Coevolution between Two Epicephala and Two Breynia Figure 3. Maximum-parsimonious (left) and maximum-likelihood (right) trees based on matK genes of 22 Phyllantheae plant species. (A) The maximum-parsimonious tree of Phyllantheae plants (length, 179; CI = 0.933; RI = 0.985). (B) The maximum-likelihood tree of Phyllantheae plants (-ln likelihood = 2922.8917; transition/transversion ratio = 2; empirical frequencies: A = 0.30688, C = 0.16491, G = 0.15206, T = 0.37615). Numbers above branches are bootstrap values. Red branches refer to the species involved in this study. doi:10.1371/journal.pone.0041657.g003 collected 28 E. lativalvaris adults and two E. mirivalvata adults in the field, and reared 109 E. lativalvaris larvae and four E. mirivalvata larvae from fruits. We observed E. lativalvaris visiting female flowers 35 times in the field, but E. mirivalvata twice only. The above statistical figures indicated that the population size of E. mirivalvata was much smaller than that of E. lativalvaris. Phylogenetic Analysis of Epicephala Moths and Breynia Plants We analyzed the COI genes of E. lativalvaris and Epicephala sp. ex B. fruticosa [11], and found that the pairwise distance between them is 0. Moreover, Epicephala sp. ex B. fruticosa would leave scars on host flowers after ovipositing, which is similar to E. lativalvaris. Based on the above information, we could conclude that the Epicephala species pollinating B. fruticosa in Kawakita and Kato [11] is E. lativalvaris. The topological structure of the maximum-parsimonious (MP) tree and the maximum-likelihood (ML) tree of plants are completely consistent (Figure 3), which indicates that B. fruticosa and B. rostrata are highly surpported to be sister taxa. Both MP and ML trees of Epicephala moths (Figure 4) showed that within five Epicephala pollinators of Breynia plants, E. mirivalvata diverged firstly from E. lativalvaris, Epicephala sp. ex B. vitis-idaea, Epicephala sp. ex B. disticha and Epicephala sp. ex B. oblongifolia, and that all of pollinators of Breynia have a close relationship with Epicephala sp. ex P. vulcani. The pollinators of Glochidion plants and three non-pollinators of Phyllanthus form a paraphyletic group. Discussion Diffuse Coevolution between Two Epicephala Species and Two Breynia Species Diffuse coevolution firstly advanced and defined by Janzen (1980) [14]. He defined coevolution as an evolutionary change in a trait of the individuals in one population in response to a trait of the individuals of a second population, followed by an evolution- ary response by the second population to the change in the first. Diffuse coevolution occurs when either or both populations in coevolution are represented by an array of populations that generate a selective pressure as a group [14]. Our study revealed that B. fruticosa and B. rostrata distributed in Hainan and Fujian were dependent on the obligate pollination of E. lativalvaris and E. mirivalvata, forming a mutualism of two2to2two species specificity. Two Epicephala species deposited eggs on female flowers before or after pollination, and their larvae exclusively fed on seeds of two Breynia species. The life history of two Epicephala species correlated with the phenology of two Breynia species; Breynia plants bloomed after dark, which coincided with the activity of Epicephala moths. According to the definition of diffuse coevolution and the classic examples of figs–fig wasps and yuccas–yucca moths, we confirm that diffuse coevolution exists between the two Epicephala species and the two Breynia species, which is firstly revealed in this study. Though a single Epicephala larva needs to consume all six seeds of each fruit to develop to maturity, enough intact fruits developed by Epicephala pollinated flowers are left to ensure the reproduction of the hosts within the whole population. PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e41657