Evolution of Reproductive Morphology in Leaf Endophytes Zheng Wang1*, Peter R. Johnston2, Zhu L. Yang3, Jeffrey P. Townsend1 1 Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, United States of America, 2 Herbarium PDD, Landcare Research, Auckland, New Zealand, 3 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China Abstract The endophytic lifestyle has played an important role in the evolution of the morphology of reproductive structures (body) in one of the most problematic groups in fungal classification, the Leotiomycetes (Ascomycota). Mapping fungal morphologies to two groups in the Leiotiomycetes, the Rhytismatales and Hemiphacidiaceae reveals significant divergence in body size, shape and complexity. Mapping ecological roles to these taxa reveals that the groups include endophytic fungi living on leaves and saprobic fungi living on duff or dead wood. Finally, mapping of the morphologies to ecological roles reveals that leaf endophytes produce small, highly reduced fruiting bodies covered with fungal tissue or dead host tissue, while saprobic species produce large and intricate fruiting bodies. Intriguingly, resemblance between asexual conidiomata and sexual ascomata in some leotiomycetes implicates some common developmental pathways for sexual and asexual development in these fungi. Citation: Wang Z, Johnston PR, Yang ZL, Townsend JP (2009) Evolution of Reproductive Morphology in Leaf Endophytes. PLoS ONE 4(1): e4246. doi:10.1371/ journal.pone.0004246 Editor: Jerome Chave, Centre National de la Recherche Scientifique, France Received September 25, 2008; Accepted December 17, 2008; Published January 22, 2009 Copyright: 2009 Wang 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 Yale University Anonymous Postdoctoral Fellowship (ZW). The funders 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: wang.zheng@yale.edu Introduction Body size and shape are traits that correlate with diverse aspects of the biology of a species, from genetics, physiology and life history to ecology. For this reason, biologists have long been interested in understanding how body size and shape evolve [1]. However, less attention has been focused on the evolution of body size of the estimated 1.5 million species of fungi, perhaps in part because they are found in varied and complex associations with many other groups of organisms in diverse ecosystems [2–3]. Until recently, studies of symbiotic relationships between higher fungi and plants have been largely relegated to underground mycorrhi- zas and root endophytes, most of which are mushroom-forming basidiomycetes. In general, fungal endophytes are fungi that live inside plants without causing apparent symptoms of infection [4], but some saprobic or pathogenic fungi can have endophytic stages in part of or even during the whole of their life history, under specific environmental conditions. Diverse fungal endophytes have been identified thriving within living leaves from all major lineages of ascomycetes, and they represent a significant proportion of total fungal diversity [4–5]. Some leaf endophytes/pathogens produce a noticeable reproductive structure (body) during sexual and asexual reproduction, and the highly specialized body shape and reduced body size of leaf endophytes makes the current classification of ascomycetes challenging [6–8]. However, form and function are often correlated through change of ecological role, and the mysterious origins of diverse leaf endophytes may be addressed by studies that relate phylogeny, ecology, and morphology to the evolutionary history of fungi. Ecological role has often been elucidated by studies of form and function evolution in animals and plants, but less frequently in fungi, due to their diversity and lability of form and unresolved phylogenies [9]. Nowhere across the fungal kingdom has this diversity and lability been more challenging to explain than in the Leotiomycetes, which includes many saprobes, plant pathogens, and endophytes [8,10–11]. Their complex reproductive morphol- ogies (sexual ascomata and asexual conidiomata) are highly variable at all taxonomic ranks. Endophytes and pathogenic endophytes (conditional pathogens) are fairly common in the Leotiomycetes, and they are traditionally classified into two groups, the Rhytismatales and Hemiphacidiaceae (Helotiales). For most species in the Rhytismatales the sexual reproductive structure develops within a dark stroma, and the stroma typically immersed within host tissue and sometimes covered by a clypeus made of fungal hyphae. The Hemiphacidiaceae contains conifer pathogens producing small ascomata beneath the leaf surface, the erumpent ascomata pushing back the covering host tissue when matured. Unlike other orders in the Leotiomycetes, classification of the Rhytismatales and Hemiphacidiaceae had been straight- forward due to their unique morphology. However, both groups have experienced many taxonomic changes as increasing amounts of molecular sequence data on relevant taxa have been gathered and analyzed [7–8,12]. To sort out the evolutionary history of various endophytic lineages and thereby the corresponding shifts in body size and shape that accompanied these transitions, we analyzed rDNA data from 77 ascomycetes, including taxonomic representation of the two endophytic groups, the Rhytismatales and Hemiphacidiaceae (Leotiomycetes). Analysis of morphology in the light of this phylogenetic data reveals a relationship between body plan and endophytic lifestyle was evident in these fungi: a highly reduced morphology is an adaptation to survival on living leaves on trees, PLoS ONE | www.plosone.org 1 January 2009 | Volume 4 | Issue 1 | e4246