Abstract
Adaptive phenotypic plasticity allows organisms to display distinct phenotypes in response to variable environments, but little is known about the genomic changes that promote the evolution of plasticity on a macroevolutionary scale. Here, combining tissue-specific transcriptomics, comparative genomics and genome editing, we show that temperature-mediated plasticity in the size of butterfly eyespot wing patterns, a derived seasonal adaptation estimated to have evolved ~60 million years ago at the base of the satyrid clade (~2,700 extant species), is fuelled by the recruitment of a Hox gene Antennapedia (Antp) to eyespot development. In satyrid butterflies, Antp regulates eyespot size in a temperature-dependent manner, increasing plasticity levels. The cooption of Antp to eyespots was driven by the evolution of a novel eyespot-specific promoter in satyrid genomes, which when disrupted in a model satyrid, Bicyclus anynana, reduced plasticity levels. We show that a taxon-specific cis-regulatory innovation in a conserved developmental gene fuelled the evolution of adaptive phenotypic plasticity across a large clade of animals.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$32.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Data availability
All data required for reproducing and extending the study are available in the main text or the Supplementary Information. Raw RNA-seq data for Bicyclus anynana are available under NCBI BioProject PRJNA1268022. Raw genome sequencing and assembly data for Junonia almana are available under NCBI BioProject PRJNA1300591. Lists of differentially expressed genes and enriched Gene Ontology terms generated in the transcriptomic analysis and lists of HCR probe sequences are available via figshare at https://doi.org/10.6084/m9.figshare.c.8026060.v1 (ref. 55). Source data are provided with this paper.
References
West-Eberhard, M. J. Phenotypic plasticity and the origins of diversity. Annu. Rev. Ecol. Syst. 20, 249–278 (1989).
Stearns, S. C. The evolutionary significance of phenotypic plasticity. Bioscience 39, 436–445 (1989).
Pfennig, D. W. et al. Phenotypic plasticity’s impacts on diversification and speciation. Trends Ecol. Evol. 25, 459–467 (2010).
Tian, S. & Monteiro, A. A transcriptomic atlas underlying developmental plasticity of seasonal forms of Bicyclus anynana butterflies. Mol. Biol. Evol. 39, msac126 (2022).
Qiu, B. et al. Canalized gene expression during development mediates caste differentiation in ants. Nat. Ecol. Evol. 6, 1753–1765 (2022).
Casasa, S., Zattara, E. E. & Moczek, A. P. Nutrition-responsive gene expression and the developmental evolution of insect polyphenism. Nat. Ecol. Evol. 4, 970–978 (2020).
Lafuente, E., Duneau, D. & Beldade, P. Genetic basis of thermal plasticity variation in Drosophila melanogaster body size. PLoS Genet. 14, e1007686 (2018).
Lafuente, E. & Beldade, P. Genomics of developmental plasticity in animals. Front. Genet. 10, 720 (2019).
van der Burg, K. R. et al. Genomic architecture of a genetically assimilated seasonal color pattern. Science 370, 721–725 (2020).
Lafuente, E., Duneau, D. & Beldade, P. Genetic basis of variation in thermal developmental plasticity for Drosophila melanogaster body pigmentation. Mol. Ecol. 33, e17294 (2024).
Brakefield, P. M. & Reitsma, N. Phenotypic plasticity, seasonal climate and the population biology of Bicyclus butterflies (Satyridae) in Malawi. Ecol. Entomol. 16, 291–303 (1991).
Brakefield, P. M. & Larsen, T. B. The evolutionary significance of dry and wet season forms in some tropical butterflies. Biol. J. Linn. Soc. 22, 1–12 (1984).
Lyytinen, A., Brakefield, P. M., Lindström, L. & Mappes, J. Does predation maintain eyespot plasticity in Bicyclus anynana?. Proc. R. Soc. Lond. Ser. B Biol. Sci. 271, 279–283 (2004).
Prudic, K. L., Stoehr, A. M., Wasik, B. R. & Monteiro, A. Eyespots deflect predator attack increasing fitness and promoting the evolution of phenotypic plasticity. Proc. R. Soc. B Biol. Sci. 282, 20141531 (2015).
Oostra, V., Brakefield, P. M., Hiltemann, Y., Zwaan, B. J. & Brattström, O. On the fate of seasonally plastic traits in a rainforest butterfly under relaxed selection. Ecol. Evol. 4, 2654–2667 (2014).
van Bergen, E. et al. Conserved patterns of integrated developmental plasticity in a group of polyphenic tropical butterflies. BMC Evol. Biol. 17, 1–13 (2017).
Bhardwaj, S. et al. Origin of the mechanism of phenotypic plasticity in satyrid butterfly eyespots. Elife 9, e49544 (2020).
Molleman, F. et al. Larval growth rate is not a major determinant of adult wing shape and eyespot size in the seasonally polyphenic butterfly Melanitis leda. PeerJ 12, e18295 (2024).
Peña, C. et al. Higher level phylogeny of Satyrinae butterflies (Lepidoptera: Nymphalidae) based on DNA sequence data. Mol. Phylogenet. Evol. 40, 29–49 (2006).
Chazot, N. et al. Conserved ancestral tropical niche but different continental histories explain the latitudinal diversity gradient in brush-footed butterflies. Nat. Commun. 12, 5717 (2021).
Oostra, V. et al. Translating environmental gradients into discontinuous reaction norms via hormone signalling in a polyphenic butterfly. Proc. R. Soc. B Biol. Sci. 278, 789–797 (2011).
Mateus, A. R. A. et al. Adaptive developmental plasticity: compartmentalized responses to environmental cues and to corresponding internal signals provide phenotypic flexibility. BMC Biol. 12, 1–15 (2014).
Monteiro, A. et al. Differential expression of ecdysone receptor leads to variation in phenotypic plasticity across serial homologs. PLoS Genet. 11, e1005529 (2015).
Kooi, R. E. & Brakefield, P. M. The critical period for wing pattern induction in the polyphenic tropical butterfly Bicyclus anynana (Satyrinae). J. Insect Physiol. 45, 201–212 (1999).
Beldade, P. & Monteiro, A. Eco-evo-devo advances with butterfly eyespots. Curr. Opin. Genet. Dev. 69, 6–13 (2021).
Matsuoka, Y. & Monteiro, A. Hox genes are essential for the development of eyespots in Bicyclus anynana butterflies. Genetics 217, iyaa005 (2021).
Banerjee, T. D. & Monteiro, A. Reuse of an insect wing venation gene-regulatory subnetwork in patterning the eyespot rings of butterflies. Preprint at bioRxiv https://doi.org/10.1101/2021.05.22.445259 (2023).
Saenko, S. V., Marialva, M. S. & Beldade, P. Involvement of the conserved Hox gene Antennapedia in the development and evolution of a novel trait. EvoDevo 2, 1–10 (2011).
Shirai, L. T. et al. Evolutionary history of the recruitment of conserved developmental genes in association to the formation and diversification of a novel trait. BMC Evol. Biol. 12, 1–11 (2012).
Oliver, J. C., Tong, X.-L., Gall, L. F., Piel, W. H. & Monteiro, A. A single origin for nymphalid butterfly eyespots followed by widespread loss of associated gene expression. PLoS Genet. 8, e1002893 (2012).
Matsuoka, Y., Murugesan, S. N., Prakash, A. & Monteiro, A. Lepidopteran prolegs are novel traits, not leg homologs. Sci. Adv. 9, eadd9389 (2023).
Murugesan, S. N. et al. Butterfly eyespots evolved via cooption of an ancestral gene-regulatory network that also patterns antennae, legs, and wings. Proc. Natl Acad. Sci. USA 119, e2108661119 (2022).
Saleh Ziabari, O. et al. Gene duplication captures morph-specific promoter usage in the evolution of aphid wing dimorphisms. Proc. Natl Acad. Sci. USA 122, e2420893122 (2025).
Demircioğlu, D. et al. A pan-cancer transcriptome analysis reveals pervasive regulation through alternative promoters. Cell 178, 1465–1477 (2019).
Saccheri, I. J. et al. The genome sequence of the squinting bush brown, Bicyclus anynana (Butler, 1879). Wellcome Open Res. 8, 280 (2023).
Phan, M. H. et al. Conservation of regulatory elements with highly diverged sequences across large evolutionary distances. Nat. Genet. 57, 1524–1534 (2025).
Tendolkar, A. et al. Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings. ELife 12, RP90846 (2024).
Andersson, R. & Sandelin, A. Determinants of enhancer and promoter activities of regulatory elements. Nat. Rev. Genet. 21, 71–87 (2020).
Jin, V. X., Singer, G. A., Agosto-Pérez, F. J., Liyanarachchi, S. & Davuluri, R. V. Genome-wide analysis of core promoter elements from conserved human and mouse orthologous pairs. BMC Bioinf. 7, 114 (2006).
Haberle, V. & Stark, A. Eukaryotic core promoters and the functional basis of transcription initiation. Nat. Rev. Mol. Cell Biol. 19, 621–637 (2018).
Özsu, N., Chan, Q. Y., Chen, B., Gupta, M. D. & Monteiro, A. Wingless is a positive regulator of eyespot color patterns in Bicyclus anynana butterflies. Dev. Biol. 429, 177–185 (2017).
Banerjee, T. D., Shan, S. K. & Monteiro, A. Optix is involved in eyespot development via a possible positional information mechanism. Preprint at bioRxiv https://doi.org/10.1101/2021.05.22.445259 (2023).
Peña, C. & Wahlberg, N. Prehistorical climate change increased diversification of a group of butterflies. Biol. Lett. 4, 274–278 (2008).
Halali, S., Brakefield, P. M. & Brattström, O. Phenotypic plasticity in tropical butterflies is linked to climatic seasonality on a macroevolutionary scale. Evolution 78, 1302–1316 (2024).
Gompel, N., Prud’homme, B., Wittkopp, P. J., Kassner, V. A. & Carroll, S. B. Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila. Nature 433, 481–487 (2005).
Chan, Y. F. et al. Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer. Science 327, 302–305 (2010).
Kvon, E. Z. et al. Progressive loss of function in a limb enhancer during snake evolution. Cell 167, 633–642. e611 (2016).
Mazo-Vargas, A. et al. Deep cis-regulatory homology of the butterfly wing pattern ground plan. Science 378, 304–308 (2022).
Moreno, J. A. et al. Emx2 underlies the development and evolution of marsupial gliding membranes. Nature 629, 127–135 (2024).
Shahandeh, M. P. et al. Circadian plasticity evolves through regulatory changes in a neuropeptide gene. Nature 635, 951–959 (2024).
Galupa, R. et al. Enhancer architecture and chromatin accessibility constrain phenotypic space during Drosophila development. Dev. Cell 58, 51–62. e54 (2023).
McDonald, J. M. & Reed, R. D. Beyond modular enhancers: new questions in cis-regulatory evolution. Trends Ecol. Evol. 39, 1035–1046 (2024).
Murugesan, S. N. & Monteiro, A. Evolution of modular and pleiotropic enhancers. J. Exp. Zool. Part B 340, 105–115 (2023).
Banerjee, T. D., Tian, S. & Monteiro, A. Laser microdissection-mediated isolation of butterfly wing tissue for spatial transcriptomics. Methods Protoc. 5, 67 (2022).
Tian, S. et al. A novel Hox gene promoter fuels the evolution of adaptive phenotypic plasticity in wing eyespots of satyrid butterflies. figshare https://doi.org/10.6084/m9.figshare.c.8026060.v1 (2025).
Choi, H. M. et al. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145, dev165753 (2018).
Bruce, H. S. et al. Hybridization chain reaction (HCR) in situ protocol. protocols.io https://doi.org/10.17504/protocols.io.bunznvf6 (2021).
Banerjee, T. D., Zhang, L. & Monteiro, A. Mapping gene expression in whole larval brains of Bicyclus anynana butterflies. Methods Protoc. 8, 31 (2025).
Banerjee, T. D. & Monteiro, A. CRISPR-Cas9 mediated genome editing in Bicyclus anynana butterflies. Methods Protoc. 1, 16 (2018).
Murugesan, S. N., Tian, S. & Monteiro, A. Genome assembly and annotation of the dark-branded bushbrown butterfly Mycalesis mineus (Nymphalidae: Satyrinae). Genome Biol. Evol. 16, evae051 (2024).
Chazot, N. et al. Conserved ancestral tropical niche but different continental histories explain the latitudinal diversity gradient in brush-footed butterflies. Zenodo https://doi.org/10.5281/zenodo.5463912 (2021).
Kumar, S. et al. TimeTree 5: an expanded resource for species divergence times. Mol. Biol. Evol. 39, msac174 (2022).
Acknowledgements
We thank J. Lee for his assistance with the promoter usage analysis. We thank K. Long Tan for helping rear the promoter knockout lines. We acknowledge National Research Foundation (NRF) Singapore award NRF-CRP20-2017-0001 (A.M.), NRF Singapore award NRF-CRP25-2020-0001 (A.M.), NRF Singapore award NRF-NRFI05-2019-0006 (A.M.) and Ministry of Education Singapore award MOE-T2EP30223-0007 (A.M.).
Author information
Authors and Affiliations
Contributions
Conceptualization: S.T. and A.M. Methodology: S.T., T.D.B. and A.M. Investigation: S.T., B.L., T.D.B. and S.N.M. Visualization: S.T., B.L. and T.D.B. Funding acquisition: A.M. Project administration: S.T. and A.M. Supervision: A.M. Writing—original draft: S.T. and A.M. Writing–review and editing: S.T., T.D.B. and A.M.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Ecology & Evolution thanks Jennifer Brisson, Sofia Casasa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Temperature-shift experiments identified a prolonged temperature sensitivity window.
Shift schemes in refined temperature-shift experiments are illustrated (left panel). See Methods and Supplementary Table 1 for detailed methods and statistics of the temperature-shift experiments. Ventral Cu1 eyespot areas (middle panel), and temperature sensitivities (right panel, only across the six non-overlapping windows) were quantified and compared across the schemes in a one-way ANCOVA (Methods). n = 35 (WS), 20 (L5-1), 20 (L5-2), 12 (L5-3), 24 (Wr), 23 (PP), 22 (P15), 23 (Wr+PP), 23 (Wr+PP + P15), 24 (L5), 27 (DS). Black dots with error bars represent mean values ± 95CI. Eyespot areas from shift schemes with the same letter are not significantly different from each other, as determined by Tukey’s test. WS, wet season; DS, dry season; L5, 5th instar larva; Wr, wanderer, PP, prepupa; P, pupa.
Extended Data Fig. 2 Co-staining of a positive eyespot marker sal in J. almana.
Sal was used as a positive eyespot marker in both immunostaining and HCR. For immunostaining and HCR, n = 3 replicates. Scale bar: 100 microns.
Extended Data Fig. 3 Calculation of Antp protein eyespot expression diameter.
Size of the Antp protein expression domain in eyespots was calculated as eyespot expression diameter. For each ventral hindwing eyespot (here we use Cu1 eyespot as an example), eyespot expression diameter was calculated as the mean of the two diameters of the Antp protein expression domain (a and b), one (a) was parallel to the proximal-distal axis of the sector (outlined in dotted lines), while the other (b) perpendicular to it. The distance between M3 and Cu1 eyespot centers (c) was constantly used as a hindwing size proxy, and entered as a covariate in statistical analyses. Scale bar: 100 microns.
Extended Data Fig. 4 Representative phenotypes of Antp mKO crispants in B. anynana.
A dotted line separates left and right sides of the same individual. Purple arrowheads denote mutant phenotypes.
Extended Data Fig. 5 Representative phenotypes of Antp mKO crispants in M. mineus.
A dotted line separates left and right sides of the same individual. Purple arrowheads denote mutant phenotypes.
Extended Data Fig. 6 Conservation of sequences and transcriptional activities of Antp promoters across B. anynana and two non-satyrid butterflies.
Antp transcript annotation and sequence conservation around the six Antp promoter regions in B. anynana and two non-satyrids, V. cardui and J. coenia. Six alternative promoters are labeled. For transcripts, coding regions (CDSs) are in black and untranslated regions (UTRs) are in gray. Antp transcript annotation for J. coenia is from37.
Extended Data Fig. 7 Antp P2 mKO crispants in B. anynana.
Two guide RNAs co-injected efficiently induced long deletions around the conserved sequences (Fig. 4d middle panel) of Antp P2 in vivo, confirmed in pooled injected eggs (upper panel). The WT bands and mutant bands are denoted by black and red arrowheads, respectively. Genomic DNA was extracted from right hindwings of eight mKO crispants, and subjected to PCR and gel electrophoresis to detect potential long deletions (middle panel). Three crispants showing clear long deletions were genotyped via Nanopore amplicon sequencing. A dotted line separates left and right sides of the same individual. Purple arrowheads denote the wings used for genotyping, and red arrowheads denote guide RNA cut sites. Sequence coverage (Cov.) across the amplicon and major KO alleles are shown (lower panel).
Extended Data Fig. 8 Antp P1 mKO crispants in B. anynana.
Two guide RNAs co-injected efficiently induced long deletions around the conserved sequences (Fig. 4d right panel) of Antp P1 in vivo, confirmed in pooled injected eggs (top panel). The WT bands and mutant bands are denoted by black and red arrowheads, respectively. A dotted line separates left and right sides of the same individual. Purple arrowheads denote mutant phenotypes.
Extended Data Fig. 9 An Antp P1 mutant line in B. anynana.
A mutant line carrying a 252 bp deletion around the conserved region (Fig. 4d right panel) of Antp P1 was generated (Methods). The gel image shows clear differentiation of PCR bands across WT, mutant heterozygotes, and mutant homozygotes. The WT bands and mutant bands are denoted by black and red arrowheads, respectively. Representative wings for each genotype in both seasonal forms are shown. A dotted line separates dorsal (left) and ventral (right) sides of the same individual.
Extended Data Fig. 10 Antp P1 mutant heterozygotes do not exhibit significant changes in plasticity levels.
Changes in eyespot size plasticity levels were assessed (two-tailed) across sib-paired WT (P1+/P1+, n = 27 (WS) and 20 (DS) individuals) and mutant heterozygotes (P1+/P1Δ252, n = 30 (WS) and 33 (DS) individuals) in a two-way ANCOVA, indicated by a significant (padj<0.05) genotype (G) x temperature (T) interaction (padj: 0.72 (Rs), 0.78 (M1), 0.80 (M2), 0.88 (M3), 1.0 (Cu1), 0.90 (Cu2), 0.82 (Pc)). Lines with error bands represent mean values ± 95CI. Detailed sample sizes are summarized in Supplementary Table 10. Full statistical results are summarized in Supplementary Table 11. P values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure. ns, not significant; *padj<0.05; **padj<0.01; ***padj<0.001.
Supplementary information
Supplementary Information
Supplementary Texts 1–3, Figs. 1–7, Unprocessed gels and Tables 1–11.
Source data
Source Data Fig. 3
Statistical source data.
Source Data Fig. 4
Statistical source data.
Source Data Fig. 5
Statistical source data.
Source Data Extended Data Fig. 1
Statistical source data.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Tian, S., Lee, B., Banerjee, T.D. et al. A novel Hox gene promoter fuels the evolution of adaptive phenotypic plasticity in wing eyespots of satyrid butterflies. Nat Ecol Evol (2025). https://doi.org/10.1038/s41559-025-02891-5
Received:
Accepted:
Published:
Version of record:
DOI: https://doi.org/10.1038/s41559-025-02891-5