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Physical interactions between pollen and pistil tissues mediate cryptic female choice in Brassica rapa

Timothée CheniniD, Estelle BarbotiD, François RoussetiD, Agnès MignotiD, Patrice DavidiD, Jeanne TonnabeliD

Abstract

Cryptic female choice - female-mediated bias in fertilization after mating - is well established in animals and can also occur in plants when multiple pollens compete on the same pistil. However, whether interactions between pollen and pistil tissues after pollen deposition contribute to this process remains unknown. Here, we experimentally test whether such interactions mediate cryptic female choice in the angiosperm Brassica rapa. We quantified fertilization success of pollen donors competing on the same pistil using paternity analyses, and in parallel, made semi-in vivo assays to measure pollen tubes trajectories emerging from the excised styles and growing toward unfertilized ovules for each donor-recipient pair. We show that pollen tube growth towards ovules predicts higher fertilization success under pollen competition. Thus, we document a previously unobserved mechanism of cryptic female choice based on physical interactions between male and female components of reproduction. In addition, different recipient plants favour different pollen donors, consistent with non-directional female choice. Plants with longer styles bias paternities more strongly towards the most successful pollen donor. Overall, our study demonstrates that interactions between pollen tubes and pistil tissues after pollen germination enable plants to bias paternity toward particular donors.

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (9 Aug 2026) · CC BY 4.0

Brassica rapa pollen tube length after growing through recipient pistil tissue is donor-by-recipient specific, predicts siring success under competition, and matters more in longer-styled recipients.

Cryptic female choice is well studied in animals, yet plant post-pollination research has focused on pollen competition, and the molecular dialogue between pollen and pistil has never been tied to the relative siring success of competing donors. Pairing paternity analysis with fully factorial semi-in vivo assays, this work tests whether pistil-conditioned pollen tube performance drives donor bias, and whether that bias is directional or compatibility-based.

Key results

  1. ShownPollen tubes were first grown through each recipient's stigma and style without competition. Donors whose tubes grew longer sired more seeds on that recipient under five-donor competition. Tube straightness and remaining distance to the nearest ovule micropyle did not predict siring.

    Multinomial logit mixed model with all three pair scores: tube length β = 0.379, p < 0.001; remaining distance to nearest micropyle β = 0.010, p = 0.866; straightness β = 0.073, p = 0.310; relative pollen production covariate β = -1.577, p = 0.002. · Pollen tube length after interaction with pistil…; Multinomial models of fertilization success; Tables

  2. SuggestedTube length after pistil passage depends on the donor-recipient pair rather than on donor identity alone, so different recipients favour different donors. This fits compatibility-based female choice rather than directional (Fisherian or good-genes) choice.

    Donor x recipient interaction was the main variance component for tube length and remaining distance (reported as 9.63% and 36.2%); donor identity explained 0.21% of tube length variance; the three terms jointly explained 38.85% (length) and 17.3% (remaining distance); straightness had only residual variance. · Pollen tube length after interaction with pistil…; Evidence for compatibility-based cryptic female…; Pair-specific pollen-pistil interaction scores

  3. ShownStyle length modulates the effect: in recipients with higher style length scores, donors with longer post-pistil tubes gained a larger siring advantage. Stigma area and ovary length did not modulate it.

    Style length score x tube length score interaction β = 0.208, p = 0.021 in a multinomial model that also tested stigma area and ovary length interactions (not significant); likelihood ratio tests with FDR correction over n = 21 tests; pistil scores were uncorrelated. · Style length modulates the fertilization…; Multinomial models of fertilization success

  4. SuggestedPair-level tube length is not explained by separately measured in vitro pollen traits or pistil morphology. This points to a pathway distinct from the in vitro trait-based female choice previously reported in B. rapa.

    No significant effect on tube length from donor in vitro tube growth rate, germination rate or pollen size, from recipient stigma area, style length or ovary length, or from their products (FDR-corrected, n = 9 tests per model). · Pollen tube length after interaction with pistil…; Individual pistil scores

  5. SuggestedThe pair-specific bias looks quantitative rather than an artefact of self-incompatibility or kin recognition: results held after removing near-excluded donors, and donor-recipient relatedness did not predict siring.

    Excluding 34 of 174 combinations (19.54%) where the donor sired <5% of seeds left conclusions unchanged; Loiselle kinship from eight microsatellite loci had no effect (β = -0.152, p = 0.748); mean within-population mate-pair relatedness 0.026. · Style length modulates the fertilization…; Evidence for compatibility-based cryptic female…; Effect of donor-recipient genetic relatedness on…

Methods, data and reproducibility

Fourteen groups of five donors and five recipients. Recipient stigmas got a five-donor pollen mix and seed paternity was assigned via microsatellites; in all but one group, each pair was also assayed alone in semi-in vivo pistil-ovule preparations. Pair scores from generalized linear mixed models predicted siring in multinomial logit mixed models.

  • Wisconsin Fast Plants (WFP) plants from five polygamous experimental source populations, arranged in fourteen mating groups of five donors and five recipients · Brassica rapa · n = 70 pollen donors and 70 pollen recipients
  • Semi-in vivo assays, one per donor-recipient pair, with eight excised ovules arranged around the excised style base on growth medium · Brassica rapa · n = 325 assays; tubes emerged in 174; 712 tubes measured
  • Hand-pollinated recipient flowers given a five-donor pollen mix, yielding fruits and seeds · Brassica rapa · n = 140 flowers; 112 fruits; 2,510 seeds
  • Offspring plantlets and parents genotyped at eight microsatellite loci for paternity assignment · Brassica rapa · n = 2,122 offspring genotyped; 985 paternities retained
  • Recipient pistil morphology (stigma area, style length, ovary length) measured from images · Brassica rapa · n = 465 pistils from 70 recipients
  • Donor in vitro pollen traits (germination rate, pollen size, tube growth rate) and stamen pollen counts · Brassica rapa · n = 70 donors; 66 stamens counted for pollen production
  • Deposited datasets and statistical analysis code · Brassica rapa · 10.5281/zenodo.21757983
Methods & tools
Semi-in vivo pollen tube assay on pollen growth medium (PGM), ImageJ with custom semi-automated macro (version 2.14.0), Multiplex microsatellite PCR (eight loci), NucleoMag Plant kit with KingFisher magnetic-bead DNA extraction, ABI 3500XL capillary sequencer with GeneMapper allele scoring, CERVUS 3.0.7 (paternity assignment), spaMM 4.6.39 (GLMMs; pois4mlogit multinomial logit mixed model), R 4.4.2, SPAGeDi 1.5 (Loiselle kinship estimator), Likelihood ratio tests with false discovery rate correction
Code
https://doi.org/10.5281/zenodo.21757983
Data & accessions
https://doi.org/10.5281/zenodo.21757983
Software & environment
R 4.4.2 with spaMM 4.6.39; CERVUS 3.0.7; SPAGeDi 1.5; ImageJ macro version 2.14.0; GeneMapper for allele calling. No container or package lockfile described.
Reproducibility gaps
The Zenodo deposit is stated to be public as of publication; other details needed for reanalysis are available from the lead contact on request. The in vitro pollen, pistil and paternity data are shared with Chenin et al. (2026).
Limitations (6)
  • The number of pollen grains each donor deposited on stigmas during hand pollination was not strictly controlled; a pollen production score from a single stamen per donor was used as a covariate instead. · stated by the authors
  • Pollen carry-over between successive pollinations meant 42.9% of offspring (13.2% to 71.9% across groups) were sired by recipient plants. These 723 paternities were discarded and, with other exclusions, only 985 of 2,122 genotyped offspring were analysed. · stated by the authors
  • The cellular and molecular mechanisms behind the pair-specific effect remain unknown. Protein expression was not compared between styles of different lengths. · stated by the authors
  • Whether this choice is adaptive is untested. Bias based on subtle relatedness differences, after self-incompatibility removes close kin, cannot be excluded; testing it needs controlled relatedness gradients plus offspring fitness data. · stated by the authors
  • Each donor-recipient pair was assayed once, on a single pistil and slide, so the pair-level interaction score cannot be separated from noise specific to that pistil or assay. This could inflate the apparent donor x recipient variance. · noted in this summary
  • The variance partitioning appears internally inconsistent. An interaction share of 36.2% is assigned to remaining distance, whose three-term total is reported as 17.3%, so the per-trait percentages may be swapped. · noted in this summary

Who should read:Read this if you study plant sexual selection, pollen-pistil interactions or cryptic female choice and want a factorial semi-in vivo plus paternity design linking pair-specific tube growth to siring success.

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