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.