Draft 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

The mound nests of F. ulkei are thermoregulatory in function and are constructed to achieve and maintain higher temperatures than adjacent undisturbed soils during the months when the ants are most active (Sherba 1962).Nests are built in exposed sites and are oriented to maximize their exposure to solar radiation (Sherba 1958); they gain heat from solar radiation in the early spring and maintain temperatures that are higher and more stable than those of surrounding soils because of the insulating properties of thatch (Sherba 1962;Frouz and Jilková 2008).This layer of organic material prevents the overheating of mounds during the warmest parts of the year in other ant species that construct thatched nests (Bollazzi and Rocces 2010;Kadochová and Frouz 2014) and it may serve the same function in F. ulkei.
Although it is recognized that mound-building ants are capable of dramatically modifying their environments and altering the chemical and physical properties of soils (Beattie and Culver 1977;Frouz and Jilková 2008;Jilková et al. 2011), few studies have explored the impact of microclimatic conditions on the composition of the communities of fungi in these soils (Ba et al. 2000;Zettler et al. 2002;Rodrigues et al. 2014).Given the availability of a large group of nests of F. ulkei in south-eastern Manitoba, we undertook a study to 1) confirm the temperature characteristics of the mound nests of this species reported in previous studies, and 2) test the hypothesis that the community of culturable fungi from soils from D r a f t nests differs from adjacent, non-nest soils.We were also interested in comparing the species richness and diversity of the communities of culturable fungi of separate mound nests of F. ulkei.

Collection of soils and temperature data
Thermocron iButton data loggers (DS1921G, Maxim Integrated Products, San Jose, USA) that had been pre-set to measure temperature every two hours were coated in Performix Plasti Dip (Plasti Dip International, Blaine, USA) to prevent moisture damage (Roznik and Alford 2012).Data loggers were buried 5 cm deep in soil on the top, south side and north side of three mound nests of Formica ulkei located on the un-forested edge of a cattle pasture that had not been grazed in approximately 10 years, south of White Mud Falls, Manitoba (UTM coordinates of mound 1 = 14U 0707355 5588945; mound 2 = 14U 0707363 5588913; mound 3 = 14U 0707367 5588908).One data logger was buried at a depth of 5 cm at one location 1 m south of each mound.Another data logger was also secured at a height of 2 m to the north (i.e., the shaded) side of a tree located in the middle of the study area to collect air temperatures.Data loggers recorded temperatures from 9 May to 18 September 2014.
Nests were sampled on 11 May and 14 July 2014 by collecting the uppermost 3 cm of soil beneath the thatch from the top and south sides of each mound.Each site on all mounds was sampled using a new plastic spoon.Soils to a depth of 3 cm were collected from adjacent non-mound soil 1 m south of nests using a soil core sampler that was sterilized in 100% ethanol and rinsed in sterile distilled water between samples.Samples were placed into separate, unused plastic freezer bags, sealed and transported in an ice cooler to the laboratory.Each sample was emptied into a clean aluminum pan, air-dried at room D r a f t temperature (18-21˚C), subjected to sieving using a 2 mm mesh to remove plant debris, and stored in a new freezer bag.

Isolation and identification of fungi
Individual soil samples were used within 3 days following collection to prepare ten-fold serial dilutions in sterile distilled water ranging from 10 -1 to 10 -7 .Each dilution was plated in triplicate on Dextrose-Peptone-Yeast Extract agar (DPYA) (Papavizas and Davey 1959) lacking oxgall and sodium propionate, and Dichloran Rose Bengal agar (DRBA) (King et al. 1979) containing 25 mg Rose Bengal, 2 mg dichloran, and KH 2 PO 4 rather than K 2 HPO 4 .Both media were supplemented with 50 mg chlortetracycline hydrochloride and 50 mg streptomycin sulphate.Duplicate sets of plates were incubated at 25˚C and 35˚C for 5 days.
All fungal colonies were transferred to Modified Leonian's agar (MLA) (Malloch 1981), incubated at room temperature and identified based on cultural and micro-morphological characters.Isolates that could be discriminated as separate taxa within genera but not identified to species were numbered.Sporulating fungi that could not be identified to the level of genus were designated as "undetermined" whereas those taxa that did not sporulate on MLA were labeled "sterile" (see supplemental Table S1).Non-filamentous fungi and Zygomycota, which were isolated in very low numbers on both DRBA and DPYA, were disregarded.Fungi recovered on DPYE were also excluded from analyses because of the high levels of bacterial contamination, particularly in soils collected in July.Dominant species of Aspergillus were characterized on Czapek Dox agar (CZ), Czapek Yeast agar (CYA), Czapek Yeast agar with 20% sucrose (CY20S) and Malt Extract agar following Klich (2002a) and on Creatine Sucrose agar (CREA) as described by Samson et al. (2014).The thermotolerances of these taxa were determined by assessing their ability to grow on CYA and MLA when incubated at 37˚C, 45˚C, and 50˚C.Cultures used for DNA D r a f t extraction were grown as described previously (Untereiner et al. 2008) and total nucleic acids were extracted from mycelia following the protocols of Lee and Taylor (1990).The nuclear ribosomal internal transcribed spacer (nucITS) region and a portion of the gene encoding the protein β-tubulin were amplified as described in Bogale et al. (2010) using the primers ITS4, ITS5 (nucITS) (White et al. 1990) and Bt2a, and Bt2b (β-tubulin) (Glass and Donaldson 1995).PCR products were cleaned using a QIAquick PCR Purification Kit (Qiagen, Mississauga, Canada).Sequencing reactions were performed using a Taq DyeDeoxy cycle sequencing kit or a BigDye Terminator cycle sequencing kit (Applied Biosystems, Inc., Foster City, USA) using the primers listed above.Confirmation of the identification of these taxa as Aspergillus navahoensis (UAMH 11867; GenBank KU310972, KU310974) and A. pseudodeflectus (UAMH 11868; GenBank KU310973, KU310975) was based on the comparison of generated DNA sequences to the nucITS and β-tubulin barcodes provided by Samson et al. (2014).

Statistical analyses
Daily temperature readings for Thermocron iButton data loggers placed in the south side of each mound were averaged per day from May 6 to September 18, 2014.Data for the tops of mounds were not included in averages because two iButtons from this location were dislodged during the course of the study.Data from the north sides of mounds were also excluded because these temperatures differed significantly from temperatures from the south sides of mounds (data not shown).A one-way analysis of variance (ANOVA) of temperature differences (mounds 1, 2, and 3, non-mounds 1, 2, and 3, and ambient temperature) was conducted using PSPP v 0.8.4 (Pfaff 2015).The same software was used to perform a posthoc Tukey HSD test.

D r a f t
Numbers of isolates on DRBA were used to calculate colony-forming units (CFU) per g of soil and the proportional abundance of each species or taxon within a group (i.e., "sterile" and "undetermined").Diversity indices (Shannon, Simpson and Simpson inverse) were calculated using BiodiversityR (Kindt and Coe 2005).Rényi diversity profiles describing the richness and evenness of sites were also generated using BiodiversityR.Between sites comparisons of species-abundance data were measured using the Morisita-Horn index of similarity in BiodiversityR.These data were converted into distance matrixes and employed to generate dendrograms using hierarchical clustering R v 3.2.2(R Core Team 2015).

Results
Maximum and mean average daily temperatures of soils from mounds exceeded those of adjacent non-mound sites (Table 1) and results of an ANOVA (F (6, 924) = 61.90,p = 0.000) (Supplemental Table S2) indicated significant differences in the mean average temperatures between sites.Post-hoc Tukey HSD multiple comparisons revealed that the average daily temperatures of mounds were higher than non-mound sites (Supplemental Table S3).The temperatures of mound 2 and 3 did not differ significantly, nor were significant differences in temperature seen among non-mound sites.All mound sites were warmer than ambient temperature whereas non-mound sites 2 and 3 were cooler.Non-mound site 1 did not differ significantly from ambient temperature.Differences in the average weekly temperatures of soils from mound and non-mound sites are illustrated in Figure 1.
Excluding non-filamentous fungi and Zygomycota, a total of 3929 isolates representing 307 taxa were recovered at all dilutions from mound nest and adjacent non-mound sites on DRBA (Table 2, Supplemental Table S1).Higher numbers of isolates and taxa were obtained from DRBA incubated at 25 C. Soils collected in July contained a larger numbers of isolates (Table 2) and had greater species richness (Table 3) than soils collected in May.

D r a f t
The abundance (CFU g -1 ), diversity, and richness of species from soils of nest mounds generally exceeded those of non-mounds, particularly in July (Table 3).Mound soils differed in richness among sites in July, as did soils from non-mounds.Species richness in May did not differ as dramatically between sites with the exception of mound 1 which was undersampled because of an error in the preparation of soil dilutions.Rényi profiles did not discriminate between mound and non-mound soils in May with respect to species diversity; soils in July differed with the exception of non-mound 2 that intersected with mound 2 and mound 3 (Figure 2).As illustrated in Figure 2, the evenness of species from soils from nonmound 2 was higher than at all other sites in May and July but the evenness of the remaining sites could not be ranked.The evenness of soil from mound 1 in May likely reflects the aforementioned under-sampling.Communities in soils from mounds were more similar to species from mounds than non-mound sites in May and July (Figure 3).This was also the case for taxa from non-mounds with the exception of the community from non-mound 1 in July that more closely resembled the mycota from mounds.
The most abundant fungi in soil from mounds were Aspergillus navahoensis (ITS 99% similarity to EF652424; β-tubulin 99% similarity to EF652248) and Aspergillus pseudodeflectus (ITS 100% similarity to EF652507; β-tubulin 100% similarity to EF652331), that represented 17.4 to 44.2% and 8.6 to 37.6% of the recovered taxa, respectively (Tables 4-5).Both species were recovered from all mounds in May and July.The proportional abundances of these species were higher in May except that A. pseudodeflectus was more abundant in mound 2 in July.Aspergillus pseudodeflectus was recovered from only a single non-mound site in May but in very low abundance (0.3%) representing a single isolate whereas A. navahoensis was never isolated from non-mound soils.Cultures of A. navahoensis conformed to the description of this species provided by Christensen and States (1982) and were distinctive in producing rapidly maturing ascomata, abundant Hülle cells, and D r a f t crystal-encrusted hyphae.Aspergillus navahoensis grew at 37˚C and at 45˚C, but showed better growth at 37˚C; it exhibited no growth at 50˚C.Aspergillus pseudodeflectus grew at 37˚C but exhibited no growth at 45˚C and 50˚C.
Additional taxa from mound soils with abundances higher than 5% included Cladosporium cladosporioides, Geomyces pannorum, Myriothecium sp. and species of Acremonium.However, these fungi were not the dominant members of the mycota of all mounds nor were they equally abundant in the same mound in both May and July.
Undetermined species were dominant members of soils from mound 3 and were more abundant in July.Sterile fungi comprised more than 5% of the isolates in soils from every mound but only in July.Species of Penicillium were dominant members of the mycota of soils from non-mound sites but were less abundant in May than in July.Other taxa from non-mound sites with abundances greater than 5% included Cladosporium cladosporioides, Geomyces pannorum, undetermined and sterile fungi, and members of the genera Acremonium, Fusarium and Phoma.However, only Geomyces pannorum, undetermined and sterile fungi, and species of Penicillium represented more than 5% of the taxa recovered at more than one site at a given sampling time.

Discussion
The results of the present study agree with Scherba (1962) who reported that the thatchcovered mound nests of Formica ulkei are warmer than surrounding undisturbed soils during the months when these ants are most active.We also observed significant differences between the temperatures of the north and south sides of mounds (data not included), a phenomenon that can likely be attributed to variations in the dimensions of mounds, the composition and density of thatch, and degree of shading (Scherba 1962;Frouz 2000; D r a f t Kadochová and Frouz 2014).
Our investigation also demonstrates that the communities of fungi in soils from nest mounds of Formica ulkei differ from non-mound soils with respect to the abundances of species, species richness, and diversity.Soils of nest mounds of F. ulkei resemble those of Solenopsis invicta (red imported fire ant) in containing greater numbers of fungal colonies than adjacent, non-nest soils (Zettler et al. 2002) but they differ in having higher levels of species richness.In July, two of the three mounds we sampled had higher levels of species diversity than non-nest soils.In contrast, culture-dependent assessments revealed that below ground nests of young colonies of Atta (leaf-cutting ants) contain lower to comparable numbers of colonies of filamentous fungi as non-nest soils but have similar levels of species diversity and richness (Rodrigues et al. 2014).

Members of the Trichocomaceae (species of Aspergillus, Paecilomyces and Penicillium)
were dominant in soils from mound nests of F. ulkei and represented 39.5% (mound 3) to 81.8% (mound 1) of the total numbers of taxa recovered.Trichocomaceae are among the most common filamentous Ascomycota isolated from the nests of mound-building and leafcutting ants (Baird et al. 2007;Zettler et al. 2002;Sharma and Sumbali 2013;Rodrigues et al. 2014) but only a single study (Zettler et al. 2002) resembles ours in recovering different representatives of this family from nests and non-nest soils.
Aspergillus accounted for more than 80% of Trichocomaceae isolated from mound nests and were represented almost exclusively by Aspergillus navahoensis (section Nidulantes) and A. pseudodeflectus (section Usti).Aspergillus navahoensis was described from soils from a cool desert shrub community in northern Arizona (Christianson and States 1982) and belongs to a section of the genus that occurs at greater than expected frequencies in desert soils (Klich 2002b).This species was recovered originally in low numbers (Christianson and States 1982) and, apart from the present study, does not appear to have been collected since it was D r a f t described.Aspergillus pseudodeflectus is an infrequently collected osmophilic species described from desert soils in Egypt (Samson and Mouchacca 1975) that was reported to be restricted to the tropics and subtropics (Christensen and Tuthill 1985).It is closely related to A. calidoustus, a more commonly encountered species known from clinical and environmental sources that is distinguished from A. pseudodeflectus based on its ecology and molecular barcodes (Samson et al. 2011(Samson et al. , 2014)).
Trichocomaceae were also abundant in soils from non-mound sites but were represented almost exclusively by species of Paecilomyces and Penicillium.These genera were consistently more abundant in non-mound soils than in soils from mounds.Members of the genus Aspergillus were absent from non-mound soils with the exception of a single colony of A. pseudodeflectus that we suspect was a contaminant.Differences in the fungal communities of the soils of nest mounds of Formica ulkei and adjacent non-nest sites likely reflect environmental factors that are influenced by nest location and architecture.For example, mound nests of F. ulkei in Illinois were shown to be restricted to drier regions along forest margins and were constructed to maximize insolation (Dreyer and Park 1932;Dreyer 1942).Nest construction also dramatically alters the physical characteristics of soil that operate to regulate the moisture content and temperatures of mounds relative to surrounding soils.Mound building can increase soil porosity and reduce the bulk density of soils, both of which influence soil aeration and soil permeability (Frouz and Jilková 2008).The moisture content in mounds of F. ulkei at 5 cm has been shown to be lower than in adjacent soils throughout the year and lower than in mounds at 30 cm during the warmer months when the ants were active (Sherba 1959).Although we did not determine the moisture content of soils at our study site, we observed that the daily temperatures of nests of F. ulkei peaked in the evening and decreased slowly during the night (data not shown) in D r a f t agreement with the description of the drier and more exposed nests of Formica polyctena (European red wood ant) (Frouz 2000).
The supposition that the nests of Formica ulkei at our study site were drier than adjacent sites is also supported by the dominance of Aspergillus in nests as compared to soils located 1 m from each mound.Species of Aspergillus are common in soils from warmer regions of the world (Domsch et al. 1993;Bills et al. 2004) and are among the most xerotolerant Ascomycota (Dix and Webster 1995;Zak and Wildman 2004).Members of this genus are particularly abundant in desert and grassland soils where they represent up to 20% of isolated species (Christensen and Tuthill 1985).Although both A. navahoensis and A. pseudodeflectus were capable of growth at the highest average daily temperatures recorded for mound and non-mound soils, only the former species was determined to be thermotolerant (i.e., it grows at temperatures below 20˚C and at 40˚C or higher).This finding, in conjunction with our observation that all Aspergillus and Paecilomyces and nearly half of the species of Penicillium were isolated at both 25˚C and 35˚C (Supplemental Table S1), suggests that water availability is also be a determinant of fungal community structure in mound nests of F.

ulkei.
Factors such as nutrient availability, soil chemistry and the physical properties of soils also likely influence the structure of fungal communities in the mound nests of F. ulkei and adjacent non-nest soils.For example, soils in ant nests have higher levels of nutrients (Frouz et al. 2005;Frouz and Jílková 2008;Ginzburg et al. 2008;Jílková et al. 2015) and differ from surrounding soils in pH, porosity, and the content of organic matter (Frouz and Jílková 2008;Jílková et al. 2011).Microbial activity is assumed to be higher in ant nests because of these differences, but the mechanisms underlying the impacts of ants on soil processes and other soil biota are not well understood (Frouz and Jílková 2008;Del Toro et al. 2012).using the approaches presented here.Our understanding of these communities would be improved with the more frequent sampling of nest mounds and adjacent non-nest soils, the isolation of fungi over a longer period of time, the use of media designed to isolate ecologically specialized taxa, and the determination of temperature differences from a larger number of sites within nest mounds.And because the enumeration methods used in our study are selective for fungi that produce abundant spores (Garrett 1981), it would be valuable to examine the diversity of culturable fungi in these soils using alternative isolation methods (described in Bills et al. 2004).The complementary use of sequence-based approaches such as environmental metagenomics would also enhance our understanding of these assemblages of fungi, particularly in recovering non-culturable species and taxa that are under-sampled employing cultured-dependent methods (Bills et al. 2004;Karst et al. 2013;Rodrigues et al. 2014).Sequence based approaches would also facilitate the identification of sterile fungi and many of the micro-morphologically simple or taxonomically challenging species present in the mound nests of Formica ulkei.b number of species within a genus or group.

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

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

Figure 3 .Figure 1 .Figure 2 .Figure 3 .
Figure 3. Dendrograms illustrating Morisita-Horn similarities between the communities of fungi proportional abundance of the ith species; b number of species within a genus or group.
a pi = proportional abundance of the ith species;

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

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

pubs Canadian Journal of Microbiology D r a f tTable 3 .
Richness and diversity estimators of fungal communities of mound (M) and non-mound (S) sites calculated in BiodiversityR. https://mc06.manuscriptcentral.

com/cjm-pubs Canadian Journal of Microbiology D r a f tTable 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.

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.