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APPENDIX 9 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

APPENDIX 9. — Phylogenetic reconstruction of ITS sequences from Amanita curtipes related specimens collected in worldwide locations (Appendix 10), using A. lepiotoides Barla as outgroup. The known epithets are next to the brackets, sp designating unknown taxa. Using a 75% coverage cutoff, a total of 584 aligned positions were analysed with the Maximum Likelihood method and the General Time Reversible substitution model (Nei & Kumar 2000), with a discrete Gamma distribution to model evolutionary rate differences among sites (5 categories [gamma parameter 0.4648]). Bootstrap support values above 0.66 (200 replicates) are shown next to the relevant nodes.

APPENDIX 10. — Additional ITS sequences used for Appendix 9 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

APPENDIX 10. — Additional ITS sequences used for Appendix 9, approximately in the same order. Abbreviations: MO, Mushroom Observer (https://mushroomobserver.org); iNAT, iNaturalist (https://www.inaturalist.org/observations).

APPENDIX 8 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

APPENDIX 8. — Examples of basidiospore outlines from the Spring 2015 Luzianes collections. Scale bar: 10 μm.

APPENDIX 4 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

APPENDIX 4. — Descriptive (mean ± SE) plots for basidiospore length (L), width (l) and L/l ratio (Q). Sample identifiers refer to the Ode samples (Spring 2015) and P01 (Spring 2010).

APPENDIX 2 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

APPENDIX 2. — Example of a matched comparison of Amanita ponderosa Malençon & R.Heim (ApoIf2-ITS4) and A. pseudovalens comb. nov., stat. nov. (ITS3- ApsIr3) for the same set of samples representing these two species, along with others of A. curtipes E.-J.Gilbert and A. lepiotoides Barla, and an unknown sample (SM-DB 61). The whole contents of each 20 µL PCR reactions were loaded, to help detect faint signals. Negative controls (neg.) with water instead of DNA. Amanita ponderosa is represented by the valens type (G73) and three samples from Spring 2010 (B07, P08, B10). Amanita pseudovalens is represented by the pseudovalens type (G74), the two MPU samples, Ode05 and Ode07. The Amanita curtipes samples (M03, M04 and M05) may produce a weak co-migrating band with the ITS3-ApsIR3 primer pair, and the A. lepiotoides samples (G08 and Eusk) were negative in both cases. Positive control reactions were not attempted because herbaria materials may be contaminated with other fungi.

FIG. 6 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

FIG. 6. — Results of PCR amplifications using discriminant probes for Amanita pseudovalens comb. nov., stat. nov.: A, ITS region: ITS3-ApsIr3 (all samples positive with concurring control with primers ITS3-ITS4, not shown); B, LSU region: NLC2R-ApsLr2 (all positive with control NLC2R-LR5); Ode15 and Ode16 were negative and positive, respectively, in another amplification (not shown); M, molecular marker; neg. designates the negative control (water); pos. designates the positive control (the P01 extract).

FIG. 1 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

FIG. 1. — Locations of the collections used in this study (see Table 1 for details). The letters designate Landscape Units: P, Alto Alentejo; R, Alentejo Central; S, Baixo Alentejo; U, Serras do Algarve e do Litoral Alentejano. The area is outlined on the inset with a yellow rectangle. Source: DGT, Carta de Unidades de Paisagem (CUP), https://www.dgterritorio.gov.pt/dados-abertos

FIG. 2 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

FIG. 2. — Phylogenetic placement of the type nrDNA sequences, in relation to ITS + LSU sequences from european Amanita Pers. taxa belonging to the Amidella clade (only the terminal epithets are shown). Using a 70% coverage cutoff, a total of 1121 aligned positions were analysed with the Maximum Likelihood method and the Tamura-Nei (Tamura & Nei 1993) substitution model, with a discrete Gamma distribution to model evolutionary rate differences among sites (five categories [gamma parameter 0.7526]), allowing for some sites to be evolutionarily invariable (40.90% sites). The percentage bootstrap support for each node (1000 replicates) is shown next to the branches. The support for each species clade is 99% (Amanita pseudovalens comb. nov., stat. nov.), 100% (Amanita curtipes E.-J.Gilbert), 100% (Amanita lepiotoides Barla) and 100% (Amanita ponderosa Malençon & R.Heim). The scale indicates 0.02 substitutions per site.

FIG. 3 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

FIG. 3. — Photographs of Amanita pseudovalens var. tartessiana var. nov. specimens from Odemira. Some images taken in the field are matched with corresponding ones taken in the laboratory: A, grouped basidiomes (Ode12) and their appearance upon arrival at the laboratory; B, outcropping away from the vegetation, showing the coarse soil texture in this case (Ode02); inset shows squamulose inner cuticle remains; C, another specimen with cuticle squamules (Ode11); D, specimen with cracked cuticle (Ode05); E, example with stipe squamules (Ode06); F, side view of an emerging basidiome (Ode03) and the same specimen showing the darkened squamules on the stipe; G, view of the hymenophore and the appendiculate pileus margin (Ode02, specimen different from B); H, details of an immature basidiome (Ode06, specimen different from E) showing the annulus and insertion of the lamellae (left), and longitudinal section (right).

FIG. 5 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

FIG. 5. — Ground view of the Luzianes locations, showing the Cistus ladanifer L. dominance: A, Luzianes A; B, Luzianes B. Both photos were taken in spring 2015 by Ana C. Silva.

FIG. 4 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E.-J.Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

FIG. 4. — Plotting of the measurements summarised in Table 5 (dots), comparing with the limits of the sporographs, based on the descriptions in Neville & Poumarat (2004) for Amanita curtipes f. pseudovalens Neville & Poumarat (continuous line) and A. ponderosa f. ponderosa Malençon & R.Heim (dashed line).

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FIGURE 3 in Amanita vladimirii (Amanitaceae, Agaricales), a new European species in section Vaginatae

FIGURE 3. Phylogenetic reconstruction of Amanita sect. Vaginatae (Maximum likelihood) from tef1-α marker. Sequences of various Amanita species outside Vaginatae (sect. Amanita and Caesareae) were designated as outgroups following Cui et al. (2018). Legends follow Hanss & Moreau (2020).

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FIGURE 2 and 3 in A new saprotrophic species of Amanita (Amanitaceae, Agaricales) from Inner Mongolia, China

FIGURE 2 and 3. Basidiomata of Amanita orientisororia on grassland (HMJAU59015) and vertical section of A. orientisororia (HMJAU59016) Bars A, B=10 mm Photos by Tolgor Bau.

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FIGURE 4. A. Basidia and subhymenium B. Basidiospores C in A new saprotrophic species of Amanita (Amanitaceae, Agaricales) from Inner Mongolia, China

FIGURE 4. A. Basidia and subhymenium B. Basidiospores C. Volval remnants on pileal surface. Bars A=10 μm, B=20 μm, C=30 μm. Drawings by Zhu L. Yang.

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FIGURE 1 in A new saprotrophic species of Amanita (Amanitaceae, Agaricales) from Inner Mongolia, China

FIGURE 1. MrBayes phylogram inferred from two-gene (ITS, nrLSU) combined dataset for Amanita. Maximum Likelihood analysis yielded the same topology. Bayesian posterior probabilities (PP) and 1000 bootstrap replicates in MP analysis were indicated as PP/MP. PP> 0.75 and MP> 70% were shown above or below each branch. New species was in bold and marked by ●.

FIGURE 3 in Amanita amplivelata, (Amanitaceae, Agaricales), a remarkable new Eurasian species of the section Vaginatae

FIGURE 3. Macromorphological features and variability of Amanita amplivelata. a. SOMF30801 (holotype), b–l. Paratypes: b–c SOMF30810, d SOMF30802, e SOMF30806, f SOMF30803, g FBozok1085, h FBozok1097, i SOMF30808, j–k PRM958042, l SOMF30804. In a, b, c, e, g and i note the prominent volva and in f and i–l the ample remnants of the general veil, which often turn ochraceous tinted. Photos: a–f, i, l B. Assyov, g–h F. Bozok, j–k J. Borovička.

FIGURE 5 in Amanita amplivelata, (Amanitaceae, Agaricales), a remarkable new Eurasian species of the section Vaginatae

FIGURE 5. Micromorphology of the volva of Amanita amplivelata. a, b. General view; c, d. External stratum detail; e, f. Main stratum detail (clusters). Photos by J.-M. Hanss. Scale bars: a = 100 μm; b–e = 50 μm; f = 20 μm.

FIGURE 2 in Amanita amplivelata, (Amanitaceae, Agaricales), a remarkable new Eurasian species of the section Vaginatae

FIGURE 2. Phylogenetic tree of Clade 5 (as defined in Hanss & Moreau 2020), based on the nrITS marker. Figures next to branches indicate Bayesian probability and Maximum Likelihood bootstrap proportions, respectively.

FIGURE 1 in Amanita amplivelata, (Amanitaceae, Agaricales), a remarkable new Eurasian species of the section Vaginatae

FIGURE 1. Phylogenetic multigene tree of Clade 5 (as defined in Hanss & Moreau 2020), based on the markers nrITS, nrLSU and TEF1α. The sequences of A. amplivelata are shown in bold. Figures next to branches indicate Bayesian probablility. The notation "T" marks type-derived sequences.