20Aim We tested the hypothesis of character release in body size among allopatric 21 populations of the carabid beetle Carabus japonicus by analysing geographical variation 22 in body size in relation to habitat temperature and sympatry/allopatry with the larger 23 congeneric species Carabus dehaanii. 24Location The main and satellite islands of Kyushu in the south-western part of the 25 Japanese archipelago. 26Methods We studied geographical variation in body length and genital size of C. 32Results While body size was positively correlated with annual mean temperature, it was 33 consistently small in sympatry but larger in some allopatric populations in warmer regions. 34The body size differences among populations were heritable. Allopatric C. japonicus 35 populations on satellite islands were derived from the sympatric mainland populations. 36In a few sympatric areas, mitochondrial haplotypes were shared between the species 37 because of introgressive hybridization, suggesting the occurrence of reproductive 38 interference between the species. We also found that genital size was not affected by 39 sympatry/allopatry but was positively correlated with body size. 40Main conclusions We demonstrated that the increased body size of allopatric C.

Interspecific variation in genital morphology contributes to prezygotic reproductive isolation in many insect species. However, developmental genes affecting genital morphology are poorly understood. We undertook larval RNA interference (RNAi) experiments with rotund (rn) gene in the ground beetle Carabus (Ohomopterus) maiyasanus to examine the knockdown effects of this gene on genital development. We injected double‐stranded RNA (dsRNA) into third instar larvae of C. maiyasanus. The injection of rn dsRNA resulted in the absence or deformation of the copulatory piece in the male genitalia, which is a characteristic part playing a role in species‐specific genital coupling between the sexes. In addition, the rn dsRNA injection resulted in shortened elytra in both sexes, which is related to the known role of rn in forewing formation in Drosophila. Our study suggests that rn plays a previously underappreciated role in male genital development and potentially contributes to genital diversity in this beetle group.