Ectomycorrhizal fungi in wood-pastures : Communities are determined by trees and soil properties, not by grazing

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/ Ectomycorrhizal fungi in wood-pastures : Communities are determined by trees and soil properties, not by grazing © 2018 Elsevier B.V. Accepted version (Final draft) Tervonen, Kaisa; Oldén, Anna; Halme, Panu Tervonen, K., Oldén, A., & Halme, P. (2019). Ectomycorrhizal fungi in wood-pastures : Communities are determined by trees and soil properties, not by grazing. Agriculture, Ecosystems and Environment, 269, 13-21. https://doi.org/10.1016/j.agee.2018.09.015 2019 Ectomycorrhizal fungi in wood-pastures: Communities are 1 determined by trees and soil properties, not by grazing 2 3 Authors: Kaisa Tervonena,b,c, Anna Oldéna,c, and Panu Halmea,c 4 5 a Department of Biological and Environmental Science, P.O. Box 35, FIN-40014 University 6 of Jyväskylä, Finland. 7 b Natural History Museum, P.O. Box 35, FIN-40014 University of Jyväskylä, Finland. 8 c School of Resource Wisdom, University of Jyväskylä, P.O. Box 35, FI-40014 University of 9 Jyväskylä, Finland. 10 Corresponding author: Kaisa Tervonen, [email protected], +358 50 5942478 11 12 Keywords: forest pastures, semi-natural, semi-open, traditional rural biotopes 13 Abstract 14 Traditional rural biotopes such as wood-pastures are species-rich environments that 15 have been created by low-intensity agriculture. Their amount has decreased 16 dramatically during the 20th century in whole Europe due to the intensification of 17 agriculture. Wood-pastures host some fungal species that prefer warm areas and are 18 adapted to semi-open conditions, but still very little is known about fungi in these 19 habitats. We studied how management, historical land-use intensity, present grazing 20 intensity, time since abandonment, and stand conditions affect the species richness 21 and community composition of ectomycorrhizal fungi. We surveyed fruit bodies on 22 three 10 m x 10 m study plots in 36 sites and repeated the surveys three times. Half of 23 the sites were currently unmanaged but had a grazing history. We measured soil pH, 24 soil moisture and the basal area of different tree species, and interviewed landowners 25 about grazing history. We found that the proportion of broadleaved trees, soil pH, and 26 soil moisture are the major drivers of the communities of ectomycorrhizal fungi in 27 boreal wood-pastures. Management or grazing intensity did not have significant 28 effects on fungal species richness, whereas historical land-use intensity seemed to 29 have a negative effect on species richness. To conclude, present stand conditions are 30 the most important factors to evaluate when planning the conservation of 31 ectomycorrhizal fungi living in semi-open forest habitats. 32 1. INTRODUCTION 33 Traditional rural biotopes are species-rich habitats that have been formed by low- 34 intensity agriculture. Wood-pastures are forested traditional rural biotopes that have 35 been grazed by domestic animals for up to hundreds of years. Long grazing history 36 has notably changed their vegetation structure. Moreover, patchy grazing pressure and 37 commonly performed selective logging have resulted in mosaic-like habitats where 38 open, semi-open and closed patches alternate (Garbarino et al., 2011; Schulman et al., 39 2008; Vainio et al., 2001; WallisDeVries et al., 1998). In the boreal zone some wood- 40 pastures have quite closed stand structure and they have also been called forest 41 pastures (sensu Takala et al., 2014). 42 The area of traditional rural biotopes has decreased steeply during the 20th 43 century in all European countries (Garbarino et al., 2011; Pykälä and Alanen, 2004). 44 Land abandonment and farming intensification are the main reasons why biodiversity 45 and the amount of these habitats have decreased. In Finland, traditional rural biotopes 46 and many species adapted to these habitats are now threatened. Less than 1 % of 47 wood-pastures remain compared to the area in the 1950’s, which was already much 48 lower than in the 1800’s (Rassi et al., 2010; Schulman et al., 2008). 49 Traditional rural biotopes host high biodiversity, which is proposed to be caused 50 by management, high habitat heterogeneity, an intermediate disturbance regime, long 51 grazing history, and variable soil properties (e.g. Benton et al., 2003; Cousins and 52 Eriksson, 2002; Oldén et al., 2016; Paltto et al., 2011; Pykälä, 2003, 2001; Saarinen 53 and Jantunen, 2005; Vujnovic et al., 2002). Currently grazed grasslands and wood- 54 pastures have been shown to have higher plant species richness than abandoned ones 55 (e.g. Dullinger et al., 2003; Oldén et al., 2016; Pykälä, 2003). Grazing benefits plant 56 species richness by removing vegetation and breaking the soil surface, and this gives 57 more space to weakly competitive species and thus increases species richness (Olff 58 and Ritchie, 1998; Pykälä, 2001). The amount of light and soil temperature are 59 increased by grazing (Olff and Ritchie, 1998; Pykälä, 2001), which improves the 60 growth conditions for many fungal species (Nitare and Sunhede, 1993). Grazing has 61 been proposed to benefit many fungal species (Jakobsson, 2005; Nauta and Jalink, 62 2001; Nitare and Sunhede, 1993). It has been found that while mowing increases 63 grassland fungal species richness (Griffith et al., 2012), grazing provides a wider 64 range of opportunities to fungal species than mowing (Nauta and Jalink, 2001). 65 Grazing increases heterogeneity by creating a mosaic of vegetation and thus it creates 66 various habitat patches for many species (Nauta and Jalink, 2001; Olff and Ritchie, 67 1998). According to the intermediate disturbance hypothesis it is expected that species 68 richness is highest at intermediate grazing intensity, where habitat heterogeneity is 69 also maximized (Grime, 1973; Milchunas et al., 1988; Mwendera et al., 1997; 70 Vujnovic et al., 2002). 71 Most species-rich traditional rural biotopes have been grazed or mowed with 72 traditional methods for a long time (Cousins and Eriksson, 2002; Myklestad and 73 Saetersdal, 2003; Pykälä, 2003). However, Oldén et al. (2016) did not find clear 74 effects of historical land-use intensity on plant species richness in wood-pastures. In 75 contrast, Lindborg and Eriksson (2004) found that historical landscape connectivity 76 has a strong effect on plant species richness in semi-natural grasslands. Many 77 characteristic grassland fungal species are dependent on continuous management that 78 has lasted for decades (Arnolds, 2001). 79 Soil properties affect species richness and communities in traditional rural 80 biotopes (Oldén et al., 2016; Raatikainen et al., 2007; Roem and Berendse, 2000). 81 Vascular plant and bryophyte species richness has been shown to increase with 82 increasing soil pH (Oldén et al., 2016; Roem and Berendse, 2000). Rousk et al. (2009) 83 found that on arable managed land fungal growth was maximized at pH 4.5 and 84 decreased both above and below that. Also, soil moisture has been shown to affect 85 fungal communities (Kaisermann et al., 2015; McHugh and Schwartz, 2016). It is 86 suggested that fungal populations are sensitive to soil moisture, and water treatments 87 decrease fungal diversity (Kaisermann et al., 2015; McHugh and Schwartz, 2016). 88 Many of the fungal studies are focused on macrofungi species in grasslands 89 (e.g. Arnolds, 2001; Nauta and Jalink, 2001; Öster, 2008). Fungal species from 90 ectomycorrhizal and coprophilous species groups cannot fruit in mowed grasslands, 91 but could have rich communities in grazed wood-pastures (Nauta and Jalink, 2001). 92 Juutilainen et al. (2016) found that the species richness of wood-inhabiting fungi in 93 wood-pastures was lower than in natural herb-rich forests, but wood-pastures hosted 94 some red-listed species and other unique species that were not found in the other 95 studied habitats. Only one study has focused on species of ectomycorrhizal fungi in 96 wooded meadows (Tedersoo et al., 2006). They found that communities of 97 mycorrhizal fungi in managed and forested old wooded meadows differ, but species 98 richness did not differ significantly. 99 The low number of studies on mycorrhizal fungi is alarming because they have 100 an important role in ecosystems (Boddy et al., 2008). It is known that at least 95% of 101 vascular plants have mycorrhizal associations (Moore et al., 2011). There are both 102 specific and non-specific associations between mycorrhizal fungi species and their 103 host trees (Molina et al., 1992; Moore et al., 2011). The reason for low number of 104 fungal studies might be that they are difficult to identify and that there are few 105 specialists who are able to conduct the studies (Boertmann, 1995; Watling, 1995). 106 In order to attain more knowledge on species of ectomycorrhizal fungi 107 inhabiting wood-pastures, we studied the effects of management, grazing history, 108 grazing intensity, time since abandonment and stand conditions on species richness 109 and communities of ectomycorrhizal fungi in wood-pastures dominated by 110 broadleaved, coniferous and mixed trees in the boreal zone. Based on earlier studies 111 we hypothesized that grazing increases fungal species richness and has an effect on 112 community composition, while species richness decreases after abandonment. We 113 also hypothesized that high historical land-use intensity increases species richness, 114 and that present intermediate grazing pressure creates highest species richness. Thus, 115 our main questions were: (1) Do grazed sites have higher species richness than 116 abandoned sites? (2) Does species richness increase with increasing historical land- 117 use intensity? (3) Does the species richness increase with grazing intensity or does it 118 peak with intermediate grazing intensity? (4) Does the species richness increase with 119 time since abandonment or does it peak after abandonment? (5) Is there a difference 120 between fungal communities among grazed and abandoned sites? and (6) How do the 121 present stand conditions affect species richness and community assembly in wood- 122 pastures? 123 2. MATERIALS AND METHODS 124 2.1. Study sites 125 We confined our study to the province of Central Finland to reduce biological and 126 geographical background variation in the data set. We studied 36 sites. The sites were 127 located in 30 farms so in each farm there were one or two study sites. 32 of the sites 128 were located in the Southern boreal and four in the Middle boreal vegetation zone 129 (Ahti et al., 1968) (Figure 1a). 130 We conducted our study in broadleaved (birch-dominated, Betula spp.), 131 coniferous (spruce-dominated, Picea abies), and mixed (with a coniferous- 132 broadleaved mixture of Picea abies, Pinus sylvestris, Betula spp., Populus tremula, 133 Alnus incana, Sorbus aucuparia or a subset of these) wood-pastures, and in each of 134 these three tree classes we included 12 sites. Half of the sites in each class were 135 currently grazed and the other had been abandoned (not grazed currently but had been 136 grazed during the recent history by domestic animals). Because our aim was to study 137 the effects of grazing, we aimed to reduce the variation caused by different stand 138 structure through selecting grazed and abandoned areas with similar tree densities 139 (mature trunks/ha). We could not control the variation in growth site type (which 140 varied from herb-rich to mesic heath) (see Hotanen et al., 2008) or the type of grazing 141 animals because of the small number of potential sites. More information on the study 142 sites is provided in Oldén et al. (2016). 143 Grazed sites were grazed yearly during the summer-autumn period by cattle, 144 horses or sheep. The grazing regime and intensity varies between sites because they 145 are managed by private farmers. In most farms, grazing started in late May or in June 146 and ended in September or when forage was depleted. Most farms use rotational 147 grazing where the animals are moved to a new pasture when forage is depleted. The 148 animals may graze the study site once or more times during the grazing season. 149 2.2. Data collection 150 At each study site we established three 10 m x 10 m square study plots based on the 151 dominant tree species and the density of mature trees. Among all study sites, the study 152 plots were at least 17 meters apart from each other. The whole selection procedure 153 was conducted without paying any attention to the ground level vegetation, and 154 during a season with almost no macrofungi producing fruit bodies (June-early July). 155 Thus, other species than trees did not affect the study plot selection. 156 Within the study plots, we recorded fungi growing on the ground and on the 157 surface of dead wood lying on the ground. We surveyed the ground very carefully by 158 pushing plants aside, but did not turn over dead wood pieces to avoid affecting the 159 fungal assemblage on the plots. We counted all the fruit bodies of stipitate 160 ectomycorrhizal macrofungi. 161 We repeated the surveys three times among all the study sites. Ten of the birch- 162 dominated study sites were surveyed three times during September-October in 2010. 163 The remaining two birch-dominated sites as well as all mixed and spruce-dominated 164 study sites were surveyed twice in August-September 2012 and once in September- 165 October 2013. We identified fungi to species level at the site when possible, but 166 collected specimens for microscopic identification if needed (altogether 1100 167 specimens). The abundance of each species in a plot was estimated by counting the 168 number of fruit bodies. With fungi it is difficult to define which fruit bodies belong to 169 one individual (Dahlberg and Mueller, 2011), so the fruit body count does not directly 170 reflect the number of individuals on the plot, but is more like a surrogate of the 171 abundance of the species. While counting the fruit bodies we removed them from the 172 ground to avoid counting the same fruit bodies during the next survey. 173 We separated mycorrhizal species from other species based on the ecological 174 information provided in Knudsen and Vesterholt (2012), Kotiranta et al. (2009), and 175 Kytövuori et al. (2005). Species that were reported in the literature to use both 176 mycorrhizal and saprotrophic strategy (Hydnum repandum, Hydnum rufescens coll., 177 etc.) were excluded except for Paxillus involutus that was reported to be mainly 178 mycorrhizal. We included only species level data in the analyses. The nomenclature 179 of agarics and boletoids follows Knudsen and Vesterholt (2012) and Aphylloporales 180 Kotiranta et al. (2009). A few exceptions in the nomenclature are indicated by 181 showing the author names in the species list (Table 1 in the Appendix B). These 182 exceptions are situations where Nordic taxonomists currently disagree with the 183 references that we used for nomenclature. The voucher specimens are preserved in the 184 herbarium of the National History Museum of University of Jyväskylä (JYV) and in 185 the personal collection of Kaisa Tervonen. 186 2.3. Background variables 187 Measuring historical land-use intensity proved to be complicated in the study area in 188 Central Finland. It was not possible to reliably measure the age of each farm, because 189 historical church records or cadastres do not specify the locations of the farms and 190 properties. Agricultural records have only been collected from the 1920's onwards. In 191 addition, in the 1800's free cattle grazing outside of fenced fields meant that cattle 192 from different farms grazed in the forests surrounding villages (Jäntti, 1945). Thus, 193 we created a surrogate for the historical land-use intensity by counting the number of 194 surrounding farms (within one kilometer buffer zone around each site) in old cadastral 195 For the three-dimensional NMDS ordinations the final stress values were 0.150 343 for all sites, 0.126 for grazed sites, and 0.104 for abandoned sites. The results for axes 344 1 and 2 are shown in Figure 3. Results for axis 3 are shown in Figure 1 in the 345 Appendix A and they only emphasize the effect of the proportion of broadleaved 346 trees. 347 4. DISCUSSION 348 4.1. Grazing-related variables did not have clear effects on the fungal 349 communities 350 Historical land-use intensity (historical number of farms surrounding the site within 351 1km) was the most important factor affecting species richness of ectomycorrhizal 352 fungi, but it did not impact community composition. High historical land-use intensity 353 had a significant negative effect on species richness among all sites, grazed sites, and 354 abandoned sites. This is surprising because one could expect that the biodiversity of 355 traditional rural biotopes in general would increase with historical land-use intensity. 356 For example, Lindborg and Eriksson (2004) found that historical landscape 357 connectivity has a strong positive effect on the present species richness of plants in 358 semi-natural grasslands. On the other hand, in our study of these same wood-pastures, 359 we did not find significant impacts of historical land-use intensity on the species 360 richness of either vascular plants or bryophytes (Oldén et al., 2016). One explanation 361 is that many of the species of boreal wood-pastures are primarily forest species 362 instead of grassland species. It seems possible that among ectomycorrhizal fungi there 363 are more species that suffer from human impacts than those that benefit from them. In 364 addition, grazing may not be the most important historical factor determining current 365 fungal assemblages, but instead other practices related to forestry and agriculture may 366 have negative impacts on local fungal diversity. Finally, we assumed that historical 367 land-use intensity correlates with historical grazing intensity, but it may not correlate 368 with the overall length of grazing history or the grazing intensity during the recent 369 decades. 370 Management situation had no effect on fungal species richness, which is in 371 contrast to our hypothesis. Management did not have a clear effect on community 372 composition either. It seems that the communities of ectomycorrhizal fungi are not 373 affected by grazing, although some individual species may respond to it. Instead, 374 vascular plants and bryophytes had higher species richness in the currently grazed 375 sites of this same setup (Oldén et al., 2016), indicating that grazing does have 376 ecological impacts in these boreal wood-pastures. 377 Present grazing intensity did not have any significant effect on species richness. 378 Thus, our result does not support our hypothesis that species richness would be 379 highest at intermediate grazing intensity. With vascular plants there are several studies 380 that show highest species richness with intermediate grazing pressure (Mwendera et 381 al., 1997; Vujnovic et al., 2002), also in these same sites (Oldén et al., 2016). 382 However, data on the stocking rates of grazers in these sites was not available, and 383 our one-time estimation of grazing intensity may not be a comprehensive estimate of 384 all the effects that grazers have on fungi throughout the grazing season and different 385 years. In addition, our results might be affected by the consumption of fruit bodies by 386 the grazers during the study. It is known, and we also noticed ourselves, that the 387 grazers eat fruit bodies (Warren and Mysterud, 1991). It is possible that in sites with 388 high grazing intensity the grazers consumed more fruit bodies, and thus fewer species 389 were observed. However, the grazers may also purposefully seek for some fruit bodies 390 over other food items (Bjugstad and Dalrymple, 1968), and in that case grazing 391 intensity does not correlate with the number of consumed fruit bodies. Grazing 392 intensity had no clear effects on fungal community composition either. Together with 393 the fact that management situation did not affect fungal species richness or 394 community composition, it is clear that trees and soil properties impact fungal 395 communities much more than grazing. 396 Time since abandonment had a positive and also slightly humped effect on 397 species richness, but it had no clear effect on community composition. Thus, 398 according to our results species richness increases slightly with time since 399 abandonment, which is opposite to our hypothesis. Many ectomycorrhizal species 400 may benefit from the increasing number of young trees during the first decades after 401 abandonment, especially if the young trees increase the number of tree species that are 402 available for mycorrhizal symbiosis. In time an abandoned wood-pasture develops 403 towards an old-growth forest, which can offer habitats for species that are dependent 404 on them (Bonsdorff et al., 2014). However, we note that more studies are needed on 405 this topic, especially because the positive effect in our data can be caused by a few 406 long-ago abandoned sites that are biodiversity hotspots due to other properties than 407 grazing. 408 4.2. Soil moisture affects species richness and community composition 409 Our result reveals that soil moisture affects the species richness of ectomycorrhizal 410 fungi. It is also one of the main drivers of ectomycorrhizal fungi community 411 composition. We found that high soil moisture in wood-pastures results in low species 412 richness of ectomycorrhizal fungi. However, the effect was significant only among 413 abandoned sites. Our recent study revealed that bryophyte species richness increases 414 with soil moisture in wood-pastures, but vascular plant species richness does not show 415 any clear responses (Oldén et al., 2016). Thus, different species groups respond 416 differently to soil moisture in wood-pastures. 417 It is clear that fungal species need moisture to grow, but one could think that the 418 mycelium of mycorrhizal fungi cannot grow properly if the soil is too moist. 419 However, Kennedy and Peay (2007) found that with increasing soil moisture plant 420 species with ectomycorrhizal associations had greater shoot biomass and 421 photosynthesis than non-mycorrhizal plants. McHugh and Schwartz (2016) instead 422 showed that water treatment decreased fungal diversity. Thus, our result supports the 423 observation of McHugh and Schwartz (2016). 424 4.3. The proportion of broadleaved trees is the main driver of community 425 composition 426 The proportion of broadleaved trees had the strongest effect on the community 427 composition of ectomycorrhizal fungi. Our result was expected, because it is known 428 that there are specific and non-specific associations between mycorrhizal fungi 429 species and their host trees (Molina et al., 1992; Moore et al., 2011). For example, the 430 fungal communities in spruce-dominated sites differed strongly from other sites, 431 which is reasonable because spruce was often the only tree species present in the 432 plots. 433 Surprisingly, tree species richness did not have a significant effect on species 434 richness of ectomycorrhizal fungi. Since many ectomycorrhizal species are 435 specialized to certain hosts, increasing tree species richness should increase 436 ectomycorrhizal species richness, through the higher number of suitable hosts for 437 different species. One reason why we did not find a significant effect might be that the 438 difference cannot be detected in such a small scale due to high overall beta diversity 439 of fungal communities on small spatial scales (Abrego et al., 2014). The difference 440 might be discovered on a larger scale. 441 4.4. Soil pH affects community composition 442 Soil pH had a strong effect on community composition of ectomycorrhizal fungi, but 443 it did not impact species richness. Soil pH correlated strongly with the proportion of 444 broadleaved trees: Most of the sites with high soil pH (max 4.9) are birch-dominated 445 herb-rich forests, while most of the low-pH sites (min 3.1) are heath forests 446 dominated by spruces or mixed trees. 447 Vascular plant and bryophyte species richness has been shown to increase with 448 increasing soil pH (Oldén et al., 2016; Roem and Berendse, 2000). According to 449 Rousk et al. (2009) fungal growth was maximized at pH 4.5 and decreased both above 450 and below that. On the other hand, fungal biomass was highest at pH 6 but decreased 451 with both increasing and decreasing pH (Rousk et al., 2009). Thus it could be 452 assumed that in our quite acidic sites species richness would increase with soil pH, 453 but we did not find significant effects on fungal species richness. 454 4.5. Survey year affected our results 455 Fungal surveys were conducted in 10 of the 12 birch-dominated sites on three visits 456 during 2010. Two birch-dominated sites and all mixed and spruce-dominated sites 457 were visited twice during 2012 and once during 2013. Autumn 2010 was quite dry 458 and this could affect our results. In 2010 the numbers of detected fruit bodies were 459 quite similar at the first and second survey visits compared to years 2012-2013, but 460 much lower at the third survey visit. Another source of bias is that many fungal 461 species do not produce fruit bodies every year (Straatsma et al., 2001), and thus a 462 higher species richness can be observed in sites that have been studied during two 463 different years, even though the number of survey visits is the same. 464 These study design problems have somewhat affected our results. Thus, we 465 cannot really be sure how strongly the communities of birch-dominated sites differ 466 from others, and how much the survey years have affected it. However, the effect of 467 the year should be small in the Bioenv-analyses where we used Chao’s dissimilarity 468 index, which should take into account the unseen species (Chao et al., 2005; Oksanen 469 et al., 2015). We also corrected for the effect of the survey year in the General Linear 470 Mixed Models by using inventory time period (2010 or 2012+2013) as a random 471 effect. 472 It is also known that studies that are only based on fruit bodies do not reveal the 473 whole fungal community, because of the species that do not produce fruit bodies 474 every year (Abrego et al., 2016; Ovaskainen et al., 2013; van der Linde et al., 2012). 475 However, we argue that even a quite large proportion of undetected species should not 476 mask the potential effects of management situation, for example. 477 5. CONCLUSIONS 478 Communities of ectomycorrhizal fungi in wood-pastures are determined by soil 479 properties and tree species composition. Based on our results, grazing-related 480 variables do not impact the communities of ectomycorrhizal fungi in boreal wood- 481 pastures, but (currently grazed and abandoned) wood-pastures may still differ in their 482 species composition from the forests that have no grazing history. Decisions on the 483 management and conservation of wood-pastures should be based on other species 484 groups that respond more clearly to management (such as vascular plants and 485 bryophytes, see Oldén et al. 2016). However, some ectomycorrhizal species or the 486 communities of saprotrophic fungi may still respond to grazing in wood-pastures. 487 More studies are needed to reveal these subjects. 488 Acknowledgements 489 We thank all the landowners for giving us permission to the field work. We also thank 490 Mika Toivonen, Anni Rintoo, Lotta Sundström, Kaisa Mustonen, Paula Salonen, and 491 Asta Vaso for field assistance. 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Ecology 85, 1840–1845. doi:10.1890/04-0367 581 McHugh, T.A., Schwartz, E., 2016. A watering manipulation in a semiarid grassland induced 582 changes in fungal but not bacterial community composition. Pedobiologia (Jena). 59, 583 121–127. doi:10.1016/j.pedobi.2016.04.003 584 691 692 Figure 2. Responses of ectomycorrhizal fungi species richness to inventory time period (ITP) 693 among a) all sites, b) grazed sites, c) abandoned sites, d) management among all sites, 694 e) the number of farms surrounding the site within 1km in the 1850s-60s (historical 695 land-use intensity), f) soil pH, g) soil moisture (% content from the ground), h) tree 696 species richness, i) grazing intensity among grazed sites (% of clipped shoots), and j) 697 time since abandonment (years) for abandoned sites. The fitted linear and quadratic 698 curves represent significant or nearly significant effects from the GLMM analyses. 699 700 701 Figure 3. Nonmetric Multidimensional Scaling (NMDS) for the community structure of ectomycorrhizal fungi species among a) all sites, b) grazed sites, and 702 c) abandoned sites with axes 1 and 2. Analyses were done with Chao’s dissimilarity index. For the categorical variables inventory time period (ITP1 in 703 2010, ITP0 in 2012-2013) and management (M1 for grazed sites, and M0 for abandoned sites) the location represents the average location of sites in 704 that category. The arrows represent the direction and strength of the a posteriori correlations between the site locations and the continuous 705 environmental variables: the historical land-use intensity (Farms), soil moisture, soil pH, the proportion of broadleaved trees (Broa), grazing intensity 706 on grazed sites, and time since abandonment on abandoned sites. Symbol size represents the proportion of broadleaved trees. 707 Appendix A for Tervonen et al.: Ectomycorrhizal fungi in wood-pastures: Communities are determined by trees and soil properties, not by grazing Authors: Kaisa Tervonen, Anna Oldén, and Panu Halme 1. Detailed information about GLMM 2. NMDS with Bray-Curtis dissimilarity index Table 1. Correlations between the environmental variables. Table 2. Results from the Bioenv analyses for mycorrhizal fungi species. Figure 1. NMDS for the community structure of mycorrhizal fungi species among all, grazed, and abandoned sites with Chao. (Axes 1 and 3) Figure 2. NMDS for the community structure of mycorrhizal fungi species among all, grazed, and abandoned sites with Bray-Curtis. (Axes 1 and 2) Figure 3. NMDS for the community structure of mycorrhizal fungi species among all, grazed, and abandoned sites with Bray-Curtis. (Axes 1 and 3) 1. Detailed information about GLMM In the General Linear Mixed Models (GLMM) we compared models by using as family Poisson or Negative Binomial and decided the best model based on Akaike's Information Criterion values. The selected family was Negative Binomial. We used “bobyqa” as optimizer in the models. We set iteration number to 100 000 with function “glmerControl”. One of the models for grazed site model had “Hessian warning”. We double-checked the results with R’s convergence -help five step instructions to see that with many different optimizers the estimates for the models were similar. Therefore we could trust our results. 2. NMDS with Bray-Curtis dissimilarity index When analyzing the data with Bray-Curtis dissimilarity index the correlations from Bioenv-analyses were almost the same, but it seems that with Chaos’s index the analysis finds a stronger effect of the proportion of broadleaved trees instead of pH (Table 2 in the Appendix). The NMDS ordinations with Chao’s and Bray-Curtis indexes are somewhat similar (Bray-Curtis NMDS ordinations in the Appendix Figure 2 and 3). Table 1. Correlations between the environmental variables. All sites Farms Moisture pH Broadleaved Moisture -0.170 pH -0.024 -0.356 * Broadleaved 0.024 0.023 0.554 *** TreesSR -0.045 -0.182 0.525 ** 0.466 ** Grazed sites Farms Moisture pH Broadleaved TreesSR Moisture -0.306 pH 0.183 -0.560 * Broadleaved -0.016 -0.127 0.581 * TreesSR 0.010 -0.339 0.592 ** 0.287 Grazing -0.010 0.034 0.104 0.113 -0.060 Abandoned sites Abandonment Farms Moisture pH Broadleaved Farms -0.176 Moisture 0.502 * -0.166 pH 0.095 -0.288 -0.137 Broadleaved 0.469 * 0.019 0.152 0.583 * TreesSR 0.151 -0.034 0.067 0.682 ** 0.717 *** ***=p<0.001, **=p<0.01, *=p<0.05 Table 2. Results from the Bioenv analyses of variables that affect mycorrhizal fungi community. Results are given for both Chao’s and Bray-Curtis dissimilarity indexes. Spearman rank correlation was used in the analyses. Inventory time period (DITP: 2010 or 2012 and 2013) and management (Dmana: grazed or abandoned sites) are set as a dummy variables. The proportion of broadleaved trees are represented as “Broadleaved” and the historical land-use intensity as “Farms”. Chao All sites Size Variables Correlation 1 Broadleaved 0.314 2 pH, Broadleaved 0.400 3 pH, Moisture, Broadleaved 0.432 4 DITP, pH, Moisture, Broadleaved 0.365 5 Dmana, DITP, pH, Moisture, Broadleaved 0.338 6 Dmana, DITP, pH, Moisture, Broadleaved, Farms 0.311 Grazed sites Size Variables Correlation 1 Moisture 0.288 2 Moisture, Broadleaved 0.427 3 pH, Moisture, Broadleaved 0.424 4 pH, Moisture, Broadleaved, Grazing 0.389 5 pH, Moisture, Broadleaved, Farms, Grazing 0.353 6 DITP, pH, Moisture, Broadleaved, Farms, Grazing 0.278 Abandoned sites Size Variables Correlation 1 Broadleaved 0.417 2 pH, Broadleaved 0.505 3 pH, Moisture, Broadleaved 0.483 4 DITP, pH, Moisture, Broadleaved 0.438 5 DITP, pH, Moisture, Broadleaved, Farms 0.372 6 DITP, pH, Moisture, Broadleaved, Farms, Abandonment 0.318 Bray-Curtis All sites Size Variables Correlation 1 pH 0.313 2 pH, Broadleaved 0.397 3 pH, Moisture, Broadleaved 0.441 4 pH, Moisture, Broadleaved, Farms 0.375 5 Dmana, DITP, pH, Moisture, Broadleaved 0.334 6 Dmana, DITP, pH, Moisture, Broadleaved, Farms 0.309 Grazed sites Size Variables Correlation 1 Moisture 0.282 2 Moisture, Broadleaved 0.361 3 pH, Moisture, Broadleaved 0.360 4 pH, Moisture, Broadleaved, Grazing 0.344 5 pH, Moisture, Broadleaved, Farms, Grazing 0.311 6 DITP, pH, Moisture, Broadleaved, Farms, Grazing 0.225 Abandoned sites Size Variables Correlation 1 pH 0.414 2 pH, Broadleaved 0.541 3 pH, Moisture, Broadleaved 0.526 4 DITP, pH, Moisture, Broadleaved 0.473 5 DITP, pH, Moisture, Broadleaved, Farms 0.397 6 DITP, pH, Moisture, Broadleaved, Farms, Abandonment 0.329 Figure 1. Nonmetric Multidimensional Scaling (NMDS) for the community structure of mycorrhizal fungi species among a) all sites, b) grazed sites, and c) abandoned sites with axes 1 and 3. Analyses were done with Chao’s dissimilarity index. For the categorical variables inventory time period (ITP1 in 2010, ITP0 in 2012-2013) and management (M1 for grazed sites, and M0 for abandoned sites) the location represents the average location of sites in that category. The arrows represent the direction and strength of the a posteriori correlations between the site locations and the continuous environmental variables: the historical land-use intensity (Farms), soil moisture, soil pH, the proportion of broadleaved trees (Broa), grazing intensity on grazed sites, and time since abandonment on abandoned sites. Symbol size represents the proportion of broadleaved trees. Figure 2. Nonmetric Multidimensional Scaling (NMDS) for the community structure of mycorrhizal fungi species among a) all sites, b) grazed sites, and c) abandoned sites with axes 1 and 2. Analyses were done with Bray-Curtis dissimilarity index. For the categorical variables inventory time period (ITP1 in 2010, ITP0 in 2012-2013) and management (M1 for grazed sites, and M0 for abandoned sites) the location represents the average location of sites in that category. The arrows represent the direction and strength of the a posteriori correlations between the site locations and the continuous environmental variables: the historical land-use intensity (Farms), soil moisture, soil pH, the proportion of broadleaved trees (Broa), grazing intensity on grazed sites, and time since abandonment on abandoned sites. Symbol size represents the proportion of broadleaved trees. Russula xerampelina coll. 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