and the adaptation is evident in the morphology of both sexual and asexual reproductive structures. Results 1. Phylogenetic analyses (Fig. 1) To infer the phylogeny of leaf endophytes and endophytic pathogens across ascomycetes, parsimony analyses using PAUP 4.0 [13] and Bayesian analyses using MrBayes [14] were performed on the 78 taxa data set, which included 77 representatives of major lineages of ascomycetes and one basidiomycete, Cotylidia sp., as the outgroup. Parsimony analyses using the rDNA data set for 78 taxa produced nine equally best trees (length = 4278, CI = 0.349, and RI = 0.509). The clade including the Leotiomycetes and the Sordariomycetes received weak support (56% BP), a result that is consistent with previous studies using diverse molecular markers. Two clades were of interest. The Rhytismatales clade includes saprobic species of Cudonia and Spathularia and endophytic species of Coccomyces, Rhytisma, and Lophodermium. The Hemiphacidiaceae clade includes saprobic species of Chlorencoelia and Hedydria, and endophytic species of Hemiphacidium, Fabrella, and Meria. These two clades were strongly supported with bootstrap values of 100% and 97% respectively. Within the Rhytismatales clade, Cudonia and Spathularia were supported as a clade (100% BP), with Coccomyces dentatus as their sister group. Within the Hemiphacidiaceae clade, species of Fabrella and Meria formed a clade with 86% BP, while Hemiphacidium longisporum was located in the clade with Chlorencoelia versiformis (57% BP). A majority rule consensus tree of the Bayesian posterior tree set presented almost the same topology as that from parsimony analyses. The Leotiomycetes - Sordariomycetes clade was strongly supported (1.00 PP). The Rhytismatales clade received a support of 1.00 PP, and the Cudonia-Spathularia clade (1.00 PP) was the sister group to Coccomyces dentatus (1.00 PP). The Hemiphacidiaceae clade was supported with 1.00 PP as well, and within the clade species of Heyderia, Hemiphacidium, Chlorencoelia formed a clade (1.00 PP) sister to the Fabrella-Meria clade (1.00 PP). 2. Analysis of correlation between leaf-living ecology and reduced morphology After a run of 1 million iterations in BayesTraits [15] to estimate the correlation between leaf-living ecology and reduced morphol- ogy, the following harmonic means of the marginal likelihood were obtained, with virtually no difference between runs: ,71.50 for the model in which leaf-living ecology and reduced morphology are independent, and ,56.50 for the model in which they are dependent. The log-Bayes Factor is twice the difference of these two harmonic means, equaling ,30, revealing very strong support (&5) for the correlation (dependent model) between the leaf-living ecology and reduced fruiting body morphology. Discussion The tar-spot symptoms on leaves of maples and oaks are typical of the highly reduced ascomata of leaf-living species in the Rhytismatales. These develop within a dark stroma immersed within host tissues. In contrast, species of the saprobic family Cudoniaceae produce large, bright ascomata on leaf duff or mossy ground. Except for a similar development style inferred from a reduced membrane covering the young fruiting body of Cudonia species, no obvious morphological characters suggest a relation- ship between the Cudoniaceae and the Rhytismatales [16]. Our results validate previous molecular studies that had suggested that the saprobic family Cudoniaceae lies within the endophytic Rhytismatales [7,12,16]. ITS sequences of Cudonia were amplified Reproductive Morphology from leaf samples in an investigation of foliar fungal endophytes, but the ITS phylogenies were not resolved for the Cudonia- Rhytismatales clade in that study [5]. Although it is very likely that certain stages in the life cycle of Cudonia species are endophytic in leaves of plants, there had been no direct evidence. For the first time, our phylogenetic placement of a bryophilous species of Cudonia found recently in Yunnan, China, ecologically links the Cudoniaceae to the endophytic/pathogenic fungi that are so characteristic of the Rhytismatales. Our data also demonstrate lability of morphology in the Hemiphacidiaceae, another family including both leaf endophytes and pathogens. Previously, all fungi placed in the Hemiphacidia- ceae produced small, highly reduced ascomata beneath the surface of leaves. In our analysis, Heyderia and Chlorencoelia species were placed within this family, despite production by both species of large and fully developed ascomata (Fig. 1). Similar morphological adaptation is also known within the basal ascomycetes (subphylum Taphrinomycotina). Saprobic Neolecta species produce large ascomata on the ground, while pathogenic species of Taphrina produce naked asci on the leaf surface [17]. The phylogenetic dependence across all taxa between leaf-living ecology and reduced morphology is strongly significant (Bayes Fac- tor = ,30). For leaf endophytes and pathogens, a small, covered ascomata is an adaptation to the ecological constraints of limited physical support, strong radiation, and rapid evaporation. For their saprobic relatives, a large and usually stalked fruiting body has been suggested to be advantageous for spore dispersal. This advantage of height and the ensuing spore production pattern may play a role in the evolution of morphology in both ascomycete and basidiomycete (mushroom) forest floor saprobes. The genetic basis of both sexual and asexual development in diverse organisms has received extensive attention. Many fungi have both asexual and sexual cycles in their life histories. In general, asexually produced conidiomata are very simple and significantly morphologically distinct from the more complex, sexually produced ascomata. But for some leotiomycetes, such as the Holwaya and Ascocoryne, the conidiomata are prominent and composed of tissues homologous to the ascomata [18]. In contrast, ascomata of endophytic species of the Rhytismatales resemble the simple conidiomata with which they are physically associated [19]. Some of the same genes or pathways may function pleiotropically in the development of both ascomata and conidiomata in these fungi. One of the central questions in evolutionary developmental biology is how the same genes and often the same pathways may be activated to perform different developmental functions in different tissues and at different times [20]. Simple morphologies without closely-related, significant evolved differences limit the usefulness of model fungi such as yeasts and Neurospora in exploring the genetic basis of the development and evolution of intricate morphological structures. Will there be another golden age for fungi in ‘‘evo-devo’’, after yeasts and Neurospora? With many genome projects being completed and new interesting discoveries about the constraints and evolvability of morphology in fungi, it seems possible. Materials and Methods 1. Taxon sampling The data matrix contained 77 taxa of the Ascomycota and one basidiomycete Cotylidia sp. as the outgroup, constructed from sequences of SSU rDNA, LSU rDNA, and 5.8S rDNA genes (table 1). All sequence data were downloaded from GenBank or were produced by the AFTOL project and used in previous study by Z.W. [8]. PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4246