rostrata in Wanshi Botanical Garden (alt. 28–201 m, 117u539– 118u259E, 24u259–24u549N) and Tianzhu Mountain National Forest Park (alt. 50–200 m, 24u359–24u399N, 117u539–117u579E) in Fujian, China. Studied Insects and Plants Epicephala lativalvaris Li, Wang & Zhang, 2012 and E. mirivalvata Li, Wang & Zhang, 2012 are two small nocturnal moths that are distinctly different in morphology [17]. Females have a different oviposition mode due to having a differently shaped ovipositor (Figure 5). However, they obligately and jointly pollinated B. fruticosa in Hainan and Fujian, and pollinated B. rostrata in Fujian. Breynia fruticosa and B. rostrata are similar in morphology: male flowers (Figure 6: A, B) have a funnel-shaped calyx with six dentations at apex; the three stamens fused into a column and concealed in calyx, which makes it hard to be touched by flower visitors except Epicephala moths; female flowers bear free calyx and styles, which is likely to be accessed by other flower visitors. The two Breynia species can be distinguished by having different calyx and stigmas: in B. fruticosa (Figure 6: C), calyx sepals radially spread into a disk, stigmas are excurved and calyx sepals are enlarged in fruit (Figure 6: D); in B. rostrata (Figure 6: E), calyx sepals are reflexed, stigmas are erect and calyx sepals are not enlarged in fruit (Figure 6: F). Methods Breynia fruticosa flowered for the 10 warmer months of the year, fruited sparsely and irregularly in Hong Kong [18]. In order to acquire detailed phenological data of B. fruticosa and B. rostrata as to compare with the life history of E. mirivalvata and E. lativalvaris, we made a tracking observation of the flower and fruit developmental stages of B. fruticosa in Hainan, as well as of B. fruticosa and B. rostrata in Fujian. We randomly selected ten individuals of B. fruticosa in Yingge Mountain Nature Reserves, and five individuals of both B. fruticosa and B. rostrata in Tianzhu Mountain National Forest Park and Wanshi Botanical Garden, respectively. We recorded the development of flowers and fruits, and counted the number of flowers, the fruits and developing fruits of the selected plant individuals once a week. To make sure whether the hybridization could happen between B. fruticosa and B. rostrata, we performed a hand-pollination hybridization experiment in Wanshi Botanical Garden. We respectively selected 50 non-pollinating female flowers of B. fruticosa and B. rostrata. We gathered pollen grains from male flowers of B. fruticosa and hand-pollinated female flowers of B. rostrata, and from male flowers of B. rostrata and hand-pollinated female flowers of B. fruticosa. We bagged these pollinated flowers with fine netting (0.5 mm mesh), and seven days later, we counted and calculated their developmental rate (Figure S2). We observed Epicephala moths during full anthesis and recorded their visiting behavior in detail, with particular attention paid to their nocturnal activities. We recorded the time that the moths spent on pollination, oviposition and pollen collection, and observed how they used their proboscis to collect pollen and pollinate flowers, and where they oviposited. We collected developing and mature fruits and put them in a cylindrical plastic box (8.5 cm612.0 cm) to rear Epicephala larvae. We then recorded how and when the larvae left fruits to cocoon and emerge. The presence of oviposition scars and eggs on fruits was considered to have a one–to–one correspondence [11]. We randomly examined 23 flowers in Hainan (Yingge Mountain) and 179 flowers in Fujian (Tianzhu Mountain and Wanshi Botanical Garden) to search for eggs (Table S3); and randomly examined oviposition scars on 1162 fruits in Hainan (Yingge Coevolution between Two Epicephala and Two Breynia Mountain, Wuzhi Mountain and Jianfeng Mountain) and 974 fruits in Fujian (Tianzhu Mountain and Wanshi Botanical Garden) to acquire the egg amount of Epicephala moths (Table S4). Adult specimens examined in this study were collected from their hosts or reared from fruits in captivity. Dissection of female flowers of Breynia plants and female genitalia of Epicephala moths was conducted under an Olympus SZ11 stereo-microscope. Photographs were taken with a Canon G10 digital camera in the field and the illustration of genitalia were prepared with an Olympus C–7070 Wide Zoom digital camera. We extracted genomic DNA of Epicephala moths from the fruits we reared, and extracted genomic DNA of B. rostrata from silica- gel dried leaves collected in Tianzhu Mountain, Fujian. The method of DNA extraction followed the CTAB procedure [19]. We PCR-amplified the COI gene fragments using primers LepF1 and LepR1 [20], and PCR-amplified the matK gene fragments using primers 570F and 1710R [21]. We analyzed the phylogenetic relationship between E. lativalvaris and E. mirivalvata based on the mitochondrial cytochrome oxidase subunit I (COI) gene of 20 Epicephala sequences, which is widely used in the phylogenetic analysis of insects [9,22,23]. The sequences of E. lativalvaris, E. mirivalvata and E. relictella were newly obtained during this study and 17 sequences were adopted from the previous studies [8–9] (Table S5). Alignment of COI gene and matK gene were aligned using Mega v. 5.05. The phylogeny was rooted with Epicephala sp. ex Flueggea suffruticosa according to the phylogenetic analysis of Epicephala and Phyllantheae [10]. We analyzed the phylogenetic relationship between B. fruticosa and B. rostrata based on the maturase K (matK) gene, which has previously been shown to be particularly useful for phylogenetic analysis of the family and tribe [8,9,21,24]. Breynia rostrata sequence was newly obtained during this study and the remaining sequences were adopted from the previous studies [8,9,21] (Table S6). The phylogeny was rooted with F. suffruticosa according to the phylogenetic analysis of Epicephala and Phyllantheae [10]. Maximum-parsimony and maximum-likelihood analyses of COI and matK were performed using PAUP v. 4.0 b10 [25]. Both maximum-parsimony and maximum-likelihood heuristic searches were conducted with equal weight, 1000 and 100 replicates of random addition analyses respectively, and tree bisection-recon- nection branch swapping. The robustness of maximum-parsimony and maximum-likelihood trees were assessed by non-parametric bootstrapping with 1000 and 100 replicates respectively. Supporting Information Figure S1 Epicephala moths sucking nectar on female flowers of Breynia fruticosa (A) and B. rostrata (B). (TIF) Figure S2 Hand-pollination hybridization experiment of Breynia fruticosa and B. rostrata. (A) flowers bagged with fine netting. (B) hand-pollinated B. rostrata with pollen of B. fruticosa. (C) non- pollinated female flower of B. fruticosa. (D) developed female flowers of B. fruticosa with pollen of B. rostrata. (E) non-pollinated female flower of B. rostrata. (F) developed female flowers of B. rostrata with pollen of B. fruticosa. (TIF) Table S1 Annual fruit stage of Breynia fruticosa and life history of Epicephala lativalvaris and E. mirivalvata in Yingge Mountain of Hainan, China. (DOC) PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e41657