Nuclear Populations of the Multinucleate Fungus of Leafcutter

Mycologia

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Nuclear populations of the multinucleate fungus of leafcutter ants can be dekaryotized and recombined to manipulate growth of nutritive hyphal nodules harvested by the ants

Alexis L. Carlson, Heather D. Ishak, James Kurian, Alexander S. Mikheyev, Isaac Gifford & Ulrich G. Mueller

To cite this article: Alexis L. Carlson, Heather D. Ishak, James Kurian, Alexander S. Mikheyev, Isaac Gifford & Ulrich G. Mueller (2017): Nuclear populations of the multinucleate fungus of leafcutter ants can be dekaryotized and recombined to manipulate growth of nutritive hyphal nodules harvested by the ants, Mycologia, DOI: 10.1080/00275514.2017.1400304 To link to this article: https://doi.org/10.1080/00275514.2017.1400304

View supplementary material Accepted author version posted online: 10 Nov 2017. Published online: 04 Jan 2018.

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Nuclear populations of the multinucleate fungus of leafcutter ants can be dekaryotized and recombined to manipulate growth of nutritive hyphal nodules harvested by the ants Alexis L. Carlsona, Heather D. Ishaka, James Kurian a, Alexander S. Mikheyev b, Isaac Gifforda, and Ulrich G. Mueller a aDepartment of Integrative Biology, University of Texas at Austin, Austin, Texas 78712; bOkinawa Institute of Science and Technology, 1919-1 Tancha, Onna-son, Kunigami, Okinawa 904-2234, Japan

ABSTRACT ARTICLE HISTORY We dekaryotized the multinucleate fungus Leucocoprinus gongylophorus, a symbiotic fungus Received 9 March 2017 cultivated vegetatively by leafcutter ants as their food. To track genetic changes resulting from Accepted 31 October 2017 dekaryotization (elimination of some nuclei from the multinuclear population), we developed two KEYWORDS multiplex microsatellite fingerprinting panels (15 loci total), then characterized the allele profiles Attamyces bromatificus; of 129 accessions generated by dekaryotization treatment. Genotype profiles of the 129 acces- Attini; fungus-growing ant; sions confirmed allele loss expected by dekaryotization of the multinucleate fungus. We found no Leucoagaricus evidence for haploid and single-nucleus strains among the 129 accessions. Microscopy of fluor- gongylophorus; escently stained dekaryotized accessions revealed great variation in nuclei number between cells Leucocoprinus of the same vegetative mycelium, with cells containing typically between 3 and 15 nuclei/cell gongylophorus; (average = 9.4 nuclei/cell; mode = 8). We distinguish four mycelial morphotypes among the multinucleate; multiplex dekaryotized accessions; some of these morphotypes had lost the full competence to produce microsatellite markers; polyploidy gongylidia (nutritive hyphal-tip swellings consumed by leafcutter ants as food). In mycelial growth confrontations between different gongylidia-incompetent accessions, allele profiles suggest exchange of nuclei between dekaryotized accessions, restoring full gongylidia competence in some of these strains. The restoration of gongylidia competence after genetic exchange between dekaryotized strains suggests the hypothesis that complementary nuclei interact, or nuclear and cytoplasmic factors interact, to promote or enable gongylidia competence.

INTRODUCTION Atta leafcutter ants grow their fungi in single-clone Leafcutter ants in the genera Atta and Acromyrmex culti- monoculture (Mueller et al. 2010) and vertically trans- vate as their main food source monocultures of fungi, mit their fungal strain from one generation to the next. called Leucocoprinus gongylophorus (Leucocoprini, When a virgin leafcutter queen leaves her natal nest for Agaricales) as sexual morph (Heim 1957; Mueller et al. her mating flight and dispersal, she maintains an inocu- 2017)orAttamyces bromatificus as asexual morph lum of her natal fungal cultivar in a storage pocket in (Kreisel 1972). The ants cultivate their fungus in sheltered her mouth, then uses this inoculum as a starter culture gardens (typically underground) and sustain fungal to initiate the garden in her newly founded nest. Such growth in these gardens by supplying their cultivars vertical transmission of clonally propagated fungal with substrate for growth (typically finely minced leaves) strains is typical for all leafcutter ants (Weber 1972; and nourishment through fecal manuring (Weber 1972; Della Lucia 2011; Marti et al. 2015; Meirelles et al. De Fine Licht and Boomsma 2010). Because the ants 2016), and fungal exchange between attine ant nests depend on their fungal garden as primary food source, appears to be constrained by incompatibilities or low and because L. gongylophorus fungi have so far not been fitness of specific ant-fungus combinations (Seal et al. found growing independently of leafcutter ants (Mueller 2012, 2014a, 2014b). However, population-genetic stu- et al. 1998;Pagnoccaetal.2001; Mueller 2002; Bacci et al. dies revealed occasional horizontal exchange of fungal 2009;Voetal.2009), the leafcutter ant–fungus association cultivars between sympatric leafcutter ant species, as appears to be an obligate symbiosis for leafcutter fungi. well as rare cases of genetic admixture between L.

CONTACT Ulrich Mueller [email protected] Alexis L. Carlson and Heather D. Ishak contributed equally to this work. Supplemental data for this article can be accessed on the publisher’s Web site. © 2018 The Mycological Society of America

Published online 04 Jan 2018 2 CARLSON ET AL.: DEKARYOTIZING MULTINUCLEATE FUNGUS OF LEAFCUTTER ANTS gongylophorus strains (Adams et al. 2000; Mueller 2002; percellinfungalsymbiontsoffivespeciesofPanamanian Mikheyev et al. 2006, 2007, 2010; Mueller et al. 2011a, leafcutter ants and that L. gongylophorus cells are highly and 2011b, 2017). obligatorily polyploid, with about 5–7 different, heterokar- Although common and well studied in plants and some yotic nuclei per cell. Polyploidy and heterokaryosis in leaf- animal lineages, polyploidy—the coexistence of multiplied cutter fungi may boost metabolism (Kooij et al. 2015)or genomes in the same nucleus—is much less frequent and help compensate for evolutionary disadvantages stemming understudied in fungi (Campbell et al. 2016). Of the known from the apparent absence or rarity of sexual reproduction polyploid fungi, most derived from evolutionarily recent via meiospore production in leafcutter fungi (Mueller 2002; transitions to polyploidy, and only two fungal polyploids of Mikheyev et al. 2006;Scottetal.2009; Mueller et al. 2011b, ancient evolutionary origin are known (Saccharomyces, 2017; Kooij et al. 2015). However, leafcutter fungi actually Rhizopus)(Maetal.2009; Albertin and Marullo 2012; do exhibit low levels of population-genetic admixture Shelest and Voigt 2014;Campbelletal.2016). Among the (Mueller et al. 2011b), most likely through the occasional Basidiomycota, the known cases of polyploidy appear to be exchange of nuclei between anastomosing mycelia (Mueller of recent evolutionary origins, including both autopoly- et al. 2011b), but true recombinatorial exchange of genetic ploid origins (e.g., Ustilago, Armillaria, Cryptococcus)and material between nuclei also seems possible. Theory pre- allopolyploid origins (e.g., Heterobasidion, Armillaria) dicts that such rare facultative genetic exchange between (Campbell et al. 2016). Some Basidiomycota can also differentiated mycelia, rare recombination, or rare ploidy have diverse populations of differentiated nuclei in cycling can generate almost the same evolutionary advan- multinucleate cells, a state called heterokaryosis. Such mul- tages of obligatory sexual reproduction (Kondrashov 1994; tinucleate cells have been observed only a few times in Otto and Lenormand 2002). species such as Phanerochaete chrysosporium (Alic et al. The multiallelic microsatellite DNA genotypes reported 1987), Heterobasidion parviporum (James et al. 2008), and for L. gongylophorus (3–5 alleles typical per locus, up to 8 several species of Agaricus and Termitomyces (Saksena et al. alleles per locus; Scott et al. 2009; Mueller et al. 2011b; Kooij 1976;DeFineLichtetal.2005;Diasetal.2008), as well as L. et al. 2015) likely underestimate the true number of coex- gongylophorus (Scott et al. 2009; Kooij et al. 2015). The isting nuclei per cell, for two reasons. First, some nuclei may number of nuclei present in the vegetative mycelium of be genetically similar or identical to each other; and differ- these species varies greatly. For example, cytological studies ences between such nuclei would not be readily apparent in of Agaricus brasiliensis determined an average of 5.8 nuclei the microsatellite marker profiles, as reported by Kooij et al. per cell (Dias et al. 2008), whereas studies of Agaricus (2015). Second, microsatellite loci may contain null alleles bisporus revealed as many as 25 nuclei per cell (Saksena (nonamplifiable alleles lacking an appropriate primer site), et al. 1976). Both polyploidy and heterokaryosis appear as leading to an inherent underestimate of the true number of multiallele genotypes in genotyping analyses (e.g., genotyp- coexisting nuclei. Despite these potential complications, ing indicates more than two alleles per locus for a single- microsatellite genotyping of L. gongylophorus has been strain mycelium), and it is therefore a challenge to deter- useful to advance understanding of population-genetic mine whether the reason for multiallele genotypes derives structure (Mikheyev et al. 2007; Mueller et al. 2011b, from polyploidy (multiple genomes per nucleus), from 2017), single-strain cultivation per nest (i.e., fungal mono- heterokaryosis (multiple, genetically differentiated nuclei culture; Mueller et al. 2010), symbiont choice (Sen et al. coexist in the same cell), or from both polyploidy and 2010), polyploidy and heterokaryon states (Kooij et al. heterokaryosis. 2015), quantitative genetics of cold tolerance (Mueller The earliest histological studies of L. gongylophorus et al. 2011a), as well as genetic admixture between closely mycelium revealed that these fungi lack clamp connections related L. gongylophorus strains (Sen et al. 2010; Mueller (Möller 1893; Hervey et al. 1977) and are multinucleate, et al. 2011b). Although the evidence for occasional sexual or with more than two nuclei per cell (Hervey et al. 1977; parasexual reproduction in natural L. gongylophorus popu- Mohali 1998). Subsequent microsatellite marker analyses lations is compelling (Mueller 2002; Mikheyev et al. 2006; of L. gongylophorus from Panama and the USA reported up Mueller et al. 2011b), it is unclear how genetic material is to five distinct alleles per L. gongylophorus strain (Scott et al. exchanged between the largely clonally propagated L. gon- 2009; Mueller et al. 2011b), suggesting that either single gylophorus strains. Interactions between established myce- nuclei are significantly polyploid (i.e., contain duplicated lium and basidiospores (Pagnocca et al. 2001; Mueller 2002; genomes), or that genetically differentiated nuclei coexist in Mueller et al. 2017) or parasexual processes after plasmo- a cell (i.e., cells are heterokaryotic), or that a combination of gamy and exchange of nuclei between nuclear populations single-nucleus ploidy and heterokaryotic nuclear popula- in different, anastomosing mycelia (Pontecorvo 1956)are tions contribute to multiallele genotypes of L. gongylo- the most plausible possibilities for genetic admixture in L. phorus.Kooijetal.(2015) recently reported 7–17 nuclei gongylophorus (Mueller et al. 2011b, 2017). MYCOLOGIA 3

To understand the heterokaryotic genetics of L. gon- UGM060511-01 listed in Mueller et al. 2011b, except gylophorus symbionts, we here (i) develop tools to UGM070517-01 was collected a year later from the dekaryotize L. gongylophorus fungi (eliminate nuclei same nest). Isolation protocols followed standard from heterokaryotic mycelium); (ii) verify successful methods for isolating attine cultivars on potato dekaryotization with multiplex microsatellite DNA fin- dextrose agar (PDA) medium (Mueller et al. 1996, gerprinting and histological methods; (iii) perform in 1998; Wang et al. 1999; Gerardo et al. 2006). vitro mycelial confrontation experiments to document plasmogamy or recombination; and (iv) elucidate the genetic basis of an evolutionarily unique morphological Dekaryotization and isolation of dekaryotized feature of L. gongylophorus, the so-called gongylidia, mycelium.— Pilot dekaryotization trial.TodekaryotizeL. which are nutritive hyphal-tip swellings produced by gongylophorus, we adapted methods originally developed L. gongylophorus in clusters (staphylae) to feed the ant for homokaryon recovery of the commercial mushroom hosts (Möller 1893; Hervey et al. 1977; Mueller 2002; Agaricus bisporus (Castle et al. 1988; Kerrigan et al. 1992; De Fine Licht et al. 2010; 2013, 2014; Mueller 2015). Zhao and Chang 1993), with the modification that we These techniques should be useful to further elucidate recovered mycelium regenerating on PDA (2% agar, 0.6 ant-fungus coevolution, determine species boundaries M mannitol; Petri plates of 100 mm diameter, 15 mm in mating experiments, and generate dekaryotized depth). We cut a 2-cm2 agar plug of gongylidia-bearing mycelium for more cost-effective whole-genome mycelium from within 5–7 mm of the growth front of 8- sequencing. wk-old cultures growing on PDA. This mycelial plug was then blended in 20 mL sterile potato dextrose broth (PDB) supplemented with 0.6 M mannitol, using a Waring METHODS Commercial Blender 700 model 33BL79 (Waring Definition of dekaryotization. —We define the term Products, Torrington, Connecticut, USA). Each blending “dekaryotization” as the experimental reduction of the cycle lasted 10 s. We repeated cycles three times, with a 2- number of genetically differentiated nuclei in a min break between each cycle to avoid overheating the population of nuclei in cells of a heterokaryotic blended liquid. To eliminate larger fragments and enrich multinucleate mycelium. The term dekaryotization the blended liquid for small particulates (i.e., protoplasts or derives from the term “dedikaryotization” used to single hyphal cells) while eliminating large particulates (i.e., produce single-nucleus cells from dikaryotic cells unmacerated mycelium), we filtered the blended (Miles and Raper 1956; McClaren 1970). Because this suspension of hyphal fragments through an autoclaved, existing term of dedikaryotization would not apply to custom-made column consisting of a 1.2-cm-long plug of the multinucleate fungi cultivated by leafcutter ants cotton packed into the tapering end of a 5-mL plastic (i.e., these fungi are not dikaryotic), we use here the pipette tip. The blended liquid was added in aliquots to more general term dekaryotization applicable to the column above the cotton plug, and liquid moved by dikaryotic and multikaryotic mycelium. gravity through the plug. For regeneration of mycelium, aliquots of 100 µL of the collected filtrate were plated onto 10 separate 2% PDA plates supplemented with 0.6 M Isolation of L. gongylophorus from gardens of Atta mannitol as osmotic stabilizer. Plates were incubated at texana. —We selected three strains of L. gongylophorus room temperature to permit regeneration of hyphal cells to develop dekaryotization methods. These three strains and any protoplasts. We did not examine the filtrate were part of a larger collection of L. gongylophorus microscopically, so we do not know whether regenerating isolated in 2007 from nests throughout the range of mycelium derived from protoplasts, single cells, minute Atta texana. The strains belong to the T-fungus hyphal fragments, or a combination of these. genotype cluster of leafcutter fungi in the USA, a Of the three L. gongylophorus strains processed, we fungal genotype only cultivated by A. texana but were successful at isolating regenerating mycelium only apparently not other leafcutter ants (Mueller et al. from strain UGM070517-01 (no live mycelium grew on 2011b). UGM070519-03 was collected from an A. the regeneration plates of UGM070519-03 and texana nest near Graham, Texas (Global Positioning UGM070316-05). We examined plates every 2–4 d for System [GPS]: N33.08, W98.73), UGM070316-05 near signs of regenerating mycelium (hyphal growth barely Montgomery, Texas (N30.32, W95.58), and visible with eye), which we then subcultured individually UGM070517-01 at the Hornsby Bend Environmental onto fresh PDA plates by cutting out the underlying plug Research Center, Texas (N30.22, W97.65; collection from the original plate and thus avert growth of regener- UGM070517-01 was collected from the same nest as ating mycelium across the original isolation plate. We 4 CARLSON ET AL.: DEKARYOTIZING MULTINUCLEATE FUNGUS OF LEAFCUTTER ANTS recovered regenerating mycelium over the course of 1 mo. Growth measurements. —To compare growth behavior In some fungi, homokaryons grow slower than hetero- of the 129 accessions of the experimental trial, we karyons (Kerrigan et al. 1992), and we therefore discarded recorded (i) the number of days until initial isolation mycelium that regenerated within 1 wk (visible after 1 wk from regeneration plates (i.e., number of days after or earlier) while retaining for further processing only plating, when the first hyphal growth of a regenerating slow-regenerating mycelia (regenerating after 2–4wk). accession became visible under a microscope) (FIG. 1a); We subcultured these isolates narrowly from their respec- and (ii) initial gongylidia vigor of each isolated accession, tive growth fronts at least one more time, but we did not defined as the number of days after the first subculturing take records to document the exact subculturing history when a subcultured accession exhibited at least 20 for each individual isolate during this pilot trial aimed at gongylidia under a microscope (FIG. 1b) (data in developing basic methods. We obtained a total of 119 SUPPLEMENTARY TABLE 6). These two isolates in the pilot trial, which we then genotyped at 10 measurements record growth behavior of regenerating microsatellite loci (methods below, Plex A) to evaluate accessions immediately after dekaryotization treatment. whether the dekaryotization procedure resulted in reduc- We terminated observations after 8 wk, and any tion of the original multiallelic state of the parental acces- accessions that did not exhibit any gongylidia growth by sion UGM070517-01. Because our methods were not yet the end of 8 wk were recorded as “na” in the “Gongylidia standardized in this pilot trial, we do not include informa- vigor” column of SUPPLEMENTARY TABLE 6 (some of tion from these pilot isolates in the Results section below these accessions produced gongylidia when older, or after (instead, we show the preliminary genotyping results later subculturing, but not during the first 8 wk). from the pilot trial in SUPPLEMENTARY TABLE 1); To compare biomass accumulation of accessions after however, the pilot trial proved valuable to develop meth- these first 8 wk, we selected 32 accessions from among odology used in the rigorously standardized experimental the 129 accessions to characterize additional growth dekaryotization trial. measures, including (iii) total area (in mm3) covered by Experimental dekaryotization trial. Because the pilot mycelium growing from a small mycelial tuft on a PDA trial was successful and produced dekaryotized geno- plate, measured 6 wk after transfer of the mycelial tuft types that differed from the genotype of the original, (FIG. 1c); and (iv) biomass (fresh weight) of a mycelial multinucleate parental strain UGM070517-01, we accession attained after 6 wk of growth (harvesting aerial repeated the dekaryotization experiment with a fresh mycelium with a spatula without underlying medium) isolate of UGM070517-01. After blending and plating (FIG. 1d). For these 32 accessions, we used flame-ster- using the methods from the pilot trial, we subcultured ilized fine-tipped forceps to subculture a small piece all regenerating mycelia over 6 wk, including this time (~1 mm3) of aerial mycelium from the growth front of also those mycelia regenerating within the first 2 wk. 32 established accessions of different genotypes. For During weeks 1–3, we checked regeneration plates daily each of these 32 accessions, we generated three such under an 80× stereomicroscope, and we excised any replicate plates. We grew cultures at room temperature. regenerating mycelium narrowly to transfer single-strain Because accessions expanded radially at about equal rate isolates onto individual PDA plate. During weeks 4–6, in all directions, we used calipers to measure two edge- we checked plates weekly for late-regenerating to-edge perpendicular diameters of the radial fungal (slow-growing) mycelium. We collected a total of 129 growth, then averaged these two diameters to derive an accessions in this experimental dekaryotization trial estimate of radial growth across the culture plate, and (SUPPLEMENTARY TABLE 2), then genotyped each then used the average diameter and standard circle cal- accession using microsatellite markers (methods culations to calculate the approximate area (in mm3). below). Observation of mycelial growth of these acces- We collected these measurements weekly for 6 wk. After sions over several weeks allowed us to categorize growth 6 wk, mycelial growth of the three replicates of each behavior into four morphotypes (Types 1–4), as accession were carefully scraped off plates with inoculat- explained below and in SUPPLEMENTARY FIG. 1. ing loops; the mycelial growth of each sample was com- Several accessions from the experimental trial developed bined and weighed in a small plastic vial. Two of the sectors of browning in older cultures. Although such accessions had one failed replicate each (the initial small discoloring can also occur in pure strains (U.G.M. and inoculum was not viable, likely because the mycelial bit H.D.I., personal observations), we genotyped mycelium was cut too small during subculturing); for these sam- from both browning mycelium and from the typical ples, we averaged areas and weights only for the two white mycelium, to evaluate whether browning could viable replicates of that accession. Researchers proces- be the result of incompatibility interactions between sing and weighing mycelia were blind to all information genetically differentiated, commingled strains. on genotypes and morphotype classifications. MYCOLOGIA 5

a. b.

c. d.

Figure 1. Growth behavior of fungal accessions after dekaryotization treatment. a and b. Growth behavior of a regenerating mycelium for 129 accessions isolated from regeneration plates, for the initial growth when subculturing these isolates immediately after dekaryotization treatment. Regenerating isolates that had lost more alleles as a result of dekaryotization treatment needed more time to (a) develop the first hyphal growth during regeneration (P < 0.0001) and (b) develop gongylidia vigor (defined as production of at least 20 gongylidia) (P < 0.001). c and d. Growth behavior for 32 dekaryotized accessions grown for 6 wk after several rounds of subculturing. Radial growth (area covered on a plate) was significantly negatively correlated with the number of alleles lost by a genotype (P = 0.031). Wet weight was also negatively correlated with the number of alleles lost by a genotype, but this trend was not statistically significant (P = 0.16). Overall, the more alleles had been lost by an accession, the poorer the (a) regeneration, (b) gongylidia production during regeneration, (c) radial growth, and (d) possibly biomass accumulation.

Multiplex microsatellite genotyping. —To improve development, we used watchmaker forceps (no. 5) to the efficiency of microsatellite genotyping using the isolate a small tuft of aerial mycelium from the L. polymerase chain reaction (PCR), we first developed gongylophorus growth front, then extracted DNA from two microsatellite multiplex PCRs that amplified, this tuft using a 10% Chelex solution, as described in respectively, 10 and 5 of the loci originally developed Mikheyev et al. (2007, 2008). After optimizing by Scott et al. (2009) for L. gongylophorus fungi. The 15 multiplex-PCR conditions, we found it most time- loci in the two multiplexes (Plexes A and B) were efficient to add the mycelial tuft directly into the PCR selected because of their high levels of allelic mix and skip this DNA extraction step (i.e., 15 min polymorphism and minimal stutter problems soaking at 94 C released the DNA, followed (SUPPLEMENTARY TABLES 1 and 2). Some primers immediately by PCR amplification). The optimized in these plexes are identical to those of Scott et al. microsatellite PCR used the Qiagen Multiplex PCR (2009), but some primers had to be redesigned to Kit (Qiagen, Germantown, Maryland, USA) and a 10 generate amplification products of desired fragment μL reaction volume, consisting of 5 μL of 2× Qiagen μ sizes. New primers were created with a desired Tm of Master Mix, 1 L aliquot of primer mix (concentrations 59–60 C, a GC content of 40–60%, at least one “GC” for each multiplexed primer are listed in clamp, and a designated fragment size range in one of SUPPLEMENTARY TABLE 3), 4 μL of nucleotide- four bin sizes (bin 1: 75–120 bp; bin 2: 130–178 bp; bin free water, plus a minute mycelial tuft (<1 mm3) 3: 188–205 bp; bin 4: 225–270 bp). Each bin size had directly added into this mix. We followed the thermal two or three loci differentiated by a fluorescent dye cycling protocol recommended by the Qiagen (FAM, HEX, or TAMRA) attached to the forward Multiplex PCR Handbook: 15 min at 95 C (release of primer 5′ end. SUPPLEMENTARY TABLE 3 lists DNA from tissue, denaturing of DNA); 35 cycles at 94 forward and reverse primer sequences, the fluorescent C for 30 s, annealing at 57 C for 30 s, and extension at dye for each primer pair, and primer concentrations 72 C for 60 s; final extension at 72 C for 10 min. We optimized for balanced amplification of alleles across all confirmed DNA amplification by visualization of PCR multiplexed loci. During early stages of multiplex-PCR products under ultraviolet (UV) light in a 2% agarose 6 CARLSON ET AL.: DEKARYOTIZING MULTINUCLEATE FUNGUS OF LEAFCUTTER ANTS gel stained with SYBR safe. For fragment analysis, we loci screened in our multiplex microsatellite mixed 1 μL of PCR product mixed with 8 μL of HiDi genotyping (see genotypes shown in the first row of (Applied Biosystems, Waltham, Massachusetts, USA) SUPPLEMENTARY TABLE 2), whereas each of the and 1.5 μL of custom-amplified size standards (CASS; confronted accessions had lost between 2 and 6 of ladder of 104R, 156R, 256R, 305R, 424R; each labeled these alleles as a result of dekaryotization treatment. with RoxF; following the methods of DeWoody et al. The accessions paired in the confrontations were 2004). We analyzed PCR products on an ABI 3100 chosen by two criteria: first, several or a large number Genetic Analyzer (Applied Biosystems, Waltham, of alleles were lost in each accession during Massachusetts, USA) in the Mueller laboratory and dekaryotization treatment; and second, the confronted scored markers (alleles) using Gene Scan 3.5 and genotypes complemented each other such that SoftGenetics GeneMarker 1.5 (State College, exchange of nuclei or parasexual recombination could Pennsylvania). potentially reconstitute the original genotype of We genotyped accessions typically only with the first UGM070517-01. SUPPLEMENTARY TABLE 4 lists multiplex of 10 loci (Plex A, SUPPLEMENTARY the accession IDs that we paired in nine confrontation TABLE 3) and if additional marker information was experiments. For a confrontation, we subcultured two needed also with the second multiplex of 5 loci (Plex agar plugs from the two accessions from their B). To verify that dekaryotized accessions showed stable respective growth fronts, then placed these plugs onto allele profiles (i.e., to rule out that allele profiles chan- a new PDA plate ~1 mm apart (but not touching) to ged over time by losing additional alleles or regaining permit rapid contact between the two growing mycelia. alleles that had been lost as a result of dekaryotization We replicated each of these nine confrontations. The treatment), we repeated genotyping two to four times confronted pairs were allowed to grow for 16 d, then over a period of several months across multiple sub- subcultured three times from the center of the zone culturing cycles for each dekaryotized accession. where confronted mycelia contacted each other (called Accessions with no missing alleles (i.e., genotypes iden- below Subculture 1, abbreviated Sub1; 2 × 3 = 6 tical to the parental fungus before dekaryotization replicate subcultures per confrontation assay; × 9 treatment) were typically regenotyped only one to two assays = 54 Sub1 plates; SUPPLEMENTARY FIG. 2). times, whereas accessions that had lost the most alleles We allowed these subcultures to grow for 13 d, then during the dekaryotization treatment (e.g., lost 9 or 10 subcultured again, but this time from the 12, 6, and 9 alleles) were regenotyped up to nine times o’clock positions along the growth front (called below (SUPPLEMENTARY TABLE 6 lists number of Subculture 2, abbreviated Sub2; 18 replicates total per regenotyping). confrontation assay; × 9 confrontations = 162 Sub2 isolates). SUPPLEMENTARY FIG. 2 shows the flowchart of the experimental set up and sample sizes Statistics. —We used regression analysis to explore the in this successive subculturing scheme to generate Sub1 relationship between (i) the number of alleles lost by an isolates (n = 54 total) and Sub2 isolates (n = 162 total). accession as a result of dekaryotization treatment and We genotyped all Sub1 isolates (n = 6 per con- (ii) the four growth rate parameters measured for each frontation) using both sets of 10+5 multiplexed individual accession (i.e., number of days until visible markers (Plexes A and B). Sub1 replicates are called regeneration; growth vigor at regeneration; area grown here “complete recombinants” if all 40 alleles of the and wet mass attained within 6 wk; see Growth original genotype UGM070517-01 were reconsti- Measurements above and FIG. 1a–d). tuted in a genetically stable mycelium and persisted across several additional subculturing cycles. Testing genetic exchange through recombination Recombinant accessions in which one or several or nuclear reassociation between mycelia.—To alleles were regained, but the full original genotype assess whether genetically stable dekaryotized was not reconstituted, are called here “partial accessions that had lost alleles could regain alleles recombinants.” To differentiate between recombina- trough nuclei exchange or recombination with other tion/nuclear exchange versus a physical mixing of dekaryotized accessions, we confronted pairs of two commingled mycelia, we subcultured and geno- accessions on PDA plates. All confronted accessions typed putative complete recombinants for four addi- had been derived through our dekaryotization tional subculturing cycles (SUPPLEMENTARY FIG. treatment from the original isolate UGM070517-01; 3). To minimize the chance of subculturing two this original isolate showed 40 alleles across the 15 commingled accessions, we subcultured mycelium MYCOLOGIA 7 with a sharpened needle, cutting the smallest possi- overlapping nuclei. To increase reliability of ble portion from the growth front. Finally, after 87 nucleus counts, therefore, two experimenters d, we again subcultured from the original confron- blindly and independently counted nuclei in each tation plate and the growth front from the 12, 3, 6, image; if counts disagreed for an image, both and 9 o’clock positions and any area of unusual experimenters independently reexamined the image morphology (called Sub1-delayed, until both reached a consensus. SUPPLEMENTARY FIG. 2). We reasoned that this additional time (“delayed”)couldgivetwocom- Estimating the number of nuclei/cell from allele mingled strains time to compete with each other, profiles of dekaryotized fungal strains.— increasing the chance that a pure dominant myce- If we lium could be isolated from the growth front after a assume that (i) each dekaryotization event (generation total of 87 d. of a cell line with less nuclei than the parental strain UGM070517-01) is associated with l nuclei lost from the original set of n nuclei in the parental strain; and (ii) if the DAPI staining and counting of nuclei per cell.—To frequency of a given allele in the parent is f,thenthe count number of nuclei per mycelial cell, we probability of this allele remaining after dekaryotization selected representative dekaryotized accessions for follows the combinatorial expression À fluorescent staining of nuclei. We used Invitrogen’s n f n À l protocol for 4′,6-diamidino-2-phenylindole (DAPI) . We can use this probability to calculate the counterstaining of nucleic acids for fluorescence n À microscopy. For each accession, we excised 1-cm2 n l pieces of aerial mycelium from representative likelihood of the observed data (SUPPLEMENTARY sectors of healthy cultures, transferred each TABLE 2) for each allele for an arbitrary set of mycelium into an individual well of a Falcon parameters under the Poisson distribution, then obtain a polystyrene tissue microplates (24 wells/plate; maximum-likelihood estimate for the parameter set. We catalog number 08-772-1H; Corning Life Sciences, implemented this approach in a custom script in R (R Tewksbury, Massachusetts, USA), allowed the Core Team 2014; SUPPLEMENTARY TABLE 7) mycelium to equilibrate in 100 µL phosphate- separately for each allele that showed presence/absence bufferedsaline(PBS;pH7.4;Gibco,Life polymorphism in SUPPLEMENTARY TABLE 2, for a Technologies, Carlsbad, California, USA) for 2 range of n from 1 to 15 that encompassed most of the min, then rinsed each mycelium twice with variation of number of nuclei/cell seen microscopically distilled water. We soaked each sample in (FIG. 2a). The 11 alleles that were never lost in the approximately 200 µL of a 1:10 dilution of 5 mg/ dekaryotization experiment were excluded in this mL DAPI solution, then incubated these in the dark likelihood estimation, because these alleles provide no at room temperature for 1 h. We washed informative data, and these alleles may even be postincubation samples twice with PBS to remove maintained by selection. The maximum-likelihood excess DAPI. For microcopy, we transferred samples modeling is therefore based on the 29 alleles that to microscope slides to observe and photograph showed allele loss as a result of the dekaryotization them with a Zeiss LSM 710 fluorescence confocal treatment (SUPPLEMENTARY TABLE 2). microscope (light filters 410–585 nm; Oberkochen, Germany) at the Microscopy Facility of the RESULTS University of Texas at Austin. We photographed only cells in which nuclei and cell boundaries were Dekaryotization. —Dekaryotization of isolate unambiguously clear. We processed images using UGM070517-01 was repeatable between the pilot and Zeiss ZEN lite 2011 SP1 1.0 (freeware; Zeiss), then experimental trials (SUPPLEMENTARY TABLES 1 and edited images with ImageJ (Preibisch et al. 2009). 2). Of the 129 accessions isolated after dekaryotization Counts of nuclei per cell were sometimes difficult to treatment in the experimental trial, 36.4% (n = 47) determine because nuclei were very close to each showed the full allelic profile of the original isolate other. Specifically, it was sometimes difficult to UGM070517-01 (i.e., un-dekaryotized mycelium was discern whether two nuclei were stacked on top of isolated), and 63.6% (n = 82) had lost at least one allele as each other, two nuclei had just been generated a result of the dekaryotization treatment through nuclear division, or a single nucleus was (SUPPLEMENTARY TABLE 2). On average, 4.8 alleles irregularly shaped to incorrectly suggest two (11%) were lost in the 82 dekaryotized accessions. The 8 CARLSON ET AL.: DEKARYOTIZING MULTINUCLEATE FUNGUS OF LEAFCUTTER ANTS

a. b. c.

d. e. f.

g. h. i. j. k. l.

Figure 2. a–f. Distributions of the number of nuclei per cell observed in L. gongylophorus mycelium of five dekaryotized accessions and the untreated L. gongylophorus isolate UGM0705017-01 from which the dekaryotized accessions were derived. Distributions are shown only for those accessions for which we were able to characterize more than 10 cells (see full data set in SUPPLEMENTARY TABLE 5). g–l. Confocal microscopy images of representative multinucleate cells (additional images are deposited at GoogleDrive; see link in Data Deposition). maximum number of alleles lost in an accession was 10 alleles that had been lost as a result of dekaryotization alleles from the original 40 alleles present in isolate treatment were later regained. Lastly, dekaryotization UGM070517-01 when multiplex genotyping with both revealed null alleles at several loci (i.e., no allele amplified Plex1 and Plex2 (SUPPLEMENTARY TABLE 2). At 8 of at a particular locus in some dekaryotized accessions, but the 15 loci, a total of 11 alleles were always present (i.e., the un-dekaryotized strain UGM070517-01 showed at least these alleles were never lost); interestingly, these were the one allele at that same locus; SUPPLEMENTARY TABLES same alleles that were always present (never absent) in a 1 and 2), indicating that our microsatellite fingerprints large population-genetic survey of more than 200 L. underestimate genetic diversity among the nuclei gongylophorus samples collected from leafcutter ants in coexisting in an L. gongylophorus cell. the USA and Mexico (Mueller et al. 2011b). We found no evidence for a haploid strain among the 129 accessions; that is, we found no dekaryotized accession showing only one Morphological variation among dekaryotized allele at each locus. Even in dekaryotized accessions that lost accessions.— We could distinguish reliably four the most alleles, most loci showed more than one allele, mycelial morphotypes (called here Types 1–4) for 127 although some loci showed only one allele of the 129 accessions growing on PDA medium (SUPPLEMENTARY TABLES 1 and 2), suggesting that (SUPPLEMENTARY FIG. 1), and only two accessions either (i) haploid strains may not be viable or (ii) single exhibited an intermediate morphology. Type 1 grew nuclei contained multiplied genomes (i.e., at least some radially in circular shape, frequently displayed fluffy nuclei were polyploid). Repeat subculturing (up to nine aerial growth, sometimes developed browning times) and multiplex genotyping of dekaryotized mycelial sectors, and produced abundant gongylidia accessions revealed that the respective genotypes were (hyphal-tip swellings that grow clustered as so-called stable over time; no additional alleles were lost and no staphylae, which are harvested by the ants for food; MYCOLOGIA 9

Weber 1972; De Fine Licht et al. 2014). Type 2 grew production (“gongylidia vigor” defined as presence of radially at a fast rate, was always circular and appressed at least 20 gongylidia) after subculturing to a new to the medium, often appeared gray or brown in plate (FIG. 1b). On average, regenerating accessions coloration, produced sometimes a few gongylidia with missing alleles took longer to show first growth initially after subculturing, but then ceased gongylidia on regeneration plates (P < 0.0001, F = 44.94, df =1, production. Type 3 grew comparatively slower as a 127; FIG. 1a) and also took longer to show vigorous dome-shaped dense mycelium, did not initially gongylidia production (P < 0.001, F = 57.47, df =1, produce gongylidia, but could eventually show some 122; FIG. 1b) than those accessions that had lost no gongylidia after several weeks or months of growth. alleles (i.e., possessing the genotype of the original Type 4 grew initially as a puffy round ball with isolate before dekaryotization treatment). abundant gongylidia after subculturing, then slowly In a second growth experiment, we selected 32 repre- spread radially across the plate while continuing to sentative accessions, subcultured and genotyped these sev- produce gongylidia. Type 4 and Type 1 were the most eral times to document stable genotypes, then measured the difficult to distinguish from each other, especially after area covered by mycelium (i.e., radial growth) and the aerial more than 3 mo of growth. The best age of a mycelium mycelial wet weight after 6 wk of growth. Area was signifi- to reveal its morphotype was 1–3 mo after subculturing cantly negatively correlated with the number of alleles lost onto PDA medium. Because Types 1 and 4 produced by a genotype (P = 0.031, F =5.98,df =1,30;FIG. 1c). Wet abundant gongylidia in both younger and older weight was also negatively correlated with the number of cultures (like the un-dekaryotized parental strain), and alleles lost by a genotype, but this trend was not statistically because Types 2 and 3 produced far fewer or no significant (P = 0.16, F = 2.05, df =1,30;FIG. 1d). gongylidia, we categorize Types 1 and 4 as Consistent with the above definitions of the growth mor- “gongylidia-competent” and Types 2 and 3 as photypes, Type 2 accessions showed the greatest radial “gongylidia-incompetent.” growth by area, but not by weight (FIG. 1c and d). Type 3 On PDA medium, the original fungal accession accessions, which consistently missed at least five alleles, UGM070517-01 displayed Type 1 mycelial growth, typically showed reduced radial growth and weight com- which was also the most common morphotype, found pared with the other three morphotypes (FIG. 1c and d). in 75.2% of the 129 accessions isolated after dekaryoti- Dataforbothgrowthexperimentsaresummarizedin zation treatment (see “Morphotype” column in SUPPLEMENTARY TABLE 6. SUPPLEMENTARY TABLE 2). The average number of alleles lost, calculated separately for each of the four morphotypes, was 2.5 (Type 1), 2.0 (Type 2), 6.8 Testing genetic exchange through recombination (Type 3), and 6.1 (Type 4). Accessions with no or nuclear reassociation between mycelia.—In observed allele losses were found among Types 1, 2, nine confrontation experiments pairing different and 4 (SUPPLEMENTARY TABLE 2). Type 2 morphs dekaryotized accessions (SUPPLEMENTARY TABLE were never found with more than five alleles missing. 4), we found four cases where the mycelial Type 3 morphs always had at least five missing alleles. confrontation generated stable and complete Types 3 and 4 are genetically the most distinct (i.e., recombinants (i.e., through multiple rounds of most alleles lost) from the original accession subculturing, recombined isolates exhibited all UGM070517-01. alleles present also in the original strain We observed no genotype differences when comparing UGM070517-01 before dekaryotization treatment; allele profiles of browning and normal (white) sectors of the SUPPLEMENTARY FIG. 3). This suggests that L. same mycelium. Browning sectors therefore do not develop gongylophorus strains have the ability to (i) because genetically distinct dekaryotized strains were com- exchange nuclei or recombine parasexually in the mingled in the same mycelium, but browning seems a laboratory and recover from allele loss after response to possible mutational variation arising in a myce- dekaryotization treatment; and (ii) possibly lium, or to unknown physiological factors. exchange genetic material therefore also under natural conditions in the field (e.g., when two L. gongylophorus strains may be cocultivated in a Growth measurements. —We recorded (i) the single garden after an ant nest raids gardens of a number of days it took for each regenerating neighboring nest; Mueller 2002). mycelium to become visible on the regeneration Most dekaryotized accessions could be confronted plates (FIG. 1a), and (ii) number of days for a without evidence of vegetative incompatibility. subcultured mycelium to show vigorous gongylidia However, although all dekaryotized accessions had 10 CARLSON ET AL.: DEKARYOTIZING MULTINUCLEATE FUNGUS OF LEAFCUTTER ANTS been derived from the same un-dekaryotized fungus, DAPI staining of dekaryotized accessions.—g Usin UGM070517-01, some confrontations of dekaryotized fluorescent microscopy, we estimated the number of accessions appeared vegetatively incompatible nuclei in the original accession UGM-0705017-01 and (SUPPLEMENTARY FIG. 5). This suggests that com- nine dekaryotized accessions (SUPPLEMENTARY plex interactions between multiple genetic factors TABLE 5; all images used in analyses are deposited at (e.g., between multiple loci) and possibly also cyto- GoogleDrive, see link in Data Deposition). Of the nine plasmic factors may modulatevegetativecompatibil- dekaryotized accessions, only five grew as sparsely spaced ity in L. gongylophorus,orthatcryptic mycelium suitable for easy visualization of single cells (i.e., incompatibility between genetic factors can arise most of the final data set in SUPPLEMENTARY TABLE 5 through mutation under the long-term asexual pro- derives from these five strains), whereas visualization of pagation of L. gongylophorus by the ants. Most inter- singlecellsfortheotherfourstrainswasmoredifficult estingly, in some cases the confrontation of two (i.e., fewer observations for these strains are listed in gongylidia-incompetent strains (e.g., a Type 2 and a SUPPLEMENTARY TABLE 5). With very few Type 3 fungus) generated stable recombinants that exceptions, cells of the same mycelium were always were gongylidia-competent (SUPPLEMENTARY FIG. multinucleate. The variation of nuclei per cell of the same 4). The restoration of gongylidia competence in mycelium was remarkably large (FIG. 2). The original recombinants suggests the possibility that comple- accession UGM-0705017 contained 0–21 nuclei/cell, with mentary nuclei interact, or nuclear and cytoplasmic an average of 9.4 nuclei, and we observed similar ranges in factors interact, to facilitate or enable gongylidia nuclei/cell in all of the five well-characterized accessions competence. (SUPPLEMENTARY TABLE 5). The spatial distribution of The confrontation experiments also revealed that a nuclei within the cytoplasm of a cell was typically not specific accession seemed to outcompete the other uniform; nuclei frequently occurred in pairs or in clusters accession when two paired accession co-occurred in of four or more nuclei. In a small number of cells of the thesamemycelium.Fouroftheconfrontation original fungus UGM-0705017 and once in accession 16-1, experiments (Mixes 4–8, SUPPLEMENTARY nucleiwereobservedinachain-likesuccessioninthecenter TABLE 4) used the same pairings of accessions (one of the cell (FIG. 2), possibly because unknown factors force parent missing six alleles and the other missing four nuclei into the cell center and away from the cell wall. alleles). Genotyping 29 subcultures of the Sub1 iso- Nuclei varied in size, ranged from spherical to lates derived from these pairings subspherical, with occasional irregularly shaped nuclei (SUPPLEMENTARY FIG. 3) yielded 21 subcultures (FIG. 2). We observed many cells in which some but not (72.4%) matching the dekaryotized strain with four all nuclei appeared to undergo mitosis, suggesting the missing alleles, 7 (24.2%) partial or full recombinants hypothesis that duplication of nuclei may not be between the confronted accessions), and only 1 sub- synchronized across all nuclei in a cell. We did not find culture (3.4%) matching the dekaryotized strain with any clamp connections among a total of 142 cells examined, six missing alleles. The accession missing four alleles confirming findings from earlier studies that likewise failed therefore was easier to isolate from the contact zone to report clamp connection in L. gongylophorus mycelium between the paired mycelia, possible because this (Möller 1893; Hervey et al. 1977;Chapelaetal.1994;see accession outcompeted the other accession in the also literature reviewed in Mueller 2002). confrontation or during the subculturing process. We observed four apical cell hyphal tips with no visible Despite this apparent advantage of specific strains nuclei, but subapical cells that did contain nuclei (FIG. 2), a in some of the confronted accessions, the delayed phenomenon also seen in the multinucleate fungus subcultures (isolated from confrontationplatesafter Termitomyces sp. (De Fine Licht et al. 2005). Two of these 3 mo) from the fungal growth front revealed in every cells with no visible nuclei were in the mycelium of the experiment (Mixes 1–9, SUPPLEMENTARY TABLE parental strain UGM0705017-01, and one each in the 4) that different genotypes can coexist commingled dekaryotized accessions 7-4 and 41-2. Not much is known alongside each other in the same mycelium for at about nuclear behavior in L. gongylophorus,butitsnuclei least 3 mo, suggesting that (i) any exchanged nuclei may be able to migrate through septal pores, allowing an may not have replicated and traveled throughout the anucleate cell to become nucleate eventually, as has been entire anastomosing mycelia; and (ii) any competi- seen in Agaricus (Kamzolkina et al. 2006). Some of the tion between confronted strains does not lead within anucleate L. gongylophorus cells showed unusual cytoplas- 3 mo to 100% representation of the competitively mic staining, possibly because these cells are older and may superior strain and to complete elimination of the have mature vacuoles displacing the cytoplasm (Timothy competitively inferior strain. James, personal communication). MYCOLOGIA 11

Estimating the number of nuclei/cell from allele from the USA is somewhat lower than the average of 12.5 profiles of dekaryotized fungal strains.— (± 0.41) calculated by Kooij et al. (2015)for14L. Maximum-likelihood estimation indicates that the gongylophorus fungi isolated from eight Atta nests and six most likely number of nuclei/cell of the parental Acromyrmex nests from Panama, suggesting possible strain UGM070517-01 prior to dekaryotization was 8 regional differences in nuclei/cell of L. gongylophorus. nuclei/cell (SUPPLEMENTARY FIG. 6). This number We did not find any haploid strain among the 82 is the same as the modal estimate of the number of dekaryotized accessions (i.e., we found no dekaryotized nuclei/cell observed in our microscopic observations accession showing only one allele at each of the 15 loci (SUPPLEMENTARY TABLE 5). screened), and the maximum number of alleles lost in a dekaryotized accession was 10 alleles (25% of the 40 alleles present prior to dekaryotization). This suggests that it is Data deposition. —Images of L. gongylophorus either difficult to reduce the large number of coexisting mycelium used in histological analyses are deposited nuclei to one nucleus per cell, haploid strains may not be at GoogleDrive: https://drive.google.com/drive/folders/ viable or have poor viability (e.g., heterokaryosis may be 0B_Zr7y_BumzDazhfNHU1MWN1OW8. beneficial because nuclei with different alleles comple- ment each other), or nuclei contain polyploid genomes. DISCUSSION Obligate polyploidy with “5–7 haplotypes on average” per nucleus was inferred by Kooij et al. (2015) for leafcutter We developed (i) a simple protocol to dekaryotize fungi from Panama. This high ploidy of L. gongylophorus multinucleate L. gongylophorus fungi cultivated by leaf- (i) suggests that most of the observed allelic diversity at a cutter ants; (ii) two panels of multiplexed microsatellite locus is due to ploidy compared with allelic diversity DNA markers (respectively 10 and 5 loci per panel) to between nuclei coexisting in a cell; and (ii) explains why track genetic changes during dekaryotization only a limited number of alleles were lost in our dekar- (SUPPLEMENTARY TABLE 3); (iii) phenotyping yotization experiments. methods to distinguish mycelial morphotypes among Of the three L. gongylophorus strains that we tried dekaryotized strains that differ in their tendency to to dekaryotize, we succeeded at isolating regenerating produce gongylidia (nutritive structures consumed by mycelium only from strain UGM070517-01. No live the ants; SUPPLEMENTARY FIG. 1); and (iv) genetic mycelium grew on the regeneration plates of two methods to combine nuclei from different dekaryotized other strains, and it is unclear why mycelium did strains into novel “recombinants.” not regenerate in these cases. All three strains had been isolated in spring 2007 from nests in central and Implications for the biology of L. gongylophorus.— north Texas, they had been maintained and subcul- Mycelium of the parental L. gongylophorus accession tured in the laboratory under the same conditions, UGM0705017-01 contained a highly variable number of and they had the same Type 1 growth morphology. nuclei per cell (average of 9.4 nuclei/cell, range: 0–21 The three strains belong to the T-fungus genotype nuclei/cell; FIG. 1, SUPPLEMENTARY TABLE 5) and cluster, a group of fungi cultivated by Atta texana showed 40 alleles across the 15 loci screened with our in central, north, and east Texas and in Louisiana, but multiplex genotyping (average of 2.7 alleles per locus; apparently absent in south Texas and Mexico range: 2–5 alleles per locus). After one round of (Mueller et al. 2011b). Whether some L. gongylo- dekaryotization, an average of 4.8 alleles were lost in 82 phorus strains are easier to dekaryotize than other dekaryotized accessions (4.8 alleles are 11% of the total of strains is not possible to say from our limited number 40 alleles present across all loci prior to dekaryotization; of dekaryotization attempts. SUPPLEMENTARY TABLES 1 and 2). We used the frequencies of allele losses to derive a maximum- likelihood estimate for the number of nuclei/cell of the Implications for future mycological analyses of L. — parental strain UGM070517-01. Our estimate of 8 nuclei/ gongylophorus. Our findings and new methods cell in the likelihood modeling (SUPPLEMENTARY FIG. suggest a series of additional mycological studies to 6)isthesameasthemodalestimateofthenumberof elucidate the biology of L. gongylophorus fungi: nuclei/cell observed in our microscopic observations (FIG. (1) To distinguish between the hypotheses explaining why 2a) and close to the overall average of 9.4 nuclei/cell of it is difficult or impossible to generate haploid strains parental strain UGM070517-01 (SUPPLEMENTARY (e.g., heterokaryosis may enhance fungal viability; sin- TABLE 5). The average of 9.4 nuclei/cell for our L. gle nuclei are highly polyploid), a future study could (i) gongylophorus isolate from the leafcutter ant Atta texana generate a far greater number of dekaryotized 12 CARLSON ET AL.: DEKARYOTIZING MULTINUCLEATE FUNGUS OF LEAFCUTTER ANTS

accessions (hundreds of accessions may have to be heterokaryotic diversity, nuclear compatibilities, generated and screened to generate eventually a sin- and nuclear-cytoplasmic interactions (including gle-nucleus strain), or (ii) reduce the number of nuclei interactions between a diverse nuclear population, per cell through multiple, successive rounds of dekar- a diverse mitochondrial population, and possible yotization treatment. If haploid strains or homokar- viruses) that determine mycelial phenotypes of yons can be generated and nuclei from genetically symbiotic fungi cultivated by diverse insect lineages differentiated strains can be combined in a heterokar- (Mueller et al. 2005). yotic mycelium, it may be possible to compare pheno- (5) Because we observed several cases in which a single cell types of homokaryotic and heterokaryotic strains (e.g., contained nuclei at different stages of mitosis (some compare tendency to produce gongylidia), paralleling nuclei were in the process of dividing, some not), it is Nobre et al. (2014)whocomparedsporeproductionof possible that L. gongylophorus may have the kind of homokaryotic and heterokaryotic mycelia in the ter- asynchronous nuclear division cycle known for several mite-cultivated fungus Termitomyces. Haploid strains, other filamentous fungi (Gladfelter et al. 2006; or strains greatly reduced in genetic diversity in a Gladfelter and Berman 2009). If L. gongylophorus has population of nuclei in a mycelium, may also be useful asynchronous nuclear division, the high nucleus to test genetic mechanisms determining fungal species counts that we observed occasionally boundaries, mating-type-dependent or vegetative (SUPPLEMENTARY TABLE 5) may represent cells compatibilities (James 2015;seealso in which nuclei divided recently, but septa had not SUPPLEMENTARY FIG. 5), as well as unilateral yet formed to divide the cells’ large populations of migration of nuclei (Harder and Aanen 2009) between nuclei. L. gongylophorus fungi may therefore offer mycelia of different L. gongylophorus genotypes. unique opportunities to study an evolutionary transi- (2) Microscopy could elucidate whether our dekaryo- tion from the presumably synchronized nuclear divi- tization treatment (blending and maceration of sion in the dikaryotic fungi of lower-attine ants to the mycelium) generates protoplasts (without cell asynchronous nuclear division in L. gongylophorus walls) from which dekaryotized hyphae regenerate, symbionts of leafcutter ants. or simply separates hyphal cells that differ in number of nuclei (FIG. 2, SUPPLEMENTARY TABLE 5), such that some of the hyphae regenerating from these separated cells show reduced allelic diversity. Implications for future studies of ant-fungus — (3) Microscopy could also characterize the number of interactions. Our new dekaryotization and nuclei/cell in dekaryotized strains immediately after multiplex-genotyping methods can be used for dekaryotized treatment (in contrast, we character- molecular-ecological studies to elucidate ant-fungus ized number of nuclei after dekaryotized strains interactions: had been maintained alive in the laboratory for 7 (6) Atta ants cultivate their fungi in monocultures y through multiple subculturing cycles). Counting (Mueller et al. 2010), but foreign cultivar strains can nuclei immediately after dekaryotization treatment enter a monoculture via import of cultivar from other could address whether the number of nuclei/cell nests, for example when workers of one attine nest steal changes over time, possibly increasing from initi- garden from a neighboring nest (Autuori 1950; ally reduced levels to the level of nuclei observed in Mintzer 1987;Rissingetal.1989; Cahan and Julian the parental strain before dekaryotization treat- 1999; Adam et al. 2000; Green et al. 2002; Mikheyev ment. Such an increase in number of nuclei/cell et al. 2006), or possibly also because wind-dispersed L. over time may exist because the dekaryotized gongylophorus spores may enter a nest via workers or strains that we examined microscopically had substrate (Pagnocca et al. 2001; Mueller 2002; Mueller approximately the same numbers and ranges of et al. 2011b, 2017).Itmaybepossibletoexperimentally nuclei as the un-dekaryotized parental strain insert garden material or spores into a leafcutter nest in UGM0705017-01 (FIG. 2, SUPPLEMENTARY the laboratory or field to study the consequences of TABLE 5). genetic exchange between genetically differentiated L. (4) L. gongylophorus mycelium shows great variation in gongylophorus strains in the presence of the ant number of nuclei per cell (SUPPLEMENTARY farmers. TABLE 5), paralleling the variable number of (7) Because of the possible import of novel L. gongylo- nuclei per cell observed in the termite-cultivated phorus strains into established leafcutter nests, two fungus Termitomyces (Nobre et al. 2014). This sug- mechanisms have been considered important in main- gests comparative investigations into regulation of taining gardens as monoculture: (i) fungus-fungus MYCOLOGIA 13

competition leading to dominance of a resident L. FUNDING gongylophorus fungus and (ii) symbiont choice by The work was supported by the National Science Foundation ants weeding out recombinant or inferior L. gongylo- (DEB-0639879, IOS-0920138, and DEB-1354666 to U.G.M.); an phorus strains (Mueller 2002; Sen et al. 2010). When Undergraduate Research Fellowship to I.G. from the College of different L. gongylophorus incocula are offered to leaf- Natural Science of the University of Texas at Austin; and the W. cutter ants in either a cafeteria-style assay or as a M. Wheeler Lost Pines Endowment. mycelial mix, Atta and Acromyrmex leafcutter ants can maintain chimeric gardens for months (Sen et al. 2010), suggesting that fungus-fungus competition ORCID appears weak, or that the ants are able to maintain a James Kurian http://orcid.org/0000-0001-6739-468X polyculture of intercropped L. gongylophorus strains by Alexander S. Mikheyev http://orcid.org/0000-0003-4369- some unknown mechanisms. However, when mycelia 1019 of different L. gongylophorus strains are mixed in equal Ulrich G. Mueller http://orcid.org/0000-0003-2677-8323 proportions in the absence of leafcutter ant farmers, one fungal strain can easily dominate the mix in short time(R.SenandU.G.Mueller,unpublisheddata), LITERATURE CITED suggesting that fungus-fungus competition can be sig- Adams RMM, Mueller UG, Green AM Narozniak JM. 2000. nificant under specific conditions. Our new methods Garden sharing and garden stealing in fungus-growing permit more refined experiments testing the relative ants. Naturwissenschaften 87: 491–493. importance of fungus-fungus competition versus sym- Albertin W, Marullo P. 2012. Polyploidy in fungi: evolution biont choice exerted by ants in maintaining garden after whole-genome duplication. Proceedings of the Royal – monoculture or polyculture, by confronting in a single Society B: Biological Sciences 279:2497 2509. Alic M, Letzring C, Gold MH. 1987. Mating system and garden un-dekaryotized natural strains, or dekaryo- basidiospore formation in the lignin-degrading basidiomy- tized strains, or both. cete Phanerochaete chrysosporium. Applied and (8) Because dekaryotized strains differ greatly in growth Environmental Microbiology 53:1464–1469. performance (FIG. 1), and because some dekaryotized Autuori M. 1950. Contribuição para o conhecimento da strains do not produce gongylidia in vitro, it should be saúva (Atta spp.-Hymenoptera-Formicidae) V. Número possible to force-switch a leafcutter colony to cultivate de formas aladas e redução dos sauveiros iniciais. 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