Context dependency, co-introductions, novel mutualisms, and host shifts shaped the ectomycorrhizal fungal communities of the alien tree Eucalyptus globulus

The identity and relevance of the ectomycorrhizal (ECM) fungal partners of Eucalyptus globulus was investigated in NW Spain, to detect which symbionts mainly support its invasiveness. Root tips of E. globulus and of three common native plant species (Quercus robur, Pinus pinaster and Halimium lasianthum) were collected in eucalypt plantations, Q. robur forests, P. pinaster plantations and shrublands. Fungal taxonomical identity was ascertained by use of rDNA and direct sequencing. We studied diversity, composition and colonization rate of the ECM fungal communities of E. globulus to determine if fungal assemblages are host specific (i.e. similar in different habitats) or more dependent on the neighbourhood context. We also identified the type of associations formed (i.e. co-introductions, familiar or novel associations). Twenty-six ECM taxa were associated with E. globulus. Most of them engaged in novel associations with eucalypts, whereas only three fungal species were co-introduced Australian aliens. Eucalypt fungal richness, diversity and colonization rate differed between habitats, being higher in native oak forests, whereas in shrublands E. globulus showed the lowest colonization rate and diversity. The Australian fungus Descolea maculata dominated the eucalypt fungal assemblage and also spread to the native host plants, in all the habitats, posing the risk of further co-invasion.

. Ectomycorrhizal fungal species found in association with Eucalyptus globulus (E), Pinus pinaster (P), Quercus robur (Q), and Halimium lasianthum (H), in four habitats (E. globulus plantation = Ep; P. pinaster plantation = Pp; Q. robur forest = Qf; and shrubland = S). Taxonomical identity was ascertained by direct sequencing of the ITS regions and BLAST search. Names in bold indicates that this is the first time that the taxon is reported in association with E. globulus. Geographical distribution have been assessed by using the available literature (see for example Vellinga et al. 2009) and the records and global mapper provided by the on-line encyclopedia of life www.discoverlife.org, the catalogue of New Zealand Fungi (http://nzfungi.landcareresearch.co.nz/html/search_index.asp), the Atlas of Living Australia website (http://www.ala.org.au/blogs-news/fungimap-putting-australian-fungi-on-the-map/), and the Global Biodiversity Information Facility web (www.gbif.org). Abbreviations in geographical distributions read as follows: Nea = Neartic region (North America, Greenland, Central Florida, highland of Mexico); Neo = Neotropical region (South Florida, lowlands of Mexico, Central America, South America, Caribbean islands); Pal = Paleartic region (Europe, North Africa, North Asia, northern and central parts of the Arabian peninsula); Afr = Afrotropical region (Africa South of the Sahara, Madagascar and the southwestern area of the Arabian peninsula); Hi = Hindomalayan region (South and Southeast Asia and southern parts of East Asia); Aus = Australasian region (Australia, New Guinea, New Zealand, New Caledonia, Tasmania, Pacific island groups). Aus* = taxon present in the Australasian region but not in the native range of E. globulus.
Association motif refers only to those species associated to Eucalyptus globulus according to Dickie et al. (2017). The term novel indicates the association between E. globulus and those ECM species which are not spread in the native range of this eucalypt (i.e. South-Eastern Australia, including Tasmania). Novel/familiar refers to the association with ECM species native in NW Spain, which status (as native or recently introduced) in the E. globulus native range is still uncertain because they have very few and recent records. The term co-introduction refers to those ECM species native in the Australian continent but alien in Europe, which were presumably co-introduced in NW Spain with E. globulus.
Non-ECM fungi were also identified, but were excluded from further analyses. They were the litter decomposers Mycena zephirus (Basidiomycota, Mycenaceae) and Hypholoma fasciculare (Basidiomycota, Strophariaceae), the ericoid mycorrhizae Meliniomyces bicolor (Ascomycota, Leotiomycetes) and Rhyzoscyphus sp. (Ascomycota, Leotiomycetes), the saprobic Coltricia sp. (Basidiomycota, Hymenochaetaceae), the soil fungus Trichosporon porosum (Basidiomycota, Trichosporonaceae), and the opportunistic, non-mycorrhizal fungus Xenasmatella sp. (Basidiomycota, Xenasmataceae). Also, some uncultured Helotiales (Ascomycota), reported as secondary colonists of ECM root tips (Tedersoo et al. 2009), were found in P. pinaster and E. globulus samples. The ericoids M. bicolor and Rhyzoscyphus have been found in a Q. robur forest patch, coming from Q. robur and E. globulus roots. Recently, ericoid mycorrhizal fungi have been described in association with Q. ilex, however, the possibility that some sharing may occur between ericaceous plants and ECM trees is still under debate (Bergero et al. 2000). 1Sequence similarity to Amanita gemmata was high (94%) but did not reach the 98% threshold for species identification. 2 Amanita gemmata has been recorded in the Australasian region, but only in New Zealand, where E. globulus is an exotic species (Whiters, 2001). For this reason, its association with E. globulus was classified as novel. 3This genus has numerous observations in both Europe and Australia (see at www.gbif.org), making it difficult to determine their native region and thus the type of interaction it establishes with E. globulus. 4Cenococcum geophilum is a cosmopolitan ECM fungus in the Holarctic realm (see at www.gbif.org) with a high adaptability to different habitats and host plants (Lo Buglio et al. 1996). Trappe (1962) showed the presence of C. graniforme, synonym of C. geophilum, mainly in the Northern Hemisphere. He also mentions the first discovery of the species in the Southern Hemisphere, in South Africa, in Stellenbosch. In regard to Australia, however, Trappe (1962) explicitly states that the species had not been encountered in Australia or New Zealand by researchers working with mycorrhizae. This leads us to conclude that recent citations of the species in Australia (Tedersoo et al. 2008, see also http://www.ala.org.au/blogs-news/fungimap-putting-australian-fungi-on-themap/) probably correspond to new introductions there. For this reason, the association between this species and E. globulus was classified as putative novel. 5 Cortinarius anomalus has one record in Australia, but in the western region, where E. globulus is not native. For this reason, its association with E. globulus was classified as novel. 6Laccaria lateritia is sometimes synonymized with L. fraterna, see at www.mycobank.org. 7Despite the genus Lactarius was generally considered not compatible with eucalypt roots in pure culture synthesis (Malajczuk et al. 1982) it was found under Eucalyptus spp. in the field in Tasmania (Tedersoo et al. 2008;Gates et al. 2011a;Gates et al. 2011b;Horton et al. 2013). In the present study we detected three species never described before in association with E. globulus. 8There are very few and recent observations of Pseudotomentella sp. in Australia, but not in the native area of E. globulus. 9Scleroderma polyrhizum has been considered native to the Northern Hemisphere by Vellinga et al. (2009), despite it has been frequently recorded in NE Australia, outside the native range of E. globulus. For this reason, its association with E. globulus was classified as novel. 10Tylospora sp. has no records in Australia, for this reason it was classified as novel.
11The Tomentella genus is a common member of the fungal community, although little information exists on its host specificity. This genus is present in Australia with T. brunneorufa Larsen (Kirk et al. 2010;Agerer & Bougher, 2001), and in Tasmania with several species (Tedersoo et al. 2008), in association with Eucalyptus. 12 Tomentella stuposa was detected in Tasmania by Tedersoo et al. (2008) in a forest dominated by E. regnans. This is the first time on our knowledge that this species is reported in association with E. globulus.
Supplementary Figure S1. Rarefied species accumulation curves of four plant species recorded in four types of habitat (beta diversity). Each curve represents the mean of 500 randomizations of sampling order. Eucalyptus globulus (E), Pinus pinaster (P), Quercus robur (Q) and Halimium lasianthum (H) in four habitats (E. globulus plantation = Ep; P. pinaster plantation = Pp; Q. robur forest = Qf; and shrubland = S).  Table S3. Observed (S obs ) and estimated (S est , with the Chao2 method) species richness and the ratio between them. Abbreviations as per figure S1.