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.

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1. Laboratory experiment with nutritionally defined P:C diets
   --hov_test: total intake of diets by ants (mg)

2. Number of variables: 4

3. Number of cases/rows: 20

4. 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.

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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).

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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

4. 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).

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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).

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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).