Molecular Ecology (2007) 16, 4822–4836 doi: 10.1111/j.1365-294X.2007.03569.x © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd Blackwell Publishing Ltd Mechanical barriers to introgressive hybridization revealed by mitochondrial introgression patterns in Ohomopterus ground beetle assemblages NOBUAKI NAGATA,* KOHEI KUBOTA,† KATSURO YAHIRO‡ and TEIJI SOTA* *Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan, †Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan, ‡Lake Biwa Museum, Oroshimo 1091, Kusatsu, Shiga 525-0001, Japan Abstract To reveal the role of diverged body size and genital morphology in reproductive isolation among closely related species, we examined patterns of, and factors limiting, introgressive hybridization between sympatric Ohomopterus ground beetles in central Japan using mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequences. We sampled 17 local assemblages that consisted of two to five species and estimated levels of interspecific gene flow using the genetic distance, D A , and maximum-likelihood estimates of gene flow. Sharing of haplotypes or haplotype lineages was detected between six of seven species that occurred in the study areas, indicating mitochondrial introgression. The intensity and direction of mitochondrial gene flow were variable among species pairs. To determine the factors affecting introgression patterns, we tested the relationships between interspecific D A and five independent variables: difference in body size, difference in genital size, phylo- genetic relatedness (nuclear gene sequence divergence), habitat difference, and species richness of the assemblage. Body and genital size differences contributed significantly to preventing gene flow. Thus, mechanical isolation mechanisms reduce the chance of intro- gressive hybridization between closely related species. Our results highlight the role of morphological divergence in speciation and assemblage formation processes through mechanical isolation. Keywords: body size, Carabus, gene flow, genitalia, prezygotic isolation, sympatric species Received 9 May 2007; revision received 20 August 2007; accepted 22 August 2007 Introduction The coexistence of closely related species requires repro- ductive isolation by premating, postmating-prezygotic, and postzygotic isolation mechanisms, of which premating mechanisms are most effective to avoid maladaptive effects of interspecific hybridization. Although premating isolation mechanisms can evolve more rapidly than postmating ones (Coyne & Orr 1989, 1997), insufficient premating isolation is frequently observed among closely related species in parapatry and even in sympatry, resulting in introgressive hybridization which is detected by intermediate phenotypes or molecular markers (Arnold 1997; Avise 2004; Bettles et al. 2005; Grant et al. 2005; Gow et al. 2006). Because premating or postmating-prezygotic isolation can be achie- ved by various behavioural, ecological, and mechanical mechanisms (Coyne & Orr 2004), evolutionary differen- tiation in any mechanism(s) will contribute to reproductive isolation. Thus, revealing the relationship between trait differences involved in isolation mechanisms and the degree of hybridization (e.g. gene introgression) will contribute to understanding the process of speciation and coexistence of species. Many cases of introgressive hybridization have been reported in ground beetles of the subtribe Carabina (Cole- optera: Carabidae; =Carabus s. lat.) using mitochondrial gene sequences or microsatellite markers (e.g. Streiff et al. 2005; Zhang et al. 2005; Garnier et al. 2006). In particular, in the subgenus Ohomopterus (genus Carabus s. str.), introgressive Correspondence: Nobuaki Nagata, Fax: +81 75 753 4100; E-mail: nagata@terra.zool.kyoto-u.ac.jp