Abstract

The origin of complex worker-caste systems in ants perplexed Darwin1 and has remained an enduring problem for evolutionary and developmental biology2,3,4,5,6. Ants originated approximately 150 million years ago, and produce colonies with winged queen and male castes as well as a wingless worker caste7. In the hyperdiverse genus Pheidole, the wingless worker caste has evolved into two morphologically distinct subcastes—small-headed minor workers and large-headed soldiers8. The wings of queens and males develop from populations of cells in larvae that are called wing imaginal discs7. Although minor workers and soldiers are wingless, vestiges or rudiments of wing imaginal discs appear transiently during soldier development7,9,10,11. Such rudimentary traits are phylogenetically widespread and are primarily used as evidence of common descent, yet their functional importance remains equivocal1,12,13,14. Here we show that the growth of rudimentary wing discs is necessary for regulating allometry—disproportionate scaling—between head and body size to generate large-headed soldiers in the genus Pheidole. We also show that Pheidole colonies have evolved the capacity to socially regulate the growth of rudimentary wing discs to control worker subcaste determination, which allows these colonies to maintain the ratio of minor workers to soldiers. Finally, we provide comparative and experimental evidence that suggests that rudimentary wing discs have facilitated the parallel evolution of complex worker-caste systems across the ants. More generally, rudimentary organs may unexpectedly acquire novel regulatory functions during development to facilitate adaptive evolution.

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All relevant data are included in the paper. The raw data for all analyses used in this study are available from the corresponding author upon request.

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Acknowledgements

We thank R. Johnson, K. Haight, A. Wild, L. Davis, R. Sanwald and A. Nisip for help collecting ants; J. Liebig for help with cuticular hydrocarbon experiments; T. Oakley and P. Ward for help with phylogenetic analyses; and Y. Tomoyasu, I. Ruvinsky, B. Hall, D. E. Wheeler, D. Schoen, A. Shingleton, V. Callier, G. Wray, S. C. Weber, members of the Abouheif Laboratory, M. J. West-Eberhard and E. O. Wilson for comments on the manuscript. We thank the McGill University Advanced BioImaging Facility for imaging support. This work was supported by KLI fellowships (Austria) to R.R. and E.A., and NSERC Discovery Grant and Steacie Fellowship (Canada) and Guggenheim Fellowship (USA) to E.A.

Author information

Author notes

    • Rajendhran Rajakumar

    Present address: Department of Genetics, Harvard Medical School, Boston, MA, USA

    • Marie-Julie Favé

    Present address: Ontario Institute for Cancer Research, Toronto, Ontario, Canada

Affiliations

  1. Department of Biology, McGill University, Montreal, Quebec, Canada

    • Rajendhran Rajakumar
    • , Sophie Koch
    • , Mélanie Couture
    • , Marie-Julie Favé
    • , Angelica Lillico-Ouachour
    • , Travis Chen
    • , Giovanna De Blasis
    • , Arjuna Rajakumar
    • , Dominic Ouellette
    •  & Ehab Abouheif

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Contributions

E.A. and R.R. conceived the project and designed experiments; E.A., R.R., A.R. and S.K. collected ants; R.R., S.K., M.C. and A.R. performed in situ hybridization and immunohistochemistry; R.R., M.-J.F., M.C., S.K. and T.C. performed RNAi; S.K. performed ablations; R.R., G.D.B., A.L.-O., M.C. and T.C. performed pheromone experiments; R.R., M.C., M.-J.F., A.L.-O., S.K., G.D.B. and T.C. performed hormone experiments; T.C. and R.R. performed semi-quantitative PCR; E.A. performed phylogenetic analyses; and D.O. mounted adult specimens. E.A. and R.R. wrote the manuscript with input from co-authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Ehab Abouheif.

Extended data figures and tables

  1. Extended Data Fig. 1 The phylogenetic correlation between presence of a soldier subcaste and presence of discrete inter-subcaste variation in rudimentary wing disc size.

    a, Pagel’s test (difference in log likelihoods = 8.43, P = 0.001) shows that there is a significant phylogenetic correlation between the presence of a soldier subcaste and the presence of discrete inter-subcaste variation in the size of rudimentary wing discs, for 21 species of ants. Grey lines indicate phylogenetic relationships, and the time scale of these relationships is indicated at the bottom in millions of years. The orange bar indicates presence of discrete inter-subcaste variation in the size of rudimentary wing discs, the green bar with two adjacent cartoons indicates presence of minor worker and soldier subcastes, and the dark green bar with three adjacent cartoons indicates presence of minor worker, soldier and supersoldier subcastes. Inter-subcaste variation in the size of rudimentary wing discs in workers (W) and/or minor workers and/or soldiers and/or supersoldiers are at the tip of each branch, where the white-circle drawings represent presence and relative size, within species, of rudimentary wing discs and asterisks indicate absence of rudimentary wing discs. Extended Data Table 1 provides references and descriptions. bk, Adults and rudimentary wing discs of minor workers and/or soldiers and/or workers of C. floridanus (be), S. geminata (fi) and M. trageri (j, k). d, e, h, i, k, Arrowheads indicate the presence of rudimentary wing discs and asterisks indicate the absence of rudimentary wing discs. All comparisons within species are to scale.

  2. Extended Data Fig. 2 Proliferation of rudimentary forewing discs in soldier-destined larvae.

    ac, Immunohistochemistry of rudimentary forewing discs in P. hyatti soldier-destined larvae. DAPI (magenta) stains all nuclei within the cells of rudimentary forewing discs, and phospho-histone H3 (PH3; yellow) stains proliferating cells. The length of the larva from which the rudimentary forewing disc was dissected is given in the bottom right corner. All images are to scale. c′ provides an increased magnification of c, to show nuclear co-localization of DAPI and PH3. Experiments were repeated twice.

  3. Extended Data Fig. 3 Imaginal disc expression of vg is restricted to wing discs during larval development in P. hyatti.

    ac, DAPI staining (a); wingless (wg) expression marking segment boundaries (b) and vestigial (vg) expression in the ventral nerve cord and in wing primordial cells in thoracic segments T2 and T3 (c) is shown for P. hyatti embryos. The three thoracic segments are labelled T1, T2 and T3. Experiments on embryos were repeated twice. df, Larval cartoons depicting leg discs and wing discs in queens (d), leg discs in minor workers (e) and leg discs and rudimentary wing discs in soldiers (f). Asterisks indicate absence of rudimentary wing discs. vg expression is indicated in purple. Lines at bottom right indicate the relative scale. go, vg expression is present in the larval (rudimentary) wing discs and is absent in the head and in leg discs of queens (n = 17) (g, j, m), minor workers (n = 20) (h, k, n) and soldiers (n = 11) (i, l, o). Black arrowheads indicate position of larval head and leg discs, and asterisks indicate absence of rudimentary wing discs. Experiments were repeated at least three times. p, Electrophoresis of PCR products obtained using vg and EF1a primers on wild-type soldier and minor-worker cDNA libraries, each constructed from three terminal-stage larvae. The 1 kb+ ladder is shown as reference and 1, 1/2, 1/4 and 1/8 represent serial dilutions of template cDNA. vg transcript (left) is detected in both soldiers and minor workers using semi-quantitative reverse-transcription PCR. The housekeeping EF1a transcript (right) is detected in both soldiers and minor workers, and levels are comparable between soldier and minor-worker cDNA libraries across dilutions. Negative controls (water with no template) show no contamination. For uncropped gel source data, see Supplementary Fig. 1. Experiments were repeated with independent biological replicates three times.

  4. Extended Data Fig. 4 vg RNAi reduces vg expression and induces apoptosis in rudimentary forewing discs.

    a, Electrophoresis of PCR products obtained using vg and NADH primers on soldier cDNA libraries each constructed from three terminal-stage larvae after vg RNAi or yfp RNAi (control RNAi) injection (Fig. 2a, red arrowhead). The 1 kb+ ladder is shown as reference and 1, 1/2, 1/4 and1/8 represent serial dilutions of template cDNA. vg transcript (left) is detected in both vg RNAi and control RNAi libraries using semi-quantitative reverse-transcription PCR. The housekeeping NADH transcript (right) is detected in both vg RNAi and control RNAi larvae, and levels are comparable between vg RNAi and control RNAi cDNA libraries across dilutions. Negative controls (water with no template) show no contamination. Experiments were repeated twice as independent biological replicates. For uncropped gel source data, see Supplementary Fig. 1. b, c, Apoptosis revealed by TUNEL assay in rudimentary forewing disc in control RNAi (b) and vg RNAi (c). Compared to control RNAi, vg RNAi induces apoptosis along the dorso-ventral margin of rudimentary forewing discs, where wild-type vg expression is normally strongest (b, c, black arrowhead). Experiments were repeated at least twice.

  5. Extended Data Fig. 5 Rudimentary forewing discs regulate size and disproportionate head-to-body scaling.

    a, Wild-type adult minor worker. b, vg RNAi intermediate adult. c, Wild-type soldier adult. Images to scale. d, Comparing ratios of log(head width (μm)) to log(body length (μm)) (log HW:log BL), between yfp RNAi (control RNAi, n = 23) and vg RNAi (n = 35); the box plot shows mean (+), interquartile range (bars), minimum-to-maximum values (whiskers); all points represent individual ants. Two-tailed Mann–Whitney U-test, U = 219, **P = 0.0031. e, Percentage change in body length (μm) versus percentage change in head width (μm) of vg RNAi compared to a 1:1 line. Each point represents (absolute(HW − HWcontrol RNAi average)/HWcontrol RNAi average) × 100 and/or (absolute(BL − BLcontrol RNAi average)/BLcontrol RNAi average) × 100. f, Comparing the percentage change in body length (μm) and head width (μm) after vg RNAi (n = 35). The box plot shows mean (+), interquartile range (bars), maximum-to-minimum values (whiskers); all points represent individual ants. One-tailed Mann–Whitney U-test, U = 470, *P = 0.0477. g, Wild-type minor worker. l, yfp RNAi (control RNAi) soldier. gl, vg RNAi individuals showing a range of intermediates between minor worker and soldier (see Fig. 2i). Wild-type minor worker is shown for reference in a, g. All image comparisons are to scale. Experiments were repeated at least three times. mt, Electrosurgical ablation of leg and rudimentary forewing discs in soldier-destined larvae (Fig. 2a, red arrowhead). m, Wild-type soldier, leg and rudimentary forewing discs. n, Site of leg disc cauterization shown by melanized cuticle. o, Ablation of leg disc (DAPI, red asterisk). p, Site of rudimentary forewing disc cauterization shown by melanized cuticle. q, Ablation of rudimentary forewing disc (DAPI, red asterisk). White or black arrowheads indicate the presence of rudimentary forewing discs. All images are to scale. r, Comparing log(head width (μm)) between leg disc ablation (n = 16) and rudimentary forewing disc ablation (n = 16); one-tailed Mann–Whitney U-test, U = 82, *P = 0.0432. s, Comparing log(body length (μm)) between leg disc ablation (n = 16) and rudimentary forewing disc ablation (n = 16). One-tailed unequal variance t-test, t = 1.77, d.f. = 22.34,*P = 0.045. t, Comparing ratio of log(head width (μm)) to log(body length(μm)), between leg disc ablation (n = 16) and rudimentary forewing disc ablation (n = 16). One-tailed unpaired t-test, t = 2.07, d.f. = 30, *P = 0.0234. The box plots in d, f, rt show mean (+), interquartile range (bars) and minimum-to-maximum values (whiskers); all points represent individual ants. Experiments were repeated at least twice.

  6. Extended Data Fig. 6 The function of rudimentary forewing discs in regulating disproportionate head-to-body scaling is specific to the soldier subcaste.

    vg RNAi on bipotential larvae (Fig. 2a, orange arrowhead). a, yfp RNAi (control RNAi) minor worker. b, vg RNAi minor worker. c, vg RNAi intermediate. d, yfp RNAi (control RNAi) soldier. Image comparisons to scale. e, log(body length (μm)) versus log(head width (μm)), comparing minor worker head-to-body scaling of yfp RNAi (control RNAi, n = 69) and vg RNAi (n = 84). ANCOVA: slope, F = 0.162, d.f. = 149, P = 0.69; y intercept, F = 0.4755, d.f. = 150, P = 0.49). f, log(body length (μm)) versus log(head width (μm)), comparing head-to-body scaling of yfp RNAi (control RNAi; n = 6) and vg RNAi (n = 4) ants that initiated soldier development. ANCOVA: F = 7.44, d.f. = 6, P = 0.0343. g, Comparing log(head width (μm)) between control RNAi (n = 69) and vg RNAi (n = 84) minor workers. Two-tailed unpaired t-test: t = 1.62, d.f. = 151, P = 0.1074. h, Comparing log(body length (μm)) between control RNAi (n = 69) and vg RNAi (n = 84) minor workers. Two-tailed unpaired t-test: t = 1.54, d.f. = 151, P = 0.1249. i, Comparing ratios of log(head width (μm)) to log(body length (μm)) between control RNAi (n = 69) and vg RNAi (n = 84) minor workers. Two-tailed unpaired t-test: t = 0.26, d.f. = 151, P = 0.80. Experiments were repeated at least three times. jp, vg RNAi on male-destined larvae (Fig. 2a, green arrowhead). j, Bar graph showing percentage of individual ants affected (red) after yfp RNAi (control RNAi; n = 0 out of 23) and vg RNAi (n = 9 out of 21). Two-tailed Fisher’s exact test: P = 0.0004. k, l, Wings from yfp RNAi (control RNAi) (k) and vg RNAi (l) males. Image comparisons are to scale. mo, Head-to-body scaling of yfp RNAi (control RNAi; n = 23) and vg RNAi males (n = 21), comparing log(head width (μm)), two-tailed Mann–Whitney U-test, U = 222.5, P = 0.6626 (m); log(body length (μm)), two-tailed Mann–Whitney U-test, U = 198, P = 0.3157 (n); and ratio of log(head width (μm)) to log(body length (μm)) (o), between control RNAi males and vg RNAi males, two-tailed unpaired t-test, t = 0.18, d.f. = 42, P = 0.86. p, log(body length (μm)) versus log(head width (μm)) comparison of head-to-body scaling, between yfp RNAi (control RNAi; n = 23) and vg RNAi males (n = 21). ANCOVA: slope, F = 3.111, d.f. = 40, P = 0.0854; y intercept, F = 0.076, d.f. = 41, P = 0.7837. Experiments were repeated at least three times. The box plots in gi and mo show mean (+), interquartile range (bars) and minimum-to-maximum values (whiskers); all points represent individual ants.

  7. Extended Data Fig. 7 Soldier inhibitory pheromone regulates size and disproportionate head-to-body scaling.

    ak, Effect of social inhibition on soldier-destined larvae (Fig. 2a, red arrowhead). a, Wild-type adult minor worker. b, Intermediate adult resulting from soldier-destined larvae being raised in colonies composed of 100% soldiers (high inhibition). c, Representative adult soldier resulting from soldier-destined larvae being raised in colonies composed of 100% minor workers (no inhibition). d, Comparing ratios of log(head width (μm)) to log(body length (μm)) between 100% minor worker (n = 24) and 100% soldier (n = 35). The box plot shows mean (+), interquartile range (bars) and minimum-to-maximum values (whiskers); all points represent individual ants. Two-tailed Mann–Whitney U-test, U = 155, ****P < 0.0001. e, Percentage change in body length (μm) versus percentage change in head width (μm) of 100% soldiers, compared to a 1:1 line. Each point represents (absolute(HW − HW100% minor worker average)/HW100% minor worker average) × 100 and/or (absolute(BL − BL100% minor worker average)/BL100% minor worker average) × 100. f, Comparing percentage change in body length (μm) and head width (μm) following high inhibition (100% soldiers; n = 35). One-tailed Mann–Whitney U-test, U = 421, *P = 0.0121. g, Wild-type minor worker. h–j, 100% soldier-raised ants showing a range of intermediates between minor workers and soldiers (see Fig. 3e). k, 100% minor-worker control soldier. All image comparisons are to scale. Experiments were repeated at least three times. lp, Application of soldier cuticular hydrocarbon extract (CHCs) to soldier-destined larvae (Fig. 2a, red arrowhead). l, Wild-type minor worker and individual ants treated with soldier CHCs and hexane solvent (control). m, Comparing slopes of hexane solvent control (n = 21) and soldier CHCs (n = 19). ANCOVA, F = 6.84, d.f. = 36, P = 0.0129. n, log(head width (μm)); one-tailed Mann–Whitney U-test, U = 114, **P = 0.0099. o, log(body length (μm)); one-tailed Mann–Whitney U-test, U = 117, *P = 0.0126. p, Ratio of log(head width (μm)) to log(body length (μm)); one-tailed Mann–Whitney U-test, U = 125, *P = 0.0221. Wild-type minor worker is shown for reference in a, g, l. The box plots in d, f, np show mean (+), interquartile range (bars), minimum-to-maximum values (whiskers); all points represent individual ants.

  8. Extended Data Fig. 8 Juvenile hormone and inhibitory pheromone regulate disc-dependent disproportionate head-to-body scaling and disc-independent proportional head:body scaling.

    Effect of juvenile-hormone activation and social inhibition on bipotential larvae (Fig. 2a, orange arrowhead). a, Absence of rudimentary wing discs in minor worker larvae exposed to solvent control with no inhibition (‘acetone + 100% MW’). a, e, i, m, Arrowheads indicate the presence of rudimentary wing discs and asterisks indicate the absence of rudimentary wing discs. b, Plot of head-to-body scaling of acetone + 100% minor worker, and juvenile-hormone activation with no inhibition (‘JH + 100% MW’); the majority of larvae treated with juvenile hormone develop into soldiers, and some develop into large minor workers. c, d, Comparing between acetone + 100% minor worker (n = 7) and juvenile hormone + 100% minor worker (n = 17) treatments of individuals that developed into the soldier size distribution. c, log(body length (μm)); two-tailed unpaired t-test; t = 5.25, d.f. = 22, ****P < 0.0001. d, log(head width (μm)); two-tailed unpaired t-test, t = 3.50, d.f. = 22, **P = 0.002. e, Initiation of growth of rudimentary forewing discs in minor worker larvae exposed to juvenile hormone + 100% minor worker. f, Plot of head-to-body scaling between acetone + 100% minor worker (n = 114) and juvenile hormone + 100% minor worker (n = 29) of individuals that developed into the minor-worker size distribution. ANCOVA: F = 7.12, d.f. = 139, P = 0.0085. g, h, Comparing between acetone + 100% minor worker (n = 114) and juvenile hormone + 100% minor worker (n = 29) treatments of individuals that developed into the minor-worker size distribution. g, log(body length (μm)); two-tailed Mann–Whitney U-test, U = 463, ****P < 0.0001. h, log(head width (μm)); two-tailed unequal variance t-test, t = 5.19, d.f. = 32, ****P < 0.0001. i, Absence of growth of rudimentary wing discs in minor worker larvae exposed to juvenile-hormone activation with high inhibition (‘JH + 100% SD’). j, Plot of head-to-body scaling between solvent control with high inhibition (‘acetone + 100% SD’; n = 88) and juvenile hormone + 100% soldiers (n = 46) of individuals that developed into the minor-worker size distribution. ANCOVA: slope, F = 0.54, d.f. = 130, P = 0.47; y intercept, F = 27.2, d.f. = 131, P < 0.0001. k, l, Comparing between acetone + 100% soldiers (n = 88) and juvenile hormone + 100% soldiers (n = 46) treatments of individuals that developed into the minor-worker size distribution. k, log(body length (μm)); two-tailed unequal variance t-test, t = 4.43, d.f. = 69, ****P < 0.0001. l, log(head width (μm)); two-tailed unequal variance t-test, t = 6.69, d.f. = 68,****P < 0.0001. m, Absence of rudimentary wing disc growth in minor worker larvae exposed to acetone + 100% soldiers. n, Plot of head-to-body scaling between acetone + 100% minor workers (n = 114) and acetone + 100% soldiers (n = 88) treatments of individuals that developed into the minor-worker size distribution. ANCOVA: slope, F = 2.84, d.f. = 198, P = 0.0937; y intercept, F = 20.77, d.f. = 199, P < 0.0001. o, p, Comparing between acetone + 100% minor workers (n = 114) and acetone + 100% soldiers (n = 88) treatments of individuals that developed into the minor-worker size distribution. o, log(body length (μm)); two-tailed Mann–Whitney U-test, U = 3178, ****P < 0.0001. p, log(head width (μm)); two-tailed unpaired t-test, t = 0.28, d.f. = 200, P = 0.7823. Bonferroni correction was applied to comparison of log(head width) and log(body length). The box plots in c, d, g, h, k, l, o, p show mean (+), interquartile range (bars) and minimum-to-maximum values (whiskers); all points represent individual ants. Plots in b, f, j, n show linear regressions in which the x axis is log(body length (μm)) and the y axis is log(head width (μm)). All images are to scale. Experiments were repeated at least three times.

  9. Extended Data Table 1 Description and references for presence or absence of soldier subcaste and discrete variation in rudimentary wing disc size across 21 ant species

Supplementary information

  1. Supplementary Figures

    This file contains Supplementary Figures 1 and 2. Supplementary Information Figure 1: Uncropped PCR agarose gel source images. Uncropped gel source images for Extended Data Fig. 3p and Extended Data Fig. 4a. Supplementary Information Figure 2: vestigial amino acid sequence alignment. To confirm orthology of the vestigial (vg) fragment, amino acid alignment of vg showing conservation, including the TONDU/vestigial domain, between Pheidole hyatti and other holometabolous insects: Solenopsis invicta, Apis mellifera, Nasonia vitripennis, Pieris rapae, Papilio xuthus and Drosophila melanogaster. This fragment used for vg in situ probe and vg dsRNA.

  2. Reporting Summary

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DOI

https://doi.org/10.1038/s41586-018-0613-1

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