Diversity of Fungi from the Mound Nests of Formica Ulkei and Adjacent Non-Nest Soils Lyndon B

562 ARTICLE Diversity of fungi from the mound nests of Formica ulkei and adjacent non-nest soils Lyndon B. Duff, Theresa M. Urichuk, Lisa N. Hodgins, Jocelyn R. Young, and Wendy A. Untereiner

Updated online 19 January 2018: The license for this article has been changed to the CC BY 4.0 license. The PDF and HTML versions of the article have been modified accordingly. Abstract: Culture-based methods were employed to recover 3929 isolates of fungi from soils collected in May and July 2014 from mound nests of Formica ulkei and adjacent non-nest sites. The abundance, diversity, and richness of species from nest mounds exceeded those of non-mound soils, particularly in July. Communities of fungi from mounds were more similar to those from mounds than non-mounds; this was also the case for non-mound soils with the exception of one non-mound site in July. Species of Aspergillus, Paecilomyces, and Penicillium were dominant in nest soils and represented up to 81.8% of the taxa recovered. Members of the genus Aspergillus accounted for the majority of Trichocomaceae from nests and were represented almost exclusively by Aspergillus navahoensis and Aspergillus pseudodeflectus. Dominant fungi from non-mound sites included Cladosporium cladosporioides, Geomyces pannorum, and species of Acremonium, Fusarium, Penicillium, and Phoma. Although mound nests were warmer than adjacent soils, the dominance of xerotolerant Aspergillus in soils from mounds and the isolation of the majority of Trichocomaceae at 25 and 35 °C suggests that both temperature and water availability may be determinants of fungal community structure in nests of F. ulkei. Key words: Aspergillus, fungal biodiversity, mound-building ant, xerotolerant. Résumé : On a eu recours a` des méthodes de culture pour recueillir 3929 isolats de champignons de sols prélevés en mai et juillet 2014 de fourmilières de Formica ulkei et de sols de sites adjacents. L’abondance, la diversité et la richesse des espèces présentes dans les fourmilières en monticule étaient supérieures a` celles de sols non issus de fourmilières, surtout en juillet. Les communautés de champignons de monticules s’apparentaient d’avantage a` celles d’autres monticuiles qu’a` celles de sols ordinaires. Ce fut la même chose pour les sols hors monticule sauf dans le cas d’un emplacement en juillet. Des espèces appartenant a` Aspergillus, Paecilomyces et Penicillium étaient dominantes dans les sols de fourmilières et représentaient jusqu’a` 81,8 % des taxons recueillis. Des membres du genre Aspergillus constituaient la majorité des trichocomacées présentes dans les nids et étaient presque exclu- sivement représentés par Aspergillus navahoensis et Aspergillus pseudodeflectus. Parmi les champignons dominants de sites hors monticule, on comptait Cladosporium cladosporioides, Geomyces pannorum et des espèces de Acremonium, Fusarium, Penicillium et Phoma. Les fourmilières étaient plus chaudes que les sols adjacents, mais la dominance d’Aspergillus xérotolérants dans les sols de fourmilières jumelée a` notre observation que la majorité des trichocomacées ont été isolées tant a` 25 qu’a` 35 °C indique que la température et la disponibilité en eau seraient des éléments détermi- nants de la structure des communautés fongiques dans les nids de F. ulkei. [Traduit par la Rédaction] Mots-clés : Aspergillus, biodiversité fongique, fourmis formant des monticules, xérotolérant.

Introduction pastures along the margins of forests and sparsely The mound-building ant Formica ulkei Emery (Hyme- wooded areas (Holmquist 1928; Dreyer and Park 1932; noptera: Formicidae) ranges from Alberta to Nova Scotia Sherba 1958). Nests are composed of excavated soil and (Canada) and southward to Illinois, Indiana, and Iowa are covered by a layer of thatch (i.e., small pieces of grass (USA) (Holmquist 1928; Sherba 1958; Glasier et al. 2013). and other plant material) (Sherba 1958, 1959; Scherba This species builds conspicuous nests in meadows and 1962).

Received 22 September 2015. Revision received 25 February 2016. Accepted 26 February 2016. L.B. Duff, T.M. Urichuk, L.N. Hodgins, J.R. Young, and W.A. Untereiner. Department of Biology, Brandon University, 270 18th Street, Brandon, MB R7A 6A9, Canada. Corresponding author: Wendy A. Untereiner (email: [email protected]). Copyright remains with the author(s) or their institution(s). This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Can. J. Microbiol. 62: 562–571 (2016) dx.doi.org/10.1139/cjm-2015-0628 Published at www.nrcresearchpress.com/cjm on 9 March 2016. Duff et al. 563

The mound nests of F. ulkei are thermoregulatory in Nests were sampled on 11 May and 14 July 2014 by function and are constructed to achieve and maintain collecting the uppermost 3 cm of soil beneath the thatch higher temperatures than adjacent undisturbed soils from the top and south sides of each mound. Each site on during the months when the ants are most active all mounds was sampled using a new plastic spoon. Soils (Scherba 1962). Nests are built in exposed sites and are to a depth of 3 cm were collected from adjacent non- oriented to maximize their exposure to solar radiation mound soil 1 m south of nests using a soil core sampler (Sherba 1958); they gain heat from solar radiation in the that was sterilized in 100% ethanol and rinsed in sterile early spring and maintain temperatures that are higher distilled water between samples. Samples were placed and more stable than those of surrounding soils because into separate, unused plastic freezer bags, sealed, and of the insulating properties of thatch (Scherba 1962; transported in an ice cooler to the laboratory. Each sam- Frouz and Jilková 2008). This layer of organic material ple was emptied into a clean aluminum pan, air-dried at prevents the overheating of mounds during the warmest room temperature (18–21 °C), subjected to sieving using a parts of the year for other ant species that construct 2 mm mesh to remove plant debris, and stored in a new thatched nests (Bollazzi and Roces 2010; Kadochová and freezer bag. Frouz 2014) and it may serve the same function in F. ulkei. Although it is recognized that mound-building ants Isolation and identification of fungi Individual soil samples were used within 3 days follow- are capable of dramatically modifying their environ- ing collection to prepare 10-fold serial dilutions in sterile ments and altering the chemical and physical properties distilled water ranging from 10−1 to 10−7. Each dilution of soils (Beattie and Culver 1977; Frouz and Jilková 2008; Jilková et al. 2011), few studies have explored the impact was plated in triplicate on dextrose – peptone – yeast of microclimatic conditions on the composition of the extract agar (DPYA) (Papavizas and Davey 1959) lacking communities of fungi in these soils (Ba et al. 2000; Zettler oxgall and sodium propionate, and on dichloran Rose et al. 2002; Rodrigues et al. 2014). Given the availability of Bengal agar (DRBA) (King et al. 1979) containing 25 mg of a large group of nests of F. ulkei in southeastern Mani- Rose Bengal, 2 mg of dichloran, and KH2PO4 rather than toba, we undertook a study to (i) confirm the tempera- K2HPO4. Both media were supplemented with 50 mg of ture characteristics of the mound nests of this species chlortetracycline hydrochloride and 50 mg of streptomy- reported in previous studies and (ii) test the hypothesis cin sulfate. Duplicate sets of plates were incubated at that the community of culturable fungi from soils from 25 and 35 °C for 5 days. nests differs from adjacent, non-nest soils. We were also All fungal colonies were transferred to modified interested in comparing the species richness and diver- Leonian’s agar (MLA) (Malloch 1981), incubated at room sity of the communities of culturable fungi of separate temperature, and identified on the basis of cultural and mound nests of F. ulkei. micromorphological characteristics. Isolates that could be discriminated as separate taxa within genera but not Materials and methods identified to species were numbered. Sporulating fungi Collection of soils and temperature data that could not be identified to the level of genus were Thermocron iButton data loggers (DS1921G, Maxim In- designated as “undetermined”, whereas those taxa that tegrated Products, San Jose, California, USA) that had did not sporulate on MLA were labeled “sterile” (see sup- been pre-set to measure temperature every 2 h were plemental Table S11). Nonfilamentous fungi and Zygomy- coated in Performix Plasti Dip (Plasti Dip International, cota, which were isolated in very low numbers on both Blaine, USA) to prevent moisture damage (Roznik and DRBA and DPYA, were disregarded. Fungi recovered on Alford 2012). Data loggers were buried 5 cm deep in soil on DPYA were also excluded from analyses because of the the top, south side, and north side of 3 mound nests of high levels of bacterial contamination, particularly in F. ulkei located on the unforested edge of a cattle pasture soils collected in July. that had not been grazed in approximately 10 years, south Dominant species of Aspergillus were characterized on of White Mud Falls, Manitoba (UTM coordinates of mound Czapek Dox agar (CZ), Czapek yeast agar (CYA), Czapek 1 = 14U 0707355 5588945; mound 2 = 14U 0707363 5588913; yeast agar with 20% sucrose (CY20S), and malt extract mound 3 = 14U 0707367 5588908). One data logger was bur- agar following Klich (2002a), and on creatine sucrose ied at a depth of 5 cm at one location 1 m south of each agar (CREA) as described by Samson et al. (2014). The mound. Another data logger was also secured at a height of thermotolerances of these taxa were determined by 2 m to the north (i.e., the shaded) side of a tree located in the assessing their ability to grow on CYA and MLA when middle of the study area to collect air temperatures. Data incubated at 37, 45, and 50 °C. Cultures used for DNA loggers recorded temperatures from 9 May to 18 September extraction were grown as described previously (Untereiner 2014. et al. 2008), and total nucleic acids were extracted from

1Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjm- 2015-0628.

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Table 1. Descriptive statistics for average daily temperatures (°C) of mound (M) and non-mound (S) sites. 95% Conﬁdence interval for mean Standard Standard Lower Upper Site N Minimum Maximum Mean deviation error bound 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

mycelia following the protocols of Lee and Taylor (1990). sured using the Morisita–Horn index of similarity in The nuclear ribosomal internal transcribed spacer BiodiversityR. These data were converted into distance (nucITS) region and a portion of the gene encoding the matrixes and employed to generate dendrograms using protein ␤-tubulin were amplified as described in Bogale hierarchical clustering R version 3.2.2 (R Core Team et al. (2010) using the primers pairs ITS4 and ITS5 (nucITS) 2015). (White et al. 1990) and Bt2a and Bt2b (␤-tubulin) (Glass and Donaldson 1995). PCR products were cleaned using a Results QIAquick PCR Purification kit (Qiagen, Mississauga, Can- Maximum and mean average daily temperatures of ada). Sequencing reactions were performed using a Taq soils from mounds exceeded those of adjacent non- DyeDeoxy cycle sequencing kit or a BigDye Terminator mound sites (Table 1), and results of an ANOVA (F[6,924] = cycle sequencing kit (Applied Biosystems, Inc., Foster 61.90, p = 0.000) (supplemental Table S21) indicated City, California, USA) using the primers listed above. significant differences in the mean average temperatures Confirmation of the identification of these taxa as Asper- between sites. Post-hoc Tukey’s HSD multiple compari- gillus navahoensis (UAMH 11867; GenBank KU310972, sons revealed that the average daily temperatures of KU310974) and Aspergillus pseudodeflectus (UAMH 11868; mounds were higher than non-mound sites (supplemen- GenBank KU310973, KU310975) was based on the com- tal Table S31). The temperatures of mounds 2 and 3 did parison of generated DNA sequences to the nucITS and not differ significantly nor were significant differences ␤-tubulin barcodes provided by Samson et al. (2014). in temperature seen among non-mound sites. All mound sites were warmer than ambient temperature, whereas Statistical analysis Daily temperature readings for Thermocron iButton non-mound sites 2 and 3 were cooler. Non-mound site 1 data loggers placed in the south side of each mound were did not differ significantly from ambient temperature. averaged per day from 6 May to 18 September 2014. Data Differences in the average weekly temperatures of soils for the tops of mounds were not included in averages from mound and non-mound sites are illustrated in because 2 iButtons from this location were dislodged Fig. 1. during the course of the study. Data from the north sides Excluding nonfilamentous fungi and Zygomycota, a of mounds were also excluded because these tempera- total of 3929 isolates representing 307 taxa were recov- tures differed significantly from temperatures from the ered at all dilutions from mound nests and adjacent non- south sides of mounds (data not shown). A 1-way analysis of mound sites on DRBA (Table 2, supplemental Table S11). variance (ANOVA) of temperature differences (mounds 1, 2, Higher numbers of isolates and taxa were obtained from and 3; non-mounds 1, 2, and 3; and ambient temperature) DRBA incubated at 25 °C. Soils collected in July contained was conducted using PSPP software version 0.8.4 (Pfaff larger numbers of isolates (Table 2) and had greater spe- 2015). The same software was used to perform a post-hoc cies richness (Table 3) than soils collected in May. Tukey’s honestly significant difference (HSD) test. The abundance (CFU/g), diversity, and richness of spe- Numbers of isolates on DRBA were used to calculate cies from soils of nest mounds generally exceeded those colony-forming units (CFU) per gram of soil and the pro- of non-mounds, particularly in July (Table 3). Mound soils portional abundance of each species or taxon within a differed in richness among sites in July, as did soils from group (i.e., “sterile” and “undetermined”). Diversity indi- non-mounds. Species richness in May did not differ as ces (Shannon, Simpson, and Simpson inverse) were cal- dramatically between sites, with the exception of mound 1, culated using BiodiversityR (Kindt and Coe 2005). Rényi which was undersampled because of an error in the prep- diversity profiles describing the richness and evenness of aration of soil dilutions. Rényi profiles did not discriminate sites were also generated using BiodiversityR. Between- between mound and non-mound soils in May with respect sites comparisons of species-abundance data were mea- to species diversity; soils in July differed, with the exception

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Fig. 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.

Table 2. Number of isolates of fungi recovered at 25 and 35 °C on DRBA from mound (M) and non-mound (S) sites in May and July 2014. Sample Incubation time temp. (°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 of non-mound 2 that intersected with mound 2 and mound 100% similarity to EF652331), which represented 17.4%– 3 (Fig. 2). As illustrated in Fig. 2, the evenness of species 44.2% and 8.6%–37.6% of the recovered taxa, respectively from soils from non-mound 2 was higher than at all other (Tables 4 and 5). Both species were recovered from all sites in May and July, but the evenness of species from the mounds in May and July. The proportional abundances remaining sites could not be ranked. The evenness of spe- of these species were higher in May except that A. pseu- cies from mound 1 in May likely reflects the aforemen- dodeflectus was more abundant in mound 2 in July. Asper- tioned undersampling. Communities in soils from mounds gillus pseudodeflectus was recovered from only a single were more similar to species from mounds than non- non-mound site in May but in very low abundance (0.3%) mound sites in May and July (Fig. 3). This was also the case representing a single isolate, whereas A. navahoensis for taxa from non-mounds, with the exception of the com- was never isolated from non-mound soils. Cultures of munity from non-mound 1 in July that more closely resem- A. navahoensis conformed to the description of this spe- bled the mycota from mounds. cies provided by Christensen and States (1982) and were The most abundant fungi in soil from mounds were distinctive in producing rapidly maturing ascomata, Aspergillus navahoensis (ITS 99% similarity to EF652424; abundant Hülle cells, and crystal-encrusted hyphae. ␤-tubulin 99% similarity to EF652248) and Aspergillus Aspergillus navahoensis grew at 37 and 45 °C, but showed pseudodeflectus (ITS 100% similarity to EF652507; ␤-tubulin better growth at 37 °C; it exhibited no growth at 50 °C.

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Table 3. Richness and diversity estimators of fungal communities of mound (M) and non-mound (S) sites calculated in BiodiversityR. Total Species Shannon Simpson Simpson CFU/g soil richness diversity diversitya inverse May 2014 M1 115.67×107 8 1.32±0.14 0.668±0.072 3.01±0.65 S1 10.83×107 44 2.75±0.07 0.871±0.014 7.72±0.81 M2 29.55×107 36 2.32±0.08 0.792±0.029 4.81±0.68 S2 36.66×107 33 2.81±0.09 0.928±0.003 13.86±0.61 M3 57.75×107 30 2.27±0.09 0.821±0.022 5.59±0.70 S3 11.40×107 37 2.21±0.09 0.800±0.022 5.00±0.55 July 2014 M1 38.70×107 49 2.11±0.08 0.781±0.021 4.57±0.43 S1 5.77×107 36 2.00±0.06 0.654±0.056 2.89±0.47 M2 42.45×107 66 3.09±0.06 0.894±0.008 9.46±0.77 S2 6.15×107 50 3.40±0.06 0.954±0.002 21.7±0.94 M3 82.88×107 85 3.33±0.05 0.924±0.005 13.2±0.80 S3 3.57×107 19 0.96±0.03 0.315±0.157 1.46±0.33

aSimpson diversity = (1 − Simpson index).

Aspergillus pseudodeflectus grew at 37 °C but exhibited no Our investigation also demonstrates that the commu- growth at 45 and 50 °C. nities of fungi in soils from nest mounds of F. ulkei differ Additional taxa from mound soils with abundances from non-mound soils with respect to the abundances of higher than 5% included Cladosporium cladosporioides, species, species richness, and diversity. Soils of nest Geomyces pannorum, Myriothecium sp., and Acremonium spp. mounds of F. ulkei resemble those of Solenopsis invicta (red However, these fungi were not the dominant members imported fire ant) in containing greater numbers of fun- of the mycota of all mounds nor were they equally abun- gal colonies than adjacent, non-nest soils (Zettler et al. dant in the same mound in both May and July. Undeter- 2002), but they differ in having higher levels of species mined species were dominant members of soils from richness. In July, 2 of the 3 mounds we sampled had mound 3 and were more abundant in July. Sterile fungi higher levels of species diversity than did non-nest comprised more than 5% of the isolates in soils from soils. In contrast, culture-dependent assessments re- every mound but only in July. vealed that below-ground nests of young colonies of Atta Species of Penicillium were dominant members of the (leaf-cutting ants) contain lower to comparable numbers mycota of soils from non-mound sites but were less of colonies of filamentous fungi as non-nest soils but abundant in May than in July. Other taxa from non- have similar levels of species diversity and richness mound sites having abundances greater than 5% in- (Rodrigues et al. 2014). cluded Cladosporium cladosporioides, Geomyces pannorum, Members of the Trichocomaceae (species of Aspergillus, undetermined and sterile fungi, and members of the Paecilomyces, and Penicillium) were dominant in soils from genera Acremonium, Fusarium, and Phoma. However, only mound nests of F. ulkei and represented 39.5% (mound 3) Geomyces pannorum, undetermined and sterile fungi, and to 81.8% (mound 1) of the total numbers of taxa recov- species of Penicillium represented more than 5% of the ered. Trichocomaceae are among the most common taxa recovered at more than one site at a given sampling filamentous Ascomycota isolated from the nests of mound- time. building and leaf-cutting ants (Zettler et al. 2002; Baird et al. 2007; Sharma and Sumbali 2013; Rodrigues et al. Discussion 2014), but only a single study (Zettler et al. 2002) resem- The results of the present study agree with Scherba bles ours in recovering different representatives of this (1962) who reported that the thatch-covered mound family from nests and non-nest soils. nests of F. ulkei are warmer than surrounding undis- Aspergillus accounted for more than 80% of Trichoco- turbed soils during the months when these ants are most maceae isolated from mound nests and was represented active. We also observed significant differences between almost exclusively by A. navahoensis (section Nidulantes) the temperatures of the north and south sides of mounds and A. pseudodeflectus (section Usti). Aspergillus navahoensis (data not included), a phenomenon that can likely be was described from soils from a cool desert shrub com- attributed to variations in the dimensions of mounds, munity in northern Arizona (Christensen and States the composition and density of thatch, and degree of 1982) and belongs to a section of the genus that occurs at shading (Scherba 1962; Frouz 2000; Kadochová and Frouz greater than expected frequencies in desert soils (Klich 2014). 2002b). This species was recovered originally in low num-

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Fig. 2. Renyi proﬁles comparing the diversity of fungi Fig. 3. Dendrograms illustrating Morisita–Horn found in mound (M1, M2, and M3) and non-mound soils similarities between the communities of fungi from (S1, S2, and S3) from (a) May 2014 and (b) July 2014. Alpha = mound (M1, M2, and M3) and non-mound (S1, S2, and S3) 0 is the species richness, alpha = 1 is the Shannon–Weiner soils in (a) May 2014 and (b) July 2014. diversity index, and alpha = 2 is the log of the reciprocal of the Simpson diversity index.

Trichocomaceae were also abundant in soils from non- mound sites but were represented almost exclusively by species of Paecilomyces and Penicillium. These genera were bers (Christensen and States 1982) and, apart from the consistently more abundant in non-mound soils than in present study, does not appear to have been collected soils from mounds. Members of the genus Aspergillus since it was described. Aspergillus pseudodeflectus is an were absent from non-mound soils with the exception of infrequently collected osmophilic species described from a single colony of A. pseudodeflectus that we suspect was a desert soils in Egypt (Samson and Mouchacca 1975) that contaminant. was reported to be restricted to the tropics and subtrop- Differences in the fungal communities of the soils of ics (Christensen and Tuthill 1985). It is closely related to nest mounds of F. ulkei and adjacent non-nest sites likely A. calidoustus, a more commonly encountered species reflect environmental factors that are influenced by nest known from clinical and environmental sources that is location and architecture. For example, mound nests of distinguished from A. pseudodeflectus based on its ecology F. ulkei in Illinois were shown to be restricted to drier and molecular barcodes (Samson et al. 2011, 2014). regions along forest margins and were constructed to

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Table 4. Colony-forming units per gram of soil (CFU/g) and the proportional abundance (pi)a of taxa recovered May 2014 from mound (M) and non-mound (S) sites. M1 S1 M2 S2 M3 S3

Taxon (n = 126)b pi (%) CFU/g pi (%) CFU/g pi (%) CFU/g pi (%) CFU/g pi (%) CFU/g pi (%) CFU/g

Acremonium spp. (9) —— 6.1 6.60×106 0.5 1.50×106 9.8 3.60×107 13.8 7.95×107 1.8 2.10×106 Aspergillus navahoensis 41.5 4.80×108 — — 44.2 1.31×108 — — 20.8 1.20×108 — — Aspergillus pseudodeﬂectus 37.6 4.35×108 — — 8.6 2.55×107 — — 34.3 1.98×108 0.3 3.00×105 Aspergillus spp. 1.3 1.50×107 — — — — — — 0.3 1.50×106 — — Aureobasidium sp. — — — — — — — — — — 0.3 3.00×105 Bipolaris spp. (2) —— — — 2.0 6.00×106 — — — — — — Cladosporium cladosporioides 1.3 1.50×107 1.1 1.20×106 9.6 2.85×107 0.8 3.00×106 2.6 1.50×107 26.3 3.00×107 Cladosporium herbarum 2.6 3.00×107 1.7 1.80×106 2.0 6.00×106 — — 0.5 3.00×106 — — Cladosporium macrocarpum — — — — — — — — — — 0.5 6.00×105 Curvularia brachyspora — — — — 0.5 1.50×106 — — — — 2.6 3.00×106 Curvularia geniculata 1.3 1.50×107 — — — — — — — — — — Curvularia spp. (2) —— — — 1.0 3.00×106 — — 1.6 9.00×106 2.6 3.00×106 Devriesia sp. — — 0.3 3.00×105 — — — — — — — — Doratomyces nanus — — 0.3 3.00×105 — — — — — — — — Fusarium spp. (4) —— 5.5 6.00×106 5.1 1.50×107 13.1 4.80×107 1.3 7.50×106 — — Geomyces sp. — — 2.7 3.00×106 — — — — — — — — Geomyces pannorum — — 16.9 1.83×107 2.5 7.50×106 8.8 3.24×107 8.3 4.80×107 34.2 3.90×107 Humicola sp. — — — — 0.5 1.50×106 — — — — — — Lecythophora sp. — — — — — — — — — — 0.5 6.00×105 Myrothecium sp. 13.0 1.50×108 0.3 3.00×105 0.5 1.50×106 — — — — — — Paecilomyces spp. (2) —— 0.3 3.00×105 — — 0.8 3.00×106 — — — — Paecilomyces marquandii — — 1.4 1.50×106 — — 8.2 3.00×107 — — 3.4 3.90×106 Penicillium spp. (20) —— 1.7 1.80×106 10.7 3.15×107 36.0 1.32×108 0.5 3.00×106 10.0 1.14×107 Phoma spp. (11) 1.4 1.67×107 33.8 3.66×107 2.5 7.50×106 — — 4.9 2.85×107 2.9 3.30×106 Sterile (20) — — 12.5 1.35×107 5.6 1.65×107 3.4 1.23×107 2.1 1.20×107 3.4 3.90×106 Tricellula sp. — — — — — — 0.8 3.00×106 — — — — Trichocladium sp. — — — — — — — — 2.6 1.50×107 — — Trichoderma spp. — — 2.7 3.00×106 0.5 1.50×106 — — — — 0.5 6.00×105 Undetermined (36) —— 12.7 1.38×107 3.6 1.05×107 18.2 6.69×107 6.5 3.75×107 10.5 1.20×107 Total Trichocomaceae 81.8 3.4 63.5 45 55.9 13.7

api is the proportional abundance of the ith species. bThe number in parentheses next to each taxon is the number of species within that genus or group. maximize insolation (Dreyer and Park 1932; Dreyer 1942). are common in soils from warmer regions of the world Nest construction also dramatically alters the physical (Domsch et al. 1993; Bills et al. 2004) and are among the characteristics of soil that operate to regulate the mois- most xerotolerant Ascomycota (Dix and Webster 1995; ture content and temperatures of mounds relative to Zak and Wildman 2004). Members of this genus are par- surrounding soils. Mound building can increase soil po- ticularly abundant in desert and grassland soils, where rosity and reduce the bulk density of soils, both of which they represent up to 20% of isolated species (Christensen influence soil aeration and soil permeability (Frouz and and Tuthill 1985). Although both A. navahoensis and Jilková 2008). The moisture content in mounds of F. ulkei A. pseudodeflectus were capable of growth at the highest at 5 cm has been shown to be lower than in adjacent soils average daily temperatures recorded for mound and throughout the year and lower than in mounds at 30 cm non-mound soils, only the former species was deter- during the warmer months when the ants were active mined to be thermotolerant (i.e., it grows at tempera- (Sherba 1959). Although we did not determine the mois- tures below 20 °C and at 40 °C or higher). This finding, in ture content of soils at our study site, we observed that conjunction with our observation that all Aspergillus and the daily temperatures of nests of F. ulkei peaked in the Paecilomyces and nearly half of the species of Penicillium evening and decreased slowly during the night (data not were isolated at both 25 and 35 °C (supplemental shown), which is in agreement with the description of Table S11), suggests that water availability is also a deter- the drier and more exposed nests of Formica polyctena minant of fungal community structure in mound nests (European red wood ant) (Frouz 2000). of F. ulkei. The supposition that the nests of F. ulkei at our study Factors such as nutrient availability, soil chemistry, site were drier than adjacent sites is also supported by and the physical properties of soils also likely influence the dominance of Aspergillus in nests as compared with the structure of fungal communities in the mound nests soils located 1 m from each mound. Species of Aspergillus of F. ulkei and adjacent non-nest soils. For example, soils

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Table 5. Colony-forming units per gram of soil (CFU/g) and the proportional abundance (pi)a of taxa recovered July 2014 from mound (M) and non-mound (S) sites. M1 S1 M2 S2 M3 S3

Taxa (n = 197)b pi (%) CFU/g pi (%) CFU/g pi (%) CFU/g pi (%) CFU/g pi (%) CFU/g pi (%) CFU/g

Acremonium spp. (12) 1.3 5.17×106 7.5 4.30×106 1.4 6.00×106 12.7 7.80×106 5.6 4.65×107 0.8 3.00×105 Acremonium-like spp. (5) —— 1.6 9.00×105 3.5 1.50×107 1.0 6.00×105 0.2 1.50×106 2.5 9.00×105 Alternaria alternata 0.0 1.67×105 — — 0.4 1.50×106 — — 1.8 1.50×107 — — Aspergillus navahoensis 36.8 1.42×108 — — 25.4 1.08×108 — — 17.4 1.44×108 — — Aspergillus niger 0.4 1.50×106 — — — — — — — — — — Aspergillus pseudodeﬂectus 22.4 8.65×107 — — 17.0 7.20×107 — — 17.2 1.43×108 — — Aspergillus spp. — — — — — — — — 1.8 1.50×107 — — Aureobasidium pullulans 0.1 5.00×105 — — 0.7 3.00×106 — — 0.9 7.50×106 — — Bipolaris sp. 0.8 3.00×106 — — — — — — — — — — Chrysosporium spp. (2) —— 1.2 6.67×105 — — 1.0 6.00×105 — — — — Cladosporium cladosporioides 0.5 2.00×106 — — 2.8 1.20×107 3.4 2.10×106 0.7 6.00×106 — — Cladosporium herbarum 1.2 4.67×106 — — 0.4 1.50×106 0.5 3.00×105 0.9 7.50×106 — — Cladosporium sphaerospermum 0.8 3.00×106 — — — — 0.5 3.00×105 — — — — Clonostachys rosea 0.8 3.00×106 — — 3.5 1.50×107 1.5 9.00×105 — — — — Clonostachys sp. — — — — 0.4 1.50×106 — — — — — — Curvularia geniculata 0.4 1.50×106 — — 0.7 3.00×106 — — 0.5 4.50×106 — — Devriesia sp. — — — — — — 1.5 9.00×105 — — — — Fusarium spp. (4) 0.0 1.67×105 0.6 3.33×105 0.4 1.50×106 1.5 9.00×105 0.4 3.30×106 0.8 3.00×105 Geomyces spp. (3) — — 0.6 3.33×105 0.4 1.50×106 — — 0.0 1.50×105 0.8 3.00×105 Geomyces pannorum 0.5 2.00×106 5.2 3.00×106 0.7 3.00×106 9.3 5.70×106 6.5 5.40×107 0.8 3.00×105 Humicola sp. — — — — — — 0.5 3.00×105 — — — — Humicola-like sp. — — — — — — 0.5 3.00×105 — — — — Idriella lunata — — — — 0.4 1.50×106 0.5 3.00×105 — — — — Myrmecridium sp. — — — — — — — — 0.5 4.50×106 — — Myrmecridium schulzeri — — 0.6 3.33×105 — — — — 0.2 1.50×106 — — Myrothecium sp. 17.5 6.77×107 — — — — — — — — — — Paecilomyces spp. (2) —— — — — — 2.4 1.50×106 — — — — Paecilomyces marquandii — — 3.5 2.00×106 0.4 1.50×106 3.9 2.40×106 0.2 1.50×106 0.8 3.00×105 Penicillium sp. (26) 1.8 6.83×106 62.8 3.62×107 8.1 3.45×107 39.0 2.40×107 2.9 2.40×107 85.7 3.06×107 Pleurostomophora sp. — — — — 0.4 1.50×106 — — 0.5 4.50×106 — — Pseudogymnoascus sp. — — — — — — 0.5 3.00×105 — — — — Ramichloridium sp. — — — — — — — — 0.2 1.50×106 — — Solosympodiella sp. — — — — — — — — 0.2 1.50×106 — — Spicellum sp. — — — — — — — — — — 0.8 3.00×105 Stachybotrys eucylindrospora — — — — — — — — 0.2 1.50×106 — — Sterile (72) 11.8 4.55×107 9.8 5.63×106 25.8 1.10×108 16.6 1.02×107 20.8 1.73×108 0.8 3.00×105 Trichoderma spp. 0.0 1.67×105 0.6 3.33×105 3.5 1.50×107 2.0 1.20×106 — — 0.8 3.00×105 Undetermined (42) 2.9 1.13×107 6.3 3.63×106 3.9 1.65×107 1.5 9.00×105 20.3 1.68×108 5.0 1.80×106 Total Trichocomaceae 61.4 66.3 50.9 45.3 39.5 86.5

api is the proportional abundance of the ith species. bThe number in parentheses next to each taxon is the number of species within that genus or group. in ant nests have higher levels of nutrients (Frouz et al. nest mounds and adjacent non-nest soils, the isolation of 2005; Frouz and Jílková 2008; Ginzburg et al. 2008; fungi over a longer period of time, the use of media Jílková et al. 2015) and differ from surrounding soils in designed to isolate ecologically specialized taxa, and the pH, porosity, and the content of organic matter (Frouz determination of temperature differences from a larger and Jílková 2008; Jílková et al. 2011). Microbial activity is number of sites within nest mounds. And because the assumed to be higher in ant nests because of these dif- enumeration methods used in our study are selective for ferences, but the mechanisms underlying the impacts of fungi that produce abundant spores (Garrett 1981), it ants on soil processes and other soil biota are not well would be valuable to examine the diversity of culturable understood (Frouz and Jílková 2008; Del Toro et al. 2012). fungi in these soils using alternative isolation methods Nests of F. ulkei are reservoirs of fungal diversity that (described in Bills et al. 2004). The complementary use of should be explored further using the approaches pre- sequence-based approaches, such as environmental meta- sented here. Our understanding of these communities genomics, would also enhance our understanding of would be improved with the more frequent sampling of these assemblages of fungi, particularly in recovering

Published by NRC Research Press 570 Can. J. Microbiol. Vol. 62, 2016 nonculturable species and taxa that are undersampled ant mounds with reference to their age. Ecology, 23(4): 486– employing cultured-dependent methods (Bills et al. 2004; 490. doi:10.2307/1930137. Dreyer, W.A., and Park, T. 1932. Local distribution of Formica Karst et al. 2013; Rodrigues et al. 2014). Sequence-based ulkei mound-nests with reference to certain ecological fac- approaches would also facilitate the identification of tors. Psyche, 39(4): 127–133. doi:10.1155/1932/28491. sterile fungi and many of the micromorphologically sim- Frouz, F., Kalcíˇk, J., and Cudlín, P. 2005. Accumulation of phos- ple or taxonomically challenging species present in the phorus in nests of red wood ants Formica s. str. Ann. Zool. mound nests of F. ulkei. Fenn. 42: 269–275. Frouz, J. 2000. The effect of nest moisture on daily temperature Acknowledgements regime in the nests of Formica polyctena wood ants. Insectes soc. 47: 229–235. doi:10.1007/PL00001708. We are indebted to Gary McNeely (Brandon University) Frouz, J., and Jílková, V. 2008. The effect of ants on soil proper- and 3 anonymous reviewers for their insightful editorial ties and processes (Hymenoptera: Formicidae). Myrmecol. comments and suggestions for the improvement of this News, 11: 191–199. paper. We also thank Dennis and Jacqueline Caya for Garrett, S.D. 1981. Soil fungi and soil fertility: an introduction to soil mycology. 2nd ed. Pergamon Press, Oxford, UK. their permission to access nest mounds located on their Ginzburg, O., Whitford, W.G., and Steinberger, Y. 2008. Effects property and David Caya for serving as a bear guard dur- of harvester ant (Messor spp.) activity on soil properties and ing soil sampling. Financial support for this study was microbial communities in a Negev Desert ecosystem. Biol. provided by a Natural Sciences and Engineering Re- Fertil. 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