ArticlePDF Available

Isotopic evidence of biotrophy and unusual nitrogen nutrition in soil-dwelling Hygrophoraceae: Hygrophoraceae 13C/15N natural abundance

Abstract and Figures

Several lines of evidence suggest that the agaricoid, non‐ectomycorrhizal members of the family Hygrophoraceae (waxcaps) are biotrophic with unusual nitrogen nutrition. However, methods for the axenic culture and lab‐based study of these organisms remain to be developed, so our current knowledge is limited to field‐based investigations. Addition of nitrogen, lime or organophosphate pesticide at an experimental field site (Sourhope) suppressed fruiting of waxcap basidiocarps. Furthermore, stable isotope natural abundance in basidiocarps were unusually high in ¹⁵N and low in ¹³C, the latter consistent with mycorrhizal nutritional status. Similar patterns were found in waxcap basidiocarps from diverse habitats across four continents. Additional data from ¹⁴C analysis of basidiocarps and ¹³C pulse label experiments suggest that these fungi are not saprotrophs but rather biotrophic endophytes, and possibly mycorrhizal. The consistently high but variable δ¹⁵N values (10‐20‰) of basidiocarps further indicate that N acquisition or processing differ from other fungi; we suggest that N may be derived from acquisition of N via soil fauna high in the food chain. This article is protected by copyright. All rights reserved.
Content may be subject to copyright.
For Peer Review Only
Isotopic evidence of biotrophy and unusual nitrogen
nutrition in soil-dwelling Hygrophoraceae
Journal:
Environmental Microbiology and Environmental Microbiology Reports
Manuscript ID
EMI-2018-0658.R2
Journal:
Environmental Microbiology
Manuscript Type:
EMI - Research article
Date Submitted by the Author:
n/a
Complete List of Authors:
Halbwachs, Hans; Bavarian Forest National Park
Easton, Gary; Aberystwyth University, IBERS
Bol, Roland; Agrosphere (IBG-3). Forschungszentrum Jülich GmbH
Hobbie, Erik; University of New Hampshire, EOS
Garnett, Mark; NERC Radiocarbon Facility
persoh, Derek; Ruhr-Universität Bochum, Department of Geobotany
Dixon, Liz; Rothamsted Research, Sustainable Soils and Grassland Systems
Ostle, Nick; Lancaster University, Lancaster Environmental Centre
Karasch, Peter
Griffith, Gareth; Aberystwyth University, IBERS
Keywords:
Biotrophy, Mutualism, Stable isotopes, 15N/13C, Nitrogen uptake,
Invertebrates, Waxcap, Mycorrhiza
Wiley-Blackwell and Society for Applied Microbiology
1
Isotopic evidence of biotrophy and unusual nitrogen nutrition in soil-dwelling
1
Hygrophoraceae
2
3
Hans Halbwachsa, Gary L. Eastonb, Roland Bolc, Erik A. Hobbied, Mark H Garnette, Derek Peršohf, Liz
4
Dixong, Nick Ostleh, Peter Karaschi & Gareth W. Griffithb*
5
6
a Bavarian Forest National Park, Freyunger Str. 2, 94481 Grafenau, Germany
7
b Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Adeilad Cledwyn,
8
Penglais, Aberystwyth, Ceredigion SY23 3DD, WALES
9
c Institute of Bio- and Geosciences, Agrosphere (IBG-3). Forschungszentrum Jülich GmbH, Wilhelm-
10
Johnen-Straße. 52428 Jülich, Germany
11
d Earth Systems Research Center, Morse Hall, University of New Hampshire, 8 College Road, Durham,
12
NH 03824-3525 USA
13
e NERC Radiocarbon Facility, Scottish Enterprise Technology Park, Rankine Avenue, East Kilbride,
14
Scotland, G75 OQF
15
f Ruhr-Universität Bochum, Department of Geobotany, Gebäude ND 03/170, Universitätsstraße 150,
16
44780 Bochum, Germany
17
g Sustainable Soils and Grassland Systems, Rothamsted Research. North Wyke, Okehampton, Devon
18
EX20 2SB
19
h Lancaster Environment Centre, Lancaster University, Lancaster UK LA1 4YQ
20
i German Mycological Society, Kirchl 78. D-94545 Hohenau, Germany
21
22
* Corresponding author: gwg@aber.ac.uk (0044-1970-622325)
23
24
Key words: Waxcap; Biotrophy; Mutualism; Stable isotopes; 15N; Nitrogen uptake; Invertebrates;
25
Mycorrhiza
26
27
28
2
Originality-Significance Statement
29
This manuscript presents the results of experiments to elucidate the nutritional biology of soil-
30
dwelling Hygrophoraceae (waxcaps). This major group of agaricoid fungi is primarily known from
31
undisturbed grassland habitats where they are dominant components of the soil fungal community,
32
more abundant than arbuscular mycorrhizal fungi (Detheridge et al., 2018). However, their resistance
33
to axenic culture has hindered study of their biology, so they are often (by default) considered as
34
saprotrophs. Here we combine field surveys assessing the effects of agricultural manipulations on
35
fruiting with isotopic (13C, 15N, and 14C) analyses to show that they are biotrophs, potentially
36
forming mycorrhizal associations with host plants. Whilst the 13C and15N patterns of their
37
basidiocarps resemble ectomycorrhizal fungi more than saprotrophs, their consistently high δ15N
38
values also indicate an unusual mode of nitrogen nutrition, for which we suggest some possible
39
mechanisms
40
41
42
Abstract
43
Several lines of evidence suggest that the agaricoid, non-ectomycorrhizal members of the family
44
Hygrophoraceae (waxcaps) are biotrophic with unusual nitrogen nutrition. However, methods for the
45
axenic culture and lab-based study of these organisms remain to be developed, so our current
46
knowledge is limited to field-based investigations. Addition of nitrogen, lime or organophosphate
47
pesticide at an experimental field site (Sourhope) suppressed fruiting of waxcap basidiocarps.
48
Furthermore, stable isotope natural abundance in basidiocarps were unusually high in 15N and low in
49
13C, the latter consistent with mycorrhizal nutritional status. Similar patterns were found in waxcap
50
basidiocarps from diverse habitats across four continents. Additional data from 14C analysis of
51
basidiocarps and 13C pulse label experiments suggest that these fungi are not saprotrophs but rather
52
biotrophic endophytes, and possibly mycorrhizal. The consistently high but variable δ15N values (10-
53
3
20‰) of basidiocarps further indicate that N acquisition or processing differ from other fungi; we
54
suggest that N may be derived from acquisition of N via soil fauna high in the food chain.
55
56
1. Introduction
57
Members of the agaric family Hygrophoraceae exhibit diverse nutritional strategies, ranging from
58
lichenised forms associated with both green algae (Lichenomphalia spp.) and cyanobacteria
59
(Dictyonema spp.) to ectomycorrhizal (ECM) taxa (Hygrophorus spp.) (Agerer, 2006; Seitzman et al.,
60
2011; Lodge et al., 2014). However, the nutritional strategies of several genera within this family are
61
less certain, notably the soil-dwelling taxa Hygrocybe, Cuphophyllus, Gliophorus, Humidicutis,
62
Chromosera, Neohygrocybe and Porpolomopsis (Griffith et al., 2002; Halbwachs et al., 2013a; Lodge
63
et al., 2014). Though associated with undisturbed grasslands in Europe (Griffith et al., 2004;
64
Halbwachs et al., 2013a), at a global level they are more commonly associated with forest habitats
65
but not with tree species that form ectomycorrhizas (Seitzman et al., 2011; Lodge et al., 2014). Here
66
we use the term ‘waxcap’ to refer to soil-dwelling Hygrophoraceae not known to be ectomycorrhizal
67
or lichenised (Boertmann, 2010).
68
69
In the European context, interest in the Hygrophoraceae stems mainly from their high conservation
70
value, since they only fruit (often abundantly) in undisturbed grasslands. Waxcap basidiocarps are
71
much rarer or absent in grasslands subject to agricultural intensification so these fungi have suffered
72
large-scale habitat loss habitats, especially in lowland areas. Recent investigation of grassland fungi
73
using DNA metabarcoding has shown that Hygrophoraceae are amongst the most abundant fungi in
74
undisturbed grassland soils (Detheridge et al., 2018). However, the effects of fertiliser or lime
75
additions, whilst widely reported to inhibit waxcap fruiting, have not been rigorously quantified.
76
Thus, a better understanding of their nutritional requirements and ecological interactions would be
77
beneficial to halt further losses.
78
79
Waxcaps are generally referred to as 'saprotrophs' in the mycological literature (Keizer, 1993;
80
Tedersoo et al., 2010) and they are currently listed as such in FUNguild (Nguyen et al., 2015)
81
4
(github.com/UMNFuN/FUNGuild), most likely by default (i.e. lack of published evidence to the
82
contrary). However, several lines of evidence now suggest that they are biotrophic, similar to their
83
lichenised and ectomycorrhizal relatives in family Hygrophoraceae, amongst which they are
84
interspersed (Seitzman et al., 2011; Lodge et al., 2014). They have fastidious nutritional
85
requirements, as evidenced by their recalcitrance to axenic culture and the failure of their spores to
86
germinate on various agar media (Beisenherz, 2000; Griffith and Roderick, 2008; Roderick, 2009).
87
They can colonise the root hairs of grassland plants and their DNA has been detected within plant
88
tissues (Halbwachs et al., 2013b; Tello et al., 2014). Furthermore, their fruiting is inhibited by the
89
killing of associated vegetation using herbicides (Griffith et al., 2014).
90
91
Evidence is accumulating that the diversity of mycorrhizal or biotrophic relationships between fungi
92
and plants is greater than previously expected, now including diverse members of Sebacinales,
93
Helotiales, Ceratobasidiales and others (Veldre et al., 2013; Behie and Bidochka, 2014; Weiss et al.,
94
2016). For example, Austroboletus forms mycorrhizal associations with Eucalyptus without root
95
penetration (Kariman et al., 2014) and Cortinarius spp. associate with Carex and other non-shrubby
96
hosts (Harrington and Mitchell, 2002). There is much discussion about whether all these should be
97
considered mycorrhizal (Heijden et al., 2015), since for the great majority the reciprocal exchange of
98
nutrients has yet to be demonstrated. Wilson (1995), in attempting to clarify the status of root-
99
associated fungi, suggested that the term 'endophyte' should be applied to fungi for which
100
morphological root adaptations and reciprocal nutrient transfer had not been demonstrated. Since
101
the exact nature of the biotrophy of waxcap fungi is unclear, here we use the term endophyte (sensu
102
Wilson), where endophytism acts as a symbiotic "waiting room" (as coined by (Selosse and Martos,
103
2014) from which tighter mycorrhizal mutualism may evolve. Indeed, paraphyletic evolution of the
104
ectomycorrhizal habit has occurred within the Hygrophoraceae, with members of Hygrophorus
105
forming typical ectomycorrhizas with both deciduous and coniferous hosts (Lodge et al., 2014). This,
106
and the phylogenetic position of waxcaps within Hygrophoraceae (Lodge et al., 2014), raises the
107
possibility that the common ancestors of Hygrophorus and waxcaps were endophytic and that the
108
former evolved the ectomycorrhizal habit following association with trees.
109
5
110
Stable isotope signatures of carbon and nitrogen (expressed as d13C and d15N) have been used to
111
examine the nutritional strategies, trophic position and feeding behaviours of diverse organisms
112
(Boecklen et al., 2011). Waxcap basidiocarps exhibit distinctive C and N isotopic natural abundance
113
patterns, being depleted in 13C and enriched in 15N relative to confirmed saprotrophic macrofungi
114
from the same habitats (Griffith et al., 2002; Seitzman et al., 2011). Ectomycorrhizal (ECM)
115
basidiomycetes also exhibit 13C depletion and 15N enrichment relative to saprotrophic species (Taylor
116
et al., 1997; Kohzu et al., 1999; Taylor et al., 2003).
117
118
For carbon isotopes, such patterns are explained by 13C partitioning within woody plants, with 13C
119
preferentially partitioned to plant cellulose relative to soluble sugars, and these representing the
120
primary carbon source for saprotrophic fungi and ECM fungi respectively (Kohzu et al., 1999). For
121
nitrogen, 14N acquired by ECM fungi from sources in the soil is preferentially transferred to hosts,
122
leading to 15N enrichment within the fungal mycelia (Hobbie and Högberg, 2012). However, 15N
123
enrichment in basidiocarps varies widely amongst the diverse groups of ECM fungi. It is also
124
noteworthy that in cases where plants obviously and heavily rely on ECM fungi as nitrogen source
125
(full and partial mycoheterotrophs among the Orchidaceae and Ericaceae), these exhibit 15N
126
enrichment rather than depletion (Gebauer and Meyer, 2003; Zimmer et al., 2007). Whilst internal
127
isotopic fractionation can partially explain the observed differences in δ13C and δ15N profiles of
128
basidiocarp tissues, isotopic analyses can additionally provide insight into which sources of C and N
129
are being accessed (Gebauer and Taylor, 1999; Preiss and Gebauer, 2008). Thus we hypothesise that
130
fungi whose tissues are particularly high in δ15N derive N from sources which are themselves high in
131
15N. This was shown using paired natural abundance and 15N tracer measurements on
132
ectomycorrhizal fungi in a pine forest (Hobbie et al., 2014).
133
134
An additional method for determining the sources from which macrofungi derive organic carbon is
135
via radiocarbon analysis. This method relies on the spike of atmospheric 14CO2 resulting from the
136
hydrogen bomb tests of 1957-1963 which declined gradually over the following 50 years to near pre-
137
6
bomb levels due to photosynthetic uptake by plants. The steep annual decline in atmospheric 14CO2
138
allows accurate estimation of the date at which the carbon present in biological tissues had been
139
fixed by photosynthesis by calibration with 14C in atmospheric CO2 and plant materials. Hobbie et al.
140
(2002) and Chapela et al. (2001) have previously used this radiocarbon approach to test the
141
mycorrhizal status of ectomycorrhizal fungi, finding that the 14C signature of accepted
142
ectomycorrhizal species was similar to that found in atmospheric CO2 0—2 yrs previously, whereas
143
the 14C signature of saprotrophic species matched that of atmospheric CO2 from 2—50 years prior to
144
sample collection.
145
146
Apart from the utility of greater understanding of the nutritional biology of waxcaps in conservation
147
efforts, their unusual patterns of 15N enrichment (Griffith et al., 2002; Seitzman et al., 2011) suggests
148
a hitherto unknown trophic strategy which merits further investigation. The negative effects of
149
agricultural soil amendments on waxcap fruiting may be linked to changes in interactions with host
150
plants or other members of the soil biota. In the present study, we present data from fruitbody
151
surveys at a grassland experimental site where several agricultural treatments were applied,
152
alongside isotopic analyses of basidiocarps from a broad range of global habitats, to test the
153
hypothesis that waxcap fungi are mutualistic root endophytes and to determine what sources of soil
154
N they may have accessed.
155
156
2. Results
157
2.1 Effect of fertiliser/lime/biocide additions on waxcap fruiting
158
The negative effects of synthetic fertilisers on the fruiting of waxcaps have been anecdotally
159
reported (Griffith et al., 2004; Boertmann, 2010) but hitherto not rigorously demonstrated.
160
Therefore, the occurrence of waxcap basidiocarps was monitored over a five-year period at the
161
Sourhope NERC Soil Biodiversity site. Twelve species of Hygrophoraceae from three genera
162
(Cuphophyllus [4], Gliophorus [2] and Hygrocybe [7]) were found on the Sourhope plots with six
163
additional species found outside plots but within the fenced 1 ha experimental area (Fig. 1; Suppdata
164
7
1). Cuphophyllus pratensis comprised 63% of the approximately 4000 basidiocarps recorded over the
165
five survey years, followed in abundance by Gliophorus laeta (17%) and Hygrocybe ceracea (12%).
166
167
Fig. 1. Effect of plot treatment on abundance of basidiocarps at Sourhope
168
Suppdata 1. Details of Sourhope surveys
169
170
All treatments clearly affected basidiocarp abundance, with a three-fold or greater reduction in
171
basidiocarp abundance relative to controls for addition with nitrogen, lime, chlorpyrifos, or nitrogen
172
plus lime (Fig. 1). Fruiting varied widely among years due to climatic variations. Basidiocarp numbers
173
between replicate plots also varied, probably due to the large size and longevity of these organisms;
174
this may have masked any differences in the response of particular species or genera to the
175
experimental treatments.
176
177
Whilst synthetic nitrogen fertiliser and the Dursban biocide were applied at standard agricultural
178
levels, lime was applied at five times the usual rate, leading to a substantial increase in pH on limed
179
plots, from 4.5 in 1999 to > 7 in 2003 (Suppdata 1). Since the waxcap species present at Sourhope are
180
also found in calcareous grasslands, the negative effect of lime application is unlikely to be due solely
181
to intolerance of high pH. However, application of lime to acid soils also mobilises key nutrients,
182
notably P, and also alters core soil processes such as nitrification or nitrogen fixation. Furthermore,
183
the additive effects of nitrogen and lime application (Fig. 1) suggest that the deleterious effect of
184
lime on fruiting related to changes in N cycling in these soils, consistent with the findings of Kuan et
185
al.(2006) at this site. Waxcap fruiting was substantially inhibited in plots with added chlorpyrifos.
186
187
2.2 15N/13C isotopic signatures of soil and vegetation.
188
To provide context for the isotopic profiles of waxcap and other basidiocarps, associated soil and
189
vegetation were collected at Sourhope and Amorbach (the other main survey site) during basidiocarp
190
field surveys. The d15N values for vegetation were all close to 0‰ (-2 to +2‰), significantly lower in
191
15N than basidiocarps, whereas d13C values resembled soil and vegetation values (Fig 2; Suppdata 2).
192
8
Soils at Sourhope and Amorbach increased in 15N and declined in total N content with greater depth
193
(Fig. 2; Fig. 3; Suppdata 2), following known patterns in higher latitudes (Evans, 2007; Clemmensen et
194
al., 2013).
195
196
Fig 2. d13C and d15N values for soil, vegetation, waxcaps and other fungi
197
Fig. 3. d15N values for soil at Amorbach and Sourhope
198
Suppdata 2. d13C and d15N values of soils and vegetation at Amorbach and Sourhope.
199
200
2.3 Natural abundance δ15N in waxcap basidiocarps
201
A) Sourhope. During field surveys at Sourhope and other sites, all basidiocarps (waxcap and any
202
others) were mapped. Representative samples from each plot and each survey visit were collected
203
and dried, and a subset of these were sent for isotopic analysis. Basidiocarp caps collected from
204
control plots at Sourhope had high d15N values, ranging from 11.7 to 20.6‰ (mean=15.2±2.2‰;
205
n=42; Table 1; Suppdata 3), substantially greater than that generally found in basidiocarps of other
206
agaric fungi that have been studied, including ECM taxa (Taylor et al., 1997; Kohzu et al., 1999;
207
Hobbie and Agerer, 2010) but consistent with earlier studies of δ15N in waxcaps (Griffith et al., 2002;
208
Seitzman et al., 2011). Known saprotrophic taxa were much lower in d15N (12 spp.; mean=1.8±3.3‰;
209
n=17; Suppdata 3), the only sample within the range of the waxcaps being Agaricus langei
210
15N=12.5‰).
211
212
Table 1. Summary of species examined from the different field sites/areas.
213
Suppdata 3. Sampling locations for basidiocarp isotope analyses
214
215
The basidiocarps analysed included samples from a range of ages (unopened caps to fully mature)
216
but δ15N did not differ significantly among different age classes (Suppdata 4a), as has been reported
217
previously for ectomycorrhizal fungi (Taylor et al., 1997). As in other agaric fruitbodies (Taylor et al.,
218
1997), the 15N enrichment was greater in cap tissue compared to stipe (Suppdata 4b), by 2.7±1.7‰.
219
This is consistent with the higher N content of caps compared to stipes (6.8±1.53% N vs 3.4±0.8) and
220
9
probably results from the greater protein content of the former, with the N component of the latter
221
mainly present in the structural polymer chitin and glycoproteins.
222
223
Suppdata4. d15N / d13C in basidiocarps of different ages between cap vs. stipe tissues.
224
225
At Sourhope, a few basidiocarps were found on plots treated with ammonium nitrate (Suppdata 3).
226
Since synthetic fertiliser made from N2 via the Haber-Bosch process has a d15N value close to zero
227
(Bateman and Kelly, 2007), it might be expected that basidiocarps formed on these plots would
228
accordingly be lower in d15N. However, C. pratensis basidiocarps showed similarly enriched 15N
229
profiles compared to those from control plots (mean 15.1±1.8‰ vs 15.2±2.2‰ respectively; Fig 4).
230
231
Fig. 4. Isotopic profiles of C. pratensis at Sourhope were unaffected by fertiliser application.
232
233
B) Other sites in Europe. Similar patterns for isotopic values of waxcap basidiocarps from Sourhope
234
were found in further analysis of samples from upland and lowland grassland sites in Wales, England,
235
Germany, Iceland and Italy (Table 1; Suppdata 3), including a wider range of species (23 species in 5
236
genera; Fig. 5). All showed very similar patterns of 15N enrichment in Wales (14.9±2.0‰; n=36; 10
237
sites), England (16.3±2.4‰; n=26; 4 sites), Bavaria (14.6±1.7‰; n=155; 2 sites) and Liguria
238
(14.2±1.5‰; n=24; 2 sites) but samples from Iceland were less highly enriched (10.2±1.7‰; n=10; 2
239
sites). Excluding Iceland, there was consistent 15N enrichment, with all but one (H. cantharellus,
240
Bavaria; 6.1‰) of the 302 basidiocarps sampled having a δ15N signature greater than 10‰ (range
241
10.9-20.9‰). There was no significant difference in d15N values for samples from the five genera and
242
21 species within grassland Hygrophoraceae, nor did grassland type significantly affect d15N (lowland
243
vs. upland).
244
245
Fig. 5. d15N / d13C values by genus across all samples related to published data
246
247
10
In Europe, waxcaps are only rarely encountered beyond grassland habitats but four samples from UK
248
woodlands were similarly enriched in 15N (14.2±1.9‰; n=4) to grassland samples. However, two
249
samples of C. lacmus from a stand of pure heather on Lundy Island were slightly lower in d15N (ca.
250
9.5‰), whilst two samples of H. cantharellus from a boggy upland site vegetated with Sphagnum
251
moss had very low δ15N values (0.5 and 1.8‰) (Suppdata 3,5). In previous work, the ectomycorrhizal
252
genus Hygrophorus (Taylor et al., 2003; Trudell et al., 2004) was much less enriched in 15N (all <9.9‰;
253
mean 3.7±4.2‰; n=21; 2 sites) than other Hygrophoraceae. This was the case here for three
254
additional UK and New Zealand Hygrophorus specimens.
255
256
Saprotrophic taxa from grassland sites across Europe were much lower in d15N (3.0±2.7‰; n=50;
257
Suppdata 3) than waxcaps, consistent with findings for saprotrophs from Sourhope.
258
259
Suppdata 5. Images of C. lacmus and H. cantharellus growing in heather/moss
260
261
C) Outside Europe. Species associated with undisturbed grasslands in northern Europe are also found
262
in other continents, usually in woodland habitats. Dried basidiocarp samples were obtained from
263
three areas outside Europe:
264
a) subtropical laurel (laurisilva) montane forest in the Canary Islands (La Palma; dominated by
265
Laurus novocanariensis and Persea indica, both Lauraceae, with low levels of ground
266
vegetation, mostly ferns, no ground-dwelling mosses);
267
b) broadleaf-podocarp forest habitats in New Zealand (Bay of Plenty region, North Island), mostly
268
secondary forest dominated by Beilschmiedia tawa, B. taraire (Lauraceae), Cyathea spp.
269
(Cyatheaceae; tree ferns), Vitex lucens (Lamiaceae), Dysoxylum spectabile (Meliaceae) or
270
Leptospermum scoparium (Myrtaceae), with some remaining canopy trees (Podocarpus spp.,
271
Dacrycarpus dacrydoides, Dacrydium cupressoides). (Barton, 1972);
272
c) primary lowland moist evergreen forest in Amazonian Ecuador (Cuyabeno Forest Reserve)
273
(Lodge and Cantrell, 1995);
274
11
Published data for waxcaps are also available for two additional sites in Africa (primary lowland
275
rainforest, Gabon; (Tedersoo et al., 2012) and coniferous forest/swamp in the USA (Harvard Forest,
276
Massachusetts; (Seitzman et al., 2011); data from these studies was also included in our analyses
277
(Table 1).
278
279
Across all five areas, δ15N values were high for waxcap basidiocarps and mean values were similar to
280
those found in European grasslands: New Zealand (11.16±5.9‰; n=32; range 1.79-20.7‰); La Palma:
281
(13.1±3.7‰; n=16; range 7.0-21.0‰); Ecuador (14.4±3.8‰; n=3; range 11.1-18.4); Gabon:
282
(14.6.±6.2‰; (range 10-22‰; n=5 from a 20 ha plot). However, in New Zealand and La Palma, both
283
areas where the vegetation is dominated by non-ectomycorrhizal Lauraceae, the range of δ15N
284
values was particularly high and included some samples where δ15N enrichment was much lower
285
than found in European grasslands. These lower (<10‰) values were all found in members of genus
286
Hygrocybe Subgenus Pseudohygrocybe, Sections Coccineae or Firmae (sensu (Lodge et al., 2014);
287
3/16 in La Palma and and 13/32 in NZ). Some of these New Zealand Hygrocybe spp. had very low
288
δ15N (<2.5; 4/32 samples).
289
290
D) Within-site variation. Within the most intensively studied northern European grassland,
291
basidiocarp δ15N values varied widely, even for a single species at a single site. For example, δ15N
292
values for C. pratensis (n=29) at Sourhope varied from 11.7 to 20.6‰ within a one ha area. At other
293
sites, δ15N varied substantially even within clusters of adjacent basidiocarps across smaller areas (1-2
294
m2): 3.4‰ range for C. virgineus (n=20), 3.0‰ for H. coccinea (n=16) and 2.0‰ for H. quieta (n=15)
295
(Suppdata 3). At Sourhope the position of all C. pratensis basidiocarps was accurately mapped, so we
296
tested whether levels of enrichment correlated with topological or other features of the Sourhope
297
site (Suppdata 6). However, no correlations were detected.
298
299
Suppdata 6. Spatial distribution of C. pratensis and C. virgineus basidiocarps at Sourhope
300
301
2.4
d
13C and 14C analyses
302
12
A) δ13C natural abundance. Waxcap basidiocarps were lower in δ13C than basidiocarp cap tissues of
303
ectomycorrhizal and saprotrophic fungi reported here and elsewhere. However, across all samples,
304
δ13C values for waxcaps were distributed within a small range (-26.5 to -31.3‰ across all European
305
samples; 4.8‰ range) (Fig. 5; Suppdata 3). The data presented here for more than 20 species in 6
306
genera and including data from several previous studies show that mean δ13C values varied relatively
307
little across the diverse range of habitats (from -27.7±0.9‰ [USA] to -30.0±0.3‰ [Spessart,
308
Germany]). Samples from New Zealand varied widely in δ13C values (-23.9 to -32.1‰) and were over-
309
represented relative to other areas amongst the more extreme δ13C values.
310
311
B) 14C radiocarbon analysis. The 14C content of three Hygrophoraceae samples were analysed,
312
alongside two samples of saprotrophic fungi (Agaricus campestris and Cystoderma amianthinum)
313
which commonly co-occur with waxcaps. Samples collected during the period 1970-1985 (from the
314
RBG Kew Fungarium) were selected, since the steeper gradient then of the atmospheric 14C curve
315
allowed more accurate dating. The calibrated midpoint ages for A. campestris and C. amianthinum
316
were 5.0 and 4.4 years, suggesting that basidiocarp carbon was derived from organic matter formed
317
several years earlier (Table 2). However, for the three Hygrophoraceae, the midpoint 14C age was
318
younger, in two cases a few months but for the third it was 1.8 years.
319
320
Table 2. 14C radiocarbon data.
321
322
3. Discussion
323
3.1 Host range of waxcap fungi
324
In Europe, waxcap fungi are predominantly associated with grassland habitats (Halbwachs et al.,
325
2013a). This partly relates to the unusual nature of European grasslands, which are largely sub-climax
326
ecosystems, in which succession to woodland has been prevented by human activity or large
327
mammalian herbivores (Vera, 2000), whereas afforestation of grassland ecosystems in other
328
continents is prevented by low or episodic rainfall. In the temperate and boreal systems dominated
329
by trees which host ECM fungi, waxcaps are encountered only rarely (Hesler and Smith, 1963).
330
13
However, forest systems at lower latitudes are generally dominated by non-ECM tree species and
331
waxcaps are widespread in such habitats. In the present study, many samples were obtained from
332
forests dominated by non-ECM (and predominantly AM-associated; (Wang and Qiu, 2006; Tedersoo
333
and Nara, 2010) Lauraceae in New Zealand and the Canary Islands. Quantitative data was not
334
obtained but waxcaps are clearly more commonly encountered here than in more northerly ECM-
335
dominated habitats. Toju et al. (2014) detected several Hygrocybe spp. in Japanese forests containing
336
significant amounts of Lauraceae (19%; Neolitsia, Litsea, Machilus), all from the roots of non-
337
fagaceous (non-ECM) plants. Thus waxcaps appear to avoid ectomycorrhizal plants (Halbwachs et al.,
338
2013a), a phenomenon also seen in some European grasslands when ectomycorrhizal shrubs such as
339
Helianthemum are present (Griffith et al., 2013). If mycorrhizal, then these observations suggest that
340
waxcaps cannot associate with ECM hosts or cannot compete with ECM fungi.
341
342
The absence of any consistent group of host plants, in contrast to ECM and ericaceous mycorrhizal
343
fungi, is the main reason that waxcaps have been classified as saprotrophs. Suggestions of their
344
putative hosts have often pointed to bryophytes (Griffith et al., 2002; Seitzman et al., 2011; Lodge et
345
al., 2014), since some waxcaps, mostly in Hygrocybe Section Cocciniae (Lodge et al., 2014), commonly
346
associate with Sphagnum, especially in boreal locations (Boertmann, 2010). The lowest δ15N (0-2‰)
347
values for European waxcaps were for two H. cantharellus samples in Sphagnum. Several of the
348
Hygrocybe samples analysed by Seitzmann et al. (2011), also from a site dominated by Sphagnum,
349
were also low in d15N (6.2-7.4). H. cantharellus is found with Sphagnum in New Zealand
350
(http://www.kaimaibush.co.nz/Fungi/Hygrocybe1.html) and several Hygrocybe spp. from this area
351
had unusually low δ15N, however, precise substrate details are not available for our samples. Thus
352
the association of certain Hygrocybe spp. with Sphagnum, and the low δ15N values for these at
353
diverse locations, suggests a biotrophic interaction atypical of those more commonly found amongst
354
waxcaps. This may relate to the important role of cyanobacteria in the nitrogen nutrition of
355
Sphagnum (Berg et al., 2013).
356
357
14
Mosses are also abundant in many European ‘waxcap’ grasslands, with Rhytidiadelphus squarrosus
358
being the second most commonly associated species (after Agrostis capillaris) in Welsh grasslands
359
(Griffith et al., 2014). At Sourhope, this moss comprised 17% of total cover in control plots (Suppdata
360
1), however, at other waxcap-rich grasslands in the UK, for example Park Grass (Silvertown et al.,
361
2006), mosses are present at only low abundance, as they are from laurisilvan forests and tropical
362
wet forests. Killing of mosses with FeSO4 did not affect waxcap fruiting (Griffith et al., 2014). In laurel
363
forests at La Palma where waxcaps are commonly found, mosses are largely absent. Taken together
364
these lines of evidence suggest that the putative mycorrhizal hosts may include both mosses and a
365
potentially diverse array of higher plants.
366
367
3.2 The biotrophic status of waxcap fungi
368
The additional δ13C natural abundance data presented here confirm for many waxcap species from
369
diverse habitats across the world that the basidiocarps of these fungi are consistently more depleted
370
in 13C (mostly -26 to -30‰) than are those of ECM (Taylor et al., 1997; Taylor et al., 2003). They are
371
also depleted in 13C relative to saprotrophic taxa examined here from grassland habitats across the
372
UK (-24.9±1.6‰), as well as saprotrophs from other, mostly woodland, habitats (Kohzu et al., 1999;
373
Taylor et al., 2003). This is consistent with the possibility that waxcaps, like other members of the
374
Hygrophoraceae, are biotrophs deriving organic C from plant hosts rather than from soil organic
375
matter.
376
377
We did not undertake extensive analyses of associated soil and vegetation but at Sourhope δ13C
378
values for the dominant plant Agrostis capillaris (mean leaf and root δ13C -27.8‰ and -27.3‰,
379
respectively; n=3; SuppData 6) and soil (-26 to -27‰ at Sourhope and increasing with soil depth; Fig.
380
2; Fig. 3; SuppData 6) were depleted in 13C relative to basidiocarps of saprotrophs at Sourhope (-
381
25.1±1.4‰; n=43), consistent with the preferential loss of 12C as CO2 during microbial catabolism of
382
organic matter (Kohzu et al., 1999). In contrast, mean d13C values for waxcap tissues at Sourhope (-
383
28.4±0.7‰) were, however, similar to vegetation and slightly depleted in 13C relative to soil by ca.
384
2‰. If waxcaps are saprotrophic, it is difficult to explain why they are depleted in 13C relative to soil,
385
15
and distinct from known saprotrophs. Thus the most parsimonious explanation for the 13C natural
386
abundance patterns reported here is that that they derive C directly from plant photosynthate.
387
388
We used the to the steep annual decline in atmospheric 14CO2 during the 1970-80s to estimate
389
accurately the date at which the carbon present in fungarium basidiocarps had been fixed by
390
photosynthesis. The 14C age of the waxcap samples analysed was 0-2 yr, whereas for two
391
saprotrophic grassland fungi, the 14C age was 4-5 years. These 14C dates for waxcaps are consistent
392
with the use of recently fixed C such as photosynthate, and not consistent with the use of soil organic
393
matter which contains C fixed many years earlier (Chapela et al., 2001; Hobbie et al., 2002). Fungal
394
colonisation of senescent or recently dead roots cannot be excluded, since this would also have a
395
recent 14C signature. Hobbie et al. (2002) found that fresh conifer needles were unexpectedly
396
enriched in 14C, dating to 0-3 years prior to collection, and they discussed the various factors that
397
could account for both this and the similar dating of 14C in basidiocarps of known mycorrhizal
398
species, including anaplerotic fixation of CO2 (Wingler et al., 1996), internal recycling of C within
399
mycelial systems and uptake of C from soil amino acids by fungi. However, Treseder et al. (2006)
400
found no evidence of transfer of 14C from labelled litter to ECM fungi.
401
402
As part of the NERC Soil Biodiversity Initiative, a 13CO2 pulse labelling facility was established at
403
Sourhope during 2000-02 (Staddon et al., 2003). We collected basidiocarps formed near the pulse
404
points to test whether any 13CO2 fixed by plants within the pulse domes was later incorporated into
405
waxcap basidiocarps formed nearby. A single heavily-labelled basidiocarp of C. pratensis (d13C
406
92.3‰; SuppData 7) was detected 20 cm from one pulse dome (14 d after the 4-6 h pulse). Johnson
407
et al. (2002) established in-growth cores (with root but not hyphal in-growth prevented by 35 µm
408
mesh) within the pulse domes and showed that 13CO2 from microbial respiration within the cores was
409
significantly reduced if cores had been rotated to disrupt hyphal connections. They ascribed this
410
respiration to arbuscular mycorrhizal fungi (AMF) but this respiration could also have been by
411
mycelia of waxcaps, a possibility supported by DNA metabarcoding studies which consistently find
412
AMF abundance (<5%) in undisturbed grasslands to be lower than that of waxcaps and other fungal
413
16
groups (Geml et al., 2014; Jumpponen and Jones, 2014; Detheridge et al., 2018). Treonis et al. (2004)
414
used the same pulsing system to monitor incorporation of 13C from labelled CO2 into microbial PLFAs
415
(phospholipid fatty acids). The most heavily labelled PLFA was 18:2w6,9 which is the dominant PLFA
416
in basidiomycetes (>80% of total PLFAs) and present at only low abundance (<2%) in AMF (Baldrian
417
et al., 2013). These observations are consistent with the other isotopic data presented above,
418
suggesting that waxcaps can access recently fixed photosynthate.
419
420
Suppdata 7. Data from the seven waxcap Basidiocarps close to pulse sites
421
422
3.3 Isotopic enrichment of 15N in waxcap basidiocarps
423
The consistently high level of 15N enrichment reported here (Table 1), significantly higher than values
424
for soil and vegetation which we also analysed (Fig. 4; Suppdata 6), is not found in any other group of
425
basidiomycetes, though similarly high values are found for some ascomycetes (Tuber spp.) associated
426
with mycoheterotrophic orchids (Schiebold et al., 2017). More than >40% of the >250 samples
427
reported here were enriched beyond 15‰ and most samples below 10‰ are either associated with
428
Sphagnum or are within Hygrocybe Section Cocciniae, as described above. ECM basidiocarps
429
enriched in δ15N are widely reported (mostly falling within the range +3 to +9‰), however, values
430
above 10‰ are unusual (Trudell et al., 2004). There has been much discussion about why ECM
431
basidiocarps are enriched in 15N, and also why ECM fungi belonging to different genera are variably
432
enriched in 15N relative to atmospheric N2 and the bulk substrate (Trudell et al., 2004; Hobbie and
433
Agerer, 2010). One probable factor for some 15N enrichment in ECM fungi is the preferential supply
434
of lighter 14N to the host due to fractionation against 15N during transamination (glutamine-
435
glutamate shuttle) at the mycorrhizal interface (Emmerton et al., 2001; Hobbie et al., 2012). Such
436
transamination reactions discriminate strongly against 15N (Handley and Raven, 1992).
437
438
Transamination from glutamine is also involved in chitin synthesis (via glucosamine:fructose-6-
439
phosphate aminotransferase; EC2.6.1.16; (Ram et al., 2004) and the fractionation against 15N during
440
glucosamine synthesis explains the lower 15N enrichment of more chitin-rich stipe tissues in waxcaps
441
17
(by 0.7-5.4‰; Suppdata 4), relative to the more protein-rich cap tissues. Similar differences in δ15N
442
between caps and stipes are reported for basidiocarps of many other fungi including ECM (Taylor et
443
al., 1997; Hobbie et al., 2012). Although the N content of chitin is low (6.3%) compared to protein
444
(ca. 15%), it comprises a significant proportion of total biomass in filamentous fungi (5-10% of cell
445
dry weight; (Free, 2013) and thus represents an important pool of N. The dynamics of chitin
446
catabolism and internal recycling in fungi are not well understood (Merzendorfer, 2011) but recent
447
genome data can provide valuable insights. For example, the model ECM species Laccaria bicolor
448
contains genes encoding chitinase/hexosaminidase (EC3.2.1.14/EC3.2.1.52) which mediates release
449
of N-acetylglucosamine monomers but it lacks the requisite genes for further breakdown (N-
450
acetylglucosamine-6-phosphate deacetylase [EC3.5.1.25] and glucosamine-6-phosphate deaminase
451
[EC3.5.99.6]) which are present in the genomes of most saprotrophic fungi hitherto studied
452
(http://www.genome.jp/kegg). This suggests, for this species at least, that once incorporated into
453
chitin, this N is not released back to the protein/inorganic pool.
454
455
To explain the wide range of taxon-specific δ15N values in the basidiocarps of ECM fungi, Hobbie and
456
Agerer (2010) correlated these differences with the nature and extent of the underlying mycelial
457
system (www.deemy.de; (Rambold and Agerer, 1997). Taxa having more extensive exploratory
458
mycelia formed basidiocarps which were more enriched in 15N (Suppdata 8). They postulated that
459
the wide variation in δ15N values could be explained using a two-pool model of distribution of N
460
within mycelial systems, one comprising immobile 15N-depleted chitin and the other mobile 15N-
461
enriched protein, as described above. They proposed that the loss of 15N-depleted N to the host
462
plant and its immobilisation in chitin leads to 15N enrichment of the protein pool which is
463
translocated for basidiocarp formation (Suppdata 8). Thus, high δ15N values for basidiocarps could be
464
explained by high levels of transfer of N to hosts or by high investment in cell wall material (i.e.
465
extensive mycelial systems). Whether this is a feature of all mycorrhizal association is unclear since
466
both (Merckx et al., 2010) and Courty et al. (2015) did not find evidence of such 15N partitioning in
467
AMF symbioses.
468
469
18
Suppdata 8. Summary of Hobbie and Agerer theoretical pattern of N isotope fractionation in
470
mycorrhizal systems
471
472
Recent DNA metabarcoding analysis of undisturbed grasslands in the UK has shown that waxcaps
473
alongside Clavariaceae comprise 40-70% of the total fungal biomass present in these soils, with
474
waxcap mycelia present at ca. 0.5-1.5 mg.g-1 dw. soil (Detheridge et al., 2018). This is consistent with
475
observations of large numbers of waxcap basidiocarps, sometimes forming large fairy rings (Griffith
476
et al., 2014). In contrast, ECM-forming Hygrophoraceae (Hygrophorus spp.) form only limited
477
mycelial networks (www.deemy.de; (Agerer, 2006), consistent with their low δ15N signatures
478
(Seitzman et al., 2011).
479
480
For some ECM, 15N enrichment of basidiocarps has been linked to 15N depletion in tissues of the host
481
plants (Högberg et al., 1996; Hobbie and Högberg, 2012). However, this may prove challenging to
482
demonstrate for waxcaps due to their apparently diverse host associations and the absence of any
483
visible soil mycelial networks (Halbwachs et al., 2013b). Nevertheless, δ15N values for European
484
waxcaps were 12.5‰ higher than those of saprotrophs inhabiting the same grasslands. The latter
485
obtain their nitrogen from plant litter and soil with lower 15N content (Gebauer and Taylor, 1999) and
486
the disparity suggests that waxcaps use nitrogen sources different from those accessed by
487
saprotrophic fungi.
488
489
3.4 N sources accessed by waxcaps
490
It has long been suggested that one reason for the drastic reduction in the numbers of ‘waxcap’
491
grasslands in Europe is the widespread use of synthetic fertilisers in agriculture. However, this has
492
not hitherto been rigorously demonstrated. At the Sourhope field experiment, application of
493
ammonium nitrate at standard agricultural rates caused a three-fold reduction in the abundance of
494
waxcap basidiocarps and even greater reduction when applied in conjunction with lime (Fig. 1).
495
496
19
The fact that these differences were consistently found over a five-year period suggests that such
497
changes are linked to long term decline of fungal mycelium in soil. ECM fruiting declined along a
498
nitrogen deposition gradient (<15 kg N ha-1 yr-1), (Lilleskov et al. (2001; 2002) with the more
499
nitrogen-sensitive species, for example Cortinarius spp., preferentially using organic N sources. The
500
authors also observed that basidiocarp δ15N values were positively correlated with mineral N levels
501
in soil. However, our data did not reveal any such correlation, with δ15N values of waxcap
502
basidiocarps similar for N-amended plots and from control plots (Fig. 4). As noted above, the species
503
examined by Lilleskov et al. (2002) were all culturable and able to use ammonium as sole N source.
504
Waxcaps remain uncultured, so it has not been possible to test directly their ability to utilise
505
inorganic N, though our field data suggest not. Nitrate or ammonium (or the redox intermediates of
506
these) could be toxic to waxcaps but it is also possible that other soil microbes outcompeted waxcaps
507
for ammonium and nitrate in the amended plots. The synergistic inhibition by nitrogen and lime on
508
waxcap fruiting points towards out-competition for resources by bacterial populations, since the
509
ratio of fungi to bacteria (F:B ratio; (Bardgett et al., 1996) is known to correlate positively with pH.
510
Dawson et al. (2003) reported a four-fold increase in bacterial numbers on N/L plots compared to
511
control plots at Sourhope, whereas populations of culturable fungi remained constant, as did fungal
512
PLFA levels (Murray et al., 2006).
513
514
The fractionation mechanisms described above and the Hobbie/Agerer two-pool model provide only
515
a partial explanation of the high δ15N values of waxcap basidiocarps. The lines of evidence set out
516
above suggest that waxcaps rely on sources of organic N. Bulk SOM at Sourhope and Amorbach were
517
enriched in 15N (6-8‰; Fig 2; Fig. 3), with the level of enrichment increasing with soil depth (linked to
518
a decrease in total N content). It is possible that waxcaps access N from aged/recalcitrant 15N-
519
enriched sources, such as complex heterocyclic polymers in deeper soil horizons (Dijkstra et al., 2006;
520
Evans, 2007; Hobbie and Ouimette, 2009). Such sources would at least partly originate from
521
organisms high in the food chain: the higher the 15N signature of an organism, the higher its position
522
in the food chain (Griffith, 2004; Boecklen et al., 2011). We consider it highly unlikely that N is
523
obtained from any plants, since these have consistently low ð15N values (-3 to +5‰; Suppdata 2;
524
20
(Temperton et al., 2012). However, more plausible alternate sources could also include microbe-
525
derived N in the form of highly recycled amino acids, amino sugars or protein-N continuously
526
recycled and mopped up by microbes (as N fragments or whole amino acids) such that the lifetime of
527
these labile compounds in soils is thereby extended (Gleixner et al., 2002; Bol et al., 2009). Such an
528
argument would explain their occurrence only in undisturbed grasslands where soil processes over
529
decadal timescales have accumulated old organic matter reserves in deeper soil strata. However, this
530
explanation ignores the higher organic N content towards the upper, more accessible surface
531
horizons.
532
533
The five-fold reduction in waxcap fruiting at Sourhope following application of the pesticide
534
chlorpyrifos offers potential insight into the N metabolism of waxcaps. This acetylcholinesterase
535
inhibitor is toxic to invertebrates, with for instance earthworms exhibiting sublethal effects at
536
standard application rates (Pelosi et al., 2014). There is no evidence of direct inhibition of fungal
537
growth/differentiation by chlorpyrifos at the levels applied here (338 mg.m-2 yr-1; Suppdata 1), so
538
this finding suggests a hitherto undescribed interaction between waxcaps and soil animals. Dawson
539
et al. (2003) quantified microbial PLFA at Sourhope and reported a substantial (7-fold) decrease in
540
fungal biomass on biocide plots. This raises the possibility that waxcaps may derive N from soil
541
invertebrates and are inhibited in fruiting (and possibly mycelial growth) by the absence of such food
542
sources. Several agaric fungi can derive N from invertebrates (Barron and Thorn, 1987), including the
543
ectomycorrhizal Laccaria laccata, which can consume collembolans and transfer N from such sources
544
to host plants (Klironomos and Hart, 2001). Similarly, Metarrhizium spp. and other claviceptaceous
545
entomopathogens can transfer insect-derived N to diverse host plants (Behie et al., 2012).
546
547
Nitrogen derived from soil animals may also explain not only the highly elevated δ15N values of
548
grassland waxcap basidiocarps but also the high level of localised variability of these δ15N patterns.
549
At Sourhope δ15N values for C. pratensis across control plots varied by 8‰ (12-20‰), similar to that
550
found for this species at a global scale. Even for basidiocarps forming part of the same fairy ring there
551
was 3-4‰ variation (Suppdata 3), suggesting a high level of spatial heterogeneity in the N sources
552
21
accessed. The only known insect-associated fungus at Sourhope was Cordyceps militaris, a pathogen
553
of lepidopteran larvae. Ascocarps of this species from control plots at Sourhope were high and
554
variable in δ15N (8.0±2.4‰, n =3; Suppdata 3).
555
556
A meta-analysis of δ15N values in soil invertebrates by Tiunov (2007) suggested that many are
557
elevated in 15N, ranging from +6.5 to +9.3‰, with predators occupying higher trophic levels in food
558
webs being more enriched in 15N (Suppdata 9). With regards to which invertebrates might be the
559
source of waxcap N, surveys of soil invertebrates in biocide plots at Sourhope found severe effects
560
on certain groups (e.g. tipulids) and lesser negative effects on other taxa (enchytraeids, nematodes,
561
tardigrades; (Dawson et al., 2003; Cole et al., 2006). The most abundant invertebrates are
562
earthworms and dipteran larvae, which together typically accounted for >70% of total animal
563
biomass (Murray et al., 2009). Bishop et al. (2008) found the endogeic species Allolobophora
564
chlorotica to be the most abundant earthworm at Sourhope (40 individuals m-2 on control plots),
565
whilst Neilson et al. (2000) found this species to be the most highly enriched in 15N (8-10‰) of all the
566
earthworm species at several of the grassland sites they studied. Similarly, Schmidt et al. (2004)
567
found that this and other endogeic earthworms and Enchytraeidae were the most highly enriched in
568
15N (11-15‰; Suppdata 9).
569
570
Suppdata 9. δ15N values in soil invertebrates from published studies
571
572
We estimated invertebrate biomass in Sourhope control plots using published data (earthworm,
573
collembolans, enchytraeid; Table 1; (Cole et al., 2006) and (tipulids; Fig. 1 in (Dawson et al., 2003) to
574
be ca 100-200 g fresh weight m-2, accounting for the substantial (>2-fold) seasonal variation that was
575
recorded. This corresponds to 1.75-3.5 g N m-2 (assuming 83% water content and 10% N content of
576
dw), ca. 1% of the total N pool (for Sourhope soils (0-10 cm; 60 kg dry weight m-2 at 0.4%N) =240 g N
577
m-2). We further estimated the total N content in waxcap biomass to be ca. 3 g.m-2 (based on 0.25
578
basidiocarps.m-2, with dry wt 2.5 g each [C. pratensis] and 5% N content (Suppdata 3), assuming 1%
579
22
of biomass in basidiocarp). These estimates suggest that the likely N demand from waxcaps and the
580
N available via soil invertebrates are of similar magnitude.
581
582
3.5 Conclusions
583
Here, we provide evidence from isotopic studies and field experiments that not only do these fungi
584
obtain C directly from plant photosynthate but that they are potentially mycorrhizal. The absence of
585
The diverse habitats occupied by waxcap fungi suggests that they can form associations with a
586
diverse range of host plants and their absence from habitats dominated by ECM suggests that they
587
are outcompeted in such habitats. The N nutrition of waxcaps is clearly unusual but their elevated
588
δ15N signatures were remarkably conserved at a global level, with rare exceptions restricted to a few
589
species within genus Hygrocybe and possible association with bryophyte hosts. Within the context of
590
their elevated δ15N signatures, we were unable to pinpoint the cause of the high variability in δ15N
591
but we propose that this is linked to their ability to utilise diverse N sources which vary in δ15N, and
592
which include soil invertebrates. However, it is also possible hitherto undiscovered uptake or loss
593
processes leading to extreme levels of 15N fractionation may be involved. In the absence of any
594
axenic/monoxenic culture system for waxcaps, it is likely that further elucidation of the mechanisms
595
involved must be obtained from genomic data and compound-specific isotopic investigations.
596
597
4. Experimental Methods
598
4.1. Field sites and plot treatments at Sourhope
599
An upland grassland (Rigg Foot, Sourhope, Kelso, Scotland; Lat/Long: 55.470°, -2.231°; altitude: 310
600
m; the focus of the NERC Soil Biodiversity Programme) was the main field site used in this study. The
601
site had previously been under pasture for at least 50 years with no fertiliser additions and is
602
classified as U4d (Festuca-Agrostis-Galium) in the UK National Vegetation classification (Suppdata 1).
603
The soils are acid brown earths, derived from andesitic lavas of Old Red Sandstone.
604
605
The experiment comprised four treatments (5 replicates each; 12×20 m plots): Control (C; no
606
additions), nitrogen fertiliser (N; granular NH4NO3 [24 g m-2 yr-1], two equal applications in May and
607
23
July); lime (L; 600 g m-2, each spring); biocide (B; Dursban 4 [Chlorpyrifos], five monthly application
608
from May to September [0.15 ml m-2]); Nitrogen/Lime (NL; as above combined) (Fitter et al., 2005).
609
Grass was cut to 6 cm at 3 week intervals (six cuts from May to September) and clippings were
610
removed (Suppdata 1). Basidiocarp surveys were conducted over a five-year period (2001-05) with 2-
611
5 visits annually (12-26 d apart) between early September and early November. Names used for
612
fungal species are in agreement with IndexFungorum.
613
614
4.2 Sampling and isotopic analyses
615
Isotopic analyses were conducted on cap (pileus) tissues of dried basidiocarps. Samples observed
616
during field surveys were picked, dried within 24 h of collection in a ventilated drying cabinet at
617
<40°C and stored desiccated at the Aberystwyth University Fungarium (code ABS). Identification of
618
samples was based on macroscopic and microscopic examination according to Boertmann (2010).
619
Subsamples of cap tissue were excised from these and ground for isotopic analysis. Further details of
620
sampling locations and the species analysed are given in Suppdata 6. At two sites (Amorbach and
621
Sourhope), soil cores were taken in a W-shaped pattern (30 cm distance between cores; 3-5
622
replicates). Samples were separated by depth into 25-30 mm segments down to the bedrock
623
(Sourhope) or up to 23 cm (Amorbach). Cores were dried overnight at 60°C and ground to a fine
624
powder. Samples of associated higher plants were also collected adjacent to soil cores, dried and
625
ground.
626
627
Isotope analyses were conducted by continuous flow-isotope ratio mass spectrometry (CF-IRMS),
628
using an automated N/C analysis-mass spectrometry (ANCA-MS) system (Europa 20/20, Crewe, UK)
629
at Lancaster, North Wyke, by Conflo III Interface. Recent samples from Italy, Liguria, La Palma and
630
northern Bavaria were analysed at the Bayreuth University (for details refer to
631
http://www.bayceer.uni-bayreuth.de/ibg/en/ausstattung/geraet/geraet_detail.php?id_obj=65905).
632
Values were referenced against atmospheric nitrogen and V-PDB limestone standards.
633
634
24
4.3. 14C analysis: To obtain basidiocarp samples corresponding to a period when atmospheric
635
radiocarbon (14C) levels were elevated, three Hygrophoraceae samples were chosen from the
636
Mycological Herbarium at the Royal Botanic Gardens (RBG), Kew. Samples were taken from grassland
637
sites but avoided urban areas, where the 14C-dead CO2 from localised fossil fuel burning could cause
638
distortion of data. The samples were analysed to quantify 14C content. Basidiocarp cap segments
639
were subjected to a weak acid wash (0.5 M HCl) to remove potential contaminants. The samples
640
were then combusted in sealed quartz tubes and sample CO2 cryogenically recovered, graphitised
641
(Slota et al., 1987) and measured for 14C concentration by accelerator mass spectrometry (AMS) at
642
the Scottish Universities Environmental Research Centre, East Kilbride, UK. Following convention, 14C
643
results were normalised to a δ13C of -25‰, and expressed as %modern (ratio of sample 14C content
644
relative to the oxalic acid international standard; (Stuiver and Polach, 1977). Radiocarbon
645
concentrations were used to determine the age of initial carbon fixation from the atmosphere by
646
matching sample %modern results with a record of atmospheric 14C content over the last 60 years.
647
This calibration was performed using ‘Calibomb’ software (Reimer et al., 2004) using the northern
648
hemisphere atmospheric 14CO2 dataset (Levin et al., 2008).
649
650
5. Acknowledgements
651
This work was funded by the Natural Environment Research Council under the Soil Biodiversity
652
Thematic Programme (Grant number: NER/T/S/2001/00143), (NERC Radiocarbon Facility
653
NRCF010001 (alloc. 1100.1004) and funding via Memorandum of Agreement from CCW, SNH, DENI,
654
NE and Plantlife to GWG. The Institute of Biological, Environmental, and Rural Sciences receives
655
strategic funding from the BBSRC. We thank Andy Stott, Darren Sleep and Helen Grant (CEH) and
656
staff in the isotope laboratories at Bayreuth and North Wyke at for efficient isotope analyses, and
657
Graham Burt-Smith and Sarah Buckland (Soil Biodiversity Site Managers) for support during field
658
sampling at Sourhope. We thank Brian Spooner (RBG Kew), Shirley Kerr, John Hedger, Debbie Evans,
659
David Mitchel for access to fungarium samples. Finally we thank Reinhard Agerer (Munich) for
660
25
inspiring discussions about the trophic mode of waxcaps during recent years and Guðríður Gyða
661
Eyjólfsdóttir (Icelandic Institute of Natural History) for her support in sampling fungi.
662
6. References
663
Agerer, R. (2006) Fungal relationships and structural identity of their ectomycorrhizae. Mycol Prog 5:
664
67-107.
665
Baldrian, P., Větrovský, T., Cajthaml, T., Dobiášová, P., Petránková, M., Šnajdr, J., and Eichlerová, I.
666
(2013) Estimation of fungal biomass in forest litter and soil. Fungal Ecol 6: 1-11.
667
Bardgett, R.D., Hobbs, P.J., and Frostegard, A. (1996) Changes in soil fungal:bacterial biomass ratios
668
following reductions in the intensity of management of an upland grassland. Biol Fertility Soils
669
22: 261-264.
670
Barron, G.L., and Thorn, R.G. (1987) Destruction of nematodes by species of Pleurotus. Can J Bot 65:
671
774-778.
672
Barton, I.L. (1972) On the vegetation of the Hunua Ranges, Auckland. N Z J Bot 10: 8-26.
673
Bateman, A.S., and Kelly, S.D. (2007) Fertilizer nitrogen isotope signatures. Isotopes Environ Health
674
Stud 43: 237-247.
675
Behie, S., Zelisko, P., and Bidochka, M. (2012) Endophytic insect-parasitic fungi translocate nitrogen
676
directly from insects to plants. Science 336: 1576-1577.
677
Behie, S.W., and Bidochka, M.J. (2014) Nutrient transfer in plantfungal symbioses. Trends Plant Sci
678
19: 734-740.
679
Beisenherz, M. (2000) Untersuchungen zur Ökologie und Systematik der Gattung Hygrocybe
680
(Agaricales). . Diss Univ Regensburg Auszüge, Regensb Mykol Schr 10: 3-65, 2002.
681
Berg, A., Danielsson, Ö., and Svensson, B.H. (2013) Transfer of fixed-N from N2-fixing cyanobacteria
682
associated with the moss Sphagnum riparium results in enhanced growth of the moss. Plant
683
Soil 362: 271-278.
684
Bishop, H.O., Grieve, I.C., Chudek, J.A., and Hopkins, D.W. (2008) Liming upland grassland: the effects
685
on earthworm communities and the chemical characteristics of carbon in casts. Eur J Soil Sci 59:
686
526-531.
687
Boecklen, W.J., Yarnes, C.T., Cook, B.A., and James, A.C. (2011) On the use of stable isotopes in
688
trophic ecology. Annu Rev Ecol, Evol Syst 42: 411-440.
689
Boertmann, D. (2010) The genus Hygrocybe: Danish Mycological Society. 2nd edition.
690
Bol, R., Gleixner, G., Poirier, N., and Balesdent, J. (2009) Molecular turnover time of SOM in particle
691
size fractions of an arable soil. Rapid Comm Mass Spect 23: 2551-2558.
692
26
Clemmensen, K., Bahr, A., Ovaskainen, O., Dahlberg, A., Ekblad, A., Wallander, H. et al. (2013) Roots
693
and associated fungi drive long-term carbon sequestration in boreal forest. Science 339: 1615-
694
1618.
695
Cole, L., Bradford, M.A., Shaw, P.J., and Bardgett, R.D. (2006) The abundance, richness and functional
696
role of soil meso-and macrofauna in temperate grasslandA case study. Appl Soil Ecol 33: 186-
697
198.
698
Courty, P.-E., Doubkova, P., Calabrese, S., Niemann, H., Lehmann, M.F., Vosatka, M., and Selosse, M.-
699
A. (2015) Species-dependent partitioning of C and N stable isotopes between arbuscular
700
mycorrhizal fungi and their C3 and C4 hosts. Soil Biol Biochem 82: 52-61.
701
Chapela, I.H., Osher, L.J., Horton, T.R., and Henn, M.R. (2001) Ectomycorrhizal fungi introduced with
702
exotic pine plantations induce soil carbon depletion. Soil Biol Biochem 33: 1733-1740.
703
Dawson, L.A., Grayston, S.J., Murray, P.J., Cook, R., Gange, A.C., Ross, J.M. et al. (2003) Influence of
704
pasture management (nitrogen and lime addition and insecticide treatment) on soil organisms
705
and pasture root system dynamics in the field. Plant Soil 255: 121-130.
706
Detheridge, A.P., Comont, D., Callaghan, T.M., Bussell, J., Brand, G., Gwynn-Jones, D. et al. (2018)
707
Vegetation and edaphic factors influence rapid establishment of distinct fungal communities on
708
former coal-spoil sites. Fungal Ecol 33: 92-103. DOI: 110.1016/j.funeco.2018.1002.1002.
709
Dijkstra, P., Ishizu, A., Doucett, R., Hart, S.C., Schwartz, E., Menyailo, O.V., and Hungate, B.A. (2006)
710
13C- and 15N natural abundance of the soil microbial biomass. Soil Biol Biochem 38: 3257-3266.
711
Emmerton, K.S., Callaghan, T.V., Jones, H.E., Leake, J.R., Michelsen, A., and Read, D.J. (2001)
712
Assimilation and isotopic fractionation of nitrogen by mycorrhizal and nonmycorrhizal subarctic
713
plants. New Phytol 151: 513-524.
714
Evans, R.D. (2007) Soil nitrogen isotope composition. Stable isotopes in Ecology and Environmental
715
Science 2: 83-98.
716
Fitter, A., Gilligan, C., Hollingworth, K., Kleczkowski, A., Twyman, R., and Pitchford, J. (2005)
717
Biodiversity and ecosystem function in soil. Funct Ecol 19: 369-377.
718
Free, S.J. (2013) Fungal cell wall organization and biosynthesis. Adv Genet 81: 33-82.
719
Gebauer, G., and Taylor, A.F.S. (1999) 15N natural abundance in fruit bodies of different functional
720
groups of fungi in relation to substrate utilization. New Phytol 142: 93-101.
721
Gebauer, G., and Meyer, M. (2003) 15N and 13C natural abundance of autotrophic and myco-
722
heterotrophic orchids provides insight into nitrogen and carbon gain from fungal association.
723
New Phytol 160: 209-223.
724
27
Geml, J., Gravendeel, B., van der Gaag, K.J., Neilen, M., Lammers, Y., Raes, N. et al. (2014) The
725
contribution of DNA metabarcoding to fungal conservation: diversity assessment, habitat
726
partitioning and mapping red-listed fungi in protected coastal Salix repens communities in the
727
Netherlands. PloS One 9: e99852.
728
Gleixner, G., Poirier, N., Bol, R., and Balesdent, J. (2002) Molecular dynamics of organic matter in a
729
cultivated soil. Org Geochem 33: 357-366.
730
Griffith, G.W. (2004) The use of stable isotopes in fungal ecology. Mycologist 18: 177-183
731
Griffith, G.W., and Roderick, K. (2008) Saprotrophic basidiomycetes in grasslands: distribution and
732
function. In Ecology of Saprotrophic Basidiomycetes British Mycological Society Symposia Series.
733
Boddy, L., Frankland, J.C., and van West, P. (eds). London: Elsevier Ltd., pp. 277-299.
734
Griffith, G.W., Easton, G.L., and Jones, A.W. (2002) Ecology and diversity of waxcap (Hygrocybe spp.)
735
fungi. Bot J Scotland 54: 7-22.
736
Griffith, G.W., Bratton, J.L., and Easton, G.L. (2004) Charismatic megafungi: the conservation of
737
waxcap grasslands. Br Wildl 15: 31-43.
738
Griffith, G.W., Graham, A., Woods, R.G., Easton, G.L., and Halbwachs, H. (2014) Effect of biocides on
739
the fruiting of waxcap fungi. Fungal Ecol 7: 67-69.
740
Griffith, G.W., Gamarra, J.P., Holden, E.M., Mitchel, D., Graham, A., Evans, D.A. et al. (2013) The
741
international conservation importance of Welsh 'waxcap' grasslands. Mycosphere 4: 969984.
742
Nguyen, N.H., Song, Z., Bates, S.T., Branco, S., Tedersoo, L., Menke, J. et al. (2015) FUNGuild: an open
743
annotation tool for parsing fungal community datasets by ecological guild. Fung Ecol
744
doi:10.1016/j.funeco.2015.06.006.
745
Halbwachs, H., Karasch, P., and Griffith, G.W. (2013a) The diverse habitats of Hygrocybepeeking
746
into an enigmatic lifestyle. Mycosphere 4: 773-792.
747
Halbwachs, H., Dentinger, B.T., Detheridge, A.P., Karasch, P., and Griffith, G.W. (2013b) Hyphae of
748
waxcap fungi colonise plant roots. Fungal Ecol 6: 487-492.
749
Handley, L.L., and Raven, J.A. (1992) The use of natural abundance of nitrogen isotopes in plant
750
physiology and ecology. Plant, Cell Environ 15: 965-985.
751
Harrington, T.J., and Mitchell, D.T. (2002) Colonization of root systems of Carex flacca and C.
752
pilulifera by Cortinarius (Dermocybe) cinnamomeus. Mycol Res 106: 452-459.
753
Heijden, M.G., Martin, F.M., Selosse, M.A., and Sanders, I.R. (2015) Mycorrhizal ecology and
754
evolution: the past, the present, and the future. New Phytol 205: 1406-1423.
755
Hesler, L.R., and Smith, A.H. (1963) North American Species of Hygrophoraceae. Knoxville, Tennessee:
756
University of Tennessee Press.
757
28
Hobbie, E.A., and Ouimette, A.P. (2009) Controls of nitrogen isotope patterns in soil profiles.
758
Biogeochemistry 95: 355-371.
759
Hobbie, E.A., and Agerer, R. (2010) Nitrogen isotopes in ectomycorrhizal sporocarps correspond to
760
belowground exploration types. Plant Soil 327: 71-83.
761
Hobbie, E.A., and Högberg, P. (2012) Nitrogen isotopes link mycorrhizal fungi and plants to nitrogen
762
dynamics. New Phytol 196: 367-382.
763
Hobbie, E.A., Sánchez, F.S., and Rygiewicz, P.T. (2012) Controls of isotopic patterns in saprotrophic
764
and ectomycorrhizal fungi. Soil Biol Biochem 48: 60-68.
765
Hobbie, E.A., Weber, N.S., Trappe, J.M., and van Klinken, G.J. (2002) Using radiocarbon to determine
766
the mycorrhizal status of fungi. New Phytol 156: 129-136.
767
Hobbie, E.A., Diepen, L.T., Lilleskov, E.A., Ouimette, A.P., Finzi, A.C., and Hofmockel, K.S. (2014)
768
Fungal functioning in a pine forest: evidence from a 15N-labeled global change experiment. New
769
Phytol 201: 1431-1439.
770
Högberg, P., Hogbom, L., Schinkel, H., Högberg, M., Johannisson, C., and Wallmark, H. (1996) 15N
771
abundance of surface soils, roots and mycorrhizas in profiles of European forest soils. Oecologia
772
108: 207-214.
773
Johnson, D., Leake, J.R., Ostle, N., Ineson, P., and Read, D.J. (2002) In situ 13CO2 pulse-labelling of
774
upland grassland demonstrates a rapid pathway of carbon flux from arbuscular mycorrhizal
775
mycelia to the soil. New Phytol 153: 327-334.
776
Jumpponen, A., and Jones, K.L. (2014) Tallgrass prairie soil fungal communities are resilient to climate
777
change. Fungal Ecol 10: 44-57.
778
Kariman, K., Barker, S.J., Jost, R., Finnegan, P.M., and Tibbett, M. (2014) A novel plant–fungus
779
symbiosis benefits the host without forming mycorrhizal structures. New Phytol 201: 1413-
780
1422.
781
Keizer, P.J. (1993) The influence of nature management on the macromycete fungi. In Fungi in
782
Europe: Investigations, Recording and Conservation. Pegler, D.N., Boddy, L., Ing, B., and Kirk,
783
P.M. (eds). Kew: Royal Botanic Gardens, pp. 251-269.
784
Klironomos, J.N., and Hart, M.M. (2001) Food-web dynamics: Animal nitrogen swap for plant carbon.
785
Nature 410: 651-652.
786
Kohzu, A., Yoshioka, T., Ando, T., Takahashi, M., Koba, K., and Wada, E. (1999) Natural 13C and 15N
787
abundance of field-collected fungi and their ecological implications. New Phytol 144: 323-330.
788
29
Kuan, H., Fenwick, C., Glover, L.A., Griffiths, B., and Ritz, K. (2006) Functional resilience of microbial
789
communities from perturbed upland grassland soils to further persistent or transient stresses.
790
Soil Biol Biochem 38: 2300-2306.
791
Levin, I., Hammer, S., Kromer, B., and Meinhardt, F. (2008) Radiocarbon observations in atmospheric
792
CO2: Determining fossil fuel CO2 over Europe using Jungfraujoch observations as background.
793
Sci Total Environ 391: 211-216.
794
Lilleskov, E.A., Fahey, T.J., and Lovett, G.M. (2001) Ectomycorrhizal fungal aboveground community
795
change over an atmospheric nitrogen deposition gradient. Ecol Appl 11: 397-410.
796
Lilleskov, E.A., Hobbie, E.A., and Fahey, T.J. (2002) Ectomycorrhizal fungal taxa differing in response
797
to nitrogen deposition also differ in pure culture organic nitrogen use and natural abundance of
798
nitrogen isotopes. New Phytol 154: 219-231.
799
Lodge, D.J., and Cantrell, S. (1995) Diversity of litter agarics at Cuyabeno, Ecuador: calibrating
800
sampling efforts in tropical rainforest. Mycologist 9: 149-151.
801
Lodge, D.J., Padamsee, M., Matheny, P.B., Aime, M.C., Cantrell, S.A., Boertmann, D. et al. (2014)
802
Molecular phylogeny, morphology, pigment chemistry and ecology in Hygrophoraceae
803
(Agaricales). Fungal Diversity 64: 1-99.
804
Merckx, V., Stöckel, M., Fleischmann, A., Bruns, T.D., and Gebauer, G. (2010) 15N and 13C natural
805
abundance of two mycoheterotrophic and a putative partially mycoheterotrophic species
806
associated with arbuscular mycorrhizal fungi. New Phytol 188: 590-596.
807
Merzendorfer, H. (2011) The cellular basis of chitin synthesis in fungi and insects: common principles
808
and differences. Eur J Cell Biol 90: 759-769.
809
Murray, P.J., Clegg, C.D., Crotty, F.V., de la Fuente Martinez, N., Williams, J.K., and Blackshaw, R.P.
810
(2009) Dissipation of bacterially derived C and N through the meso-and macrofauna of a
811
grassland soil. Soil Biol Biochem 41: 1146-1150.
812
Murray, P.J., Cook, R., Currie, A.F., Dawson, L.A., Gange, A.C., Grayston, S.J., and Treonis, A.M. (2006)
813
Interactions between fertilizer addition, plants and the soil environment: Implications for soil
814
faunal structure and diversity. Appl Soil Ecol 33: 199-207.
815
Neilson, R., Boag, B., and Smith, M. (2000) Earthworm δ13C and δ15N analyses suggest that putative
816
functional classifications of earthworms are site-specific and may also indicate habitat diversity.
817
Soil Biol Biochem 32: 1053-1061.
818
Pelosi, C.l., Barot, S.b., Capowiez, Y., Hedde, M.l., and Vandenbulcke, F. (2014) Pesticides and
819
earthworms. A review. Agronomy Sust Dev 34: 199-228.
820
30
Preiss, K., and Gebauer, G. (2008) A methodological approach to improve estimates of nutrient gains
821
by partially myco-heterotrophic plants. Isotopes Environ Health Stud 44: 393-401.
822
Ram, A.F.J., Arentshorst, M., Damveld, R.A., Klis, F.M., and van den Hondel, C.A. (2004) The cell wall
823
stress response in Aspergillus niger involves increased expression of the glutamine: fructose-6-
824
phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell
825
wall. Microbiology 150: 3315-3326.
826
Rambold, G., and Agerer, R. (1997) DEEMY: The concept of a characterization and determination
827
system for ectomycorrhizae. Mycorrhiza 7: 113-116.
828
Reimer, P.J., Brown, T.A., and Reimer, R.W. (2004) Discussion: Reporting and calibration of post-
829
bomb 14C data. Radiocarbon 46: 1299-1304.
830
Roderick, K. (2009) The Ecology of Grassland Macrofungi. In PhD thesis IBERS: Aberystwyth
831
University, p. 275.
832
Schiebold, J.M.-I., Bidartondo, M.I., Karasch, P., Gravendeel, B., and Gebauer, G. (2017) You are what
833
you get from your fungi: nitrogen stable isotope patterns in Epipactis species. Ann Bot 119:
834
1085-1095.
835
Schmidt, O., Curry, J.P., Dyckmans, J., Rota, E., and Scrimgeour, C.M. (2004) Dual stable isotope
836
analysis (δ13C and δ15N) of soil invertebrates and their food sources. Pedobiologia 48: 171-180.
837
Seitzman, B.H., Ouimette, A., Mixon, R.L., Hobbie, E.A., and Hibbett, D.S. (2011) Conservation of
838
biotrophy in Hygrophoraceae inferred from combined stable isotope and phylogenetic
839
analyses. Mycologia 103: 280-290
840
Selosse, M.-A., and Martos, F. (2014) Do chlorophyllous orchids heterotrophically use mycorrhizal
841
fungal carbon? Trends Plant Sci 19: 683-685.
842
Silvertown, J., Poulton, P.R., Johnston, E., Edwards, G., Heard, M., and Biss, P.M. (2006) The Park
843
Grass Experiment 1856-2006: its contribution to ecology. J Ecol 94: 801-814.
844
Slota, P., Jull, A.J.T., Linick, T., and Toolin, L.J. (1987) Preparation of small samples for 14C accelerator
845
targets by catalytic reduction of CO. Radiocarbon 29: 303-306.
846
Staddon, P.L., Ramsey, C.B., Ostle, N., Ineson, P., and Fitter, A.H. (2003) Rapid turnover of hyphae of
847
mycorrhizal fungi determined by AMS microanalysis of C-14. Science 300: 1138-1140.
848
Stuiver, M., and Polach, H.A. (1977) Reporting of 14C data. Radiocarbon 19: 355-363.
849
Taylor, A.F.S., Fransson, P.M., Högberg, P., Högberg, M.N., and Plamboeck, A.H. (2003) Species level
850
patterns in 13C and 15N abundance of ectomycorrhizal and saprotrophic fungal sporocarps. New
851
Phytol 159: 757-774.
852
31
Taylor, A.F.S., Högbom, L., Högberg, M., Lyon, A.J.E., Nasholm, T., and Högberg, P. (1997) Natural 15N
853
abundance in fruit bodies of ectomycorrhizal fungi from boreal forests. New Phytol 136: 713-
854
720.
855
Tedersoo, L., and Nara, K. (2010) General latitudinal gradient of biodiversity is reversed in
856
ectomycorrhizal fungi. New Phytol 185: 351-354.
857
Tedersoo, L., Naadel, T., Bahram, M., Pritsch, K., Buegger, F., Leal, M. et al. (2012) Enzymatic
858
activities and stable isotope patterns of ectomycorrhizal fungi in relation to phylogeny and
859
exploration types in an afrotropical rain forest. New Phytol 195: 832-843.
860
Tedersoo, L., Nilsson, R.H., Abarenkov, K., Jairus, T., Sadam, A., Saar, I. et al. (2010) 454
861
Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but
862
reveal substantial methodological biases. New Phytol 188: 291-301.
863
Tello, S.A., Silva-Flores, P., Agerer, R., Halbwachs, H., Beck, A., and Peršoh, D. (2014) Hygrocybe
864
virginea is a systemic endophyte of Plantago lanceolata. Mycol Prog 13: 471-475.
865
Temperton, V.M., Märtin, L.L., Luecke, A., Röder, D., and Kiehl, K. (2012) Effects of four different
866
restoration treatments on the natural abundance of 15N stable isotopes in plants. Frontiers in
867
plant science 3: 70.
868
Tiunov, A.V. (2007) Stable isotopes of carbon and nitrogen in soil ecological studies. Biol Bull 34: 395-
869
407.
870
Toju, H., Sato, H., and Tanabe, A.S. (2014) Diversity and spatial structure of belowground plant
871
fungal symbiosis in a mixed subtropical forest of ectomycorrhizal and arbuscular mycorrhizal
872
plants. PloS one 9: e86566.
873
Treonis, A.M., Ostle, N.J., Stott, A.W., Primrose, R., Grayston, S.J., and Ineson, P. (2004) Identification
874
of groups of metabolically-active rhizopshere microorganisms by stable isotope probing of
875
PLFAs. Soil Biol Biochem 36: 533-537.
876
Treseder, K.K., Torn, M.S., and Masiello, C.A. (2006) An ecosystem-scale radiocarbon tracer to test
877
use of litter carbon by ectomycorrhizal fungi. Soil Biol Biochem 38: 1077-1082.
878
Trudell, S.A., Rygiewicz, P.T., and Edmonds, R.L. (2004) Patterns of nitrogen and carbon stable
879
isotope ratios in macrofungi, plants and soils in two old growth conifer forests. New Phytol 164:
880
317-335.
881
Veldre, V., Abarenkov, K., Bahram, M., Martos, F., Selosse, M.-A., Tamm, H. et al. (2013) Evolution of
882
nutritional modes of Ceratobasidiaceae (Cantharellales, Basidiomycota) as revealed from
883
publicly available ITS sequences. Fungal Ecol 6: 256-268.
884
Vera, F.W.M. (2000) Grazing Ecology and Forest History. New York: CABI Publishing.
885
32
Wang, B., and Qiu, Y.L. (2006) Phylogenetic distribution and evolution of mycorrhizas in land plants.
886
Mycorrhiza 16: 299-363.
887
Weiss, M., Waller, F., Zuccaro, A., and Selosse, M. (2016) Sebacinales-One thousand and one
888
interactions with land plants. New Phytol 211: 20-40.
889
Wingler, A., Wallenda, T., and Hampp, R. (1996) Mycorrhiza formation on Norway spruce (Picea
890
abies) roots affects the pathway of anaplerotic CO2 fixation. Physiol Plant 96: 699-705.
891
Wilson, D. (1995) Endophyte: the evolution of a term, and clarification of its use and definition.
892
Oikos: 274-276.
893
Zimmer, K., Hynson, N.A., Gebauer, G., Allen, E.B., Allen, M.F., and Read, D.J. (2007) Wide
894
geographical and ecological distribution of nitrogen and carbon gains from fungi in pyroloids
895
and monotropoids (Ericaceae) and in orchids. New Phytol 175: 166-175.
896
897
Table 1. Summary of δ15Nand δ13Canalyses.Data for 55 species from 12 countries are presented below (across six genera of
Hygrophoraceae;10 samples were not identified to species level).The total of 387 samples include 25 (in italic font) from two
published studies (Gabon, Ted e rs oo et al., 2012;USA, Seitzmann et al., 2010). δ15Nare shown first, then δ13C, with replicate number
in brackets.Samples with δ15Nvalues below 7.5‰, and below 5‰ are indicated in bold font and light or dark grey respectively.
Tab l e 2. Radiocarbon data for UK samples of three Hygrophoraceae and two
grassland saprotrophs
0 0 0 0
Fig. 1. Effect of plot treatment on abundance of basidiocarps at Sourhope over the
course of 18 autumn surveys in 2001-05.(B =Biocide, C1/C2 = control plots, L =
lime, N = nitrogen and NL =nitrogen +lime; n=5 per treatment). Error bars indicate
standard deviation.Inset table shows treatment effect Pvalues using
PERMANOVA.
Mean no. basidiocarps per 240 m2plot
Treatment
Fig. 2. Comparison of δ15N and δ13C values in waxcap basidiocarps to
ectomycorrhizal Hygrophoraceae (Hygrophorus spp.) and mean values for
saprotrophic fungi at Sourhope and Amorbach. Mean values for soil (0-5 cm) and
plant tissues are also shown. Raw isotopic data for basidiocarps is provided in
Suppdata 2 and for soil plants in Suppdata 3. Error bars indicate standard error.
Fig. 3. δ15N enrichment patterns (‰) along a soil depth gradient. Vertical bars
indicate soil depth range sampled at Amorbach (grey bars) and Sourhope (black
bars). X-axis error bars indicate standard deviation.
Soil depth (cm)
δ15N (‰)
d13C ()
d15N ()
SH[C]
WF
SH[N] 8.84
1.72
Fig. 4. Isotopic profiles of Cuphophyllus pratensis are unaffected by fertiliser
application (with a δ15N value of zero) suggesting that these fungi are unable to
utilise inorganic-N. Open and filled diamond symbols indicate samples from
Sourhope, from control (SH [C]) and nitrogen-treated (SH [N]) plots respectively.
Open circles indicate samples from an unfertilised sheep-grazed meadow in Wales
(Waunfawr, Aberystwyth), shown for comparison. The X and Y error bars illustrate
standard deviation for all samples.
Fig. 5. Comparison of intergeneric δ15N and δ13C variations in Hygrophoraceae
genera across all sites and average δ15N and δ13C for ectomycorrhizal and
saprotrophic fungi from 16 published woodland studies. Genera of family
Hygrophoraceae are shown with inverted black triangles; Mean for grassland
saprotrophic fungi (GS) is indicated with green square. Mean values for
ectomycorrhizal fungi (blue circles) and woodland saprotrophic fungi (red triangles)
from published studies are also shown. Error bars in grey indicate standard error.
For Hygrophoraceae genera are labelled as follows: Cuphophyllus (Cu), Gliophorus
(Gl), Humidicutis (Hu), Hygrocybe (Hc), Hygrophorus (Hp), Neohygrocybe (Ne),
Porpolomopsis (Po). Raw data and keys to published study labels (including
numbers of replicates) are presented in Suppdata 10.
Po
δ15N (‰)
δ13C (‰)
Hc
Hu
Cu
Gl
Hp
Ag
H1
Av
Av
Cl
Cl
H5
H5
Ma
Ha
Tp
Wi
Wi
TpHe
Ko
He
Ta
Ko
Se
Se
H5
Ta
Ze
Ze
H9
Tr
Ma
H1
Tr
Ag H9
Ne
Ha
GS
Po
Suppdata 1: Details of Sourhope surveys
Details of vegetation analyses and site management regimes at the Sourhope site are given in
Burt-Smith, G. (2001;Report III.Results from the Sourhope Field Experiment:1999-2001.
unpublished;24pp;available from http://soilbionerc.ac.uk/Download/Report - 3 - allpdf) and
Burt-Smith, G. (2003;NERC Soil Biodiversity Thematic Programme.Report III.Results from the
Sourhope field experiment 1999-2003.http://users.aber.ac.uk/gwg/pdf/NERC Soil Biodiversity
Programme 1998-2004 datasets.zip) respectively.
Suppdata 2. δ13C and δ15N values of associated (A) soil and (B)plants tissues from
waxcap grasslands in UK, Germany and Iceland.
Suppdata 3. Raw data for isotopic analyses
Genus Species Country Lat/Long Coll Date
Cap
δ15N (‰)
Cap
d13C (‰)
Cap %
total N
Cap %
total C
Cap
C/N
ratio
Notes
SOURHOPE
1Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 26-Sep-02 16.04 P-29.2 6.21 44.22 7.12 13C-pulse e nriched
2Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 11-Oct-02 16.28 P-28.7 5.86 42.93 7.33 13 C-pulse e nriched
3Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 11-Oct-02 16.77 P-28.96 6.45 35.11 5.44 13 C-pulse e nriched
4Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 11-Oct-02 16.54 P-28.77 7.46 41.24 5.53 13 C-pulse e nriched
5Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 16.09 -2 7. 66 6.40 41.78 6.53
6Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 14.23 -2 7. 93 5.60 41.57 7.43
7Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 26-Sep-02 14.28 P-92.79 5.56 38.63 6.95 13C-pulse e nriched
8Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 25-Oct-02 14.63 P-28.26 4.50 41.26 9.17 13 C-pulse e nriched
9Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 11-Oct-02 19.81 P-29.18 7.18 43.43 6.05 13 C-pulse e nriched
10 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 16.17 -2 8. 04 6.39 41.84 6.55
11 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 17.52 -2 8. 75 7.18 43.33 6.04
12 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 15.58 -2 9. 43 4.88 42.83 8.78
13 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 11.72 -2 8. 17 6.28 44.03 7.01
14 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 28-Aug-02 14.14 -28 .6 1 5.19 43.83 8.45
15 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 02-Oct-01 16.58 -2 9. 37 7.59 44.73 5.89
16 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 02-Oct-01 14.44 -2 8. 36 7.08 45.48 6.42
17 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 02-Oct-01 18.08 -2 9. 37 6.40 45.60 7.13
18 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 18-Oct-01 F1 4.8 9 -27 .6 2 7.12 42.02 5.90 Fertili ser plot
19 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 18-Oct-01 F1 5.6 1 -28 .3 8 6.40 45.64 7.13 Fertili ser plot
20 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 18-Oct-01 F1 5.7 3 -28 .6 3 5.19 43.91 8.47 Fertili ser plot
21 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 13-Sep-01 F1 6.4 3 -29 .2 1 6.69 44.19 6.60 Fertili ser plot
22 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 01-Oct-01 12.15 -2 9. 10 6.28 43.08 6.86
23 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 12-Sep-02 18.23 -29 .2 9 7.29 43.13 5.92
24 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 14.78 -2 8. 54 5.05 42.95 8.50
25 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 15.08 -2 8. 24 5.60 43.31 7.73
26 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 15.22 -2 8. 13 5.92 43.89 7.41
27 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 15.73 -2 8. 55 6.85 41.66 6.08
28 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 20.56 -2 9. 24 6.83 42.85 6.27
29 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 16.50 -2 9. 19 6.20 43.95 7.09
30 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 12.82 -2 8. 80 6.64 41.37 6.23
31 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 13.23 -2 8. 84 6.41 42.91 6.69
32 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 13.97 -2 7. 81 7.01 42.59 6.08
33 Cupho phyllus p ratensis Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 14.38 -2 8. 50 5.98 41.16 6.89
34 Cupho phyllus virgine us Sco tland Sourhope 55.4700, -2.2312 18 -Oct-01 F17. 07 -2 6.8 6 6.16 42.11 6.83 Fe rtiliser plot
35 Cupho phyllus virgine us Sco tland Sourhope 55.4700, -2.2312 01 -Oct-01 F16. 53 -2 7.9 9 6.08 43.76 7.20 Fe rtiliser plot
36 Cupho phyllus virgine us Sco tland Sourhope 55.4700, -2.2312 18 -Oct-01 16.32 -27 .66 5.44 45.77 8.41
37 Gliophorus laet us Sco tland Sourhope 55.4700, -2.2312 02 -Oct-01 16.78 -27 .5 9 5.49 42.02 7.65
38 Gliophorus laet us Sco tland Sourhope 55.4700, -2.2312 02 -Oct-01 14.77 -28 .5 6 5.85 48.52 8.29
39 Gliophorus laet us Sco tland Sourhope 55.4700, -2.2312 01 -Oct-01 F13 .11 -2 7. 89 5.39 43.30 8.04 Fertilis er plot
40 Gliophorus laet us Sco tland Sourhope 55.4700, -2.2312 18 -Oct-01 F14 .99 -2 8. 17 4.17 41.70 10.00 Fertilis er plot
41 Gliophorus laet us Sco tland Sourhope 55.4700, -2.2312 02 -Oct-01 14.12 -28 .9 6 4.95 48.61 9.82
42 Gliophorus psittacinu s Scotlan d Sourhope 55.4700, -2.2312 26-Sep-02 16.57 -27 .6 1 5.62 44.40 7.90
43 Gliophorus psittacinu s Scotlan d Sourhope 55.4700, -2.2312 13-Sep-01 18.99 -27 .2 1 5.75 41.89 7.29
44 Gliophorus psittacinu s Scotlan d Sourhope 55.4700, -2.2312 30-Oct-01 15.45 -2 7. 72 6.07 41.68 6.87
45 Hygrocybe cera cea Scotlan d Sourhope 55.4700, -2.2312 1 1-Oct-02 11.58 nd 5.81 nd nd
46 Hygrocybe cera cea Scotlan d Sourhope 55.4700, -2.2312 27-Sep-05 13.06 -27 .7 4 7.19 42.81 5.95
47 Hygrocybe cera cea Scotlan d Sourhope 55.4700, -2.2312 0 1-Oct-01 F11 .48 -2 8 .48 5.45 43.60 8.00 Fertili ser plot
48 Hygrocybe con ica Scotlan d Sourhope 55.4700, -2.2312 0 2-Oct-01 14.23 -28 .8 5 5.96 45.86 7.69
49 Hygrocybe con ica Scotlan d Sourhope 55.4700, -2.2312 0 2-Oct-01 11.57 -29 .2 8 5.45 49.19 9.03
50 Hygrocybe con ica Scotlan d Sourhope 55.4700, -2.2312 0 2-Oct-01 12.49 -30 .0 9 5.41 47.66 8.81
51 Hygrocybe sple ndidissima S cotlan d Sourhope 55.4700, -2.2312 02 -Oct-01 12.09 -28 .4 2 6.05 48.66 8.04
Mea n 15.23 -28 .4 4 6.08 43.44 7.27
SD 2.16 0.69 0.78 2.48 1.10
COUNT 42 43 51 50 50
Min 11.57 -30 .0 9 4.17 35.11 5.44
Max 20.56 -2 6.8 6 7.59 49.19 10.00
NORTHERN EUROPE A (WALES) UK
52 Cupho phyllus lacmu s Wale s Maes Meddyg on, Rh iwlas 53.1610,-4.1223 09-Nov-10 13.70 -2 9.6 2 3.29 39.13 11.90
53 Cuphophyllus pratensis Wale s Bron ydd Mawr 51.9840,-3.6318 31-Oct-02 13.54 -2 9. 51 6.16 42.70 6.93
54 Cuphophyllus pratensis Wale s Bron ydd Mawr 51.9840,-3.6318 31-Oct-02 15.53 -2 9. 48 5.42 42.82 7.90
55 Cuphophyllus pratensis Wale s Bron ydd Mawr 51.9840,-3.6318 31-Oct-02 15.71 -3 0. 24 5.95 44.08 7.41
56 Cuphophyllus pratensis Wale s Wa un fa wr 52.4149,-4.0476 02-Nov-01 14.14 -2 7.9 5 6.10 44.45 7.29
57 Cuphophyllus pratensis Wale s Wa un fa wr 52.4149,-4.0476 02-Nov-01 15.16 -2 8.2 3 7.84 45.26 5.77
58 Cuphophyllus pratensis Wale s Wa un fa wr 52.4149,-4.0476 02-Nov-01 15.33 -2 8.5 5 6.21 41.61 6.70
59 Cuphophyllus pratensis Wale s Wa un fa wr 52.4149,-4.0476 02-Nov-01 15.46 -2 8.9 1 5.25 42.89 8.17
60 Cuphophyllus pratensis Wale s Wa un fa wr 52.4149,-4.0476 02-Nov-01 16.37 -2 8.1 6 7.33 44.49 6.07
61 Cuphophyllus pratensis Wale s Ystu mtuen 52.4021,-3.8672 06-Nov-02 16.04 -29 .5 9 5.25 43.97 8.38
62 Cuphophyllus pratensis Wale s Ystu mtuen 52.4021,-3.8672 06-Nov-02 16.91 -29 .5 2 5.12 43.25 8.45
63 Cuphophyllus pratensis Wale s Ystu mtuen 52.4021,-3.8672 06-Nov-02 17.40 -29 .3 0 7.05 42.39 6.01
64 Cuphophyllus virgineus Wa les B ronydd Mawr 51.9840,-3.6318 16-Oct-03 14.44 -30 .0 2 4.50 45.65 10.14
65 Cuphophyllus virgineus Wa les B ronydd Mawr 51.9840,-3.6318 16-Oct-03 14.51 -30 .4 0 5.97 43.80 7.34
66 Cuphophyllus virgineus Wa les B ronydd Mawr 51.9840,-3.6318 16-Oct-04 15.39 -29 .8 1 5.81 46.51 8.01
67 Cuphophyllus virgineus Wa les B ronydd Mawr 51.9840,-3.6318 16-Oct-03 17.06 -28 .5 5 5.66 45.06 7.96
68 Cuphophyllus virgineus Wa les Wa u nf awr 52.4149,-4.0476 02-Nov-01 10.90 -26 .54 5.16 41.83 8.11
69 Cuphophyllus virgineus Wa les Wa u nf awr 52.4149,-4.0476 02-Nov-01 11.77 -27 .73 4.61 45.62 9.89
70 Cuphophyllus virgineus Wa les Wa u nf awr 52.4149,-4.0476 02-Nov-01 12.11 -27 .04 6.07 44.10 7.26
71 Cuphophyllus virgineus Wa les Wa u nf awr 52.4149,-4.0476 02-Nov-01 12.76 -28 .17 4.80 44.27 9.22
72 Cuphophyllus virgineus Wa les Porthmadog 52.9115,-4.0997 20-Sep-03 W14. 7 7 W-26. 56 W5.3 6 W43 .3 0 W8 .0 8 WOODL AND
73 Hygrocybe can tha rellus Wa les L lyn Cwm Dulyn 53.0214,-4.2421 05-Sep-10 M0.5 0 M-2 8. 78 M8 .0 0 M43.9 1 M5. 90 MOSS
74 Hygrocybe can tha rellus Wa les L lyn Cwm Dulyn 53.0214,-4.2421 05-Sep-10 M1.8 2 M-2 7. 23 M10.0 7 M43 .7 3 M4. 34 MOS S
75 Hygrocybe chlo ropha na Wa le s Abe rystwyth University 52.4194,-4.0683 01-Oct-01 14.19 -29 .0 5 8.78 44.80 5.10
76 Hygrocybe chlo ropha na Wa le s Abe rystwyth University 52.4194,-4.0683 01-Oct-01 15.97 -28 .7 5 7.88 41.74 5.30
77 Hygrocybe chlo ropha na Wa le s Abe rystwyth University 52.4194,-4.0683 01-Oct-01 16.48 -28 .7 4 8.75 44.61 5.10
78 Hygrocybe chlo ropha na Wa le s Abe rystwyth University 52.4194,-4.0683 01-Oct-01 17.09 -29 .2 4 9.12 44.10 4.84
79 Hygrocybe chlo ropha na Wa le s Llanigon 52.0527,-3.1463 05-Jun-04 W12. 21 W-25 . 79 W7 .6 0 W45 .2 2 W5 .9 5 WOODL AND
80 Hygrocybe chlo ropha na Wa le s Bangor 53.2179,-4.1664 14-May-04 W1 3. 22 W-2 9. 01 W7 .8 9 W45 .9 1 W5 .8 2 WOODL AND
81 Hygrocybe citrinoviren s Wale s Ystu mtuen 52.4021,-3.8672 17-Oct-02 13.63 -28 .8 5 5.89 42.17 7.16
82 Hygrocybe coccin ea Wa le s Neb o 52.2652,-4.1317 27-Nov-02 10.93 -30 .1 4 6.16 43.32 7.03
83 Hygrocybe coccin ea Wa le s Wau nf a wr 52.4149,-4.0476 05-Dec-02 11.13 -28 .6 9 7.14 46.38 6.50
84 Hygrocybe coccin ea Wa le s Ystumtu en 52.4021,-3.8672 06-Nov-02 13.62 -27. 94 7.24 43.26 5.98
85 Hygrocybe glutinipes Wale s Wa un fa wr 52.4149,-4.0476 11-Feb-01 19.52 -28 .0 6 5.43 44.74 8.24
86 Hygrocybe quieta Wale s Llanigon 52.0527,-3.1463 10-Aug-03 W16. 4 8 W-2 7. 27 W6 .2 3 W4 4. 97 W7 .2 2 WOODL AN D
87 Neohygrocybe nitrata Wales Bangor 53.1629, -4.1236 11-Oct-06 15.90 -29.52 8.45 43.40 5.1
88 Porpolomopsis calyptriformis Wales Abergorlech 51.9834,-4.0615 16-Nov-02 17.07 -29.06 6.96 43.76 6.29
89 Porpolomopsis calyptriformis Wales Aberystwyth University 52.4194,-4.0683 4-Dec-01 14.93 -27.98 5.33 42.46 7.97
90 Porpolomopsis calyptriformis Wales Aberystwyth University 52.4194,-4.0683 4-Dec-01 15.48 -27.14 7.41 43.13 5.82
91 Porpolomopsis calyptriformis Wales Disgwylfa, Epynt 52.0829,-3.4680 23-Nov-02 16.55 -29.55 7.27 44.65 6.14
92 Porpolomopsis calyptriformis Wales Waunfawr 52.4149,-4.0476 2-Nov-01 14.23 -28.97 4.60 45.13 9.82
93 Porpolomopsis calyptriformis Wales Y Fron 53.0657,-4.2355 12-Oct-02 14.27 -29.44 7.57 43.62 5.76
Mean 14.87 -28.85 6.32 43.75 7.25
SD 1.97 0.92 1.37 1.48 1.62
COUNT 36 36 36 36 36
Min 10.90 -30.40 3.29 39.13 4.84
Max 19.52 -26.54 9.12 46.51 11 .9 0
NORTHERN EUROPE B (ENGLAND) UK
94 Cuphophyllus lacmus England Lundy 51.1999,-4.6744 11-Nov-04 H9.47 H-28.92 H6.16 H43.11 H6.99 IN HEATHER
95 Cuphophyllus lacmus England Lundy 51.1999,-4.6738 11-Nov-04 H9.66 H-27.50 H5.83 H39.77 H6.82 IN HEATHER
96 Cuphophyllus pratensis England Cocklepark 55.2174,-1.6838 20-Oct-04 16.23 -29.56 5.64 43.63 7.74
97 Cuphophyllus pratensis England Park Grass 51.8040,-0.3722 19-Nov-04 17.20 -29.72 5.27 43.41 8.24
98 Cuphophyllus pratensis England Park Grass 51.8040,-0.3722 19-Nov-04 18.77 -29.45 6.52 41.92 6.43
99 Cuphophyllus pratensis England Clitheroe 53.8067,-2.4294 24-Oct-02 18.82 -28.29 7.17 43.06 6.01
100 Cuphophyllus pratensis England Park Grass 51.8040,-0.3722 19-Nov-04 19.64 -30.28 6.35 43.08 6.78
101 Cuphophyllus pratensis England Clitheroe 53.8067,-2.4294 10-Oct-02 19.71 -27.92 6.35 42.98 6.77
102 Cuphophyllus pratensis England Clitheroe 53.8067,-2.4294 24-Oct-02 19.76 -29.77 7.43 42.39 5.71
103 Cuphophyllus virgineus England Cocklepark 55.2174,-1.6838 4-Nov-03 14.26 -28.54 4.97 43.79 8.81
104 Cuphophyllus virgineus England Cocklepark 55.2174,-1.6838 4-Nov-03 14.82 -29.65 4.83 45.34 9.39
105 Cuphophyllus virgineus England Cocklepark 55.2174,-1.6838 20-Oct-04 15.96 -30.21 5.92 42.94 7.25
106 Cuphophyllus virgineus England Lundy 51.1694,-4.6720 10-Nov-04 16.37 -27.39 5.26 43.93 8.35
107 Gliophorus psittacinus England Cocklepark 55.2174,-1.6838 26-Nov-07 15.85 -29.61 4.84 31.36 6.5
108 Gliophorus psittacinus England Cocklepark 55.2174,-1.6838 26-Nov-07 20.94 -29.26 8.21 45.70 5.6
109 Hygrocybe citrinovirens England Clitheroe 53.8067,-2.4294 9-Nov-02 13.68 -28.33 6.90 42.19 6.11
110 Hygrocybe coccinea England Cocklepark 55.2174,-1.6838 20-Oct-04 14.19 -28.99 7.05 44.43 6.30
111 Hygrocybe punicea England Cocklepark 55.2174,-1.6838 20-Oct-04 11 .8 6 -28.59 6.35 45.33 7.14
112 Hygrocybe punicea England Park Grass 51.8040,-0.3722 19-Nov-04 13.09 -29.62 6.98 43.02 6.16
113 Hygrocybe punicea England Park Grass 51.8040,-0.3722 19-Nov-04 13.81 -29.48 6.72 45.64 6.79
114 Hygrocybe punicea England Park Grass 51.8040,-0.3722 5-Nov-08 13.93 -29.59 8.86 48.09 5.4
115 Hygrocybe punicea England Park Grass 51.8040,-0.3722 5-Nov-08 14.01 -29.84 9.72 43.62 4.5
116 Hygrocybe punicea England Park Grass 51.8040,-0.3722 5-Nov-08 14.83 -30.59 8.35 44.69 5.4
117 Hygrocybe punicea England Park Grass 51.8040,-0.3722 19-Nov-04 15.20 -28.21 7.24 45.21 6.24
118 Hygrocybe punicea England Park Grass 51.8040,-0.3722 5-Nov-08 16.90 -29.73 8.72 44.91 5.1
119 Hygrocybe punicea England Lundy 51.1694,-4.6720 11-Nov-04 18.01 -28.12 7.49 45.69 6.10
120 Porpolomopsis calyptriformis England Clitheroe 53.8067,-2.4294 10-Oct-02 16.71 -30.20 5.66 41.88 7.40
121 Porpolomopsis calyptriformis England Clitheroe 53.8067,-2.4294 10-Oct-02 17.83 -28.77 7.09 44.81 6.32
Mean 16.25 -29.22 6.77 43.58 6.63
SD 2.40 0.83 1.30 2.88 1.16
COUNT 26 26 26 26 26
Min 11. 86 -30.59 4.83 31.36 4.49
Max 20.94 -27.39 9.72 48.09 9.39
NORTHERN EUROPE (C) GERMANY
122 Cuphophyllus flavipes Germany Amorbach 49.6460, 9.2010 1-Oct-08 14.39 -30.12 5.80 41.67 7.19
123 Cuphophyllus fornicata Germany Amorbach 49.6460, 9.2010 2-Oct-08 19.67 -29.98 6.44 43.70 6.78
124 Cuphophyllus pratensis Germany Amorbach 49.6460, 9.2010 4-Oct-08 15.23 -29.86 6.80 41.61 6.12
125 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 8-Oct-08 13.71 -28.89 6.89 44.60 6.47
126 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 6-Oct-08 14.82 -29.16 6.09 44.17 7.26
127 Gliophorus psittacinus Germany Amorbach 49.6460, 9.2010 12-Oct-08 15.99 -28.56 7.07 45.86 6.49
128 Hygrocybe aurantiosplendens Germany Amorbach 49.6460, 9.2010 1-Oct-08 13.08 -28.93 6.87 39.80 5.79
129 Hygrocybe chlorophana Germany Amorbach 49.6460, 9.2010 7-Oct-08 13.62 -29.00 8.78 43.95 5.01
130 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 10-Oct-08 13.81 -28.86 8.18 45.73 5.59
131 Hygrocybe insipida Germany Amorbach 49.6460, 9.2010 11-Oct-08 12.10 -28.74 7.57 42.86 5.66
132 Hygrocybe mucronella Germany Amorbach 49.6460, 9.2010 9-Oct-08 19.23 -28.89 6.51 45.71 7.02
133 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 17.43 -30.79 4.94 44.78 9.07 Transect1 A1
134 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 16.90 -30.48 5.90 43.81 7.42 Transect1 A2
135 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 15.31 -30.06 4.67 44.82 9.60 Transect1 A3
136 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.67 -30.55 4.65 44.08 9.49 Transect1 A4
137 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 15.62 -30.58 4.86 44.70 9.20 Transect1 A5
138 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 15.62 -30.14 5.44 43.94 8.08 Transect1 A6
139 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.24 -30.13 4.82 44.21 9.18 Transect1 A7
140 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 15.32 -29.97 5.63 43.79 7.77 Transect1 A8
141 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 15.11 -30.02 4.96 44.14 8.90 Transect1 A9
142 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.70 -29.76 5.34 44.25 8.29 Transect1 A10
143 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 15.69 -30.07 5.74 44.10 7.68 Transect1 A11
144 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.56 -30.05 4.64 43.29 9.34 Transect1 A12
145 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.30 -30.07 4.75 44.71 9.42 Transect1 A13
146 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.28 -30.24 4.86 45.71 9.41 Transect1 A14
147 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 15.03 -29.88 5.36 44.31 8.26 Transect1 A15
148 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 12.99 -30.47 4.97 43.74 8.79 Transect2 B1
149 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.90 -30.58 4.80 42.32 8.81 Transect2 B2
150 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.08 -30.39 5.04 44.38 8.80 Transect2 B3
151 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.41 -30.62 5.02 44.22 8.81 Transect2 B4
152 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.19 -30.58 5.20 44.63 8.58 Transect2 B5
153 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.37 -30.83 5.14 44.27 8.61 Transect2 B6
154 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 12.96 -31.11 4.86 44.85 9.23 Transect2 B7
155 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 12.62 -30.80 4.44 44.52 10.02 Transect2 B8
156 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 12.83 -30.97 4.92 45.29 9.21 Transect2 B9
157 Cuphophyllus virgineus Germany Amorbach 49.6460, 9.2010 7-Nov-15 11 .5 3 -30.93 5.99 45.14 7.53 Transect2 B10
158 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.16 -30.65 6.45 46.40 7.19 Fairy ring R1
159 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 13.85 -30.32 6.91 45.85 6.63 Fairy ring R2
160 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 13.67 -30.48 6.98 45.12 6.46 Fairy ring R3
161 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 13.25 -30.88 6.21 45.56 7.34 Fairy ring R4
162 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 11. 77 -30.65 6.11 44.89 7.34 Fairy ring R5
163 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.29 -31.29 5.41 42.89 7.93 Fairy ring R6
164 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 11. 68 -30.70 6.92 46.65 6.74 Fairy ring R7
165 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 12.05 -31.02 6.01 42.63 7.10 Fairy ring R8
166 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 12.89 -30.78 6.52 45.93 7.04 Fairy ring R9
167 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 14.40 -30.45 6.46 45.89 7.11 Fairy ring R10
168 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 13.50 -30.46 6.62 45.88 6.93 Fairy ring R11
169 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 13.17 -31.04 5.78 44.84 7.76 Fairy ring R12
170 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 11. 76 -30.85 6.00 45.68 7.61 Fairy ring R13
171 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 12.37 -31.12 6.02 45.40 7.54 Fairy ring R14
172 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 13.46 -30.40 7.42 45.87 6.18 Fairy ring R15
173 Hygrocybe coccinea Germany Amorbach 49.6460, 9.2010 7-Nov-15 11. 41 -30.61 6.13 45.09 7.36 Fairy ring R16
174 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.34 -29.06 4.20 41.28 9.82 Fairy ring
175 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.75 -29.36 4.72 42.19 8.93 Fairy ring
176 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 12.51 -29.51 4.27 41.54 9.74 Fairy ring
177 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 11. 98 -29.60 3.76 41.00 10.91 Fairy ring
178 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.20 -29.03 4.37 42.54 9.74 Fairy ring
179 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 14.21 -29.79 4.33 41.59 9.61 Fairy ring
180 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.00 -29.26 4.28 42.70 9.97 Fairy ring
181 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.14 -28.98 4.07 41.07 10.10 Fairy ring
182 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.05 -29.64 4.41 42.11 9.55 Fairy ring
183 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 12.38 -29.08 4.47 42.15 9.44 Fairy ring
184 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.17 -29.05 4.52 42.41 9.37 Fairy ring
185 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 12.91 -29.28 4.38 42.79 9.78 Fairy ring
186 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 12.57 -29.51 3.87 42.50 10.97 Fairy ring
187 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 12.95 -28.98 4.37 42.52 9.73 Fairy ring
188 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 15.21 -29.50 5.04 41.63 8.27 Fairy ring
189 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.33 -29.22 4.46 41.78 9.36 Fairy ring
190 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 14.73 -29.45 5.01 42.77 8.54 Fairy ring
191 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.82 -29.62 4.33 42.30 9.77 Fairy ring
192 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 13.57 -29.30 4.52 41.55 9.19 Fairy ring
193 Cuphophyllus virgineus (ring) Germany Amorbach 49.6452,9.2007 31/10/15 15.39 -28.96 4.59 41.57 9.06 Fairy ring
194 Cuphophyllus virgineus Germany Amorbach 49.6452,9.2007 31/10/15 16.54 -28.70 4.61 42.19 9.16
195 Cuphophyllus virgineus Germany Amorbach 49.6452,9.2007 31/10/15 17.52 -28.48 4.77 41.86 8.78
196 Cuphophyllus virgineus Germany Amorbach 49.6452,9.2007 31/10/15 15.36 -28.65 4.41 42.84 9.72
197 Cuphophyllus virgineus Germany Amorbach 49.6452,9.2007 31/10/15 15.00 -28.25 4.18 42.87 10.25
198 Cuphophyllus virgineus Germany Amorbach 49.6452,9.2007 31/10/15 17.01 -28.64 4.17 41.60 9.97
199 Cuphophyllus virgineus Germany Amorbach 49.6452,9.2007 31/10/15 14.52 -28.24 4.26 42.75 10.03
200 Cuphophyllus virgineus Germany Amorbach 49.6452,9.2007 31/10/15 15.66 -29.81 4.39 41.41 9.43
201 Cuphophyllus virgineus Germany Amorbach 49.6452,9.2007 31/10/15 13.19 -28.81 3.91 42.52 10.87
202 Cuphophyllus virgineus Germany Amorbach 49.6452,9.2007 31/10/15 17.12 -28.73 4.69 41.91 8.94
203 Cuphophyllus virgineus Germany Amorbach 49.6464,9.2019 7/11/15 14.67 -27.50 4.94 40.02 8.10
204 Cuphophyllus virgineus Germany Amorbach 49.6464,9.2019 7/11/15 16.57 -28.12 4.74 41.01 8.66
205 Cuphophyllus virgineus Germany Amorbach 49.6464,9.2019 7/11/15 16.61 -28.36 4.54 41.04 9.03
206 Cuphophyllus virgineus Germany Amorbach 49.6464,9.2019 7/11/15 16.54 -28.55 4.93 42.27 8.57
207 Cuphophyllus virgineus Germany Amorbach 49.6464,9.2019 7/11/15 14.92 -28.27 4.64 40.17 8.66
208 Cuphophyllus virgineus Germany Amorbach 49.6464,9.2019 7/11/15 16.91 -27.71 5.60 42.88 7.65
209 Cuphophyllus virgineus Germany Amorbach 49.6464,9.2019 7/11/15 17.50 -28.27 5.00 41.20 8.23
210 Cuphophyllus virgineus Germany Amorbach 49.6464,9.2019 7/11/15 16.31 -28.10 4.27 40.89 9.58
211 Cuphophyllus virgineus Germany Amorbach 49.6464,9.2019 7/11/15 17.57 -28.29 4.95 41.77 8.44
212 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 15.90 -28.85 5.28 41.37 7.84
213 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 15.20 -28.21 5.39 42.10 7.81
214 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 15.70 -28.70 5.16 42.81 8.30
215 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 15.83 -28.40 4.87 40.97 8.41
216 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 14.68 -29.58 4.43 42.32 9.55
217 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 13.90 -31.19 3.83 39.10 10.22
218 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 15.50 -29.08 4.03 41.96 10.40
219 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 14.55 -28.67 3.71 42.01 11 . 33
220 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 14.65 -29.11 3.58 39.18 10.96
221 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 13.76 -28.94 3.70 40.74 11 . 02
222 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 15.20 -29.03 5.17 43.00 8.31
223 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 15.89 -28.33 5.01 41.99 8.39
224 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 14.20 -28.91 3.81 42.32 11 . 12
225 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 14.87 -28.93 4.43 44.70 10.09
226 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 14.51 -30.09 4.99 42.24 8.47
227 Cuphophyllus virgineus Germany Amorbach 49.6453,9.2009 7/11/15 13.69 -29.35 3.81 40.42 10.61
228 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 13.53 -29.66 5.56 42.78 7.69
229 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 13.19 -29.68 5.80 43.35 7.47
230 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 14.08 -29.31 5.69 44.07 7.75
231 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 14.14 -29.44 4.80 43.04 8.96
232 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 12.79 -29.73 5.39 42.95 7.98
233 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 13.72 -29.39 4.93 41.69 8.45
234 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 13.43 -27.97 7.09 43.56 6.14
235 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 13.77 -29.68 6.30 44.55 7.07
236 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 13.89 -29.09 5.40 41.95 7.77
237 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 14.36 -29.57 5.92 43.80 7.39
238 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 14.45 -29.37 5.88 43.00 7.31
239 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 13.58 -29.37 6.78 44.83 6.62
240 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 13.73 -29.53 5.77 43.16 7.48
241 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 12.77 -29.50 6.74 43.88 6.51
242 Hygrocybe coccinea Germany Amorbach 49.6453,9.2009 7/11/15 13.23 -29.06 6.27 42.45 6.77
243 Hygrocybe chlorophana Germany Amorbach 49.6453,9.2009 7/11/15 14.53 -28 .56 7.21 42.97 5.96
244 Hygrocybe chlorophana Germany Amorbach 49.6453,9.2009 7/11/15 15.15 -28 .29 6.87 41.61 6.06
245 Hygrocybe chlorophana Germany Amorbach 49.6453,9.2009 7/11/15 14.78 -29 .48 6.44 40.54 6.30
246 Hygrocybe chlorophana Germany Amorbach 49.6453,9.2009 7/11/15 15.24 -28 .96 7.56 42.88 5.67
247 Hygrocybe chlorophana Germany Amorbach 49.6453,9.2009 7/11/15 14.71 -29 .27 7.10 42.04 5.92
248 Hygrocybe chlorophana Germany Amorbach 49.6453,9.2009 7/11/15 14.89 -28 .94 7.98 42.29 5.30
249 Hygrocybe cantharellus Germany Amorbach 49.6452,9.2010 25/10/14 6.08 -31.16 5.91 42.85 7.25
250 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 17.05 -29.94 4.50 41.57 9.24
251 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 15.99 -30.36 5.22 41.06 7.86
252 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.73 -29.93 4.44 41.21 9.28
253 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 15.58 -30.11 4.11 40.15 9.77
254 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.65 -29.85 4.17 41.74 10.02
255 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.73 -29.84 4.33 45.69 10.54
256 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 15.50 -30.07 4.25 40.85 9.61
257 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 15.23 -29.89 4.90 41.23 8.41
258 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.53 -29.80 4.03 41.43 10.29
259 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.55 -29.86 4.39 41.57 9.47
260 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.75 -29.78 4.55 41.83 9.20
261 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 15.56 -29.95 3.82 40.67 10.65
262 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 17.09 -30.45 5.23 42.87 8.20
263 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.99 -29.71 4.17 40.57 9.73
264 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 17.77 -29.34 5.39 41.68 7.73
265 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.39 -29.99 4.23 41.19 9.74
266 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.03 -30.18 5.80 41.59 7.16
267 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 16.55 -30.34 4.64 41.55 8.96
268 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 14.93 -30.31 3.90 41.27 10.57
269 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/11/15 15.22 -30.09 4.13 40.81 9.87
270 Cuphophyllus pratensis Germany Bischbrunn, Spessart 49.8882,9.4678 6/1