Canadian Journal of Microbiology
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, JocelynDraft R.; Brandon University, Biology Untereiner, Wendy; Brandon University, Biology
Keyword: Aspergillus, fungal biodiversity, xerotolerant, mound-building ant
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Duff et al.; Fungi from nests of Formica ulkei 1
1 Diversity of fungi from the mound nests of Formica ulkei and adjacent non-nest soils
2
3 Lyndon B. Duff, Theresa M. Urichuk, Lisa N. Hodgins, Jocelyn R. Young, and Wendy A.
4 Untereiner 1
5 Department of Biology, Brandon University, 270 18 th Street, Brandon, Manitoba, R7A 6A9,
6 Canada
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8 L.B. Duff ([email protected])
9 T.M. Urichuk ([email protected])
10 L.N. Hodgins ([email protected])
11 J.R. Young ([email protected])Draft
12 W.A. Untereiner ([email protected])
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22 1 Corresponding author
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Duff et al.; Fungi from nests of Formica ulkei 2 23 Abstract
24 Culture based methods were employed to recover 3929 isolates of fungi from soils collected
25 in May and July 2014 from mound nests of Formica ulkei and adjacent non nest sites. The
26 abundance, diversity, and richness of species from nest mounds exceeded those of non
27 mound soils, particularly in July. Communities of fungi from mounds were more similar to
28 those from mounds than non mounds; this was also the case for non mound soils with the
29 exception of one non mound site in July. Species of Aspergillus , Paecilomyces and
30 Penicillium were dominant in nest soils and represented up to 81.8% of the taxa recovered.
31 Members of the genus Aspergillus accounted for the majority of Trichocomaceae from nests
32 and were represented almost exclusively by Aspergillus navahoensis and A. pseudodeflectus . 33 Dominant fungi from non mound sites Draftincluded Cladosporium cladosporioides , Geomyces 34 pannorum and species of Acremonium , Fusarium , Penicillium and Phoma. Although mound
35 nests were warmer than adjacent soils, the dominance of xerotolerant Aspergillus in soils
36 from mounds and the isolation of the majority of Trichocomaceae at 25˚C and 35˚C suggests
37 that both temperature and water availability may be determinants of fungal community
38 structure in nests of F. ulkei .
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41 Key words: Aspergillus , fungal biodiversity, mound building ant, xerotolerant
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Duff et al.; Fungi from nests of Formica ulkei 3 48 Introduction
49 The mound building ant Formica ulkei Emery (Hymenoptera: Formicidae) ranges from Alberta
50 to Nova Scotia (Canada) and southward to Illinois, Indiana and Iowa (USA) (Holmquist 1928;
51 Sherba 1958; Glasier et al. 2013). This species builds conspicuous nests in meadows and
52 pastures along the margins of forests and sparsely wooded areas (Holmquist 1928; Dreyer
53 and Park 1932; Sherba 1958). Nests are composed of excavated soil and covered by a layer
54 of thatch (i.e., small pieces of grass and other plant material) (Sherba 1958, 1959, 1962).
55 The mound nests of F. ulkei are thermoregulatory in function and are constructed to
56 achieve and maintain higher temperatures than adjacent undisturbed soils during the months
57 when the ants are most active (Sherba 1962). Nests are built in exposed sites and are 58 oriented to maximize their exposure toDraft solar radiation (Sherba 1958); they gain heat from 59 solar radiation in the early spring and maintain temperatures that are higher and more stable
60 than those of surrounding soils because of the insulating properties of thatch (Sherba 1962;
61 Frouz and Jilková 2008). This layer of organic material prevents the overheating of mounds
62 during the warmest parts of the year in other ant species that construct thatched nests
63 (Bollazzi and Rocces 2010; Kadochová and Frouz 2014) and it may serve the same function
64 in F. ulkei .
65 Although it is recognized that mound building ants are capable of dramatically modifying
66 their environments and altering the chemical and physical properties of soils (Beattie and
67 Culver 1977; Frouz and Jilková 2008; Jilková et al. 2011), few studies have explored the
68 impact of microclimatic conditions on the composition of the communities of fungi in these
69 soils (Ba et al. 2000; Zettler et al. 2002; Rodrigues et al. 2014). Given the availability of a
70 large group of nests of F. ulkei in south eastern Manitoba, we undertook a study to 1) confirm
71 the temperature characteristics of the mound nests of this species reported in previous
72 studies, and 2) test the hypothesis that the community of culturable fungi from soils from
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Duff et al.; Fungi from nests of Formica ulkei 4 73 nests differs from adjacent, non nest soils. We were also interested in comparing the species
74 richness and diversity of the communities of culturable fungi of separate mound nests of F.
75 ulkei .
76
77 Materials and Methods
78 Collection of soils and temperature data
79 Thermocron iButton data loggers (DS1921G, Maxim Integrated Products, San Jose, USA)
80 that had been pre set to measure temperature every two hours were coated in Performix
81 Plasti Dip (Plasti Dip International, Blaine, USA) to prevent moisture damage (Roznik and
82 Alford 2012). Data loggers were buried 5 cm deep in soil on the top, south side and north side 83 of three mound nests of Formica ulkei Draftlocated on the un forested edge of a cattle pasture that 84 had not been grazed in approximately 10 years, south of White Mud Falls, Manitoba (UTM
85 coordinates of mound 1 = 14U 0707355 5588945; mound 2 = 14U 0707363 5588913; mound
86 3 = 14U 0707367 5588908). One data logger was buried at a depth of 5 cm at one location 1
87 m south of each mound. Another data logger was also secured at a height of 2 m to the north
88 (i.e., the shaded) side of a tree located in the middle of the study area to collect air
89 temperatures. Data loggers recorded temperatures from 9 May to 18 September 2014.
90 Nests were sampled on 11 May and 14 July 2014 by collecting the uppermost 3 cm of
91 soil beneath the thatch from the top and south sides of each mound. Each site on all mounds
92 was sampled using a new plastic spoon. Soils to a depth of 3 cm were collected from
93 adjacent non mound soil 1 m south of nests using a soil core sampler that was sterilized in
94 100% ethanol and rinsed in sterile distilled water between samples. Samples were placed into
95 separate, unused plastic freezer bags, sealed and transported in an ice cooler to the
96 laboratory. Each sample was emptied into a clean aluminum pan, air dried at room
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Duff et al.; Fungi from nests of Formica ulkei 5 97 temperature (18 21˚C), subjected to sieving using a 2 mm mesh to remove plant debris, and
98 stored in a new freezer bag.
99
100 Isolation and identification of fungi
101 Individual soil samples were used within 3 days following collection to prepare ten fold serial
102 dilutions in sterile distilled water ranging from 10 1 to 10 7. Each dilution was plated in triplicate
103 on Dextrose Peptone Yeast Extract agar (DPYA) (Papavizas and Davey 1959) lacking oxgall
104 and sodium propionate, and Dichloran Rose Bengal agar (DRBA) (King et al. 1979)
105 containing 25 mg Rose Bengal, 2 mg dichloran, and KH 2PO 4 rather than K 2HPO 4. Both media
106 were supplemented with 50 mg chlortetracycline hydrochloride and 50 mg streptomycin 107 sulphate. Duplicate sets of plates wereDraft incubated at 25˚C and 35˚C for 5 days. 108 All fungal colonies were transferred to Modified Leonian’s agar (MLA) (Malloch 1981),
109 incubated at room temperature and identified based on cultural and micro morphological
110 characters. Isolates that could be discriminated as separate taxa within genera but not
111 identified to species were numbered. Sporulating fungi that could not be identified to the level
112 of genus were designated as “undetermined” whereas those taxa that did not sporulate on
113 MLA were labeled “sterile” (see supplemental Table S1). Non filamentous fungi and
114 Zygomycota, which were isolated in very low numbers on both DRBA and DPYA, were
115 disregarded. Fungi recovered on DPYE were also excluded from analyses because of the
116 high levels of bacterial contamination, particularly in soils collected in July.
117 Dominant species of Aspergillus were characterized on Czapek Dox agar (CZ), Czapek
118 Yeast agar (CYA), Czapek Yeast agar with 20% sucrose (CY20S) and Malt Extract agar
119 following Klich (2002a) and on Creatine Sucrose agar (CREA) as described by Samson et al.
120 (2014). The thermotolerances of these taxa were determined by assessing their ability to
121 grow on CYA and MLA when incubated at 37˚C, 45˚C, and 50˚C. Cultures used for DNA
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Duff et al.; Fungi from nests of Formica ulkei 6 122 extraction were grown as described previously (Untereiner et al. 2008) and total nucleic acids
123 were extracted from mycelia following the protocols of Lee and Taylor (1990). The nuclear
124 ribosomal internal transcribed spacer (nucITS) region and a portion of the gene encoding the
125 protein β tubulin were amplified as described in Bogale et al. (2010) using the primers ITS4,
126 ITS5 (nucITS) (White et al. 1990) and Bt2a, and Bt2b (β tubulin) (Glass and Donaldson
127 1995). PCR products were cleaned using a QIAquick PCR Purification Kit (Qiagen,
128 Mississauga, Canada). Sequencing reactions were performed using a Taq DyeDeoxy cycle
129 sequencing kit or a BigDye Terminator cycle sequencing kit (Applied Biosystems, Inc., Foster
130 City, USA) using the primers listed above. Confirmation of the identification of these taxa as
131 Aspergillus navahoensis (UAMH 11867; GenBank KU310972, KU310974) and A. 132 pseudodeflectus (UAMH 11868; GenBankDraft KU310973, KU310975) was based on the 133 comparison of generated DNA sequences to the nucITS and β tubulin barcodes provided by
134 Samson et al. (2014).
135
136 Statistical analyses
137 Daily temperature readings for Thermocron iButton data loggers placed in the south side of
138 each mound were averaged per day from May 6 to September 18, 2014. Data for the tops of
139 mounds were not included in averages because two iButtons from this location were
140 dislodged during the course of the study. Data from the north sides of mounds were also
141 excluded because these temperatures differed significantly from temperatures from the south
142 sides of mounds (data not shown). A one way analysis of variance (ANOVA) of temperature
143 differences (mounds 1, 2, and 3, non mounds 1, 2, and 3, and ambient temperature) was
144 conducted using PSPP v 0.8.4 (Pfaff 2015). The same software was used to perform a post
145 hoc Tukey HSD test.
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Duff et al.; Fungi from nests of Formica ulkei 7 146 Numbers of isolates on DRBA were used to calculate colony forming units (CFU) per g
147 of soil and the proportional abundance of each species or taxon within a group (i.e., “sterile”
148 and “undetermined”). Diversity indices (Shannon, Simpson and Simpson inverse) were
149 calculated using BiodiversityR (Kindt and Coe 2005). Rényi diversity profiles describing the
150 richness and evenness of sites were also generated using BiodiversityR. Between sites
151 comparisons of species abundance data were measured using the Morisita Horn index of
152 similarity in BiodiversityR. These data were converted into distance matrixes and employed to
153 generate dendrograms using hierarchical clustering R v 3.2.2 (R Core Team 2015).
154
155 Results 156 Maximum and mean average daily temperaturesDraft of soils from mounds exceeded those of 157 adjacent non mound sites (Table 1) and results of an ANOVA ( F(6, 924) = 61.90, p = 0.000)
158 (Supplemental Table S2) indicated significant differences in the mean average temperatures
159 between sites. Post hoc Tukey HSD multiple comparisons revealed that the average daily
160 temperatures of mounds were higher than non mound sites (Supplemental Table S3). The
161 temperatures of mound 2 and 3 did not differ significantly, nor were significant differences in
162 temperature seen among non mound sites. All mound sites were warmer than ambient
163 temperature whereas non mound sites 2 and 3 were cooler. Non mound site 1 did not differ
164 significantly from ambient temperature. Differences in the average weekly temperatures of
165 soils from mound and non mound sites are illustrated in Figure 1.
166 Excluding non filamentous fungi and Zygomycota, a total of 3929 isolates representing
167 307 taxa were recovered at all dilutions from mound nest and adjacent non mound sites on
168 DRBA (Table 2, Supplemental Table S1). Higher numbers of isolates and taxa were obtained
169 from DRBA incubated at 25 C. Soils collected in July contained a larger numbers of isolates
170 (Table 2) and had greater species richness (Table 3) than soils collected in May.
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Duff et al.; Fungi from nests of Formica ulkei 8 171 The abundance (CFU g 1), diversity, and richness of species from soils of nest mounds
172 generally exceeded those of non mounds, particularly in July (Table 3). Mound soils differed
173 in richness among sites in July, as did soils from non mounds. Species richness in May did
174 not differ as dramatically between sites with the exception of mound 1 which was under
175 sampled because of an error in the preparation of soil dilutions. Rényi profiles did not
176 discriminate between mound and non mound soils in May with respect to species diversity;
177 soils in July differed with the exception of non mound 2 that intersected with mound 2 and
178 mound 3 (Figure 2). As illustrated in Figure 2, the evenness of species from soils from non
179 mound 2 was higher than at all other sites in May and July but the evenness of the remaining
180 sites could not be ranked. The evenness of soil from mound 1 in May likely reflects the 181 aforementioned under sampling. CommunitiesDraft in soils from mounds were more similar to 182 species from mounds than non mound sites in May and July (Figure 3). This was also the
183 case for taxa from non mounds with the exception of the community from non mound 1 in
184 July that more closely resembled the mycota from mounds.
185 The most abundant fungi in soil from mounds were Aspergillus navahoensis (ITS 99%
186 similarity to EF652424; β tubulin 99% similarity to EF652248) and Aspergillus
187 pseudodeflectus (ITS 100% similarity to EF652507; β tubulin 100% similarity to EF652331),
188 that represented 17.4 to 44.2% and 8.6 to 37.6% of the recovered taxa, respectively (Tables
189 4 5). Both species were recovered from all mounds in May and July. The proportional
190 abundances of these species were higher in May except that A. pseudodeflectus was more
191 abundant in mound 2 in July. Aspergillus pseudodeflectus was recovered from only a single
192 non mound site in May but in very low abundance (0.3%) representing a single isolate
193 whereas A. navahoensis was never isolated from non mound soils. Cultures of A.
194 navahoensis conformed to the description of this species provided by Christensen and States
195 (1982) and were distinctive in producing rapidly maturing ascomata, abundant Hülle cells, and
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Duff et al.; Fungi from nests of Formica ulkei 9 196 crystal encrusted hyphae. Aspergillus navahoensis grew at 37˚C and at 45˚C, but showed
197 better growth at 37˚C; it exhibited no growth at 50˚C. Aspergillus pseudodeflectus grew at
198 37˚C but exhibited no growth at 45˚C and 50˚C.
199 Additional taxa from mound soils with abundances higher than 5% included
200 Cladosporium cladosporioides , Geomyces pannorum , Myriothecium sp. and species of
201 Acremonium . However, these fungi were not the dominant members of the mycota of all
202 mounds nor were they equally abundant in the same mound in both May and July.
203 Undetermined species were dominant members of soils from mound 3 and were more
204 abundant in July. Sterile fungi comprised more than 5% of the isolates in soils from every
205 mound but only in July. 206 Species of Penicillium were dominantDraft members of the mycota of soils from non mound 207 sites but were less abundant in May than in July. Other taxa from non mound sites with
208 abundances greater than 5% included Cladosporium cladosporioides , Geomyces pannorum ,
209 undetermined and sterile fungi, and members of the genera Acremonium , Fusarium and
210 Phoma. However, only Geomyces pannorum , undetermined and sterile fungi, and species of
211 Penicillium represented more than 5% of the taxa recovered at more than one site at a given
212 sampling time.
213
214 Discussion
215 The results of the present study agree with Scherba (1962) who reported that the thatch
216 covered mound nests of Formica ulkei are warmer than surrounding undisturbed soils during
217 the months when these ants are most active. We also observed significant differences
218 between the temperatures of the north and south sides of mounds (data not included), a
219 phenomenon that can likely be attributed to variations in the dimensions of mounds, the
220 composition and density of thatch, and degree of shading (Scherba 1962; Frouz 2000;
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Duff et al.; Fungi from nests of Formica ulkei 10 221 Kadochová and Frouz 2014).
222 Our investigation also demonstrates that the communities of fungi in soils from nest
223 mounds of Formica ulkei differ from non mound soils with respect to the abundances of
224 species, species richness, and diversity. Soils of nest mounds of F. ulkei resemble those of
225 Solenopsis invicta (red imported fire ant) in containing greater numbers of fungal colonies
226 than adjacent, non nest soils (Zettler et al. 2002) but they differ in having higher levels of
227 species richness. In July, two of the three mounds we sampled had higher levels of species
228 diversity than non nest soils. In contrast, culture dependent assessments revealed that below
229 ground nests of young colonies of Atta (leaf cutting ants) contain lower to comparable
230 numbers of colonies of filamentous fungi as non nest soils but have similar levels of species 231 diversity and richness (Rodrigues et al.Draft 2014). 232 Members of the Trichocomaceae (species of Aspergillus , Paecilomyces and Penicillium )
233 were dominant in soils from mound nests of F. ulkei and represented 39.5% (mound 3) to
234 81.8% (mound 1) of the total numbers of taxa recovered. Trichocomaceae are among the
235 most common filamentous Ascomycota isolated from the nests of mound building and leaf
236 cutting ants (Baird et al. 2007; Zettler et al. 2002; Sharma and Sumbali 2013; Rodrigues et al.
237 2014) but only a single study (Zettler et al. 2002) resembles ours in recovering different
238 representatives of this family from nests and non nest soils.
239 Aspergillus accounted for more than 80% of Trichocomaceae isolated from mound nests
240 and were represented almost exclusively by Aspergillus navahoensis (section Nidulantes ) and
241 A. pseudodeflectus (section Usti ). Aspergillus navahoensis was described from soils from a
242 cool desert shrub community in northern Arizona (Christianson and States 1982) and belongs
243 to a section of the genus that occurs at greater than expected frequencies in desert soils
244 (Klich 2002b). This species was recovered originally in low numbers (Christianson and States
245 1982) and, apart from the present study, does not appear to have been collected since it was
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Duff et al.; Fungi from nests of Formica ulkei 11 246 described. Aspergillus pseudodeflectus is an infrequently collected osmophilic species
247 described from desert soils in Egypt (Samson and Mouchacca 1975) that was reported to be
248 restricted to the tropics and subtropics (Christensen and Tuthill 1985). It is closely related to
249 A. calidoustus , a more commonly encountered species known from clinical and environmental
250 sources that is distinguished from A. pseudodeflectus based on its ecology and molecular
251 barcodes (Samson et al. 2011, 2014).
252 Trichocomaceae were also abundant in soils from non mound sites but were
253 represented almost exclusively by species of Paecilomyces and Penicillium . These genera
254 were consistently more abundant in non mound soils than in soils from mounds. Members of
255 the genus Aspergillus were absent from non mound soils with the exception of a single colony 256 of A. pseudodeflectus that we suspect Draftwas a contaminant. 257 Differences in the fungal communities of the soils of nest mounds of Formica ulkei and
258 adjacent non nest sites likely reflect environmental factors that are influenced by nest location
259 and architecture. For example, mound nests of F. ulkei in Illinois were shown to be restricted
260 to drier regions along forest margins and were constructed to maximize insolation (Dreyer and
261 Park 1932; Dreyer 1942). Nest construction also dramatically alters the physical
262 characteristics of soil that operate to regulate the moisture content and temperatures of
263 mounds relative to surrounding soils. Mound building can increase soil porosity and reduce
264 the bulk density of soils, both of which influence soil aeration and soil permeability (Frouz and
265 Jilková 2008). The moisture content in mounds of F. ulkei at 5 cm has been shown to be
266 lower than in adjacent soils throughout the year and lower than in mounds at 30 cm during the
267 warmer months when the ants were active (Sherba 1959). Although we did not determine the
268 moisture content of soils at our study site, we observed that the daily temperatures of nests of
269 F. ulkei peaked in the evening and decreased slowly during the night (data not shown) in
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Duff et al.; Fungi from nests of Formica ulkei 12 270 agreement with the description of the drier and more exposed nests of Formica polyctena
271 (European red wood ant) (Frouz 2000).
272 The supposition that the nests of Formica ulkei at our study site were drier than adjacent
273 sites is also supported by the dominance of Aspergillus in nests as compared to soils located
274 1 m from each mound. Species of Aspergillus are common in soils from warmer regions of the
275 world (Domsch et al. 1993; Bills et al. 2004) and are among the most xerotolerant
276 Ascomycota (Dix and Webster 1995; Zak and Wildman 2004). Members of this genus are
277 particularly abundant in desert and grassland soils where they represent up to 20% of isolated
278 species (Christensen and Tuthill 1985). Although both A. navahoensis and A.
279 pseudodeflectus were capable of growth at the highest average daily temperatures recorded 280 for mound and non mound soils, only theDraft former species was determined to be thermotolerant 281 (i.e., it grows at temperatures below 20˚C and at 40˚C or higher). This finding, in conjunction
282 with our observation that all Aspergillus and Paecilomyces and nearly half of the species of
283 Penicillium were isolated at both 25˚C and 35˚C (Supplemental Table S1), suggests that
284 water availability is also be a determinant of fungal community structure in mound nests of F.
285 ulkei .
286 Factors such as nutrient availability, soil chemistry and the physical properties of soils
287 also likely influence the structure of fungal communities in the mound nests of F. ulkei and
288 adjacent non nest soils. For example, soils in ant nests have higher levels of nutrients (Frouz
289 et al. 2005; Frouz and Jílková 2008; Ginzburg et al. 2008; Jílková et al. 2015) and differ from
290 surrounding soils in pH, porosity, and the content of organic matter (Frouz and Jílková 2008;
291 Jílková et al. 2011). Microbial activity is assumed to be higher in ant nests because of these
292 differences, but the mechanisms underlying the impacts of ants on soil processes and other
293 soil biota are not well understood (Frouz and Jílková 2008; Del Toro et al. 2012).
294 Nests of Formica ulkei are reservoirs of fungal diversity that should be explored further
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Duff et al.; Fungi from nests of Formica ulkei 13 295 using the approaches presented here. Our understanding of these communities would be
296 improved with the more frequent sampling of nest mounds and adjacent non nest soils, the
297 isolation of fungi over a longer period of time, the use of media designed to isolate
298 ecologically specialized taxa, and the determination of temperature differences from a larger
299 number of sites within nest mounds. And because the enumeration methods used in our
300 study are selective for fungi that produce abundant spores (Garrett 1981), it would be
301 valuable to examine the diversity of culturable fungi in these soils using alternative isolation
302 methods (described in Bills et al. 2004). The complementary use of sequence based
303 approaches such as environmental metagenomics would also enhance our understanding of
304 these assemblages of fungi, particularly in recovering non culturable species and taxa that 305 are under sampled employing cultured dependentDraft methods (Bills et al. 2004; Karst et al. 306 2013; Rodrigues et al. 2014). Sequence based approaches would also facilitate the
307 identification of sterile fungi and many of the micro morphologically simple or taxonomically
308 challenging species present in the mound nests of Formica ulkei.
309
310 Acknowledgments
311 We are indebted to Gary McNeely (Brandon University) and three anonymous reviewers for
312 their insightful editorial comments and suggestions for the improvement of this paper. We also
313 thank Dennis and Jacqueline Caya for their permission to access nest mounds located on
314 their property and David Caya for serving as a bear guard during soil sampling. Financial
315 support for this study was provided by a Natural Sciences and Engineering Research Council
316 (NSERC) of Canada Discovery Grant to W.A.U. Funding in the form of NSERC
317 Undergraduate Summer Research Awards to L.B.D. (2014, 2015), T.M.U. (2014) and J.R.Y.
318 (2015) is very gratefully acknowledged.
319
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Duff et al.; Fungi from nests of Formica ulkei 21 492 Figure captions
493 Figure 1. Weekly averages of ambient temperatures and temperatures of mound (M1, M2 and
494 M3) and non mound (S1, S2 and S3) soils. Markers indicate the dates when soils were
495 collected.
496
497 Figure 2. Renyi profiles comparing the diversity of fungi found in mound and non mound soils
498 from a) May 2014 and b) July 2014. Alpha = 0 is the species richness, alpha = 1 is the
499 Shannon Weiner diversity index, and alpha = 2 is the log of the reciprocal of the Simpson
500 diversity index.
501 502 Figure 3. Dendrograms illustrating Morisita HornDraft similarities between the communities of fungi 503 from mound (M1, M2 and M3) and non mound (S1, S2 and S3) soils in a) May 2014 and b)
504 July 2014.
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Draft 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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Draft
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.
Std. 95% Confidence interval for mean Site N Minimum Maximum Mean Std. error deviation 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
Draft
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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 Temperature of M1 S1 M2 S2 M3 S3 Total time incubation (°C) 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
Draft
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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
7 M1 115.67 x 10 8 1.32 ± 0.14 0.668 ± 0.072 3.01 ± 0.65
7 1 10.83 x 10 44 2.75 ± 0.07 0.871 ± 0.014 7.72 ± 0.81
7 M2 29.55 x 10 36 2.32 ± 0.08 0.792 ± 0.029 4.81 ± 0.68
7 S2 36.66 x 10 33 2.81 ± 0.09 0.928 ± 0.003 13.86 ± 0.61
7 M3 57.75 x 10 30 Draft2.27 ± 0.09 0.821 ± 0.022 5.59 ± 0.70 7 S3 11.4 x 10 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
7 M1 38.7 x 10 49 2.11 ± 0.08 0.781 ± 0.021 4.57 ± 0.43
7 S1 5.77 x 10 36 2.00 ± 0.06 0.654 ± 0.056 2.89 ± 0.47
7 M2 42.45 x 10 66 3.09 ± 0.06 0.894 ± 0.008 9.46 ± 0.77
7 S2 6.15 x 10 50 3.40 ± 0.06 0.954 ± 0.002 21.7 ± 0.94
7 M3 82.88 x 10 85 3.33 ± 0.05 0.924 ± 0.005 13.2 ± 0.80
7 S3 3.57 x 10 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 abundance a of taxa recovered May 2014 from mound (M) and non mound (S) sites. M1 S1 M2 S2 M3 S3 Taxa (n = 126) p p p p p p i CFU/g i CFU/g i CFU/g i CFU/g i CFU/g i 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 10 8 − − 44.2 1.31 x 10 8 − − 20.8 1.20 x 10 8 − − Aspergillus pseudodeflectus 37.6 4.35 x 10 8 − − 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 10 7 − − − − − − 0.3 1.50 x 10 6 − − Aureobasidium sp. − − − − − − − − − − 0.3 3.00 x 10 5 Bipolaris spp. (2) − − − − 2.0 6.00 x 10 6 − − − − − − Cladosporium cladosporioides 1.3 1.50 x 10 7 1.1 1.20 x 10 6 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 10 7 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 10 5 Curvularia brachyspora − − − − 0.5 1.50 x 10 6 − − − − 2.6 3.00 x 10 6 Curvularia geniculatus 1.3 1.50 x 10 7 − − − − − − − − − − Draft 6 6 6 Curvularia spp. (2) − − − − 1.0 3.00 x 10 − − 1.6 9.00 x 10 2.6 3.00 x 10 Devresia sp. − − 0.3 3.00 x 10 5 − − − − − − − − Doratomyces nanus − − 0.3 3.00 x 10 5 − − − − − − − − Fusarium spp. (4) − − 5.5 6.00 x 10 6 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 10 6 − − − − − − − − Geomyces pannorum − − 16.9 1.83 x 10 7 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 10 6 − − − − − − Lecythophora sp. − − − − − − − − − − 0.5 6.00 x 10 5 Myrothecium sp. 13.0 1.50 x 10 8 0.3 3.00 x 10 5 0.5 1.50 x 10 6 − − − − − − Paecilomyces spp. (2) − − 0.3 3.00 x 10 5 − − 0.8 3.00 x 10 6 − − − − Paecilomyces marquandii − − 1.4 1.50 x 10 6 − − 8.2 3.00 x 10 7 − − 3.4 3.90 x 10 6 Penicillium spp. (20) − − 1.7 1.80 x 10 6 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 10 7 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 10 7 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 10 6 − − − − Trichocladium sp. − − − − − − − − 2.6 1.50 x 10 7 − − Trichoderma spp. − − 2.7 3.00 x 10 6 0.5 1.50 x 10 6 − − − − 0.5 6.00 x 10 5 Undetermined (36) − − 12.7 1.38 x 10 7 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 b pi = proportional abundance of the ith species; 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 abundance a of taxa recovered July 2014 from mound (M) and non mound (S) sites. M1 S1 M2 S2 M3 S3 Taxa (n = 197) p p p p p p i CFU/g i CFU/g i CFU/g i CFU/g i CFU/g i 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 10 5 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 10 5 − − 0.4 1.50 x 10 6 − − 1.8 1.50 x 10 7 − − Aspergillus navahoensis 36.8 1.42 x 10 8 − − 25.4 1.08 x 10 8 − − 17.4 1.44 x 10 8 − − Aspergillus niger 0.4 1.50 x 10 6 − − − − − − − − − − Aspergillus pseudodeflectus 22.4 8.65 x 10 7 − − 17.0 7.20 x 10 7 − − 17.2 1.43 x 10 8 − − Aspergillus spp. − − − − − − − − 1.8 1.50 x 10 7 − − Aureobasidium pullulans 0.1 5.00 x 10 5 − − 0.7 3.00 x 10 6 − − 0.9 7.50 x 10 6 − − Bipolaris sp. 0.8 3.00 x 10 6 − − − − − − − − − − Chrysosporium spp. (2) − − 1.2 6.67 x 10 5 − − 1.0 6.00 x 10 5 − − − − Cladosporium cladosporioides 0.5 2.00 x 10 6 − − 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 10 6 − − 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 10 6 − − − − 0.5 3.00 x 10 5 − − − − Clonostachys rosea 0.8 3.00 x 10 6 − − 3.5 1.50 x 10 7 1.5 9.00 x 10 5 − − − − Draft 6 Clonostachys sp. − − − − 0.4 1.50 x 10 − − − − − − Curvularia geniculatus 0.4 1.50 x 10 6 − − 0.7 3.00 x 10 6 − − 0.5 4.50 x 10 6 − − Deverisea sp. − − − − − − 1.5 9.00 x 10 5 − − − − Fusarium spp. (4) 0.0 1.67 x 10 5 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 10 5 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 10 6 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 10 5 − − − − Humicola like sp. − − − − − − 0.5 3.00 x 10 5 − − − − Idriella lunata − − − − 0.4 1.50 x 10 6 0.5 3.00 x 10 5 − − − − Myrmecridium sp. − − − − − − − − 0.5 4.50 x 10 6 − − Myrmecridium schulzeri − − 0.6 3.33 x 10 5 − − − − 0.2 1.50 x 10 6 − − Myrothecium sp. 17.5 6.77 x 10 7 − − − − − − − − − − Paecilomyces spp. (2) − − − − − − 2.4 1.50 x 10 6 − − − − Paecilomyces marquandii − − 3.5 2.00 x 10 6 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 10 6 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 10 6 − − 0.5 4.50 x 10 6 − − Pseudogymnoascus sp. − − − − − − 0.5 3.00 x 10 5 − − − − Ramichloridium sp. − − − − − − − − 0.2 1.50 x 10 6 − − Solosympodiella sp. − − − − − − − − 0.2 1.50 x 10 6 − − Spicellum sp. − − − − − − − − − − 0.8 3.00 x 10 5 Stachybotrys eucylindriospora − − − − − − − − 0.2 1.50 x 10 6 − − Sterile (72) 11.8 4.55 x 10 7 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 10 5 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 10 7 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 b pi = proportional abundance of the ith species; number of species within a genus or group.
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