DOI: 10.5061/dryad.b5mkkwhk5
title: Reciprocal nutritional provisioning between leafcutter ants and their fungal cultivar mediates performance of symbiotic farming systems

description: Optimized food acquisition is challenging because foraged diet items are
 chemically complex and often nutritionally imbalanced. These challenges
 are likely magnified when foraged foods are used to provision others
 (e.g., offspring, nestmates, symbionts) with different nutritional
 requirements. We used a theoretical framework of nutritional niches to
 study these provisioning challenges in leafcutter ants that cultivate a
 fungal symbiont with nutrients derived from freshly foraged plant
 fragments. While the leaf-cutting behaviours of free-ranging foragers are
 well studied, little is known about how colonies use these plant fragments
 to produce their fungal crop within underground nest chambers. For
 instance, gardener ants are known to convert vegetation into a nutritional
 mulch that they plant on the fungus garden. However, it remains poorly
 understood how the ants use this mulch to target the specific nutritional
 needs of their fungal crop, and whether the cultivar signals if
 provisioned mulch meets its nutritional needs. Towards answers, we
 performed three experiments to assess the precision and specificity of
 nutritional regulation in farming systems of the Panamanian leafcutter ant
 Acromyrmex echinatior. A laboratory feeding experiment with nutritionally
 defined diets showed that ant farmers collect a specific intake target for
 protein and carbohydrates and then linked strict protein regulation by
 foragers to the cultivar’s fundamental niche for protein.  An in
 vitro experiment with the fungal cultivar in isolation did not detect a
 signal of protein stress that could be used by the ants to regulate their
 provisioning behaviour, but it did identify an elevated fatty acid that
 may reinforce optimal nutritional provisioning if detected by gardening
 ants. A feeding experiment with isotopically labelled diets then revealed
 nutrient-specific and caste-specific allocation timelines, with nitrogen
 being assimilated into the cultivar’s nutritional rewards before being
 exclusively consumed by developing brood. In turn, these combined results
 help resolve the integrated behaviours that give rise to resilient
 leafcutter farming productivity.  These results show how
 nutritional niches can help disentangle reciprocal provisioning dynamics
 between symbionts while providing a framework to explore the nutritional
 transactions that mediate symbiotic stability (e.g., sanctioning,
 screening, policing).
description: Do ants differentially regulate
 nutrients? We established<strong>
 </strong>ten<strong> </strong>queenless <em>A.
 echinatior</em> subcolonies (<em>hereafter
 </em>colonies) from five parent colonies that were collected
 in Soberanía Park (Panama) and maintained at the University
 of Copenhagen in a climate-controlled room (25°C, 70–75% RH) (Table S1).
 We used a diet-choice
 experiment to test if ants regulate intake targets for protein (P) and/or
 carbohydrates (C). Each day for ten days, colonies were
 provided two pre-weighed nutritionally-defined agar-based diets
 (<em>ca.</em> 15 g) in separate Petri dishes containing
 protein:carbohydrate (P:C) ratios of either 1:6 or 2:1 and P+C dilutions
 of 30 g/L (modified from Dussutour & Simpson, 2008; Shik et al.,
 2018) (Fig. 1<em>a</em>, Table S2). These ratios and the
 concentration of nutrients were chosen based on published results showing
 the fungal cultivar’s needs for these macronutrients and concentrations of
 these macronutrients in typically foraged plant fragments (Shik et al.,
 2021). Cumulative diet intake was calculated as dry diet mass collected by
 foragers each day, using control diet dry:wet ratios to estimate initial
 diet dry mass (as per Kay et al., 2012; Krabbe et al., 2019). P and C
 intake levels were calculated using diet recipes to determine their dry
 weight fractions.  We performed Bartlett’s homogeneity of variance
 test to test for differences in the regulation of P and C intake, a
 Wilcoxon signed-rank test to test for differences in diet (1:6 vs. 2:1)
 and nutrient (cumulative protein vs. cumulative carbohydrate) intake
 across colonies, and a one-sample <em>t</em>-test to test
 whether the P:C ratio foraged by colonies differed from the P:C ratios
 provided by each diet. These and all subsequent statistical analyses were
 performed using R 4.2.1 (R Core
 team, 2022). Additional methodological and analytical details
 for this experiment and all described below are provided in Supplementary
 Appendix S1. Do colonies regulate nutrients relative to
 cultivar needs? We tested
 whether nutritional foraging choices of <em>A. echinatior
 </em>colonies are related to the FNN dimensions of their fungal
 cultivar. As part of a recently published comparative study, an <em>in vitro
 </em>experiment was used to measure the FNN for hyphal growth across
 ratios and concentrations of P and C for the <em>L.
 gongylophorus</em> fungal cultivar isolated from three colonies of
 <em>A. echinatior </em>(Shik et al., 2021). In
 that experiment, the three cultivar isolates were confined to Petri dishes
 with 36 diet treatments (P:C ratios: 1:9,1:6, 1:3, 1:2, 1:1, 2:1, 3:1,
 6:1, 9:1; P+C dilutions: 8, 20, 40, 60 g/L) and eight replicates per diet
 treatment (N = 864 total Petri dishes). After a period of growth, two
 cultivar performance traits were measured (growth area
 (mm<sup>2</sup>), and staphyla density (number of staphylae
 per mm<sup>2</sup>)). Here, we replot the resulting cultivar
 FNN heatmaps in a new way and use them to complement the colony-level
 feeding experiments described above (Appendix S1). 
 Heatmaps were generated using the fields package
 in R studio (Nychka et al., 2015) where blue areas indicate low
 performance and yellow areas indicate high performance. Performance
 isoclines were generated using non-parametric thin-plate
 splines. Least-square regressions were used to assess the
 significance of the linear and quadratic terms (and their linear
 interaction) of variation in the dependent variables (growth area,
 staphyla density) across the P and C diet treatments. This analysis was
 performed at the level of each isolate and on the mean values across
 isolates used to generate the figure (Table S3). 
 Does the cultivar signal its nutritional
 needs? We used an untargeted approach to test for
 changes in profiles of 37 fatty acids putatively involved in signalling
 (Table S4)<strong> </strong>when fungal cultivars from five
 <em>A. echinatior </em>colonies were confined to Petri dishes
 with C-biased (1:3) and P-biased (3:1) P:C media (N = 45 Petri dishes; n =
 3 plates per treatment per colony). Based on the FNN results, we selected
 P:C diets at the 8 g/L P + C dilution using the diet recipe from
 Crumière et al.
 (2021). This analysis included a test for the presence
 of linoleic acid (9,12 octadecadienoic acid (Z,Z)-, methyl ester, Table
 S4) due to its previous identification as an important fatty acid
 metabolized by <em>L. gongylophorus </em>and its association
 with behavioural modification of leafcutter ants (Khadempour et al., 2021).
 After 42 days of
 <em>in vitro </em>growth, fungal tissue was collected from the
 surface of growth media, flash frozen in liquid nitrogen and stored at -80
 ºC for lipid extractions and analysis with gas chromatography
 mass-spectrometry. Values were adjusted relative to results from a control
 media treatment cultured in the same way but without being inoculated with
 the fungal cultivar.  To test for P:C diet effects on lipid profiles,
 we performed a Principal Coordinate Analysis (PCoA) using the vegan
 package version 2.6-2 (Oksanen
 et al., 2022) with Bray-Curtis dissimilarity and a PERMANOVA
 in the vegan package with colony of origin included as a random effect to
 test for statistical significance. Candidate compounds were
 identified using a random forest analysis, which is a supervised
 classification and variable selection method for multivariate data, in the
 randomForest package V4.7-1.1 (Liaw & Wiener, 2022).
 Do worker castes play distinct roles in
 nutritional regulation? The function of three putative adult
 worker castes in <em>A. echinatior </em>colonies remains
 uncertain (Bot & Boomsma, 1996; Larsen et
 al., 2014) but knowledge of task discretization among these castes can
 help interpret whether measures of nutritional regulation described in
 sections 2.5 and 2.6 (see below) are sufficiently
 precise. We thus combined morphological,
 behavioural, and isotopic data to test for differences among minor, media
 and major workers. We
 first collected 10 workers of the body size categories representing each
 putative caste from each of six colonies (N = 180 workers) and measured
 their head widths (mm) and dry weight (mg). We then used a mixed model
 log-log scaling approach with colony identity as a random factor to test
 whether caste exhibit continuous variation in head with (HW) with body
 mass (M). If caste regressions exhibit different slopes, this provides
 evidence for ‘break points’ in body shape that define ‘physical caste’
 systems (Oster & Wilson, 1979). We next used a modified ethogram
 approach (Santos et al., 2005; Calheiros et al.,
 2019) to identify and quantify three
 fungus-gardening behaviours in standardized nest fragments
 (fungus inspection, fungus licking, fungus reconstruction). We tested for
 differences among the three body size categories in the frequency of task
 performance using mixed models with colony identity as a random factor
 followed by posthoc tests (Table S5).  Does the cultivar exhibit nutrient-specific
 assimilation and allocation? We performed an isotopic tracer
 experiment on Day 0 of the diet choice experiment described above (section
 2.1) where the P:C diets were<em> </em>enriched with known
 amounts of the heavy isotopes of carbon
 (<sup>13</sup>C-enriched glucose) and nitrogen
 (<sup>15</sup>N-enriched ammonium nitrate) (Table S2).
 Just before providing
 colonies with enriched diets on Day 0, we collected samples from each
 colony to quantify baseline enrichment of <sup>13</sup>C and
 <sup>15</sup>N (i.e., natural abundance). Following the heavy
 isotope ‘pulse’, we thereafter provided colonies ‘unenriched’ P:C diets
 and then collected samples for isotopic analyses on Days 1, 2, 5 and 10
 (<em>as per</em>
 Shik et al., 2018). On each sampling day, we collected samples of
 fungus gardens from each colony at three vertical layers (top, middle,
 bottom), and from two tissue types (hyphae (all three levels), staphylae
 (middle layer)). At each sampling day, we also sampled adult ants by
 pooling individuals of three putative castes from each colony (n = 5
 minor; n = 5 media; n = 3 major). We separately analysed ant body segments
 to distinguish between assimilation (head-thorax) and
 transport/nestmate-dissemination/cultivar-provisioning (abdomen)
 (<em>as per</em> Shik et al.,
 2018). How does the fungal cultivar regulate
 nutritional provisioning of its workforce?  
 The isotopic data were used to
 test for caste-specific timelines of nutrient ingestion and allocation.
 Developing ants (larvae) are also called a digestive caste that, among
 other functions, likely mediates nutrient exchange within fungus gardens
 (Erthal et al., 2007). We thus also analysed enrichment timelines in
 larvae and pupae collected from the middle layer of the fungus garden of
 each colony on each sampling day. We further tested for nutrient-specific
 colony waste disposal by collecting trash samples from each colony every
 day. All samples were
 dried at 50°C for ≥ 24 hours and were then crushed, homogenized, and added
 in ca. 0.15 mg aliquots into pre-weighed tin capsules. Isotopic analyses of
 <sup>15</sup>N/<sup>14</sup>N and
 <sup>13</sup>C/<sup>12</sup>C were conducted using
 an Isoprime isotope mass spectrometer (Cheadle Hulme, UK) coupled to a
 Eurovector CN analyser (Pavia, Italy). For subsequent mixed-model
 statistical analyses, we calculated excess μg <sup>15</sup>N
 and μg <sup>13</sup>C per g dry mass of each sample (hereafter
 <sup>15</sup>N and <sup>13</sup>C as per Shik et
 al., 2018) (Table S6). 
description: This README file was generated on 2023-09-08 by Jonathan Shik GENERAL
 INFORMATION 1. Reciprocal nutritional provisioning between leafcutter ants
 and their fungal cultivar mediates performance of symbiotic farming
 systems 2. Corresponding author information Name: Jonathan Shik
 Institution: University of Copenhagen Address: Universitetsparken 15, 2100
 Copenhagen Email: jonathan.shik@bio.ku.dk 3. Usage notes: These datasets
 were generated in at the University of Copenhagen based on colonies
 leafcutter ant colonies (Acromyrmex echinatior) collected at the
 Smithsonian Tropical Research Institute in Panama (Soberenia Park). 4.
 Excel spreadsheet and associated text files with datasets used for all
 statistical analyses described in the Methods and reported in the Results.
 Datasets included: 1. Excel spreadsheet: A) hov\_test B) IT\_stats C)
 cumu\_in\_box D) in\_segement E) FNN heatmaps F) Isolate FNN heatmaps G)
 caste\_scaling H) behav box plot means I) behav analysis 2. Separate text
 files A) Cumulative\_intake.txt B) fungus\_food.txt C) intake\_by\_day.txt
 D) nutrient\_allocation.txt E) nutrient\_processing.txt F)
 waste\_disposal.txt Also included with raw data is all R code for
 executing all analyses and raw versions of figures. ########## 1.
 Laboratory experiment with nutritionally defined P:C diets --hov\_test:
 total intake of diets by ants (mg) 1. Number of variables: 4 2. Number of
 cases/rows: 20 3. Variable List: * parent\_colony: colony from which
 subcolonies were made * col\_ID: any of the 10 experimental colonies *
 nutrient: the macronutrient measured for intake (carb or prot) * intake:
 the cumulative amount of dry macronutrient intake (mg) --IT\_stats:
 Protein annd carbohydrate intake and P:C ratios 1. Number of variables: 7
 2. Number of cases/rows: 10 3. Variable List: * parent\_colony: colony
 from which subcolonies were made * col\_ID: any of the 10 experimental
 colonies * prot: the protein cumulative intake (mg, dry) * carb: the
 carbohydrate cumulative intake (mg, dry) * p\_c\_ratio: the ratio of
 protein to carbohydrate (P:C) * tot\_o\_s: total cumulative amount of 1:6
 P:C diet intake over 10 days (mg) * tot\_t\_o: total cumulative amount of
 3:1 P:C diet intake over 10 days (mg) --cumu\_in\_box: total intake of
 protein and carbohydrates by colonies 1. Number of variables: 5 2. Number
 of cases/rows: 20 3. Variable List: * parent\_colony: colony from which
 subcolonies were made * col\_ID: any of the 10 experimental colonies * dv:
 the Protein or Carbohydrate variable * pt: dummy variable coding
 macronutrient * intake: total cumulative amount of diet intake over 10
 days --in\_segement: cumulative intake of each colony day by day 1. Number
 of variables: 5 2. Number of cases/rows: 100 3. Variable List: * col\_ID:
 any of the 10 experimental colonies * day: day 0 to 9 during the
 experiment * col\_time: combined day by colony grouping variable *
 cumu\_p: cumulative protein intake added per day (mg) * cumu\_c:
 cumulative carbohydrate intake added per day (mg) METHODS: We established
 ten queenless A. echinatior subcolonies (hereafter colonies) from five
 parent colonies that were collected in Soberanía Park (Panama) and
 maintained at the University of Copenhagen in a climate-controlled room
 (25°C, 70-75% RH) (Table S1). We used a diet-choice experiment to test if
 ants regulate intake targets for protein (P) and/or carbohydrates (C).
 Each day for ten days, colonies were provided two pre-weighed
 nutritionally-defined agar-based diets (ca. 15 g) in separate Petri dishes
 containing protein:carbohydrate (P:C) ratios of either 1:6 or 2:1 and P+C
 dilutions of 30 g/L (modified from Dussutour & Simpson, 2008; Shik
 et al., 2018) (Fig. 1a, Table S2). These ratios and the concentration of
 nutrients were chosen based on published results showing the fungal
 cultivar’s needs for these macronutrients and concentrations of these
 macronutrients in typically foraged plant fragments (Shik et al., 2021).
 Cumulative diet intake was calculated as dry diet mass collected by
 foragers each day, using control diet dry:wet ratios to estimate initial
 diet dry mass (as per Kay et al., 2012; Krabbe et al., 2019). P and C
 intake levels were calculated using diet recipes to determine their dry
 weight fractions. We performed Bartlett’s homogeneity of variance test to
 test for differences in the regulation of P and C intake, a Wilcoxon
 signed-rank test to test for differences in diet (1:6 vs. 2:1) and
 nutrient (cumulative protein vs. cumulative carbohydrate) intake across
 colonies, and a one sample t-test to test whether the P:C ratio foraged by
 colonies differed from the P:C ratios provided by each diet. These and all
 subsequent statistical analyses were performed using R 4.2.1 (R Core team,
 2022). Additional methodological and analytical details for this
 experiment and all described below are provided in Supplementary Appendix
 S1. ######## 2) In vitro experiment with the fungal cultivar to measure
 the fundamental nutritional niche --FNN heatmaps: data for figures where
 area (mm^2) and staphyla density (staphylae number per area) were mapped
 across gradients of percent protein and carbohydrates (mean values across
 fungi from 3 colonies) 1. Number of variables: 5 2. Number of cases/rows:
 36 3. Variable List: * dillution\_diet: the P+C dillution (8,20,40,60
 mg/L) and diet ratio (P:C: 1:1, 1:2. 1:3, 1:6, 1:9, 2:1, 3:1, 6:1, 9:1) *
 area: fungal growth area (mm^2) * staphdens: staphyla density (#/mm^2) *
 pct\_prot: percent protein in diet * pct\_carb: percent carbohydrate in
 diet --Isolate FNN heatmaps: FNN data for statistics tables (each colony
 provided, all raw data) 1. Number of variables: 7 2. Number of cases/rows:
 36 3. Variable List: * plate\_id: combined variable of colony, dillution,
 diet, plate replicate (1-8) * colony\_id: the three colonies in the
 experiment (Ae\_1, Ae\_2, Ae\_3) * dillution\_diet: the P+C dillution
 (8,20,40,60 mg/L) and diet ratio (P:C: 1:1, 1:2. 1:3, 1:6, 1:9, 2:1, 3:1,
 6:1, 9:1) * area: fungal growth area (mm^2) * staphdens: staphyla density
 (#/mm^2) * pct\_prot: percent protein in diet * pct\_carb: percent
 carbohydrate in diet METHODS: We tested whether nutritional foraging
 choices of A. echinatior colonies are related to the FNN dimensions of
 their fungal cultivar. As part of a recently published comparative study,
 an in vitro experiment was used to measure the FNN for hyphal growth
 across ratios and concentrations of P and C for the L. gongylophorus
 fungal cultivar isolated from three colonies of A. echinatior (Shik et
 al., 2021). In that experiment, the three cultivar isolates were confined
 to Petri dishes with 36 diet treatments (P:C ratios: 1:9,1:6, 1:3, 1:2,
 1:1, 2:1, 3:1, 6:1, 9:1; P+C dilutions: 8, 20, 40, 60 g/L) and eight
 replicates per diet treatment (N = 864 total Petri dishes). After a period
 of growth, two cultivar performance traits were measured (growth area
 (mm2), and staphyla density (number of staphylae per mm2)). Here, we
 replot the resulting cultivar FNN heatmaps in a new way and use them to
 complement the colony-level feeding experiments described above (Appendix
 S1). Heatmaps were generated using the fields package in R studio (Nychka
 et al., 2015) where blue areas indicate low performance and yellow areas
 indicate high performance. Performance isoclines were generated using
 non-parametric thin-plate splines. Least-square regressions were used to
 assess the significance of the linear and quadratic terms (and their
 linear interaction) of variation in the dependent variables (growth area,
 staphyla density) across the P and C diet treatments. This analysis was
 performed at the level of each isolate and on the mean values across
 isolates used to generate the figure (Table S3). ####### 3) Caste
 morphology scaling --caste\_scaling: scaling figure and plot where log
 head width plotted against log body mass of workers for three putative
 castes (minor, media, major) 1. Number of variables: 4 2. Number of
 cases/rows: 180 3. Variable List: * col\_ID: 10 workers of the body size
 categories representing each putative caste from each of 6 colonies (N =
 180 workers) * caste: three putative morphological castes (minor, media,
 major) * log\_mass: Log10 dry weight (mg) of individual workers * log\_hw:
 Log10 dry head widths (mm) of individual workers 4. Analysis of gardening
 behavior data --behav box plot means: data for behavioral box plots where
 specific behavioral types are compared across castes and colonies (percent
 mean activity) 1. Number of variables: 4 2. Number of cases/rows: 54 3.
 Variable List: * col\_ID: workers analyzed from 6 colonies (N = 180
 workers) * caste: three putative morphological castes (minor, media,
 major) * behav: the three gardening behaviors analyzed (inspecting fungus,
 licking fungus, reconstructing fungus) * mean\_pct\_activity: the mean
 percent activity dedicated to each task 5. Data for stats of behavioral
 data --behav analysis: data for stats comparing 3 behaviors (inspect
 fungus, lick fungus, reconstruct fungus) across 3 putative castes and
 colonies 1. Number of variables: 6 2. Number of cases/rows: 91 3. Variable
 List: * col: workers analyzed from 6 colonies (N = 180 workers) *
 replicate: 1-5 replicate observations per colony * caste: three putative
 morphological castes (minor, media, major) * inspect\_fungus: inspecting
 fungus by gardeners (percent) * lick\_fungus: licking fungus by gardeners
 (percent) * reconstruct\_fungus: reconstructing fungus by gardeners
 (percent) METHODS The function of three putative adult worker castes in A.
 echinatior colonies remains uncertain (Bot & Boomsma, 1996; Larsen
 et al., 2014) but knowledge of task discretization among these castes can
 help interpret whether measures of nutritional regulation described in
 sections 2.5 and 2.6 (see below) are sufficiently precise. We thus
 combined morphological, behavioural, and isotopic data to test for
 differences among minor, media and major workers. We first collected 10
 workers of the body size categories representing each putative caste from
 each of six colonies (N = 180 workers) and measured their head widths (mm)
 and dry weight (mg). We then used a mixed model log-log scaling approach
 with colony identity as a random factor to test whether caste exhibit
 continuous variation in head with (HW) with body mass (M). If caste
 regressions exhibit different slopes, this provides evidence for ‘break
 points’ in body shape that define ‘physical caste’ systems (Oster
 & Wilson, 1979). We next used a modified ethogram approach (Santos
 et al., 2005; Calheiros et al., 2019) to identify and quantify three
 fungus-gardening behaviours in standardized nest fragments (fungus
 inspection, fungus licking, fungus reconstruction). We tested for
 differences among the three body size categories in the frequency of task
 performance using mixed models with colony identity as a random factor
 followed by posthoc tests (Table S5). ######## 6) Data for analyses of
 fatty acids produced by the fungus in vitro grown under nutritionally
 optimal (1:3 P:C) and suboptimal (3:1 P:C) conditions
 --Sample\_info\_no17\_PC: matrix heading data for R annalyses 1. Number of
 variables: 3 2. Number of cases/rows: 27 3. Variable List: * sample:
 colony\_ID, P:C diet ratio (1:3, 3:1), replicate * colony: 5 parent
 colonies from which fungi were isolated * medium: P:C diet ratio on which
 colony were grown (1:3, 3:1) --Matrix PC: Matrix of data from 37 fatty
 acids for multivariate analyses and PCoA figure 1. Number of variables: 38
 2. Number of cases/rows: 27 3. Variable List: * \[column A\]: colony\_ID\,
 P:C diet ratio \(1:3\, 3:1\)\, replicate * [column B-AL] each of the 37
 fatty acids has it's own column in this matrix (measurement
 spectrum). The fatty acids are listed in Supplementary Table S4 METHODS We
 used an untargeted approach to test for changes in profiles of 37 fatty
 acids putatively involved in signalling (Table S4) when fungal cultivars
 from five A. echinatior colonies were confined to Petri dishes with
 C-biased (1:3) and P-biased (3:1) P:C media (N = 45 Petri dishes; n = 3
 plates per treatment per colony). Based on the FNN results, we selected
 P:C diets at the 8 g/L P + C dilution using the diet recipe from Crumière
 et al. (2021). This analysis included a test for the presence of linoleic
 acid (9,12 octadecadienoic acid (Z,Z)-, methyl ester, Table S4) due to its
 previous identification as an important fatty acid metabolized by L.
 gongylophorus and its association with behavioural modification of
 leafcutter ants (Khadempour et al., 2021). After 42 days of in vitro
 growth, fungal tissue was collected from the surface of growth media,
 flash frozen in liquid nitrogen and stored at -80 ºC for lipid extractions
 and analysis with gas chromatography mass-spectrometry. Values were
 adjusted relative to results from a control media treatment cultured in
 the same way but without being inoculated with the fungal cultivar. To
 test for P:C diet effects on lipid profiles, we performed a Principal
 Coordinate Analysis (PCoA) using the vegan package version 2.6-2 (Oksanen
 et al., 2022) with Bray-Curtis dissimilarity and a PERMANOVA in the vegan
 package with colony of origin included as a random effect to test for
 statistical significance. Candidate compounds were identified using a
 random forest analysis, which is a supervised classification and variable
 selection method for multivariate data, in the randomForest package
 V4.7-1.1 (Liaw & Wiener, 2022). ####### 7) Data from stable
 isotope enrichment experiment in separate text files: 1. Variable List
 common to all isotope text files: * id: dummy id overall * sampleid: dummy
 id for sample * colony\_ID:each of the 10 experimental colonies * Initial
 harvest: the dry mass of the isotopically enriched diet consumed (mg) on
 Day 0 * Time: sample day (0-10) of colony tissues for isotopic analysis *
 Nutrient: either 15N [N] or 13C [C] * enrich: excess amount of isotopic
 enrichment (relative to pre-enriched baseline and sample dry mass) of ug
 15N or ug 13C \*\*\*\*\*Missing values from the isotope enrichment
 datasets are denoted by NA. These few missing values were lost during the
 isotopic analyses. 2.1 Do ants differentially regulate nutrients?
 INDIVIDUAL text files: --fungus\_food.txt 1. Number of variables: 9 2.
 Number of cases/rows: 150 3. Variable List: * Fungus tissue: (gongylidia
 vs. surrounding middle layer hyphae) * Replicate: technical replicate 1-2
 --fungus\_layers.txt 1. Number of variables: 9 2. Number of cases/rows:
 300 3. Variable List: * Fungus\_layer: (Bottom, Middle, Top) three layers
 of fungus garden sampled * Replicate: technical replicate 1-2
 --nutrient\_allocation.txt 1. Number of variables: 8 2. Number of
 cases/rows: 230 3. Variable List: * Caste: (Minor, Media, Major, larvae,
 pupae) 5 ant castes sampled (head-thorax only for adults)
 --nutrient\_processing.txt 1. Number of variables: 9 2. Number of
 cases/rows: 296 3. Variable List: * Adultcast: (Minor, Media, Major) ant
 castes * Ant\_tissue: the body segment sampled (head-thorax vs. gaster)
 --waste\_disposal.txt 1. Number of variables: 8 2. Number of cases/rows:
 110 3. Variable List: * sampletype: trash only METHODS We performed an
 isotopic tracer experiment on Day 0 of the diet choice experiment
 described above (section 2.1) where the P:C diets were enriched with known
 amounts of the heavy isotopes of carbon (13C-enriched glucose) and
 nitrogen (15N-enriched ammonium nitrate) (Table S2). Just before providing
 colonies with enriched diets on Day 0, we collected samples from each
 colony to quantify baseline enrichment of 13C and 15N (i.e., natural
 abundance). Following the heavy isotope ‘pulse’, we thereafter provided
 colonies ‘unenriched’ P:C diets and then collected samples for isotopic
 analyses on Days 1, 2, 5 and 10 (as per Shik et al., 2018). On each
 sampling day, we collected samples of fungus gardens from each colony at
 three vertical layers (top, middle, bottom), and from two tissue types
 (hyphae (all three levels), staphylae (middle layer)). At each sampling
 day, we also sampled adult ants by pooling individuals of three putative
 castes from each colony (n = 5 minor; n = 5 media; n = 3 major). We
 separately analysed ant body segments to distinguish between assimilation
 (head-thorax) and transport/nestmate-dissemination/cultivar-provisioning
 (abdomen) (as per Shik et al., 2018). The isotopic data were used to test
 for caste-specific timelines of nutrient ingestion and allocation.
 Developing ants (larvae) are also called a digestive caste that, among
 other functions, likely mediates nutrient exchange within fungus gardens
 (Erthal et al., 2007). We thus also analysed enrichment timelines in
 larvae and pupae collected from the middle layer of the fungus garden of
 each colony on each sampling day. We further tested for nutrient-specific
 colony waste disposal by collecting trash samples from each colony every
 day. All samples were dried at 50°C for ≥ 24 hours, and were then crushed,
 homogenized, and added in ca. 0.15 mg aliquots into pre-weighed tin
 capsules. Isotopic analyses of 15N/14N and 13C/12C were conducted using an
 Isoprime isotope mass spectrometer (Cheadle Hulme, UK) coupled to a
 Eurovector CN analyser (Pavia, Italy). For subsequent mixed-model
 statistical analyses, we calculated excess μg 15N and μg 13C per g dry
 mass of each sample (hereafter 15N and 13C as per Shik et al., 2018)
 (Table S6). ####