Coevolution between Two Epicephala and Two Breynia Figure 4. Maximum-parsimonious (left) and maximum-likelihood (right) trees based on COI genes of 19 Epicephala moths species. (A) The maximum-parsimonious tree of Epicephala moths (length, 486; CI = 0.519; RI = 0.518). (B) The maximum-likelihood tree of Epicephala moths (- ln likelihood = 1628.20777; transition/transversion ratio = 2; empirical frequencies: A = 0.29923, C = 0.16930, G = 0.14101, T = 0.39046). Numbers above branches are bootstrap values. Red branches refer to the species involved in this study. doi:10.1371/journal.pone.0041657.g004 The impact of the phenology of the allopatric B. fruticosa on the life history of Epicephala moths was obvious. Different climates in Hainan and Fujian led to different phenology of B. fruticosa, which resulted in different life history of Epicephala species. In Hainan B. fruticosa has six fruit periods without dormant stage, so do two Epicephala species (Table S1). While in Fujian, pollinated female flowers of B. fruticosa were dormant in winter, so a minority of Epicephala individuals overwintered in eggs in female flowers and a majority overwintered in pupas until hosts bloom in the following year (Table S2). Breynia fruticosa and B. rostrata are sister taxa, which suggests that the pollinators they are sharing might have pollinated their ancestor, and that this pollination system maintained after the speciation of B. fruticosa and B. rostrata. Hybridization of Two Breynia Plants Induced by Epicephala Moths The study of the genetic structure of closely related figs and their associated pollinators in the Neotropical Region showed that shared use of host figs and colonization of novel hosts by the wasps may result in hybridization and genetic introgression across different fig species [15]. We found similar phenomena in Breynia plants. Among the four species of Breynia plants in China, B. fruticosa and B. rostrata are closely related in morphology. Stamens concealed in closing calyx distinguish them from B. retusa, and free calyx and styles distinguish them from B. vitis-idaea. The phylogenetic analysis based on matK gene also revealed that B. fruticosa and B. rostrata are sister taxa (Figure 3). Breynia fruticosa and B. rostrata distributed in Wanshi Botanical Garden and Tianzhu Mountain have similar phenology. When Epicephala moths collected pollen grains from male flowers of B. fruticosa, they are likely to deposit pollen grains on female flowers of the sympatric B. rostrata, hence resulting in the hybridization of B. fruticosa and B. rostrata, and vice versa. As B. fruticosa and B. rostrata are closely related, their hybrid offsprings could survive and possess double characters of both species. This hypothesis is supported by the fact that some plant individuals in Tianzhu Mountain and Wanshi Botanical Garden have double characters of both B. fruticosa and B. rostrata (Figure 1). Breynia rostrata was recorded to have distribution in Hainan [16], but we did not find it in this study. We only found three individuals with characters of both B. rostrata and B. fruticosa in Yingge Mountain, which indicated that the population of B. rostrata was either very small or has disappeared. The plants with double characters in Hainan may imply that B. rostrata once existed there and might have hybridized with B. fruticosa in nature. Relationship between E. Lativalvaris and E. Mirivalvata In Breynia–Epicephala diffuse coevolution mutualism, Epicephala lativalvaris and E. mirivalvata overlap in the ecological niche, and are competitive for resources. Epicephala mirivalvata has a much smaller population size than E. lativalvaris does and plays a secondary role in this mutualism. This is closely associated with female ovipositor structures and oviposition mode. Epicephala lativalvaris has an apically spine-shaped ovipositor (Figure 5: A), which could pierce PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41657