The microbial garden of fungus-growing ants: Distinct lignocellulosic profile and spatial structure, yet a similar microbiota

The microbial garden of fungus-growing ants: Distinct lignocellulosic profile and spatial structure, yet a similar microbiota | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The microbial garden of fungus-growing ants: Distinct lignocellulosic profile and spatial structure, yet a similar microbiota Mariana O. Barcoto, Rodrigo H. S. Garcia, Gabriel G. P. Pennachioni, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9554856/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Obligate microbial gardeners, attine ants cultivate fungi for food using mostly plant-derived substrates for nourishing their fungal crops. The fungal crop grows together with its associated microbiota then forming the microbial garden, a spongy structure scaffolded by substrates. Attines differ in their cultivation systems, in part, by foraging for a diverse set of plant substrates. Here, we hypothesize that the garden lignocellulosic profile, microbiota composition, and garden spatial structure differ between attine species according to their substrate preferences. Results We sampled young and old garden regions, as well as waste material from attines representing the lower fungiculture ( Apterostigma sp., Mycetophylax sp., and Mycocepurus sp.), the higher fungiculture ( Mycetomoellerius sp.), and leaf-cutting fungiculture ( Acromyrmex coronatus and Atta sexdens ). Then, we determined the garden chemical composition via 13 C NMR, the microbiota composition by 16S rRNA sequencing, and spatial distribution using SEM. We found plant-derived fragments representing the majority of recognizable substrates, providing the spatial framework of the microbial garden. In consonance, it exhibits a lignocellulosic nature, which is modified to a greater extent in gardens of lower fungiculture systems than in the higher and leaf-cutting ones. In lower attine, Burkholderia is an abundant microbial member, considered part of the fungiculture core microbiota and a biomarker of lower and higher fungiculture systems. In higher and leaf-cutting gardens, otherwise, Mesoplasma is abundant, taken as a signature of leaf-cutting systems. Group-wise comparisons revealed only At. sexdens differing from other fungiculture systems by showing lower diversity. Conclusions All attine gardens, but At. sexdens , share a similar taxonomic composition. Our findings unveil biofilms as intrinsic and ubiquitous components of attine microbial gardens. Essentially, each fungiculture has its characteristic architecture, emphasizing the substrate role in shaping the garden’s spatial organization. Fungiculture Plant substrates Biofilm Attine Symbiosis Microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background From insects [ 1 , 2 ] to humans [ 3 ], a wide diversity of animals feed on fungi. These include gastropod mollusks [ 4 , 5 ], reptiles [ 6 ], birds [ 7 ], terrestrial and arboreal mammals [ 8 – 11 ], all of them benefiting from fungi’s nutritional content [ 12 ]. Fungi are enriched in protein, dietary fiber, ergosterol, minerals (such as sulphur, iron, zinc, sodium, copper, selenium, manganese, calcium, phosphorus), vitamins (e.g., B2 and vitamin D), and all the essential amino acids and polyunsaturated fatty acids [ 3 , 13 , 14 ]. Mycophagy spectrum varies from obligate to accidental fungal consumption, depending on how dependent the animal is on fungi as food resources [ 15 ]. Fungus-feeding behavior is globally widespread [ 16 , 17 ] and occurs either seasonally (when other resources are scarce) or year-round [ 15 ]. However, among the many mycophagous animals, obligatory fungal cultivation was recognized only in fungus-growing insects: macrotermitine termites, attine ants, platypodinae, and scolytinae beetles. These insects independently evolved mechanisms that allow advanced practices for fungal crop cultivation, known as fungiculture. In advanced fungiculture systems, insects inoculate, cultivate, harvest, and vector fungi, establishing a nutritional dependence [ 18 – 20 ]. Advanced fungal cultivation by ants evolved once in the subtribe Attina (Hymenoptera: Formicidae: Myrmicinae: Attini, the “attine” ants), an event linked to one of the astronomical catastrophes of Earth’s evolution [ 21 – 23 ]. When the Chicxulub asteroid collided with the current site of the Yucatán Peninsula about 66 million years ago, the impact energy of about 10 23 joules had several environmental and paleontological consequences [ 24 – 26 ]. With gigatons of asteroid and Earth’s material being ejected at more than five kilometers per second, a dust cloud took only hours to cover the planet’s surface with soot sulfate aerosols. Such a dusty atmosphere blocked sunlight, cooled the planet, and interrupted photosynthesis [ 26 ]. Although this event caused mass-mortality at the Cretaceous-Paleogene limits, some life forms were able to thrive. Fungi, by consuming the organic matter from dead animals and plants, obtain energy independently of light, thus peaking at the period [ 25 , 27 ]. Serendipitously at the Cretaceous-Paleogene limits (~ 66.65 ± 13.28 million years ago), somewhere in a South American rainforest, ant fungiculture arose. Earth ecosystems, back then, favored a symbiotic lifestyle in which ants foraged for organic matter, brought it to the fungus, which digested it, providing labile nutrients to the ants. Ant-fungus partners surpassed the catastrophic conditions, coevolved, diversified into 20 genera, 247 extant species, and radiated (primarily) through the Neotropics [ 22 , 23 , 28 , 29 ]. All attines are obligate fungal farmers, though they have evolved different fungiculture practices and have achieved different scales. Four phylogenetically related groups of attine cultivate four groups of basidiomycetous fungi, thus four fungiculture systems are recognized: the lower, the yeast, the coral, and the higher (of which ramifies the leaf-cutting) fungicultures. Lower fungicultures are the ancestral system, closest to the early stages of fungal cultivation. “Lower attines”, a paraphyletic group of 85 attine species distributed in 11 genera, cultivate fungal species in the genera Leucocoprinus and Leucoagaricus ( Basidiomycota: Agaricales: Agaricaceae: Leucocoprineae [ 21 , 23 , 29 ]). They inhabit small and slow-growing colonies, with one or multiple queens and a few hundred tiny monomorphic workers. These nourish the fungal crop using dry and dead plant parts, floral parts, seeds, husks, insect frass, and carcasses, then take their own nutrients from fungal mycelium. Considered not fully domesticated, their cultivars are thought to be able to sustain a free-living, detached from the symbiosis with ants [ 30 – 35 ]. In the yeast fungiculture, fungal species in the tribe Leucocoprineae grow in yeast-like phase when associated with a clade of 19 species in the Cyphomyrmex rimosus group. Workers are thought to forage for nectar and sap, regurgitating these fluids for nourishing the crop [ 21 , 23 , 29 , 36 ]. The coral fungiculture is the only system in which the fungal crop does not belong to Agaricaceae ; instead, the clade of 30 species in the Apterostigma pilosum group cultivate species in the coral-fungus genus Myrmecopterula ( Basidiomycota: Agaricales: Pterulaceae ) [ 21 , 23 , 29 , 37 ]. Ants in the pilosum group use decaying wood, plant debris, flowers, seeds, husks, and feces to nourish their fungal crop [ 38 ]. Higher fungiculture is the derived system where agaricaceous fungal crops were not yet observed living apart from the 113 ant species that cultivate them using diverse plant parts [ 21 , 23 , 29 , 35 ]. Fungal crops are multinucleate or polyploid [ 39 ]; they form vacuolated swollen cells named gongylidia, which store metabolites from autophagic processes for recycling cellular contents. Filling the gongylidia, a fluid enriched in essential amino acids, lipids, free sugars, polysaccharides, and other macromolecules, becomes available to “higher attines” [ 40 – 42 ]. A subgroup of 52 higher attine species underwent a major transition, enabling them to cut fresh leaves as food for their fungal crop, which subsequently evolved into one of the main herbivores in the Neotropics. Achieving one of the highest complexities in animal society, the leaf-cutting fungiculture is often regarded as a fifth type of fungiculture. Most leaf-cutting ants cultivate L. gongylophorus , a highly domesticated fungus with nutritional and morphological adaptations for its symbiotic life with ants [ 23 , 28 , 29 , 43 – 45 ]. Leaf-cutting ants of the genera Atta and Acromyrmex represent the most extensively studied fungiculture systems [ 46 ], and consequently, current knowledge of fungus garden structure and metabolism is biased toward these taxa. When metabolizing the substrate, the fungus forms the “microbial garden”: an intertwined mesh of hyphae scaffolded by the substrate reminiscences, together with a microbial community that colonizes both the hyphae and the substrate (Fig. 1 A [ 47 ]). The garden structure then follows a longitudinal continuum of substrate degradation and nutritional patches [ 47 – 52 ]. Sites where the substrate has been recently added, i.e., “young gardens”, tend to be darker, where the substrate is relatively uncolonized by fungal hyphae and microbiota. As soon as the substrate is taken and consumed by the crop and microbiota, hyphal abundance increases, and the garden achieves clearer tones of beige-brown, which is characteristic of “old gardens”. In higher attines, a whitish “central garden” enriched in gongylidia may be found between the young and old regions [ 44 , 53 ]. Garden portions with no nutritional value to the ants are removed to “waste” dumps, in which contaminated garden pieces, harmful substrates, and dead ants are piled up [ 54 , 55 ]. A microbial garden comes into existence when a queen-to-be leaves her parental ant colony, carrying a garden piece inside a pouch of her pharynx, known as the infrabuccal pocket [ 30 ]. From queen to queen, the fungal crop lineage continues, which is the main way we know the fungus reproduces [ 19 ]. Carried from the parental nest, the first garden piece brings, together with the fungal crop, the garden microbiota (Fig. 1 B) [ 52 , 56 , 57 ]. Exhibiting characteristic taxonomic and functional profiles, the microbiota appears to be convergently adapted to the fungiculture environment, distinguishing it from other symbiotic systems [ 58 , 59 ]. Added substrates form the framework of the garden spatial structure, shaping fungal growth patterns and microbiota establishment [ 47 ]. In gardens of the leaf-cutting Atta sexdens , distinct sets of substrates not only changed the lignocellulosic profile, but also the microbiota composition, the fungal crop development, the biofilm spatial distribution, and even colony survivorship [ 60 ]. Since distinct fungiculture systems are nourished by substrates of distinct nature, including plant parts and insect frass/carcasses [ 21 ], we hypothesized that the garden chemical profile, microbiota composition, and spatial structure would differ according to the substrate. To tackle this query, we sampled young and old garden regions, as well as waste material from different fungiculture systems across the attine phylogeny (Fig. 1 C): i) Lower fungiculture, represented by the ant species Apterostigma sp., Mycetophylax sp., and Mycocepurus sp.; ii) Higher fungiculture, represented by the species Mycetomoellerius sp.; and iii) Leaf-cutting fungiculture, represented by the species Acromyrmex coronatus and At. sexdens. Then, we analyzed the substrate morphology and the microbiota spatial distribution via Scanning Electron Microscopy (SEM). The garden chemical profile was determined using 13 C Solid-state Nuclear Magnetic Resonance ( 13 C ssNMR), and the microbiota taxonomic composition was investigated by 16S rRNA sequencing. Besides providing further details on the substrates foraged by attine ants and the chemical composition of their garden, our findings shed light on the microbiota composition and biofilm spatial organization across different garden regions, under distinct fungiculture practices. Methods Colonies sampling and maintenance Attine colonies were collected in Atlantic rainforest areas at Parque Estadual Serra do Mar, Núcleo Picinguaba (S23°21’ W44°49’), Ubatuba-SP, between April 18–22, 2023, and November 24–28, 2023. Fungus-growing ants were sampled both at lowland ombrophile forests (characterized by clay-sandy soils, and a canopy of trees higher than 20 meters, epiphytes and lianas), and at coastal plain forest (“restinga”, with a canopy of trees and shrubs, and an understory of ground herbs and shrubs [ 61 , 62 ]). Field collections were also carried out in rainforest areas at Parque Nacional do Itatiaia (S22°27'; W44°36'), Itatiaia-RJ, between January 12–16, 2024. Colonies were sampled in highland ombrophile forests with continuous or partially interrupted canopy (originated naturally or anthropogenically [ 63 ]). Mycocepurus sp., Mycetophylax sp. (at the “restinga”), Mycetomoellerius sp., Acromyrmex sp., and Atta sp. colonies were collected by excavating a trench surrounding the nest area until exposing the garden chamber [ 64 ]. Colonies of Apterostigma sp. and Mycetophylax sp. (at the highland rainforest) were collected by carefully revolving the litter and breaking decomposing trunks. We collected three colonies per attine species ( Table S1 ). Garden and waste fragments were aseptically collected in sterilized recipients until further processing. Whenever possible, young and old regions of the garden were set apart, as well as the waste. We used entomological forceps for manually removing workers, eggs, pupae, and larvae from young and old gardens for 13 C ssNMR, metabarcoding, and SEM analyses. Ant workers taxonomic identification Ants were identified both by morphology (following Feitosa and Dias [ 65 ]) and by sequencing the Cytochrome Oxidase I mitochondrial gene ( COI ), a DNA barcoding locus able to tell apart ant species [ 66 , 67 ]. For COI sequencing, genomic DNA was extracted from attine workers kept in 96% ethanol sampling vouchers, by boiling ants’ legs and/or thorax in 150 µL of 10% Chelex X-100 (Bio-Rad, Hercules, 1422822) at 55°C for 30 minutes, then at 99°C for 60 minutes [ 68 ]. Primers Jerry [ 69 ] and Ben [ 70 ] were used to amplify a COI region of about 400bp, while the primers LEP-F1 and LEP-R1 [ 71 ] were employed for amplifying a COI region of about 700bp, totaling a region of ~ 1100pb [ 72 ]. PCR amplifications were prepared for a final volume of 25 µL, containing: 1.25 mM each of each dNTP (thus 4 µL of dNTPs), 5 µL of 5X buffer, 1 µL of BSA (1 mg mL –1 ), 2 µL of MgCl 2 (25 mM), 1 µL of each primer (10 µM), 0.5 µL of Taq polymerase (5 U µL –1 ), 2 µL of diluted genomic DNA (1:10), and 8.5 µL of sterile ultrapure water. Jerry/Ben amplification cycle included: initial denaturation at 95°C for 5 min, 25 cycles of denaturation at 95°C for 30s, annealing at 55°C for 45s, extension at 72°C for 1 min, then 20 cycles of denaturation at 95°C for 30s, annealing at 55°C for 45s, extension at 72°C for 1 min and a final extension step at 72°C for 10 min. LEP amplification was carried out as follows: 94ºC for 1min, six cycles of [94ºC for 1 min; 45ºC for 1:30 min], 36 cycles of [72ºC for 1:15min; 94ºC for 1 min; 51ºC for 1:30min; 72ºC for 1:15 min]; then 72º for 5min [ 72 ]. Amplicons were purified using FastAP thermosensitive alkaline phosphatase (Thermo Scientific), following the manufacturer’s protocol. Forward and reverse sequences were produced on an ABI3500 Sequencer (Life Technologies), with consensus sequences being assembled with Unipro UGENE v.52.0 [ 73 ]. Contigs matching at the genus level were searched in NCBI-GenBank through BLASTn, and these contigs are deposited in GenBank. 13 C Solid-state Nuclear Magnetic Resonance ( 13 C ssNMR) For being highly sensitive to modifications in chemical composition and changes in local microstructures [ 74 – 76 ], 13 C ssNMR can tell apart garden regions and waste, as well as lignocellulosic variations in response to ants foraging [ 60 ]. Since lower attine colonies did not yield enough biomass for 13 C ssNMR analysis, this was carried out using pooled gardens and waste samples. Colonies' samples were pooled by attine species (n = 3 colonies per species) and region (young, old, waste), resulting in 18 samples (3 regions × 6 attine species). Samples were freeze-dried and analyzed using a Bruker Avance 400 spectrometer, equipped with a Bruker 4-mm MAS double-resonance probe head, at 13 C and 1 H frequencies of 100.5 MHz and 400.0 MHz, respectively. We followed the literature for attributing typical spectra of lignocellulosic material to polysaccharide and lignin peaks [ 74 ]. High-resolution 13 C solid-state NMR spectra were acquired using 1 H- 13 C cross-polarization excitation, under magic angle spinning (MAS) and high-power 1 H decoupling ( 13 C-CPMAS). Experimental parameters included: cross-polarization time of 1 ms; recycle delay of 2 s; 1 H and 13 C pulse lengths of 3.3 µs and 4.0 µs, respectively; 1 H decoupling amplitude of gB 1 /2π = 70 kHz (SPINAL-64 pulse scheme). The 13 C-CPMAS method is not considered quantitative on the absolute number of chemical components, because of its sensitivity to local mobility and abundance of 1 H around the 13 C nuclei. When samples have a similar overall composition and identical parameters are used for acquiring signals, normalizing the spectra by the total spectral intensity (spectral area) may reveal changes in components. By directly comparing intensities within the same spectral regions, i.e., changes in the intensity profile, changes in the relative amounts of each type of component may be identified. In this work, the procedure consisted of calculating the area of selected spectral regions associated with different sample components and normalizing them by the total spectral area. All measurements were performed using identical acquisition parameters. DNA extraction, sequencing, and diversity analyses The microbial taxonomic composition was analyzed for three colonies per attine species (following the protocols from Barcoto et al. [ 60 ]). Separated by regions, the total DNA of 58 samples (3 colonies x 3 regions x 6 diets + one extra colony of Atta sp.) was extracted using the PowerLyzer PowerSoil DNA isolation kit (Qiagen). DNA concentration was quantified by a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific). Sequencing was carried out on an Illumina HiSeq 2500 platform, generating 2 × 100 bp (200 cycles) paired-end reads, with at least 100,000 sequences per sample at Novogene Corporation Inc. For the bacterial community, the region v3-v4 of 16S rRNA was amplified using the primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT), generating amplicons of approximately 450–550 base pairs. Sequences were preprocessed using QUIIME 2 (v2024.5.0) [ 77 ]. Demultiplexing and quality control checking were performed by DADA2 [ 78 ], employing consensus methods for filtering out chimeric sequences and low-quality ones (< q20). 16S rRNA sequencing yielded approximately 9,429,000 reads, with an average of 162,566 per sample. Sequences were later rarefied to 92,000 based on the lowest number of sequences per sample, with singletons and doubletons being removed. Taxonomic classification of the 16S rRNA regions was carried out using the SILVA Database (version 138), with 97% of similarity [ 79 ]. ASVs assigned as “mitochondria” and “chloroplasts” were removed. Taxonomic profiling and diversity analysis were carried out in MicrobiomeAnalyst [ 80 – 82 ], both by the webserver and by the R package MicrobiomeAnalystR. Features with low count (minimum count = 4, prevalence in samples = 10%) and low variance (10%, based on inter-quantile range) were filtered out, and the data were normalized to total sum scaling. Relative abundance was determined at the phylum, class, and genus levels. Heat tree analysis to depict taxonomic differences between microbial communities was performed at the genus and species level, using the non-parametric Wilcoxon Rank- Sum test (p-value cutoff = 0.05). Alpha-diversity was calculated at the genus level, for the following diversity metrics: observed features (richness), Shannon, Simpson, and Fisher, using Mann-Whitney/Kruskal-Wallis and Wilcoxon test for post hoc pairwise comparisons. Beta-Diversity was calculated at the genus level, using Principal Coordinates Analysis (PCoA) as ordination method, based on Bray-Curtis distances; statistical significance of data distribution and pairwise comparisons were defined by PERMANOVA. P -values were corrected by the Benjamini–Hochberg method, and a false discovery rate (FDR) ≤ 0.05 was considered statistically significant. Scanning Electron Microscopy (SEM) Samples were pooled as described for 13 C NMR analysis and prepared for imaging acquisition as formerly described [ 47 ]. Briefly, samples were fixed for at minimum 24 hours in Karnovsky solution (2.5% glutaraldehyde and 2% paraformaldehyde in 0.05 M cacodylate buffer, pH 7.2), maintained at 4°C. Fixed samples were dehydrated by ethanol gradual washing series (30%, 50%, 70%, 90%, and three times in 100%) and critical point-dried (EM CPD 300). Fragments of dried samples, no larger than 1 mm 3 in size, were mounted in aluminum stubs, adding up to nine fragments per stub, which were sputter-coated with colloidal gold (Baltec SCD 050). Images were visualized and digitally registered using a JEOL IT300 SEM. We recorded about 15–25 images per sample, ranging from the following magnifications: i) 100x-700x to observe hyphal density, substrate visual deterioration, and colonization patterns); ii) 700x-1,500x to detail microbial spatial organization; iii) 1,500x- 3,000x to describe physical interactions (in particular, biofilm structure); iv) 3,000x-4,000x to focus on informative microbial assemblages. The resulting images were visually analyzed to morphologically identify substrate structures and to characterize garden and waste features. These features included: 1) Presence of coiled and thick hyphae; 2) Presence of clamp connections; 3) Hyphal density; 4) Presence and abundance of gongylidia; 5) Gongylidia microbiota; 6) Hyphae microbiota; 7) Spread biofilm; 8) 3D biofilm; 9) Biofilm structure; 10) Yeasts and/or conidia; 11) Degree of substrate deterioration. Each feature was categorized, according to its approximate frequency, as: 0 - not observed; 1 - extremely rare (spotted once or a few times); 2 - observed in low abundance; 3 - observed in moderate abundance; 4 - observed in abundance; 5 - extremely common. Results Plant parts are dominant substrates in attine microbial gardens By employing SEM imaging, we identified substrates’ microstructures in a three-dimensional perspective, allowing us to hypothesize on their importance for the garden spatial structure. These substrates were pre-processed by the ants and already decayed when added to the garden, which, along with microbial overgrowth, narrows the substrate identification at a fine level. Surpassing these limitations, visually analyzing the resulting 1144 SEM images indicates that plant-derived fragments account for most incorporated substrates (Fig. 2 ). Leaf parts, discerned by mesophyll, epidermal surface remnants, stomata, and trichomes (Fig. 2 A [ 84 – 87 ]), were observed in all attine gardens, but predominantly in Mycetomoellerius sp. and At. sexdens gardens (Fig. 2 B). Silica phytoliths, distinguished as narrow long, cylindrical long, spherical and spheroid echinate phytoliths (Fig. 2 A [ 88 , 89 ]), were slightly more abundant in Apterostigma sp. and At. sexdens gardens (Fig. 2 B ) . Vessel elements, spotted by annular thickenings and pitted thickening walls (Fig. 2 A; [ 90 , 91 ]), were noticed in all gardens, though in larger proportions in Mycetomoellerius sp. (Fig. 2 B ) . Floral buds and anthetic floral parts, distinguished by dehiscent anthers, endothecial thickening remnants, and pollen grains (Fig. 2 A [ 92 – 96 ]) were more frequently observed in gardens of Apterostigma sp. and Mycocepurus sp., and in the waste of Mycetophylax sp. (Fig. 2 B ) . In summary, SEM images reaffirm that leaves are a common item in the diet of higher and leaf-cutting attines, while flower parts are customarily foraged by lower attines. Curiously, insect carcasses (Fig. 2 A) were only found in Mycetophylax sp. gardens (Fig. 2 B), as this would also be expected in gardens of other lower attines. Attine microbial gardens form a longitudinal continuum of lignocellulosic degradation Mirroring the abundance of plant-derived fragments as substrates, the chemical profile revealed by 13 C ssNMR indicates the microbial garden exhibiting a lignocellulosic nature. This is evidenced by the typical spectral profile of lignocellulosic materials, in which different spectral regions exhibit predominant contributions from polysaccharides (mainly cellulose and hemicellulose) and lignin, along with additional aliphatic signal contributions (as indicated by horizontal bars along the spectra in Fig. 3 , colored by each of these components). Due to the strong spectral overlap typical of 13 C ssNMR of lignocellulosic materials, trends were evaluated by calculating spectral areas, normalized by the total spectral area, within regions dominated by polysaccharides (cellulose and hemicellulose), lignin, and aliphatic components. These values are shown as inset bar graphs in Fig. 3 , where the lighter-toned color accounts for the aliphatic components, the middle-toned color to lignin, and the dark-toned color to the polysaccharides. Comparison across samples provided an estimate of the relative abundance of each component. Overall, the signals across the spectral regions suggest a slight degradation of lignocellulosic polysaccharide components, along with the accumulation of their byproducts in the form of aliphatic compounds, progressively increasing from the young garden to the waste samples (Fig. 3 ). Young gardens tend to display similar lignocellulosic compositions, with polysaccharides as the dominant fraction, with Apterostigma sp., and to a lesser extent, Acromyrmex coronatus , exhibiting higher lignin content. Specifically, signals in the 0–45 ppm region, associated with aliphatic compounds derived from lipids and proteins, show a gradual increase. In contrast, signals in the aromatic (110–160 ppm) and methoxy (~ 56 ppm) regions, predominantly attributed to lignin, do not exhibit a clear trend from the young garden to the waste. Some of these signals remain relatively intense in the waste, suggesting incomplete lignin metabolism across the garden. Signals attributed to cellulose and hemicellulose, spanning the 60–110 ppm and 160–200 ppm regions, show a general tendency to decrease from the young garden to the waste, being markedly reduced in most cases. This pattern was consistently found in gardens of Mycocepurus sp., Mycetomoellerius sp., Acromyrmex coronatus , and Atta sexdens (Fig. 3 A, 3 D, 3 E, 3 F). The lower attine Apterostigma sp. is the main exception, since lignin signals are characteristically high in young gardens, along with higher peaks to hemicelluloses (101.8 ppm and 172 ppm, Fig. 3 B). Waste is chemically similar to old gardens, except for a higher content of aliphatic compounds and a decrease in polysaccharide signals, while lignin remains relatively prominent. Another distinct pattern is observed for the lower attine Mycetophylax sp., where young and old gardens exhibit very similar spectral features. In this case, the waste shows only a slight decrease in polysaccharide signals accompanied by an increase in aliphatic signals and relatively persistent lignin-related contributions, suggesting that these components are only partially degraded within the garden and tend to accumulate in the waste (Fig. 3 C). Concisely, we observed distinct fungiculture systems varying in lignocellulosic composition, reflecting distinct foraging patterns of attine species. From different plant species and plant organs, discrete lignocellulose profiles give rise to continuums of degradation in the microbial garden, in a way that each fungiculture is a unique system for biomass conversion. Lignocellulose modification is achieved to a greater extent in gardens of lower fungiculture systems than in the higher and leaf-cutting ones. All attine gardens, but Atta sexdens , share a similar taxonomic composition Bacterial communities were investigated by amplicon sequencing of 16S rRNA genes, which yielded 27,528 amplicon sequence variants (ASVs) assigned to Bacteria . In brief, the microbiota assembles spatiotemporally along the degradation continuum: young and old regions are similarly rich and diverse; the waste, otherwise, is the richest and most diverse region (Supplementary material, Figures S1 -S3 ). The bacterial phyla Proteobacteria , Firmicutes , Actinobacteria , and Bacteroidota are abundant in the garden and in the waste, though varying in proportions by ant species ( Figure S4 ). The bacterial genus Burkholderia tends to be more abundant in lower attine gardens, and Mesoplasma is more abundant in higher and leaf-cutting gardens, though Burkholderia becomes abundant in the waste of almost all attine systems (Fig. 4 ). In consonance, Burkholderia / Paraburkholderia / Cabaleronia integrate the core microbiota of lower and higher attines gardens, only becoming part of the A. sexdens (but not of the Ac. coronatus ) core microbiota in waste samples ( Figure S5-S7 ). Biomarker determination based on linear discriminant analysis effect size (LEfSe) reveals that, except for Mycetophylax sp., Burkholderia is a biomarker of lower and higher fungicultures, while Mesoplasma is a signature of leaf-cutting systems ( Figure S8 ). By comparing distinct garden regions and attine fungicultures, we identified the microbiota of At. sexdens as the only differing from other attine species, characteristically having several bacterial genera in lower abundance (Fig. 4 , Table 1 , Figures S9-S24 ). Table 1 Bacterial genera differentially abundant in each attine fungiculture and garden regions, as determined by heat tree analysis [ 97 ] comparing group-wise relative abundances (Wilcoxon test, P < 0.05). Some abundant genera are shared between attine species (indicated by letters representing the fungiculture which the genera are shared with: Ap Apterostigma , Mp Mycocepurus , Mc Mycetophylax , Mm Mycetomoellerius , Ac Acromyrmex coronatus , At Atta sexdens ). Genus-level heat trees denote only At. sexdens fungiculture differing significantly from other attine, as At. sexdens have diverse genera at lower abundance ( Figures S10-S24 ). Thus, the bacterial genera listed here are those whose abundance significantly differs from At. sexdens . Mycocepurus sp. Young garden Old garden Waste Butyricicoccus (Ap, Mm) Duganella (Mm) Marmoricola Paludicola (Mc) Paraclostridium Pseudonocardia (Mc, Ac) Robinsoniella (Mc) Solirubrobacter Tumebacillus Acrocarpospora Anaerocolumna Catenibacillus (Mc) Duganella (Mm) Nocardia (Ap) Paludicola (Mm) Pseudonocardia (Mc) Raoultibacter (Mm) Tumebacillus Tsukamurella Hyphomicrobium (Mc, Mm) Labrys (Mc) Solirubrobacter (Mc) Streptomyces (Mc) Terrimicrobium (Mc) Apterostigma sp. Acidibacter Acidothermus Akkermansia (Mc) Butyricicoccus (Mp, Mm) Clostridium sensu stricto Paucilactobacillus Roseiarcus Ruminococcus Acidibacter Acidipila-Silvibacterium Acidothermus Conexibacter Edaphobacter Humibacter Inquilinus Mycobacterium Nocardia (Mp) Rhodanobacter Roseiarcus Solirubrobacter Terracidiphilus Humibacter Rhodanobacter Rhodomicrobium (Mm) Terriglobus Mycetophylax sp. Akkermansia (Ap) Anaerocolumna Anaerofustis Anaerovorax Catenibacillus Clostridium methylpentosum group (Mm) Clostridium sensu stricto Eubacterium coprostanoligenes group (Mm ) Friedmanniella (Mm) Jatrophihabitans (Mm, Ac) Odoribacter Paludicola (Mp) Pseudonocardia (Mp, Ac) Raoultibacter (Mm) Robinsoniella (Mp) Serratia Yokenella Akkermansia Anaerovorax Catenibacillus (Mp) Methylocella Pseudonocardia (Mp) Ruminococcus Afipia Amaricoccus Bauldia (Mm) Bordetella Catenulispora Cellvibrio Dongia Hephaestia Hylemonella Hyphomicrobium (Mp, Mm) Jatrophihabitans (Mm) Labrys (Mp) Luteimonas Marmoricola Methylocella Mesorhizobium Mumia (Mm) Nordella (Mc) Panacagrimonas Parapedobacter Pigmentiphaga (Mm) Pseudaminobacter Pseudoduganella Pseudohongiella Pseudolabrys Pseudonocardia (Mm) Rhizobacter Rhodopila Rhodoplanes (Mm) Saccharothrix Solirubrobacter (Mp, Mm) Sporichthya Streptomyces (Mp) Terriglobus Terrimicrobium (Mp) Thermomonas Mycetomoellerius sp. Amycolatopsis Butyricicoccus (Mp,Ap) Clostridium methylpentosum group (Mc) Duganella (Mp) Eubacterium coprostanoligenes group (Mc) Friedmanniella (Mc) Herbaspirillum Jatrophihabitans (Mc, Ac) Methylocella Raoultibacter (Mc) Rugosimonospora Actinomycetospora Aureimonas (Ac) Butyricicoccus Clostridium methylpentosum group Duganella (Mp) Eubacterium coprostanoligenes group Friedmanniella Jatrophihabitans (Ac) Luedemannella Paludicola (Mp) Raoultibacter (Mp) Amycolatopsis Aquicella Bauldia (Mc) Ciceribacter Dactylosporangium Duganella Edaphobacter Friedmanniella Herbaspirillum Hyphomicrobium (Mp, Mc) Jatrophihabitans (Mc, Ac) Legionella (Ac) Mumia (Mc) Nocardia Nordella (Mc, Ac) Pigmentiphaga (Mm) Pseudonocardia (Mc) Ralstonia Rhodomicrobium (Ap, Ac) Rhodoplanes (Mc) Rugosimonospora Solirubrobacter (Mp, Mc) Streptosporangium Acromyrmex coronatus Afipia Glutamicibacter Jatrophihabitans (Mc, Mm) Nakamurella Ottowia Patulibacter Pseudonocardia (Mp, Mc) Aureimonas (Mm) Glutamicibacter Jatrophihabitans (Mm) Klenkia Methylocella Jatrophihabitans (Mc, Ac) Legionella (Mm) Luteipulveratus Nordella (Mc, Mm) Ottowia Rhodoblastus Rhodomicrobium (Ap, Mm) Roseiarcus In young gardens, Proteobacteria , Firmicutes , and Actinobacteriota are the dominant phyla, predominantly in the class Gammaproteobacteria , Bacilli , and Alphaproteobacteria ( Figure S25) . At the genus level, most young gardens’ ASVs (10–60%) were not assigned or assigned to less abundant genera (gathered as “others”; Fig. 4 A). In Mycocepurus sp., assigned ASVs were mostly attributed to Bacillus and Burkholderia ; in Apterostigma sp., to Burkholderia; in Mycetophylax sp., to Burkholderia and Tyzzerella ; in Mycetomoellerius sp., to Mesoplasma , Pullulanibacillus , and Burkholderia ; in Ac. coronatus to Mesoplasma and Glutamicibacter ; and in At. sexdens , to Mesoplasma and uncultured Lachnospiraceae (Fig. 4 A). Young gardens did not differ in richness and alpha diversity indices (Wilcoxon test, FDR-adjusted P < 0.05, Fig. 4 B; Fig S26 ). Beta diversity analysis based on Bray-Curtis dissimilarity indicates that the community does not vary according to attine species, yielding no differences in pairwise comparisons (Fig. 4 C, PERMANOVA, FDR-adjusted P > 0.05). Taxa contributing to the dissimilarity between young gardens include Mesoplasma (7.2–24.56%), uncultured Lachnospiraceae (7.9–10.83%), Glutamicibacter (7.48–9.85), and Bacillus (6.34–8.74%; SIMPER analysis based on Bray–Curtis, Supplementary Material). Proteobacteria , Firmicutes , and Actinobacteriota continue as dominant phyla in old gardens, mainly in the class Gammaproteobacteria , Bacilli , and Clostridia ( Figure S27) . Like for the young garden, most old gardens’ ASVs (10–80%) were not assigned at the genus level or assigned to less abundant genera (“others”). Assigned ASVs at the genus level, in Mycocepurus sp., were mostly attributed to Candidatus Soleaferrea , Bacillus , and Tyzzerella ; in Apterostigma sp., to Burkholderia ; in Mycetophylax sp., to Candidatus Soleaferrea , and Tyzzerella ; in Mycetomoellerius sp., to Pullulanibacillus , Burkholderia , and Tyzzerella ; in Ac . coronatus , to Mesoplasma and uncultured Lachnospiraceae ; and in At. sexdens , to Mesoplasma , Burkholderia , and uncultured Lachnospiraceae (Fig. 4 A). Pairwise comparisons of alpha-diversity indices showed no significant differences between attine old gardens (Wilcoxon test, FDR-adjusted P < 0.05, Fig. 4 B; Fig S28 ). Beta diversity analysis based on Bray-Curtis implies the community does not vary with the attine species (PERMANOVA, FDR-adjusted P < 0.05), reinforced by pairwise comparisons yielding no differences between attine old gardens (Fig. 4 C). Dissimilarity between old gardens is attributed mostly to Mesoplasma (3.20-20.31%), Pullulanibacillus (16.08–18.61%), Burkholderia (4.34–14.71%), uncultured Lachnospiraceae (9.02–11.38%), and Candidatus Soleaferrea (3.9–8.15%; SIMPER analysis based on Bray–Curtis, Supplementary Material). In waste samples, Proteobacteria , Actinobacteriota , and Bacteroidota are the most abundant bacterial phyla, with the majority in the class Gammaproteobacteria , Alphaproteobacteria , and Actinobacteria ( Figure S29) . Between 20–70% of waste’s ASVs were either not assigned at the genus level or assigned to “others” (not abundant genera). In the Mycocepurus sp. waste, assigned ASVs at the genus level were primarily traced to Burkholderia and Rhizobium ; in the Apterostigma sp. waste, to Burkholderia and Pullulanibacillus ; in the Mycetophylax sp. waste, to Flavobacterium and Pseudomonas ; in the Mycetomoellerius sp. waste, to Burkholderia , Rhizobium , and Sphingobacterium ; in the Ac . coronatus waste, to uncultured Microbacteriaceae , Stenotrophomonas , and Sphingobacterium ; and in the waste of At . sexdens , to Burkholderia , Stenotrophomonas , and Flavobacterium (Fig. 4 A). Pairwise comparisons of alpha-diversity indices yielded no differences between attine waste samples (Wilcoxon test, FDR-adjusted P < 0.05, Fig. 4 B; Figure S30 ). Beta-diversity analysis based on Bray-Curtis signals that the community does not vary according to attine species (PERMANOVA, FDR-adjusted P < 0.05), which is supported by no differences between attine gardens obtained in pairwise comparisons (Fig. 4 C). Pullulanibacillus (15.14–17.72%), Burkholderia (8.04–12.44%), Mesoplasma (6.5–7.43%), and Rhizobium (5.03–6.35%) are the taxa contributing the most to the dissimilarity between attine waste (SIMPER analysis based on Bray–Curtis, Supplementary Material). Microbiota and biofilms are ubiquitous components of attine fungiculture systems We visually analyzed 1144 SEM images to determine garden and waste spatial organization and microbiota distribution. Each fungiculture has its own characteristic spatial structure, especially differing in hyphae morphology and growth patterns (Fig. 5 A). Still, we found among them analogous patterns of substrate deterioration, microbiota-substrate, and microbiota-hyphae physical interactions were detected across the systems. From the young garden to the waste, it follows a progressive increase in substrate deterioration, colonization by hyphae, microbiota, and biofilm complexity (Fig. 5 B, Figure S31 ). Accordingly, young regions tend to hold less deteriorated substrates, yet are sparsely colonized by hyphae and microbiota. Fungal hyphae frequently start colonizing the substrate at its edges, furrows, and indentations. The microbiota tends to be scattered at this point, occurring in isolated or as small communities, present at the substrate surface and interiors, also surrounding fungal hyphae. The hyphal density varies in old regions according to the fungiculture system, with hyphae often surrounded by biofilm. Gongylidia are commonly observed across the gardens of higher and leaf-cutting attines, frequently populated by the microbiota. Substrates in the old garden are more deteriorated, which is evidenced by exposed remnants of vessels, anthers, and mesophyll. Substrates in old gardens cannot be dissociated from thick microbial biofilms in their broken-down structure. In the waste, substrates are rarely identifiable due to their extensive deterioration, coupled with an exuberant biofilm. There, a multi-morphological microbial community, embedded in a matrix, appears to engage in physical interactions constantly. It is worthy pointing out that differences in young and old garden regions are much more evident in leaf-cutting gardens, from which emerge a “longitudinally stratified” garden. In lower attines and even in the higher attine Mycetomoellerius sp., the longitudinal patterns of substrate degradation, hyphae, and microbiota colonization are way more subtle (Fig. 5 , Figure S31 ). Lower attine gardens present some distinguishable features, including: gongylidia-like structures in Mycocepurus sp.; clamp connections characterizing the Apterostigma sp. fungal crop (as expected for fungi in the Pterulaceae family); and gardens of Mycetophylax sp. assembling as a set of wool balls with almost indistinguishable young and old regions (Fig. 5 , Figure S31 ). Discussion In the macro scale, it is known for long that community diversity and organization patterns respond to environmental conditions [ 98 ]. In the micron scale, likewise, patterns of distribution in microbial ecosystems determine much of their functioning and responses [ 99 – 101 ]. Microbiota organization follows environmental cues and interactions within its neighborhood, in a way that ecological functions are, by far, correlated to changes in spatial patterning [ 102 – 104 ]. This put the microbiota spatial organization side by side with the taxonomic composition in determining ecosystem properties [ 99 , 101 , 105 ]. As a consequence, a deeper comprehension of ecological processes shaping ecosystems requires a better knowledge of their spatial patterns and their correlation to environmental conditions [ 100 , 106 , 107 ]. Here, we investigated the architecture of the microbial garden, the microbial ecosystem housed in attine ant colonies. Using physical-chemical, sequencing, and imaging tools, we determined how nutrients, fungal crops, and microbial biofilms are spatially organized in the fungiculture systems of ant species distributed across the attine phylogeny (Fig. 1 ). These attine foraged predominantly for plant parts (Fig. 2 ), yielding microbial gardens with a lignocellulosic nature (Fig. 3 ). As the fungal crop and the microbiota breakdown and consume the substrates, a characteristic continuum of lignocellulose degradation is established [ 60 ]. Following this gradient, there exists a microbiota with similar taxonomic composition among all attine gardens, except for At. sexdens (Fig. 4 ). In fact, a biofilm-forming microbiota is an ever-present element of the microbial garden architecture (Fig. 5 ). So far, the composition of attine colonies’ diet has been mostly described from observational data on ant foraging behavior, by recording and identifying substrate fragments acquired by the ants. Typically, these fragments are morphologically categorized before entering the colony, and include leaf and floral parts, seeds, husks, insect frass, and carcasses [ 35 , 108 ]. Alternatively, dietary DNA (dDNA) was employed to specify the foraging choices of the higher attines Trachymyrmex septentrionalis and Mycetomoellerius turrifex , by sequencing a region of the chloroplast trnL intron and COI markers directly from the garden [ 109 ]. We followed a third route, adapting microscopical methods to ascertain the diet of herbivorous mammals [ 110 – 112 ] to characterize substrates after ants had added them to the garden, that is, while being digested. Diverse plant fragments were detected as substrates in young gardens, i.e., where the substrate was recently added by ants, supporting that attine fungiculture systems assume the ecological role of herbivores (Fig. 2 [ 21 , 30 ]). Although overlapping in some foraging choices, attine ants in distinct fungiculture systems tend to prefer distinct plant species and plant organs [ 21 , 29 , 35 , 109 , 113 , 114 ], yielding differences in the chemical profile of each microbial garden (Fig. 3 ). This is illustrated by floral buds and anthetic floral parts, such as anther wall remnants (endothecial thickenings) and pollen grains, being often observed in gardens of Apterostigma sp. and Mycocepurus sp., and in the waste of Mycetophylax sp. (Fig. 2 ). Layers of lignin compose mature endothecium, which is crucial for anther dehiscence and pollen release [ 115 , 116 ], and pollen themselves are a prominent source of lignin [ 117 , 118 ]. One could expect that breaking down these lignin-enriched substrates along the garden to render chemical profiles essentially different from those of fungiculture systems fed mostly with leaf parts (Fig. 3 ). Regardless of the substrate with which they were nourished, attine microbial gardens tend to form a continuum of substrate degradation. From young regions, passing by old regions, until the waste, there is a progressive increase in aliphatic compounds reflecting an accumulation of lipids and proteins (Fig. 3 [ 119 , 120 ]). Lignin-derived compounds slightly decrease from the young garden to the waste, though, since they are still present in the waste, lignin seems to be incompletely metabolized in the garden. The fate of cellulose and hemicelluloses varied widely according to fungiculture systems: while in some of them cellulose and hemicelluloses are reduced in old gardens (and even more in the waste), in others these signals did not evidence substantial changes (Fig. 3 [ 49 , 121 , 122 ]. Although previously debated [ 123 , 124 ], the cellulose-degrading capacity is coded by the L. gongylophorus genome [ 45 , 49 , 53 , 125 , 126 ], being supported by enzymatic, chemical, ultrastructural, and transcriptional data [ 49 , 121 , 127 , 128 ]. By exhibiting a lignocellulolytic potential, the garden microbiota is thought to complementarily contribute to cellulose decomposition [ 52 , 59 ]. Thus, cellulose degradation in the leaf-cutting ants’ garden (Fig. 3 ) is possibly a fungus-microbiota teamwork. Lignocellulose modification seems to be achieved to a greater extent in gardens of lower fungiculture systems than at the higher and leaf-cutting ones (Fig. 3 ), which is conceivably related to reductions in the fungal lignocellulolytic capacity along the symbiosis evolution [ 45 , 129 ]. As lignocellulosic components are modified and their end products are accumulated [ 119 , 130 ], a gradient of physicochemical conditions could be formed along the garden regions, and with that, a profusion of microbial niches is established [ 60 , 131 – 133 ]. The microbiota follows the degradation continuum, with Proteobacteria , Actinobacteria , and Firmicutes mainly inhabiting young ( Figure S25 ) and old regions ( Figure S27 ), then Bacteroidota replacing Firmicutes in the waste ( Figure S29 ). In lower attine gardens and the waste of almost all attine systems, Burkholderia is an abundant microbial member, then considered part of the fungiculture core microbiota and a biomarker of lower and higher fungiculture systems ( Figure S5-S8 ). Our sequencing data does not allow us to infer the metabolic potential of these bacterial genera, and we do not intend to suggest putative roles for the microbiota in the garden based only on the literature. Besides, we also consider plausible the scenario where they are transiently inhabiting the garden. Keeping this in mind, knowing in which environments the abundant and characteristic genera in the microbial garden were already reported may assist future experiments targeting their function and ecological roles in attine fungiculture. In that sense, Burkholderia (taxonomically revised and having some strains reassigned to Paraburkholderia and Caballeronia [ 134 ]) is an environmentally widespread bacterium with lignocellulolytic capacity [ 135 , 136 ], also a well-known insect symbiont [ 137 , 138 ]. Not so abundant in gardens of the lower attine Mycocepurus smithii [ 139 ], Burkholderia is commonly found in leaf-cutting attine gardens, where it is thought of as a defensive symbiont, protecting the system against fungal contaminants [ 140 , 141 ]. Mesoplasma , otherwise, is abundant in higher and leaf-cutting gardens, taken as a signature of leaf-cutting systems (Fig. 4 ; Figure S8 ). This common member of At . sexdens fungiculture (Fig. 4 ) participates in the garden response to diet, characterizing a state of diet-induced dysbiosis [ 60 ]. Mesoplasma strains isolated from Trachymyrmex septentrionalis metabolize glucose, fructose, arginine, and N-acetylglucosamine, suggestively producing ammonia for the ants [ 142 ]. Curiously, Mesoplasma was abundantly found in the Atta workers’ body [ 56 , 143 , 144 ], raising the question of whether/how the ant microbiota participates in the microbial garden assembly. Leaf-cutting ants apply fecal droplets, replete with gongylidia-produced enzymes, over the freshly added substrate as a pretreatment [ 145 – 150 ]. It is yet to be investigated if this fluid is enriched in microorganisms, and if it is, whether these microbes play any role in plant decomposition. It is also to be determined whether the ants somehow shape the microbiota, directly by filtering out harmful microbes, or indirectly by selecting suitable substrates or applying antimicrobial compounds [ 46 , 151 ]. In this perspective, At . sexdens hygienic processes for garden cleaning, such as applying glandular chemical compounds and behavioral strategies for pathogen removal [ 46 , 151 , 152 ], could explain their garden microbiota standing out with a lower microbial abundance (Fig. 4 ; Figures S9-S24 ). With At. sexdens being an exception, all attine gardens share a similar taxonomic composition ( Figures S10-S24 ). In fact, fungus-growing insects are thought to harbor a characteristic microbiota, with marked similarities in taxonomic composition and functional profile. Such evidence for host-microbiota convergence at higher hierarchical levels (e.g., phylum and class) seems to be applied to bacterial genera as well [ 58 , 59 ]. It is not clear what makes the microbiota members thrive in the fungiculture ecosystem, though some of them could be favored by an environment concomitantly enriched in plant and fungal biomass. Fungal metabolites are selective forces assembling the microbiota of the rhizosphere [ 153 ] and different parts of mushroom-forming fungus, possibly behind the compositional and functional convergence of mushroom microbiota as well [ 154 ]. From this, one may hypothesize that the fungal crop metabolism could shape the microbiota composition, especially considering that some Burkholderia strains are capable of – and benefit from – using fungal metabolites. Besides surpassing fungal defenses, Burkholderia surround fungal hyphae with biofilm, then comigrate with fungi in soil [ 155 – 157 ]. On the other hand, bacteria in leaf-cutting fungiculture seem to influence the fungal crop by promoting its growth, also influencing phosphate solubilization, biofilm formation, cellulose, and chitin degradation [ 158 ]. Yet, fungal-microbiota interactions in microbial gardens are a research window wide open: there are diverse microbial members often found in attine gardens that are plant-associated, and future experiments could be designed to investigate their role and application possibilities. These include Glutamicibacter (reported as denitrifiers and fungal-growth promoters [ 159 , 160 ]), Tyzzerella (reported to be favored by plant fiber [ 161 ]), and Pullulanibacillus (reported as a biofilm-forming bacterium [ 162 , 163 ]). We looked closely at the microbiota architecture by SEM, as if we were accompanying the microbial community structuring according to chemical modifications in the substrate (Fig. 5 ). Therein lies the continuum of lignocellulose degradation, in a way that each fungiculture seems to constitute a unique system for biomass conversion (Figs. 2 , 3 , 5 [ 129 ]). Chemical particularities possibly reflect particular metabolic processes required for substrate decomposition, yielding particular nutritional niches for the microbiota to assemble [ 133 , 164 , 165 ]. Our findings reinforce that the microbiota, spatially organized in biofilms, are ubiquitous components of attine fungiculture systems (Fig. 5 ). Through biofilms, the microbiota can cross-communicate, adhere, interact with the substrate, and avoid contacting toxins. The biofilm matrix retains lignocellulolytic enzymes, which convert the entire structure - including non-degradative members - into a digestive one [ 166 – 170 ]. Whether, when, and where microbiota-fungus and microbiota-ant interactions occur, and how important they are for attine ecology and evolution, are open windows of research. Attine microbial gardens offer countless possibilities for fruitful microbial ecology research, integrating multidisciplinary approaches for unveiling such a multidimensional microbial ecosystem. Conclusions Attine microbial gardens are primarily herbivorous, with distinct fungiculture systems varying in lignocellulosic composition according to foraging patterns, that is, select for different plant species and plant organs. Gardens form a continuum of lignocellulosic degradation, which is followed by the microbiota: Proteobacteria , Actinobacteria , and Firmicutes mainly inhabiting young and old regions, then Bacteroidota replaces Firmicutes in the waste. In lower attine gardens and the waste of almost all attine systems, Burkholderia is an abundant microbial member, considered part of the fungiculture core microbiota and a biomarker of lower and higher fungiculture systems. In higher and leaf-cutting gardens, otherwise, Mesoplasma is abundant, taken as a signature of leaf-cutting systems. Each attine garden constitutes a unique system for biomass conversion, in turn yielding nutritional niches for the microbiota to assemble, in the majority as biofilms. Declarations Declaration of generative AI and AI-assisted technologies in the writing process The authors declare that no AI and AI-assisted technologies were used for the writing process. Acknowledgements The authors would like to thank Elliot Watanabe Kitajima for constructive comments on SEM analysis, Renato Barbosa Salaroli for technical assistance, Raquel Lima de Sousa for field assistance, and Manoela Ramalho, Mario Tyago Murakami, Simone Raposo Cotta, and Rodrigo Gouvêa Taketani for their constructive insights and suggestions. We would like to thank Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) for providing the collecting permit (# 74585). We also thank Conselho Nacional de Gestão do Patrimônio Genético (CGen) for providing a permit to access the genetic heritage (#SISGen AA39A6D). Author contributions Mariana de Oliveira Barcoto - Conceptualization, Data curation, Investigation, Methodology, Project administration, Formal analysis (metabarcoding data and SEM), Visualization, Writing – original draft, and Writing – review & editing. Rodrigo Henrique dos Santos Garcia - Formal analysis (NMR data). Gabriel Giorgio Pressuto Pennachioni - Formal analysis (ant workers identification). João Gabriel da Silva Soares - Formal analysis (NMR data). Eduardo Ribeiro de Azevedo - Formal analysis (NMR data), Resources, Validation, Writing – review & editing. Lucas William Mendes - Formal analysis (metabarcoding data), Software, Resources, Writing – review & editing. Alessandra Ike Coan - Formal analysis (plant anatomy), Writing – review & editing. Mauricio Bacci Jr - Funding acquisition, Writing – review & editing. Andre Rodrigues - Conceptualization, Methodology, Project administration, Supervision, Resources, Funding acquisition, Writing – review & editing. Conflicts of interest The authors declare no competing interests. Funding The research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant #2019/03746-0). MOB received PhD scholarships from FAPESP (# 2021/08013-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. AR received a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; #305269/2018). MB received a research grant from FAPESP (CEPID #21/10639-5). Data availability Datasets supporting the conclusions of this article are available in the FigShare repository, under the Project “The most famous gardeners”. NMR spectra may be accessed by https://doi.org/10.6084/m9.figshare.31378345, SEM images by https://doi.org/10.6084/m9.figshare.31376602. ASV sequences are available at the NCBI repository, under the BioProject ID PRJNA1430830, and COI sequences used for ants identification are under GenBank accession numbers PZ094865-PZ094893 Ethics approval and consent to participate Fieldwork was carried out with collecting permit emitted by Chico Mendes de Conservação da Biodiversidade (ICMBio, permit #74585). Access of genetic heritage was permitted by Conselho Nacional de Gestão do Patrimônio Genético (CGen) (permit #SISGen AA39A6D). References Kimura MT, Tuno N. Phylogeny, ecology, and evolution of mycophagous Drosophilidae (Diptera). Biol J Linn Soc. 2024;142(3):247–60. https://doi.org/10.1093/biolinnean/blad140 . Kozel P, Lejeune B, Lepoint G, et al. Stable isotopes of saproxylic beetles reveal low differences among trophic guilds and suggest a high dependence on fungi. Ecol Monogr. 2025;95(1):e70002. https://doi.org/10.1002/ecm.70002 . O'Regan HJ, Lamb AL, Wilkinson DM. The missing mushrooms: Searching for fungi in ancient human dietary analysis. J Archaeol Sci. 2016;75:139–43. https://doi.org/10.1016/j.jas.2016.09.009 . Ori F, Menotta M, Leonardi M. Effect of slug mycophagy on Tuber aestivum spores. Fungal Biol. 2021;125(10):796–805. https://doi.org/10.1016/j.funbio.2021.05.002 . Barahona-Segovia RM, Gallardo‐Pillancari E, Orrego G, Osorio F. Mycophagy and feeding on other native land snails by the black snail Macrocyclis peruvianus (Gastropoda, Macrocyclidae). Austral Ecol. 2024;49(2):e13491. https://doi.org/10.1111/aec.13491 . Elliott TF, Jusino MA, Trappe JM, et al. A global review of the ecological significance of symbiotic associations between birds and fungi. Fungal Divers. 2019;98(1):161–94. https://doi.org/10.1007/s13225-019-00436-3 . Elliot TF, Bower DS, Vernes K. Reptilian Mycophagy: A global review of mutually beneficial associations between reptiles and macrofungi. Mycosphere. 2019;10(1):776–97. https://doi:10.5943/mycosphere/10/1/18 . Cork SJ, Kenagy GJ. Nutritional value of hypogeous fungus for a forest-dwelling ground squirrel. Ecology. 1989;70(3):577–86. https://doi.org/10.2307/1940209 . Launchbaugh KL, Urness PJ. Mushroom consumption (mycophagy) by North American cervids. Great Basin Naturalist. 1992;52(4):321–7. http://www.jstor.org/stable/41712738 . Claridge AW, Trappe JM, Cork SJ, Claridge DL. Mycophagy by small mammals in the coniferous forests of North America: nutritional value of sporocarps of Rhizopogon vinicolor , a common hypogeous fungus. J Comp Physiol. 1999;169(3):172–8. https://doi.org/10.1007/s003600050208 . Hanson AM, Hodge KT, Porter LM. Mycophagy among primates. Mycologist. 2003;17(1):6–10. https://doi.org/10.1017/S0269915X0300106X . Wallis IR, Claridge AW, Trappe JM. Nitrogen content, amino acid composition and digestibility of fungi from a nutritional perspective in animal mycophagy. Fungal Biol. 2012;116(5):590–602. https://doi.org/10.1016/j.funbio.2012.02.007 . Derbyshire EJ, Delange J. Fungal protein–what is it and what is the health evidence? A systematic review focusing on mycoprotein. Front Sustain Food Syst. 2021;5:581682. https://doi.org/10.3389/fsufs.2021.581682 . Ahmad MI, Farooq S, Alhamoud Y, et al. A review on mycoprotein: History, nutritional composition, production methods, and health benefits. Trends Food Sci Technol. 2022;121:14–29. https://doi.org/10.1016/j.tifs.2022.01.027 . Claridge AW, Trappe JM. Sporocarp mycophagy: nutritional, behavioral, evolutionary, and physiological aspects. Mycol Ser. 2005;23:599. Fogel R, Trappe JM. Fungus consumption (mycophagy) by small animals. Northwest Sci. 1978;52(1):1–31. Elliott TF, Truong C, Jackson SM, et al. Mammalian mycophagy: a global review of ecosystem interactions between mammals and fungi. Fungal Syst Evol. 2022;9(1):99–159. https://doi.org/10.3114/fuse.2022.09.07 . Martin MM. The evolution of insect-fungus associations: from contact to stable symbiosis. Am Zool. 1992;32(4):593–605. https://doi.org/10.1093/icb/32.4.593 . Mueller UG, Gerardo NM, Aanen DK. The evolution of agriculture in insects. Annu Rev Ecol Evol Syst. 2005;36(1):563–95. https://doi.org/10.1146/annurev.ecolsys.36.102003.152626 . Biedermann PH, Vega FE. Ecology and evolution of insect–fungus mutualisms. Annu Rev Entomol. 2020;65(1):431–. https://doi.org/10.1146/annurev-ento-011019-024910 . 55. Schultz TR, Brady SG. Major evolutionary transitions in ant agriculture. PNAS. 2008;105(14):5435–40. https://doi.org/10.1073/pnas.0711024105 . Branstetter MG, Ješovnik A, Sosa-Calvo J, et al. Dry habitats were crucibles of domestication in the evolution of agriculture in ants. Proc Biol Sci. 2017;284(1852):20170095. https://doi.org/10.1098/rspb.2017.0095 . Schultz TR, Sosa-Calvo J, Kweskin MP, et al. The coevolution of fungus-ant agriculture. Science. 2024;386(6717):105–10. https://doi.org/10.1126/science.adn7179 . Van den Bergh S. Astronomical catastrophes in Earth history. Publ Astron Soc Pac. 1994;106(701):689–95. https://doi.org/10.1086/133431 . Schulte P, Alegret L, Arenillas I, et al. The Chicxulub asteroid impact and mass extinction at the Cretaceous-Paleogene boundary. Science. 2010;327(5970):1214–18. https://doi.org/10.1126/science.1177265 . Morgan JV, Bralower TJ, Brugger J, Wünnemann K. The Chicxulub impact and its environmental consequences. Nat Rev Earth Environ. 2022;3(5):338–54. https://doi.org/10.1038/s43017-022-00283-y . Berry K. Evidence for fungal proliferation following the Cretaceous/Paleogene mass-extinction event, based on chemostratigraphy in the Raton and Powder River basins, western North America. Acta Palaeobot. 2020;60(1):134–42. https://doi.org/10.35535/acpa-2020-0005 . Mehdiabadi NJ, Schultz TR. Natural history and phylogeny of the fungus-farming ants (Hymenoptera: Formicidae: Myrmicinae: Attini). Myrmecological News. 2010;13:37–55. Schultz TR, Gawne R, Peregrine PN. The convergent evolution of agriculture in humans and insects. MIT Press; 2022. Weber NA. The fungus-culturing behavior of ants. Am Zool. 1972;12(3):577–. https://doi.org/10.1093/icb/12.3.577 . 87. Mueller UG, Rehner SA, Schultz TR. The evolution of agriculture in ants. Science. 1998;281(5385):2034–8. https://doi.org/10.1126/science.281.5385.2034 . Fernández-Marín H, Zimmerman JK, Wcislo WT, Rehner SA. Colony foundation, nest architecture and demography of a basal fungus‐growing ant, Mycocepurus smithii (Hymenoptera, Formicidae). J Nat Hist. 2005;39(20):1735–43. https://doi.org/10.1080/00222930400027462 . Diehl-Fleig E, Diehl E. Nest architecture and colony size of the fungus-growing ant Mycetophylax simplex Emery, 1888 (Formicidae, Attini). Insectes Sociaux. 2007;54(3):242–7. https://doi.org/10.1007/s00040-007-0936-7 . Klingenberg C, Brandão CRF, Engels W. Primitive nest architecture and small monogynous colonies in basal Attini inhabiting sandy beaches of southern Brazil. Stud Neotrop Fauna Environ. 2007;42(2):121–6. https://doi.org/10.1080/01650520601065509 . de Fine Licht HH, Boomsma JJ. Forage collection, substrate preparation, and diet composition in fungus-growing ants. Ecol Entomol. 2010;35(3):259–69. https://doi.org/10.1111/j.1365-2311.2010.01193.x . Murakami T, Higashi S. Social organization in two primitive attine ants, Cyphomyrmex rimosus and Myrmicocrypta ednaella , with reference to their fungus substrates and food sources. J Ethol. 1997;15(1):17–25. https://doi.org/10.1007/BF02767322 . Leal-Dutra CA, Griffith GW, Neves MA et al. Reclassification of Pterulaceae Corner (Basidiomycota: Agaricales) introducing the ant-associated genus Myrmecopterula gen. nov., Phaeopterula Henn. and the corticioid Radulomycetaceae fam. nov. IMA fungus 2020;11(1):2. https://doi.org/10.1186/s43008-019-0022-6 Fernandez-Marin H, Zimmerman JK, Wcislo WT. Ecological traits and evolutionary sequence of nest establishment in fungus-growing ants (Hymenoptera, Formicidae, Attini). Biol J Linn Soc. 2004;81(1):39–48. https://doi.org/10.1111/j.1095-8312.2004.00268.x . Kooij PW, Aanen DK, Schiøtt M, Boomsma JJ. Evolutionarily advanced ant farmers rear polyploid fungal crops. J Evol Biol. 2015;28(11):1911–24. https://doi.org/10.1111/jeb.12718 . Shik JZ, Rytter W, Arnan X, Michelsen A. Disentangling nutritional pathways linking leafcutter ants and their co-evolved fungal symbionts using stable isotopes. Ecology. 2018;99(9):1999–2009. https://doi.org/10.1002/ecy.2431 . Conlon BH, O'Tuama D, Michelsen A. A fungal symbiont converts provisioned cellulose into edible yield for its leafcutter ant farmers. Biol Lett. 2022;18(4):20220022. https://doi.org/10.1098/rsbl.2022.0022 . Leal-Dutra CA, Yuen LM, Guedes BAM, et al. Evidence that the domesticated fungus Leucoagaricus gongylophorus recycles its cytoplasmic contents as nutritional rewards to feed its leafcutter ant farmers. IMA fungus. 2023;14(1):19. https://doi.org/10.1186/s43008-023-00126-5 . Hölldobler B, Wilson EO. The Ants. Harvard University Press; 1990. De Fine Licht HH, Boomsma JJ, Tunlid A. Symbiotic adaptations in the fungal cultivar of leaf-cutting ants. Nat Commun. 2014;5(1):5675. https://doi.org/10.1038/ncomms6675 . Nygaard S, Hu H, Li C, et al. Reciprocal genomic evolution in the ant–fungus agricultural symbiosis. Nat Commun. 2016;7(1):12233. https://doi.org/10.1038/ncomms12233 . Fernández-Marín H, Zimmerman JK, Nash DR, et al. Reduced biological control and enhanced chemical pest management in the evolution of fungus farming in ants. Proc R Soc B: Biol Sci. 2009;276(1665):2263–9. https://doi.org/10.1098/rspb.2009.0184 . Barcoto MO, Silva RAP, Kitajima EW, Rodrigues A. Microbiota of attine ants' gardens: Visualizing a microbial landscape by Scanning Electron Microscopy. J Vis Exp (JoVE). 2024;212:e67334. https://doi.org/10.3791/67334 . Craven SE, Dix MW, Michaels GE. Attine fungus gardens contain yeasts. Science. 1970;169(3941):184–6. https://doi.org/10.1126/science.169.3941.184 . Grell MN, Linde T, Nygaard S, et al. The fungal symbiont of Acromyrmex leaf-cutting ants expresses the full spectrum of genes to degrade cellulose and other plant cell wall polysaccharides. BMC Genom. 2013;14(1):928. https://doi.org/10.1186/1471-2164-14-928 . Lange L, Grell MN. The prominent role of fungi and fungal enzymes in the ant–fungus biomass conversion symbiosis. Appl Microbiol Biotechnol. 2014;98(11):4839–51. https://doi.org/10.1007/s00253-014-5708-5 . Huang EL, Aylward FO, Kim YM, et al. The fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation. Environ Microbiol Rep. 2014;6(4):389–95. https://doi.org/10.1111/1758-2229.12163 . Moreira-Soto RD, Sanchez E, Currie CR, Pinto-Tomás AA. Ultrastructural and microbial analyses of cellulose degradation in leaf-cutter ant colonies. Microbiology. 2017;163(11):1578–89. https://doi.org/10.1099/mic.0.000546 . Aylward FO, Khadempour L, Tremmel DM, et al. Enrichment and broad representation of plant biomass-degrading enzymes in the specialized hyphal swellings of Leucoagaricus gongylophorus , the fungal symbiont of leaf-cutter ants. PLoS ONE. 2015;10(8):e0134752. https://doi.org/10.1371/journal.pone.0134752 . North RD, Jackson CW, Howse PE. Communication between the fungus garden and workers of the leaf-cutting ant, Atta sexdens rubropilosa , regarding choice of substrate for the fungus. Physiol Entomol. 1999;24(2):127–33. https://doi.org/10.1046/j.1365-3032.1999.00122.x . Bot AN, Currie CR, Hart AG, Boomsma JJ. Waste management in leaf-cutting ants. Ethol Ecol Evol. 2001;13(3):225–37. https://doi.org/10.1080/08927014.2001.9522772 . Meirelles LA, McFrederick QS, Rodrigues A, et al. Bacterial microbiomes from vertically transmitted fungal inocula of the leaf-cutting ant Atta texana . Environ Microbiol Rep. 2016;8(5):630–40. https://doi.org/10.1111/1758-2229.12415 . Sadowski VA, Sapountzis P, Kooij PW, et al. Bacterial communities of fungus-growing ant queens are species-specific and suggest vertical transmission. PLoS ONE. 2025;20(10):e0306011. https://doi.org/10.1371/journal.pone.0306011 . Aylward FO, Suen G, Biedermann PH et al. Convergent bacterial microbiotas in the fungal agricultural systems of insects. mBio 2014;5(6):10-1128. https://doi.org/10.1128/mbio.02077-14 Barcoto MO, Carlos-Shanley C, Fan H, et al. Fungus-growing insects host a distinctive microbiota apparently adapted to the fungiculture environment. Sci Rep. 2020;10(1):12384. https://doi.org/10.1038/s41598-020-68448-7 . Barcoto MO, de Sousa RL, Soares JGDS, et al. You are what your fungus eats: diet shapes the microbial garden of a fungus-growing ant. npj Biofilms Microbiomes. 2025;12:20. https://doi.org/10.1038/s41522-025-00876-7 . Joly CA, Martinelli LA, Alves LF, et al. As parcelas permanentes do projeto temático Biota Gradiente Funcional: composição florística, estrutura e funcionamento da floresta ombrófila densa dos Núcleos Picinguaba e Santa Virgínia do Parque Estadual da Serra do Mar. Brasil: Estado de São Paulo; 2008. pp. 109–48. Marques MC, Silva SM, Liebsch D. Coastal plain forests in southern and southeastern Brazil: ecological drivers, floristic patterns and conservation status. Brazilian J Bot. 2015;38(1):1–18. https://doi.org/10.1007/s40415-015-0132-3 . Santos RD. Mapeamentos temáticos do Parque Nacional do Itatiaia. Brasília: IBAMA/FBDS, 1998;40 – 8. Rodrigues A, Cable RN, Mueller UG, et al. Antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf-cutting ants. Antonie Van Leeuwenhoek. 2009;96(3):331–42. https://doi.org/10.1007/s10482-009-9350-7 . Feitosa RM, Dias AM. An illustrated guide for the identification of ant subfamilies and genera in Brazil. Insect Syst Evol. 2024;55(5):451–571. https://doi.org/10.1163/1876312X-bja10062 . Smith MA, Hallwachs W, Janzen DH. Diversity and phylogenetic community structure of ants along a Costa Rican elevational gradient. Ecography. 2014;37(8):720–31. https://doi.org/10.1111/j.1600-0587.2013.00631.x . Ronque MU, Azevedo-Silva M, Mori GM, et al. Three ways to distinguish species: using behavioural, ecological, and molecular data to tell apart two closely related ants, Camponotus renggeri and Camponotus rufipes (Hymenoptera: Formicidae). Zool J Linn Soc. 2016;176(1):170–81. https://doi.org/10.1111/zoj.12303 . Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 1991;10(4);506 – 13. Simon C, Frati F, Beckenbach A, et al. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am. 1994;87(6):651–701. https://doi.org/10.1093/aesa/87.6.651 . Moreau CS, Bell CD, Vila R, et al. Phylogeny of the ants: diversification in the age of angiosperms. Science. 2006;312(5770):101–4. https://doi.org/10.1126/science.1124891 . Hebert PD, Penton EH, Burns JM, et al. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator . Proc Natl Aca Sci USA. 2004;101(41):14812–7. https://doi.org/10.1073/pnas.0406166101 . Kooij PW, Dentinger BM, Donoso DA et al. Cryptic diversity in Colombian edible leaf-cutting ants (Hymenoptera: Formicidae). Insects 2018;9(4):191. https://doi.org/10.3390/insects9040191 Okonechnikov K, Golosova O, Fursov M, Ugene Team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012;28(8):1166–7. https://doi.org/10.1093/bioinformatics/bts091 . Rezende CA, De Lima MA, Maziero P, et al. Chemical and morphological characterization of sugarcane bagasse submitted to a delignification process for enhanced enzymatic digestibility. Biotechnol Biofuels. 2011;4(1):54. https://doi.org/10.1186/1754-6834-4-54 . Bernardinelli OD, Lima MA, Rezende CA, et al. Quantitative 13 C MultiCP solid-state NMR as a tool for evaluation of cellulose crystallinity index measured directly inside sugarcane biomass. Biotechnol Biofuels. 2015;8(1):110. https://doi.org/10.1186/s13068-015-0292-1 . Simmons TJ, Mortimer JC, Bernardinelli OD, et al. Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR. Nat Commun. 2016;7(1):13902. https://doi.org/10.1038/ncomms13902 . Bolyen E, Rideout JR, Dillon MR, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotech. 2019;37(8):852–7. https://doi.org/10.1038/s41587-019-0209-9 . Callahan BJ, McMurdie PJ, Rosen MJ, et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Meth. 2016;13(7):581–3. https://doi.org/10.1038/nmeth.3869 . Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2012;41(D1):D590–6. https://doi.org/10.1093/nar/gks1219 . Dhariwal A, Chong J, Habib S, et al. MicrobiomeAnalyst: a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data. Nucleic Acids Res. 2017;45(W1):W180–8. https://doi.org/10.1093/nar/gkx295 . Chong J, Liu P, Zhou G, Xia J. Using MicrobiomeAnalyst for comprehensive statistical, functional, and meta-analysis of microbiome data. Nat Protoc. 2020;15(3):799–821. https://doi.org/10.1038/s41596-019-0264-1 . Lu Y, Zhou G, Ewald J, et al. MicrobiomeAnalyst 2.0: comprehensive statistical, functional and integrative analysis of microbiome data. Nucleic Acids Res. 2023;51(W1). https://doi.org/10.1093/nar/gkad407 . W310-8. Eichi VR, Tyerman SD, Wirthensohn MG. Water relations and mesophyll anatomy in almond leaves. J Am Soc Hortic Sci. 2014;139(6):627–33. https://doi.org/10.21273/JASHS.139.6.627 . Borsuk AM, Roddy AB, Théroux-Rancourt G, Brodersen CR. Structural organization of the spongy mesophyll. New Phytol. 2022;234(3):946–60. https://doi.org/10.1111/nph.17971 . Polizel AM, Medri ME, Nakashima K, et al. Molecular, anatomical and physiological properties of a genetically modified soybean line transformed with rd29A: AtDREB1A for the improvement of drought tolerance. Genet Mol Res. 2011;10(4):3641–56. Watts S, Kariyat R. Morphological characterization of trichomes shows enormous variation in shape, density and dimensions across the leaves of 14 Solanum species. AoB Plants. 2021;13(6):plab071. https://doi.org/10.1093/aobpla/plab071 . Giuliani C, Giovanetti M, Lupi D, et al. Tools to tie: Flower characteristics, VOC emission profile, and glandular trichomes of two Mexican salvia species to attract bees. Plants. 2020;9(12):1645. https://doi.org/10.3390/plants9121645 . Lentfer CJ, Boyd WE, Gojak D. Hope Farm Windmill: Phytolith analysis of cereals in early colonial Australia. J Archaeol Sci. 1997;24(9):841–56. https://doi.org/10.1006/jasc.1996.0164 . George N, Antony A, Ramachandran T, et al. Microscopic investigations of silicification and lignification suggest their coexistence in tracheary phytoliths in date fruits ( Phoenix dactylifera L). Front Plant Sci. 2020;11:977. https://doi.org/10.3389/fpls.2020.00977 . Peritore-Galve FC, Tancos MA, Smart CD. Bacterial canker of tomato: revisiting a global and economically damaging seedborne pathogen. Plant Dis. 2021;105(6):1581–95. https://doi.org/10.1094/PDIS-08-20-1732-FE . Xu T, Li Z, Bao S, Su Y, et al. Xylem vessel type and structure influence the water transport characteristics of. Panax notoginseng PLoS One. 2023;18(3):e0281080. https://doi.org/10.1371/journal.pone.0281080 . Guimond CM, Andrews PK, Lang GA. Scanning electron microscopy of floral initiation in sweet cherry. J Am Soc Hortic Sci. 1998;123(4):509–12. Gotelli MM, Galati BG, Zarlavsky G. Pollen development and anther morphology in 14 species of Rhamnaceae. Plant Syst Evol. 2016;302(10):1433–44. https://doi.org/10.1007/s00606-016-1342-2 . Polowick PL, Sawhney VK. A scanning electron microscopic study on the initiation and development of floral organs of Brassica napus (cv. Westar). Am J Bot. 1986;73(2):254–63. https://doi.org/10.1002/j.1537-2197.1986.tb08528.x . Keijzer CJ, Hoek IHS, Willeemse MTM. The processes of anther dehiscence and pollen dispersal III. The dehydration of the filament tip and the anther in three monocotyledonous species. New Phytol. 1987;106(2):281–7. https://doi.org/10.1111/j.14698137.1987.tb00143.x . Ermolaev A, Mardini M, Buravkov S, et al. A simple and user-friendly method for high-quality preparation of pollen grains for Scanning Electron Microscopy (SEM). Plants. 2024;13(15):2140. https://doi.org/10.3390/plants13152140 . Foster ZS, Sharpton TJ, Grünwald NJ, Metacoder. An R package for visualization and manipulation of community taxonomic diversity data. PLoS Comput Biol. 2017;13(2):e1005404. https://doi.org/10.1371/journal.pcbi.1005404 . Von Humboldt A, Bonpland A. Essai sur la géographie des plantes. Chez Levrault, Schoell et Campagnie, Libraries, Paris, 1805. Wimpenny JW. Spatial order in microbial ecosystems. Biol Rev. 1981;56(3):295–342. https://doi.org/10.1111/j.1469-185X.1981.tb00352.x . Martiny JBH, Bohannan BJ, Brown JH, et al. Microbial biogeography: putting microorganisms on the map. Nat Rev Microbiol. 2006;4(2):102–12. https://doi.org/10.1038/nrmicro1341 . McCallum G, Tropini C. The gut microbiota and its biogeography. Nat Rev Microbiol. 2024;22(2):105–. https://doi.org/10.1038/s41579-023-00969-0 . 18. Dejea CM, Wick EC, Hechenbleikner EM, et al. Microbiota organization is a distinct feature of proximal colorectal cancers. Proc Natl Aca Sci USA. 2014;111(51):18321–26. https://doi.org/10.1073/pnas.1406199111 . Welch JLM, Rossetti BJ, Rieken CW, et al. Biogeography of a human oral microbiome at the micron scale. Proc Natl Aca Sci USA. 2016;113(6):E791–800. https://doi.org/10.1073/pnas.1522149113 . Kim D, Barraza JP, Arthur RA, et al. Spatial mapping of polymicrobial communities reveals a precise biogeography associated with human dental caries. Proc Natl Aca Sci USA. 2020;117(22):12375–86. https://doi.org/10.1073/pnas.1919099117 . Lamont RJ, Hajishengallis G, Koo H. Social networking at the microbiome-host interface. Infect Immun. 2023;91(9):e00124–23. https://doi.org/10.1128/iai.00124-23 . Adade EE, Al Lakhen K, Lemus AA, Valm AM. Recent progress in analyzing the spatial structure of the human microbiome: Distinguishing biogeography and architecture in the oral and gut communities. Curr Opin Endocr Metab Res. 2021;18:275–83. https://doi.org/10.1016/j.coemr.2021.04.005 . Azimi S, Lewin GR, Whiteley M. The biogeography of infection revisited. Nat Rev Microbiol. 2022;20(10):579–. https://doi.org/10.1038/s41579-022-00683-3 . 92. Shik JZ, Kooij PW, Donoso DA, et al. Nutritional niches reveal fundamental domestication trade-offs in fungus-farming ants. Nat Ecol Evol. 2021;5(1):122–34. https://doi.org/10.1038/s41559-020-01314-x . Richards-Perhatch VM, Boshers E, Greenwold M, Seal JN. Forage preference in two geographically co-occurring fungus gardening ants: A dietary DNA approach. PLoS ONE. 2025;20(12):e0336150. https://doi.org/10.1371/journal.pone.0336150 . Storr GM. Microscopic analysis of faeces, a technique for ascertaining the diet of herbivorous, mammals. Aust J Biol Sci. 1961;14(1):157–64. https://doi.org/10.1071/BI9610157 . Stewart DRM. Analysis of plant epidermis in faeces: a technique for studying the food preferences of grazing herbivores. J Appl Ecol. 1967;4(1):83–111. https://doi.org/10.2307/2401411 . Stewart DRM, Stewart J. Food preference data by faecal analysis for African plains ungulates. Afr Zool. 1970;5(1):115–29. https://hdl.handle.net/10520/AJA00445096_238 . Wirth R, Herz H, Ryel RJ, Beyschlag W, Hölldobler B. Herbivory of leaf-cutting ants: a case study on Atta colombica in the tropical rainforest of Panama. Springer; 2003. Leal IR, Wirth R, Tabarelli M. The multiple impacts of leaf-cutting ants and their novel ecological role in human‐modified neotropical forests. Biotropica. 2014;46(5):516–28. https://doi.org/10.1111/btp.12126 . Yadav S. Lignin biosynthesis in plants and its role in anther dehiscence in Brassicaceae. Botanica. 2018;68:48–59. Borghi M, Takeda-Kimura Y. Synthesis and physiological function of lignin in the reproductive structures of angiosperms. Phytochem Rev. 2025;24(4):2539–51. https://doi.org/10.1007/s11101-024-10043-z . Leopold EB, Nickmann R, Hedges JI, Ertel JR. Pollen and lignin records of late Quaternary vegetation, Lake Washington. Science. 1982;218(4579):1305–7. https://doi.org/10.1126/science.218.4579.1305 . Hu FS, Hedges JI, Gordon ES, Brubaker LB. Lignin biomarkers and pollen in postglacial sediments of an Alaskan lake. Geochim Cosmochim Acta. 1999;63(9):1421–30. https://doi.org/10.1016/S0016-7037(99)00100-3 . Sharma RK, Arora DS. Fungal degradation of lignocellulosic residues: an aspect of improved nutritive quality. Crit Rev Microbiol. 2015;41(1):52–60. https://doi.org/10.3109/1040841X.2013.791247 . Wang Y, Luo Y, Luo L, et al. Enhancement of the nutritional value of fermented corn stover as ruminant feed using the fungi Pleurotus spp. Sci Rep. 2021;11(1):11961. https://doi.org/10.1038/s41598-021-90236-0 . Moller IE, De Fine Licht HH, Harholt J, et al. The dynamics of plant cell-wall polysaccharide decomposition in leaf-cutting ant fungus gardens. PLoS ONE. 2011;6(3):e17506. https://doi.org/10.1371/journal.pone.0017506 . Somera AF, Lima AM, dos Santos-Neto ÁJ, et al. Leaf-cutter ant fungus gardens are biphasic mixed microbial bioreactors that convert plant biomass to polyols with biotechnological applications. Appl Environ Microbiol. 2015;81(13):4525–35. https://doi.org/10.1128/AEM.00046-15 . Abril AB, Bucher EH. Evidence that the fungus cultured by leaf-cutting ants does not metabolize cellulose. Ecol Lett. 2002;5(3):325–8. https://doi.org/10.1046/j.1461-0248.2002.00327.x . Abril AB, Bucher EH. Nutritional sources of the fungus cultured by leaf-cutting ants. Appl Soil Ecol. 2004;26(3):243–. https://doi.org/10.1016/j.apsoil.2003.12.008 . 47. Aylward FO, Burnum-Johnson KE, Tringe SG, et al. Leucoagaricus gongylophorus produces diverse enzymes for the degradation of recalcitrant plant polymers in leaf-cutter ant fungus gardens. Appl Environ Microbiol. 2013;79(12):3770–8. https://doi.org/10.1128/AEM.03833-12 . Suen G, Scott JJ, Aylward FO, et al. An insect herbivore microbiome with high plant biomass-degrading capacity. PLoS Gen. 2010;6(9):e1001129. https://doi.org/10.1371/journal.pgen.1001129 . Nagamoto NS, Garcia MG, Forti LC, et al. Microscopic evidence supports the hypothesis of high cellulose degradation capacity by the symbiotic fungus of leaf-cutting ants. J Biol Research-Thessaloniki. 2011;16:308–12. Vigueras G, Paredes-Hernández D, Revah S, et al. Growth and enzymatic activity of Leucoagaricus gongylophorus , a mutualistic fungus isolated from the leaf‐cutting ant Atta mexicana , on cellulose and lignocellulosic biomass. Lett Appl Microbiol. 2017;65(2):173–81. https://doi.org/10.1111/lam.12759 . DeMilto AM, Rouquette M Jr, Mueller UG, et al. Effects of substrate, ant and fungal species on plant fiber degradation in a fungus-gardening ant symbiosis. J Insect Physiol. 2017;98:301–8. https://doi.org/10.1016/j.jinsphys.2017.02.001 . Lena GD, Patroni E, Quaglia GB. Improving the nutritional value of wheat bran by a white-rot fungus. Int J Food Sci Technol. 1997;32(6):513–9. https://doi.org/10.1111/j.1365-2621.1997.tb02125.x . Donaldson GP, Lee SM, Mazmanian SK. Gut biogeography of the bacterial microbiota. Nat Rev Microbiol. 2016;14(1):20–32. https://doi.org/10.1038/nrmicro3552 . Deehan EC, Duar RM, Armet AM, et al. Modulation of the gastrointestinal microbiome with nondigestible fermentable carbohydrates to improve human health. Microbiol Spectr. 2017;5(5):10–1128. https://doi.org/10.1128/microbiolspec.bad-0019-2017 . Veličković M, Wu R, Gao Y, et al. Mapping microhabitats of lignocellulose decomposition by a microbial consortium. Nat Chem Biol. 2024;20(8):1033–43. https://doi.org/10.1038/s41589-023-01536-7 . Dobritsa AP, Samadpour M. Transfer of eleven species of the genus Burkholderia to the genus Paraburkholderia and proposal of Caballeronia gen. nov. to accommodate twelve species of the genera Burkholderia and Paraburkholderia . Int J Syst Evol Microbiol. 2016;66(8):2836–46. https://doi.org/10.1099/ijsem.0.001065 . Bugg TD, Ahmad M, Hardiman EM, Singh R. The emerging role for bacteria in lignin degradation and bio-product formation. Curr Opin Biotechnol. 2011;22(3):394–400. https://doi.org/10.1016/j.copbio.2010.10.009 . Akita H, Kimura ZI, Yusoff MZM, et al. Identification and characterization of Burkholderia multivorans CCA53. BMC Res Notes. 2017;10(1):249. https://doi.org/10.1186/s13104-017-2565-1 . Eilmus S, Heil M. Bacterial associates of arboreal ants and their putative functions in an obligate ant-plant mutualism. Appl Environ Microbiol. 2009;75(13):4324–32. https://doi.org/10.1128/AEM.00455-09 . Kaltenpoth M, Flórez LV. Versatile and dynamic symbioses between insects and Burkholderia bacteria. Annu Rev Entomol. 2020;65(1):145–70. https://doi.org/10.1146/annurev-ento-011019-025025 . Kellner K, Ishak HD, Linksvayer TA, Mueller UG. Bacterial community composition and diversity in an ancestral ant fungus symbiosis. FEMS Microbiol Ecol. 2015;91(7):fiv073. https://doi.org/10.1093/femsec/fiv073 . Santos AV, Dillon RJ, Dillon VM, et al. Ocurrence of the antibiotic producing bacterium Burkholderia sp. in colonies of the leaf-cutting ant Atta sexdens rubropilosa . FEMS Microbiol Lett. 2004;239(2):319–23. https://doi.org/10.1016/j.femsle.2004.09.005 . Francoeur CB, May DS, Thairu MW, et al. Burkholderia from fungus gardens of fungus-growing ants produces antifungals that inhibit the specialized parasite Escovopsis . Appl Environ Microbiol. 2021;87(14):e00178–21. https://doi.org/10.1128/AEM.00178-21 . Green EA, Klepacki I, Klassen JL. Isolation and characterization of mollicute symbionts from a fungus-growing ant reveals high niche overlap leading to co-exclusion. mBio. 2025;16(7):e00893–25. https://doi.org/10.1128/mbio.00893-25 . Ramalho MDO, Martins C, Morini MSC, Bueno OC. What can the bacterial community of Atta sexdens (Linnaeus, 1758) tell us about the habitats in which this ant species evolves? Insects 2020;11(6):332. https://doi.org/10.3390/insects11060332 Zani RDOA, Ferro M, Bacci M Jr. Three phylogenetically distinct and culturable diazotrophs are perennial symbionts of leaf-cutting ants. Ecol Evol. 2021;11(24):17686–99. https://doi.org/10.1002/ece3.8213 . Martin MM, Martin JS. The presence of protease activity in the rectal fluid of primitive attine ants. J Insect Physiol. 1971;17(10):1897–906. https://doi.org/10.1016/0022-1910(71)90134-X . Boyd ND, Martin MM. Faecal proteinases of the fungus-growing ant, Atta texana : their fungal origin and ecological significance. J Insect Physiol. 1975;21(11):1815–20. https://doi.org/10.1016/0022-1910(75)90247-4 . De Fine Licht HH, Schiøtt M, Rogowska-Wrzesinska A, et al. Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts. Proc Natl Aca Sci USA. 2013;110(2):583–7. https://doi.org/10.1073/pnas.1212709110 . Kooij PW, Rogowska-Wrzesinska A, Hoffmann D, et al. Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate. ISME J. 2014;8(5):1032–40. https://doi.org/10.1038/ismej.2013.231 . Kooij PW, Pullens JW, Boomsma JJ, Schiøtt M. Ant mediated redistribution of a xyloglucanase enzyme in fungus gardens of Acromyrmex echinatior . BMC Microbiol. 2016;16(1):81. https://doi.org/10.1186/s12866-016-0697-4 . Schiøtt M, Boomsma JJ. Proteomics reveals synergy between biomass degrading enzymes and inorganic Fenton chemistry in leaf-cutting ant colonies. eLlife. 2021;10:e61816. https://doi.org/10.7554/eLife.61816 . Currie CR, Stuart AE. Weeding and grooming of pathogens in agriculture by ants. Proc R Soc Lond B Biol Sci. 2001;268(1471):1033–9. https://doi.org/10.1098/rspb.2001.1605 . Yek SH, Boomsma JJ, Poulsen M. Towards a better understanding of the evolution of specialized parasites of fungus-growing ant crops. Psyche J Entom. 2012;2012(1):239392. https://doi.org/10.1155/2012/239392 . Bae IH, Kim H, Kim SM, Lee YH. Multi-meta-omics reveal unique symbiotic synchronization between ectomycorrhizal fungus and soil microbiome in Tricholoma matsutake habitat. Microbiome. 2026;14(1):23. https://doi.org/10.1186/s40168-025-02292-7 . Yoo S, Seo CW, Lim YW. Functionally distinct core microbes of Tricholoma matsutake revealed by cross-study analysis. Microbiome. 2026;14:58. https://doi.org/10.1186/s40168-025-02329-x . Warmink JA, Van Elsas JD. Migratory response of soil bacteria to Lyophyllum sp. strain Karsten in soil microcosms. Appl Environ Microbiol. 2009;75(9):2820–30. https://doi.org/10.1128/AEM.02110-08 . Nazir R, Warmink JA, Voordes DC, et al. Inhibition of mushroom formation and induction of glycerol release - Ecological strategies of Burkholderia terrae BS001 to create a hospitable niche at the fungus Lyophyllum sp. strain Karsten. Microb Ecol. 2013;65(1):245–54. https://doi.org/10.1007/s00248-012-0100-4 . Stopnisek N, Zühlke D, Carlier A, et al. Molecular mechanisms underlying the close association between soil Burkholderia and fungi. ISME J. 2016;10(1):253–64. https://doi.org/10.1038/ismej.2015.73 . Martiarena MJS, Deveau A, Montoya QV, et al. The hyphosphere of leaf-cutting ant cultivars is enriched with helper bacteria. Microb Ecol. 2023;86(3):1773–88. https://doi.org/10.1007/s00248-023-02187-w . Chen M, He T, Wu Q, et al. Enhanced heterotrophic nitrification and aerobic denitrification performance of Glutamicibacter arilaitensis EM-H8 with different carbon sources. Chemosphere. 2023;323:138266. https://doi.org/10.1016/j.chemosphere.2023.138266 . Kumari S, Naraian R. Enhanced growth and yield of oyster mushroom by growth-promoting bacteria Glutamicibacter arilaitensis MRC119. J Basic Microbiol. 2021;61(1):45–54. https://doi.org/10.1002/jobm.202000379 . Cheng J, Ye K, Fu C et al. Comprehensive assessment of rice bran dietary fiber on gut microbiota composition and metabolism during in vitro fermentation. Food Res Int 2024:197:115231. https://doi.org/10.1016/j.foodres.2024.115231 Niu L, Tang T, Song L, et al. Pullulanibacillus pueri sp. nov., isolated from Pu'er tea. Int J Syst Evol Microbiol. 2015;65(Pt7):2167–71. https://doi.org/10.1099/ijs.0.000239 . Aizawa T, Sato J, Saito S, et al. An extracellular polysaccharide is involved in the aluminum tolerance of Pullulanibacillus sp. CA42, a newly isolated strain from the Chinese water chestnut growing in an actual acid sulfate soil area in Vietnam. Front Microbiol. 2023;14:1241244. https://doi.org/10.3389/fmicb.2023.1241244 . Gharechahi J, Sarikhan S, Han JL, et al. Functional and phylogenetic analyses of camel rumen microbiota associated with different lignocellulosic substrates. npj Biofilms Microbiomes. 2022;8(1):46. https://doi.org/10.1038/s41522-022-00309-9 . Tom LM, Aulitto M, Wu YW, et al. Low-abundance populations distinguish microbiome performance in plant cell wall deconstruction. Microbiome. 2022;10(1):183. https://doi.org/10.1186/s40168-022-01377-x . Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol. 2010;8(9):623–. https://doi.org/10.1038/nrmicro2415 . 33. Flemming HC, Wingender J, Szewzyk U, et al. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol. 2016;14(9):563–75. https://doi.org/10.1038/nrmicro.2016.94 . Dragoš A, Kiesewalter H, Martin M, et al. Division of labor during biofilm matrix production. Curr Biol. 2018;28(12):1903–13. https://doi.org/10.1016/j.cub.2018.04.046 . Chatterjee S, Samal B, Singh P, et al. Transition of a solitary to a biofilm community life style in bacteria: a survival strategy with division of labour. Int J Dev Biol. 2020;64:259–65. https://doi.org/10.1387/ijdb.190176sc . Flemming HC, van Hullebusch ED, Neu TR, et al. The biofilm matrix: multitasking in a shared space. Nat Rev Microbiol. 2023;21(2):70–86. https://doi.org/10.1038/s41579-022-00791-0 . Additional Declarations No competing interests reported. Supplementary Files GardenersSupplementaryEnvironMicro.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 15 May, 2026 Editor assigned by journal 30 Apr, 2026 Submission checks completed at journal 30 Apr, 2026 First submitted to journal 28 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9554856","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":632449080,"identity":"ea084e8d-1a6b-4288-adb6-518dd1b5c7ac","order_by":0,"name":"Mariana O. Barcoto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYFACHijN3gChDYjVIsHAc4BkLRIJRGrRbeA9+LmixqaOX/KN2YMPf+4wmEsfwK/F7ABfsuSZY2kSkrNzzA1ntj1jsOxLIKSFx0CyseGwhMHtHDNp3obDDAZnCDgMqMX4J1jLzTNm0n/+EKfFDGLLDR4zaQY2YrQc5kuzbDiWJjmzJ61MsrftGY9lDyEtx3sP32yoseHnZz+8TeLHnzty5jwEtDAwo3IPENSAAQ6QrGMUjIJRMAqGPwAAYQ4/sEivyQAAAAAASUVORK5CYII=","orcid":"","institution":"São Paulo State University - UNESP","correspondingAuthor":true,"prefix":"","firstName":"Mariana","middleName":"O.","lastName":"Barcoto","suffix":""},{"id":632449083,"identity":"40c6ff2c-11fb-47b7-87d5-b6f7c8f0e2a8","order_by":1,"name":"Rodrigo H. S. Garcia","email":"","orcid":"","institution":"University of São Paulo - USP","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"H. S.","lastName":"Garcia","suffix":""},{"id":632449084,"identity":"bc8c2ab8-4b10-468f-8ea2-d0fa9c2488b4","order_by":2,"name":"Gabriel G. P. Pennachioni","email":"","orcid":"","institution":"São Paulo State University - UNESP","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"G. P.","lastName":"Pennachioni","suffix":""},{"id":632449085,"identity":"b275f80b-cb33-4fae-b812-9bda53861dba","order_by":3,"name":"João G. S. Soares","email":"","orcid":"","institution":"University of São Paulo - USP","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"G. S.","lastName":"Soares","suffix":""},{"id":632449091,"identity":"7eebaeda-aa8b-4f62-8f64-933d0f659baa","order_by":4,"name":"Eduardo R. deAzevedo","email":"","orcid":"","institution":"University of São Paulo - USP","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"R.","lastName":"deAzevedo","suffix":""},{"id":632449097,"identity":"d1064fea-5159-40b0-8f2d-a5ff67f34f36","order_by":5,"name":"Lucas W. Mendes","email":"","orcid":"","institution":"University of São Paulo - USP","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"W.","lastName":"Mendes","suffix":""},{"id":632449101,"identity":"d072f9d3-0d8f-49da-ab9e-458c8663afda","order_by":6,"name":"Alessandra I. Coan","email":"","orcid":"","institution":"São Paulo State University - UNESP","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"I.","lastName":"Coan","suffix":""},{"id":632449102,"identity":"c4ef80ef-2505-47eb-805f-1685889b6e42","order_by":7,"name":"Mauricio Bacci Jr","email":"","orcid":"","institution":"São Paulo State University - UNESP","correspondingAuthor":false,"prefix":"","firstName":"Mauricio","middleName":"","lastName":"Bacci","suffix":"Jr"},{"id":632449103,"identity":"35a2a7e6-217d-41e1-91ca-b695816c352d","order_by":8,"name":"Andre Rodrigues","email":"","orcid":"","institution":"São Paulo State University - UNESP","correspondingAuthor":false,"prefix":"","firstName":"Andre","middleName":"","lastName":"Rodrigues","suffix":""}],"badges":[],"createdAt":"2026-04-28 13:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9554856/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9554856/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108494028,"identity":"e11232af-ef2e-4a5f-add7-a1f166be6221","added_by":"auto","created_at":"2026-05-05 10:02:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1993912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAttine microbial garden. A)\u003c/strong\u003e Schematic illustration of the gradient of plant decomposition that characterizes leaf-cutting ants, the most well-known so far. The longitudinal continuum of substrate degradation forms the nutritional patches known as young and old garden, and waste piles. \u003cstrong\u003eB) \u003c/strong\u003e\u003cem\u003eAtta sexdens\u003c/em\u003e microbial garden, yet incipient (we estimate it was two to three months old, with possibly the first generation of workers), maintained by the queen. As evidenced by the squared sites in the scanning electron microscopy images, the bacterial community is already present at this early stage of the garden life, forming biofilms over and amidst the fungal hyphae.\u003cstrong\u003e C)\u003c/strong\u003e We sampled young and old garden regions, as well as waste material from lower (\u003cem\u003eApterostigma \u003c/em\u003esp.,\u003cem\u003e Mycetophylax \u003c/em\u003esp.,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eMycocepurus \u003c/em\u003esp.), higher (\u003cem\u003eMycetomoellerius \u003c/em\u003esp.),\u003cem\u003e \u003c/em\u003eand leaf-cutting (\u003cem\u003eAcromyrmex coronatus \u003c/em\u003eand \u003cem\u003eAtta sexdens\u003c/em\u003e) attines\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1Intro.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9554856/v1/d1f36a54678bd0b0a293835c.jpg"},{"id":108494182,"identity":"75b78837-65af-441f-9551-ee26093e0a6b","added_by":"auto","created_at":"2026-05-05 10:02:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2112332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFood items in microbial gardens. \u003c/strong\u003eSEM allowed the identification of some substrates in fungiculture systems \u003cstrong\u003eA)\u003c/strong\u003e Schematic illustration of substrates with features that assist their identification, with respective examples of SEM images. \u003cstrong\u003eB) \u003c/strong\u003eRelative frequency in which substrate features were observed in SEM analysis, gathered as heatmaps for each fungiculture. Illustrations by Mariana Barcoto.\u003c/p\u003e","description":"","filename":"Fig2SEM.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9554856/v1/a7044beb0353fe2b5f36d65b.jpg"},{"id":108483939,"identity":"747207c7-6b5d-4060-8cf8-0cc3058d7349","added_by":"auto","created_at":"2026-05-05 08:33:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGarden lignocellulosic composition of fungiculture systems.\u003c/strong\u003e \u003csup\u003e13\u003c/sup\u003eC ssNMR was employed to distinguish the lignocellulosic profile of young and old gardens, as well as the waste. Fungiculture systems vary in lignocellulosic composition, since attine species exhibit distinct foraging patterns. Signals in different spectral regions suggest the degradation of lignocellulosic components, accompanied by the accumulation of aliphatic byproducts. These changes generally progress from the young garden to the waste, forming a continuum of lignocellulosic degradation. Spectral regions associated with polysaccharides (cellulose and hemicellulose), lignin, and aliphatic compounds are indicated by colored horizontal bars along the spectra. The relative contributions of these components were estimated by integrating the spectral areas within these regions and normalizing by the total spectral area. These results are shown as inset bar graphs for each sample, with the lighter-toned color representing the aliphatic components, the middle-toned color representing lignin, and the dark-toned color representing polysaccharides. \u003cstrong\u003eA)\u003c/strong\u003e \u003cem\u003eMycocepurus\u003c/em\u003e sp. (lower attine); \u003cstrong\u003eB)\u003c/strong\u003e \u003cem\u003eApterostigma\u003c/em\u003e sp. (lower attine); \u003cstrong\u003eC)\u003c/strong\u003e \u003cem\u003eMycetophylax\u003c/em\u003e sp. (lower attine); \u003cstrong\u003eD)\u003c/strong\u003e \u003cem\u003eMycetomoellerius\u003c/em\u003e sp. (higher attine); \u003cstrong\u003eE)\u003c/strong\u003e \u003cem\u003eAcromyrmex coronatus\u003c/em\u003e (leaf-cutting attine); and \u003cstrong\u003eF) \u003c/strong\u003e\u003cem\u003eAtta sexdens\u003c/em\u003e (leaf-cutting attine).\u003c/p\u003e","description":"","filename":"Fig3NMR.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9554856/v1/2f70dd2eb31d9d9e8864ff5f.jpg"},{"id":108494019,"identity":"ccb47937-c0c1-400a-9d8b-013b685d5f91","added_by":"auto","created_at":"2026-05-05 10:02:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":957213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAttine fungiculture systems, except for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtta sexdens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, have a similar bacterial composition. A) \u003c/strong\u003eRelative abundance (%) of bacterial genera (depicting only genera with relative abundance \u0026gt; 1%), for the different attine fungiculture systems. \u003cstrong\u003eB) \u003c/strong\u003eAlpha diversity indices at the genus level based on the Shannon index. Pairwise comparisons were carried out using Mann-Whitney/Kruskal-Wallis, with the Wilcoxon as post hoc test, estimating no significant differences between fungiculture systems in each garden region and waste (other diversity metrics are presented at the Supplementary Material). \u003cstrong\u003eC) \u003c/strong\u003eBeta diversity estimated with Principal Coordinates Analysis (PCoA) ordination, based on Bray-Curtis distances. Statistical significance of data distribution and pairwise comparisons, estimated by PERMANOVA, found no significant differences between fungiculture systems in each garden region and waste. Both alpha- and beta diversity were calculated on the total number of genera.\u003c/p\u003e","description":"","filename":"Fig416S.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9554856/v1/d36d5614162766909166733d.jpg"},{"id":108483941,"identity":"26dd4403-11f1-4268-8028-f46648fcf786","added_by":"auto","created_at":"2026-05-05 08:33:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2254405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDissecting the fungiculture spatial organization. A) \u003c/strong\u003eFeatures used to describe the garden structure, fungal crop, microbiota, and biofilm spatial distribution across attine fungiculture systems, based on SEM imaging. Such features were schematically illustrated by the side of respective SEM images where they were identified. \u003cstrong\u003eB) \u003c/strong\u003eEach feature was categorized as 0 to 5, from not observed at all to extremely common. The estimated abundance in those features, spotted by SEM analysis, were gathered as heatmaps for each attine fungiculture, by garden region.\u003c/p\u003e","description":"","filename":"Fig5Biofilm.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9554856/v1/bc6aaf49ac8f9481264cc0b8.jpg"},{"id":108804155,"identity":"35c8f0dd-a18e-4290-a88a-1a5b25e75e98","added_by":"auto","created_at":"2026-05-08 15:16:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8497845,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9554856/v1/ec99aa0d-c110-4387-87d5-e59db338798b.pdf"},{"id":108483936,"identity":"9d0d324d-a77a-457a-8876-4629bbd2ce37","added_by":"auto","created_at":"2026-05-05 08:33:57","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":28482478,"visible":true,"origin":"","legend":"","description":"","filename":"GardenersSupplementaryEnvironMicro.docx","url":"https://assets-eu.researchsquare.com/files/rs-9554856/v1/0e31ea93bf2c82b664c2a527.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The microbial garden of fungus-growing ants: Distinct lignocellulosic profile and spatial structure, yet a similar microbiota","fulltext":[{"header":"Background","content":"\u003cp\u003eFrom insects [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] to humans [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], a wide diversity of animals feed on fungi. These include gastropod mollusks [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], reptiles [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], birds [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], terrestrial and arboreal mammals [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], all of them benefiting from fungi\u0026rsquo;s nutritional content [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Fungi are enriched in protein, dietary fiber, ergosterol, minerals (such as sulphur, iron, zinc, sodium, copper, selenium, manganese, calcium, phosphorus), vitamins (e.g., B2 and vitamin D), and all the essential amino acids and polyunsaturated fatty acids [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Mycophagy spectrum varies from obligate to accidental fungal consumption, depending on how dependent the animal is on fungi as food resources [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Fungus-feeding behavior is globally widespread [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and occurs either seasonally (when other resources are scarce) or year-round [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, among the many mycophagous animals, obligatory fungal cultivation was recognized only in fungus-growing insects: macrotermitine termites, attine ants, platypodinae, and scolytinae beetles. These insects independently evolved mechanisms that allow advanced practices for fungal crop cultivation, known as fungiculture. In advanced fungiculture systems, insects inoculate, cultivate, harvest, and vector fungi, establishing a nutritional dependence [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdvanced fungal cultivation by ants evolved once in the subtribe Attina (Hymenoptera: Formicidae: Myrmicinae: Attini, the \u0026ldquo;attine\u0026rdquo; ants), an event linked to one of the astronomical catastrophes of Earth\u0026rsquo;s evolution [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. When the Chicxulub asteroid collided with the current site of the Yucat\u0026aacute;n Peninsula about 66\u0026nbsp;million years ago, the impact energy of about 10\u003csup\u003e23\u003c/sup\u003e joules had several environmental and paleontological consequences [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. With gigatons of asteroid and Earth\u0026rsquo;s material being ejected at more than five kilometers per second, a dust cloud took only hours to cover the planet\u0026rsquo;s surface with soot sulfate aerosols. Such a dusty atmosphere blocked sunlight, cooled the planet, and interrupted photosynthesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although this event caused mass-mortality at the Cretaceous-Paleogene limits, some life forms were able to thrive. Fungi, by consuming the organic matter from dead animals and plants, obtain energy independently of light, thus peaking at the period [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Serendipitously at the Cretaceous-Paleogene limits (~\u0026thinsp;66.65\u0026thinsp;\u0026plusmn;\u0026thinsp;13.28\u0026nbsp;million years ago), somewhere in a South American rainforest, ant fungiculture arose. Earth ecosystems, back then, favored a symbiotic lifestyle in which ants foraged for organic matter, brought it to the fungus, which digested it, providing labile nutrients to the ants. Ant-fungus partners surpassed the catastrophic conditions, coevolved, diversified into 20 genera, 247 extant species, and radiated (primarily) through the Neotropics [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll attines are obligate fungal farmers, though they have evolved different fungiculture practices and have achieved different scales. Four phylogenetically related groups of attine cultivate four groups of basidiomycetous fungi, thus four fungiculture systems are recognized: the lower, the yeast, the coral, and the higher (of which ramifies the leaf-cutting) fungicultures. Lower fungicultures are the ancestral system, closest to the early stages of fungal cultivation. \u0026ldquo;Lower attines\u0026rdquo;, a paraphyletic group of 85 attine species distributed in 11 genera, cultivate fungal species in the genera \u003cem\u003eLeucocoprinus\u003c/em\u003e and \u003cem\u003eLeucoagaricus\u003c/em\u003e (\u003cem\u003eBasidiomycota: Agaricales: Agaricaceae: Leucocoprineae\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]). They inhabit small and slow-growing colonies, with one or multiple queens and a few hundred tiny monomorphic workers. These nourish the fungal crop using dry and dead plant parts, floral parts, seeds, husks, insect frass, and carcasses, then take their own nutrients from fungal mycelium. Considered not fully domesticated, their cultivars are thought to be able to sustain a free-living, detached from the symbiosis with ants [\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the yeast fungiculture, fungal species in the tribe \u003cem\u003eLeucocoprineae\u003c/em\u003e grow in yeast-like phase when associated with a clade of 19 species in the \u003cem\u003eCyphomyrmex rimosus\u003c/em\u003e group. Workers are thought to forage for nectar and sap, regurgitating these fluids for nourishing the crop [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The coral fungiculture is the only system in which the fungal crop does not belong to \u003cem\u003eAgaricaceae\u003c/em\u003e; instead, the clade of 30 species in the \u003cem\u003eApterostigma pilosum\u003c/em\u003e group cultivate species in the coral-fungus genus \u003cem\u003eMyrmecopterula\u003c/em\u003e (\u003cem\u003eBasidiomycota: Agaricales: Pterulaceae\u003c/em\u003e) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Ants in the \u003cem\u003epilosum\u003c/em\u003e group use decaying wood, plant debris, flowers, seeds, husks, and feces to nourish their fungal crop [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Higher fungiculture is the derived system where agaricaceous fungal crops were not yet observed living apart from the 113 ant species that cultivate them using diverse plant parts [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Fungal crops are multinucleate or polyploid [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]; they form vacuolated swollen cells named gongylidia, which store metabolites from autophagic processes for recycling cellular contents. Filling the gongylidia, a fluid enriched in essential amino acids, lipids, free sugars, polysaccharides, and other macromolecules, becomes available to \u0026ldquo;higher attines\u0026rdquo; [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. A subgroup of 52 higher attine species underwent a major transition, enabling them to cut fresh leaves as food for their fungal crop, which subsequently evolved into one of the main herbivores in the Neotropics. Achieving one of the highest complexities in animal society, the leaf-cutting fungiculture is often regarded as a fifth type of fungiculture. Most leaf-cutting ants cultivate \u003cem\u003eL. gongylophorus\u003c/em\u003e, a highly domesticated fungus with nutritional and morphological adaptations for its symbiotic life with ants [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLeaf-cutting ants of the genera \u003cem\u003eAtta\u003c/em\u003e and \u003cem\u003eAcromyrmex\u003c/em\u003e represent the most extensively studied fungiculture systems [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and consequently, current knowledge of fungus garden structure and metabolism is biased toward these taxa. When metabolizing the substrate, the fungus forms the \u0026ldquo;microbial garden\u0026rdquo;: an intertwined mesh of hyphae scaffolded by the substrate reminiscences, together with a microbial community that colonizes both the hyphae and the substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]). The garden structure then follows a longitudinal continuum of substrate degradation and nutritional patches [\u003cspan additionalcitationids=\"CR48 CR49 CR50 CR51\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Sites where the substrate has been recently added, i.e., \u0026ldquo;young gardens\u0026rdquo;, tend to be darker, where the substrate is relatively uncolonized by fungal hyphae and microbiota. As soon as the substrate is taken and consumed by the crop and microbiota, hyphal abundance increases, and the garden achieves clearer tones of beige-brown, which is characteristic of \u0026ldquo;old gardens\u0026rdquo;. In higher attines, a whitish \u0026ldquo;central garden\u0026rdquo; enriched in gongylidia may be found between the young and old regions [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Garden portions with no nutritional value to the ants are removed to \u0026ldquo;waste\u0026rdquo; dumps, in which contaminated garden pieces, harmful substrates, and dead ants are piled up [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. A microbial garden comes into existence when a queen-to-be leaves her parental ant colony, carrying a garden piece inside a pouch of her pharynx, known as the infrabuccal pocket [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. From queen to queen, the fungal crop lineage continues, which is the main way we know the fungus reproduces [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Carried from the parental nest, the first garden piece brings, together with the fungal crop, the garden microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Exhibiting characteristic taxonomic and functional profiles, the microbiota appears to be convergently adapted to the fungiculture environment, distinguishing it from other symbiotic systems [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdded substrates form the framework of the garden spatial structure, shaping fungal growth patterns and microbiota establishment [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In gardens of the leaf-cutting \u003cem\u003eAtta sexdens\u003c/em\u003e, distinct sets of substrates not only changed the lignocellulosic profile, but also the microbiota composition, the fungal crop development, the biofilm spatial distribution, and even colony survivorship [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Since distinct fungiculture systems are nourished by substrates of distinct nature, including plant parts and insect frass/carcasses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], we hypothesized that the garden chemical profile, microbiota composition, and spatial structure would differ according to the substrate. To tackle this query, we sampled young and old garden regions, as well as waste material from different fungiculture systems across the attine phylogeny (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC): i) Lower fungiculture, represented by the ant species \u003cem\u003eApterostigma\u003c/em\u003e sp., \u003cem\u003eMycetophylax\u003c/em\u003e sp., and \u003cem\u003eMycocepurus\u003c/em\u003e sp.; ii) Higher fungiculture, represented by the species \u003cem\u003eMycetomoellerius\u003c/em\u003e sp.; and iii) Leaf-cutting fungiculture, represented by the species \u003cem\u003eAcromyrmex coronatus\u003c/em\u003e and \u003cem\u003eAt. sexdens.\u003c/em\u003e Then, we analyzed the substrate morphology and the microbiota spatial distribution via Scanning Electron Microscopy (SEM). The garden chemical profile was determined using \u003csup\u003e13\u003c/sup\u003eC Solid-state Nuclear Magnetic Resonance (\u003csup\u003e13\u003c/sup\u003eC ssNMR), and the microbiota taxonomic composition was investigated by 16S rRNA sequencing. Besides providing further details on the substrates foraged by attine ants and the chemical composition of their garden, our findings shed light on the microbiota composition and biofilm spatial organization across different garden regions, under distinct fungiculture practices.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eColonies sampling and maintenance\u003c/h2\u003e \u003cp\u003eAttine colonies were collected in Atlantic rainforest areas at Parque Estadual Serra do Mar, N\u0026uacute;cleo Picinguaba (S23\u0026deg;21\u0026rsquo; W44\u0026deg;49\u0026rsquo;), Ubatuba-SP, between April 18\u0026ndash;22, 2023, and November 24\u0026ndash;28, 2023. Fungus-growing ants were sampled both at lowland ombrophile forests (characterized by clay-sandy soils, and a canopy of trees higher than 20 meters, epiphytes and lianas), and at coastal plain forest (\u0026ldquo;restinga\u0026rdquo;, with a canopy of trees and shrubs, and an understory of ground herbs and shrubs [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]). Field collections were also carried out in rainforest areas at Parque Nacional do Itatiaia (S22\u0026deg;27'; W44\u0026deg;36'), Itatiaia-RJ, between January 12\u0026ndash;16, 2024. Colonies were sampled in highland ombrophile forests with continuous or partially interrupted canopy (originated naturally or anthropogenically [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]). \u003cem\u003eMycocepurus\u003c/em\u003e sp., \u003cem\u003eMycetophylax\u003c/em\u003e sp. (at the \u0026ldquo;restinga\u0026rdquo;), \u003cem\u003eMycetomoellerius\u003c/em\u003e sp., \u003cem\u003eAcromyrmex\u003c/em\u003e sp., and \u003cem\u003eAtta\u003c/em\u003e sp. colonies were collected by excavating a trench surrounding the nest area until exposing the garden chamber [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Colonies of \u003cem\u003eApterostigma\u003c/em\u003e sp. and \u003cem\u003eMycetophylax\u003c/em\u003e sp. (at the highland rainforest) were collected by carefully revolving the litter and breaking decomposing trunks. We collected three colonies per attine species (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Garden and waste fragments were aseptically collected in sterilized recipients until further processing. Whenever possible, young and old regions of the garden were set apart, as well as the waste. We used entomological forceps for manually removing workers, eggs, pupae, and larvae from young and old gardens for \u003csup\u003e13\u003c/sup\u003eC ssNMR, metabarcoding, and SEM analyses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnt workers taxonomic identification\u003c/h3\u003e\n\u003cp\u003eAnts were identified both by morphology (following Feitosa and Dias [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]) and by sequencing the Cytochrome Oxidase I mitochondrial gene (\u003cem\u003eCOI\u003c/em\u003e), a DNA barcoding locus able to tell apart ant species [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. For \u003cem\u003eCOI\u003c/em\u003e sequencing, genomic DNA was extracted from attine workers kept in 96% ethanol sampling vouchers, by boiling ants\u0026rsquo; legs and/or thorax in 150 \u0026micro;L of 10% Chelex X-100 (Bio-Rad, Hercules, 1422822) at 55\u0026deg;C for 30 minutes, then at 99\u0026deg;C for 60 minutes [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Primers Jerry [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] and Ben [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] were used to amplify a COI region of about 400bp, while the primers LEP-F1 and LEP-R1 [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] were employed for amplifying a COI region of about 700bp, totaling a region of ~\u0026thinsp;1100pb [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. PCR amplifications were prepared for a final volume of 25 \u0026micro;L, containing: 1.25 mM each of each dNTP (thus 4 \u0026micro;L of dNTPs), 5 \u0026micro;L of 5X buffer, 1 \u0026micro;L of BSA (1 mg mL\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), 2 \u0026micro;L of MgCl\u003csub\u003e2\u003c/sub\u003e (25 mM), 1 \u0026micro;L of each primer (10 \u0026micro;M), 0.5 \u0026micro;L of Taq polymerase (5 U \u0026micro;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), 2 \u0026micro;L of diluted genomic DNA (1:10), and 8.5 \u0026micro;L of sterile ultrapure water. Jerry/Ben amplification cycle included: initial denaturation at 95\u0026deg;C for 5 min, 25 cycles of denaturation at 95\u0026deg;C for 30s, annealing at 55\u0026deg;C for 45s, extension at 72\u0026deg;C for 1 min, then 20 cycles of denaturation at 95\u0026deg;C for 30s, annealing at 55\u0026deg;C for 45s, extension at 72\u0026deg;C for 1 min and a final extension step at 72\u0026deg;C for 10 min. LEP amplification was carried out as follows: 94\u0026ordm;C for 1min, six cycles of [94\u0026ordm;C for 1 min; 45\u0026ordm;C for 1:30 min], 36 cycles of [72\u0026ordm;C for 1:15min; 94\u0026ordm;C for 1 min; 51\u0026ordm;C for 1:30min; 72\u0026ordm;C for 1:15 min]; then 72\u0026ordm; for 5min [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Amplicons were purified using FastAP thermosensitive alkaline phosphatase (Thermo Scientific), following the manufacturer\u0026rsquo;s protocol. Forward and reverse sequences were produced on an ABI3500 Sequencer (Life Technologies), with consensus sequences being assembled with Unipro UGENE v.52.0 [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Contigs matching at the genus level were searched in NCBI-GenBank through BLASTn, and these contigs are deposited in GenBank.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e13\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eC Solid-state Nuclear Magnetic Resonance (\u003c/b\u003e \u003csup\u003e \u003cb\u003e13\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eC ssNMR)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor being highly sensitive to modifications in chemical composition and changes in local microstructures [\u003cspan additionalcitationids=\"CR75\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], \u003csup\u003e13\u003c/sup\u003eC ssNMR can tell apart garden regions and waste, as well as lignocellulosic variations in response to ants foraging [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Since lower attine colonies did not yield enough biomass for \u003csup\u003e13\u003c/sup\u003eC ssNMR analysis, this was carried out using pooled gardens and waste samples. Colonies' samples were pooled by attine species (n\u0026thinsp;=\u0026thinsp;3 colonies per species) and region (young, old, waste), resulting in 18 samples (3 regions \u0026times; 6 attine species). Samples were freeze-dried and analyzed using a Bruker Avance 400 spectrometer, equipped with a Bruker 4-mm MAS double-resonance probe head, at \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e1\u003c/sup\u003eH frequencies of 100.5 MHz and 400.0 MHz, respectively. We followed the literature for attributing typical spectra of lignocellulosic material to polysaccharide and lignin peaks [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. High-resolution \u003csup\u003e13\u003c/sup\u003eC solid-state NMR spectra were acquired using \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC cross-polarization excitation, under magic angle spinning (MAS) and high-power \u003csup\u003e1\u003c/sup\u003eH decoupling (\u003csup\u003e13\u003c/sup\u003eC-CPMAS). Experimental parameters included: cross-polarization time of 1 ms; recycle delay of 2 s; \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC pulse lengths of 3.3 \u0026micro;s and 4.0 \u0026micro;s, respectively; \u003csup\u003e1\u003c/sup\u003eH decoupling amplitude of gB\u003csub\u003e1\u003c/sub\u003e/2π\u0026thinsp;=\u0026thinsp;70 kHz (SPINAL-64 pulse scheme).\u003c/p\u003e \u003cp\u003eThe \u003csup\u003e13\u003c/sup\u003eC-CPMAS method is not considered quantitative on the absolute number of chemical components, because of its sensitivity to local mobility and abundance of \u003csup\u003e1\u003c/sup\u003eH around the \u003csup\u003e13\u003c/sup\u003eC nuclei. When samples have a similar overall composition and identical parameters are used for acquiring signals, normalizing the spectra by the total spectral intensity (spectral area) may reveal changes in components. By directly comparing intensities within the same spectral regions, i.e., changes in the intensity profile, changes in the relative amounts of each type of component may be identified. In this work, the procedure consisted of calculating the area of selected spectral regions associated with different sample components and normalizing them by the total spectral area. All measurements were performed using identical acquisition parameters.\u003c/p\u003e\n\u003ch3\u003eDNA extraction, sequencing, and diversity analyses\u003c/h3\u003e\n\u003cp\u003eThe microbial taxonomic composition was analyzed for three colonies per attine species (following the protocols from Barcoto et al. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]). Separated by regions, the total DNA of 58 samples (3 colonies x 3 regions x 6 diets\u0026thinsp;+\u0026thinsp;one extra colony of \u003cem\u003eAtta\u003c/em\u003e sp.) was extracted using the PowerLyzer PowerSoil DNA isolation kit (Qiagen). DNA concentration was quantified by a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific). Sequencing was carried out on an Illumina HiSeq 2500 platform, generating 2 \u0026times; 100 bp (200 cycles) paired-end reads, with at least 100,000 sequences per sample at Novogene Corporation Inc. For the bacterial community, the region v3-v4 of 16S rRNA was amplified using the primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT), generating amplicons of approximately 450\u0026ndash;550 base pairs. Sequences were preprocessed using QUIIME 2 (v2024.5.0) [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Demultiplexing and quality control checking were performed by DADA2 [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], employing consensus methods for filtering out chimeric sequences and low-quality ones (\u0026lt;\u0026thinsp;q20). 16S rRNA sequencing yielded approximately 9,429,000 reads, with an average of 162,566 per sample. Sequences were later rarefied to 92,000 based on the lowest number of sequences per sample, with singletons and doubletons being removed. Taxonomic classification of the 16S rRNA regions was carried out using the SILVA Database (version 138), with 97% of similarity [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. ASVs assigned as \u0026ldquo;mitochondria\u0026rdquo; and \u0026ldquo;chloroplasts\u0026rdquo; were removed. Taxonomic profiling and diversity analysis were carried out in MicrobiomeAnalyst [\u003cspan additionalcitationids=\"CR81\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e], both by the webserver and by the R package MicrobiomeAnalystR. Features with low count (minimum count\u0026thinsp;=\u0026thinsp;4, prevalence in samples\u0026thinsp;=\u0026thinsp;10%) and low variance (10%, based on inter-quantile range) were filtered out, and the data were normalized to total sum scaling. Relative abundance was determined at the phylum, class, and genus levels. Heat tree analysis to depict taxonomic differences between microbial communities was performed at the genus and species level, using the non-parametric Wilcoxon Rank- Sum test (p-value cutoff\u0026thinsp;=\u0026thinsp;0.05). Alpha-diversity was calculated at the genus level, for the following diversity metrics: observed features (richness), Shannon, Simpson, and Fisher, using Mann-Whitney/Kruskal-Wallis and Wilcoxon test for \u003cem\u003epost hoc\u003c/em\u003e pairwise comparisons. Beta-Diversity was calculated at the genus level, using Principal Coordinates Analysis (PCoA) as ordination method, based on Bray-Curtis distances; statistical significance of data distribution and pairwise comparisons were defined by PERMANOVA. \u003cem\u003eP\u003c/em\u003e-values were corrected by the Benjamini\u0026ndash;Hochberg method, and a false discovery rate (FDR)\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003ch3\u003eScanning Electron Microscopy (SEM)\u003c/h3\u003e\n\u003cp\u003eSamples were pooled as described for \u003csup\u003e13\u003c/sup\u003eC NMR analysis and prepared for imaging acquisition as formerly described [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Briefly, samples were fixed for at minimum 24 hours in Karnovsky solution (2.5% glutaraldehyde and 2% paraformaldehyde in 0.05 M cacodylate buffer, pH 7.2), maintained at 4\u0026deg;C. Fixed samples were dehydrated by ethanol gradual washing series (30%, 50%, 70%, 90%, and three times in 100%) and critical point-dried (EM CPD 300). Fragments of dried samples, no larger than 1 mm\u003csup\u003e3\u003c/sup\u003e in size, were mounted in aluminum stubs, adding up to nine fragments per stub, which were sputter-coated with colloidal gold (Baltec SCD 050). Images were visualized and digitally registered using a JEOL IT300 SEM. We recorded about 15\u0026ndash;25 images per sample, ranging from the following magnifications: i) 100x-700x to observe hyphal density, substrate visual deterioration, and colonization patterns); ii) 700x-1,500x to detail microbial spatial organization; iii) 1,500x- 3,000x to describe physical interactions (in particular, biofilm structure); iv) 3,000x-4,000x to focus on informative microbial assemblages. The resulting images were visually analyzed to morphologically identify substrate structures and to characterize garden and waste features. These features included: 1) Presence of coiled and thick hyphae; 2) Presence of clamp connections; 3) Hyphal density; 4) Presence and abundance of gongylidia; 5) Gongylidia microbiota; 6) Hyphae microbiota; 7) Spread biofilm; 8) 3D biofilm; 9) Biofilm structure; 10) Yeasts and/or conidia; 11) Degree of substrate deterioration. Each feature was categorized, according to its approximate frequency, as: 0 - not observed; 1 - extremely rare (spotted once or a few times); 2 - observed in low abundance; 3 - observed in moderate abundance; 4 - observed in abundance; 5 - extremely common.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlant parts are dominant substrates in attine microbial gardens\u003c/h2\u003e \u003cp\u003eBy employing SEM imaging, we identified substrates\u0026rsquo; microstructures in a three-dimensional perspective, allowing us to hypothesize on their importance for the garden spatial structure. These substrates were pre-processed by the ants and already decayed when added to the garden, which, along with microbial overgrowth, narrows the substrate identification at a fine level. Surpassing these limitations, visually analyzing the resulting 1144 SEM images indicates that plant-derived fragments account for most incorporated substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Leaf parts, discerned by mesophyll, epidermal surface remnants, stomata, and trichomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA [\u003cspan additionalcitationids=\"CR85 CR86\" citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]), were observed in all attine gardens, but predominantly in \u003cem\u003eMycetomoellerius\u003c/em\u003e sp. and \u003cem\u003eAt. sexdens\u003c/em\u003e gardens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Silica phytoliths, distinguished as narrow long, cylindrical long, spherical and spheroid echinate phytoliths (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]), were slightly more abundant in \u003cem\u003eApterostigma\u003c/em\u003e sp. and \u003cem\u003eAt. sexdens\u003c/em\u003e gardens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Vessel elements, spotted by annular thickenings and pitted thickening walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA; [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]), were noticed in all gardens, though in larger proportions in \u003cem\u003eMycetomoellerius\u003c/em\u003e sp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Floral buds and anthetic floral parts, distinguished by dehiscent anthers, endothecial thickening remnants, and pollen grains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA [\u003cspan additionalcitationids=\"CR93 CR94 CR95\" citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]) were more frequently observed in gardens of \u003cem\u003eApterostigma\u003c/em\u003e sp. and \u003cem\u003eMycocepurus\u003c/em\u003e sp., and in the waste of \u003cem\u003eMycetophylax\u003c/em\u003e sp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. In summary, SEM images reaffirm that leaves are a common item in the diet of higher and leaf-cutting attines, while flower parts are customarily foraged by lower attines. Curiously, insect carcasses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) were only found in \u003cem\u003eMycetophylax\u003c/em\u003e sp. gardens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), as this would also be expected in gardens of other lower attines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAttine microbial gardens form a longitudinal continuum of lignocellulosic degradation\u003c/h3\u003e\n\u003cp\u003eMirroring the abundance of plant-derived fragments as substrates, the chemical profile revealed by \u003csup\u003e13\u003c/sup\u003eC ssNMR indicates the microbial garden exhibiting a lignocellulosic nature. This is evidenced by the typical spectral profile of lignocellulosic materials, in which different spectral regions exhibit predominant contributions from polysaccharides (mainly cellulose and hemicellulose) and lignin, along with additional aliphatic signal contributions (as indicated by horizontal bars along the spectra in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, colored by each of these components). Due to the strong spectral overlap typical of \u003csup\u003e13\u003c/sup\u003eC ssNMR of lignocellulosic materials, trends were evaluated by calculating spectral areas, normalized by the total spectral area, within regions dominated by polysaccharides (cellulose and hemicellulose), lignin, and aliphatic components. These values are shown as inset bar graphs in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, where the lighter-toned color accounts for the aliphatic components, the middle-toned color to lignin, and the dark-toned color to the polysaccharides. Comparison across samples provided an estimate of the relative abundance of each component. Overall, the signals across the spectral regions suggest a slight degradation of lignocellulosic polysaccharide components, along with the accumulation of their byproducts in the form of aliphatic compounds, progressively increasing from the young garden to the waste samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eYoung gardens tend to display similar lignocellulosic compositions, with polysaccharides as the dominant fraction, with \u003cem\u003eApterostigma\u003c/em\u003e sp., and to a lesser extent, \u003cem\u003eAcromyrmex coronatus\u003c/em\u003e, exhibiting higher lignin content. Specifically, signals in the 0\u0026ndash;45 ppm region, associated with aliphatic compounds derived from lipids and proteins, show a gradual increase. In contrast, signals in the aromatic (110\u0026ndash;160 ppm) and methoxy (~\u0026thinsp;56 ppm) regions, predominantly attributed to lignin, do not exhibit a clear trend from the young garden to the waste. Some of these signals remain relatively intense in the waste, suggesting incomplete lignin metabolism across the garden. Signals attributed to cellulose and hemicellulose, spanning the 60\u0026ndash;110 ppm and 160\u0026ndash;200 ppm regions, show a general tendency to decrease from the young garden to the waste, being markedly reduced in most cases. This pattern was consistently found in gardens of \u003cem\u003eMycocepurus\u003c/em\u003e sp., \u003cem\u003eMycetomoellerius\u003c/em\u003e sp., \u003cem\u003eAcromyrmex coronatus\u003c/em\u003e, and \u003cem\u003eAtta sexdens\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The lower attine \u003cem\u003eApterostigma\u003c/em\u003e sp. is the main exception, since lignin signals are characteristically high in young gardens, along with higher peaks to hemicelluloses (101.8 ppm and 172 ppm, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Waste is chemically similar to old gardens, except for a higher content of aliphatic compounds and a decrease in polysaccharide signals, while lignin remains relatively prominent. Another distinct pattern is observed for the lower attine \u003cem\u003eMycetophylax\u003c/em\u003e sp., where young and old gardens exhibit very similar spectral features. In this case, the waste shows only a slight decrease in polysaccharide signals accompanied by an increase in aliphatic signals and relatively persistent lignin-related contributions, suggesting that these components are only partially degraded within the garden and tend to accumulate in the waste (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Concisely, we observed distinct fungiculture systems varying in lignocellulosic composition, reflecting distinct foraging patterns of attine species. From different plant species and plant organs, discrete lignocellulose profiles give rise to continuums of degradation in the microbial garden, in a way that each fungiculture is a unique system for biomass conversion. Lignocellulose modification is achieved to a greater extent in gardens of lower fungiculture systems than in the higher and leaf-cutting ones.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAll attine gardens, but\u003c/b\u003e \u003cb\u003eAtta sexdens\u003c/b\u003e, \u003cb\u003eshare a similar taxonomic composition\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBacterial communities were investigated by amplicon sequencing of 16S rRNA genes, which yielded 27,528 amplicon sequence variants (ASVs) assigned to \u003cem\u003eBacteria\u003c/em\u003e. In brief, the microbiota assembles spatiotemporally along the degradation continuum: young and old regions are similarly rich and diverse; the waste, otherwise, is the richest and most diverse region (Supplementary material, \u003cb\u003eFigures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S3\u003c/b\u003e). The bacterial phyla \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eBacteroidota\u003c/em\u003e are abundant in the garden and in the waste, though varying in proportions by ant species (\u003cb\u003eFigure S4\u003c/b\u003e). The bacterial genus \u003cem\u003eBurkholderia\u003c/em\u003e tends to be more abundant in lower attine gardens, and \u003cem\u003eMesoplasma\u003c/em\u003e is more abundant in higher and leaf-cutting gardens, though \u003cem\u003eBurkholderia\u003c/em\u003e becomes abundant in the waste of almost all attine systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In consonance, \u003cem\u003eBurkholderia / Paraburkholderia / Cabaleronia\u003c/em\u003e integrate the core microbiota of lower and higher attines gardens, only becoming part of the \u003cem\u003eA. sexdens\u003c/em\u003e (but not of the \u003cem\u003eAc. coronatus\u003c/em\u003e) core microbiota in waste samples (\u003cb\u003eFigure S5-S7\u003c/b\u003e). Biomarker determination based on linear discriminant analysis effect size (LEfSe) reveals that, except for \u003cem\u003eMycetophylax\u003c/em\u003e sp., \u003cem\u003eBurkholderia\u003c/em\u003e is a biomarker of lower and higher fungicultures, while \u003cem\u003eMesoplasma\u003c/em\u003e is a signature of leaf-cutting systems (\u003cb\u003eFigure S8\u003c/b\u003e). By comparing distinct garden regions and attine fungicultures, we identified the microbiota of \u003cem\u003eAt. sexdens\u003c/em\u003e as the only differing from other attine species, characteristically having several bacterial genera in lower abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eFigures S9-S24\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBacterial genera differentially abundant in each attine fungiculture and garden regions, as determined by heat tree analysis [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e] comparing group-wise relative abundances (Wilcoxon test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Some abundant genera are shared between attine species (indicated by letters representing the fungiculture which the genera are shared with: \u003cem\u003eAp Apterostigma\u003c/em\u003e, \u003cem\u003eMp Mycocepurus\u003c/em\u003e, \u003cem\u003eMc Mycetophylax\u003c/em\u003e, \u003cem\u003eMm Mycetomoellerius\u003c/em\u003e, \u003cem\u003eAc Acromyrmex coronatus\u003c/em\u003e, \u003cem\u003eAt Atta sexdens\u003c/em\u003e). Genus-level heat trees denote only \u003cem\u003eAt. sexdens\u003c/em\u003e fungiculture differing significantly from other attine, as \u003cem\u003eAt. sexdens\u003c/em\u003e have diverse genera at lower abundance (\u003cb\u003eFigures S10-S24\u003c/b\u003e). Thus, the bacterial genera listed here are those whose abundance significantly differs from \u003cem\u003eAt. sexdens\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eMycocepurus\u003c/b\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYoung garden\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOld garden\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWaste\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eButyricicoccus\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Ap, Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eDuganella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMarmoricola\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePaludicola\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eParaclostridium\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudonocardia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc, Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRobinsoniella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eSolirubrobacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTumebacillus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAcrocarpospora\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAnaerocolumna\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eCatenibacillus\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eDuganella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eNocardia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Ap)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePaludicola\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudonocardia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRaoultibacter\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTumebacillus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTsukamurella\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHyphomicrobium\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc, Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eLabrys\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eSolirubrobacter\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eStreptomyces\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTerrimicrobium\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eApterostigma\u003c/b\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAcidibacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAcidothermus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAkkermansia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eButyricicoccus\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp, Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eClostridium sensu stricto\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePaucilactobacillus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRoseiarcus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRuminococcus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAcidibacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAcidipila-Silvibacterium\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAcidothermus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eConexibacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eEdaphobacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eHumibacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eInquilinus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMycobacterium\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eNocardia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhodanobacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRoseiarcus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eSolirubrobacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTerracidiphilus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHumibacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhodanobacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhodomicrobium\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTerriglobus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMycetophylax\u003c/b\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAkkermansia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Ap)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAnaerocolumna\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAnaerofustis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAnaerovorax\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eCatenibacillus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eClostridium methylpentosum\u003c/em\u003e group \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eClostridium sensu stricto\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eEubacterium coprostanoligenes\u003c/em\u003e group \u003csup\u003e\u003cem\u003e(Mm\u003c/em\u003e)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eFriedmanniella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eJatrophihabitans\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm, Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eOdoribacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePaludicola\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudonocardia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp, Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRaoultibacter\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRobinsoniella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eSerratia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eYokenella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAkkermansia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAnaerovorax\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eCatenibacillus\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMethylocella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudonocardia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRuminococcus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAfipia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAmaricoccus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eBauldia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eBordetella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eCatenulispora\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eCellvibrio\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eDongia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eHephaestia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eHylemonella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eHyphomicrobium\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp, Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eJatrophihabitans\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eLabrys\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eLuteimonas\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMarmoricola\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMethylocella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMesorhizobium\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMumia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eNordella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePanacagrimonas\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eParapedobacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePigmentiphaga\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudaminobacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudoduganella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudohongiella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudolabrys\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudonocardia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhizobacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhodopila\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhodoplanes\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eSaccharothrix\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eSolirubrobacter\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp, Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eSporichthya\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eStreptomyces\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTerriglobus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eTerrimicrobium\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eThermomonas\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMycetomoellerius\u003c/b\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAmycolatopsis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eButyricicoccus\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp,Ap)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eClostridium methylpentosum\u003c/em\u003e group \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eDuganella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eEubacterium coprostanoligenes\u003c/em\u003e group \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eFriedmanniella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eHerbaspirillum\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eJatrophihabitans\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc, Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMethylocella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRaoultibacter\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRugosimonospora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eActinomycetospora\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAureimonas\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eButyricicoccus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eClostridium methylpentosum\u003c/em\u003e group\u003c/p\u003e \u003cp\u003e\u003cem\u003eDuganella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eEubacterium coprostanoligenes\u003c/em\u003e group\u003c/p\u003e \u003cp\u003e\u003cem\u003eFriedmanniella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eJatrophihabitans\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eLuedemannella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePaludicola\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRaoultibacter\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAmycolatopsis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAquicella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eBauldia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eCiceribacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eDactylosporangium Duganella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eEdaphobacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eFriedmanniella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eHerbaspirillum\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eHyphomicrobium\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp, Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eJatrophihabitans\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc, Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eLegionella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMumia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eNocardia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eNordella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc, Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePigmentiphaga\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudonocardia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRalstonia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhodomicrobium\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Ap, Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhodoplanes\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRugosimonospora\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eSolirubrobacter\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp, Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eStreptosporangium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcromyrmex coronatus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAfipia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eGlutamicibacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eJatrophihabitans\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc, Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eNakamurella\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eOttowia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePatulibacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003ePseudonocardia\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mp, Mc)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAureimonas\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eGlutamicibacter\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eJatrophihabitans\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eKlenkia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eMethylocella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eJatrophihabitans\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc, Ac)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eLegionella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eLuteipulveratus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eNordella\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Mc, Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eOttowia\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhodoblastus\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRhodomicrobium\u003c/em\u003e \u003csup\u003e\u003cem\u003e(Ap, Mm)\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eRoseiarcus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn young gardens, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eActinobacteriota\u003c/em\u003e are the dominant phyla, predominantly in the class \u003cem\u003eGammaproteobacteria\u003c/em\u003e, \u003cem\u003eBacilli\u003c/em\u003e, and \u003cem\u003eAlphaproteobacteria\u003c/em\u003e (\u003cb\u003eFigure S25)\u003c/b\u003e. At the genus level, most young gardens\u0026rsquo; ASVs (10\u0026ndash;60%) were not assigned or assigned to less abundant genera (gathered as \u0026ldquo;others\u0026rdquo;; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In \u003cem\u003eMycocepurus\u003c/em\u003e sp., assigned ASVs were mostly attributed to \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eBurkholderia\u003c/em\u003e; in \u003cem\u003eApterostigma\u003c/em\u003e sp., to \u003cem\u003eBurkholderia;\u003c/em\u003e in \u003cem\u003eMycetophylax\u003c/em\u003e sp., to \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eTyzzerella\u003c/em\u003e; in \u003cem\u003eMycetomoellerius\u003c/em\u003e sp., to \u003cem\u003eMesoplasma\u003c/em\u003e, \u003cem\u003ePullulanibacillus\u003c/em\u003e, and \u003cem\u003eBurkholderia\u003c/em\u003e; in \u003cem\u003eAc. coronatus\u003c/em\u003e to \u003cem\u003eMesoplasma\u003c/em\u003e and \u003cem\u003eGlutamicibacter\u003c/em\u003e; and in \u003cem\u003eAt. sexdens\u003c/em\u003e, to \u003cem\u003eMesoplasma\u003c/em\u003e and uncultured \u003cem\u003eLachnospiraceae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Young gardens did not differ in richness and alpha diversity indices (Wilcoxon test, FDR-adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; \u003cb\u003eFig S26\u003c/b\u003e). Beta diversity analysis based on Bray-Curtis dissimilarity indicates that the community does not vary according to attine species, yielding no differences in pairwise comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, PERMANOVA, FDR-adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Taxa contributing to the dissimilarity between young gardens include \u003cem\u003eMesoplasma\u003c/em\u003e (7.2\u0026ndash;24.56%), uncultured \u003cem\u003eLachnospiraceae\u003c/em\u003e (7.9\u0026ndash;10.83%), \u003cem\u003eGlutamicibacter\u003c/em\u003e (7.48\u0026ndash;9.85), and \u003cem\u003eBacillus\u003c/em\u003e (6.34\u0026ndash;8.74%; SIMPER analysis based on Bray\u0026ndash;Curtis, Supplementary Material).\u003c/p\u003e \u003cp\u003e \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eActinobacteriota\u003c/em\u003e continue as dominant phyla in old gardens, mainly in the class \u003cem\u003eGammaproteobacteria\u003c/em\u003e, \u003cem\u003eBacilli\u003c/em\u003e, and \u003cem\u003eClostridia\u003c/em\u003e (\u003cb\u003eFigure S27)\u003c/b\u003e. Like for the young garden, most old gardens\u0026rsquo; ASVs (10\u0026ndash;80%) were not assigned at the genus level or assigned to less abundant genera (\u0026ldquo;others\u0026rdquo;). Assigned ASVs at the genus level, in \u003cem\u003eMycocepurus\u003c/em\u003e sp., were mostly attributed to \u003cem\u003eCandidatus Soleaferrea\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003eTyzzerella\u003c/em\u003e; in \u003cem\u003eApterostigma\u003c/em\u003e sp., to \u003cem\u003eBurkholderia\u003c/em\u003e; in \u003cem\u003eMycetophylax\u003c/em\u003e sp., to \u003cem\u003eCandidatus Soleaferrea\u003c/em\u003e, and \u003cem\u003eTyzzerella\u003c/em\u003e; in \u003cem\u003eMycetomoellerius\u003c/em\u003e sp., to \u003cem\u003ePullulanibacillus\u003c/em\u003e, \u003cem\u003eBurkholderia\u003c/em\u003e, and \u003cem\u003eTyzzerella\u003c/em\u003e; in \u003cem\u003eAc\u003c/em\u003e. \u003cem\u003ecoronatus\u003c/em\u003e, to \u003cem\u003eMesoplasma\u003c/em\u003e and uncultured \u003cem\u003eLachnospiraceae\u003c/em\u003e; and in \u003cem\u003eAt. sexdens\u003c/em\u003e, to \u003cem\u003eMesoplasma\u003c/em\u003e, \u003cem\u003eBurkholderia\u003c/em\u003e, and uncultured \u003cem\u003eLachnospiraceae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Pairwise comparisons of alpha-diversity indices showed no significant differences between attine old gardens (Wilcoxon test, FDR-adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; \u003cb\u003eFig S28\u003c/b\u003e). Beta diversity analysis based on Bray-Curtis implies the community does not vary with the attine species (PERMANOVA, FDR-adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reinforced by pairwise comparisons yielding no differences between attine old gardens (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Dissimilarity between old gardens is attributed mostly to \u003cem\u003eMesoplasma\u003c/em\u003e (3.20-20.31%), \u003cem\u003ePullulanibacillus\u003c/em\u003e (16.08\u0026ndash;18.61%), \u003cem\u003eBurkholderia\u003c/em\u003e (4.34\u0026ndash;14.71%), uncultured \u003cem\u003eLachnospiraceae\u003c/em\u003e (9.02\u0026ndash;11.38%), and \u003cem\u003eCandidatus Soleaferrea\u003c/em\u003e (3.9\u0026ndash;8.15%; SIMPER analysis based on Bray\u0026ndash;Curtis, Supplementary Material).\u003c/p\u003e \u003cp\u003eIn waste samples, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eActinobacteriota\u003c/em\u003e, and \u003cem\u003eBacteroidota\u003c/em\u003e are the most abundant bacterial phyla, with the majority in the class \u003cem\u003eGammaproteobacteria\u003c/em\u003e, \u003cem\u003eAlphaproteobacteria\u003c/em\u003e, and \u003cem\u003eActinobacteria\u003c/em\u003e (\u003cb\u003eFigure S29)\u003c/b\u003e. Between 20\u0026ndash;70% of waste\u0026rsquo;s ASVs were either not assigned at the genus level or assigned to \u0026ldquo;others\u0026rdquo; (not abundant genera). In the \u003cem\u003eMycocepurus\u003c/em\u003e sp. waste, assigned ASVs at the genus level were primarily traced to \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eRhizobium\u003c/em\u003e; in the \u003cem\u003eApterostigma\u003c/em\u003e sp. waste, to \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003ePullulanibacillus\u003c/em\u003e; in the \u003cem\u003eMycetophylax\u003c/em\u003e sp. waste, to \u003cem\u003eFlavobacterium\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e; in the \u003cem\u003eMycetomoellerius\u003c/em\u003e sp. waste, to \u003cem\u003eBurkholderia\u003c/em\u003e, \u003cem\u003eRhizobium\u003c/em\u003e, and \u003cem\u003eSphingobacterium\u003c/em\u003e; in the \u003cem\u003eAc\u003c/em\u003e. \u003cem\u003ecoronatus\u003c/em\u003e waste, to \u003cem\u003euncultured Microbacteriaceae\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e, and \u003cem\u003eSphingobacterium\u003c/em\u003e; and in the waste of \u003cem\u003eAt\u003c/em\u003e. \u003cem\u003esexdens\u003c/em\u003e, to \u003cem\u003eBurkholderia\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e, and \u003cem\u003eFlavobacterium\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Pairwise comparisons of alpha-diversity indices yielded no differences between attine waste samples (Wilcoxon test, FDR-adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; \u003cb\u003eFigure S30\u003c/b\u003e). Beta-diversity analysis based on Bray-Curtis signals that the community does not vary according to attine species (PERMANOVA, FDR-adjusted \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which is supported by no differences between attine gardens obtained in pairwise comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). \u003cem\u003ePullulanibacillus\u003c/em\u003e (15.14\u0026ndash;17.72%), \u003cem\u003eBurkholderia\u003c/em\u003e (8.04\u0026ndash;12.44%), \u003cem\u003eMesoplasma\u003c/em\u003e (6.5\u0026ndash;7.43%), and \u003cem\u003eRhizobium\u003c/em\u003e (5.03\u0026ndash;6.35%) are the taxa contributing the most to the dissimilarity between attine waste (SIMPER analysis based on Bray\u0026ndash;Curtis, Supplementary Material).\u003c/p\u003e\n\u003ch3\u003eMicrobiota and biofilms are ubiquitous components of attine fungiculture systems\u003c/h3\u003e\n\u003cp\u003eWe visually analyzed 1144 SEM images to determine garden and waste spatial organization and microbiota distribution. Each fungiculture has its own characteristic spatial structure, especially differing in hyphae morphology and growth patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Still, we found among them analogous patterns of substrate deterioration, microbiota-substrate, and microbiota-hyphae physical interactions were detected across the systems. From the young garden to the waste, it follows a progressive increase in substrate deterioration, colonization by hyphae, microbiota, and biofilm complexity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003eFigure S31\u003c/b\u003e). Accordingly, young regions tend to hold less deteriorated substrates, yet are sparsely colonized by hyphae and microbiota. Fungal hyphae frequently start colonizing the substrate at its edges, furrows, and indentations. The microbiota tends to be scattered at this point, occurring in isolated or as small communities, present at the substrate surface and interiors, also surrounding fungal hyphae. The hyphal density varies in old regions according to the fungiculture system, with hyphae often surrounded by biofilm. Gongylidia are commonly observed across the gardens of higher and leaf-cutting attines, frequently populated by the microbiota. Substrates in the old garden are more deteriorated, which is evidenced by exposed remnants of vessels, anthers, and mesophyll. Substrates in old gardens cannot be dissociated from thick microbial biofilms in their broken-down structure. In the waste, substrates are rarely identifiable due to their extensive deterioration, coupled with an exuberant biofilm. There, a multi-morphological microbial community, embedded in a matrix, appears to engage in physical interactions constantly. It is worthy pointing out that differences in young and old garden regions are much more evident in leaf-cutting gardens, from which emerge a \u0026ldquo;longitudinally stratified\u0026rdquo; garden. In lower attines and even in the higher attine \u003cem\u003eMycetomoellerius\u003c/em\u003e sp., the longitudinal patterns of substrate degradation, hyphae, and microbiota colonization are way more subtle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cb\u003eFigure S31\u003c/b\u003e). Lower attine gardens present some distinguishable features, including: gongylidia-like structures in \u003cem\u003eMycocepurus\u003c/em\u003e sp.; clamp connections characterizing the \u003cem\u003eApterostigma\u003c/em\u003e sp. fungal crop (as expected for fungi in the \u003cem\u003ePterulaceae\u003c/em\u003e family); and gardens of \u003cem\u003eMycetophylax\u003c/em\u003e sp. assembling as a set of wool balls with almost indistinguishable young and old regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cb\u003eFigure S31\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the macro scale, it is known for long that community diversity and organization patterns respond to environmental conditions [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. In the micron scale, likewise, patterns of distribution in microbial ecosystems determine much of their functioning and responses [\u003cspan additionalcitationids=\"CR100\" citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e]. Microbiota organization follows environmental cues and interactions within its neighborhood, in a way that ecological functions are, by far, correlated to changes in spatial patterning [\u003cspan additionalcitationids=\"CR103\" citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e]. This put the microbiota spatial organization side by side with the taxonomic composition in determining ecosystem properties [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. As a consequence, a deeper comprehension of ecological processes shaping ecosystems requires a better knowledge of their spatial patterns and their correlation to environmental conditions [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e, \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e, \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. Here, we investigated the architecture of the microbial garden, the microbial ecosystem housed in attine ant colonies. Using physical-chemical, sequencing, and imaging tools, we determined how nutrients, fungal crops, and microbial biofilms are spatially organized in the fungiculture systems of ant species distributed across the attine phylogeny (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These attine foraged predominantly for plant parts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), yielding microbial gardens with a lignocellulosic nature (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As the fungal crop and the microbiota breakdown and consume the substrates, a characteristic continuum of lignocellulose degradation is established [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Following this gradient, there exists a microbiota with similar taxonomic composition among all attine gardens, except for \u003cem\u003eAt. sexdens\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In fact, a biofilm-forming microbiota is an ever-present element of the microbial garden architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSo far, the composition of attine colonies\u0026rsquo; diet has been mostly described from observational data on ant foraging behavior, by recording and identifying substrate fragments acquired by the ants. Typically, these fragments are morphologically categorized before entering the colony, and include leaf and floral parts, seeds, husks, insect frass, and carcasses [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e]. Alternatively, dietary DNA (dDNA) was employed to specify the foraging choices of the higher attines \u003cem\u003eTrachymyrmex septentrionalis\u003c/em\u003e and \u003cem\u003eMycetomoellerius turrifex\u003c/em\u003e, by sequencing a region of the chloroplast \u003cem\u003etrnL\u003c/em\u003e intron and COI markers directly from the garden [\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. We followed a third route, adapting microscopical methods to ascertain the diet of herbivorous mammals [\u003cspan additionalcitationids=\"CR111\" citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e] to characterize substrates after ants had added them to the garden, that is, while being digested. Diverse plant fragments were detected as substrates in young gardens, i.e., where the substrate was recently added by ants, supporting that attine fungiculture systems assume the ecological role of herbivores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]). Although overlapping in some foraging choices, attine ants in distinct fungiculture systems tend to prefer distinct plant species and plant organs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e, \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e, \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e], yielding differences in the chemical profile of each microbial garden (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This is illustrated by floral buds and anthetic floral parts, such as anther wall remnants (endothecial thickenings) and pollen grains, being often observed in gardens of \u003cem\u003eApterostigma\u003c/em\u003e sp. and \u003cem\u003eMycocepurus\u003c/em\u003e sp., and in the waste of \u003cem\u003eMycetophylax\u003c/em\u003e sp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Layers of lignin compose mature endothecium, which is crucial for anther dehiscence and pollen release [\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e, \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e], and pollen themselves are a prominent source of lignin [\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e, \u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e]. One could expect that breaking down these lignin-enriched substrates along the garden to render chemical profiles essentially different from those of fungiculture systems fed mostly with leaf parts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegardless of the substrate with which they were nourished, attine microbial gardens tend to form a continuum of substrate degradation. From young regions, passing by old regions, until the waste, there is a progressive increase in aliphatic compounds reflecting an accumulation of lipids and proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e [\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e, \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e]). Lignin-derived compounds slightly decrease from the young garden to the waste, though, since they are still present in the waste, lignin seems to be incompletely metabolized in the garden. The fate of cellulose and hemicelluloses varied widely according to fungiculture systems: while in some of them cellulose and hemicelluloses are reduced in old gardens (and even more in the waste), in others these signals did not evidence substantial changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e, \u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e122\u003c/span\u003e]. Although previously debated [\u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e, \u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e124\u003c/span\u003e], the cellulose-degrading capacity is coded by the \u003cem\u003eL. gongylophorus\u003c/em\u003e genome [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e, \u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e], being supported by enzymatic, chemical, ultrastructural, and transcriptional data [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e, \u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e, \u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e]. By exhibiting a lignocellulolytic potential, the garden microbiota is thought to complementarily contribute to cellulose decomposition [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Thus, cellulose degradation in the leaf-cutting ants\u0026rsquo; garden (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) is possibly a fungus-microbiota teamwork. Lignocellulose modification seems to be achieved to a greater extent in gardens of lower fungiculture systems than at the higher and leaf-cutting ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which is conceivably related to reductions in the fungal lignocellulolytic capacity along the symbiosis evolution [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e]. As lignocellulosic components are modified and their end products are accumulated [\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e, \u003cspan citationid=\"CR130\" class=\"CitationRef\"\u003e130\u003c/span\u003e], a gradient of physicochemical conditions could be formed along the garden regions, and with that, a profusion of microbial niches is established [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan additionalcitationids=\"CR132\" citationid=\"CR131\" class=\"CitationRef\"\u003e131\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e133\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe microbiota follows the degradation continuum, with \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eFirmicutes\u003c/em\u003e mainly inhabiting young (\u003cb\u003eFigure S25\u003c/b\u003e) and old regions (\u003cb\u003eFigure S27\u003c/b\u003e), then \u003cem\u003eBacteroidota\u003c/em\u003e replacing \u003cem\u003eFirmicutes\u003c/em\u003e in the waste (\u003cb\u003eFigure S29\u003c/b\u003e). In lower attine gardens and the waste of almost all attine systems, \u003cem\u003eBurkholderia\u003c/em\u003e is an abundant microbial member, then considered part of the fungiculture core microbiota and a biomarker of lower and higher fungiculture systems (\u003cb\u003eFigure S5-S8\u003c/b\u003e). Our sequencing data does not allow us to infer the metabolic potential of these bacterial genera, and we do not intend to suggest putative roles for the microbiota in the garden based only on the literature. Besides, we also consider plausible the scenario where they are transiently inhabiting the garden. Keeping this in mind, knowing in which environments the abundant and characteristic genera in the microbial garden were already reported may assist future experiments targeting their function and ecological roles in attine fungiculture. In that sense, \u003cem\u003eBurkholderia\u003c/em\u003e (taxonomically revised and having some strains reassigned to \u003cem\u003eParaburkholderia\u003c/em\u003e and \u003cem\u003eCaballeronia\u003c/em\u003e [\u003cspan citationid=\"CR134\" class=\"CitationRef\"\u003e134\u003c/span\u003e]) is an environmentally widespread bacterium with lignocellulolytic capacity [\u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e135\u003c/span\u003e, \u003cspan citationid=\"CR136\" class=\"CitationRef\"\u003e136\u003c/span\u003e], also a well-known insect symbiont [\u003cspan citationid=\"CR137\" class=\"CitationRef\"\u003e137\u003c/span\u003e, \u003cspan citationid=\"CR138\" class=\"CitationRef\"\u003e138\u003c/span\u003e]. Not so abundant in gardens of the lower attine \u003cem\u003eMycocepurus smithii\u003c/em\u003e [\u003cspan citationid=\"CR139\" class=\"CitationRef\"\u003e139\u003c/span\u003e], \u003cem\u003eBurkholderia\u003c/em\u003e is commonly found in leaf-cutting attine gardens, where it is thought of as a defensive symbiont, protecting the system against fungal contaminants [\u003cspan citationid=\"CR140\" class=\"CitationRef\"\u003e140\u003c/span\u003e, \u003cspan citationid=\"CR141\" class=\"CitationRef\"\u003e141\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eMesoplasma\u003c/em\u003e, otherwise, is abundant in higher and leaf-cutting gardens, taken as a signature of leaf-cutting systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; \u003cb\u003eFigure S8\u003c/b\u003e). This common member of \u003cem\u003eAt\u003c/em\u003e. \u003cem\u003esexdens\u003c/em\u003e fungiculture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) participates in the garden response to diet, characterizing a state of diet-induced dysbiosis [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. \u003cem\u003eMesoplasma\u003c/em\u003e strains isolated from \u003cem\u003eTrachymyrmex septentrionalis\u003c/em\u003e metabolize glucose, fructose, arginine, and N-acetylglucosamine, suggestively producing ammonia for the ants [\u003cspan citationid=\"CR142\" class=\"CitationRef\"\u003e142\u003c/span\u003e]. Curiously, \u003cem\u003eMesoplasma\u003c/em\u003e was abundantly found in the \u003cem\u003eAtta\u003c/em\u003e workers\u0026rsquo; body [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR143\" class=\"CitationRef\"\u003e143\u003c/span\u003e, \u003cspan citationid=\"CR144\" class=\"CitationRef\"\u003e144\u003c/span\u003e], raising the question of whether/how the ant microbiota participates in the microbial garden assembly. Leaf-cutting ants apply fecal droplets, replete with gongylidia-produced enzymes, over the freshly added substrate as a pretreatment [\u003cspan additionalcitationids=\"CR146 CR147 CR148 CR149\" citationid=\"CR145\" class=\"CitationRef\"\u003e145\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR150\" class=\"CitationRef\"\u003e150\u003c/span\u003e]. It is yet to be investigated if this fluid is enriched in microorganisms, and if it is, whether these microbes play any role in plant decomposition. It is also to be determined whether the ants somehow shape the microbiota, directly by filtering out harmful microbes, or indirectly by selecting suitable substrates or applying antimicrobial compounds [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR151\" class=\"CitationRef\"\u003e151\u003c/span\u003e]. In this perspective, \u003cem\u003eAt\u003c/em\u003e. \u003cem\u003esexdens\u003c/em\u003e hygienic processes for garden cleaning, such as applying glandular chemical compounds and behavioral strategies for pathogen removal [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR151\" class=\"CitationRef\"\u003e151\u003c/span\u003e, \u003cspan citationid=\"CR152\" class=\"CitationRef\"\u003e152\u003c/span\u003e], could explain their garden microbiota standing out with a lower microbial abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; \u003cb\u003eFigures S9-S24\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eWith \u003cem\u003eAt. sexdens\u003c/em\u003e being an exception, all attine gardens share a similar taxonomic composition (\u003cb\u003eFigures S10-S24\u003c/b\u003e). In fact, fungus-growing insects are thought to harbor a characteristic microbiota, with marked similarities in taxonomic composition and functional profile. Such evidence for host-microbiota convergence at higher hierarchical levels (e.g., phylum and class) seems to be applied to bacterial genera as well [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. It is not clear what makes the microbiota members thrive in the fungiculture ecosystem, though some of them could be favored by an environment concomitantly enriched in plant and fungal biomass. Fungal metabolites are selective forces assembling the microbiota of the rhizosphere [\u003cspan citationid=\"CR153\" class=\"CitationRef\"\u003e153\u003c/span\u003e] and different parts of mushroom-forming fungus, possibly behind the compositional and functional convergence of mushroom microbiota as well [\u003cspan citationid=\"CR154\" class=\"CitationRef\"\u003e154\u003c/span\u003e]. From this, one may hypothesize that the fungal crop metabolism could shape the microbiota composition, especially considering that some \u003cem\u003eBurkholderia\u003c/em\u003e strains are capable of \u0026ndash; and benefit from \u0026ndash; using fungal metabolites. Besides surpassing fungal defenses, \u003cem\u003eBurkholderia\u003c/em\u003e surround fungal hyphae with biofilm, then comigrate with fungi in soil [\u003cspan additionalcitationids=\"CR156\" citationid=\"CR155\" class=\"CitationRef\"\u003e155\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR157\" class=\"CitationRef\"\u003e157\u003c/span\u003e]. On the other hand, bacteria in leaf-cutting fungiculture seem to influence the fungal crop by promoting its growth, also influencing phosphate solubilization, biofilm formation, cellulose, and chitin degradation [\u003cspan citationid=\"CR158\" class=\"CitationRef\"\u003e158\u003c/span\u003e]. Yet, fungal-microbiota interactions in microbial gardens are a research window wide open: there are diverse microbial members often found in attine gardens that are plant-associated, and future experiments could be designed to investigate their role and application possibilities. These include \u003cem\u003eGlutamicibacter\u003c/em\u003e (reported as denitrifiers and fungal-growth promoters [\u003cspan citationid=\"CR159\" class=\"CitationRef\"\u003e159\u003c/span\u003e, \u003cspan citationid=\"CR160\" class=\"CitationRef\"\u003e160\u003c/span\u003e]), \u003cem\u003eTyzzerella\u003c/em\u003e (reported to be favored by plant fiber [\u003cspan citationid=\"CR161\" class=\"CitationRef\"\u003e161\u003c/span\u003e]), and \u003cem\u003ePullulanibacillus\u003c/em\u003e (reported as a biofilm-forming bacterium [\u003cspan citationid=\"CR162\" class=\"CitationRef\"\u003e162\u003c/span\u003e, \u003cspan citationid=\"CR163\" class=\"CitationRef\"\u003e163\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eWe looked closely at the microbiota architecture by SEM, as if we were accompanying the microbial community structuring according to chemical modifications in the substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therein lies the continuum of lignocellulose degradation, in a way that each fungiculture seems to constitute a unique system for biomass conversion (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e [\u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e]). Chemical particularities possibly reflect particular metabolic processes required for substrate decomposition, yielding particular nutritional niches for the microbiota to assemble [\u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e133\u003c/span\u003e, \u003cspan citationid=\"CR164\" class=\"CitationRef\"\u003e164\u003c/span\u003e, \u003cspan citationid=\"CR165\" class=\"CitationRef\"\u003e165\u003c/span\u003e]. Our findings reinforce that the microbiota, spatially organized in biofilms, are ubiquitous components of attine fungiculture systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Through biofilms, the microbiota can cross-communicate, adhere, interact with the substrate, and avoid contacting toxins. The biofilm matrix retains lignocellulolytic enzymes, which convert the entire structure - including non-degradative members - into a digestive one [\u003cspan additionalcitationids=\"CR167 CR168 CR169\" citationid=\"CR166\" class=\"CitationRef\"\u003e166\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR170\" class=\"CitationRef\"\u003e170\u003c/span\u003e]. Whether, when, and where microbiota-fungus and microbiota-ant interactions occur, and how important they are for attine ecology and evolution, are open windows of research. Attine microbial gardens offer countless possibilities for fruitful microbial ecology research, integrating multidisciplinary approaches for unveiling such a multidimensional microbial ecosystem.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e Attine microbial gardens are primarily herbivorous, with distinct fungiculture systems varying in lignocellulosic composition according to foraging patterns, that is, select for different plant species and plant organs. Gardens form a continuum of lignocellulosic degradation, which is followed by the microbiota: \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eFirmicutes\u003c/em\u003e mainly inhabiting young and old regions, then \u003cem\u003eBacteroidota\u003c/em\u003e replaces \u003cem\u003eFirmicutes\u003c/em\u003e in the waste. In lower attine gardens and the waste of almost all attine systems, \u003cem\u003eBurkholderia\u003c/em\u003e is an abundant microbial member, considered part of the fungiculture core microbiota and a biomarker of lower and higher fungiculture systems. In higher and leaf-cutting gardens, otherwise, \u003cem\u003eMesoplasma\u003c/em\u003e is abundant, taken as a signature of leaf-cutting systems. Each attine garden constitutes a unique system for biomass conversion, in turn yielding nutritional niches for the microbiota to assemble, in the majority as biofilms.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no AI and AI-assisted technologies were used for the writing process.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Elliot Watanabe Kitajima for constructive comments on SEM analysis, Renato Barbosa Salaroli for technical assistance, Raquel Lima de Sousa for field assistance, and Manoela Ramalho, Mario Tyago Murakami, Simone Raposo Cotta, and Rodrigo Gouv\u0026ecirc;a Taketani for their constructive insights and suggestions. We would like to thank Instituto Chico Mendes de Conserva\u0026ccedil;\u0026atilde;o da Biodiversidade (ICMBio) for providing the collecting permit (# 74585). We also thank Conselho Nacional de Gest\u0026atilde;o do Patrim\u0026ocirc;nio Gen\u0026eacute;tico (CGen) for providing a permit to access the genetic heritage (#SISGen AA39A6D). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMariana de Oliveira Barcoto - \u003c/strong\u003eConceptualization, Data curation, Investigation, Methodology, Project administration, Formal analysis (metabarcoding data and SEM), Visualization, Writing \u0026ndash; original draft, and Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eRodrigo Henrique dos Santos Garcia\u003c/strong\u003e - Formal analysis (NMR data). Gabriel Giorgio Pressuto Pennachioni - Formal analysis (ant workers identification). \u003cstrong\u003eJo\u0026atilde;o Gabriel da Silva Soares\u003c/strong\u003e - Formal analysis (NMR data). \u003cstrong\u003eEduardo Ribeiro de Azevedo\u003c/strong\u003e - Formal analysis (NMR data), Resources, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eLucas William Mendes\u003c/strong\u003e - Formal analysis (metabarcoding data), Software, Resources, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eAlessandra Ike Coan\u003c/strong\u003e - Formal analysis (plant anatomy), Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eMauricio Bacci Jr\u003c/strong\u003e - Funding acquisition, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eAndre Rodrigues - \u003c/strong\u003eConceptualization, Methodology, Project administration, Supervision, Resources, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was funded by Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP, grant #2019/03746-0). MOB received PhD scholarships from FAPESP (# 2021/08013-0) and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brazil (CAPES) - Finance Code 001. AR received a research fellowship from Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq; #305269/2018). MB received a research grant from FAPESP (CEPID #21/10639-5). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDatasets supporting the conclusions of this article are available in the FigShare repository, under the Project \u0026ldquo;The most famous gardeners\u0026rdquo;. NMR spectra may be accessed \u003c/p\u003e\n\u003cp\u003eby https://doi.org/10.6084/m9.figshare.31378345, SEM images by https://doi.org/10.6084/m9.figshare.31376602. ASV sequences are available at the NCBI repository, under the BioProject ID PRJNA1430830, and COI sequences used for ants identification are under GenBank accession numbers PZ094865-PZ094893\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFieldwork was carried out with collecting permit emitted by Chico Mendes de Conserva\u0026ccedil;\u0026atilde;o da Biodiversidade (ICMBio, permit #74585). Access of genetic heritage was permitted by Conselho Nacional de Gest\u0026atilde;o do Patrim\u0026ocirc;nio Gen\u0026eacute;tico (CGen) (permit #SISGen AA39A6D).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKimura MT, Tuno N. Phylogeny, ecology, and evolution of mycophagous Drosophilidae (Diptera). Biol J Linn Soc. 2024;142(3):247\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/biolinnean/blad140\u003c/span\u003e\u003cspan address=\"10.1093/biolinnean/blad140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozel P, Lejeune B, Lepoint G, et al. Stable isotopes of saproxylic beetles reveal low differences among trophic guilds and suggest a high dependence on fungi. Ecol Monogr. 2025;95(1):e70002. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ecm.70002\u003c/span\u003e\u003cspan address=\"10.1002/ecm.70002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Regan HJ, Lamb AL, Wilkinson DM. The missing mushrooms: Searching for fungi in ancient human dietary analysis. J Archaeol Sci. 2016;75:139\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jas.2016.09.009\u003c/span\u003e\u003cspan address=\"10.1016/j.jas.2016.09.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOri F, Menotta M, Leonardi M. Effect of slug mycophagy on \u003cem\u003eTuber aestivum\u003c/em\u003e spores. Fungal Biol. 2021;125(10):796\u0026ndash;805. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.funbio.2021.05.002\u003c/span\u003e\u003cspan address=\"10.1016/j.funbio.2021.05.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarahona-Segovia RM, Gallardo‐Pillancari E, Orrego G, Osorio F. Mycophagy and feeding on other native land snails by the black snail \u003cem\u003eMacrocyclis peruvianus\u003c/em\u003e (Gastropoda, Macrocyclidae). Austral Ecol. 2024;49(2):e13491. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/aec.13491\u003c/span\u003e\u003cspan address=\"10.1111/aec.13491\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElliott TF, Jusino MA, Trappe JM, et al. A global review of the ecological significance of symbiotic associations between birds and fungi. Fungal Divers. 2019;98(1):161\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13225-019-00436-3\u003c/span\u003e\u003cspan address=\"10.1007/s13225-019-00436-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElliot TF, Bower DS, Vernes K. Reptilian Mycophagy: A global review of mutually beneficial associations between reptiles and macrofungi. Mycosphere. 2019;10(1):776\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.5943/mycosphere/10/1/18\u003c/span\u003e\u003cspan address=\"https://doi:10.5943/mycosphere/10/1/18\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCork SJ, Kenagy GJ. Nutritional value of hypogeous fungus for a forest-dwelling ground squirrel. Ecology. 1989;70(3):577\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/1940209\u003c/span\u003e\u003cspan address=\"10.2307/1940209\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaunchbaugh KL, Urness PJ. Mushroom consumption (mycophagy) by North American cervids. Great Basin Naturalist. 1992;52(4):321\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.jstor.org/stable/41712738\u003c/span\u003e\u003cspan address=\"http://www.jstor.org/stable/41712738\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClaridge AW, Trappe JM, Cork SJ, Claridge DL. Mycophagy by small mammals in the coniferous forests of North America: nutritional value of sporocarps of \u003cem\u003eRhizopogon vinicolor\u003c/em\u003e, a common hypogeous fungus. J Comp Physiol. 1999;169(3):172\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s003600050208\u003c/span\u003e\u003cspan address=\"10.1007/s003600050208\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanson AM, Hodge KT, Porter LM. Mycophagy among primates. Mycologist. 2003;17(1):6\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0269915X0300106X\u003c/span\u003e\u003cspan address=\"10.1017/S0269915X0300106X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWallis IR, Claridge AW, Trappe JM. Nitrogen content, amino acid composition and digestibility of fungi from a nutritional perspective in animal mycophagy. Fungal Biol. 2012;116(5):590\u0026ndash;602. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.funbio.2012.02.007\u003c/span\u003e\u003cspan address=\"10.1016/j.funbio.2012.02.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDerbyshire EJ, Delange J. Fungal protein\u0026ndash;what is it and what is the health evidence? A systematic review focusing on mycoprotein. Front Sustain Food Syst. 2021;5:581682. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fsufs.2021.581682\u003c/span\u003e\u003cspan address=\"10.3389/fsufs.2021.581682\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad MI, Farooq S, Alhamoud Y, et al. A review on mycoprotein: History, nutritional composition, production methods, and health benefits. Trends Food Sci Technol. 2022;121:14\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tifs.2022.01.027\u003c/span\u003e\u003cspan address=\"10.1016/j.tifs.2022.01.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClaridge AW, Trappe JM. Sporocarp mycophagy: nutritional, behavioral, evolutionary, and physiological aspects. Mycol Ser. 2005;23:599.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFogel R, Trappe JM. Fungus consumption (mycophagy) by small animals. Northwest Sci. 1978;52(1):1\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElliott TF, Truong C, Jackson SM, et al. Mammalian mycophagy: a global review of ecosystem interactions between mammals and fungi. Fungal Syst Evol. 2022;9(1):99\u0026ndash;159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3114/fuse.2022.09.07\u003c/span\u003e\u003cspan address=\"10.3114/fuse.2022.09.07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin MM. The evolution of insect-fungus associations: from contact to stable symbiosis. Am Zool. 1992;32(4):593\u0026ndash;605. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/icb/32.4.593\u003c/span\u003e\u003cspan address=\"10.1093/icb/32.4.593\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMueller UG, Gerardo NM, Aanen DK. The evolution of agriculture in insects. Annu Rev Ecol Evol Syst. 2005;36(1):563\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev.ecolsys.36.102003.152626\u003c/span\u003e\u003cspan address=\"10.1146/annurev.ecolsys.36.102003.152626\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiedermann PH, Vega FE. Ecology and evolution of insect\u0026ndash;fungus mutualisms. Annu Rev Entomol. 2020;65(1):431\u0026ndash;. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-ento-011019-024910\u003c/span\u003e\u003cspan address=\"10.1146/annurev-ento-011019-024910\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u0026thinsp;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchultz TR, Brady SG. Major evolutionary transitions in ant agriculture. PNAS. 2008;105(14):5435\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.0711024105\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0711024105\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBranstetter MG, Ješovnik A, Sosa-Calvo J, et al. Dry habitats were crucibles of domestication in the evolution of agriculture in ants. Proc Biol Sci. 2017;284(1852):20170095. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rspb.2017.0095\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2017.0095\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchultz TR, Sosa-Calvo J, Kweskin MP, et al. The coevolution of fungus-ant agriculture. Science. 2024;386(6717):105\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.adn7179\u003c/span\u003e\u003cspan address=\"10.1126/science.adn7179\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan den Bergh S. Astronomical catastrophes in Earth history. Publ Astron Soc Pac. 1994;106(701):689\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1086/133431\u003c/span\u003e\u003cspan address=\"10.1086/133431\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchulte P, Alegret L, Arenillas I, et al. The Chicxulub asteroid impact and mass extinction at the Cretaceous-Paleogene boundary. Science. 2010;327(5970):1214\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1177265\u003c/span\u003e\u003cspan address=\"10.1126/science.1177265\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorgan JV, Bralower TJ, Brugger J, W\u0026uuml;nnemann K. The Chicxulub impact and its environmental consequences. Nat Rev Earth Environ. 2022;3(5):338\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s43017-022-00283-y\u003c/span\u003e\u003cspan address=\"10.1038/s43017-022-00283-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerry K. Evidence for fungal proliferation following the Cretaceous/Paleogene mass-extinction event, based on chemostratigraphy in the Raton and Powder River basins, western North America. Acta Palaeobot. 2020;60(1):134\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.35535/acpa-2020-0005\u003c/span\u003e\u003cspan address=\"10.35535/acpa-2020-0005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehdiabadi NJ, Schultz TR. Natural history and phylogeny of the fungus-farming ants (Hymenoptera: Formicidae: Myrmicinae: Attini). Myrmecological News. 2010;13:37\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchultz TR, Gawne R, Peregrine PN. The convergent evolution of agriculture in humans and insects. MIT Press; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeber NA. The fungus-culturing behavior of ants. Am Zool. 1972;12(3):577\u0026ndash;. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/icb/12.3.577\u003c/span\u003e\u003cspan address=\"10.1093/icb/12.3.577\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u0026thinsp;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMueller UG, Rehner SA, Schultz TR. The evolution of agriculture in ants. Science. 1998;281(5385):2034\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.281.5385.2034\u003c/span\u003e\u003cspan address=\"10.1126/science.281.5385.2034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFern\u0026aacute;ndez-Mar\u0026iacute;n H, Zimmerman JK, Wcislo WT, Rehner SA. Colony foundation, nest architecture and demography of a basal fungus‐growing ant, \u003cem\u003eMycocepurus smithii\u003c/em\u003e (Hymenoptera, Formicidae). J Nat Hist. 2005;39(20):1735\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/00222930400027462\u003c/span\u003e\u003cspan address=\"10.1080/00222930400027462\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiehl-Fleig E, Diehl E. Nest architecture and colony size of the fungus-growing ant \u003cem\u003eMycetophylax simplex\u003c/em\u003e Emery, 1888 (Formicidae, Attini). Insectes Sociaux. 2007;54(3):242\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00040-007-0936-7\u003c/span\u003e\u003cspan address=\"10.1007/s00040-007-0936-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlingenberg C, Brand\u0026atilde;o CRF, Engels W. Primitive nest architecture and small monogynous colonies in basal Attini inhabiting sandy beaches of southern Brazil. Stud Neotrop Fauna Environ. 2007;42(2):121\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/01650520601065509\u003c/span\u003e\u003cspan address=\"10.1080/01650520601065509\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Fine Licht HH, Boomsma JJ. Forage collection, substrate preparation, and diet composition in fungus-growing ants. Ecol Entomol. 2010;35(3):259\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2311.2010.01193.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2311.2010.01193.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurakami T, Higashi S. Social organization in two primitive attine ants, \u003cem\u003eCyphomyrmex rimosus\u003c/em\u003e and \u003cem\u003eMyrmicocrypta ednaella\u003c/em\u003e, with reference to their fungus substrates and food sources. J Ethol. 1997;15(1):17\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF02767322\u003c/span\u003e\u003cspan address=\"10.1007/BF02767322\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeal-Dutra CA, Griffith GW, Neves MA et al. Reclassification of Pterulaceae Corner (Basidiomycota: Agaricales) introducing the ant-associated genus \u003cem\u003eMyrmecopterula\u003c/em\u003e gen. nov., \u003cem\u003ePhaeopterula\u003c/em\u003e Henn. and the corticioid \u003cem\u003eRadulomycetaceae\u003c/em\u003e fam. nov. \u003cem\u003eIMA fungus\u003c/em\u003e 2020;11(1):2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s43008-019-0022-6\u003c/span\u003e\u003cspan address=\"10.1186/s43008-019-0022-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandez-Marin H, Zimmerman JK, Wcislo WT. Ecological traits and evolutionary sequence of nest establishment in fungus-growing ants (Hymenoptera, Formicidae, Attini). Biol J Linn Soc. 2004;81(1):39\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1095-8312.2004.00268.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1095-8312.2004.00268.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKooij PW, Aanen DK, Schi\u0026oslash;tt M, Boomsma JJ. Evolutionarily advanced ant farmers rear polyploid fungal crops. J Evol Biol. 2015;28(11):1911\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jeb.12718\u003c/span\u003e\u003cspan address=\"10.1111/jeb.12718\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShik JZ, Rytter W, Arnan X, Michelsen A. Disentangling nutritional pathways linking leafcutter ants and their co-evolved fungal symbionts using stable isotopes. Ecology. 2018;99(9):1999\u0026ndash;2009. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ecy.2431\u003c/span\u003e\u003cspan address=\"10.1002/ecy.2431\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConlon BH, O'Tuama D, Michelsen A. A fungal symbiont converts provisioned cellulose into edible yield for its leafcutter ant farmers. Biol Lett. 2022;18(4):20220022. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rsbl.2022.0022\u003c/span\u003e\u003cspan address=\"10.1098/rsbl.2022.0022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeal-Dutra CA, Yuen LM, Guedes BAM, et al. Evidence that the domesticated fungus \u003cem\u003eLeucoagaricus gongylophorus\u003c/em\u003e recycles its cytoplasmic contents as nutritional rewards to feed its leafcutter ant farmers. IMA fungus. 2023;14(1):19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s43008-023-00126-5\u003c/span\u003e\u003cspan address=\"10.1186/s43008-023-00126-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH\u0026ouml;lldobler B, Wilson EO. The Ants. Harvard University Press; 1990.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Fine Licht HH, Boomsma JJ, Tunlid A. Symbiotic adaptations in the fungal cultivar of leaf-cutting ants. Nat Commun. 2014;5(1):5675. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms6675\u003c/span\u003e\u003cspan address=\"10.1038/ncomms6675\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNygaard S, Hu H, Li C, et al. Reciprocal genomic evolution in the ant\u0026ndash;fungus agricultural symbiosis. Nat Commun. 2016;7(1):12233. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms12233\u003c/span\u003e\u003cspan address=\"10.1038/ncomms12233\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFern\u0026aacute;ndez-Mar\u0026iacute;n H, Zimmerman JK, Nash DR, et al. Reduced biological control and enhanced chemical pest management in the evolution of fungus farming in ants. Proc R Soc B: Biol Sci. 2009;276(1665):2263\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rspb.2009.0184\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2009.0184\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarcoto MO, Silva RAP, Kitajima EW, Rodrigues A. Microbiota of attine ants' gardens: Visualizing a microbial landscape by Scanning Electron Microscopy. J Vis Exp (JoVE). 2024;212:e67334. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3791/67334\u003c/span\u003e\u003cspan address=\"10.3791/67334\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCraven SE, Dix MW, Michaels GE. Attine fungus gardens contain yeasts. Science. 1970;169(3941):184\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.169.3941.184\u003c/span\u003e\u003cspan address=\"10.1126/science.169.3941.184\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrell MN, Linde T, Nygaard S, et al. The fungal symbiont of \u003cem\u003eAcromyrmex\u003c/em\u003e leaf-cutting ants expresses the full spectrum of genes to degrade cellulose and other plant cell wall polysaccharides. BMC Genom. 2013;14(1):928. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1471-2164-14-928\u003c/span\u003e\u003cspan address=\"10.1186/1471-2164-14-928\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLange L, Grell MN. The prominent role of fungi and fungal enzymes in the ant\u0026ndash;fungus biomass conversion symbiosis. Appl Microbiol Biotechnol. 2014;98(11):4839\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-014-5708-5\u003c/span\u003e\u003cspan address=\"10.1007/s00253-014-5708-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang EL, Aylward FO, Kim YM, et al. The fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation. Environ Microbiol Rep. 2014;6(4):389\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1758-2229.12163\u003c/span\u003e\u003cspan address=\"10.1111/1758-2229.12163\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreira-Soto RD, Sanchez E, Currie CR, Pinto-Tom\u0026aacute;s AA. Ultrastructural and microbial analyses of cellulose degradation in leaf-cutter ant colonies. Microbiology. 2017;163(11):1578\u0026ndash;89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/mic.0.000546\u003c/span\u003e\u003cspan address=\"10.1099/mic.0.000546\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAylward FO, Khadempour L, Tremmel DM, et al. Enrichment and broad representation of plant biomass-degrading enzymes in the specialized hyphal swellings of \u003cem\u003eLeucoagaricus gongylophorus\u003c/em\u003e, the fungal symbiont of leaf-cutter ants. PLoS ONE. 2015;10(8):e0134752. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0134752\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0134752\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorth RD, Jackson CW, Howse PE. Communication between the fungus garden and workers of the leaf-cutting ant, \u003cem\u003eAtta sexdens rubropilosa\u003c/em\u003e, regarding choice of substrate for the fungus. Physiol Entomol. 1999;24(2):127\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-3032.1999.00122.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-3032.1999.00122.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBot AN, Currie CR, Hart AG, Boomsma JJ. Waste management in leaf-cutting ants. Ethol Ecol Evol. 2001;13(3):225\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/08927014.2001.9522772\u003c/span\u003e\u003cspan address=\"10.1080/08927014.2001.9522772\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeirelles LA, McFrederick QS, Rodrigues A, et al. Bacterial microbiomes from vertically transmitted fungal inocula of the leaf-cutting ant \u003cem\u003eAtta texana\u003c/em\u003e. Environ Microbiol Rep. 2016;8(5):630\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1758-2229.12415\u003c/span\u003e\u003cspan address=\"10.1111/1758-2229.12415\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadowski VA, Sapountzis P, Kooij PW, et al. Bacterial communities of fungus-growing ant queens are species-specific and suggest vertical transmission. PLoS ONE. 2025;20(10):e0306011. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0306011\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0306011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAylward FO, Suen G, Biedermann PH et al. Convergent bacterial microbiotas in the fungal agricultural systems of insects. \u003cem\u003emBio\u003c/em\u003e 2014;5(6):10-1128. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mbio.02077-14\u003c/span\u003e\u003cspan address=\"10.1128/mbio.02077-14\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarcoto MO, Carlos-Shanley C, Fan H, et al. Fungus-growing insects host a distinctive microbiota apparently adapted to the fungiculture environment. Sci Rep. 2020;10(1):12384. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-68448-7\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-68448-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarcoto MO, de Sousa RL, Soares JGDS, et al. You are what your fungus eats: diet shapes the microbial garden of a fungus-growing ant. npj Biofilms Microbiomes. 2025;12:20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41522-025-00876-7\u003c/span\u003e\u003cspan address=\"10.1038/s41522-025-00876-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoly CA, Martinelli LA, Alves LF, et al. As parcelas permanentes do projeto tem\u0026aacute;tico Biota Gradiente Funcional: composi\u0026ccedil;\u0026atilde;o flor\u0026iacute;stica, estrutura e funcionamento da floresta ombr\u0026oacute;fila densa dos N\u0026uacute;cleos Picinguaba e Santa Virg\u0026iacute;nia do Parque Estadual da Serra do Mar. Brasil: Estado de S\u0026atilde;o Paulo; 2008. pp. 109\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarques MC, Silva SM, Liebsch D. Coastal plain forests in southern and southeastern Brazil: ecological drivers, floristic patterns and conservation status. Brazilian J Bot. 2015;38(1):1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s40415-015-0132-3\u003c/span\u003e\u003cspan address=\"10.1007/s40415-015-0132-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantos RD. Mapeamentos tem\u0026aacute;ticos do Parque Nacional do Itatiaia. Bras\u0026iacute;lia: IBAMA/FBDS, 1998;40\u0026thinsp;\u0026ndash;\u0026thinsp;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodrigues A, Cable RN, Mueller UG, et al. Antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf-cutting ants. Antonie Van Leeuwenhoek. 2009;96(3):331\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10482-009-9350-7\u003c/span\u003e\u003cspan address=\"10.1007/s10482-009-9350-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeitosa RM, Dias AM. An illustrated guide for the identification of ant subfamilies and genera in Brazil. Insect Syst Evol. 2024;55(5):451\u0026ndash;571. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1163/1876312X-bja10062\u003c/span\u003e\u003cspan address=\"10.1163/1876312X-bja10062\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith MA, Hallwachs W, Janzen DH. Diversity and phylogenetic community structure of ants along a Costa Rican elevational gradient. Ecography. 2014;37(8):720\u0026ndash;31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1600-0587.2013.00631.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1600-0587.2013.00631.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRonque MU, Azevedo-Silva M, Mori GM, et al. Three ways to distinguish species: using behavioural, ecological, and molecular data to tell apart two closely related ants, \u003cem\u003eCamponotus renggeri\u003c/em\u003e and \u003cem\u003eCamponotus rufipes\u003c/em\u003e (Hymenoptera: Formicidae). Zool J Linn Soc. 2016;176(1):170\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/zoj.12303\u003c/span\u003e\u003cspan address=\"10.1111/zoj.12303\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 1991;10(4);506\u0026thinsp;\u0026ndash;\u0026thinsp;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimon C, Frati F, Beckenbach A, et al. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am. 1994;87(6):651\u0026ndash;701. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aesa/87.6.651\u003c/span\u003e\u003cspan address=\"10.1093/aesa/87.6.651\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreau CS, Bell CD, Vila R, et al. Phylogeny of the ants: diversification in the age of angiosperms. Science. 2006;312(5770):101\u0026ndash;4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1124891\u003c/span\u003e\u003cspan address=\"10.1126/science.1124891\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHebert PD, Penton EH, Burns JM, et al. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly \u003cem\u003eAstraptes fulgerator\u003c/em\u003e. Proc Natl Aca Sci USA. 2004;101(41):14812\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.0406166101\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0406166101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKooij PW, Dentinger BM, Donoso DA et al. Cryptic diversity in Colombian edible leaf-cutting ants (Hymenoptera: Formicidae). \u003cem\u003eInsects\u003c/em\u003e 2018;9(4):191. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects9040191\u003c/span\u003e\u003cspan address=\"10.3390/insects9040191\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkonechnikov K, Golosova O, Fursov M, Ugene Team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012;28(8):1166\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/bts091\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/bts091\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRezende CA, De Lima MA, Maziero P, et al. Chemical and morphological characterization of sugarcane bagasse submitted to a delignification process for enhanced enzymatic digestibility. Biotechnol Biofuels. 2011;4(1):54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1754-6834-4-54\u003c/span\u003e\u003cspan address=\"10.1186/1754-6834-4-54\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernardinelli OD, Lima MA, Rezende CA, et al. Quantitative \u003csup\u003e13\u003c/sup\u003eC MultiCP solid-state NMR as a tool for evaluation of cellulose crystallinity index measured directly inside sugarcane biomass. Biotechnol Biofuels. 2015;8(1):110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13068-015-0292-1\u003c/span\u003e\u003cspan address=\"10.1186/s13068-015-0292-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimmons TJ, Mortimer JC, Bernardinelli OD, et al. Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR. Nat Commun. 2016;7(1):13902. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms13902\u003c/span\u003e\u003cspan address=\"10.1038/ncomms13902\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolyen E, Rideout JR, Dillon MR, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotech. 2019;37(8):852\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41587-019-0209-9\u003c/span\u003e\u003cspan address=\"10.1038/s41587-019-0209-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCallahan BJ, McMurdie PJ, Rosen MJ, et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Meth. 2016;13(7):581\u0026ndash;3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.3869\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.3869\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2012;41(D1):D590\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gks1219\u003c/span\u003e\u003cspan address=\"10.1093/nar/gks1219\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhariwal A, Chong J, Habib S, et al. MicrobiomeAnalyst: a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data. Nucleic Acids Res. 2017;45(W1):W180\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkx295\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkx295\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChong J, Liu P, Zhou G, Xia J. Using MicrobiomeAnalyst for comprehensive statistical, functional, and meta-analysis of microbiome data. Nat Protoc. 2020;15(3):799\u0026ndash;821. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41596-019-0264-1\u003c/span\u003e\u003cspan address=\"10.1038/s41596-019-0264-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Y, Zhou G, Ewald J, et al. MicrobiomeAnalyst 2.0: comprehensive statistical, functional and integrative analysis of microbiome data. Nucleic Acids Res. 2023;51(W1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkad407\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkad407\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. W310-8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEichi VR, Tyerman SD, Wirthensohn MG. Water relations and mesophyll anatomy in almond leaves. J Am Soc Hortic Sci. 2014;139(6):627\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21273/JASHS.139.6.627\u003c/span\u003e\u003cspan address=\"10.21273/JASHS.139.6.627\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorsuk AM, Roddy AB, Th\u0026eacute;roux-Rancourt G, Brodersen CR. Structural organization of the spongy mesophyll. New Phytol. 2022;234(3):946\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.17971\u003c/span\u003e\u003cspan address=\"10.1111/nph.17971\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePolizel AM, Medri ME, Nakashima K, et al. Molecular, anatomical and physiological properties of a genetically modified soybean line transformed with rd29A: AtDREB1A for the improvement of drought tolerance. Genet Mol Res. 2011;10(4):3641\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatts S, Kariyat R. Morphological characterization of trichomes shows enormous variation in shape, density and dimensions across the leaves of 14 \u003cem\u003eSolanum\u003c/em\u003e species. AoB Plants. 2021;13(6):plab071. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aobpla/plab071\u003c/span\u003e\u003cspan address=\"10.1093/aobpla/plab071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiuliani C, Giovanetti M, Lupi D, et al. Tools to tie: Flower characteristics, VOC emission profile, and glandular trichomes of two Mexican salvia species to attract bees. Plants. 2020;9(12):1645. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants9121645\u003c/span\u003e\u003cspan address=\"10.3390/plants9121645\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLentfer CJ, Boyd WE, Gojak D. Hope Farm Windmill: Phytolith analysis of cereals in early colonial Australia. J Archaeol Sci. 1997;24(9):841\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/jasc.1996.0164\u003c/span\u003e\u003cspan address=\"10.1006/jasc.1996.0164\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeorge N, Antony A, Ramachandran T, et al. Microscopic investigations of silicification and lignification suggest their coexistence in tracheary phytoliths in date fruits (\u003cem\u003ePhoenix dactylifera\u003c/em\u003e L). Front Plant Sci. 2020;11:977. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2020.00977\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2020.00977\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeritore-Galve FC, Tancos MA, Smart CD. Bacterial canker of tomato: revisiting a global and economically damaging seedborne pathogen. Plant Dis. 2021;105(6):1581\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PDIS-08-20-1732-FE\u003c/span\u003e\u003cspan address=\"10.1094/PDIS-08-20-1732-FE\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu T, Li Z, Bao S, Su Y, et al. Xylem vessel type and structure influence the water transport characteristics of. Panax notoginseng PLoS One. 2023;18(3):e0281080. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0281080\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0281080\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuimond CM, Andrews PK, Lang GA. Scanning electron microscopy of floral initiation in sweet cherry. J Am Soc Hortic Sci. 1998;123(4):509\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGotelli MM, Galati BG, Zarlavsky G. Pollen development and anther morphology in 14 species of Rhamnaceae. Plant Syst Evol. 2016;302(10):1433\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00606-016-1342-2\u003c/span\u003e\u003cspan address=\"10.1007/s00606-016-1342-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePolowick PL, Sawhney VK. A scanning electron microscopic study on the initiation and development of floral organs of \u003cem\u003eBrassica napus\u003c/em\u003e (cv. Westar). Am J Bot. 1986;73(2):254\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/j.1537-2197.1986.tb08528.x\u003c/span\u003e\u003cspan address=\"10.1002/j.1537-2197.1986.tb08528.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeijzer CJ, Hoek IHS, Willeemse MTM. The processes of anther dehiscence and pollen dispersal III. The dehydration of the filament tip and the anther in three monocotyledonous species. New Phytol. 1987;106(2):281\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.14698137.1987.tb00143.x\u003c/span\u003e\u003cspan address=\"10.1111/j.14698137.1987.tb00143.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErmolaev A, Mardini M, Buravkov S, et al. A simple and user-friendly method for high-quality preparation of pollen grains for Scanning Electron Microscopy (SEM). Plants. 2024;13(15):2140. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants13152140\u003c/span\u003e\u003cspan address=\"10.3390/plants13152140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoster ZS, Sharpton TJ, Gr\u0026uuml;nwald NJ, Metacoder. An R package for visualization and manipulation of community taxonomic diversity data. PLoS Comput Biol. 2017;13(2):e1005404. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pcbi.1005404\u003c/span\u003e\u003cspan address=\"10.1371/journal.pcbi.1005404\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVon Humboldt A, Bonpland A. Essai sur la g\u0026eacute;ographie des plantes. Chez Levrault, Schoell et Campagnie, Libraries, Paris, 1805.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWimpenny JW. Spatial order in microbial ecosystems. Biol Rev. 1981;56(3):295\u0026ndash;342. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1469-185X.1981.tb00352.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1469-185X.1981.tb00352.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartiny JBH, Bohannan BJ, Brown JH, et al. Microbial biogeography: putting microorganisms on the map. Nat Rev Microbiol. 2006;4(2):102\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro1341\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro1341\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCallum G, Tropini C. The gut microbiota and its biogeography. Nat Rev Microbiol. 2024;22(2):105\u0026ndash;. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41579-023-00969-0\u003c/span\u003e\u003cspan address=\"10.1038/s41579-023-00969-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u0026thinsp;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDejea CM, Wick EC, Hechenbleikner EM, et al. Microbiota organization is a distinct feature of proximal colorectal cancers. Proc Natl Aca Sci USA. 2014;111(51):18321\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1406199111\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1406199111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWelch JLM, Rossetti BJ, Rieken CW, et al. Biogeography of a human oral microbiome at the micron scale. Proc Natl Aca Sci USA. 2016;113(6):E791\u0026ndash;800. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1522149113\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1522149113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim D, Barraza JP, Arthur RA, et al. Spatial mapping of polymicrobial communities reveals a precise biogeography associated with human dental caries. Proc Natl Aca Sci USA. 2020;117(22):12375\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1919099117\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1919099117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamont RJ, Hajishengallis G, Koo H. Social networking at the microbiome-host interface. Infect Immun. 2023;91(9):e00124\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/iai.00124-23\u003c/span\u003e\u003cspan address=\"10.1128/iai.00124-23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdade EE, Al Lakhen K, Lemus AA, Valm AM. Recent progress in analyzing the spatial structure of the human microbiome: Distinguishing biogeography and architecture in the oral and gut communities. Curr Opin Endocr Metab Res. 2021;18:275\u0026ndash;83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.coemr.2021.04.005\u003c/span\u003e\u003cspan address=\"10.1016/j.coemr.2021.04.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzimi S, Lewin GR, Whiteley M. The biogeography of infection revisited. Nat Rev Microbiol. 2022;20(10):579\u0026ndash;. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41579-022-00683-3\u003c/span\u003e\u003cspan address=\"10.1038/s41579-022-00683-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u0026thinsp;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShik JZ, Kooij PW, Donoso DA, et al. Nutritional niches reveal fundamental domestication trade-offs in fungus-farming ants. Nat Ecol Evol. 2021;5(1):122\u0026ndash;34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41559-020-01314-x\u003c/span\u003e\u003cspan address=\"10.1038/s41559-020-01314-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichards-Perhatch VM, Boshers E, Greenwold M, Seal JN. Forage preference in two geographically co-occurring fungus gardening ants: A dietary DNA approach. PLoS ONE. 2025;20(12):e0336150. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0336150\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0336150\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStorr GM. Microscopic analysis of faeces, a technique for ascertaining the diet of herbivorous, mammals. Aust J Biol Sci. 1961;14(1):157\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1071/BI9610157\u003c/span\u003e\u003cspan address=\"10.1071/BI9610157\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStewart DRM. Analysis of plant epidermis in faeces: a technique for studying the food preferences of grazing herbivores. J Appl Ecol. 1967;4(1):83\u0026ndash;111. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/2401411\u003c/span\u003e\u003cspan address=\"10.2307/2401411\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStewart DRM, Stewart J. Food preference data by faecal analysis for African plains ungulates. Afr Zool. 1970;5(1):115\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hdl.handle.net/10520/AJA00445096_238\u003c/span\u003e\u003cspan address=\"https://hdl.handle.net/10520/AJA00445096_238\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWirth R, Herz H, Ryel RJ, Beyschlag W, H\u0026ouml;lldobler B. Herbivory of leaf-cutting ants: a case study on \u003cem\u003eAtta colombica\u003c/em\u003e in the tropical rainforest of Panama. Springer; 2003.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeal IR, Wirth R, Tabarelli M. The multiple impacts of leaf-cutting ants and their novel ecological role in human‐modified neotropical forests. Biotropica. 2014;46(5):516\u0026ndash;28. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/btp.12126\u003c/span\u003e\u003cspan address=\"10.1111/btp.12126\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav S. Lignin biosynthesis in plants and its role in anther dehiscence in Brassicaceae. Botanica. 2018;68:48\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorghi M, Takeda-Kimura Y. Synthesis and physiological function of lignin in the reproductive structures of angiosperms. Phytochem Rev. 2025;24(4):2539\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11101-024-10043-z\u003c/span\u003e\u003cspan address=\"10.1007/s11101-024-10043-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeopold EB, Nickmann R, Hedges JI, Ertel JR. Pollen and lignin records of late Quaternary vegetation, Lake Washington. Science. 1982;218(4579):1305\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.218.4579.1305\u003c/span\u003e\u003cspan address=\"10.1126/science.218.4579.1305\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu FS, Hedges JI, Gordon ES, Brubaker LB. Lignin biomarkers and pollen in postglacial sediments of an Alaskan lake. Geochim Cosmochim Acta. 1999;63(9):1421\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0016-7037(99)00100-3\u003c/span\u003e\u003cspan address=\"10.1016/S0016-7037(99)00100-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma RK, Arora DS. Fungal degradation of lignocellulosic residues: an aspect of improved nutritive quality. Crit Rev Microbiol. 2015;41(1):52\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/1040841X.2013.791247\u003c/span\u003e\u003cspan address=\"10.3109/1040841X.2013.791247\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Luo Y, Luo L, et al. Enhancement of the nutritional value of fermented corn stover as ruminant feed using the fungi \u003cem\u003ePleurotus\u003c/em\u003e spp. Sci Rep. 2021;11(1):11961. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-021-90236-0\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-90236-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoller IE, De Fine Licht HH, Harholt J, et al. The dynamics of plant cell-wall polysaccharide decomposition in leaf-cutting ant fungus gardens. PLoS ONE. 2011;6(3):e17506. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0017506\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0017506\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSomera AF, Lima AM, dos Santos-Neto \u0026Aacute;J, et al. Leaf-cutter ant fungus gardens are biphasic mixed microbial bioreactors that convert plant biomass to polyols with biotechnological applications. Appl Environ Microbiol. 2015;81(13):4525\u0026ndash;35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.00046-15\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00046-15\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbril AB, Bucher EH. Evidence that the fungus cultured by leaf-cutting ants does not metabolize cellulose. Ecol Lett. 2002;5(3):325\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1461-0248.2002.00327.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1461-0248.2002.00327.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbril AB, Bucher EH. Nutritional sources of the fungus cultured by leaf-cutting ants. Appl Soil Ecol. 2004;26(3):243\u0026ndash;. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.apsoil.2003.12.008\u003c/span\u003e\u003cspan address=\"10.1016/j.apsoil.2003.12.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u0026thinsp;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAylward FO, Burnum-Johnson KE, Tringe SG, et al. \u003cem\u003eLeucoagaricus gongylophorus\u003c/em\u003e produces diverse enzymes for the degradation of recalcitrant plant polymers in leaf-cutter ant fungus gardens. Appl Environ Microbiol. 2013;79(12):3770\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.03833-12\u003c/span\u003e\u003cspan address=\"10.1128/AEM.03833-12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuen G, Scott JJ, Aylward FO, et al. An insect herbivore microbiome with high plant biomass-degrading capacity. PLoS Gen. 2010;6(9):e1001129. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pgen.1001129\u003c/span\u003e\u003cspan address=\"10.1371/journal.pgen.1001129\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagamoto NS, Garcia MG, Forti LC, et al. Microscopic evidence supports the hypothesis of high cellulose degradation capacity by the symbiotic fungus of leaf-cutting ants. J Biol Research-Thessaloniki. 2011;16:308\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVigueras G, Paredes-Hern\u0026aacute;ndez D, Revah S, et al. Growth and enzymatic activity of \u003cem\u003eLeucoagaricus gongylophorus\u003c/em\u003e, a mutualistic fungus isolated from the leaf‐cutting ant \u003cem\u003eAtta mexicana\u003c/em\u003e, on cellulose and lignocellulosic biomass. Lett Appl Microbiol. 2017;65(2):173\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/lam.12759\u003c/span\u003e\u003cspan address=\"10.1111/lam.12759\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeMilto AM, Rouquette M Jr, Mueller UG, et al. Effects of substrate, ant and fungal species on plant fiber degradation in a fungus-gardening ant symbiosis. J Insect Physiol. 2017;98:301\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2017.02.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jinsphys.2017.02.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLena GD, Patroni E, Quaglia GB. Improving the nutritional value of wheat bran by a white-rot fungus. Int J Food Sci Technol. 1997;32(6):513\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2621.1997.tb02125.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2621.1997.tb02125.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonaldson GP, Lee SM, Mazmanian SK. Gut biogeography of the bacterial microbiota. Nat Rev Microbiol. 2016;14(1):20\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro3552\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro3552\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeehan EC, Duar RM, Armet AM, et al. Modulation of the gastrointestinal microbiome with nondigestible fermentable carbohydrates to improve human health. Microbiol Spectr. 2017;5(5):10\u0026ndash;1128. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/microbiolspec.bad-0019-2017\u003c/span\u003e\u003cspan address=\"10.1128/microbiolspec.bad-0019-2017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeličković M, Wu R, Gao Y, et al. Mapping microhabitats of lignocellulose decomposition by a microbial consortium. Nat Chem Biol. 2024;20(8):1033\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41589-023-01536-7\u003c/span\u003e\u003cspan address=\"10.1038/s41589-023-01536-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDobritsa AP, Samadpour M. Transfer of eleven species of the genus \u003cem\u003eBurkholderia\u003c/em\u003e to the genus \u003cem\u003eParaburkholderia\u003c/em\u003e and proposal of \u003cem\u003eCaballeronia\u003c/em\u003e gen. nov. to accommodate twelve species of the genera \u003cem\u003eBurkholderia\u003c/em\u003e and \u003cem\u003eParaburkholderia\u003c/em\u003e. Int J Syst Evol Microbiol. 2016;66(8):2836\u0026ndash;46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/ijsem.0.001065\u003c/span\u003e\u003cspan address=\"10.1099/ijsem.0.001065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBugg TD, Ahmad M, Hardiman EM, Singh R. The emerging role for bacteria in lignin degradation and bio-product formation. Curr Opin Biotechnol. 2011;22(3):394\u0026ndash;400. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.copbio.2010.10.009\u003c/span\u003e\u003cspan address=\"10.1016/j.copbio.2010.10.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkita H, Kimura ZI, Yusoff MZM, et al. Identification and characterization of \u003cem\u003eBurkholderia multivorans\u003c/em\u003e CCA53. BMC Res Notes. 2017;10(1):249. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13104-017-2565-1\u003c/span\u003e\u003cspan address=\"10.1186/s13104-017-2565-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEilmus S, Heil M. Bacterial associates of arboreal ants and their putative functions in an obligate ant-plant mutualism. Appl Environ Microbiol. 2009;75(13):4324\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.00455-09\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00455-09\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaltenpoth M, Fl\u0026oacute;rez LV. Versatile and dynamic symbioses between insects and \u003cem\u003eBurkholderia\u003c/em\u003e bacteria. Annu Rev Entomol. 2020;65(1):145\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-ento-011019-025025\u003c/span\u003e\u003cspan address=\"10.1146/annurev-ento-011019-025025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKellner K, Ishak HD, Linksvayer TA, Mueller UG. Bacterial community composition and diversity in an ancestral ant fungus symbiosis. FEMS Microbiol Ecol. 2015;91(7):fiv073. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/femsec/fiv073\u003c/span\u003e\u003cspan address=\"10.1093/femsec/fiv073\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantos AV, Dillon RJ, Dillon VM, et al. Ocurrence of the antibiotic producing bacterium \u003cem\u003eBurkholderia\u003c/em\u003e sp. in colonies of the leaf-cutting ant \u003cem\u003eAtta sexdens rubropilosa\u003c/em\u003e. FEMS Microbiol Lett. 2004;239(2):319\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.femsle.2004.09.005\u003c/span\u003e\u003cspan address=\"10.1016/j.femsle.2004.09.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrancoeur CB, May DS, Thairu MW, et al. \u003cem\u003eBurkholderia\u003c/em\u003e from fungus gardens of fungus-growing ants produces antifungals that inhibit the specialized parasite \u003cem\u003eEscovopsis\u003c/em\u003e. Appl Environ Microbiol. 2021;87(14):e00178\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.00178-21\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00178-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen EA, Klepacki I, Klassen JL. Isolation and characterization of mollicute symbionts from a fungus-growing ant reveals high niche overlap leading to co-exclusion. mBio. 2025;16(7):e00893\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mbio.00893-25\u003c/span\u003e\u003cspan address=\"10.1128/mbio.00893-25\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamalho MDO, Martins C, Morini MSC, Bueno OC. What can the bacterial community of \u003cem\u003eAtta sexdens\u003c/em\u003e (Linnaeus, 1758) tell us about the habitats in which this ant species evolves? \u003cem\u003eInsects\u003c/em\u003e 2020;11(6):332. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects11060332\u003c/span\u003e\u003cspan address=\"10.3390/insects11060332\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZani RDOA, Ferro M, Bacci M Jr. Three phylogenetically distinct and culturable diazotrophs are perennial symbionts of leaf-cutting ants. Ecol Evol. 2021;11(24):17686\u0026ndash;99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ece3.8213\u003c/span\u003e\u003cspan address=\"10.1002/ece3.8213\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin MM, Martin JS. The presence of protease activity in the rectal fluid of primitive attine ants. J Insect Physiol. 1971;17(10):1897\u0026ndash;906. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0022-1910(71)90134-X\u003c/span\u003e\u003cspan address=\"10.1016/0022-1910(71)90134-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyd ND, Martin MM. Faecal proteinases of the fungus-growing ant, \u003cem\u003eAtta texana\u003c/em\u003e: their fungal origin and ecological significance. J Insect Physiol. 1975;21(11):1815\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0022-1910(75)90247-4\u003c/span\u003e\u003cspan address=\"10.1016/0022-1910(75)90247-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Fine Licht HH, Schi\u0026oslash;tt M, Rogowska-Wrzesinska A, et al. Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts. Proc Natl Aca Sci USA. 2013;110(2):583\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1212709110\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1212709110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKooij PW, Rogowska-Wrzesinska A, Hoffmann D, et al. \u003cem\u003eLeucoagaricus gongylophorus\u003c/em\u003e uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate. ISME J. 2014;8(5):1032\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ismej.2013.231\u003c/span\u003e\u003cspan address=\"10.1038/ismej.2013.231\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKooij PW, Pullens JW, Boomsma JJ, Schi\u0026oslash;tt M. Ant mediated redistribution of a xyloglucanase enzyme in fungus gardens of \u003cem\u003eAcromyrmex echinatior\u003c/em\u003e. BMC Microbiol. 2016;16(1):81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12866-016-0697-4\u003c/span\u003e\u003cspan address=\"10.1186/s12866-016-0697-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchi\u0026oslash;tt M, Boomsma JJ. Proteomics reveals synergy between biomass degrading enzymes and inorganic Fenton chemistry in leaf-cutting ant colonies. eLlife. 2021;10:e61816. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7554/eLife.61816\u003c/span\u003e\u003cspan address=\"10.7554/eLife.61816\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurrie CR, Stuart AE. Weeding and grooming of pathogens in agriculture by ants. Proc R Soc Lond B Biol Sci. 2001;268(1471):1033\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rspb.2001.1605\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2001.1605\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYek SH, Boomsma JJ, Poulsen M. Towards a better understanding of the evolution of specialized parasites of fungus-growing ant crops. Psyche J Entom. 2012;2012(1):239392. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2012/239392\u003c/span\u003e\u003cspan address=\"10.1155/2012/239392\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBae IH, Kim H, Kim SM, Lee YH. Multi-meta-omics reveal unique symbiotic synchronization between ectomycorrhizal fungus and soil microbiome in \u003cem\u003eTricholoma matsutake\u003c/em\u003e habitat. Microbiome. 2026;14(1):23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40168-025-02292-7\u003c/span\u003e\u003cspan address=\"10.1186/s40168-025-02292-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoo S, Seo CW, Lim YW. Functionally distinct core microbes of \u003cem\u003eTricholoma matsutake\u003c/em\u003e revealed by cross-study analysis. Microbiome. 2026;14:58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40168-025-02329-x\u003c/span\u003e\u003cspan address=\"10.1186/s40168-025-02329-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarmink JA, Van Elsas JD. Migratory response of soil bacteria to \u003cem\u003eLyophyllum\u003c/em\u003e sp. strain Karsten in soil microcosms. Appl Environ Microbiol. 2009;75(9):2820\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.02110-08\u003c/span\u003e\u003cspan address=\"10.1128/AEM.02110-08\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNazir R, Warmink JA, Voordes DC, et al. Inhibition of mushroom formation and induction of glycerol release - Ecological strategies of \u003cem\u003eBurkholderia terrae\u003c/em\u003e BS001 to create a hospitable niche at the fungus \u003cem\u003eLyophyllum\u003c/em\u003e sp. strain Karsten. Microb Ecol. 2013;65(1):245\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00248-012-0100-4\u003c/span\u003e\u003cspan address=\"10.1007/s00248-012-0100-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStopnisek N, Z\u0026uuml;hlke D, Carlier A, et al. Molecular mechanisms underlying the close association between soil Burkholderia and fungi. ISME J. 2016;10(1):253\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ismej.2015.73\u003c/span\u003e\u003cspan address=\"10.1038/ismej.2015.73\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartiarena MJS, Deveau A, Montoya QV, et al. The hyphosphere of leaf-cutting ant cultivars is enriched with helper bacteria. Microb Ecol. 2023;86(3):1773\u0026ndash;88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00248-023-02187-w\u003c/span\u003e\u003cspan address=\"10.1007/s00248-023-02187-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen M, He T, Wu Q, et al. Enhanced heterotrophic nitrification and aerobic denitrification performance of \u003cem\u003eGlutamicibacter arilaitensis\u003c/em\u003e EM-H8 with different carbon sources. Chemosphere. 2023;323:138266. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemosphere.2023.138266\u003c/span\u003e\u003cspan address=\"10.1016/j.chemosphere.2023.138266\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumari S, Naraian R. Enhanced growth and yield of oyster mushroom by growth-promoting bacteria \u003cem\u003eGlutamicibacter arilaitensis\u003c/em\u003e MRC119. J Basic Microbiol. 2021;61(1):45\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jobm.202000379\u003c/span\u003e\u003cspan address=\"10.1002/jobm.202000379\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng J, Ye K, Fu C et al. Comprehensive assessment of rice bran dietary fiber on gut microbiota composition and metabolism during in vitro fermentation. Food Res Int 2024:197:115231. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodres.2024.115231\u003c/span\u003e\u003cspan address=\"10.1016/j.foodres.2024.115231\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu L, Tang T, Song L, et al. \u003cem\u003ePullulanibacillus pueri\u003c/em\u003e sp. nov., isolated from Pu'er tea. Int J Syst Evol Microbiol. 2015;65(Pt7):2167\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/ijs.0.000239\u003c/span\u003e\u003cspan address=\"10.1099/ijs.0.000239\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAizawa T, Sato J, Saito S, et al. An extracellular polysaccharide is involved in the aluminum tolerance of \u003cem\u003ePullulanibacillus\u003c/em\u003e sp. CA42, a newly isolated strain from the Chinese water chestnut growing in an actual acid sulfate soil area in Vietnam. Front Microbiol. 2023;14:1241244. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2023.1241244\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2023.1241244\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGharechahi J, Sarikhan S, Han JL, et al. Functional and phylogenetic analyses of camel rumen microbiota associated with different lignocellulosic substrates. npj Biofilms Microbiomes. 2022;8(1):46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41522-022-00309-9\u003c/span\u003e\u003cspan address=\"10.1038/s41522-022-00309-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTom LM, Aulitto M, Wu YW, et al. Low-abundance populations distinguish microbiome performance in plant cell wall deconstruction. Microbiome. 2022;10(1):183. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40168-022-01377-x\u003c/span\u003e\u003cspan address=\"10.1186/s40168-022-01377-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol. 2010;8(9):623\u0026ndash;. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro2415\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro2415\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u0026thinsp;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlemming HC, Wingender J, Szewzyk U, et al. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol. 2016;14(9):563\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro.2016.94\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro.2016.94\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDragoš A, Kiesewalter H, Martin M, et al. Division of labor during biofilm matrix production. Curr Biol. 2018;28(12):1903\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cub.2018.04.046\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2018.04.046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChatterjee S, Samal B, Singh P, et al. Transition of a solitary to a biofilm community life style in bacteria: a survival strategy with division of labour. Int J Dev Biol. 2020;64:259\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1387/ijdb.190176sc\u003c/span\u003e\u003cspan address=\"10.1387/ijdb.190176sc\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlemming HC, van Hullebusch ED, Neu TR, et al. The biofilm matrix: multitasking in a shared space. Nat Rev Microbiol. 2023;21(2):70\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41579-022-00791-0\u003c/span\u003e\u003cspan address=\"10.1038/s41579-022-00791-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fungiculture, Plant substrates, Biofilm, Attine, Symbiosis, Microbiota","lastPublishedDoi":"10.21203/rs.3.rs-9554856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9554856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eObligate microbial gardeners, attine ants cultivate fungi for food using mostly plant-derived substrates for nourishing their fungal crops. The fungal crop grows together with its associated microbiota then forming the microbial garden, a spongy structure scaffolded by substrates. Attines differ in their cultivation systems, in part, by foraging for a diverse set of plant substrates. Here, we hypothesize that the garden lignocellulosic profile, microbiota composition, and garden spatial structure differ between attine species according to their substrate preferences.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe sampled young and old garden regions, as well as waste material from attines representing the lower fungiculture (\u003cem\u003eApterostigma\u003c/em\u003e sp., \u003cem\u003eMycetophylax\u003c/em\u003e sp., and \u003cem\u003eMycocepurus\u003c/em\u003e sp.), the higher fungiculture (\u003cem\u003eMycetomoellerius\u003c/em\u003e sp.), and leaf-cutting fungiculture (\u003cem\u003eAcromyrmex coronatus\u003c/em\u003e and \u003cem\u003eAtta sexdens\u003c/em\u003e). Then, we determined the garden chemical composition via \u003csup\u003e13\u003c/sup\u003eC NMR, the microbiota composition by 16S rRNA sequencing, and spatial distribution using SEM. We found plant-derived fragments representing the majority of recognizable substrates, providing the spatial framework of the microbial garden. In consonance, it exhibits a lignocellulosic nature, which is modified to a greater extent in gardens of lower fungiculture systems than in the higher and leaf-cutting ones. In lower attine, \u003cem\u003eBurkholderia\u003c/em\u003e is an abundant microbial member, considered part of the fungiculture core microbiota and a biomarker of lower and higher fungiculture systems. In higher and leaf-cutting gardens, otherwise, \u003cem\u003eMesoplasma\u003c/em\u003e is abundant, taken as a signature of leaf-cutting systems. Group-wise comparisons revealed only \u003cem\u003eAt. sexdens\u003c/em\u003e differing from other fungiculture systems by showing lower diversity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAll attine gardens, but \u003cem\u003eAt. sexdens\u003c/em\u003e, share a similar taxonomic composition. Our findings unveil biofilms as intrinsic and ubiquitous components of attine microbial gardens. Essentially, each fungiculture has its characteristic architecture, emphasizing the substrate role in shaping the garden\u0026rsquo;s spatial organization.\u003c/p\u003e","manuscriptTitle":"The microbial garden of fungus-growing ants: Distinct lignocellulosic profile and spatial structure, yet a similar microbiota","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-05 08:33:51","doi":"10.21203/rs.3.rs-9554856/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-15T09:36:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T12:27:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T09:25:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Microbiome","date":"2026-04-28T13:21:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a62bd117-30cc-4db8-9e5e-e292b97d8e05","owner":[],"postedDate":"May 5th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-15T09:36:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T12:27:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T09:25:52+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T14:09:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-05 08:33:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9554856","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9554856","identity":"rs-9554856","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}