmanita muscaria\u003c/em\u003e, an ectomycorrhizal fungus native to the Northern Hemisphere, has been presumably introduced to Patagonia via exotic pine plantations. Recent findings confirm its presence in pure \u003cem\u003eNothofagus\u003c/em\u003e forests within several National Parks. Morphological and molecular analyses verified its association with \u003cem\u003eN. dombeyi\u003c/em\u003e and \u003cem\u003eN. pumilio\u003c/em\u003e, showing its capacity to invade native tree roots. Although its distribution in \u003cem\u003eNothofagus\u003c/em\u003e forests is still limited to small patches, its expansion could threaten the biodiversity in protected areas and other native forests of high landscape, ecological and economic value in Argentine Patagonia. Early detection and management strategies, critical to mitigate its spread, are discussed.\u003c/p\u003e","manuscriptTitle":"Molecular analysis confirms the invasion of Amanita muscaria in native Nothofagus forests from Patagonia, Argentina","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 11:06:38","doi":"10.21203/rs.3.rs-6523152/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-05-11T09:56:47+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-05T08:57:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Biological Invasions","date":"2025-05-04T21:18:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-26T12:25:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Invasions","date":"2025-04-24T14:36:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-invasions","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"binv","sideBox":"Learn more about [Biological Invasions](https://www.springer.com/journal/10530)","snPcode":"10530","submissionUrl":"https://submission.nature.com/new-submission/10530/3","title":"Biological Invasions","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a55833ab-3592-4cfd-9f8a-3f2e24ad56e7","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:08:48+00:00","versionOfRecord":{"articleIdentity":"rs-6523152","link":"https://doi.org/10.1007/s10530-025-03712-3","journal":{"identity":"biological-invasions","isVorOnly":false,"title":"Biological Invasions"},"publishedOn":"2025-11-18 15:58:07","publishedOnDateReadable":"November 18th, 2025"},"versionCreatedAt":"2025-05-08 11:06:38","video":"","vorDoi":"10.1007/s10530-025-03712-3","vorDoiUrl":"https://doi.org/10.1007/s10530-025-03712-3","workflowStages":[]},"version":"v1","identity":"rs-6523152","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6523152","identity":"rs-6523152","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}