Urichuk, Theresa M.; Brandon University, Biology Keyword ...81 Plasti Dip (Plasti Dip International, Blaine, USA) to prevent moisture damage (Roznik and 82 Alford 2012). Data loggers

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Diversity of fungi from the mound nests of Formica ulkei

and adjacent non-nest soils

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2015-0628.R2

Manuscript Type: Article

Date Submitted by the Author: 25-Feb-2016

Complete List of Authors: Duff, Lyndon B.; Brandon University, Biology Urichuk, Theresa M.; Brandon University, Biology Hodgins, Lisa N.; Brandon University, Biology Young, Jocelyn R.; Brandon University, Biology Untereiner, Wendy; Brandon University, Biology

Keyword: Aspergillus, fungal biodiversity, xerotolerant, mound-building ant

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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Duff et al.; Fungi from nests of Formica ulkei 1

Diversity of fungi from the mound nests of Formica ulkei and adjacent non-nest soils 1

2

Lyndon B. Duff, Theresa M. Urichuk, Lisa N. Hodgins, Jocelyn R. Young, and Wendy A. 3

Untereiner1 4

Department of Biology, Brandon University, 270 18th Street, Brandon, Manitoba, R7A 6A9, 5

Canada 6

7

L.B. Duff ([email protected]) 8

T.M. Urichuk ([email protected]) 9

L.N. Hodgins ([email protected]) 10

J.R. Young ([email protected]) 11

W.A. Untereiner ([email protected]) 12

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14

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16

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20

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1 Corresponding author 22

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Abstract 23

Culture-based methods were employed to recover 3929 isolates of fungi from soils collected 24

in May and July 2014 from mound nests of Formica ulkei and adjacent non-nest sites. The 25

abundance, diversity, and richness of species from nest mounds exceeded those of non-26

mound soils, particularly in July. Communities of fungi from mounds were more similar to 27

those from mounds than non-mounds; this was also the case for non-mound soils with the 28

exception of one non-mound site in July. Species of Aspergillus, Paecilomyces and 29

Penicillium were dominant in nest soils and represented up to 81.8% of the taxa recovered. 30

Members of the genus Aspergillus accounted for the majority of Trichocomaceae from nests 31

and were represented almost exclusively by Aspergillus navahoensis and A. pseudodeflectus. 32

Dominant fungi from non-mound sites included Cladosporium cladosporioides, Geomyces 33

pannorum and species of Acremonium, Fusarium, Penicillium and Phoma. Although mound 34

nests were warmer than adjacent soils, the dominance of xerotolerant Aspergillus in soils 35

from mounds and the isolation of the majority of Trichocomaceae at 25˚C and 35˚C suggests 36

that both temperature and water availability may be determinants of fungal community 37

structure in nests of F. ulkei. 38

39

40

Key words: Aspergillus, fungal biodiversity, mound-building ant, xerotolerant 41

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Introduction 48

The mound-building ant Formica ulkei Emery (Hymenoptera: Formicidae) ranges from Alberta 49

to Nova Scotia (Canada) and southward to Illinois, Indiana and Iowa (USA) (Holmquist 1928; 50

Sherba 1958; Glasier et al. 2013). This species builds conspicuous nests in meadows and 51

pastures along the margins of forests and sparsely wooded areas (Holmquist 1928; Dreyer 52

and Park 1932; Sherba 1958). Nests are composed of excavated soil and covered by a layer 53

of thatch (i.e., small pieces of grass and other plant material) (Sherba 1958, 1959, 1962). 54

The mound nests of F. ulkei are thermoregulatory in function and are constructed to 55

achieve and maintain higher temperatures than adjacent undisturbed soils during the months 56

when the ants are most active (Sherba 1962). Nests are built in exposed sites and are 57

oriented to maximize their exposure to solar radiation (Sherba 1958); they gain heat from 58

solar radiation in the early spring and maintain temperatures that are higher and more stable 59

than those of surrounding soils because of the insulating properties of thatch (Sherba 1962; 60

Frouz and Jilková 2008). This layer of organic material prevents the overheating of mounds 61

during the warmest parts of the year in other ant species that construct thatched nests 62

(Bollazzi and Rocces 2010; Kadochová and Frouz 2014) and it may serve the same function 63

in F. ulkei. 64

Although it is recognized that mound-building ants are capable of dramatically modifying 65

their environments and altering the chemical and physical properties of soils (Beattie and 66

Culver 1977; Frouz and Jilková 2008; Jilková et al. 2011), few studies have explored the 67

impact of microclimatic conditions on the composition of the communities of fungi in these 68

soils (Ba et al. 2000; Zettler et al. 2002; Rodrigues et al. 2014). Given the availability of a 69

large group of nests of F. ulkei in south-eastern Manitoba, we undertook a study to 1) confirm 70

the temperature characteristics of the mound nests of this species reported in previous 71

studies, and 2) test the hypothesis that the community of culturable fungi from soils from 72

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nests differs from adjacent, non-nest soils. We were also interested in comparing the species 73

richness and diversity of the communities of culturable fungi of separate mound nests of F. 74

ulkei. 75

76

Materials and Methods 77

Collection of soils and temperature data 78

Thermocron iButton data loggers (DS1921G, Maxim Integrated Products, San Jose, USA) 79

that had been pre-set to measure temperature every two hours were coated in Performix 80

Plasti Dip (Plasti Dip International, Blaine, USA) to prevent moisture damage (Roznik and 81

Alford 2012). Data loggers were buried 5 cm deep in soil on the top, south side and north side 82

of three mound nests of Formica ulkei located on the un-forested edge of a cattle pasture that 83

had not been grazed in approximately 10 years, south of White Mud Falls, Manitoba (UTM 84

coordinates of mound 1 = 14U 0707355 5588945; mound 2 = 14U 0707363 5588913; mound 85

3 = 14U 0707367 5588908). One data logger was buried at a depth of 5 cm at one location 1 86

m south of each mound. Another data logger was also secured at a height of 2 m to the north 87

(i.e., the shaded) side of a tree located in the middle of the study area to collect air 88

temperatures. Data loggers recorded temperatures from 9 May to 18 September 2014. 89

Nests were sampled on 11 May and 14 July 2014 by collecting the uppermost 3 cm of 90

soil beneath the thatch from the top and south sides of each mound. Each site on all mounds 91

was sampled using a new plastic spoon. Soils to a depth of 3 cm were collected from 92

adjacent non-mound soil 1 m south of nests using a soil core sampler that was sterilized in 93

100% ethanol and rinsed in sterile distilled water between samples. Samples were placed into 94

separate, unused plastic freezer bags, sealed and transported in an ice cooler to the 95

laboratory. Each sample was emptied into a clean aluminum pan, air-dried at room 96

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temperature (18-21˚C), subjected to sieving using a 2 mm mesh to remove plant debris, and 97

stored in a new freezer bag. 98

99

Isolation and identification of fungi 100

Individual soil samples were used within 3 days following collection to prepare ten-fold serial 101

dilutions in sterile distilled water ranging from 10-1 to 10-7. Each dilution was plated in triplicate 102

on Dextrose-Peptone-Yeast Extract agar (DPYA) (Papavizas and Davey 1959) lacking oxgall 103

and sodium propionate, and Dichloran Rose Bengal agar (DRBA) (King et al. 1979) 104

containing 25 mg Rose Bengal, 2 mg dichloran, and KH2PO4 rather than K2HPO4. Both media 105

were supplemented with 50 mg chlortetracycline hydrochloride and 50 mg streptomycin 106

sulphate. Duplicate sets of plates were incubated at 25˚C and 35˚C for 5 days. 107

All fungal colonies were transferred to Modified Leonian’s agar (MLA) (Malloch 1981), 108

incubated at room temperature and identified based on cultural and micro-morphological 109

characters. Isolates that could be discriminated as separate taxa within genera but not 110

identified to species were numbered. Sporulating fungi that could not be identified to the level 111

of genus were designated as “undetermined” whereas those taxa that did not sporulate on 112

MLA were labeled “sterile” (see supplemental Table S1). Non-filamentous fungi and 113

Zygomycota, which were isolated in very low numbers on both DRBA and DPYA, were 114

disregarded. Fungi recovered on DPYE were also excluded from analyses because of the 115

high levels of bacterial contamination, particularly in soils collected in July. 116

Dominant species of Aspergillus were characterized on Czapek Dox agar (CZ), Czapek 117

Yeast agar (CYA), Czapek Yeast agar with 20% sucrose (CY20S) and Malt Extract agar 118

following Klich (2002a) and on Creatine Sucrose agar (CREA) as described by Samson et al. 119

(2014). The thermotolerances of these taxa were determined by assessing their ability to 120

grow on CYA and MLA when incubated at 37˚C, 45˚C, and 50˚C. Cultures used for DNA 121

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extraction were grown as described previously (Untereiner et al. 2008) and total nucleic acids 122

were extracted from mycelia following the protocols of Lee and Taylor (1990). The nuclear 123

ribosomal internal transcribed spacer (nucITS) region and a portion of the gene encoding the 124

protein β-tubulin were amplified as described in Bogale et al. (2010) using the primers ITS4, 125

ITS5 (nucITS) (White et al. 1990) and Bt2a, and Bt2b (β-tubulin) (Glass and Donaldson 126

1995). PCR products were cleaned using a QIAquick PCR Purification Kit (Qiagen, 127

Mississauga, Canada). Sequencing reactions were performed using a Taq DyeDeoxy cycle 128

sequencing kit or a BigDye Terminator cycle sequencing kit (Applied Biosystems, Inc., Foster 129

City, USA) using the primers listed above. Confirmation of the identification of these taxa as 130

Aspergillus navahoensis (UAMH 11867; GenBank KU310972, KU310974) and A. 131

pseudodeflectus (UAMH 11868; GenBank KU310973, KU310975) was based on the 132

comparison of generated DNA sequences to the nucITS and β-tubulin barcodes provided by 133

Samson et al. (2014). 134

135

Statistical analyses 136

Daily temperature readings for Thermocron iButton data loggers placed in the south side of 137

each mound were averaged per day from May 6 to September 18, 2014. Data for the tops of 138

mounds were not included in averages because two iButtons from this location were 139

dislodged during the course of the study. Data from the north sides of mounds were also 140

excluded because these temperatures differed significantly from temperatures from the south 141

sides of mounds (data not shown). A one-way analysis of variance (ANOVA) of temperature 142

differences (mounds 1, 2, and 3, non-mounds 1, 2, and 3, and ambient temperature) was 143

conducted using PSPP v 0.8.4 (Pfaff 2015). The same software was used to perform a post-144

hoc Tukey HSD test. 145

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Numbers of isolates on DRBA were used to calculate colony-forming units (CFU) per g 146

of soil and the proportional abundance of each species or taxon within a group (i.e., “sterile” 147

and “undetermined”). Diversity indices (Shannon, Simpson and Simpson inverse) were 148

calculated using BiodiversityR (Kindt and Coe 2005). Rényi diversity profiles describing the 149

richness and evenness of sites were also generated using BiodiversityR. Between sites 150

comparisons of species-abundance data were measured using the Morisita-Horn index of 151

similarity in BiodiversityR. These data were converted into distance matrixes and employed to 152

generate dendrograms using hierarchical clustering R v 3.2.2 (R Core Team 2015). 153

154

Results 155

Maximum and mean average daily temperatures of soils from mounds exceeded those of 156

adjacent non-mound sites (Table 1) and results of an ANOVA (F(6, 924) = 61.90, p = 0.000) 157

(Supplemental Table S2) indicated significant differences in the mean average temperatures 158

between sites. Post-hoc Tukey HSD multiple comparisons revealed that the average daily 159

temperatures of mounds were higher than non-mound sites (Supplemental Table S3). The 160

temperatures of mound 2 and 3 did not differ significantly, nor were significant differences in 161

temperature seen among non-mound sites. All mound sites were warmer than ambient 162

temperature whereas non-mound sites 2 and 3 were cooler. Non-mound site 1 did not differ 163

significantly from ambient temperature. Differences in the average weekly temperatures of 164

soils from mound and non-mound sites are illustrated in Figure 1. 165

Excluding non-filamentous fungi and Zygomycota, a total of 3929 isolates representing 166

307 taxa were recovered at all dilutions from mound nest and adjacent non-mound sites on 167

DRBA (Table 2, Supplemental Table S1). Higher numbers of isolates and taxa were obtained 168

from DRBA incubated at 25 C. Soils collected in July contained a larger numbers of isolates 169

(Table 2) and had greater species richness (Table 3) than soils collected in May. 170

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The abundance (CFU g-1), diversity, and richness of species from soils of nest mounds 171

generally exceeded those of non-mounds, particularly in July (Table 3). Mound soils differed 172

in richness among sites in July, as did soils from non-mounds. Species richness in May did 173

not differ as dramatically between sites with the exception of mound 1 which was under-174

sampled because of an error in the preparation of soil dilutions. Rényi profiles did not 175

discriminate between mound and non-mound soils in May with respect to species diversity; 176

soils in July differed with the exception of non-mound 2 that intersected with mound 2 and 177

mound 3 (Figure 2). As illustrated in Figure 2, the evenness of species from soils from non-178

mound 2 was higher than at all other sites in May and July but the evenness of the remaining 179

sites could not be ranked. The evenness of soil from mound 1 in May likely reflects the 180

aforementioned under-sampling. Communities in soils from mounds were more similar to 181

species from mounds than non-mound sites in May and July (Figure 3). This was also the 182

case for taxa from non-mounds with the exception of the community from non-mound 1 in 183

July that more closely resembled the mycota from mounds. 184

The most abundant fungi in soil from mounds were Aspergillus navahoensis (ITS 99% 185

similarity to EF652424; β-tubulin 99% similarity to EF652248) and Aspergillus 186

pseudodeflectus (ITS 100% similarity to EF652507; β-tubulin 100% similarity to EF652331), 187

that represented 17.4 to 44.2% and 8.6 to 37.6% of the recovered taxa, respectively (Tables 188

4-5). Both species were recovered from all mounds in May and July. The proportional 189

abundances of these species were higher in May except that A. pseudodeflectus was more 190

abundant in mound 2 in July. Aspergillus pseudodeflectus was recovered from only a single 191

non-mound site in May but in very low abundance (0.3%) representing a single isolate 192

whereas A. navahoensis was never isolated from non-mound soils. Cultures of A. 193

navahoensis conformed to the description of this species provided by Christensen and States 194

(1982) and were distinctive in producing rapidly maturing ascomata, abundant Hülle cells, and 195

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crystal-encrusted hyphae. Aspergillus navahoensis grew at 37˚C and at 45˚C, but showed 196

better growth at 37˚C; it exhibited no growth at 50˚C. Aspergillus pseudodeflectus grew at 197

37˚C but exhibited no growth at 45˚C and 50˚C. 198

Additional taxa from mound soils with abundances higher than 5% included 199

Cladosporium cladosporioides, Geomyces pannorum, Myriothecium sp. and species of 200

Acremonium. However, these fungi were not the dominant members of the mycota of all 201

mounds nor were they equally abundant in the same mound in both May and July. 202

Undetermined species were dominant members of soils from mound 3 and were more 203

abundant in July. Sterile fungi comprised more than 5% of the isolates in soils from every 204

mound but only in July. 205

Species of Penicillium were dominant members of the mycota of soils from non-mound 206

sites but were less abundant in May than in July. Other taxa from non-mound sites with 207

abundances greater than 5% included Cladosporium cladosporioides, Geomyces pannorum, 208

undetermined and sterile fungi, and members of the genera Acremonium, Fusarium and 209

Phoma. However, only Geomyces pannorum, undetermined and sterile fungi, and species of 210

Penicillium represented more than 5% of the taxa recovered at more than one site at a given 211

sampling time. 212

213

Discussion 214

The results of the present study agree with Scherba (1962) who reported that the thatch-215

covered mound nests of Formica ulkei are warmer than surrounding undisturbed soils during 216

the months when these ants are most active. We also observed significant differences 217

between the temperatures of the north and south sides of mounds (data not included), a 218

phenomenon that can likely be attributed to variations in the dimensions of mounds, the 219

composition and density of thatch, and degree of shading (Scherba 1962; Frouz 2000; 220

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Kadochová and Frouz 2014). 221

Our investigation also demonstrates that the communities of fungi in soils from nest 222

mounds of Formica ulkei differ from non-mound soils with respect to the abundances of 223

species, species richness, and diversity. Soils of nest mounds of F. ulkei resemble those of 224

Solenopsis invicta (red imported fire ant) in containing greater numbers of fungal colonies 225

than adjacent, non-nest soils (Zettler et al. 2002) but they differ in having higher levels of 226

species richness. In July, two of the three mounds we sampled had higher levels of species 227

diversity than non-nest soils. In contrast, culture-dependent assessments revealed that below 228

ground nests of young colonies of Atta (leaf-cutting ants) contain lower to comparable 229

numbers of colonies of filamentous fungi as non-nest soils but have similar levels of species 230

diversity and richness (Rodrigues et al. 2014). 231

Members of the Trichocomaceae (species of Aspergillus, Paecilomyces and Penicillium) 232

were dominant in soils from mound nests of F. ulkei and represented 39.5% (mound 3) to 233

81.8% (mound 1) of the total numbers of taxa recovered. Trichocomaceae are among the 234

most common filamentous Ascomycota isolated from the nests of mound-building and leaf-235

cutting ants (Baird et al. 2007; Zettler et al. 2002; Sharma and Sumbali 2013; Rodrigues et al. 236

2014) but only a single study (Zettler et al. 2002) resembles ours in recovering different 237

representatives of this family from nests and non-nest soils. 238

Aspergillus accounted for more than 80% of Trichocomaceae isolated from mound nests 239

and were represented almost exclusively by Aspergillus navahoensis (section Nidulantes) and 240

A. pseudodeflectus (section Usti). Aspergillus navahoensis was described from soils from a 241

cool desert shrub community in northern Arizona (Christianson and States 1982) and belongs 242

to a section of the genus that occurs at greater than expected frequencies in desert soils 243

(Klich 2002b). This species was recovered originally in low numbers (Christianson and States 244

1982) and, apart from the present study, does not appear to have been collected since it was 245

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described. Aspergillus pseudodeflectus is an infrequently collected osmophilic species 246

described from desert soils in Egypt (Samson and Mouchacca 1975) that was reported to be 247

restricted to the tropics and subtropics (Christensen and Tuthill 1985). It is closely related to 248

A. calidoustus, a more commonly encountered species known from clinical and environmental 249

sources that is distinguished from A. pseudodeflectus based on its ecology and molecular 250

barcodes (Samson et al. 2011, 2014). 251

Trichocomaceae were also abundant in soils from non-mound sites but were 252

represented almost exclusively by species of Paecilomyces and Penicillium. These genera 253

were consistently more abundant in non-mound soils than in soils from mounds. Members of 254

the genus Aspergillus were absent from non-mound soils with the exception of a single colony 255

of A. pseudodeflectus that we suspect was a contaminant. 256

Differences in the fungal communities of the soils of nest mounds of Formica ulkei and 257

adjacent non-nest sites likely reflect environmental factors that are influenced by nest location 258

and architecture. For example, mound nests of F. ulkei in Illinois were shown to be restricted 259

to drier regions along forest margins and were constructed to maximize insolation (Dreyer and 260

Park 1932; Dreyer 1942). Nest construction also dramatically alters the physical 261

characteristics of soil that operate to regulate the moisture content and temperatures of 262

mounds relative to surrounding soils. Mound building can increase soil porosity and reduce 263

the bulk density of soils, both of which influence soil aeration and soil permeability (Frouz and 264

Jilková 2008). The moisture content in mounds of F. ulkei at 5 cm has been shown to be 265

lower than in adjacent soils throughout the year and lower than in mounds at 30 cm during the 266

warmer months when the ants were active (Sherba 1959). Although we did not determine the 267

moisture content of soils at our study site, we observed that the daily temperatures of nests of 268

F. ulkei peaked in the evening and decreased slowly during the night (data not shown) in 269

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agreement with the description of the drier and more exposed nests of Formica polyctena 270

(European red wood ant) (Frouz 2000). 271

The supposition that the nests of Formica ulkei at our study site were drier than adjacent 272

sites is also supported by the dominance of Aspergillus in nests as compared to soils located 273

1 m from each mound. Species of Aspergillus are common in soils from warmer regions of the 274

world (Domsch et al. 1993; Bills et al. 2004) and are among the most xerotolerant 275

Ascomycota (Dix and Webster 1995; Zak and Wildman 2004). Members of this genus are 276

particularly abundant in desert and grassland soils where they represent up to 20% of isolated 277

species (Christensen and Tuthill 1985). Although both A. navahoensis and A. 278

pseudodeflectus were capable of growth at the highest average daily temperatures recorded 279

for mound and non-mound soils, only the former species was determined to be thermotolerant 280

(i.e., it grows at temperatures below 20˚C and at 40˚C or higher). This finding, in conjunction 281

with our observation that all Aspergillus and Paecilomyces and nearly half of the species of 282

Penicillium were isolated at both 25˚C and 35˚C (Supplemental Table S1), suggests that 283

water availability is also be a determinant of fungal community structure in mound nests of F. 284

ulkei. 285

Factors such as nutrient availability, soil chemistry and the physical properties of soils 286

also likely influence the structure of fungal communities in the mound nests of F. ulkei and 287

adjacent non-nest soils. For example, soils in ant nests have higher levels of nutrients (Frouz 288

et al. 2005; Frouz and Jílková 2008; Ginzburg et al. 2008; Jílková et al. 2015) and differ from 289

surrounding soils in pH, porosity, and the content of organic matter (Frouz and Jílková 2008; 290

Jílková et al. 2011). Microbial activity is assumed to be higher in ant nests because of these 291

differences, but the mechanisms underlying the impacts of ants on soil processes and other 292

soil biota are not well understood (Frouz and Jílková 2008; Del Toro et al. 2012). 293

Nests of Formica ulkei are reservoirs of fungal diversity that should be explored further 294

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using the approaches presented here. Our understanding of these communities would be 295

improved with the more frequent sampling of nest mounds and adjacent non-nest soils, the 296

isolation of fungi over a longer period of time, the use of media designed to isolate 297

ecologically specialized taxa, and the determination of temperature differences from a larger 298

number of sites within nest mounds. And because the enumeration methods used in our 299

study are selective for fungi that produce abundant spores (Garrett 1981), it would be 300

valuable to examine the diversity of culturable fungi in these soils using alternative isolation 301

methods (described in Bills et al. 2004). The complementary use of sequence-based 302

approaches such as environmental metagenomics would also enhance our understanding of 303

these assemblages of fungi, particularly in recovering non-culturable species and taxa that 304

are under-sampled employing cultured-dependent methods (Bills et al. 2004; Karst et al. 305

2013; Rodrigues et al. 2014). Sequence based approaches would also facilitate the 306

identification of sterile fungi and many of the micro-morphologically simple or taxonomically 307

challenging species present in the mound nests of Formica ulkei. 308

309

Acknowledgments 310

We are indebted to Gary McNeely (Brandon University) and three anonymous reviewers for 311

their insightful editorial comments and suggestions for the improvement of this paper. We also 312

thank Dennis and Jacqueline Caya for their permission to access nest mounds located on 313

their property and David Caya for serving as a bear guard during soil sampling. Financial 314

support for this study was provided by a Natural Sciences and Engineering Research Council 315

(NSERC) of Canada Discovery Grant to W.A.U. Funding in the form of NSERC 316

Undergraduate Summer Research Awards to L.B.D. (2014, 2015), T.M.U. (2014) and J.R.Y. 317

(2015) is very gratefully acknowledged. 318

319

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Figure captions 492

Figure 1. Weekly averages of ambient temperatures and temperatures of mound (M1, M2 and 493

M3) and non-mound (S1, S2 and S3) soils. Markers indicate the dates when soils were 494

collected. 495

496

Figure 2. Renyi profiles comparing the diversity of fungi found in mound and non-mound soils 497

from a) May 2014 and b) July 2014. Alpha = 0 is the species richness, alpha = 1 is the 498

Shannon-Weiner diversity index, and alpha = 2 is the log of the reciprocal of the Simpson 499

diversity index. 500

501

Figure 3. Dendrograms illustrating Morisita-Horn similarities between the communities of fungi 502

from mound (M1, M2 and M3) and non-mound (S1, S2 and S3) soils in a) May 2014 and b) 503

July 2014. 504

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Figure 1. Weekly averages of ambient temperatures and temperatures of mound (M1, M2 and M3) and non-mound (S1, S2 and S3) soils. Markers indicate the dates when soils were collected.

577x347mm (96 x 96 DPI)

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Figure 2. Renyi profiles comparing the diversity of fungi found in mound and non-mound soils from a) May 2014 and b) July 2014. Alpha = 0 is the species richness, alpha = 1 is the Shannon-Weiner diversity index,

and alpha = 2 is the log of the reciprocal of the Simpson diversity index.

397x725mm (96 x 96 DPI)

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Figure 3. Dendrograms illustrating Morisita-Horn similarities between the communities of fungi from mound (M1, M2 and M3) and non-mound (S1, S2 and S3) soils in a) May 2014 and b) July 2014.

133x236mm (300 x 300 DPI)

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Table 1. Descriptive statistics for average daily temperatures (°C) of mound (M) and non-mound (S) sites.

Site N Minimum Maximum Mean Std.

deviation Std. error

95% Confidence interval for mean

Lower bound Upper bound

M1 133 7.03 23.08 17.11 3.77 0.33 16.46 17.75

M2 133 8.52 29.08 22.26 5.13 0.45 21.38 23.14

M3 133 7.10 25.17 18.83 4.34 0.38 18.09 19.58

S1 133 7.46 19.92 14.96 3.03 0.26 14.44 15.48

S2 133 6.17 18.96 14.40 3.10 0.27 13.87 14.93

S3 133 6.25 18.58 13.94 2.82 0.24 13.46 14.43

All sites 798 6.17 29.08 16.92 4.70 0.17 16.58 17.25

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Table 2. Number of isolates of fungi recovered at 25 °C and 35 °C on DRBA from mound (M) and non-mound (S) sites in May and July 2014.

Sample time

Temperature of incubation (°C)

M1 S1 M2 S2 M3 S3 Total

May 25 134 135 191 80 391 237 1168 May 35 54 2 152 152 116 20 496 July 25 416 59 267 139 552 1 1434 July 35 328 59 152 35 240 17 831

Total − 932 255 762 406 1299 275 3929

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Table 3. Richness and diversity estimators of fungal communities of mound (M) and non-mound (S) sites calculated in BiodiversityR.

May 2014 Total CFU/g soil Species richness Shannon diversity Simpson diversity a Simpson inverse

M1 115.67 x 107 8 1.32 ± 0.14 0.668 ± 0.072 3.01 ± 0.65

1 10.83 x 107 44 2.75 ± 0.07 0.871 ± 0.014 7.72 ± 0.81

M2 29.55 x 107 36 2.32 ± 0.08 0.792 ± 0.029 4.81 ± 0.68

S2 36.66 x 107 33 2.81 ± 0.09 0.928 ± 0.003 13.86 ± 0.61

M3 57.75 x 107 30 2.27 ± 0.09 0.821 ± 0.022 5.59 ± 0.70

S3 11.4 x 107 37 2.21 ± 0.09 0.800 ± 0.022 5.00 ± 0.55

July 2014 Total CFU/g soil Species richness Shannon diversity Simpson diversity Simpson inverse

M1 38.7 x 107 49 2.11 ± 0.08 0.781 ± 0.021 4.57 ± 0.43

S1 5.77 x 107 36 2.00 ± 0.06 0.654 ± 0.056 2.89 ± 0.47

M2 42.45 x 107 66 3.09 ± 0.06 0.894 ± 0.008 9.46 ± 0.77

S2 6.15 x 107 50 3.40 ± 0.06 0.954 ± 0.002 21.7 ± 0.94

M3 82.88 x 107 85 3.33 ± 0.05 0.924 ± 0.005 13.2 ± 0.80

S3 3.57 x 107 19 0.955 ± 0.03 0.315 ± 0.157 1.46 ± 0.33 a Simpson diversity = (1-Simpson index)

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Table 4. Colony forming units per gram of soil (CFU/g) and the proportional abundancea of taxa recovered May 2014 from mound (M) and non-mound (S) sites.

Taxa (n = 126)

M1 S1 M2 S2 M3 S3

pi (%)

CFU/g pi

(%) CFU/g

pi (%)

CFU/g pi

(%) CFU/g

pi (%)

CFU/g pi

(%) CFU/g

Acremonium spp. (9)b − − 6.1 6.60 x 10

6 0.5 1.50 x 10

6 9.8 3.60 x 10

7 13.8 7.95 x 10

7 1.8 2.10 x 10

6

Aspergillus navahoensis 41.5 4.80 x 108 − − 44.2 1.31 x 10

8 − − 20.8 1.20 x 10

8 − −

Aspergillus pseudodeflectus 37.6 4.35 x 108 − − 8.6 2.55 x 10

7 − − 34.3 1.98 x 10

8 0.3 3.00 x 10

5

Aspergillus spp. 1.3 1.50 x 107 − − − − − − 0.3 1.50 x 10

6 − −

Aureobasidium sp. − − − − − − − − − − 0.3 3.00 x 105

Bipolaris spp. (2) − − − − 2.0 6.00 x 106 − − − − − −

Cladosporium cladosporioides 1.3 1.50 x 107

1.1 1.20 x 106 9.6 2.85 x 10

7 0.8 3.00 x 10

6 2.6 1.50 x 10

7 26.3 3.00 x 10

7

Cladosporium herbarum 2.6 3.00 x 107 1.7 1.80 x 10

6 2.0 6.00 x 10

6 − − 0.5 3.00 x 10

6 − −

Cladosporium macrocarpum − − − − − − − − − − 0.5 6.00 x 105

Curvularia brachyspora − − − − 0.5 1.50 x 106 − − − − 2.6 3.00 x 10

6

Curvularia geniculatus 1.3 1.50 x 107 − − − − − − − − − −

Curvularia spp. (2) − − − − 1.0 3.00 x 106 − − 1.6 9.00 x 10

6 2.6 3.00 x 10

6

Devresia sp. − − 0.3 3.00 x 105 − − − − − − − −

Doratomyces nanus − − 0.3 3.00 x 105 − − − − − − − −

Fusarium spp. (4) − − 5.5 6.00 x 106 5.1 1.50 x 10

7 13.1 4.80 x 10

7 1.3 7.50 x 10

6 − −

Geomyces sp. − − 2.7 3.00 x 106 − − − − − − − −

Geomyces pannorum − − 16.9 1.83 x 107 2.5 7.50 x 10

6 8.8 3.24 x 10

7 8.3 4.80 x 10

7 34.2 3.90 x 10

7

Humicola sp. − − − − 0.5 1.50 x 106 − − − − − −

Lecythophora sp. − − − − − − − − − − 0.5 6.00 x 105

Myrothecium sp. 13.0 1.50 x 108 0.3 3.00 x 10

5 0.5 1.50 x 10

6 − − − − − −

Paecilomyces spp. (2) − − 0.3 3.00 x 105 − − 0.8 3.00 x 10

6 − − − −

Paecilomyces marquandii − − 1.4 1.50 x 106 − − 8.2 3.00 x 10

7 − − 3.4 3.90 x 10

6

Penicillium spp. (20) − − 1.7 1.80 x 106 10.7 3.15 x 10

7 36.0 1.32 x 10

8 0.5 3.00 x 10

6 10.0 1.14 x 10

7

Phoma spp. (11) 1.4 1.67 x 107 33.8 3.66 x 10

7 2.5 7.50 x 10

6 − − 4.9 2.85 x 10

7 2.9 3.30 x 10

6

Sterile (20) − − 12.5 1.35 x 107 5.6 1.65 x 10

7 3.4 1.23 x 10

7 2.1 1.20 x 10

7 3.4 3.90 x 10

6

Tricellula sp. − − − − − − 0.8 3.00 x 106 − − − −

Trichocladium sp. − − − − − − − − 2.6 1.50 x 107 − −

Trichoderma spp. − − 2.7 3.00 x 106 0.5 1.50 x 10

6 − − − − 0.5 6.00 x 10

5

Undetermined (36) − − 12.7 1.38 x 107 3.6 1.05 x 10

7 18.2 6.69 x 10

7 6.5 3.75 x 10

7 10.5 1.20 x 10

7

Total Trichocomaceae 81.8 3.4 63.5 45 55.9 13.7 a pi = proportional abundance of the ith species;

b number of species within a genus or group.

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Table 5. Colony forming units per gram of soil (CFU/g) and the proportional abundancea of taxa recovered July 2014 from mound (M) and non-mound (S) sites.

Taxa (n = 197)

M1 S1 M2 S2 M3 S3

pi (%)

CFU/g pi

(%) CFU/g

pi (%)

CFU/g pi

(%) CFU/g

pi (%)

CFU/g pi

(%) CFU/g

Acremonium spp. (12)b 1.3 5.17 x 10

6 7.5 4.30 x 10

6 1.4 6.00 x 10

6 12.7 7.80 x 10

6 5.6 4.65 x 10

7 0.8 3.00 x 10

5

Acremonium-like spp. (5) − − 1.6 9.00 x 105 3.5 1.50 x 10

7 1.0 6.00 x 10

5 0.2 1.50 x 10

6 2.5 9.00 x 10

5

Alternaria alternata 0.0 1.67 x 105 − − 0.4 1.50 x 10

6 − − 1.8 1.50 x 10

7 − −

Aspergillus navahoensis 36.8 1.42 x 108 − − 25.4 1.08 x 10

8 − − 17.4 1.44 x 10

8 − −

Aspergillus niger 0.4 1.50 x 106 − − − − − − − − − −

Aspergillus pseudodeflectus 22.4 8.65 x 107 − − 17.0 7.20 x 10

7 − − 17.2 1.43 x 10

8 − −

Aspergillus spp. − − − − − − − − 1.8 1.50 x 107 − −

Aureobasidium pullulans 0.1 5.00 x 105 − − 0.7 3.00 x 10

6 − − 0.9 7.50 x 10

6 − −

Bipolaris sp. 0.8 3.00 x 106 − − − − − − − − − −

Chrysosporium spp. (2) − − 1.2 6.67 x 105 − − 1.0 6.00 x 10

5 − − − −

Cladosporium cladosporioides 0.5 2.00 x 106 − − 2.8 1.20 x 10

7 3.4 2.10 x 10

6 0.7 6.00 x 10

6 − −

Cladosporium herbarum 1.2 4.67 x 106 − − 0.4 1.50 x 10

6 0.5 3.00 x 10

5 0.9 7.50 x 10

6 − −

Cladosporium sphaerospermum 0.8 3.00 x 106 − − − − 0.5 3.00 x 10

5 − − − −

Clonostachys rosea 0.8 3.00 x 106 − − 3.5 1.50 x 10

7 1.5 9.00 x 10

5 − − − −

Clonostachys sp. − − − − 0.4 1.50 x 106 − − − − − −

Curvularia geniculatus 0.4 1.50 x 106 − − 0.7 3.00 x 10

6 − − 0.5 4.50 x 10

6 − −

Deverisea sp. − − − − − − 1.5 9.00 x 105 − − − −

Fusarium spp. (4) 0.0 1.67 x 105 0.6 3.33 x 10

5 0.4 1.50 x 10

6 1.5 9.00 x 10

5 0.4 3.30 x 10

6 0.8 3.00 x 10

5

Geomyces spp. (3) − − 0.6 3.33 x 105 0.4 1.50 x 10

6 − − 0.0 1.50 x 10

5 0.8 3.00 x 10

5

Geomyces pannorum 0.5 2.00 x 106 5.2 3.00 x 10

6 0.7 3.00 x 10

6 9.3 5.70 x 10

6 6.5 5.40 x 10

7 0.8 3.00 x 10

5

Humicola sp. − − − − − − 0.5 3.00 x 105 − − − −

Humicola-like sp. − − − − − − 0.5 3.00 x 105 − − − −

Idriella lunata − − − − 0.4 1.50 x 106 0.5 3.00 x 10

5 − − − −

Myrmecridium sp. − − − − − − − − 0.5 4.50 x 106 − −

Myrmecridium schulzeri − − 0.6 3.33 x 105 − − − − 0.2 1.50 x 10

6 − −

Myrothecium sp. 17.5 6.77 x 107 − − − − − − − − − −

Paecilomyces spp. (2) − − − − − − 2.4 1.50 x 106 − − − −

Paecilomyces marquandii − − 3.5 2.00 x 106 0.4 1.50 x 10

6 3.9 2.40 x 10

6 0.2 1.50 x 10

6 0.8 3.00 x 10

5

Penicillium sp. (26) 1.8 6.83 x 106 62.8 3.62 x 10

7 8.1 3.45 x 10

7 39.0 2.40 x 10

7 2.9 2.40 x 10

7 85.7 3.06 x 10

7

Pleurostomophora sp. − − − − 0.4 1.50 x 106 − − 0.5 4.50 x 10

6 − −

Pseudogymnoascus sp. − − − − − − 0.5 3.00 x 105 − − − −

Ramichloridium sp. − − − − − − − − 0.2 1.50 x 106 − −

Solosympodiella sp. − − − − − − − − 0.2 1.50 x 106 − −

Spicellum sp. − − − − − − − − − − 0.8 3.00 x 105

Stachybotrys eucylindriospora − − − − − − − − 0.2 1.50 x 106 − −

Sterile (72) 11.8 4.55 x 107 9.8 5.63 x 10

6 25.8 1.10 x 10

8 16.6 1.02 x 10

7 20.8 1.73 x 10

8 0.8 3.00 x 10

5

Trichoderma spp. 0.0 1.67 x 105 0.6 3.33 x 10

5 3.5 1.50 x 10

7 2.0 1.20 x 10

6 − − 0.8 3.00 x 10

5

Undetermined (42) 2.9 1.13 x 107 6.3 3.63 x 10

6 3.9 1.65 x 10

7 1.5 9.00 x 10

5 20.3 1.68 x 10

8 5.0 1.80 x 10

6

Total Trichocomaceae 61.4 66.3 50.9 45.3 39.5 86.5 a pi = proportional abundance of the ith species;

b number of species within a genus or group.

Page 29 of 29



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