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. 2023 May 26;14:3041. doi: 10.1038/s41467-023-38722-z

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Single-fly oviposition assays testing H₂O versus 0.1% hexanoic acid and H₂O versus 0.5% hexanoic acid in instant medium. Left: egg-laying rate. ***P < 0.001; *P < 0.05; NS P > 0.05 (Kruskal-Wallis rank sum test with Nemenyi post-hoc test). Right: oviposition preference indices are only shown for genotypes that laid at least 2 eggs in these assays. Statistically-significant differences from 0 (no preference) are indicated: ***P < 0.001; **P < 0.01; NS P > 0.05 (Wilcoxon test with Bonferroni correction for multiple comparisons); N = 30–83 flies across 1–3 technical replicates. b Mean number (± SEM) of mature eggs per pair of ovaries per fly. NS P > 0.05 (two-sample t-test); N = 9 flies. c Quantification of the number of eggs and indentations on different substrates of the indicated genotypes; N = 30 flies in 1 technical replicate. d Single-fly oviposition assays testing 0.05% hexanoic acid versus 0.05% butyric acid of D. melanogaster Ir75b mutant and rescue genotypes. Left: egg-laying rate for Ir75b-Gal4 control (w;Ir75b-Gal4/+;Ir75b^DsRed), UAS-DmelIr75b control (w;UAS-DmelIr75b/+;Ir75b^DsRed), UAS-DsecIr75b control (w;UAS-DsecIr75b/+;Ir75b^DsRed), DmelIr75b rescue (w;Ir75b-Gal4/UAS-DmelIr75b;Ir75b^DsRed), and DsecIr75b rescue (w;Ir75b-Gal4/UAS-DsecIr75b;Ir75b^DsRed). Both rescue strains showed small but significant reduction in the number of eggs compared to DmelIr75b-Gal4 control. Right: oviposition preference indices for these genotypes. No significant differences were detected between Ir75b-Gal4 control, UAS-DmelIr75b control and DmelIr75b rescue strains. The DsecIr75b rescue strain showed a significant shift in preference toward 0.05% hexanoic acid compared to both DmelIr75b-Gal4 and UAS-DsecIr75b controls. ***P < 0.001; **P < 0.01 (Wilcoxon tests with Bonferroni correction for multiple comparisons); N = 28–55 flies per genotype measured across at least 2 technical replicates. e Schematic summarizing the contributions of different olfactory pathways to niche specialization in D. sechellia. Exact P values for the statistical comparisons are provided in the Source Data files.