Role of Sex-Concordant Gene Expression in the Coevolution of Exaggerated Male and Female Genitalia in a Beetle Group

Abstract

Some sexual traits, including genitalia, have undergone coevolutionary diversification toward exaggerated states in both sexes among closely related species, but the underlying genetic mechanisms that allow correlated character evolution between the sexes are poorly understood. Here, we studied interspecific differences in gene expression timing profiles involved in the correlated evolution of corresponding male and female genital parts in three species of ground beetle in Carabus (Ohomopterus). The male and female genital parts maintain morphological matching, whereas large interspecific variation in genital part size has occurred in the genital coevolution between the sexes toward exaggeration. We analyzed differences in gene expression involved in the interspecific differences in genital morphology using whole transcriptome data from genital tissues during genital morphogenesis. We found that the gene expression variance attributed to sex was negligible for the majority of differentially expressed genes, thus exhibiting sex-concordant expression, although large variances were attributed to stage and species differences. For each sex, we obtained co-expression gene networks and hub genes from differentially expressed genes between species that might be involved in interspecific differences in genital morphology. These gene networks were common to both sexes, and both sex-discordant and sex-concordant gene expression were likely involved in species-specific genital morphology. In particular, the gene expression related to exaggerated genital size showed no significant intersexual differences, implying that the genital sizes in both sexes are controlled by the same gene network with sex-concordant expression patterns, thereby facilitating the coevolution of exaggerated genitalia between the sexes while maintaining intersexual matching.

Keywords: character evolution; interspecific differences; sexual traits; transcriptome; weighted gene co-expression network analysis.

Fig. 1.

The species of Carabus (Ohomopterus) studied. (A) Phylogenic relationships of Carabus iwawakianus, C. maiyasanus, and C. uenoi with the outgroup C. yaconinus (Fujisawa et al. 2019). Photographs of male and female genitalia and male (left) and female (right) beetles are shown. (B) Interspecific differences and matching between the sexes of genital part sizes in C. iwawakianus, C. maiyasanus, and C. uenoi (data from Sasabe et al. 2010). Black dots indicate copulatory piece length (CPL) and vaginal appendix length (VAL); horizontal and vertical bars indicate copulatory piece width (CPW) and vaginal appendix width (VAW), respectively. IvM denotes the comparison between C. iwawakianus and C. maiyasanus and UvIM the comparison between C. uenoi and C. iwawakianus/C. maiyasanus. (C) Developmental stages and timing of RNAlater fixation in the third instar and pupal stages. Abdominal parts of a third instar larva, and male and female pupae are shown to indicate the dissected portions for RNA extraction.

Fig. 2.

(A, B) Comparison of interspecific DEGs in males and females at each developmental stage. (A) IvM comparison (between Carabus iwawakianus and C. maiyasanus). For each sex, I-up and M-up indicate up-regulation in C. iwawakianus and C. maiyasanus, respectively; nonDEG, not differentially expressed between the species. (B) UvIM comparison (between C. uenoi and C. iwawakianus/C. maiyasanus). For each sex, U-up indicates up-regulation in C. uenoi versus both C. iwawakianus and C. maiyasanus, and IM-up indicates up-regulation in both C. iwawakianus and C. maiyasanus versus C. uenoi; nonDEG, not differentially expressed between the species between C. uenoi and C. iwawakianus/C. maiyasanus or oppositely regulated between C. uenoi versus C. iwawakianus and C. uenoi versus C. maiyasanus comparisons. (C, D) Violin plots showing the distribution of percentages of the expression variance explained by stage, species, and sex differences for 3,895 DEGs in the IvM comparison (C) and for 7,031 DEGs in the UvIM comparison (D).

Fig. 3.

The expression profiles of the top hub genes in male and female modules of the IvM comparison. For each top hub gene, expression profiles in both sexes are shown. Male modules (top hub gene in parentheses): M2 (XLOC 17554), M5 (scra), and M8 (XLOC 17722); female modules: F2 (XLOC 1508), F4 (C901), and F6 (Cdk1). Green and red lines indicate the expression profiles of Carabus iwawakianus and C. maiyasanus, respectively. Asterisks indicate significantly different expression levels between species at each stage (P > 0.05). Modules with the same daggers (†) or double daggers (‡) share hub genes and are common modules between the sexes.

Fig. 4.

The expression profiles of the top hub genes in male and female modules of the UvIM comparison. For each top hub gene, expression profiles of both sexes are shown. Male modules (top hub gene in parentheses): M1 (Rbf), M3 (Skeletor) and M5 (amos), and M10 (Ndf); female module: F2 (XLOC 13636), F3 (XLOC 1508) and F6 (unc-13), and F7 (Ndf). Note that F7 shares the same top hub gene with M10. Green, red, and blue lines indicate the expression profiles of Carabus iwawakianus, C. maiyasanus, and C. uenoi, respectively. Expression levels with the same letter (a, b) are not significantly different from one another (P > 0.05) among species at each stage by the multiple comparison test. Modules with the same daggers (†) or double daggers (‡) share hub genes and represent common modules between the sexes.