Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Forest System For Syst 2171-9845 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) 19998 10.5424/fs/2023322-19998 RESEARCH ARTICLE Guapirioid ectomycorrhiza: a novel fungus-plant subtype is described associated to Guapira opposita (Nyctaginaceae) in the Brazilian restinga https://orcid.org/0000-0001-9596-7553 Furtado Ariadne N. M. Departamento de Botânica, Universidade Federal de Santa Catarina, Campus Universitário Reitor João David Ferreira Lima, 88040-960, Florianópolis (SC), Brazil. https://orcid.org/0000-0003-3502-4232 Leonardi Marco Dipartimento di Scienze della Vita, della Salute e dell'Ambiente, Università degli Studi di L’Aquila, I-67100, Coppito (AQ), Italy. https://orcid.org/0000-0001-5642-3537 Comandini Ornella Department of Biomedical Sciences, University of Cagliari, I-09042, Monserrato (CA), Italy. https://orcid.org/0000-0002-9352-1037 Rinaldi Andrea C. Department of Biomedical Sciences, University of Cagliari, I-09042, Monserrato (CA), Italy. https://orcid.org/0000-0002-1810-4890 Neves Maria Alice Departamento de Botânica, Universidade Federal de Santa Catarina, Campus Universitário Reitor João David Ferreira Lima, 88040-960, Florianópolis (SC), Brazil. 12 06 2023 04 2023 32 2 e009 22 11 2022 12 06 2023 12 06 2023 ©2023 CSIC 2023 This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License.

This article belongs to the Special Issue “Forests: Reservoirs of Global Mycocultural Heritage and Mycological Resources".https://revistas.inia.es/index.php/fs/si1

Aim of study:

Despite its recognized ecosystemic importance, knowledge about mycorrhizal associations in Brazil is still dearth, and the diversity of the native mycorrhizal fungi remains unknown. In this study, we characterized morpho-anatomically and molecularly the ectomycorrhizae found associated with Guapira opposita in the restinga, vegetation established on sandy soil.

Area of study:

Coastal of the Atlantic Forest of southern Brazil, Florianópolis: Parque Natural Municipal das Dunas da Lagoa da Conceição, Monumento Natural Municipal da Lagoa do Peri and Parque Natural Municipal da Lagoa do Jacaré das Dunas do Santinho.

Material and methods:

Ten morphotypes were identified associated with G. opposita, including the native threatened species Austroboletus festivus. Members of the clade /Tomentella-thelephora were the most representative in our study, with six taxa. Based on particular characteristics, such as short, and simple or long and thin branched ectoMycorrhizal systems, close connections between the layered ectomycorrhizal mantle and the cortical root cells, absence of a Hartig net and other fungal elements in the cortex, we propose the name ‘Guapirioid ectomycorrhiza’ for this new morphology.

Main results:

Paradoxically, this is the first ethnomycological study that addresses a complete study of fungi with biocultural importance in the ethnic group where ethnomycology emerged as a discipline. Previous studies related to mushrooms with Mazatec people focused exclusively on entheogenic mushrooms leaving aside paramount issues related to traditional mycological knowledge of the fungal local resources, including their edibility and cultural relevance. All interviewees consumed mushrooms, indicating high levels of mycophilia. Twenty-seven species, all of them having a native Mazatec name, were identified. Species of Pleurotus, Auricularia, Cantharellus, and Schizophyllum spp. showed the highest cultural value indexes.

Research highlights:

The results presented in this work confirm the presence of ectomycorrhizae in the restinga and suggest the existence of a diversity, above and below-ground, much greater than previously known. Furthermore, our data confirm the morphological and possibly ecological differences of tropical ectomycorrhizae from those known from temperate forests.

 Atlantic Forest; Basidiomycota; Brazilian Funga; diversity; mycorrhizal symbiosis; nurse plant; Coordenação de Aperfeiçoamento Pessoal de Nível Superior – Brazil Finance Code 001 (CAPES-DS and PDSE scholarships to the first author) Introduction

Mycorrhizal fungi establish symbioses with approximately 95% of the plants, stimulating plant growth, drawing nutrients from the soil, and making plants less susceptible to environmental stresses such as drought and salinity (Dighton, 2016). Based on morpho-anatomical structural features, several types of mycorrhizal symbioses are recognized (Martin et al., 2016), with four main fungal groups forming the two major associations: arbuscular mycorrhizae (AM) and ectomycorrhizae. The AM are formed mainly by the Glomeromycota and have emerged with early land plants (Schüßler et al., 2001), while fungi forming ectomycorrhizae have a more recent evolutionary history and are spread across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota (Brundrett & Tedersoo, 2018). Although much information has been produced about taxa that form AM associations, there is less knowledge about ectomycorrhizae, especially in tropical and neotropical ecosystems (Corrales et al., 2018). In ectomycorrhiza, Hartig net and fungal mantle are the main structural characteristics, but these may be incompletely developed or patchy, as often seen in the ectomycorrhizae of herbs and shrubs (Smith & Read, 2008).

Investigations with ectomycorrhizal fungi in native ecosystems in Brazil began in the 1960s with Singer’s research in the Amazon (Singer & Morello, 1960; Singer et al., 1983). After these, other studies continued to reveal the diversity of ectomycorrhizal fungi in Brazilian forests (Roy et al., 2016; Sulzbacher et al., 2020). Still, the relative scarcity of knowledge about ectomycorrhizae and many other belowground interactions stress the need for new research in the neotropics.

It is likely that in neotropical ecosystems, ectomycorrhizae optimize plant fitness and enhance their nutrient acquisition and allow the exchange of nutrients and signals between plants through common mycelium network (Huang et al., 2019). This is especially true where there are conditions limiting the decomposition process (Smith & Read, 2008), such as the restinga, an ecosystem that occupy 79% of the coastal Atlantic Forest. Restinga has sandy, acidic and nutrient-poor soils, often with high salinity and high irradiance (SOS Mata Atlântica, 2021). Several plant species manage to establish and survive in the restinga, and ectomycorrhizal fungi may be a key component that allows it (Weidlich et al., 2020).

Restinga and mangroves are essential as a buffer between the ocean and the forest, and to shelter many living beings, serving as habitat and refuge for several species in at least one part of their life cycles (Steiner et al., 2015). The restinga also stores rainwater, controls flooding and regulates the water cycle (Lessa et al., 2000). Sadly, anthropic activities are negatively affecting the diversity and functionality of the ectomycorrhizal community in forest soils due to soil erosion, changes in land use, inorganic toxins, fire, and invasion of introduced non-native plants (Asmelash et al., 2016). Recently, the ˈParque Municipal Natural das Dunas da Lagoa da Conceiçãoˈ, one of the largest fragments of restinga in Florianópolis, Santa Catarina, Brazil, and one of the places where we carried out this study, suffered an environmental disaster. The disruption of a water and sewage treatment system released excess organic matter and a large amount of nitrogen and phosphorus in the area, leaving the ecosystem out of balance.

Guapira opposita (Vell.) Reitz (Nyctaginaceae) is a polymorphic species with a wide distribution in Brazil. In southern Brazil, the species grows in the rainforest and in the restinga within the Atlantic Forest domain (Magnago et al., 2012). Members of the species can reach 25 m tall but are often shorter when growing in shallow or rocky soils. In restinga, plants of G. opposita grow as short trees and have leaves that are slightly shorter and more coriaceous than the plants growing in the rainforest. The fruits are fleshy, dark red to purple, and attract birds that act as dispersers (Reitz, 1970). Guapira opposita acts as nurse plant (Castanho et al., 2012), promoting the growth of other plants around itself and therefore contributing to regeneration processes in restinga by increasing biodiversity and helping to maintain other plant species (Dalotto et al., 2018).

Since the restinga constitutes a fragmented ecosystem marked by an intense and accelerated process of deforestation and territorial decrease (SOS Mata Atlântica, 2021), studies that aim to understand the fungi of this ecosystem are important to help develop conservation and restauration projects. Indeed, a more extensive knowledge of ectomycorrhizal symbiosis in the restinga might be pivotal for its ecological restoration, as recently noted by Weidlich et al. (2020). In this work, we present the morpho-anatomical and genetic characterization of Guapirioid ectomycorrhizae formed between G. opposita and the fungi from restinga, including new records of symbiotic associations in the Atlantic Forest.

 Material and methods Collecting site and fungal sampling

Collections of G. opposita roots and fungi basidiomata were made between October 2017 and May 2019 in three restinga areas in Florianópolis, Brazil: ˈParque Natural Municipal das Dunas da Lagoa da Conceiçãoˈ (-27.694028, -48.506587), ˈMonumento Natural Municipal da Lagoa do Periˈ (-27.728243, -48.510175) and ˈParque Natural Municipal da Lagoa do Jacaré das Dunas do Santinhoˈ (-27.467783, -48.393395). The vegetation in these areas is associated with Quaternary coastal sand deposits and rocky coastal habitats (Magnago et al., 2012) and grows on sandy soil between the ocean and forest. Besides G. opposita, members of the following potential ectomycorrhizal families grow in restinga: Fabaceae, Moraceae, Myrtaceae, Nyctaginaceae, Polygonaceae, and Salicaceae (Binfaré & Falkenberg, 2017). Soil samples (approx. 20 cm3) were collected below the basidiomata and left in water overnight. The isolated roots were further cleaned and carefully selected under a stereomicroscope. The roots that had an ectomycorrhizal mantle were morphotyped. From each root system with a mantle, several tips were transferred to 70% ethanol and stored at -20ºC for subsequent DNA extraction. Also, part of the root system with the mantle was fixed in 4% glutaraldehyde for morphological analyses. Voucher material of the basidiomata, mycorrhizae and the plants are deposited in the FLOR herbarium and fungarium and the permanent collection of the mycology laboratory (Micolab) at the Universidade Federal de Santa Catarina (UFSC), Florianópolis, Brazil.

The basidiomata were identified by comparing them with the morphology described in the literature (Menolli Jr. et al., 2009; Magnago & Neves, 2014) and by using DNA barcoding of the ITS region. The specimens were collected next to G. opposita plants. After making morpho-anatomical analyses, the basidiomata were dried at 40 °C for further preservation. The identification of the plant was made by a botanist and confirmed by DNA barcoding (ITS region) (Cheng et al., 2015).

 Morpho-anatomical analysis of mycorrhizae

Mantle preparations of ectomycorrhizae fixed in glutaraldehyde were made under a Leica MZ6 stereomicroscope and fixed on microscope slides for observation under a light optical microscope. Observations and descriptions were made with a Zeiss Axioplan 2 microscope, at 1000 × total magnification, at the University of Cagliari, Italy. Images were taken using a digital camera attached to the microscope. Illustrations of microscopic features from the mantle layers were based on observations under the light microscope. The morphotype descriptions followed the standard methodology and terminology used for studying ectomycorrhizae (Agerer, 1991; 1995). Munsell Soil Colour Charts (Munsell Color Co., Grand Rapids, MI, USA) were used as references to describe the colors of ectomycorrhizae. The ectomycorrhizae descriptions are based on 6-10 root tips of each morphotype recorded.

The anatomical analyses of the ectomycorrhizae were conducted at the University of L’Aquila (UnivAq), Italy. For these, mycorrhizal tips were embedded in LR White resin (Multilab Supplies, Surrey, UK), sectioned at 2.5 μm thick with a ultramicrotome (Ultracut R, Leica), stained with 1% toluidine blue in 1% sodium borate buffer for 10-15 s at 60ºC, observed with a microscope, and photographed using a digital camera. Ultrathin sections (60-80 nm) were made with a diamond knife, mounted on copper grids, and stained with UranyLess EM (Electron Microscopy Sciences, Hatfield, PA, USA). The sections were examined using a transmission electron microscope (CM100, Philips, Eindhoven, Netherlands) operating at 80 kV and photographed with a digital camera (PHURONA, Emsis, Münster, Germany).

 Molecular and phylogenetic analysis

The molecular characterization of the ectomycorrhizae was done at Laboratório Multiusuário de Estudos de Biologia, Florianópolis, Brazil. We accessed fungal DNA (from the basidiomata and ectomycorrhizal mantle) using two approaches:

1) Genomic DNA was extracted from the basidiomata using a PowerPlantPro DNA Isolation Kit (MO BIO Laboratories, Inc.), following the manufacturer’s protocol adapted for fungi. The internal transcribed spacer of ribosomal nuclear DNA (nrITS) region was amplified using the primers ITS1F and ITS4 (Gardes & Bruns, 1993) and the following cycling parameters: an initial denaturation at 94ºC for 2 min; 40 cycles of 30 s at 94ºC, 45 s at 55ºC and 1 min at 72ºC; and a final extension at 72ºC for 7 min.

2) A direct PCR approach was applied to amplify the ITS region from the ectomycorrhizal tips isolated from soil samples (Iotti & Zambonelli, 2006). The ectomycorrhizal tips were selected and directly amplified using the same pair of primers (ITS1F/ITS4) (Gardes & Bruns, 1993). Two microliters of 20 mg/mL BSA (bovine serum albumine) solution was added to each reaction tube to prevent PCR inhibition. The parameters applied to the PCR cycles followed Leonardi et al. (2013).

To identify the plant host, plant DNA was extracted from the ectomycorrhizal root tips using an isolation kit (see above), and the barcoding region (ITS) for the plant was amplified using the ITS-u1 and ITS-u4 primer pair (Cheng et al., 2015) and the following cycle parameters: an initial denaturation at 94ºC for 4 min; 34 cycles of 30 s at 94ºC, 40 s at 55ºC and 1 min at 72ºC; and a final extension at 72ºC for 10 min. The DNA extracted from G. opposita leaves was used as a positive control. PCR products were purified using PEG (polyethylene glycol, Sambrook et al., 1989). Sanger sequencing was performed with a BigDye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) at the company Myleus Biotecnologia, in Minas Gerais, Brazil, following the manufacturer’s instructions and using the same primers cited above for the correspondent symbionts. When needed, the generated sequences and their respective chromatograms were manually edited with Geneious R9 (Kearse et al., 2012).

The consensus sequences were used as operational taxonomic units (OTUs) and they were accessed into Genbank. The taxonomic identity of the newly generated sequences from G. opposita leaves, basidiomata and the ectomycorrhizal roots with the morphotypes was determined by phylogenetic affinity of the consensus sequences with homologous sequences from Genbank database (https://www.ncbi.nlm.nih.gov/nucleotide/) using a Blast search (Altschul et al., 1990). New sequences generated during this work were included in GenBank (Sayers et al., 2009) and the accession numbers for all sequences used are shown in Table 1. Sequenced ectomycorrhizal vouchers are in bold in the section “Studied material” following the description of the equivalent Guapirioid ectomycorrhiza.

Identification of ectomycorrhizal morphotypes by their phylogenetic affinity in GenBank.
Species Accession No. Voucher E[1] %Id[2] Best BLAST match Symbiont of best match (potential) Location of best match
Amanita viscidolutea MW000471 M70 0.0 99.37 Amanita sp. (MN123998) - Brazil
Austroboletus festivus OP819290 M41 0.0 97.56 Austroboletus festivus (KY886203) - Brazil
Inocybe sp. OP819291 M51 0.0 90.20 Uncultured Inocybe (JN663843) Dicymbe corimbosa Guyana
2e-174 87.68 Inocybe amazoniensis (JN642232) Dicymbe sp. Guyana
Thelephoraceae OP819292 M53 0.0 96.71 Uncultured Thelephoraceae (AY667418) Neea sp. Ecuador
Tomentella sp1 OP819288 M36 0.0 90.25 Tomentella longisterigmata (MG136837) - Finland
0.0 91.38 Tomentella sp. (KX398760) Coccoloba uvifera Caribbean region
Tomentella sp2 OP819289 M36’ 0.0 91.32 Uncultured fungus (LC622630) Pinus tabuliformis China
0.0 90.52 Tomentella sp. (MW027950) Quercus pubescens -
Tomentella sp3 OP819293 M57 2e-157 91.79 Uncultured Tomentella cf. stuposa (FJ013054) Pinus pinaster Spain
1e-154 91.67 Uncultured fungus (KT289957) Dicymbe corimbosa Guyana
Tomentella sp4 OP819286 M30 0.0 92.73 Tomentella sp. (JX548271) Pisonia taina Puerto Rico
Tomentella sp5 OP819287 M34 0.0 97.85 Uncultured ectomycorrhizal fungus - Tomentella (FN557576) Coccoloba sp. Ecuador
0.0 92.75 Tomentella sp. (KF472145) Coccoloba uvifera France/ Guadaloupe
Tomentella sp6 OP819294 M68 0.0 92.99 Fungal sp. (MG761298) Pinus ponderosa USA
0.0 93.40 Uncultured Tomentella sp. (EU625804) Corallorhiza odontorhiza USA

[1] e-value: indicates the probability of similarity in the identification between the sequences generated in our study and the GenBank sequences. [2] % Id: percentage of similarity between the sequences generated in our study and the GenBank sequences.

 Results

Ten ectomycorrhizal morphotypes were found associated with G. opposita roots corresponding to Basidiomycota taxa (Fig. 1), based on the barcoding (molecular studies). The best represented genus was Tomentella with six taxa (Table 1). Some interesting findings of this work include the collection of Amanita viscidolutea and Austroboletus festivus (Fig. 2) basidiomata, two native species from the restinga of the Atlantic Forest.

Ectomycorrhizal morphotypes associated with <em>Guapira opposita</em> roots in the restinga of Santa Catarina Island, Brazil. A, <em>Amanita viscidolutea</em> (morphotype M70). B, <em>Austroboletus festivus</em> (M41). C, <em>Inocybe</em> sp. (M51). D, <em>Thelephoraceae</em> (M53). E, <em>Tomentella</em> sp1 and <em>Tomentella</em> sp2 (M36 and M36’, respectively). F, <em>Tomentella</em> sp3 (M57). G, <em>Tomentella</em> sp4 (M30). H, <em>Tomentella</em> sp5 (M34). I, <em>Tomentella</em> sp6 (M68). Basidiomata of <em>Austroboletus festivus</em> collected in restinga in Santa Catarina Island, Brazil. Description of ectomycorrhizae

— Amanita viscidolutea Menolli, Capelari & Baseia (Figs. 1A, 3A).

For a complete description of the ectomycorrhiza of A. viscidolutea on G. opposita, see Furtado et al. (2022).

— Austroboletus festivus (Singer) Wolfe (Fig. 1B)

Morphological characters: Mycorrhizal system simple, unramified, mostly dense and long, short-distance exploration system. Main axis 1.5-4.0(−7.0) mm long and 0.6-0.9 mm in diameter, sometimes tortuous, but mostly straight to bent. Mantle surface grainy to woolly, with some portions stringy, strong brown (HUE 7.5YR 4/6 and 5/8) with yellowish red parts (HUE 5YR 4/6). Older parts dusky red (HUE 10R 3/4). Hyphal strands not observed. Emanating hyphae abundant. Sclerotia not observed.

Anatomical characters of mantle in surface views:> Outer mantle layer plectenchymatous, a net of branching hyphae in a regular ring-like arrangement. Hyphae densely incrusted with small crystals, rough, frequently branched, hiding any deeper structure, only some hyphal tips or cystidia-like end-cells visible at the margins. Hyphae (3.5–)4.0-4.6(−6.5) μm thick, brownish yellow, hyphal walls slightly thick, less than 1 μm thick; clampless. Middle mantle layer as a transitional type between plectenchymatous and pseudoparenchymatous, irregularly shaped hyphae from a coarse net, hyphae brownish yellow, 6.0-12 µm thick, thin-walled, cell-walls slightly rough, some crystals, anastomoses mostly with a septum. Inner mantle layerpseudoparenchymatous, hyphae rusty brown, shape of the hyphae variable, 4-5 cells in a square of 20 × 20 µm, 7.0-15 µm long, 5.0-6.5 μm in diameter, slightly thick-walled, cell walls often agglutinated, lacking crystals, anastomoses open.

Anatomical characters of emanating elements: Hyphal strands lacking. Emanating hyphae abundant, rusty brown, long, 4.0-5.5 (6.0) µm in diameter, ramification Y-shaped frequent, clampless; cell walls densely incrusted with small colorless crystals, cell walls up to 1.0 thick; awl-shaped, apical ends simple to bent, walls at the tips thick as remaining walls; diverticulate hyphae frequent. Cystidia like end-cells, clavate to cylindrical, abundant, (10–)12-19(–22) × (4.0–)6.0-7.0(–7.8) µm, slightly thick-walled, colorless to light yellow; cell walls rough, mostly lacking crystals, but less dense and smaller crystal layer can also be found (Fig. 4A).

Anatomical characters, longitudinal section: Mantle (10–)15-18(–25) µm thick, at the very tip up to 20 µm thick, plectenchymatous throughout, very compact, no layers discernable; hyphal cells roundish, 5.0-6.0(–7.0) × 2.0-3.5 µm, slightly thick-walled (up to 1.0 µm). Tannin cells not observed. Cortical cells non-differentiated, in 2-3(–4) rows, elongated, (40–)50-75(–83) × (13–)15-23(–25) µm, tangentially oriented. Hartig net not present (Fig. 3B).

Longitudinal section of <em>Guapira opposita</em> ectomycorrhizae with <em>Amanita viscidolutea</em> (A), <em>Austroboletus festivus</em> (B), <em>Tomentella</em> sp1. (C) and <em>Tomentella</em> sp2. (D) showing the conspicuous ectomycorrhizal mantles, with the particularities of each species, covering the cortical cells of host plant root, without the establishment of the Hartig net. No intercellular or intracellular fungal penetration can be observed in the root cortex. Cor = cortical layer; Man = mantle. Bars: 5 µm.

Studied material: BRAZIL. Santa Catarina: Florianópolis, Monumento Natural Municipal da Lagoa do Peri, -27.728243, -48.510175, 03 Feb 2019, A.N.M. Furtado (M41(A)R208, M41(B)R209, M41(C)R210, M41(D)R211, M41(E)R212, M41(F)R213, M41(G)R214, M41(H)R217, M41(I)R218, M41(J)R220, M41(K)R277, M41(L)R287).

— Inocybe sp. (Fig. 1C)

Morphological characters: Mycorrhizal system simple, unramified, contact exploration system. Main axis (0.5–)0.7-4.5(−5.5) mm long and 0.1-0.3 mm in diameter, straight, rarely bent. Mantle surface smooth, slightly silver, mostly dense, light-yellow brown (HUE 2.5Y 6/4) to light gray (HUE 5YR 7/1); purplish gray (HUE 10PB 8/4) at the very tip. Older mycorrhizae with a uniform pale-yellow color (HUE 2.5Y 7/4). Hyphal strands absent. Emanating hyphae abundant, not specially distributed. Sclerotia not observed.

Anatomical characters of mantle in surface views: Outer mantle layer plectenchymatous, hyphae rather irregularly arranged, no special pattern discernable, with a gelatinous matrix between the hyphae. Hyphae hyaline, but sometimes membranaceous yellowish, cylindrical, constricted at the septa, abundant oleaginous contents, 4.0-5.0(–7.0) μm thick, slightly inflated in middle portion, Y-connections frequent, thin-walled, smooth; clampless. Middle mantle layer plectenchymatous as well as the outer layer but hyphae often growing in longitudinal directions regarding root orientation. Hyphae cylindrical, hyaline, 3.5-4.5 μm thick, thin-walled; clampless. Inner mantle layer plectenchymatous, with a ring-like arrangement of hyphae bundles. Hyphae cylindrical, 3.0-4.0(–6.0) μm thick, sometimes slightly inflated in middle portion, squarrosely branched, smooth, thin-walled; clampless.

Anatomical characters of emanating elements. Hyphal strands lacking. Emanating hyphae awl-shaped but with bent tips, sometimes curved (sickled-shaped), (–87)94-112 µm long, (–3.0)4.0-5.0 µm thick, membranaceous yellowish brown, smooth, not striking, thin-walled, slightly constricted at the septa, clamped; oleaginous contents covering the surface. Cystidia absent.

Studied materials: BRAZIL. Santa Catarina: Florianópolis, Monumento Natural Municipal da Lagoa do Peri, -27.728243, -48.510175, 04 Feb 2019, A.N.M. Furtado (M51(A)R230, M51(B)R232, M51(C)R234, M51(D)R237, M51(E)R240, M51(F)R243, M51(G)R257).

— Thelephoraceae sp1 (Fig. 1D)

Morphological characters: Mycorrhizal system strongly ramified, irregularly pinnate, 7.0-9.5 mm long, contact exploration system. Main axis 0.15-0.3 mm in diameter. Mantle surface smooth, becoming stringy with age, dark reddish brown (HUE 2.5YR 2.5/4) to dark brown (HUE 7.5YR 3/4) in older parts with silvered patches all over the surface. Unramified ends tortuous, beaded in some parts, (2.3−)2.9-4.7 mm long and 0.21-0.27 mm in diameter. Hyphal strands absent. Emanating hyphae present, abundant, slightly cottony, all over the surface but concentrated in the middle portion of the branch. Sclerotia not observed.

Anatomical characters of mantle in surface views: Outer mantle layer plectenchymatous, with a ring-like arrangement of hyphae bundles. Hyphae 5.0-6.5(–8.0) μm thick, membranaceous brownish yellow, smooth, thick-walled (up to 1.0 µm thick), anastomoses at contact-septum, very agglutinated; clampless. Middle mantle layer as a transition between plectenchymatous and pseudoparenchymatous layers. Hyphae irregularly shaped forming a coarse net, (16–)18-22 µm long, 5.0-7.0 µm in diameter, thin-walled; clampless. Inner mantle layer loosely plectenchymatous, hyphae arranged net-like. Hyphae membranaceous brownish yellow, smooth, thin-walled, (3.0–)4.0-5.5 µm thick, densely agglutinated; clampless.

Anatomical characters of emanating elements. Hyphal strands absent. Emanating hyphae hyaline, ornamented with irregularly shaped crystals (except at the apical end), thin-walled, 80-126 µm long, (1.5–)2-2.5 µm in diameter, not ramified; clamped, anastomosis with a clamp, bent at the apical end, strongly glutinous. Cystidia lacking.

Studied materials: BRAZIL. Santa Catarina: Florianópolis, Monumento Natural Municipal da Lagoa do Peri, -27.728243, -48.510175, 04 Feb 2019, A.N.M. Furtado (M53(A)R235, M53(B)R236, M53(C)R239, M53(D)R243).

— Tomentella sp1 (Fig. 1E)

Morphological characters: Mycorrhizal system simple, unramified, sometimes with short monopodial pinnate portions, mostly dense, frequently starting from a distinct yellowish-brown mycorrhiza, short-distance exploration system. Main axis 3.2-6.0 mm long and 0.2-0.4 mm in diameter, sometimes tortuous, but mostly bent. Mantle surface mostly reddish black (HUE 10R 2.5/1) to very dusky red (HUE 10R 2.5/2) and dark reddish brown (HUE 5YR 2.5/2) with some darker grains that give the mantle’s spongy texture. Unramified ends straight to bent, 0.2-0.3(−1.0) mm long and 0.2-0.3 mm in diameter. Hyphal strands not observed. Emanating hyphae frequent, sparse over the mantle. Sclerotia not observed.

Anatomical characters of mantle in surface views: Outer mantle layer pseudoparenchymatous, composed by angular to epidermoid cells, of various shapes and dimensions. Hyphae reddish brown to yellowish, 5-6 cells in a square of 20 × 20 µm, (9.0–)12-18(−22) µm long, (7.0–)10-15 μm in diameter, slightly thick-walled, smooth, cell walls often agglutinated, aggregating soil particles; clampless. Middle mantle layer very similar to the Outer mantle layer but here hyphae are much more branched than in the outer part. Inner mantle layer pseudoparenchymatous, more compact and organized than the outer layer, formed by variable shaped cells, some containing droplets, 4-5 cells in a square of 20 × 20 µm, 7.0-18(–22) μm long, 5.0-10(–13) μm in diameter, brownish, slightly thick-walled.

Anatomical characters of emanating elements. Hyphal strands lacking. Emanating hyphae similar to elongated cystidia, 5.0-6.0 µm in diameter, not ramified, clamped; cell walls smooth, thick-walled, yellowish brown, sometimes filled with homogeneous yellow contents; straight, apical ends simple or inflated, sometimes flattened, walls at tips as thick as remaining walls. Cystidia abundant, bottle-shaped with straight to bent neck, 59-67(–82) µm long, at basis 7.0-10(–12) µm in diameter and 2.0-3.0 µm in diameter apically, smooth, thick-walled, slightly yellow, clampless (Fig. 4B).

Cystidia diversity in the mantle of Guapirioid ectomycorrhizae from the Brazilian restinga. A. Clavate to cylindrical cystidia (black arrows) of <em>Austroboletus festivus</em>. B. Bottle-shaped with straight neck cystidia (black arrows) of <em>Tomentella</em> sp1. C. Lageniform cystidia (black arrows) of <em>Tomentella</em> sp2. D. Clavate cystidia (black arrows) of <em>Tomentella</em> sp4. E. Bottle-shaped with straight neck (red arrows) and flask-shaped (black arrow) cystidia of <em>Tomentella</em> sp5. F. Lageniform (light blue arrows) and bottle-shaped with straight neck (red arrows) cystidia; and cystidia like a normal clamped hypha but often twisted like a cork-screw (black arrows) of <em>Tomentella</em> sp6. Bars: 10 µm.

Anatomical characters, longitudinal section: Mantle 35-40(–50) µm thick, hyphae thick-walled, often with two discernible layers: outer layer 22-28 µm thick, formed by densely arranged hyphae, hyphal cells roundish to different shapes, 2.5-6.5 × 3.5-6.0 µm; inner layer 7.5-12.5(–15) µm thick, just as compact as the outer layer, hyphae narrow and longitudinally elongated, 12-15 × 3.7-6.0 µm. Tannin cells not observed. Cortical cells non-differentiated, in 3-4 rows, longitudinally rectangular, (50–)55-70(–82) × (15–)22-32(–37) µm, tangentially oriented. Hartig net not present (Fig. 3C).

Studied materials: BRAZIL. Santa Catarina: Florianópolis, Monumento Natural Municipal da Lagoa do Peri, -27.728243, -48.510175, 03 Feb 2019, A.N.M. Furtado (M36(A)R200, M36(B)R202, M36(C)R203, M36(D)R204, M36(E)R210, M36(F)R212, M36(G)R215, M36(H)R216, M36(I)R220).

— Tomentella sp2 (Fig. 1E)

Morphological characters: Mycorrhizal system simple, unramified, often making the connection between a distinct dark brown ectomycorrhiza and the root without the mantle, medium-distance exploration system, smooth subtype. Main axis 14-25 mm long, 0.2-0.5 mm in diameter, straight to slightly curved. Mantle surface frequently stringy, sometimes cottony, olive yellow (HUE 2.5Y 6/6) to olive brown (HUE 2.5 4/3 to 4/4). Hyphal strands present, relatively abundant, sparse, branched, connected in a restricted point to the mantle; pale yellow (HUE 2.5Y 8/3). Emanating hyphae abundant, like hyphal fans. Sclerotia not observed.

Anatomical characters of mantle in surface views: Outer mantle layer pseudoparenchymatous with angular cells bearing a delicate hyphal net of irregularly shaped angular-triangular or barrel-like cells on surface. Hyphae slightly yellow, 3-4 cells in a square of 20 × 20 µm, 4.5-5.5(−7.0) µm long, 12-15 μm in diameter, thin-walled, smooth; clampless. Middle mantle layer not clearly observable. Inner mantle layer as a transition type between plectenchymatous and pseudoparenchymatous, compacted, formed by polygonal, elongated or irregular hyphal segments, 4.0-6.0 μm thick, slightly yellow, thick-walled (up to 1.0 µm thick); clampless.

Anatomical characters of emanating elements. Hyphal strands (35–)50-70(–85) × 7.0-8.5(–10) µm, with smooth margins; ramification with one or two side branches at nodia. Hyphae slightly differentiated, densely interwined and aggregated: central hyphae clamped, smooth, 5.0-5.5 µm in diameter, thick-walled (2 µm thick), membranaceous yellow; peripheral hyphae clamped, smooth, 3.0-4.5 µm in diameter, hyphae wall up to 1.0 µm thick, slightly tortuous, loosely woven, repeatedly branched, hyaline. Emanating hyphae similar to elongated cystidia, 3.0-5.0 µm in diameter, secondarily septate, clamped; ramification Y-shaped bellow the clamps; cell walls smooth, slightly thick-walled (up to 0.5 µm thick), hyaline, filled with oil yellow contents; bent, apical ends simple or inflated, sometimes undifferentiated. Cystidia on Hyphal strands, rare, lageniform, clamped, 12-17 µm long, at basis 3.0-4.5 µm in diameter and 2.0-2.5 µm apically, smooth, hyphae wall up to 1.0 µm thick, hyaline (Fig. 4C).

Anatomical characters, longitudinal section: Mantle (25–)32-42(–45) µm thick, hyphae slightly thick-walled, two hardly discernible layers: outer layer (in some places weakly developed) 17.5-22.5 µm thick, hyphal cells often elongated, (10–)12-18(–20) × 2.0-3.0 (–4.5) µm; inner layer (10–)12.5-15 µm thick, more compact than the outer layer, hyphae of varied shapes, 3.0-4.0(–5.5) µm thick. Tannin cells not observed. Cortical cells non-differentiated, in 3-4 rows, longitudinally rectangular, (45–)57-125 × 10-12.5(–23) µm, tangentially oriented. Hartig net not present (Fig. 3D).

Studied materials: BRAZIL. Santa Catarina: Florianópolis, Monumento Natural Municipal da Lagoa do Peri, -27.728243, -48.510175, 03 Feb 2019, A.N.M. Furtado (M36’(A)R200, M36’(B)R202, M36’(C)R203, M36’(D)R204, M36’(E)R210, M36’(F)R212, M36’(G)R215, M36’(H)R216, M36’(I)R220).

— Tomentella sp3 (Fig. 1F)

Morphological characters: Mycorrhizal system irregularly pinnate, dichotomous like, sometimes simples (unramified), 11.5-13 mm long, contact exploration system. Main axis 0.1-0.2 mm in diameter. Mantle surface visible, decaying when older, smooth, loosely grainy becoming stringy with age, yellowish red (HUE 5YR 4/6) becoming dark reddish brown (HUE 2.5YR 2.5/4) aging. Unramified ends straight, (0.6−)1.3-3.0(−5.0) mm long and 0.1-0.2 mm in diameter. Hyphal strands and Emanating hyphae absent. Sclerotia not observed.

Anatomical characters of mantle in surface views: Outer mantle layer pseudoparenchymatous, composed of angular cells. Hyphae compact, membranaceous light brown, smooth, thin-walled (up to 0.5 µm thick), 2-4 cells in a square of 20 × 20 µm, 11-15(−17) µm long, 10-12 μm in diameter; clampless. Middle mantle layer as a transitional type between plectenchymatous and pseudoparenchymatous, irregularly shaped hyphae forming a coarse net, looser than the outer layer. Hyphae smooth, thin-walled, plasmatical light brown, 11-12.5(−17) µm long, (6.0–)9.0-11 μm in diameter; clampless. Inner mantle layer plectenchymatous, hyphae rather irregularly arranged, but sometimes arranged net-like. Hyphae hyaline, smooth, thin-walled, 4.0-6.0 µm thick; anastomosing with a clamp.

Anatomical characters of emanating elements. Hyphal strands absent. Emanating hyphae absent, although when old the outer mantle becomes loose from the mycorrhiza surface imitating these structures. Cystidia lacking.

Studied materials: BRAZIL. Santa Catarina: Florianópolis, Parque Natural Municipal das Dunas da Lagoa da Conceição, -27.694028, -48.506587, 04 Feb 2019, A.N.M. Furtado (M57(A)R247, M57(B)R248, M57(C)R249, M57(D)R250, M57(E)R252, M57(F)R253, M57(G)R254, M57(H)R255, M57(I)R256, M57(J)R257, M57(K)R258, M57(L)R259, M57(M)R261).

— Tomentella sp4 (Fig. 1G)

Morphological characters: Mycorrhizal system simple, sometimes irregularly pinnate, 2.5-4.0 mm long, medium distance exploration system, smooth subtype. Main axis 0.1-0.25 mm in diameter. Mantle surface visible, smooth, dense becoming spongy with age, shiny, yellowish brown (HUE 10YR 5/4) to dark yellowish brown (HUE 10YR 4/4) in older parts. Unramified ends mostly straight, (0.4−)1.3-3.7(−4.5) mm long and 0.15-0.25 mm in diameter. Hyphal strands present but infrequent, growing in flat angles, mostly in the proximal and median portions. Emanating hyphae scarce, like subtle hyphal fans, more concentrated in the proximal portion of the Mycorrhizal system. Sclerotia not observed.

Anatomical characters of mantle in surface views: Outer mantle layer plectenchymatous, hyphae arranged net-like, sometimes with a weak ring-like pattern and prominent cystidia departing from it. Hyphae 5.0-7.0(–10) μm thick, yellowish, smooth, thick-walled (1.0 µm thick); clampless. Middle mantle layer as a thin layer, representing a transitional type between plectenchymatous and pseudoparenchymatous systems. Hyphae yellowish brown, smooth, 12-16 × 4.5-7.0 µm, thin-walled. Inner mantle layer denser than the outer layer, pseudoparenchymatous, irregularly shaped hyphae form a coarse net, puzzle-like. Hyphae brownish yellow, thick-walled (1.0 µm thick), 7-9 cells in a square of 20 × 20 µm, 9.6-11(–13) µm long, 4.0-6.0 µm in diameter; clampless.

Anatomical characters of emanating elements. Hyphal strands present, albeit rare, sparsely distributed, never at the very tip, 20-24 µm thick, becoming tapered towards the apex; reversely oriented ramification, several hyphae grow out of the margin. Very agglutinate sticking soil particles on the surface. Hyphae undifferentiated rather loosely woven of uniform diameter, secondarily septate, thick-walled (1.0 µm thick), membranaceous brownish yellow, irregularly shaped crystals on the surface, 3.0-5.0 µm in diameter; clamped, slightly enlarged near the septum. Emanating hyphae rare, awl-shaped (bristle-like) with a slightly bent apex, brownish yellow, 119-197(–310) µm long, 3.0-5.0 µm in diameter, abundant secondary septation, Y-ramifications present, thin-walled (<0.5 µm thick), smooth but agglutinated, holding soils particles on the surface; clamped irregularly, but anastomoses with the septum. Cystidia abundant, slightly clavate, 37-42(–72) µm long, at basis 4.0-6.0(–7.0) µm in diameter and 4.0-5.0 µm apically, smooth, thick-walled (1.0µm thick), brownish yellow; clampless (Fig. 4D).

Studied materials: BRAZIL. Santa Catarina: Florianópolis, Monumento Natural Municipal da Lagoa do Peri, -27.728243, -48.510175, 20 Mar 2018, A.N.M. Furtado (M1(R2)R172, M1(S2)R173, M30(A)R173).

— Tomentella sp5 (Fig. 1H)

Morphological characters: Mycorrhizal system simple, unramified, contact exploration system. Main axis 0.6-1.5 mm long and 0.15-0.2 mm in diameter, bent ends. Mantle surface visible, felty, slightly floury, very pale brown (HUE 10YR 8/4) to brownish yellow (HUE 10YR 6/6) aging. Hyphal strands and Emanating hyphae absent. Sclerotia not observed.

Anatomical characters of mantle in surface views: Outer mantle layer plectenchymatous, hyphae rather irregularly arranged, no special pattern discernable, but hyphae often growing in longitudinal directions. Hyphae glutinous, 3.0-4.0 μm thick, slightly yellow, smooth, thin-walled; clampless. Middle mantle layer as a transition between plectenchymatous and pseudoparenchymatous layers, hyphae irregularly shaped forming a coarse net. Hyphae hyaline, thin-walled; clampless. Inner mantle layer plectenchymatous, hyphae arranged hash-like. Hyphae hyaline, strongly glutinous, smooth, thin-walled, 3.5-5.0 μm thick; clampless.

Anatomical characters of emanating elements. Hyphal strands and Emanating hyphae absent. Cystidia present, but rare, parting from the inner layer, smooth, strong brown, slightly thick-walled (0.9 µm thick), bottle shaped with a straight neck [(11.5–)13-15 µm long, at basis 4.5-5.5 µm in diameter and 2.5-3.0(–4.5) µm apically] or flask-shaped [19-23 µm long, 3.0-4.0 µm in a diameter]; clampless (Fig. 4E).

Studied materials: BRAZIL. Santa Catarina: Florianópolis, Parque Natural Municipal da Lagoa do Jacaré das Dunas do Santinho -27.467783, -48.393395, 08 Nov 2018, A.N.M. Furtado (M34(A)R180, M34(B)R183, M34(C)R185, M34(D)R187, M34(E)R198).

— Tomentella sp6 (Fig. 1I)

Morphological characters: Mycorrhizal system monopodial-pinnate, 12.9-14 mm long, contact exploration system. Main axis 0.18-0.27 mm in diameter. Mantle surface visible, densely grainy (warty) to short spiny, shiny and coppery, dark reddish brown (HUE 5YR 2.5/2) to red (HUE 2.5YR 4/8) in older parts. Unramified ends straight with some becoming bent, (3.6−)5.0-12.8(−17) mm long and 0.26-0.31 mm in diameter. Hyphal strands absent. Emanating hyphae frequent, branched, yellowish red (HUE YR 5/8). Sclerotia not observed.

Anatomical characters of mantle in surface views: Outer mantle layer pseudoparenchymatous, composed by roundish cells from which emerge cystidia and long Emanating hyphae. Hyphae light brown, smooth, thick-walled (1.0 µm thick), 4-6 cells in a square of 20 × 20 µm, 10-14(−16) µm long, 9.0-11(–14) μm in diameter; clampless. Middle mantle layer pseudoparenchymatous, composed of angular hyphae, with large dimensions. Hyphae brownish yellow, smooth, thick-walled (up to 2.0 µm thick), 3-4 cells in a square of 20 × 20 µm, (19−)23-25 µm long, (15–)17-20 μm in diameter; clampless. Inner mantle layer plectenchymatous, hyphae rather irregularly arranged, no special pattern discernable, but hyphae often growing in longitudinal directions regarding root orientation. Hyphae 3.0-6.0 µm thick, hyaline, smooth, thin-walled, with granular content and a gelatinous matrix between the hyphae; clamped, with Y-ramifications frequent.

Anatomical characters of emanating elements. Hyphal strands absent. Emanating hyphae 4.0-6.0 µm thick, bent in the apex, thick-walled (1.0 µm thick), brownish yellow, secondarily septate; clamped in all septa. Hyphae distributed all over the Mantle surface but concentrated in middle and distal portions of the root. Cystidia abundant, of variable shapes, thin-walled (0.5 µm thick); lageniform [17-23(–26) µm long, at basis (7.0–)9.0-13 µm in diameter and 3.0-4.0 µm apically], bottle-shaped with a straight neck [25-36 µm long, at basis 9.0-11 µm in diameter and 3.0-5.0 µm apically] or like a normal hypha but often twisted like a cork-screw [27-33(–40) × 4.0-6.0] and in this case, clamped (Fig. 4F).

Studied materials: BRAZIL. Santa Catarina: Florianópolis, Parque Natural Municipal das Dunas da Lagoa da Conceição, -27.694028, -48.506587, 25 Mar 2019, A.N.M. Furtado (M68(A)R291, M68(B)R292).

The results presented here endorse the existence of morpho-anatomical particularities of ectomycorrhizae associated with G. opposita. Despite these genera being widely known to be ectomycorrhizal and usually having typical anatomical structures (mantle, Hartig net and extraradicular hyphae), the ectomycorrhizae of A. viscidolutea (Furtado et al., 2022), A. festivus, Tomentella sp1 and Tomentella sp2 exhibit particular characteristics (Fig. 3). It is possible that the lack of the Hartig net is common among other taxa that grow in restinga. The anatomy of the ectomycorrhizae studied revealed close connections between the layered ectomycorrhizal mantle and the cortical root cells. However, no intercellular or intracellular fungal penetration was observed in the root cortex. Additionally, the epidermal cells of the root of G. opposita, where the mantle is established, are absent. Although we understand that this is not a taxonomic character for the symbiosis, we are working to understand if the absence of the epidermis may be an evolutionary step that helped the establishment of the association or maybe a physiological response of G. opposita to the fungal colonization.

 Discussion

We have described the occurrence of ectomycorrhizae in the roots of G. opposita and propose the name Guapirioid ectomycorrhiza based on the particularities of the morphology. The short and simple or long and thin branched ectoMycorrhizal systems, close connections between the layered mantle and the cortical cells, absence of a Hartig net and other fungal elements in the cortex are diagnostic characteristics that distinguish the Guapirioid from other ectomycorrhizae.

All the Basidiomycota fungi sequenced from G. opposita in this work belong to putatively ectomycorrhizal lineages (Table 1) and form visible ectomycorrhizal morphotypes with several layers of hyphae composing a distinguishable mantle. Some of the Guapirioid ectomycorrhizae are discussed below.

Sequences of the morphotype M70 had 99.37% (Table 1) similarity with sequences of Amanita viscidolutea basidiomata. This species was originally described for the northeast coast of Brazil, in Rio Grande do Norte, and it has been collected in Paraíba and Santa Catarina, probably having a distribution along the restinga. Records based on field observations made by other researchers suggested Coccoloba (Polygonaceae) (Menolli Jr. et al., 2009) or an unnamed Nyctaginaceae species (Wartchow et al., 2012) as possible symbionts. These suggestions have not been confirmed but are possibly not wrong. However, we proved that A. viscidolutea forms an association with G. opposita, and the morpho-anatomical features of this symbiosis have been recently described (Furtado et al., 2022). This was the first description of an Amanita ectomycorrhiza on Guapira.

An interesting species recorded in our site collection was A. festivus. Sequences from the ectomycorrhizal root tips (morphotype M41) had 97.56% (Table 1) of similarity with Brazilian sequences of A. festivus basidiomata. Austroboletus festivus was originally described from Pernambuco, northeastern Brazil (Singer, 1970). The species was also recorded in Paraná and Santa Catarina (Magnago & Neves, 2014) and probably has a wide distribution on the restinga. This is the first characterization of a native Austroboletus ectomycorrhiza with G. opposita in the restinga. Currently, and as contribution from our work about the ectomycorrhizae from the restinga, both A. festivus and Amanita viscidolutea were considered in The IUCN Red List of Threatened Species and included in the vulnerable category according to IUCN criteria (Neves & Furtado, 2020; Neves et al., 2020; see also http://iucn.ekoo.se/en/iucn/welcome).

One Guapirioid ectomycorrhiza (morphotype 51 – Inocybe sp.) had 90.20% of similarity with an unknown Inocybe ectomycorrhiza associated with Dicymbe corimbosa in Guyana. Ectomycorrhizae of an unknown species of Inocybe were collected during our research. However, there are almost no voucher sequences with similarity greater than 90% in Genbank. Inocybe has ca. 750 species described (Matheny et al., 2020), but the taxon is undersampled in Brazil. There are only seven species known for Brazilian forests: I. austrolilacina Wartchow & RM Silveira, I. cavalcantiae Wartchow, I. curvipes P. Karst., I. hyperhytra Rick, I. lepidospara Wartchow, I. martinica Pegler and I. violaceolamellata Rick (Wartchow, 2018). The mycorrhizal potencial of Inocybe was not cited, but the authors suggest members of Nyctaginaceae, Euphorbiaceae and Fabaceae subfam. Caesalpinoideae as potential partners. The color and anatomical features of the Inocybe ectomycorrhizae is useful to distinguish morphotypes (Seress et al., 2015). The Inocybe ectomycorrhiza described here was compared to the seventeen descriptions previously published for the genus (Seress et al., 2015; DEEMY website http://www.deemy.de/). The Inocybe Guapirioid ectomycorrhiza can be differentiated mainly by two characters: (i) presence of a contact exploration system, as opposed to a short-distance system frequent in the other species of the genus; and (ii) the purple tip, in contrast to the white tip found in all other Inocybe ectomycorrhizae.

Representatives of /Tomentella-thelephora clade were the most frequently encountered taxa in our study (M53 – Thelephoraceae; Tomentella sp1 – M36; Tomentella sp2 – M36’; Tomentella sp3 – M57; Tomentella sp4 – M30; Tomentella sp5 – M34; and Tomentella sp6 – M68 morphotypes), as expected and reported in previous studies (Tedersoo et al., 2014). Indeed, studies based on root and soil analyses often find that the /Tomentella–thelephora clade is diverse and dominant in neotropical habitats, but usually they are undersampled due to their inconspicuous basidiomata (Tedersoo et al., 2014), making it difficult to identify the sampled taxon. Unlike what has been reported in other works made in Latin America (Haug et al., 2005; Alvarez-Majarrez et al., 2017), the Tomentella ectomycorrhizae found in restinga often form morphotypes with a contact exploration system, or even with a medium distance exploration system with a smooth subtype.

Among the various Tomentella species found during this survey and linked to G. opposita, Tomentella sp1 (morphotype M36) and Tomentella sp2 (morphotype M36’) were the most interesting. These taxa were frequent in our study and grew on the same root segment. Considering that G. opposita has secondary roots, with frequently smaller diameters that branch off from its main, larger-diameter root, we observe: Tomentella sp2 (morphotype M36’) growing in the most proximal portion of the secondary root segment, and Tomentella sp1 (morphotype M36) growing more distal to the main root on the same root segment (Fig. 1E). Even though we do not have information about the biology of these species, there is possibly a functional codependency between them that could be explained if their growth and development could be monitored. Sequences of Tomentella sp1 had 91.38% similarity with a Tomentella mycorrhiza isolated from the root of Coccoloba uvifera from the Caribbean region and had 90.25% similarity with basidiomata sequences of Tomentella longisterigmata from coniferous forests from Finland (Table 1). Tomentella sp2 had 91.32% similarity with an ectomycorrhiza associated with Pinus tabuliformis from China (Lu et al., 2018) and 90.52% similarity with a Tomentella associated with Quercus pubescens (Mrak et al., 2021) (Table 1). Tomentella is a widespread genus, distributed throughout the temperate and tropical regions worldwide, forming abundant ectomycorrhizae in coniferous and deciduous forests. At least 100 species are currently known, and several have been described in recent years, thanks to molecular tools that allow to ascertain the existence of numerous cryptic species in a genus where basidiomata are inconspicuous and rarely found (Lu & Yuan, 2021). In extreme ecosystems like the restinga the constant disturbances, anthropogenic or biotic, can also affect the production of basidiomata.

Two morphotypes (M34 – Tomentella sp5 and M53 – Thelephoraceae) of the /Tomentella-thelephora ectomycorrhizae had high similarity with samples from Ecuador. The morphotype M34 presented 96.71% similarity with the sequence of an ectomycorrhiza isolated from the roots of Neea sp. (Haug et al., 2005). The morphotype M53 had 97.85% similarity with an ectomycorrhiza isolated from the roots of Coccoloba sp. (Polygonaceae) from Ecuador (Tedersoo et al., 2010).

There are records of Guapira and Neea species forming mycorrhiza with a single fungal taxon member of Thelephoraceae, where a fine root system made of only long roots was observed with an incomplete hyphal mantle development and with no suppression of root hairs formation (Moyersoen, 1993; Haug et al., 2005). However, we found in G. opposita that there were at least four different fungal species on the root system, including taxa from phylogenetically distant orders, e.g. Austroboletus festivus (M41) sharing the same root system with Tomentella sp1 (M36) and Tomentella sp2 (M36’). Also, even though G. opposita has a fine root system composed by long roots, there was a complete formation of the ectomycorrhizal mantle, as well as the suppression of the root hairs formation.

The morphotype M30 (Tomentella sp4) had a 92.73% similarity with a Tomentella sp. ectomycorrhiza isolated from the roots of Pisonia taina from Puerto Rico (Hayward & Horton, 2014), but there are no basidiomata sequences with similarity greater than 90% in Genbank. The morphotype M57 (Tomentella sp3) had a 91.79% similarity to Tomentella stuposa associated to Pinus pinaster roots, from Spain, but when the morpho-anatomical characteristics were compared it was clear that they were not the same (Jakucs et al., 2005). Both have a similar general organization of the mantle layers; however, the arrangement of the hyphae differs (it is a star-like structure in T. stuposa morphotype and a net-like arrangement in M57 morphotype). Furthermore, the morphotype of T. stuposa has emanating elements and Hyphal strands, features that are absent in the M57 morphotype. These are some of the characteristics that allow us to consider them as ectomycorrhizae from distinct species. An interesting particularity of the morphotype M57 is that even though it often occupies the entire root system, it is possible to find other morphotypes starting from it, such as M51 (Inocybe sp.) and M53 (Thelephoraceae sp.) morphotypes. The highest percentage of similarity for the Tomentella sp6 (morphotype M68 - 93.40%) was with an ectomycorrhiza of Tomentella sp. isolated from the rhizome of the orchid Corallorhiza odontorhiza (McCormick et al., 2009). Although orchids are often found forming endomycorrhizal associations with the genus Rhizoctonia, the ectomycorrhizal association with taxa of Thelephoraceae has been previously reported (Bidartondo & Read, 2008; Roy et al., 2013).

The Guapirioid ectomycorrhizae have potential to be used in G. opposita seedlings in restoration projects in restinga (Weidlich et al., 2020). The increase in the plant survival rate in the field would be directly related to the fact that both symbionts would be adapted to local conditions, thus without the risk of introducing alien plant hosts and/or fungal species (Dalotto et al., 2018). To use the Guapirioid ectomycorrhizal fungi in restoration projects the biology, physiology and ecology of the fungal community need to be better understood. Also, the fungi need to be isolated to be used as inocula, which has proven to be a great challenge, since many ectomycorrhizal species do not grow easily on culture media (Weidlich et al., 2020).

 Conclusion

Our study showed that G. opposita forms symbiosis with at least ten taxa of ectomycorrhizal fungi in the restinga. We are just starting to explore the biological and functional complexity of the Guapirioid ectomycorrhiza and in future experiments we aim to better understanding how G. opposita acts as a nurse plant. We want to understand how the plant is affected by its fungal partners and how the mycelial network shared with neighboring host plants works, considering its importance in restinga. The apparent generalist status of G. opposita as a symbiont is probably due to its role as a nurse plant, but more studies on the mycorrhizal status and identification of the mycobionts are needed to draw conclusions about the evolution and potential host specificity in this and other species of Nyctaginaceae.

The collection of basidiomata of Tomentella and other Thelephoraceae continues to be a big challenge for those who study the species in Thelephoraceae. During this study we did not find Tomentella basidiomata to check their correspondence with the ectomycorrhizae found in G. opposita, but we continue to search.

It is currently understood that ecosystem recovery is favored by the greater accessibility of disposable macronutrients in mycorrhizae-rich environments. The results obtained from this work make it possible to discuss the impact that these associations have on the formation and maintenance of the ecosystem and on the establishment of seedlings in sandy soils (Weidlich et al., 2020).

This work is the first to characterize the ectomycorrhizae native to restinga and to propose Guapirioid ectomycorrhiza as the first type described for the Atlantic Forest in Brazil. Unfortunately, the environmental disaster that happened in the study area and cited in the Introduction may have caused an irreversible disruption of the ecological communities in that point of the restinga. It is important to continue to study the fungal diversity above and belowground in the restinga so better conservation and restauration projects can be proposed.

 Acknowledgments

This article is a part of the PhD thesis of ANMF. The authors thank LAMEB (UFSC, Brazil) for the support during the molecular work. We are indebted to Dr. Maria Giammatteo (UnivAq, Italy) for kindly providing microscopy images. We also thank the following: Dr. Mayara K. Caddah (UFSC, Brazil) for helping identify the plant; Dr. Savio T. Farias for helping during field expeditions; and those at the FLOR herbarium and fungarium (UFSC, Brazil) for their support.

Authors’ contributions Conceptualization: A. N. M. Furtado, M. Leonardi, O. Comandini, A. C. Rinaldi, M. A. Neves. Data curation: A. N. M. Furtado, M. Leonardi, O. Comandini, A. C. Rinaldi, M. A. Neves. Formal analysis: A. N. M. Furtado, M. Leonardi, O. Comandini, A. C. Rinaldi, M. A. Neves. Funding acquisition: A. N. M. Furtado, A. C. Rinaldi, M. A. Neves. Investigation: A. N. M. Furtado, M. Leonardi, O. Comandini, A. C. Rinaldi, M. A. Neves. Methodology: A. N. M. Furtado, M. Leonardi, O. Comandini, A. C. Rinaldi, M. A. Neves. Project administration: A. N. M. Furtado, M. Leonardi, O. Comandini, A. C. Rinaldi, M. A. Neves. Resources: M. Leonardi, O. Comandini, A. C. Rinaldi, M. A. Neves. Software: Not applicable. Supervision: A. N. M. Furtado, A. C. Rinaldi, M. A. Neves. Validation: A. N. M. Furtado, M. Leonardi, O. Comandini, A. C. Rinaldi, M. A. Neves. Visualization: A. N. M. Furtado, M. Leonardi, O. Comandini, A. C. Rinaldi, M. A. Neves. Writing – original draft: A. N. M. 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