The development of extremely large male genitalia under spatial limitation.

Extensive research in evolutionary biology has focused on the exaggeration of sexual traits; however, the developmental basis of exaggerated sexual traits has only been determined in a few cases. The evolution of exaggerated sexual traits may involve the relaxation of constraints or developmental processes mitigating constraints. Ground beetles in the subgenus Ohomopterus (genus Carabus) have species-specific genitalia that show coevolutionary divergence between the sexes. Here, we examined the morphogenesis of the remarkably enlarged male and female genitalia of Carabus uenoi by X-ray microcomputed tomography. The morphogenetic processes generating the male and female genitalia at the pupal stage were qualitatively similar to those in closely related species with standard genital sizes. Higher growth rates contributed to the exaggeration of both the male and female genital parts of C. uenoi, possibly related to a gene network commonly upregulated in both sexes. Additionally, the length of the copulatory piece (CP), the enlarged male genital part stored in the aedeagus (AD), reached close to that of the AD at the later developmental stages and thereafter decelerated to grow in parallel with the AD, suggesting a structural constraint on the CP by the outer AD. Then, unlike related species, the lengths of the CP and AD increased at eclosion, suggesting a mechanism leading to further elongation of the male genitalia. These observations suggest that a developmental process allows continuous growth of the male genitalia even under the spatial limitation. These results revealed the spatio-temporal dynamics of the development of exaggerated genital structures under structural constraints.

Reproductive isolation via divergent genital morphology due to cascade reinforcement in Ohomopterus ground beetles.

Secondary contact between incipient species and selection against maladaptive hybridization can drive reinforcement between populations in contact and result in reproductive character displacement (RCD). Resultant divergence in mating traits within a species may generate downstream reproductive isolation between populations with displaced and non-displaced traits, referred to as the cascade reinforcement hypothesis. We examined this hypothesis using three allopatric populations of the ground beetle Carabus maiyasanus with a genital lock-and-key system. This species shows RCD in male and female genital morphologies in populations in contact with the sister species C. iwawakianus. In a reciprocal mating experiment using three allopatric populations with differences in male and female genital sizes, insemination failure increased as the difference in genital size increased. Based on the reproductive isolation index, insemination failure was the major postmating-prezygotic isolation barrier, at least in one population pair with comparable total isolation to those of other species pairs. By contrast, there was only incomplete premating isolation among populations. These results suggest that RCD in genital morphologies drives incipient allopatric speciation, supporting the cascade reinforcement hypothesis. These findings provide insight into the roles of interspecific interactions and subsequent trait diversification in speciation processes.

Reproductive Character Displacement in Genital Morphology in Ohomopterus Ground Beetles.

AbstractGenital morphology reveals rapid diversification among species, and species-specific divergence in genital morphology may result in reproductive isolation and promote speciation. Natural selection against maladaptive hybridization may cause species-specific genital divergence. In this context, divergence in mating traits is expected to be greater between sympatric populations than between allopatric populations in a pair of species, known as reproductive character displacement (RCD). However, there are few examples of RCD in the genital morphology of closely related species. Additionally, processes leading to RCD have rarely been inferred. In this study, we examined RCD and its underlying mechanisms by focusing on species-specific genital morphologies of closely related Ohomopterus ground beetle species. A morphological analysis showed patterns of RCD in species-specific genital parts in both sexes. Interspecific hybridization was confirmed by a mate choice experiment and by a population genetic analysis indicating extensive interspecific gene flow, suggesting that reinforcement is the most plausible process underlying the observed RCD. We found variation in the degree of displacement in contact zones, which may correspond with the ongoing process of genital evolution and speciation. Our results provide support for the lock-and-key hypothesis of genital evolution in closely related Ohomopterus species.

Heterochrony and growth rate variation mediate the development of divergent genital morphologies in closely related Ohomopterus ground beetles.

The rapid divergence of genital morphology is well studied in the context of sexual selection and speciation; however, little is known about the developmental mechanisms underlying divergence in genitalia. Ground beetles in the subgenus Ohomopterus genus Carabus have species-specific genitalia that show coevolutionary divergence between the sexes. In this study, using X-ray microcomputed tomography, we examined the morphogenesis of male and female genitalia in two closely related Ohomopterus species with divergent genital morphologies. The morphogenetic processes generating the male and female genitalia at the pupal stage were qualitatively similar in the two species. The male aedeagus and internal sac and female bursa copulatrix were partially formed at pupation and developed gradually thereafter. The species-specific genital parts, male copulatory piece, and female vaginal appendix differed in the timing and rate of development. The relatively long copulatory piece of Carabus maiyasanus began to develop earlier, but subsequent rates of growth were similar in the two species. The timing of the formation of the vaginal appendix and initial growth rates were similar, but subsequent rapid growth led to a longer vaginal appendix in C. maiyasanus. Thus, substantial interspecific differences in the size of genital parts were mediated by different underlying developmental mechanisms between the sexes (i.e., a shift in the developmental schedule in males and a change in growth rate in females). These results revealed the spatio-temporal dynamics of species-specific genital structure development, providing a novel platform for evo-devo studies of the diversification of genital morphologies.