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Genetic basis of camouflage in an alpine plant and its long-term co-evolution with an insect herbivore

Nature Ecology & Evolution volume 9, pages 628–638 (2025) Cite this article

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Abstract

Camouflage through colour change can involve reversible or permanent changes in response to cyclic predator or herbivore pressures. The evolution of background matching in camouflaged phenotypes partly depends on the genetics of the camouflage trait, but this has received little attention in plants. Here we clarify the genetic pathway underlying the grey-leaved morph of fumewort, Corydalis hemidicentra, of the Qinghai-Tibet Plateau that by being camouflaged escapes herbivory from caterpillars of host-specialized Parnassius butterflies. Field experiments show that camouflaged grey leaves matching the surrounding scree habitat experience reduced oviposition by female butterflies and herbivory by caterpillars, resulting in higher fruit set than that achieved by green-leaved plants. The defence is entirely visual. Multi-omics data and functional validation reveal that a 254-bp-inserted transposon causes anthocyanin accumulation in leaves, giving them a rock-like grey colour. Demographic analyses of plant and butterfly effective population sizes over the past 500 years indicate that plant populations have been more stable at sites with camouflage than at sites with only green-leaved plants. In the recent past, populations of Parnassius butterflies have declined at sites with camouflaged plants. These findings provide insights into the genetics of a plant camouflage trait and its potential role in the rapidly changing dynamics of plant–herbivore interactions.

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Fig. 1: Study sites, habitat and leaf colour morphs of C. hemidicentra and observational and experimental data on Parnassius herbivory and its costs.
The alternative text for this image may have been generated using AI.
Fig. 2: Identification of the causal mutations underlying the camouflaged morph at the NW study sites.
The alternative text for this image may have been generated using AI.
Fig. 3: Enhanced expression of the bHLH35 gene triggered by the insert SV and increased targeting of downstream anthocyanin genes.
The alternative text for this image may have been generated using AI.
Fig. 4: Divergence of C. hemidicentra lineages and demographic histories of interacting plant and P. cephalus populations at study sites in the NW and SE of the QTP.
The alternative text for this image may have been generated using AI.

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Data availability

DNA sequencing reads generated in this project are available from the NCBI SRA database and through BioProject ID PRJNA793921, PRJNA1107342 and PRJNA1037772. RNA and ChIP sequencing data are available with BioProject ID PRJNA904070. The genome assembly is available at the China National Center for Bioinformation and Genome Warehouse and can be accessed with BioProject accession no. PRJCA013415. The genome annotation files and ChIP–seq graph files are available via Figshare at https://doi.org/10.6084/m9.figshare.26891578.v2 (ref. 66). Source data are provided with this paper.

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Acknowledgements

This project was supported equally by grants from the Second Tibetan Plateau Scientific Expedition and Research (STEP) (2019QZKK0502), the Science and Technology Major Project of Xizang (XZ202402ZD0005) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB31000000), additionally by the Natural Science Foundation of China (32030006, 91731311, 91731301 and 31590821), the Fundamental Research Funds for the Central Universities (lzujbky-2022-it16) to J.L. and the National Youth Talent Support Program and Natural Science Foundation of China (31670214 and 31971569) to Y.N. All the computation works were supported by the Big Data Computing Platform for Western Ecological Environment and Regional Development and the Supercomputing Center of Lanzhou University.

Author information

Author notes

  1. These authors contributed equally: Han Zhang, Pan Zhang, Yang Niu, Tongzhou Tao, Gang Liu.

Authors and Affiliations

  1. State Key Laboratory of Herbage Improvement and Grassland Agro-ecosystems, College of Ecology, Lanzhou University, Lanzhou, China

    Han Zhang, Pan Zhang, Gang Liu, Congcong Dong, Zeyu Zheng, Zengzhu Zhang, Ying Li, Zhimin Niu, Wenyu Liu, Minjie Li & Jianquan Liu

  2. State Key Laboratory of Plant Diversity and Specialty Crops, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China

    Yang Niu, Zemin Guo & Hang Sun

  3. Key Laboratory for Bio-resource and Eco-environment of Ministry of Education & Sichuan Zoige Alpine Wetland Ecosystem National Observation and Research Station, College of Life Science, Sichuan University, Chengdu, China

    Tongzhou Tao & Jianquan Liu

  4. University of Chinese Academy of Sciences, Beijing, China

    Zemin Guo

  5. Yunnan Key Laboratory of International Rivers and Transboundary Eco-security, Yunnan University, Kunming, China

    Shaoji Hu

  6. Institute of International Rivers and Eco-security, Yunnan University, Kunming, China

    Shaoji Hu

  7. Building No. 10, Anwai Xiaoguanbeili, Chaoyang District, Beijing, China

    Yang Yang

  8. Department of Biology, Washington University, Saint Louis, MO, USA

    Susanne S. Renner

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Contributions

J.L. led the project. J.L., S.S.R., H.S. and M.L designed the project. H.Z. performed all genome data analyses. T.T., Z. Zheng and W.L. finished the demographic analyses. P.Z. and Z.N. performed molecular experiments. Y.N., M.L., C.D., Z. Zhang, Y.L., G.L., Z.G., S.H. and Y.Y. carried out field experiments and collected plants and butterflies. J.L., S.S.R. and H.Z. co-wrote the paper. All authors revised the paper.

Corresponding authors

Correspondence to Minjie Li, Hang Sun, Susanne S. Renner or Jianquan Liu.

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Extended data

Extended Data Fig. 1 Four behavioral ecological experiments about the selection of Parnassius on Corydalis hemidicentra.

a, Setup of a constructed net used to attract Parnassius cephalus to visit artificially created decoy leaves, with grey leaves on the right and green leaves on the left. b, Quadrats used to observe P. cephalus’ preference for ovipositing, with the decoy green or grey leaves strategically placed. Red arrows pointed out the camouflaged grey decoy leaves. c, Observing the feeding preferences of Parnassius caterpillars on the two morphs of C. hemidicentra through analyzing the frequency of feeding selection between green and camouflaged fresh leaves at consistent intervals (n = 5 animals). Data represent the mean ± SD and P value is from two-sided Student’s t test. d, Comparing of feeding time on similar sample leaves, scale bar = 1 cm. Each dot represents the measurements (n = 8–14) of each individual (n = 3 animals) and vertical line represents the median of each group. P values are from two-sided Student’s t test.

Source data

Extended Data Fig. 2 Comparative metabolome analysis of two morphs of Corydalis hemidicentra.

a, PCA score plot of metabolism. b, OPLS-DA score plot of metabolism, the horizontal axis represents the predicted principal component score of the first principal component, and the vertical axis represents the orthogonal principal component score. c, KEGG enrichment of differentially expressed metabolites (DEMs). VIP ≥ 1 and P_value < 0.05 indicate important differences in metabolites between the morphs. Enrichment P values are from Fisher’s exact test. d, Heatmap showing the abundance patterns of 16 differentially expressed (mean metabolism level ≥ 0.1) flavonoid metabolites identified by wide-targeted metabolomics, with the columns showing results from three replicates for each of the morphs. e, Eight anthocyanins showing differential accumulation between green and reddish grey leaves. Data represent the mean ± SD from four replicates. P values are from two-sided Student’s t test.

Extended Data Fig. 3 Comparative transcriptome analysis of the two leaf color morphs of Corydalis hemidicentra.

a, PCA score plot of transcript profiles. b, Scatter plot of transcriptome levels. Fold change ≥ 2 and false discovery rate (FDR) ≤ 0.05 indicate significant difference of differentially expressed genes (DEGs). c, The expression pattern of DEGs. d, KEGG enrichment of DEGs. Enrichment P values are from Fisher’s exact test. The number of DEGs associated with each category is indicated in brackets after the term description, and enrichment values are indicated by color, as shown in the color scale. e, Expression levels of 12 anthocyanin genes in the two morphs. PAL phenylalanine ammonia-lyase, C4H cinnamate-4-hydroxylase, 4CL 4-coumarate CoA ligase 4, CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone 3-hydroxylase, F3′H flavonoid 3′-hydroxylase, F3′5′H flavonoid 3′,5′-hydroxylase, DFR dihydroflavonol 4-reductase, ANS anthocyanidin synthase, UFGT flavonoid-3-O-glucosyltransferase, FLS flavonol synthase, FG2 flavonol-3-O-glucoside L-rhamnosyltransferase. Data represent the mean ± SD from four replicates. P values are from two-sided Student’s t test.

Extended Data Fig. 4 Haplotype structure of SNPs around the GWAS peak in Corydalis hemidicentra individuals and PCR validation of bHLH35-ins in green and camouflaged morphs.

a, SNP schematic around the GWAS peak (P_value < 0.01). b, LD heatmap of the magnified region (bHLH35-ins ±10 kb). c, The correlation between LD (R2) and SV/SNPs located in the 10 kb region surrounding the bHLH35-ins (r = 0.55, P = 7.9×10−4). The solid orange line represents the linear regression line, while the grey shadows indicate the 95% confidence intervals. P value is from Pearson correlation test. d, Position and haplotype structure of the bHLH35 gene within 2 kb upstream and downstream. e, Agarose gel electrophoresis of PCR amplification of bHLH35-ins among green (n = 15) and camouflaged (n = 10) sampled leaves.

Source data

Extended Data Fig. 5 Expression and structural analysis of anthocyanin-related bHLH transcription factors in Corydalis hemidicentra.

a, Expression profiles of anthocyanin-related transcription factors from the bHLH gene family. The color scale from blue (low) to red (high) represents the FPKM (Min-Max normalization) of genes. b, Phylogenetic tree and multiple motifs of the group III bHLH family in C. hemidicentra, phylogenetic comparisons and subdivision of bHLH transcription factors are based on A. thaliana. c, bHLH binding sites detected in 2-kb-upstream sequences of twelve core genes in the anthocyanin biosynthetic pathway. d, A diagram illustrating the structure of the LUC fusion construct to proving the regulatory effect of bHLH35 on anthocyanin structural genes.

Extended Data Fig. 6 Validation of the bHLH35 functions in anthocyanin accumulation in Arabidopsis thaliana and different patterns of anthocyanin accumulation in A. thaliana and Corydalis hemidicentra.

a, Stable overexpression of the orthologous AtbHLH35 (AT5G57150.4) in A. thaliana, scale bars = 1 cm. Transgenic plant were treated with normal ( ~ 150 μmol m−2s−1) or high light ( ~ 400 μmol m−2s−1) stress for seven days. b, Anthocyanin, chlorophyll and carotenoids concentrations for different transgenic lines. Data represent the mean ± SD from four biological replicates. P values are from two-sided Student’s t test. c, qRT-PCR for early (CHS and CHI), late (F3H, DFR and ANS) and final (UFGT) anthocyanin biosynthesis pathway genes in the rosette leaves of A. thaliana. Data represent the mean ± SD from three biological replicates. d, The proportion of anthocyanin categories. Anthocyanidins are differentially substituted on the anthocyanidin B-ring as follows: Cyanidin R1 = OH, R2 = H; Delphinidin R1 = R2 = OH; Malvidin R1 = R2 = OMe; Pelargonidin R1 = R2 = H; Peonidin R1 = OMe, R2 = H Petunidin R1 = OH, R2 = OMe. e, Absolute quantitation of anthocyanins in A. thaliana (n = 3) and C. hemidicentra (n = 4). Data represent the mean ± SD. P values are from two-sided Student’s t test.

Source data

Extended Data Fig. 7 Population genetic structure of Corydalis hemidicentra.

a, Maximum-likelihood phylogenies of the six populations of C. hemidicentra and the outgroup (Corydalis calcicola). Caishichang (CSC), Huaguangfeng (HGF), Pujinlangba (PJ) come from the Northwest lineage, and Dongbaxueshan (DB), Tianbaoshan (TBS), Shikaxueshan (SK) belong to the Southeast lineage. b, The ML tree inferred from the chloroplast genome sequence. Sample numbers in green font represent green-leaved individuals, those in grey font camouflaged individuals. c, Distribution of cross validation error in the ADMIXTURE analyses, each K conducted 10 independent runs. The box represents the 25th and 75th percentiles, with the middle line indicating the median, while the whiskers extend up to 1.5 times the interquartile range. d, ADMIXTURE results based on whole-genome SNPs with K = 2 to 6. e, Principal component analysis of PC1 and PC2.

Extended Data Fig. 8 Population-genomic analyses of Corydalis hemidicentra.

a, Estimated haplotype sharing among 157 individuals from the six study populations. Heatmap colors indicate the total length of IBD blocks for each pairwise lineage comparison. b, The four scenarios modelled using Fastsimcoal2. Model (IV) fits the data best under the Akaike Information Criterion.

Extended Data Fig. 9 Modelled historic effective population sizes (Ne) for Corydalis hemidicentra (Che) and Parnassius cephalus (Pce) populations at the NW and SE study sites.

a, Population split times among linages inferred with MSMC2. The relative cross-coalescence rate (RCCR) of C. hemidicentra reached a value of 0.5 at ~732,900 to 937,300 years ago. The RCCR of P. cephalus reached a value of 0.5 at ~1,069,000 to 1,367,000 years ago. b, Demographic histories of C. hemidicentra populations deduced from Stairway Plot 2. The grey shading indicates the LGM (the last glaciation maximum) and Xixiabangma glacial periods.

Extended Data Fig. 10 Relationships and population-genetic structure of Parnassius cephalus individuals.

a, Maximum-likelihood tree and admixture under the K = 2 and K = 3 models, using samples from Bayankalashan, Maduo, Qinghai as the outgroup. Samples with the letters PJ (Pujinlangba) come from the northwestern study sites and samples with the letters TBS (Tianbaoshan) and SK (Shikaxueshan) from the southeastern study sites. b, PCA of re-sequenced individuals. c, Estimated haplotype sharing among all 25 individuals from the three populations. Heatmap colors indicate the total length of IBD blocks for each pairwise lineage comparison.

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Supplementary Notes 1–6 and Figs. 1–11.

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Parnassius visiting, ovipositing and feeding on C. hemidicentra.

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Zhang, H., Zhang, P., Niu, Y. et al. Genetic basis of camouflage in an alpine plant and its long-term co-evolution with an insect herbivore. Nat Ecol Evol 9, 628–638 (2025). https://doi.org/10.1038/s41559-025-02653-3

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