Supplementary material for Man against machine: Do fungal fruitbodies and eDNA give similar biodiversity assessments across broad environmental gradients? Tobias Guldberg Frøslev, Rasmus Kjøller, Hans Henrik Bruun, Rasmus Ejrnæs, Anders Johannes Hansen, Thomas Læssøe, Jacob Heilmann- Clausen 1 Supplementary methods. This study was part of the Biowide project, and many aspects are presented and discussed in more detail in Brunbjerg et al. (2017). Environmental variables. Soil samples (0-10 cm, 5 cm diameter) were collected within 4 subplots of the 130 sites and separated in organic (Oa) and mineral (A/B) soil horizons. Across all sites, a total of 664 soil samples were collected. Organic horizons were separated from the mineral horizons when both were present. Soil pH was measured on 10g soil in 30 ml deionized water, shaken vigorously for 20 seconds, and then settling for 30 minutes. Measurements were done with a Mettler Toledo Seven Compact pH meter. Soil pH of the 0-10 cm soil layer was calculated weighted for the proportion of organic matter to mineral soil (average of samples taken in 4 subplots). Organic matter content was measured as the percentage of the 0-10 cm core that was organic matter. 129 of the total samples were measured for carbon content (LECO elemental analyzer) and total phosphorus content (H2SO4-Se digestion and colorimetric analysis). NIR was used to analyze each sample for total carbon and phosphorus concentrations. Reflectance spectra was analyzed within a range of 10000-4000 cm-1 with a Antaris II NIR spectrophotometer (Thermo Fisher Scientific). A partial least square regression was used to test for a correlation between the NIR data and the subset reference analyses to calculate total carbon and phosphorous (see Brunbjerg et al. 2017 for more details). 2 Supplementary Figures a Dataset 50 Both Fruitbody OTU Fruitbody meth. only only OTU 3340 3874 obs. obs. 0 6099 obs. 2992 50 Frequency (in 130 sites) obs. 428 sp./ 556 OTU 930 species 1965 OTUs 100 1000 2000 3000 Species/OTUs b c 60 d r = 0.78, p < 2.2e−16 40 y = 6.78 + 0.822 x 60 0 40 40 40 Frequency (in 130 sites) 20 richness OTU 20 Registered as fruitbody RickenellaMycena Mycenafibula galopus speireaLaccaria laccata Mycena cinerella 0 0 Clitopilus hobsonii Phallus impudicus Galerina Mycenavittiformis epipterygiaEntolomaRussula sericeum ochroleuca Mycena leptocephalaCoprinellus micaceus Mycena sanguinolenta Hypholoma fasciculare 0 20 40 60 0 20 40 60 Radulomyces confluens Ganoderma applanatum Registered as OTU Fruitbody richness e Dataset 50 Both Fruitbody OTU Fruitbody meth. only only OTU 2220 2114 obs. obs. 0 2598 obs. 1909 50 Frequency (in 130 sites) obs. 279 sp./ 358 OTU 568 species 935 OTUs 100 500 1000 1500 Species/OTUs f g 60 h r = 0.68, p < 2.2e−16 40 40 y = 5.71 + 0.774 x 0 30 40 40 20 Frequency (in 130 sites) 20 richness OTU Registered as fruitbody 10 Mycena metata 0 MycenaMycena galopus speireaLaccaria laccata Mycena cinerella 0 Clitopilus Galerinahobsonii vittiformis Mycena epipterygiaMycena galericulataDelicatula Entolomaintegrella sericeum Mycena leptocephala Coprinellus micaceus Mycena sanguinolenta Hypholoma fasciculare 0 20 40 60 10 20 30 40 Radulomyces confluensEntoloma conferendum Registered as OTU Fruitbody richness Supplementary Figure 1. Frequency of species and OTUs among the 130 sampling sites, restricted to Agaricomycetes (a-e) and Agaricales (f-h). a) Frequency of Agaricomycetes species and OTUs sorted by decreasing frequency, and grouped by 3 species/OTUs recorded with both methods or only by either DNA or as fruitbody, y- axis indicates the number of sampling sites (of 130) in which a species or OTU was found, number of species/OTUs and number of observations are indicated for each group. b) Top 10 most frequent Agaricomycetes species recorded with either method. c) scatterplot of fruitbody-based frequency vs DNA frequency of the 428 Agaricomycetes species recorded by both methods. d) Species/OTU richness of the 130 sites as recorded with fruitbody or DNA for the Agaricomycetes 428 species recorded with both methods. e) Frequency of Agaricales species and OTUs sorted by decreasing frequency, and grouped by species/OTUs recorded with both methods or only by either DNA or as fruitbody, y-axis indicates the number of sampling sites (of 130) in which a species or OTU was found, number of species/OTUs and number of observations are indicated for each group. f) Top 10 most frequent Agaricales species recorded as with either method. g) scatterplot of fruitbody-based frequency vs DNA frequency of the 279 Agaricales species recorded by both methods. h) Species/OTU richness of the 130 sites as recorded with fruitbody or DNA for the 279 Agaricales species recorded with both methods. 4 a 1000 Species/OTUs 10 Basidiomycota b Ascomycota 1000 Rozellomycota Glomeromycota Species/OTUs 10 Chytridiomycota Mucoromycota Agaricomycetes Mortierellomycota Dothideomycetes Leotiomycetes c Sordariomycetes Olpidiomycota Eurotiomycetes 1000 Tremellomycetes Pezizomycetes Archaeorhizomycetes Entorrhizomycota Glomeromycetes Microbotryomycetes Mortierellomycetes Orbiliomycetes Entomophthoromycota Lecanoromycetes Archaeosporomycetes Mucoromycetes Species/OTUs Saccharomycetes Kickxellomycota Rhizophydiomycetes 10 Umbelopsidomycetes Pucciniomycetes Monoblepharomycota Dataset Geoglossomycetes Rozellomycotina_pp Cystobasidiomycetes OTU Endogonomycetes Zoopagomycota Fruitbody Exobasidiomycetes Entorrhizomycetes Olpidiomycetes Agaricales Spizellomycetes Calcarisporiellomycota Rhizophlyctidomycetes d Russulales Ustilaginomycetes GS27 Taphrinomycetes 100 Polyporales Kickxellomycetes Paraglomeromycetes Thelephorales Xylonomycetes Basidiobolomycetes Agaricostilbomycetes Cantharellales Dacrymycetes Zoopagomycetes Boletales Monoblepharidomycetes 10 Chytridiomycetes Species/OTUs Atractiellomycetes Sebacinales Malasseziomycetes Pezizomycotina_pp Hymenochaetales GS14 Dataset GS18 GS37 Atheliales Entomophthoromycetes OTU Geminibasidiomycetes Fruitbody 1 Incertae_sedis Trechisporales Calcarisporiellomycetes GS17 Cortinarius Auriculariales Mucoromycotina_pp Inocybe Tritirachiomycetes Entoloma SupplementaryMycena Figure 2. Taxonomic compositio Corticiales Psathyrella Coprinopsis Gomphales fruitbody data. a) NumberGalerina of species in each phylum. b) Number of species in each Hygrocybe Geastrales Clavaria Conocybe Hebeloma Amylocorticiales Coprinellus Amanita Phallales Pluteus Tricholoma Agaricomycetes_pp Agaricus Ramariopsis Dataset Clitocybe Gloeophyllales Clavulinopsis OTU Cuphophyllus Hysterangiales Gymnopus Fruitbody Lycoperdon Incertae_sedis Deconica Clitopilus Hemimycena Tremellodendropsidales Laccaria Typhula Pholiota Hypholoma Marasmius Parasola Flagelloscypha Lepiota Naucoria Panaeolus Gliophorus n of Hygrophorus Arrhenia Crepidotus 5 DNA metabarcodingAgrocybe Hymenogaster Dataset Lepista Cystoderma OTU Gymnopilus Hohenbuehelia Fruitbody Coprinus Mycenella Pholiotina Mallocybe Pseudobaeospora and class. c) Number of species in each order of Agaricomycetes. d) Number of species in each genus of Agaricales (restricted to genera with at least 5 species recorded). Y- axis is logarithmic. Agaricomycetes Agaricales r = 0.74, p < 2.2e−16 r = 0.8, p < 2.2e−16 y = 113 + 1.75 x y = 114 + 3.47 x 400 300 OTU richness (full data) OTU 200 100 0 40 80 120 160 0 40 80 120 160 OTU richness (restricted data) Supplementary figure 3. Correlation between OTU richness based on taxonomic subsets. Blue lines represent the linear regression of OTU richness of the taxonomically restricted dataset against OTU richness based on the full data, while the dotted line shows the identity line (x=y). Correlations are shown for the taxonomic subsets Agaricomycetes and Agaricales). 6 a Full data Agaricomycetes Agaricales 3+ 10 21 11 10 21 9 6 10 8 All taxa 1−2 28 21 6 24 22 5 32 18 2 0 22 11 27 12 41 13 3+ 7 16 7 7 15 7 3 6 6 db − restricted 1−2 25 21 10 21 24 7 24 19 4 Number of redlist species (fruitbody, all) Number of redlist species (fruitbody, 0 28 16 33 16 52 16 0 1−2 3+ 0 1−2 3+ 0 1−2 3+ Number of redlist species (DNA) b Full data Agaricomycetes Agaricales 3+ 5 9 9 4 10 8 4 10 8 All taxa 1−2 22 24 6 20 25 4 22 17 2 0 33 20 2 37 20 2 53 14 3+ 4 5 7 3 5 7 3 5 6 db − restricted 1−2 16 25 7 14 25 5 13 19 4 0 Number of redlist species (fruitbody, soil) Number of redlist species (fruitbody, 40 23 3 44 25 2 63 17 0 1−2 3+ 0 1−2 3+ 0 1−2 3+ Number of redlist species (DNA) Supplementary figure 4. Red-listed species. Number of red-listed species recorded at each site with either method. Upper panel shows data for all species, lower panel is restricted to red-listed species present in the DNA reference database (and thus possible to identify with both methods), a) shows data for the full fruitbody dataset, b) shows data for soil fruitbody data only. 7 Supplementary Tables Supplementary table 1. Number of species per phylum Phylum OTU Fruitbody total Soil frb. Non-soil frb. Ascomycota 3143 (39 %) 348 (20 %) 105 (10 %) 243 (36 %) Basidiomycota 3221 (40 %) 1398 (80 %) 958 (90 %) 440 (64 %) Calcarisporiellomycota 1 (0 %) - - - Chytridiomycota 163 (2 %) - - - Entomophthoromycota 15 (0 %) - - - Entorrhizomycota 22 (0 %) - - - Glomeromycota 267 (3 %) 3 (0 %) 3 (0 %) - Kickxellomycota 13 (0 %) - - - Monoblepharomycota 9 (0 %) - - - Mortierellomycota 144 (2 %) - - - Mucoromycota 144 (2 %) 2 (0 %) 1 (0 %) 1 (0 %) Olpidiomycota 23 (0 %) - - - Rozellomycota 322 (4 %) - - - unidentified 614 (8 %) - - - Zoopagomycota 9 (0 %) - - - 8 Supplementary table 2. Number of species per class Class OTU Fruitbody total