Coevolution between Two Epicephala and Two Breynia Figure 1. Female flowers and fruits with double characters of Breynia plants. Female flowers (A) and fruits (B) with erect stigmas and flat calyx sepals. Female flowers (C) and fruits (D) with excurved stigmas and reflexed calyx sepals. (cs) calyx sepals. (s) stigma. doi:10.1371/journal.pone.0041657.g001 generations correspondingly, lasting from the first generation in middle January to the sixth generation in late December (Table S1). In Fujian, both B. fruticosa and B. rostrata have 4–5 fruit periods per year, which lasted from the first period in early June to the fifth period in late April of the following year. A large number of flowers withered in winter due to absence of pollinators. A small amount of flowers that were pollinated during middle to late November were dormant and no seeds were formed until middle or late March of the following year, and the fifth fruit period lasted from middle to late April. Both E. lativalvaris and E. mirivalvata have 4–5 generations correspondingly, which lasted from the first generation in middle June to the fifth generation in middle May of the following year. Larvae that pupated during early to middle November could emerge into adults, pollinate mature female flowers and lay eggs of the fifth generation during middle to late November. Larvae that pupated in late November overwintered in pupas, and they began to emerge in late April and disappeared in middle May. Pollinated flowers and Epicephala eggs in flowers were dormant until middle or late March of the following year, and the adults of the fifth generation emerged in middle May (Table S2). We smelled flower fragrance of both B. fruticosa and B. rostrata at night only, which suggests that they were blooming at night. A single larva of both E. lativalvaris and E. mirivalvata needs to consume all six seeds within a fruit to develop into maturity. Mature larvae (the last instar) gnawed a hole in the fruit wall and exited, then produced cocoons and turned into pupae on leaves of hosts or the ambient plants. Pupal stage of E. lativalvaris lasted 9– 15 days, and pupal stage of E. mirivalvata lasted 9–12 days. Adults of both Epicephala species could survive 3–5 days by sucking nectar for nutrition (Figure S1). Hybridization of Breynia Species We found some plant individuals are difficult to identify, because they possess double morphological characters of both B. fruticosa and B. rostrata: three individuals (Figure 1: A, B) in Hainan with erect stigmas (characteristic of B. rostrata) and flat calyx sepals (characteristic of B. fruticosa); nineteen individuals (Figure 1: C, D) in Fujian with excurved stigmas (characteristic of B. fruticosa) and reflexed calyx sepals (characteristic of B. rostrata). The result of the hand-pollination hybridization experiment showed that there are no reproductive barriers between B. fruticosa and B. rostrata. 90% B. fruticosa female flowers (n = 50) with pollen grains from B. rostrata developed, so did 94% B. rostrata female flowers (n = 50) with pollen grains from B. fruticosa (Table S7). PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41657