Coevolution between Two Epicephala and Two Breynia Figure 2. Behavior of female Epicephala moths. Epicephala lativalvaris collecting pollen grains on male flower of Breynia fruticosa (A) and actively pollinating for Breynia fruticosa (B) and B. rostrata (C). E. lativalvaris inserting ovipositor through calyx lobe and ovary to lay eggs on B. fruticosa (D) and B. rostrata (E). (F) Egg of E. lativalvaris. E. mirivalvata laying eggs between ovary and calyx lobe on B. fruticosa (G) and B. rostrata (H). (I) Egg of E. mirivalvata. (e) egg. doi:10.1371/journal.pone.0041657.g002 Behavior of Epicephala Adults Epicephala moths became active after dark, which was in accordance with the blossom of B. fruticosa and B. rostrata flowers. Female adults of the two Epicephala species actively collected pollen from male flowers and pollinated female flowers of both Breynia species at night. Female Epicephala adults pushed proboscis tip into the dehiscent pit of calyx lobes, rubbed against stamen column to collect pollen grains, and sometimes they pushed the whole proboscis into the pit (Figure 2: A). During pollen collection, female adults intermittently spread proboscis several times to get more pollen grains. They only collected pollen grains in one male flower each time effectively, which could pollinate more than ten female flowers in the same or different plants. Epicephala adults repeatedly rubbed proboscis against stigma to deposit pollen grains on all stigmas (Figure 2: B, C). They would pace back and forth to pollinate all flowers on one branch, and female flowers were pollinated in this way as to pollinate most efficiently. The two Epicephala species exhibited similar behavior of pollen collection and pollination, but their oviposition behavior was visibly different. Epicephala lativalvaris females bent abdomen to basal calyx on ventral surface and inserted ovipositor through calyx lobes and ovary to lay eggs into ovary tissue (Figure 2: D–F). Epicephala mirivalvata females bent abdomen to basal calyx on dorsal surface to deposit eggs between ovary and calyx lobes (Figure 2: G–I). After oviposition, Epicephala moths often gave complemen- tary pollination to make sure there are enough pollen grains for female flowers. On rare occasion, they would not lay eggs if disturbed. Number of Epicephala Eggs and Consumption of Breynia Seeds Epicephala lativalvaris laid eggs by piercing through calyx lobes and ovary wall, leaving oviposition scars on fruits and calyx lobes. We could acquire their egg amount by counting scars on each fruit. In all sampled fruits (n = 1269 in B. fruticosa, n = 867 in B. rostrata), 72.0% in B. fruticosa (n = 914) and 88.6% in B. rostrata (n = 768) with oviposition scars loaded E. lativalvaris eggs (Table S3). Epicephala mirivalvata laid eggs between calyx lobes and ovary wall. Therefore their eggs could only be observed by dissecting female flowers. Only 2.6% female flowers in B. fruticosa (n = 2) and 4.0% in B. rostrata (n = 5) loaded E. mirivalvata eggs in all dissected flowers (n = 76 in B. fruticosa, n = 126 in B. rostrata) (Table S4). We PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41657