DOI: 10.5061/dryad.kh18932jj
title: Data from: Divergence in cuticular wax profiles generates partial behavioural isolation between leaf beetle populations with different dispersal traits

description: Sex pheromones are among the most studied insect mating signals, with
 their extensive diversity underscoring their crucial role in promoting
 behavioural isolation during speciation. In Chrysomelidae, cuticular wax
 (CW), a hydrophobic layer covering the insect cuticle, is a potential
 barrier that facilitates behavioural isolation. Male leaf beetles use
 female CW as a mating signal, and their species-specific profiles prevent
 heterospecific mating between closely related species, implying that
 divergence in CW profiles promotes reproductive isolation, and, hence,
 contributes to speciation. However, the role of CW as an isolating barrier
 remains unclear owing to limited knowledge regarding intraspecific
 divergence in female CW and its coevolution with male preferences. Through
 chemical analysis and behavioural experiments, we demonstrated that
 intraspecific divergence in female CW profiles contributes to partial
 behavioural isolation between leaf beetle populations with different
 dispersal traits: the flight-capable macropterous and flightless
 brachypterous forms of Galerucella grisescens (Coleoptera: Chrysomelidae).
 To the best of our knowledge, this is the first study on the coevolution
 of female CW and male preferences at the intraspecific level in
 Coleoptera. Our results support the potential role of CW as an isolating
 barrier in Chrysomelidae and are consistent with previous findings that
 loss of flight enhances beetle diversification.
description: (a) Insects The macropterous population
 originated from Ogata, Akita, Japan (40°0ʹ N, 139°56ʹ E), and the
 brachypterous population originated from Sendai, Miyagi, Japan (38°16ʹ N,
 140°52ʹ E). Each population was reared under the same conditions to avoid
 the potential effects of extrinsic factors (humidity, temperature, and
 nutrition) on CW profiles and to investigate genetically based divergence
 in phenotypes. Both populations were maintained in a climate chamber at 25
 ± 1 ℃ and 60% relative humidity under a 16L:8D photoperiod and reared on
 fresh leaves of <em>Rumex obtusifolius</em>. Newly emerged
 adults were sexed and kept separately from each other. Unmated adults at
 7–8 days post-emergence were used in the experiments.
 (b) Hindwing polymorphism To describe hindwing
 polymorphism, we examined the development of hindwings in females and
 males from each macropterous and brachypterous population
 (<em>n</em> = 25). After the beetles were freeze-killed, their
 right forewings and hindwings were dissected. Images were captured using a
 camera (Axiocam 305 Colour; Zeiss Microscopy, Jena, Germany) on a
 stereomicroscope (SteREO Discovery.V12, Zeiss Microscopy). Each forewing
 and hindwing area was measured using ImageJ version 1.53t. To correct wing
 size variations caused by body size variations, the development of
 hindwings was evaluated by calculating the ratios of hindwing areas to
 forewing areas. (c) CW extraction
 Female and male CW were extracted from 100 individuals of each
 macropterous and brachypterous population per extraction. After beetles
 were freeze-killed at –20 ℃, they were thawed for 15 min at room
 temperature. They were then immersed in two sequential 20 mL aliquots of
 <em>n</em>-hexane for 30 min each. The two extracts were
 mixed, and the solvent was evaporated at 39 ℃ using a rotary vacuum
 evaporator (N1200A‐S, EYELA, Tokyo, Japan). The extracts were dried under
 a gentle stream of nitrogen gas. The obtained CW was used for chemical
 analysis and mating experiments. (d) Chemical
 analysis To investigate the divergence in the CW
 profiles, we conducted a chemical analysis. The CW samples were
 reconstituted in 500 µL of <em>n</em>-hexane containing 2 µg
 of <em>n</em>-pentadecane as an internal standard. Three
 samples were prepared from females and males from each macropterous and
 brachypterous population. An aliquot of 1 µL of each sample was injected
 manually into a gas chromatography-mass spectrometry (GC‐MS) (GS-MS-QP2010
 Ultra, Shimadzu, Kyoto, Japan) equipped with a DB‐5ms column (30 m × 0.25
 mm i.d., 0.25 µm film thickness, J&W Scientific, CA, USA). The
 analytical conditions were partly based on those used in previous studies
 (Sugeno et al. 2006; Chiba et al. 2023). Helium was used as the carrier
 gas. The inlet temperature was 280 ℃, and injection was performed in split
 mode (1:10). The oven temperature program was started at 50 ℃ and heated
 to 200 ℃ at a rate of 30 ℃/min, followed by an increase to 320 ℃ at a rate
 of 2 ℃/min, and finally maintained for 15 min. The mass spectrometer was
 operated in an electron impact mode at 70 eV with a source temperature of
 230 ℃. Saturated hydrocarbons, the major components in the CW of
 <em>G. grisescens</em>, were identified by their mass spectra
 and Kovats indices. The Kovats indices of the detected compounds were
 calculated from the retention times of a standard mixture
 of <em>n</em>-alkanes (C9–C40) (GL Sciences, Tokyo,
 Japan). (e) Mating experiment To
 elucidate whether male preferences diverged between populations, we
 conducted mating experiments. All the experiments below were conducted at
 25 ± 1 ℃ during the light period of the rearing condition.
 (i) Male preference for live females in a two-choice
 design First, we explored male mating preferences
 through two-choice experiments. Macropterous and brachypterous females
 were released together on a 48‐mm‐diameter glass Petri dish lined with
 filter paper, after which a male was released in the same test arena. We
 recorded which females were first chosen by males (<em>n</em>
 = 60). When males attempted aedeagal insertion, we regarded it as ‘chose’.
 The experiments were terminated when the males chose either the female or
 did not attempt aedeagal insertion within 3 h. (ii)
 Male preference for female CW We investigated male
 preferences for female CW to elucidate whether female CW mediated male
 mate preferences. A male was released on a 48‐mm‐diameter glass Petri dish
 lined with filter paper, on which a macropterous and brachypterous female
 was fixed with double-sided tape on the same centreline passing a circle
 at the centre of the Petri dish, 1 cm away from the wall. To prevent
 potential positional bias, the locations of the two females were exchanged
 for each replicate. The females were subjected to one of three treatments:
 1) dead, 2) reapplied, or 3) exchanged. The procedure was based on a
 previous study (Chiba et al. 2023). Briefly, for Treatment 1, live females
 were freeze-killed at –20 ℃ for 30 min, following which they were thawed
 for 15 min at room temperature. For Treatment 2, the CW of the
 freeze-killed females was removed by immersing them
 in <em>n</em>-hexane twice for 30 min each, and then the CW
 was reapplied by pipetting one female equivalent of the
 <em>n</em>-hexane solution of CW that had been extracted from
 females of the same wing morph. For Treatment 3,
 <em>n</em>-hexane-washed females were covered with one female
 equivalent of CW extracted from females of different wing morph. We
 recorded which females were first chosen by males (<em>n</em>
 = 60). The experiments were terminated when the males chose either the
 female or did not attempt aedeagal insertion within 3 h.
 (iii) Male preferences for live females under a no-choice
 design When reproductive isolation is well established,
 mate preferences can be observed under both two- and no-choice designs.
 Thus, we evaluated male mate preferences in a no-choice situation. A
 macropterous or brachypterous female was released on a 48‐mm‐diameter
 glass Petri dish lined with filter paper, followed by a male in the same
 test arena (<em>n</em> = 30). We measured the time taken by
 males to copulate with paired females. In addition, because <em>G.
 grisescens</em> males inspect potential mates while mounting them
 and decide whether to attempt subsequent aedeagal insertion, we counted
 the number of times they dismounted from paired females without aedeagal
 insertion. The experiments were terminated when males copulated with
 paired females or when 3 h had elapsed since the start of the
 observations. (f) Statistical analysis
 The CW profiles between populations were compared by hierarchical
 clustering analysis using z-scores calculated for the peak areas of major
 CW components (peaks occurring at a mean relative abundance of over 0.5%
 in either or both populations). The analysis was based on the Euclidean
 distance measure and Ward’s clustering method. Male preferences for live
 females in the two-choice design were analysed using binomial tests. Mate
 preferences between treatments were compared using Fisher’s exact test
 with Holm correction to investigate male preferences for female CW. The
 latency to copulate with paired females was compared using log-rank tests
 when male mate preferences were investigated under a no-choice design. The
 number of times males dismounted from paired females without copulatory
 attempts was compared using the Mann–Whitney <em>U</em> test.
 All analyses were performed using R version 4.4.1.
description: # Data from: Divergence in cuticular wax profiles generates partial
 behavioural isolation between leaf beetle populations with different
 dispersal traits Dataset DOI:
 [10.5061/dryad.kh18932jj](10.5061/dryad.kh18932jj) ## **Description of the
 data and file structure** We obtained the data on hindwing polymorphism,
 chemical composition of cuticular wax, and male mate preferences under
 two-choice and no-choice situation. The dataset contains 4 folders: ###
 **Folder: wing_sizes.zip** #### **Description:**  This folder contains
 data on wing sizes to evaluate hindwing polymorphism in *Galerucella
 grisescens*. ##### **File: wing_sizes_data.csv** **·**
 **rep.**: Replicates. **·** **forewing (mm2):** The sizes of forewings.
 **·** **hindwing (mm2):** The sizes of hindwings. **·** **ratio (h/f):**
 The ratios of hindwing sizes to forewing sizes. To correct wing size
 variations caused by body size variations, we used the ratios for
 evaluating hindwing polymorphism. ### **Folder: gcms.zip** ####
 **Description:**  This folder contains data on GC-MS analysis of female
 and male cuticular wax, as well as the formatted data and R scripts for
 hierarchical clustering analysis. ##### **File: gcms_data.csv**
 **Description:**  This file contains data on peak areas of detected
 cuticular wax components. **·** **Peak No.**: Peak numbers. **·** **RT
 (min)**: Retention time. **·** **Compound**: The name of detected
 compounds. **·** **KI**: Kovats indices. **·** **BF**: Brachypterous
 females. **·** **BM**: Brachypterous males. **·** **MF**: Macropterous
 females. **·** **MM**: Macropterous males. **·** **rep1-3**: Replicates.
 ##### **File: female_cw_for_clustering.csv** ##### **File:
 male_cw_for_clustering.csv** **Description:**  These files contain the
 formatted data for hierarchical clustering analysis. Each value represents
 z-scores calculated for the peak areas of detected cuticular wax
 components. **·** **BF**: Brachypterous females. **·** **BM**:
 Brachypterous males. **·** **MF**: Macropterous females. **·** **MM**:
 Macropterous males. **·** **rep1-3**: Replicates. #####
 **File:** **Rcode_clustering_analysis_female_cw.R** ##### **File:
 Rcode_clustering_analysis_male_cw.R** **Description:**  These files are R
 scripts for hierarchical clustering analysis. ###
 **Folder: two_choice_experiments.zip** #### **Description:**  This folder
 contains data of two-choice mating experiments and R scripts for
 statistics. ##### **File: two_choice_data.csv** **Description:**  This
 file contains data on male mate preferences under two-choice design. **·**
 **rep.**: Replicates. **·** **choice:** Females chosen by males. B and M
 represents brachypterous and macropterous females, respectively. “no
 copulation” means that males did not attempt aedeagal insertion within 3
 h. **·** **liveF:** Live females. **·** **deadF:** Dead females. **·**
 **reappliedF:** Reapplied females. **·** **exchangedF:** Exchanged
 females. **·** **BM**: Brachypterous males. **·** **MM**: Macropterous
 males. ##### **File: Rcode_two_choice_bm_binomial.R** ##### **File:
 Rcode_two_choice_mm_binomial.R** **Description:**  These files are R
 scripts for binomial tests to analyze male preferences for live females
 under two-choice design. ##### **File: Rcode_two_choice_bm_fisher.R**
 ##### **File: Rcode_two_choice_mm_fisher.R** **Description:**  These files
 are R scripts for Fisher’s exact tests with Holm correction to analyze
 male preferences for female cuticular wax. ### **Folder:
 no_choice_experments.zip** #### **Description:**  This folder contains
 data of no-choice mating experiments, as well as the formatted data and R
 scripts for statistical analysis. ##### **File: no_choice_data.csv**
 **Description:**  This file contains data on male mate preferences under
 no-choice design. **·** **rep.**: Replicates. **·** **copulation (min)**:
 Latency to copulate with paired females. “no copulation” means that males
 did not attempt aedeagal insertion within 3 h. **·** **dismounting**:
 Number of times that males dismounted from paired females without
 attempting aedeagal insertion. **·** **BF**: Brachypterous females. **·**
 **BM**: Brachypterous males. **·** **MF**: Macropterous females. **·**
 **MM**: Macropterous males. ##### **File: bm_logrank.csv** ##### **File:
 mm_logrank.csv** **Description:**  These files contain the formatted data
 for log-rank tests to analyze copulation latency. **·** **time**: Latency
 (min) to copulate with paired females. For the pairs in which males did
 not attempt aedeagal insertion within 3 h timeframe of the experiments, we
 regarded it as censored data and give “180”. **·** **cens**: “1”
 represents that males copulated with paired females within 3h. “0”
 represents censored data (males did not attempt aedeagal insertion within
 3 h). **·** **treat**: “1” represents pairs of males and brachypterous
 females. “2” represents pairs of males and macropterous females. #####
 **File: bm_mannwhitneyu.csv** ##### **File: mm_mannwhitneyu.csv**
 **Description:**  These files contain the formatted data for Mann-Whitney
 *U* tests to analyze the number of times that males dismounted from paired
 females without attempting aedeagal insertion. **·** **values**: The
 number of times that males dismounted from paired females without
 attempting aedeagal insertion. **·** **treat**: “1” represents pairs of
 males and brachypterous females. “2” represents pairs of males and
 macropterous females. ##### **File: Rcode_bm_logrank.R** ##### **File:
 Rcode_mm_logrank.R** **Description:**  R scripts for log-rank tests to
 analyze copulation latency. ##### **File: Rcode_bm_mannwhitneyu.R** #####
 **File: Rcode_mm_mannwhitneyu.R** **Description:**  R scripts for
 Mann-Whitney *U* tests to analyze the number of times that males
 dismounted from paired females without attempting aedeagal insertion. ##
 Access information Other publicly accessible locations of the data: * N/A
 Data was derived from the following sources: * N/A