Diffuse Coevolution between Two Epicephala Species (Gracillariidae) and Two Breynia Species (Phyllanthaceae) Jing Zhang . , Shuxia Wang . , Houhun Li*., Bingbing Hu, Xiaofei Yang, Zhibo Wang College of Life Sciences, Nankai University, Tianjin, China Abstract The diffuse coevolution between two moth species (Epicephala lativalvaris and E. mirivalvata) and two plant species (Breynia fruticosa and B. rostrata) is reported based on field observations and indoor experiments conducted in Hainan and Fujian, China. Study results showed that the two Epicephala species jointly pollinated the two Breynia species, which led to a unique obligate pollination mutualism of two2to2two species specificity. A single Epicephala larva exclusively fed on seeds of host plants and developed to maturity by consuming all six seeds of each fruit, whereas a fraction of intact fruits were left to ensure the reproduction of plants within the whole population. Larvae of the two Epicephala species are competitive for resources; the population of E. mirivalvata is much smaller than that of E. lativalvaris, which has resulted from the differences in the female ovipositor structures and oviposition mode. The life history of Epicephala species highly coincides with the phenology of Breynia plants, and different phenology of B. fruticosa resulted in the different life history of the two Epicephala species in Hainan and Fujian. The natural hybridization of two host plants, possibly induced by the alternate pollination of two Epicephala species, is briefly discussed. Citation: Zhang J, Wang S, Li H, Hu B, Yang X, et al. (2012) Diffuse Coevolution between Two Epicephala Species (Gracillariidae) and Two Breynia Species (Phyllanthaceae). PLoS ONE 7(7): e41657. doi:10.1371/journal.pone.0041657 Editor: Dmitry A. Filatov, University of Oxford, United Kingdom Received February 14, 2012; Accepted June 25, 2012; Published July 27, 2012 Copyright: 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the National Natural Science Foundation of China (No. 30930014). Funder’s website: http://www.nsfc.gov.cn/. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lihouhun@nankai.edu.cn . These authors contributed equally to this work. Introduction The obligate pollination relationship between plants and their seed-parasitic pollinators is perhaps one of the most specialized cases in mutualism between insects and plants [1–4]. Ehrlich and Raven defined coevolution as the interaction between two major groups of organisms with a close and evident ecological relation- ship, such as plants and herbivores [5]. In the known classical obligate pollination mutualism between figs–fig wasps and yuccas– yucca moths, figs and yuccas depend exclusively on pollination of fig wasps and yucca moths respectively, while sacrificing some seeds for larvae of fig wasps and yucca moths to feed [6,7]. The obligate pollination mutualism between Epicephala moths of Gracillariidae and Glochidion plants of Euphorbiaceae was discov- ered in 2003 [3]. Three pollination relationships were proposed [8]: (1) one Epicephala species obligately pollinates one Glochidion species, (2) two Epicephala species jointly pollinate one Glochidion species, and (3) two Epicephala species jointly pollinate two closely related parapatric Glochidion species. The most distinctive floral features associated with the pollination mode are the structures of the pistils and stamens: styles are usually reduced to entire tips and medially fused, or filaments and anthers are variously fused [9– 10]. An obligate pollination relationship between Breynia vitis-idaea and an unnamed Epicephala species was discovered in 2004 [11]. This Epicephala species actively pollinated B. vitis-idaea and was observed loading numerous pollen grains on proboscis. Another Epicephala species on leaves of B. fruticosa was also observed carrying numerous pollen grains on proboscis, which led to a deduction that this species was an active pollinator of B. fruticosa [11]. Okamoto et al. [12] suggested the floral scent of Glochidion is one of the important key signals that mediate the encounters of the species-specific partners in the Glochidion–Epicephala mutualism. In tightly coevolved mutualism between Breynia vitis-idaea and Epicephala species, system-specific chemistry is not necessary for efficient host location to Epicephala moths [13]. By means of field observations and indoor experiments, we studied the phenology, the phylogenetic relationship and the morphological characters of the two Breynia species, as well as the morphological characters, the behavior, the life history, the phylogenetic relationship and the population size of the two Epicephala species in Hainan and Fujian. Based on related literature and results of our study, we discussed herein: (1) the impact of the different phenology of the allopatric B. fruticosa populations on the life history of the Epicephala moths in coevolution; (2) the influence of moth pollinators on the hybridization of Breynia species; and (3) whether the relationship between the pollinators is stable in this diffuse coevolution. Results Life History of Epicephala Moths and Phenology of Breynia Plants In Hainan, B. fruticosa has six fruit periods per year, lasting from the first period in early January to the sixth period in late November. Epicephala lativalvaris and E. mirivalvata have six PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41657