Nutritional niches reveal fundamental domestication trade-offs in fungus-farming ants

Abstract

During crop domestication, human farmers traded greater productivity for higher crop vulnerability outside specialized cultivation conditions. We found a similar domestication trade-off across the major co-evolutionary transitions in the farming systems of attine ants. First, the fundamental nutritional niches of cultivars narrowed over ~60 million years of naturally selected domestication, and laboratory experiments showed that ant farmers representing subsequent domestication stages strictly regulate protein harvest relative to cultivar fundamental nutritional niches. Second, ants with different farming systems differed in their abilities to harvest the resources that best matched the nutritional needs of their fungal cultivars. This was assessed by quantifying realized nutritional niches from analyses of items collected from the mandibles of laden ant foragers in the field. Third, extensive field collections suggest that among-colony genetic diversity of cultivars in small-scale farms may offer population-wide resilience benefits that species with large-scale farming colonies achieve by more elaborate and demanding practices to cultivate less diverse crops. Our results underscore that naturally selected farming systems have the potential to shed light on nutritional trade-offs that shaped the course of culturally evolved human farming.

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Fig. 1: Assessment of domestication trade-offs across transitions in mutualistic farming practices of fungus-farming ants.
Fig. 2: Heatmaps of cultivar growth used to describe cultivar FNNs.
Fig. 3: Laboratory experiments comparing foraging decisions of colonies of three representative attine ant species.
Fig. 4: Fungus-farming ants representing different stages of cultivar domestication and organizational complexity navigate nutritional landscapes to harvest RNNs relative to their cultivar’s FNNs.
Fig. 5: Comparison of two fully domesticated farming systems with different operational scales and ecological impacts.
Fig. 6: Colonies of P. cornetzi co-exist within nutritional foraging environments despite farming different cultivar haplotypes with variable properties of nutrient yield and growth.

Data availability

The DNA sequences generated during this study are available from the National Center for Biotechnology Information’s GenBank database. This includes the data for the 215 ant specimens (accession codes for the cytochrome c oxidase I sequences are provided in Supplementary Table 13), the 99 fungal cultivar samples (accession codes for the LSU and ITS sequences are provided in Supplementary Table 14) and plant sample ITS1 sequences harvested by colonies of A. colombica (accession codes are provided in Supplementary Table 8). Ant sequence datasets and supporting information are also deposited in BOLD66 under the project titled DS-ATTINENG “Nutritional niches reveal fundamental domestication trade-offs in fungus-farming ants” (http://dx.doi.org/10.5883/DS-ATTINENG).

Code availability

No custom code was generated for this study. The sequence alignment matrices and Newick files used to generate the phylogenies shown in Fig. 5 (both fungal and ant trees) and Extended Data Fig. 8 are available as text files in Supplementary Data 13. All of the code used to generate the results will be made available upon request.

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Acknowledgements

STRI provided support and access to facilities in Gamboa. The Autoridad Nacional del Ambiente y el Mar (ANAM) gave permission to the laboratory groups of J.J.B. and J.Z.S. to sample attine ants in Panama and export them to Denmark. We thank students of the Tropical Behavioural Ecology and Evolution field course in 2015 for additional assistance with collecting colonies. J.Z.S. was supported by a postdoctoral fellowship via a Smithsonian Institution Competitive Grant to W.T.W., J.J.B. and J.Z.S., an EU Marie Sklodowska-Curie International Incoming Fellowship (327940), the Centre for Social Evolution at the University of Copenhagen (Danish National Research Foundation: DNRF57), an ERC Advanced grant (ANTS: 323085) to J.J.B. and an ERC Starting Grant to J.Z.S. (ELEVATE: 757810). J.C.S. was supported by SJU start-up funds and NSF-DEB number 2016372.

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Contributions

J.Z.S., J.J.B. and W.T.W. conceived of and designed the study. J.Z.S., E.B.G., M.F., A.J.J.C., D.A.D. and P.W.K. performed the fieldwork, collected the colonies, isolated the fungal cultivars and performed the in vitro experiments with fungal cultivars. D.A.D., J.Z.S., P.W.K. and A.J.J.C. extracted DNA and performed DNA barcoding analyses for ants and fungal cultivars. P.W.K., J.Z.S. and J.H. performed the microsatellite analyses. X.A. and J.Z.S. performed the statistical analyses. J.C.S. performed the phylogenetics analyses. J.Z.S. and J.J.B. wrote the initial draft of the manuscript. J.Z.S., P.W.K., D.A.D., J.C.S., A.J.J.C., X.A., J.H., W.T.W. and J.J.B. contributed to interpreting the data and editing subsequent drafts of the manuscript.

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Correspondence to Jonathan Z. Shik.

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Extended data

Extended Data Fig. 1 Cultivars exhibited consistent FNNs for hyphal growth when isolated from different colonies of each attine species, supporting that the heatmaps based on species means (Fig. 2, Fig. 4b,c) accurately represent each cultivar’s FNN.

Additional details about how these heatmaps were generated and how they were interpreted are provided in Fig. 2. Heatmaps are provided here for attine species where multiple colonies were sampled. Additional colony-level heatmaps for P. cornetzi and A. colombica are provided in Fig. 6a. Collection IDs corresponding to experiment IDs were: Ae_3 [177625], Ae_2 [177624], Ae_1 [177609], Tz_30 [177632], Tz_32 [177634], Sa_23 [177623], Sa_21 [177614], Ad_5 [177629], Ad_6 [177630], Ad_7 [177631], Cc_12 [37861], Cc_13 [37862], and Cc14 [37864] (Supplementary Table 3). As in Fig. 2, least-square regressions showed that each of the response surface regressions producing the heatmap colour-gradients was significant (Supplementary Table 1).

Extended Data Fig. 2 Cultivars of the higher-neoattine genera Sericomyrmex, Mycetomoellerius, Paratrachymyrmex (purple) and the leafcutter genera Acromyrmex (light green) and Atta (dark green) often exhibited mortality when confined to media with macronutrient mixtures outside their FNNs.

As shown in the figure legend, dark red indicates 100% survival and dark blue indicates 0% survival (a clear inoculation plug or a failure to colonize the agar plate from the inoculation plug). Percent mortality data were averaged across cultivars isolated from colonies of the same attine species (n = 2, M. zeteki; n = 3, P. cornetzi; n = 1, S. amabilis; n = 3, A. echinatior; n = 3, A. colombica, Supplementary Table 2). Mortality data were not recorded for the S. amabilis colony Sa_23. Small white areas on some of the plots indicate 100% survival.

Extended Data Fig. 3 The emergence of large-scale fungus farming by Atta leafcutter ants cannot be explained solely by an increase in intrinsic cultivar growth rate.

Coloured bars (corresponding to the farming transitions in the schematic tree of Fig. 1a) represent in vitro growth rates of cultivars during 30 days on a standard PDA medium. Vertical lines separate the major farming transitions that distinguish ten attine species sympatrically inhabiting the Panamanian rainforest of Soberanía park (see caption Fig. 1a). ANOVA showed that attine species farmed cultivars with significantly different growth rates after 30-days (F7,41 = 24.42, p < 0.0001) and letters indicate pairwise differences that were significant at P < 0.05 in post-hoc Tukey tests. Numbers inside bars indicate numbers of colonies from which cultivars were isolated and used to calculate mean growth rates (+ SE) (Supplementary Table 3). The cultivars of C. rimosus and C. longiscapus were not included in the statistical analyses because we had no replicate colonies for these two ant species.

Extended Data Fig. 4 Cumulative amounts of protein and carbohydrates collected over 15 days by whole colonies of a) P. cornetzi and b) A. colombica.

While colonies of P. cornetzi and M. smithii30 avoided protein-biased substrates with ratios above 1:3 P:C, colonies of A. colombica collected large amounts of the 1:1 P:C agar that P. cornetzi and M. smithii avoided. Colonies of A. colombica further collected statistically similar levels of carbohydrates on 1:1, 1:3, and 1:6 P:C substrates (Supplementary Table 4). This tolerance of higher protein levels enables A. colombica to sustain higher carbohydrate intake levels on 1:1 P:C diets. Letters indicate pairwise differences that were significant at P < 0.05 (post-hoc Tukey tests) (Supplementary Table 4). These intake plots (means + SE) provide an alternative single-nutrient presentation of the bi-variate intake data presented in Fig. 3b.

Extended Data Fig. 5 Colonies of P. cornetzi increasingly faced crop failure when confined to P:C macronutrient mixtures with excess protein relative to carbohydrates (χ²4 = 17.9; p = 0.001).

While similar results were observed for M. smithii30, no colonies of A. colombica experienced crop failure, even when confined to the same protein-biased substrates. Cultivar survival probabilities were estimated with a Cox proportional hazards model where substrate treatment was the explanatory variable, initial garden mass was a covariate, and days remaining in the feeding experiment was the response variable.

Extended Data Fig. 6 STRUCTURE analyses of 9 microsatellite loci indicated that workers from 28 Atta colombica colonies in the Gamboa area of Soberanía park represent a single interbreeding population.

Specifically, the mean log-likelihood was highest at K = 1 (although ΔK cannot be assessed at K = 1). When K was set to two, no individuals could be assigned to a single cluster. This Structure plot included a burn-in of 250,000, MCMC reps of 500,000 and 25 iterations per K.

Extended Data Fig. 7 STRUCTURE analyses of nuclear genetic marker data from nine microsatellite loci from 297 Paratrachymyrmex and Mycetomoellerius workers, from 194 and 103 colonies respectively, indicate that P. cornetzi and P. ADG3274 represent distinct ant species within the total community of eight species belonging to these two ant genera in Soberanía Park.

a, A STRUCTURE analysis across all samples (N = 297) indicated that the most likely population subdivision is two species because K = 2 reached the highest (ΔK) resolution (burn-in 200000, MCMC reps = 5000000, 25 iterations per K). This analysis separated samples that were all previously identified as P. cornetzi using morphological characters. It also grouped three Barcode of Life Data System66 (www.boldsystems.org) haplotypes (AAP2324, ABY9919, ADG3341) separately from the newly recognized cryptic species P. ADG3274, that otherwise clustered with the remaining species (light blue). Although ΔK cannot be calculated for K = 1, the log-likelihood at K = 1 (−11121.864) was substantially lower than the one at K = 2 (−9397.616), suggesting that ‘no population subdivision’ is unlikely, consistent with all other species being morphologically distinct. b, A similar STRUCTURE analysis using those individuals assigned to cluster 1 in panel a (N = 156) indicated that three barcoded haplotypes (AAP2324, ABY9919, ADG3341) located in the BOLD database represent a single interbreeding population within Soberanía Park called P. cornetzi. Specifically, ΔK was highest at 2 (1305.79) but the mean log-likelihood was similar for K = 1 (−3849.092) and K = 2 (−3698.860), suggesting that any existing population structure here is weak. The haplotypes also do not separate the dark blue and light blue bars across our sampling sites consistent with failure to reliably assign individual workers to clusters 1 or 2 as expected under high admixture rates. One sample was identified as P. ADG3274 by barcoding analysis (177340) (Fig. 5, Extended Data Fig. 8), but could not be reliably distinguished from P. cornetzi by this STRUCTURE analysis. c, A STRUCTURE plot generated for the remaining (non-P. cornetzi) individuals (N = 156), indicated that K = 8 was the most likely subdivision, with little to no admixture between these species. The cryptic species P. ADG3274 forms a distinct cluster in this analysis. These genetic species assignments correspond well to species identities obtained by morphological criteria and barcoding (Extended Data Fig. 8) and included four currently named species (P. bugnioni, M. isthmicus, M. opulentus, and M. zeteki), and three unnamed species (M. PAN004 and M. PAN002, and P. ADG3274). The ants M. PAN004 and M. PAN002 were placed in the genus Mycetomoellerius35 since they grouped with M. isthmicus, M. opulentus, and M. zeteki in the CO1 barcoding tree (Extended Data Fig. 8, Supplementary Table 13) and because they exhibited the required morphological characters to justify this decision. This STRUCTURE analysis also suggested there are potentially more than one species within the M. zeteki and M. PAN002 clusters, and that six stray samples (yellow bars) may also be distinct despite being morphologically grouped similar to P. bugnioni (n = 2 samples), M. isthmicus (n = 1), T. PAN004 (n = 1), and M. zeteki (n = 2). Overall, the results of this analysis confirm that perhaps only ~ half of the species are known even in a well-studied insect group in the tropics. The potential taxonomic implications of the P. cornetzi and P. ADG3274 species complex are beyond the scope of the present study. Samples labelled with “no barcode” were ants that were identified as belonging to Paratrachymyrmex and Mycetomoellerius using morphological characters but were not included in barcoding analyses.

Extended Data Fig. 8 A majority-rule consensus tree based on COI sequences of 185 Paratrachymyrmex and Mycetomoellerius ants supported our microsatellite analyses as the same eight species were recognized as co-occurring across Soberanía National Park.

Three of these species belonged to the genus Paratrachymyrmex (P. cornetzi [n = 130], P. ADG3274 [n = 15], P. bugnioni [n = 2]) and five belonged to the genus Mycetomoellerius (M. zeteki [n = 8], M. opulentus [n = 2], M. isthmicus [n = 7], M. PAN002 [n = 10], M. PAN004 [n = 11]; Supplementary Table 13)22,35 which is sister to the genus Sericomyrmex. This tree contains 9 additional public P. cornetzi COI sequences deposited at the Barcode of Life Data System database66 (BOLD, www.boldsystems.org) that supported that the main P. cornetzi haplotype occurs at least from Ecuador to Costa Rica (N = 3 specimens), and that the cryptic P. cornetzi haplotype [ADG3274] is distributed at least from the Canal Zone in Panama to La Selva forest in Costa Rica (N = 6 specimens). The tree was rooted by a worker of M. smithii.

Extended Data Fig. 9 Each of the five fungal haplotypes cultivated by P. cornetzi was widely distributed across Soberanía National Park.

For a map of these sampling locations, see Extended Data Fig. 10, Supplementary Table 14. We combined cultivars from La Seda plots 1 and 2 for this figure since these plots were within 200 m of each other. Four of the five fungal haplotypes that we obtained from these Panama study plots matched cultivar haplotypes sampled from Brazilian colonies of Paratrachymyrmex and Sericomyrmex by Solomon et al.35, suggesting broad geographic distributions extending across Central and South America. Additional Soberanía Park sampling localities where no specific plots were assigned (so no labels in Extended Data Fig. 10) included La Laguna (N 9.1196, W −79.6942), Horse Patch (N 9.11990, W −79.70730), and Barro Colorado Island (BCI: N 9.15744, W −79.83523).

Extended Data Fig. 10 We mapped local-scale higher neoattine colony distributions within the six 20-m2 study plots distributed across Soberanía National Park.

This totalled 263 colonies (9 species) that were mapped by tracking foraging workers back to their nests after which pinned specimens were screened in microsatellite (Extended Data Fig. 7) DNA barcoding analyses (Extended Data Fig. 8, Supplementary Table 13), as well as with morphological identification. We located colonies by placing bait (polenta) in the leaf litter and observing foragers for 27 ± 3 searching hours per plot (N = 163 total searching hours). The ant M. opulentus was recorded within the forest, but not within any of the 20 m2 plots. Nests were marked by flags at nest entrances, which are inconspicuous holes under the leaf litter the circumference of a pencil. Within 20-m2 plots, we observed 43 ± 17 (range: 21–66) higher-neoattine colonies representing 5 ± 1 (range: 4–7) higher-neoattine species. Dashed lines indicate boundaries of 20m2 plots, and an additional 5 m is provided because some colonies were marked despite occurring just beyond the 20-m2 plot border lines. Trees > 1 m circumference dbh were also mapped. Plot abbreviations were as follows: BP1: Bird Plot [N 9.16324, W −79.74553], GP1: Gamboa Plot [N 9.11489, W −79.69784], JG1: Juan Grande [N 9.13528, W −79.72141], LS1: La Seda [N 9.15451, W −79.73583], LS2: La Seda [N 9.15624, W −79.73472], TB1: Thomas Barbour [N 9.15744; W −79.83523]). The satellite image of the Panama Canal Zone is from Google Maps (credit to the images data supplier’s used in the satellite image (Billeder © CNES / Airbus, Landsat / Copernicus, Maxar Technologies, U.S. Geological Survey, Kortdata © 2020)).

Supplementary information

Supplementary Information

Supplementary methods, Figs. 1–3, Tables 1–12 and references.

Reporting Summary

Supplementary Tables 13 and 14

Supplementary Table 13 Supporting information for the worker ant samples used in the DNA barcoding analyses, along with the associated GenBank accession codes for the cytochrome c oxidase I DNA sequences. Supplementary Table 14 Supporting information for the fungal samples used in the DNA barcoding analyses, along with the associated GenBank accession codes for the ITS and LSU DNA sequences.

Supplementary Data 1

Supplementary Data 1 Ant sequence matrix and tree for Fig. 5 (sequence matrix–aligned sequence data).

Supplementary Data 2

Supplementary Data 2 Fungus sequence matrix and tree for Fig. 5 (a pruned version is used in Fig. 6b) (sequence matrix–aligned sequence data).

Supplementary Data 3

Supplementary Data 3 Ant sequence matrix and tree for Extended Data Fig. 8 (sequence matrix–aligned sequence data).

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Shik, J.Z., Kooij, P.W., Donoso, D.A. et al. Nutritional niches reveal fundamental domestication trade-offs in fungus-farming ants. Nat Ecol Evol (2020). https://doi.org/10.1038/s41559-020-01314-x

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