Data from: Mate recognition in orchid bees is mediated by acquired chemical signals
Data files
Sep 25, 2026 version files 183.45 MB
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2023_2024perfumefiles.zip
164.66 MB
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Combineddisplay2023and2024.csv
1.67 KB
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Display_data_2024.csv
37.80 KB
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Displaydataplots2023_24.Rmd
6.06 KB
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GLMs_ht.Rmd
4.68 KB
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info.csv
1.34 KB
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Mateplot.R
1.84 KB
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Matrix_ht_20260115.csv
10.09 KB
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Metadata_beeids_2024.csv
2.95 KB
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Mirrored_bar_graphfile.csv
2.12 KB
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mirrorplotprop.Rmd
5.46 KB
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nMDS_ht.Rmd
5.11 KB
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README.md
8.07 KB
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Spermatheca_images.zip
18.70 MB
Sep 30, 2026 version files 183.45 MB
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2023_2024perfumefiles.zip
164.66 MB
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Combineddisplay2023and2024.csv
1.67 KB
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Display_data_2024.csv
37.80 KB
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Displaydataplots2023_24.Rmd
6.06 KB
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GLMs_ht.Rmd
4.68 KB
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info.csv
1.34 KB
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Mateplot.R
1.84 KB
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Matrix_ht_20260115.csv
10.09 KB
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Metadata_beeids_2024.csv
2.95 KB
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Mirrored_bar_graphfile.csv
2.12 KB
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mirrorplotprop.Rmd
5.46 KB
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nMDS_ht.Rmd
5.11 KB
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README.md
9.18 KB
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Spermatheca_images.zip
18.70 MB
Abstract
The evolution of prezygotic reproductive barriers plays a central role in speciation and is often mediated by divergence in mating signals. Male orchid bees collect volatile compounds from the environment to concoct species-specific perfumes that function as sexual chemical signals during courtship display. While perfumes are thought to convey species recognition, their direct role in assortative mating remains unresolved. Here, we experimentally tested whether perfume composition may facilitate mate recognition between two recently diverged and sympatric sister species, Euglossa dilemma and E. viridissima. We collected perfumes from males in sympatric regions of Mexico and experimentally supplemented E. dilemma males with either conspecific (E. dilemma) or heterospecific (E. viridissima) perfumes. We conducted single-choice mating assays and found that the rate of male courtship display did not differ between treatments. However, females of E. dilemma exhibited a strong preference for males with conspecific perfumes. While 77% of males loaded with E. dilemma perfume mated, only 8% of males loaded with E. viridissima perfume mated. Our results support the hypothesis that perfume composition alone can mediate reproductive isolation in orchid bees. These findings highlight the role of environmentally acquired signals as extended phenotypes that can rapidly generate behavioral isolation and contribute to speciation.
Dataset DOI: 10.5061/dryad.12jm63zc3
Description of the data and file structure
Cleaned behavioral data and R files for Behavioral Ecology manuscript "Mate recognition in orchid bees is mediated by acquired chemical signals" by
Marissa C. Sandovala, Janosch Dohrsb, Thomas Chouvencc, Thomas Eltzb, Santiago R. Ramíreza
*equal contributors
aUniversity of California, Davis
bRuhr-Universität Bochum
cUniversity of Florida Ft. Lauderdale Research and Education Center
*equal contributors
Corresponding author: mcsandoval@ucdavis.edu
The data are from the mate choice experiments of Euglossa dilemma bees in flight cages. Data include a matrix of perfume compounds collected from wild caught male bees of E. dilemma and E. viridissima (collected in field sites in Mexico summer and fall of 2023 and 2024), behavioral records of male courtship behavior in the cages (performed at UF-FLREC, Davie, Florida in August - November 2024, mating assays in supplemental info performed at UF-FLREC, Davie, Florida August - November 2023), and spermatheca images of females involved in the mating assays (August - November 2024, UF-FLREC, Davie, Florida).
Files and variables
File: Spermatheca_images.zip
Description: Zip file of the images of dissected female spermathecas post mating assay with female bee code (e.g., MXD1F) as file name.
File: Display_data_2024.csv
Description: Observation of courtship display data for single-choice mating assays. Raw data and computed average for each observation round (i.e., three trials).
Variables
- Bee ID: Code given to male bees in the experiment (e.g., MXD1)
- Date (once perfume injected): Date of introduction into flight cage. Formatted as "Date.Month.Year"
- Trial 1: First round of courtship observations with binary data (0 is no display and 1 is display)
- Trial 2: Second round of courtship observations with binary data (0 is no display and 1 is display)
- Trial 3: Third round of courtship observations with binary data (0 is no display and 1 is display)
- Average: Average "amount" of courtship across the three observation trial rounds
- Perfume: Orchid bee species perfume source (E. dilemma or E. viridissima)
- Cage: Flight cage ID
- female mated: Yes/No binary
File: Matrix_ht_20260115.csv
Description: Chemical matrix of perfume compounds injected for each individual male bee in the experiment.
Variables
- masterRT: Retention time (Units correspond to time for a compound to travel through the column, expressed in decimal minutes. Time zero would refer to time of compound injection and larger RT numbers therefore indicate longer time to travel through the column)
- Mitko#: Reference for internal compound library
- Substance type: Classification (P = perfume, ISTD = internal standard, LGS = labial gland secretions , CHC = cuticular hydrocarbons)
- Substance Group: Compound class
- Name: Individual compound name (question marks reflect uncertainty in P compounds, and blank cells of non-P compounds also reflect unidentified LGS and CHC compounds)
- MW: Molecular weight (question marks and blank cells reflect uncertainty or unknown value, respectively, of MW for given compound)
- Columns to the right of MW correspond to an individual male's perfume injection and have the header corresponding to that male's ID (e.g., MXV10_ht). The values correspond to the summed ion currents.
File: Mirrored_bar_graphfile.csv
Description: Formatted file of perfume compounds from the chemical matrix to easily produce the mirror plot (Figure 5) in the manuscript. This file is semicolon-delimited, rather than comma-delimited, as there is molecular weight information embedded which utilizes commas for compounds with unknown identity (e.g., m/z: 88,76,171,41,77,227,213,39,195, 104).
Variables
- masterRT: Retention time
- Mitko#: Reference for internal compound library
- Substance Group: Compound class
- Compound_name: Individual compound name
- AvrgProportionDilemma: Average proportion of a given compound for E. dilemma treatment males
- AvrgProportionViridissima: Average proportion of a given compound for E. viridissima treatment males
- AvrgProportionAll: Average proportion of a given compound across all treatment males
File: Metadata_beeids_2024.csv
Description: Meta data for the individual female and male bees involved in the single-choice mating assays.
Variables
- ID: Code given to male bees in the experiment (e.g., MXD1)
- Perfume: Orchid bee species perfume source (E. dilemma or E. viridissima)
- Perfume_code: Perfume ID given when collecting from wild caught males in Mexico
- Nest_Origin: Source nest ID
- M_Mark: Scratch marked on a male's thorax to confirm identity upon removal from cage. Possible marks included the following symbols: "X", "V", "+", "N", "/", "Z", " 'square' ", " 'triangle' ", and " 'star' "
- Cage: Flight cage ID
- Date_introduction: Date of male introduction to the flight cage. Formatted as "Date.Month.Year"
- Date_injection: Date of perfume injection of treatment male. Formatted as "Date.Month.Year"
- Date_sampling: Date of removal of male from the flight cage. Formatted as "Date.Month.Year"
- Female_ID: Code given to female bees in the experiment (e.g., MXD1F)
- F_Nest_Origin: Source nest ID
- F_Mark: Female Otolith tag ID
- F_introduced: Date female introduced to flight cage
- female_mated: Yes/No binary
File: mirrorplotprop.Rmd
Description: R markdown file for producing the mirror plot in the manuscript.
File: info.csv
Description: CSV file which provides species info for each individual bee (i.e., Bee ID) sample, whether pre- or post-experiment. The species values with "ht" refer to post-experiment (hind tibiae) and without correspond to pre-experiment (i.e., perfume pool we used to inject into a given male's hind tibiae).
File: nMDS_ht.Rmd
Description: R markdown for the nMDS plot which visualizes the perfume in the hind tibiae of the males post-experiment. To execute this script the following files are needed: "Matrix_ht_20260115.csv" and "info.csv".
File: GLMs_ht.Rmd
Description: R markdown to run the binary outcome generalized linear mixed models of the E. dilemma treatment in the Supplement. Uses the "Matrix_ht_20260115.csv" chemical matrix file to see how the effect of proportion of HNDB, perfume richness, and amount of perfume on mating status.
File: Combineddisplay2023and2024.csv
Description: Observation data for single- and dual-choice courtship displays of male bees, visualized in the Supplement.
Variables
- ID: Code given to male bees in 2023 and 2024
- Year: 2023 or 2024
- Perfume: Orchid bee species perfume source (E. dilemma or E. viridissima)
- summed_display_rate: Sum of each individual's average display rate over the experimental days (3-5). For example, if a male only displayed 4 times in the first day's 30 observational periods (trials), then his daily average would be 0.13 for the first day.
- average_display_rate: summed_display_rate divided by the number of experimental days (3-5)
File: Displaydataplots2023_24.Rmd
Description: R markdown file for producing the courtship display plots in the Supplement which compares the single- and dual-choice experimental design effects on courtship behavior.
File: Mateplot.R
Description: R markdown file for mating bar plot.
File: 2023_2024perfumefiles.zip
Description: Zip folder with raw GC-MS perfume files (Agilent .D files). An Agilent .D file is multi-file container directory which contains information about the raw instrument signals, sequence parameters, and acquisition metadata for a single analytical run. Each folder is organized by perfume pool with various subfiles within. For example, "Edil_5_23splitless" (perfume pool used for male "MXD12"). To view and extract the raw integration data, the authors used the Agilent MSD ChemStation software (Version G1701DA Revision D.01.00). Peaks were identified and cross-referenced using a user mass-spectral library (Eltz, unpublished) and a commercially available spectral library (Adams, 2007). An open source alternative software that supports .D files is OpenChrom by Lablicate.
Code/software
Analyses were run using R version 4.3.0 with the following packages:
dplyr v. 1.1.2
tidyr v. 1.3.0
ggplot2 v. 3.5.1
stats v 4.3.0
vegan v 2.6-4
tidyverse v 2.0.0
ad34 v 1.7-22
ggtext v 0.1.2
ecodist v 2.0.9
scales v 1.3.0
ggpubr v 1.0.0
All of the chromatograms, mass spectra, and retention times were analyzed with the Agilent MSD ChemStation software (Version G1701DA Revision D.01.00). Peaks were identified and cross-referenced using a user mass-spectral library (Eltz, unpublished) and a commercially available spectral library (Adams, 2007).
Change Log
Changes after September 25, 2026: Minor updates to README only. No files changed.