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Species in the fungal genus Sticta form symbiotic associations primarily with either green algae or cyanobacteria, but tripartite associations or photosymbiodemes involving both types of photobionts occur in some species. Sticta is known to associate with green algae in the genus Symbiochloris. However, previous studies have shown that algae from other genera, such as Heveochlorella, may also be suitable partners for Sticta. We examined the diversity of green algal partners in the genus Sticta and assessed the patterns of association between the host fungus and its algal symbiont. We used multi-locus sequence data from multiple individuals collected in Australia, Cuba, Madagascar, Mauritius, New Zealand, Reunion and South America to infer phylogenies for fungal and algal partners and performed tests of congruence to assess coevolution between the partners. In addition, event-based methods were implemented to examine which cophylogenetic processes have led to the observed association patterns in Sticta and its green algal symbionts. Our results show that in addition to Symbiochloris, Sticta associates with green algae from the genera Chloroidium, Coccomyxa, Elliptochloris and Heveochlorella, the latter being the most common algal symbiont associated with Sticta in this study. Geography plays a strong role in shaping fungal-algal association patterns in Sticta as mycobionts associate with different algal lineages in different geographic locations. While fungal and algal phylogenies were mostly congruent, event-based methods did not find any evidence for cospeciation between the partners. Instead, the association patterns observed in Sticta and associated algae, were largely explained by other cophylogenetic events such as host-switches, losses of symbiont and failure of the symbiont to diverge with its host. Our results also show that tripartite associations with green algae evolved multiple times in Sticta., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ecological preferences, partner compatibility, or partner availability are known to be important factors shaping obligate and intimate lichen symbioses. We considered a complex of Cladonia species, traditionally differentiated by the extent of sexual reproduction and the type of vegetative propagules, to assess if the reproductive and dispersal strategies affect mycobiont-photobiont association patterns. In total 85 lichen thalli from 72 European localities were studied, two genetic markers for both Cladonia mycobionts and Asterochloris photobionts were analyzed. Variance partitioning analysis by multiple regression on distance matrices was performed to describe and partition variance in photobiont genetic diversity. Asexually reproducing Cladonia in our study were found to be strongly specific to their photobionts, associating with only two closely related Asterochloris species. In contrast, sexually reproducing lichens associated with seven unrelated Asterochloris lineages, thus being photobiont generalists. The reproductive mode had the largest explanatory power, explaining 44% of the total photobiont variability. Reproductive and dispersal strategies are the key factors shaping photobiont diversity in this group of Cladonia lichens. A strict photobiont specialisation observed in two studied species may steer both evolutionary flexibility and responses to ecological changes of these organisms, and considerably limit their distribution ranges., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The class Laboulbeniomycetes comprises biotrophic parasites associated with arthropods and fungi. Two orders are currently recognized, Pyxidiophorales and Laboulbeniales. Herpomyces is an isolated genus of Laboulbeniales, with species that exclusively parasitize cockroaches (Blattodea). Here, we evaluate 39 taxa of Laboulbeniomycetes with a three-locus phylogeny (nrSSU, ITS, nrLSU) and propose a new order in this class. Herpomycetales accommodates a single genus, Herpomyces, with currently 26 species, one of which is described here based on morphological and molecular data. Herpomyces shelfordellae is found on Shelfordella lateralis cockroaches from Hungary, Poland, and the USA. We also build on the six-locus dataset from the Ascomycota Tree of Life paper (Schoch and colleagues, 2009) to confirm that Laboulbeniomycetes and Sordariomycetes are sister classes, and we apply laboulbeniomyceta as a rankless taxon for the now well-resolved node that describes the most recent common ancestor of both classes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A major challenge to evolutionary biologists is to understand how biodiversity is distributed through space and time and across the tree of life. Diversification of organisms is influenced by many factors that act at different times and geographic locations but it is still not clear which have a significant impact and how drivers interact. To study diversification, we chose the lichen genus Sticta, by sampling through most of the global range and producing a time tree. We estimate that Sticta originated about 30 million years ago, but biogoegraphic analysis was unclear in estimating the origin of the genus. Furthermore, we investigated the effect of dispersal ability finding that Sticta has a high dispersal rate, as collections from Hawaii showed that divergent lineages colonized the islands at least four times. Symbiont interactions were investigated using BiSSE to understand if green-algal or cyanobacterial symbiont interactions influenced diversification, only to find that the positive results were driven almost completely by Type I error. On the other hand, another BiSSE analysis found that an association with Andean tectonic activity increases the speciation rate of species., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lichen biodiversity and its generative evolutionary processes are practically unknown in the MIOI (Madagascar and Indian Ocean Islands) biodiversity hotspot. We sought to test the hypothesis that lichenized fungi in this region have undergone a rapid radiation, following a single colonization event, giving rise to narrow endemics, as is characteristic of other lineages of plants. We extensively sampled specimens of the lichen genus Sticta in the Mascarene archipelago (mainly Réunion) and in Madagascar, mainly in the northern range (Amber Mt and Marojejy Mt) and produced the fungal ITS barcode sequence for 148 thalli. We further produced a four-loci data matrix for 68 of them, representing the diversity and geographical distribution of ITS haplotypes. We reconstructed the phylogenetic relationships within this group, established species boundaries with morphological context, and estimated the date of the most recent common ancestor. Our inferences resolve a robust clade comprising 31 endemic species of Sticta that arose from the diversification following a single recent (c. 11 Mya) colonization event. All but three species have a very restricted range, endemic to either the Mascarene archipelago or a single massif in Madagascar. The first genus of lichens to be studied with molecular data in this region underwent a recent radiation, exhibits micro-endemism, and thus exemplifies the biodiversity characteristics found in other taxa in Madagascar and the Mascarenes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Synteny can be maintained for certain genomic regions across broad phylogenetic groups. In these homologous genomic regions, sites that are under relaxed purifying selection, such as intergenic regions, could be used broadly as markers for population genetic and phylogenetic studies on species complexes. To explore the potential of this approach, we found 125 Collinear Orthologous Regions (COR) ranging from 1 to >10kb across nine genomes representing the Lecanoromycetes and Eurotiomycetes (Pezizomycotina, Ascomycota). Twenty-six of these COR were found in all 24 eurotiomycete genomes surveyed for this study. Given the high abundance and availability of fungal genomes we believe this approach could be adopted for other large groups of fungi outside the Pezizomycotina. Asa proof of concept, we selected three Collinear Orthologous Regions (COR1b, COR3, and COR16), based on synteny analyses of several genomes representing three classes of Ascomycota: Eurotiomycetes, Lecanoromycetes, and Lichinomycetes. COR16, for example, was found across these three classes of fungi. Here we compare the resolving power of these three new markers with five loci commonly used in phylogenetic studies of fungi, using section Polydactylon of the cyanolichen-forming genus Peltigera (Lecanoromycetes) - a clade with several challenging species complexes. Sequence data were subjected to three species discovery and two validating methods. COR markers substantially increased phylogenetic resolution and confidence, and highly contributed to species delimitation. The level of phylogenetic signal provided by each of the COR markers was higher than the commonly used fungal barcode ITS. High cryptic diversity was revealed by all methods. As redefined here, most species represent lineages that have relatively narrower, and more homogeneous biogeographical ranges than previously understood. The scabrosoid clade consists of ten species, seven of which are new. For the dolichorhizoid clade, twenty-two new species were discovered for a total of twenty-nine species in this clade., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The precise boundary delineations between taxa in symbiotic associations are very important for evolutionary and ecophysiological studies. Growing evidence indicates that in many cases, the use of either morphological characters or molecular markers results in diversity underestimation. In lichen symbioses, Trebouxia is the most common genus of lichen phycobionts, however, the diversity within this genus has been poorly studied and as such there is no clear species concept. This study constitutes a multifaceted approach incorporating aspects of ultrastructural characterization by TEM and phylogenomics to evaluate the morphological and genetic diversity of phycobionts within the sexually reproducing lichen Ramalina fraxinea in the context of Mediterranean and temperate populations. Results reveal an association with at least seven different Trebouxia lineages belonging to at least two species, T. decolorans and T. jamesii, and diverse combinations of such lineages coexisting within the same thallus depending on the analyzed sample. Some of these lineages are shared by several other non-related lichen taxa. Our findings indicate the existence of a highly diverse assemblage of Trebouxia algae associating with R. fraxinea and suggest a possible incipient speciation within T. decolorans rendering a number of lineages or even actual species. This study stresses the importance of coordinated ultrastructural and molecular analyses to improve estimates of diversity and reveal the coexistence of more than one Trebouxia species within the same thallus. It is also necessary to have clearer species delimitation criteria within the genus Trebouxia and microalgae in general., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Symbiotic fungi living in plants as endophytes, and in lichens as endolichenic fungi, cause no apparent symptoms to their hosts. They are ubiquitous, ecologically important, hyperdiverse, and represent a rich source of secondary compounds for new pharmaceutical and biocontrol products. Due in part to the lack of visible reproductive structures and other distinctive phenotypic traits for many species, the diversity and phylogenetic affiliations of these cryptic fungi are often poorly known. The goal of this study was to determine the phylogenetic placement of representative endophytes within the Eurotiomycetes (Pezizomycotina, Ascomycota), one of the most diverse and evolutionarily dynamic fungal classes, and to use that information to infer processes of macroevolution in trophic modes. Sequences of a single locus marker spanning the nuclear ribosomal internal transcribed spacer region (nrITS) and 600 base pairs at the 5' end of the nuclear ribosomal large subunit (nrLSU) were obtained from previous studies of >6000 endophytic and endolichenic fungi from diverse biogeographic locations and hosts. We conducted phylum-wide phylogenetic searches using this marker to determine which fungal strains belonged to Eurotiomycetes and the results were used as the basis for a class-wide, seven-locus phylogenetic study focusing on endophytic and endolichenic Eurotiomycetes. Our cumulative supermatrix-based analyses revealed that representative endophytes within Eurotiomycetes are distributed in three main clades: Eurotiales, Chaetothyriales and Phaeomoniellales ord. nov., a clade that had not yet been described formally. This new order, described herein, is sister to the clade including Verrucariales and Chaetothyriales. It appears to consist mainly of endophytes and plant pathogens. Morphological characters of endophytic Phaeomoniellales resemble those of the pathogenic genus Phaeomoniella. This study highlights the capacity of endophytic and endolichenic fungi to expand our understanding of the ecological modes associated with particular clades, and provides a first estimation of their phylogenetic relationships in the Eurotiomycetes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Lecanoromycetes is the largest class of lichenized Fungi, and one of the most species-rich classes in the kingdom. Here we provide a multigene phylogenetic synthesis (using three ribosomal RNA-coding and two protein-coding genes) of the Lecanoromycetes based on 642 newly generated and 3329 publicly available sequences representing 1139 taxa, 317 genera, 66 families, 17 orders and five subclasses (four currently recognized: Acarosporomycetidae, Lecanoromycetidae, Ostropomycetidae, Umbilicariomycetidae; and one provisionarily recognized, 'Candelariomycetidae'). Maximum likelihood phylogenetic analyses on four multigene datasets assembled using a cumulative supermatrix approach with a progressively higher number of species and missing data (5-gene, 5+4-gene, 5+4+3-gene and 5+4+3+2-gene datasets) show that the current classification includes non-monophyletic taxa at various ranks, which need to be recircumscribed and require revisionary treatments based on denser taxon sampling and more loci. Two newly circumscribed orders (Arctomiales and Hymeneliales in the Ostropomycetidae) and three families (Ramboldiaceae and Psilolechiaceae in the Lecanorales, and Strangosporaceae in the Lecanoromycetes inc. sed.) are introduced. The potential resurrection of the families Eigleraceae and Lopadiaceae is considered here to alleviate phylogenetic and classification disparities. An overview of the photobionts associated with the main fungal lineages in the Lecanoromycetes based on available published records is provided. A revised schematic classification at the family level in the phylogenetic context of widely accepted and newly revealed relationships across Lecanoromycetes is included. The cumulative addition of taxa with an increasing amount of missing data (i.e., a cumulative supermatrix approach, starting with taxa for which sequences were available for all five targeted genes and ending with the addition of taxa for which only two genes have been sequenced) revealed relatively stable relationships for many families and orders. However, the increasing number of taxa without the addition of more loci also resulted in an expected substantial loss of phylogenetic resolving power and support (especially for deep phylogenetic relationships), potentially including the misplacements of several taxa. Future phylogenetic analyses should include additional single copy protein-coding markers in order to improve the tree of the Lecanoromycetes. As part of this study, a new module ("Hypha") of the freely available Mesquite software was developed to compare and display the internodal support values derived from this cumulative supermatrix approach., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The phylum Ascomycota is by far the largest group in the fungal kingdom. Ecologically important mutualistic associations such as mycorrhizae and lichens have evolved in this group, which are regarded as key innovations that supported the evolution of land plants. Only a few attempts have been made to date the origin of Ascomycota lineages by using molecular clock methods, which is primarily due to the lack of satisfactory fossil calibration data. For this reason we have evaluated all of the oldest available ascomycete fossils from amber (Albian to Miocene) and chert (Devonian and Maastrichtian). The fossils represent five major ascomycete classes (Coniocybomycetes, Dothideomycetes, Eurotiomycetes, Laboulbeniomycetes, and Lecanoromycetes). We have assembled a multi-gene data set (18SrDNA, 28SrDNA, RPB1 and RPB2) from a total of 145 taxa representing most groups of the Ascomycota and utilized fossil calibration points solely from within the ascomycetes to estimate divergence times of Ascomycota lineages with a Bayesian approach. Our results suggest an initial diversification of the Pezizomycotina in the Ordovician, followed by repeated splits of lineages throughout the Phanerozoic, and indicate that this continuous diversification was unaffected by mass extinctions. We suggest that the ecological diversity within each lineage ensured that at least some taxa of each group were able to survive global crises and rapidly recovered., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

In lichen-forming fungi, traditional taxonomical concepts are frequently in conflict with molecular data, and identifying appropriate taxonomic characters to describe phylogenetic clades remains challenging in many groups. The selection of suitable markers for the reconstruction of solid phylogenetic hypotheses is therefore fundamental. The lichen genus Usnea is highly diverse, with more than 350 estimated species, distributed in polar, temperate and tropical regions. The phylogeny and classification of Usnea have been a matter of debate, given the lack of phenotypic characters to describe phylogenetic clades and the low degree of resolution of phylogenetic trees. In this study, we investigated the phylogenetic relationships of 52 Usnea species from across the genus, based on ITS rDNA, nuLSU, and two protein-coding genes RPB1 and MCM7. ITS comprised several highly variable regions, containing substantial genetic signal, but also susceptible to causing bias in the generation of the alignment. We compared several methods of alignment of ITS and found that a simultaneous optimization of alignment and phylogeny (using BAli-phy) improved significantly both the topology and the resolution of the phylogenetic tree. However the resolution was even better when using protein-coding genes, especially RPB1 although it is less variable. The phylogeny based on the concatenated dataset revealed that the genus Usnea is subdivided into four highly-supported clades, corresponding to the traditionally circumscribed subgenera Eumitria, Dolichousnea, Neuropogon and Usnea. However, characters that have been used to describe these clades are often homoplasious within the phylogeny and their parallel evolution is suggested. On the other hand, most of the species were reconstructed as monophyletic, indicating that combinations of phenotypic characters are suitable discriminators for delimitating species, but are inadequate to describe generic subdivisions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recent molecular systematic studies have indicated that the traits currently used for generic delimitation in the jelly lichens (Collemataceae s. str.), may not characterize monophyletic groups. Here we reconstruct the phylogeny of Collemataceae using Bayesian and maximum likelihood analyses based on mitochondrial (mtSSU rDNA) and nuclear (nuLSU rDNA, Beta-tubulin and MCM7) markers of 70 Collemataceae species. We studied the evolution of four morphological and ecological characters traditionally used to delimit genera and infra-generic groups. Finally, we tested if differences in branch-lengths between clades are due to differences in rates of molecular evolution. Eleven strongly supported groups were recovered in the resulting well-resolved and well-supported phylogeny. The presence/absence of a eucortex, which is currently used as the cardinal character to define genera in the group, does not characterize monophyletic groups corresponding to the genera as currently circumscribed. Ancestral state reconstruction indicates that the most recent common ancestor of the jelly lichens most likely was saxicolous/terricolous, lacked a tomentum, and had transversally septate ascospores. Although the cortex state could not be reconstructed for the ancestor of the family, our observations indicate that a lack of cortex may have an evolutionary advantage in saxicolous/terricolous species in semi-arid environments, as non-corticate species tends to be larger and occur in higher frequency and abundance in such regions, compared to corticate species. A significant evidence for faster evolutionary rates was found in a lineage mainly including taxa that occur in the wet tropics and humid temperate regions, compared to other lineages. We suggest that this can explain the greater diversity of Collemataceae in tropical and humid areas., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Accurate species circumscriptions are central for many biological disciplines and have critical implications for ecological and conservation studies. An increasing body of evidence suggests that in some cases traditional morphology-based taxonomy have underestimated diversity in lichen-forming fungi. Therefore, genetic data play an increasing role for recognizing distinct lineages of lichenized fungi that it would otherwise be improbable to recognize using classical phenotypic characters. Melanohalea (Parmeliaceae, Ascomycota) is one of the most widespread and common lichen-forming genera in the northern Hemisphere. In this study, we assess traditional phenotype-based species boundaries, identify previously unrecognized species-level lineages and discuss biogeographic patterns in Melanohalea. We sampled 487 individuals worldwide, representing 18 of the 22 described Melanohalea species, and generated DNA sequence data from mitochondrial, nuclear ribosomal, and protein-coding markers. Diversity previously hidden within traditional species was identified using a genealogical concordance approach. We inferred relationships among sampled species-level lineages within Melanohalea using both concatenated phylogenetic methods and a coalescent-based multilocus species tree approach. Although lineages identified from genetic data are largely congruent with traditional taxonomy, we found strong evidence supporting the presence of previously unrecognized species in six of the 18 sampled taxa. Strong nodal support and overall congruence among independent loci suggest long-term reproductive isolation among most species-level lineages. While some Melanohalea taxa are truly widespread, a limited number of clades appear to have much more restricted distributional ranges. In most instances the concatenated gene tree and multilocus species tree approaches provided similar estimates of relationships. However, nodal support was generally higher in the phylogeny estimated from concatenated data, and relationships among taxa within one major clade were largely unresolved in the species tree. This study contributes to our understanding of diversity and evolution in common lichen-forming fungi by incorporating multiple locus sequence data to circumscribe morphologicallly cryptic lineages and infer relationships within a coalescent-based species tree approach., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Through a culture-based survey of living sapwood and leaves of rubber trees (Hevea spp.) in remote forests of Peru, we discovered a new major lineage of Ascomycota, equivalent to a class rank. Multilocus phylogenetic analyses reveal that this new lineage originated during the radiation of the 'Leotiomyceta', which resulted not only in the evolution of the Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Geoglossomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes, and Sordariomycetes, but also of the majority of hyperdiverse foliar endophytes. Because its origin is nested within this major burst of fungal diversification, we could not recover strong support for its phylogenetic relationship within the 'Leotiomyceta'. Congruent with their long phylogenetic history and distinctive preference for growing in sapwood, this new lineage displays unique morphological, physiological, and ecological traits relative to known endophytes and currently described members of the 'Leotiomyceta'. In marked contrast to many foliar endophytes, the strains we isolated fail to degrade cellulose and lignin in vitro. Discovery of the new class, herein named Xylonomycetes and originally mis-identified by ITSrDNA sequencing alone, highlights the importance of inventorying tropical endophytes from unexplored regions, using multilocus data sets to infer the phylogenetic placement of unknown strains, and the need to sample diverse plant tissues using traditional methods to enhance efforts to discover the evolutionary, taxonomic, and functional diversity of symbiotrophic fungi., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Although significant progress has been made resolving deep branches of the fungal tree of life, many fungal systematists are interested in species-level questions to both define species and assess fungal biodiversity. Fungal genome sequences are a useful resource to systematic biologists for developing new phylogenetic markers that better represent the whole genome. Here we report primers for two newly identified single-copy protein-coding genes, FG1093 and MS204, for use with ascomycetes. Although fungi were the focus of this study, this methodological approach could be easily applied to marker development for studies of other organisms. The tests used here to assess phylogenetic informativeness are computationally rapid, require only rudimentary datasets to evaluate existing or newly developed markers, and can be applied to other non-model organisms to assist in experimental design of phylogenetic studies. Phylogenetic utility of the markers was tested in two genera, Gnomoniopsis and Ophiognomonia (Gnomoniaceae, Diaporthales). The phylogenetic performance of β-tubulin, ITS, and tef-1α was compared with FG1093 and MS204. Phylogenies inferred from FG1093 and MS204 were largely in agreement with β-tubulin, ITS, and tef-1α although some topological conflict was observed. Resolution and support for branches differed based on the combination of markers used for each genus. Based on two independent tests of phylogenetic performance, FG1093 and MS204 were determined to be equal to or better than β-tubulin, ITS, and tef-1α in resolving species relationships. Differences were found in site-specific rate of evolution in all five markers. In addition, isolates from 15 orders and 22 families of Ascomycota were screened using primers for FG1093 and MS204 to demonstrate primer utility across a wide diversity of ascomycetes. The primer sets for the newly identified genes FG1093 and MS204 and methods used to develop them are useful additions to the ascomycete systematists' toolbox., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The resolution of the phylogenetic relationships within the order Teloschistales (Ascomycota, lichen-forming-fungi), with nearly 2000 known species and outstanding phenotypic diversity, has been hindered by the limitation in the resolving power that single-locus or two-locus phylogenetic studies have provided to date. In this context, an extensive taxon sampling within the Teloschistales with more loci (especially nuclear protein-coding genes) was needed to confront the current taxonomic delimitations and to understand evolutionary trends within this order. Comprehensive maximum likelihood and bayesian analyses were performed based on seven loci using a cumulative supermatrix approach, including protein-coding genes RPB1 and RPB2 in addition to nuclear and mitochondrial ribosomal RNA-coding genes. We included 167 taxa representing 12 of the 15 genera recognized within the currently accepted Teloschistineae, 22 of the 43 genera within the Physciineae, 49 genera of the closely related orders Lecanorales, Lecideales, and Peltigerales, and the dubiously placed family Brigantiaeaceae and genus Sipmaniella. Although the progressive addition of taxa (cumulative supermatrix approach) with increasing amounts of missing data did not dramatically affect the loss of support and resolution, the monophyly of the Teloschistales in the current sense was inconsistent, depending on the loci-taxa combination analyzed. Therefore, we propose a new, but provisional, classification for the re-circumscribed orders Caliciales and Teloschistales (previously referred to as Physciineae and Teloschistineae, respectively). We report here that the family Brigantiaeaceae, previously regarded as incertae sedis within the subclass Lecanoromycetidae, and Sipmaniella, are members of the Teloschistales in a strict sense. Within this order, one lineage led to the diversification of the mostly epiphytic crustose Brigantiaeaceae and Letrouitiaceae, with a circumpacific center of diversity and found mostly in the tropics. The other main lineage led to another epiphytic crustose family, mostly tropical, and with an Australasian center of diversity--the Megalosporaceae--which is sister to the mainly rock-inhabiting, cosmopolitan, and species rich Teloschistaceae, with a diversity of growth habits ranging from crustose to fruticose. Our results confirm the use of a cumulative supermatrix approach as a viable method to generate comprehensive phylogenies summarizing relationships of taxa with multi-locus to single locus data., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A molecular phylogeny of combined mtSSU, nuLSU, and RPB2 data revealed previously unrecognized levels of parallel evolution and phenotypic divergence in the lichen family Graphidaceae. Five clades were supported within the family: the Fissurina, Ocellularia, Graphis, Topeliopsis, and Thelotrema clades, containing 33 of the 42 currently accepted genera within the family. The results for the first time provide a fully resolved phylogeny of this family and confirm the synonymy of Graphidaceae and Thelotremataceae. Ancestral character state reconstruction using likelihood, Bayesian, and parsimony approaches indicate that lirellate ascomata evolved independently in each of the five clades. Carbonized ascomata evolved independently in at least four of the five clades. An unexpected result was the independent evolution of columella structures in the Fissurina and Ocellularia clades. Besides these more general findings, we document several cases in which evolution of several traits in parallel resulted in striking look-alikes within unrelated lineages, such as Topeliopsismuscigena and Chapsameridensis in the Topeliopsis and Thelotrema clades, Leptotremawightii, Myriotremalaeviusculum, and Leucodectonphaeosporum in the Ocellularia and Thelotrema clades, Ocellulariastylothecia and Melanotremameiosporum in the Fissurina and Ocellularia clades, and Myriotremapycnoporellum, Myriotremaclandestinum and Wirthiotremaglaucopallens in the Fissurina, Ocellularia, and Topeliopsis clades. Pagel's test of independent character evolution suggested that at least for some of the traits involved in these cases, ecological constraints may have caused their evolution in parallel. The most intriguing find is the correlation between gall-forming thalli and vertical columns of calcium oxalate crystals, suggesting that these crystals do not function as light distributors, as previously assumed, but instead stabilize the thalli which are usually hollow beneath, similar to a dome-shaped structure. Ancestral character state reconstruction together with an approach to visualize the phenotype of putative ancestral lineages suggested the alpha-Graphidaceae to resemble some of the extant species currently classified in Myriotrema s.lat., with pore-like ascomata, and non-amyloid ascospores with lens-shaped lumina., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mounting evidence suggests many morphology-based species circumscriptions in lichenized ascomycetes misrepresent fungal diversity. The lichenized ascomycete genus Xanthoparmelia includes over 800 described species displaying a considerable range of morphological and chemical variation. Species circumscriptions in this genus have traditionally been based on thallus morphology, medullary chemistry, and the presence or absence of sexual or asexual reproductive structures. Notwithstanding concerted effort on the part of taxonomists to arrive at a natural classification, modern taxonomic concepts for the most part remain unclear. Here we assess the evolution of characters traditionally regarded as taxonomically important by reconstructing a phylogenetic hypothesis based on sequence data from four nuclear ribosomal markers as well as fragments from two protein-coding nuclear loci. A total of 414 individuals were tested, representing 19 currently accepted species. Most sampled species, as currently circumscribed, were recovered as polyphyletic, suggesting that major diagnostic characters have evolved in a homoplasious manner. The vagrant growth form, distinct medullary chemistries, and production of vegetative diaspores appear to have evolved independently multiple times. Application of a population assignment test resulted in the recognition of 21 species-level genetic clusters, each of which was supported by a comparison of genetic distances as well as a Bayesian species delimitation method calculating probabilities associated with speciation events. Inferred clusters are largely incongruent with traditionally circumscribed species due to the prevalence of cryptic diversity and, in some cases, high levels of intraspecific morphological and chemical variation. These results call for a major taxonomic revision of Xanthoparmelia species in western North America., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Neofusicoccum is a recently described genus of common endophytes and pathogens of woody hosts, previously placed in the genus Botryosphaeria. Many morphological characteristics routinely used to describe species overlap in Neofusicoccum, and prior to the use of molecular phylogenetics, isolates from different hosts and locations were often misidentified. Two cryptic species Neofusicoccum ribis and Neofusicoccum parvum were initially described from different continents and recently another four species within this complex were described using fixed nucleotide polymorphisms for differentiation. In a survey of eucalypt cankers in eastern Australia, a collection of morphologically similar Neofusicoccum isolates were obtained. This collection was analysed within the framework of the morphological (MSRC), ecological (ESRC) and phylogenetic (PSRC) species recognition concepts. Morphological data based on spore measurements (MSRC), together with pathogenicity trials (ESRC) were considered alongside molecular analysis (PSRC), which included multiple gene phylogenies constructed from four nuclear gene regions. We also used the Genealogical Sorting Index method to provide objective evidence for the status of terminal taxa in the phylogenetic analysis. The isolates examined exhibited overlapping morphological and culture characteristics, similar pathogenicity to excised stems and shared hosts within the same locations. Phylogenetic analysis separated isolates into 8 clades corresponding to six described species: N. ribis, N. parvum, Neofusicoccum kwambonambiense, Neofusicoccum cordaticola, Neofusicoccum umdonicola, Neofusicoccum batangarum, and two new species. GSI support indicated combined phylogenetic data were monophyletic for all clades and all p-values were significant allowing us to reject the null hypothesis that all groups were from a single mixed group. Consequently the description of Neofusicoccum occulatum is presented., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A growing body of evidence indicates that in some cases morphology-based species circumscription of lichenized fungi misrepresents the number of existing species. The cosmopolitan "rock posy" lichen (Rhizoplaca melanophthalma) species-complex includes a number of morphologically distinct species that are both geographically and ecologically widespread, providing a model system to evaluate speciation in lichen-forming ascomycetes. In this study, we assembled multiple lines of evidence from nuclear DNA sequence data, morphology, and biochemistry for species delimitation in the R. melanophthalma species-complex. We identify a total of ten candidate species in this study, four of which were previously recognized as distinct taxa and six previously unrecognized lineages found within what has been thus far considered a single species. Candidate species are supported using inferences from multiple empirical operational criteria. Multiple instances of sympatry support the view that these lineages merit recognition as distinct taxa. Generally, we found little corroboration between morphological and chemical characters, and previously unidentified lineages were morphologically polymorphic. However, secondary metabolite data supported one cryptic saxicolous lineage, characterized by orsellinic-derived gyrophoric and lecanoric acids, which we consider to be taxonomically significant. Our study of the R. melanophthalma species-complex indicates that the genus Rhizoplaca, as presently circumscribed, is more diverse in western North American than originally perceived, and we present our analyses as a working example of species delimitation in morphologically cryptic and recently diverged lichenized fungi., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Lichens are a prominent feature of northern conifer forests and a large number of species are thought to be circumboreal. Whether or not circumboreal lichen species really constitute monophyletic groups has seldom been tested. We investigated molecular phylogenetic patterns in the mycobiont of Mycoblastus sanguinarius, a well known epiphytic lichen species of the boreal forest, based on material collected from across the high latitude northern hemisphere. A three-locus dataset of internal transcribed spacer rDNA, translation elongation factor 1-α and replication licensing factor Mcm7 DNA sequences revealed that material treated until now as belonging to M. sanguinarius does indeed form a monophyletic group within the genus and is distinct from a strongly supported Mycoblastus affinis. The M. sanguinarius complex appears closely related to the rare Mycoblastus glabrescens, which is currently known only from the Pacific Northwest and was rediscovered during the present study. However, within M. sanguinarius s.lat. in the northern hemisphere, two deeply divergent and morphologically coherent species can be recovered, one of which matches the southern hemisphere species Mycoblastus sanguinarioides and turns out to be widespread in North America and Asia, and one of which corresponds to M. sanguinarius s.str. Both M. sanguinarius and M. sanguinarioides exhibit additional low-level genetic differentiation into geographically structured clades, the most prominent of which are distributed in East Asia/eastern North America and western North America/Europe, respectively. Individuals from these lowest-level clades are morphologically indistinguishable but chemical analyses by thin layer chromatography revealed that each clade possesses its own fatty acid profile, suggesting that chemical differentiation precedes morphological differentiation and may be a precursor to speciation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The species delimitation in fungi is currently in flux. A growing body of evidence shows that the morphology-based species circumscription underestimates the number of existing species. The large and ever growing number of DNA sequence data of fungi makes it possible to use these to identify potential cases of hidden species, which then need to be studied with extensive taxon samplings. We used Parmeliaceae, one of the largest families of lichenized fungi as a model. Intra- and interspecific distances derived from maximum-likelihood phylogenetic trees inferred from 491 nuclear ITS rDNA sequences were examined for five major clades of parmelioid lichens. The intra- and interspecific distances were well separated in most cases allowing the calculation of a threshold, with exceptions of highly deviating distances in a few cases. These situations are shown to be taxa in which the current delimitation needs revision. Thus the analysis of the distance distributions is shown to be a powerful tool for identifying species complexes., (Copyright 2010 Elsevier Inc. All rights reserved.)

Many lichen fungi form symbioses with filamentous Nostoc cyanobacteria, which cause the lichen to swell and become extremely gelatinous when moist. Within the Lecanoromycetes, such gelatinous lichens are today mainly classified in the Collemataceae (Peltigerales, Ascomycota). We performed Bayesian MCMC, maximum likelihood, and maximum parsimony analyses of three independent markers (mtSSU rDNA, nuLSU rDNA, and RPB1), to improve our understanding of the phylogeny and classification in the Peltigerales, as well as the evolution of morphological characters that have been used for classification purposes in this group. The Collemataceae and the non-gelatinous Pannariaceae are paraphyletic but can be re-circumscribed as monophyletic if Leciophysma, Physma, Ramalodium and Staurolemma are transferred to the Pannariaceae. The gelatinous taxa transferred to the Pannariaceae deviate from other Collemataceae in having simple ascospores, and several also have a ring-shaped exciple as in other Pannariaceae, rather than the disc-shaped exciple found in the typical Collemataceae. Both Collema and Leptogium are non-monophyletic. The re-circumscribed Collemataceae shares a distinct ascus type with the sister group Placynthiaceae and the Coccocarpiaceae, whereas Pannariaceae includes a variety of structures. All Pannariaceae have one-celled ascospores, whereas all Collemataceae have two- or multi-celled spores. Reconstructions of the number of character state transformations in exciple structure, thallus gelatinosity, and ascus apex structure indicate that the number of transformations is distinctly higher than the minimum possible. Most state transformations in the exciple took place from a ring-shaped to a disc-shaped exciple. Depending on the reconstruction method, most or all transformations in thallus structure took place from a non-gelatinous to a gelatinous thallus. Gains and losses of internal structures in the ascus apex account for all or a vast majority of the number of transformations in the ascus, whereas direct transformations between asci with internal structures appear to have been rare.

Fruiting bodies are responsible for the effective dispersal of meiospores in ascomycetes. Different fruiting body types include open (apothecia) or closed (perithecia, cleistothecia) forms, which have traditionally been used as key paradigms for ascomycete classification. Molecular phylogenies show that most fruiting body types have multiple phylogenetic origins within the phylum, and are not suitable for the circumscription of classes. One exception are perithecia that are restricted in non-lichenized fungi to the monophyletic class Sordariomycetes. However, lichenized fungi with perithecioid fruiting bodies were found to belong to three other classes unrelated to Sordariomycetes. One of these is Lecanoromycetes, which includes the bulk of lichenized fungi. To understand the evolution of perithecioid fruiting bodies in the mostly apotheciate Lecanoromycetes, we assembled a combined data set of nuclear and mitochondrial ribosomal, and RPB1 DNA sequences, and traced the evolution of two morphological characters (fruiting body type and fruiting body development). We reconstructed ancestral character states using maximum likelihood and Bayesian methods. Additionally, we tested for correlation of character changes in a combined Bayesian/maximum likelihood framework. The results suggest that perithecia have evolved in unrelated groups of lichen-forming fungi. Within Lecanoromycetes they have evolved independently several times from apotheciate ancestors. Further, our analyses support a correlation between the type of fruiting body and the type of ascoma ontogeny. The evolution of angiocarpous ascoma development in Lecanoromycetes is a pre-adaptation for the repeated gain of perithecia. This finding is consistent with the hypothesis of a neotenic origin of perithecioid fruiting bodies in Lecanoromycetes.

This study focuses on a pair of fungal species, Moelleriella libera and M. raciborskii (Ascomycota: Clavicipitaceae) from the neotropics and paleotropics, respectively, that are phenotypically nearly indistinguishable. Molecular analyses based on DNA sequences from RNA polymerase II subunit 2 (RPB2), translation elongation factor 1-alpha (EF1-alpha) and beta-tubulin genes confirm that they are recently derived sister species. Speciation appears to have followed an historical transoceanic dispersal event. Models of population structure and migration from TCS, IM, and coalescent-based analyses suggest there is little gene flow between the two species. The direction of dispersal, investigated using the progression rule and coalescent-based gene genealogies, was likely from the New World to the Old World.

Rhynchosporium secalis is an important pathogen of barley globally. Fourteen polymorphic microsatellites were analyzed for 1664 R. secalis isolates sampled from 37 field populations to infer their demographic history. The results falsified the hypothesis that R. secalis co-evolved with its barley host in the Middle East. Populations from Scandinavia had significantly higher allelic diversities, the greatest number of private alleles and the highest genotypic diversities. All but three of the analyzed populations had an excess of gene diversity compared to the number of alleles, consistent with a recent population bottleneck. The remaining populations had a gene diversity deficit consistent with a population expansion following a recent population bottleneck in the last +/-100 years. A coalescent analysis revealed that the effective population sizes based on theta, of the analyzed populations were small relative to their ancestral population sizes, indicating that only a fraction of the diversity present in the ancestral populations was transmitted into current populations. These findings are consistent with the hypothesis that the pathogen population on barley experienced a selection bottleneck imposed by the host and/or are founder populations. The mean estimate of migration rates was 2.2 (avg 90% confidence interval=1.3-3.1). Major migration routes were identified among populations separated by long distances, eg between South Africa and Australia, as well as among North Africa, the Middle East and California, suggesting contemporary exchange of infected barley seed. In contrast with earlier findings, most populations exhibited significant gametic disequilibrium, probably as a result of genetic drift. We conclude that the majority of R. secalis populations have experienced human-mediated migration that led to numerous and relatively recent founder events around the world.

Neofusicoccum parvum and N. ribis (Botryosphaeriaceae, Ascomycetes) are closely related, plant pathogenic fungi with a world-wide distribution on a wide range of woody hosts. Species boundaries in the N. parvum/N. ribis complex have eluded definition, despite the application of various tools for characterisation. In this study, we test the hypothesis that only one species exists amongst isolates from the N. parvum/N. ribis complex, identified from Syzygiumcordatum trees across their native distribution in South Africa. Genealogical concordance phylogenetic species recognition (GCPSR) was applied based on concordance of genealogies obtained from DNA sequence data for five nuclear loci. These data showed that the single species hypothesis must be rejected. Rather, all analyses support the existence of three previously unrecognised, cryptic species within the N. parvum/N.ribis complex from S. cordatum, in addition to N. parvum and N. ribis. The three lineages reflecting these cryptic taxa are sympatric across their geographical range, indicating barriers to gene flow other than geographic isolation. Phenotypic characters failed to detect all the species uncovered by the GCPSR. Sequence data of the Internal Transcribed Spacer (ITS) of the ribosomal DNA locus, which is thought to be useful for barcoding in fungi, did not distinguish all the species with confidence. RNA polymerase II subunit (RPB2) was the most informative to distinguish all the species a posteriori to the application of GCPSR. The results reflect the critical importance of using multiple gene genealogies and adequate sampling to identify cryptic species and to characterise the true diversity within the Botryosphaeriaceae.

Fungi are one of the most diverse groups of Eukarya and play essential roles in terrestrial ecosystems as decomposers, pathogens and mutualists. This study unifies disparate reports of unclassified fungal sequences from soils of diverse origins and anchors many of them in a well-supported clade of the Ascomycota equivalent to a subphylum. We refer to this clade as Soil Clone Group I (SCGI). We expand the breadth of environments surveyed and develop a taxon-specific primer to amplify 2.4kbp rDNA fragments directly from soil. Our results also expand the known range of this group from North America to Europe and Australia. The ancient origin of SCGI implies that it may represent an important transitional form among the basal Ascomycota groups. SCGI is unusual because it currently represents the only major fungal lineage known only from sequence data. This is an important contribution towards building a more complete fungal phylogeny and highlights the need for further work to determine the function and biology of SCGI taxa.

Three anamorph genera of the Botryosphaeriaceae namely Diplodia, Lasiodiplodia and Dothiorella have typically dark, ovoid conidia with thick walls, and are consequently difficult to distinguish from each other. These genera are well-known pathogens of especially pine species. We generated a multiple gene genealogy to resolve the phylogenetic relationships of Botryosphaeriaceae with dark conidial anamorphs, and mapped host associations based on this phylogeny. The multiple gene genealogy separated Diplodia, Lasiodiplodia and Dothiorella and it revealed trends in the patterns of host association. The data set was expanded to include more lineages of the Botryosphaeriaceae, and included all isolates from different host species for which ITS sequence data are available. Results indicate that Diplodia species occur mainly on gymnosperms, with a few species on both gymnosperms and angiosperms. Lasiodiplodia species occur equally on both gymnosperms and angiosperms, Dothiorella species are restricted to angiosperms and Neofusicoccum species occur mainly on angiosperms with rare reports on Southern Hemisphere gymnosperms. Botryosphaeria species with Fusicoccum anamorphs occur mostly on angiosperms with rare reports on gymnosperms. Ancestral state reconstruction suggests that a putative ancestor of the Botryosphaeriaceae most likely evolved on the angiosperms. Another interesting observation was that both host generalist and specialist species were observed in all the lineages of the Botryosphaeriaceae, with little evidence of host associated co-evolution.

Multi-gene phylogenetic analyses were conducted to address the evolution of Clavicipitaceae (Ascomycota). Data are presented here for approximately 5900 base pairs from portions of seven loci: the nuclear ribosomal small and large subunit DNA (nrSSU and nrLSU), beta-tubulin, elongation factor 1alpha (EF-1alpha), the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2), and mitochondrial ATP Synthase subunit 6 (mtATP6). These data were analyzed in a complete 66-taxon matrix and 91-taxon supermatrix that included some missing data. Separate phylogenetic analyses, with data partitioned according to genes, produced some conflicting results. The results of separate analyses from RPB1 and RPB2 are in agreement with the combined analyses that resolve a paraphyletic Clavicipitaceae comprising three well-supported clades (i.e., Clavicipitaceae clade A, B, and C), whereas the tree obtained from mtATP6 is in strong conflict with the monophyly of Clavicipitaceae clade B and the sister-group relationship of Hypocreaceae and Clavicipitaceae clade C. The distribution of relative contribution of nodal support for each gene partition was assessed using both partitioned Bremer support (PBS) values and combinational bootstrap (CB) analyses, the latter of which analyzed bootstrap proportions from all possible combinations of the seven gene partitions. These results suggest that CB analyses provide a more consistent estimate of nodal support than PBS and that combining heterogeneous gene partitions, which individually support a limited number of nodes, results in increased support for overall tree topology. Analyses of the 91-taxa supermatrix data sets revealed that some nodes were more strongly supported by increased taxon sampling. Identifying the localized incongruence of mtATP6 and analyses of complete and supermatrix data sets strengthen the evidence for rejecting the monophyly of Clavicipitaceae and much of the current subfamilial classification of the family. Although the monophyly of the grass-associated subfamily Clavicipitoideae (e.g., Claviceps, Balansia, and Epichloë) is strongly supported, the subfamily Cordycipitoideae (e.g., Cordyceps and Torrubiella) is not monophyletic. In particular, species of the genus Cordyceps, which are pathogens of arthropods and truffles, are found in all three clavicipitaceous clades. These results imply that most characters used in the current familial classification of Clavicipitaceae are not diagnostic of monophyly.

The resolving power and statistical support provided by two protein-coding (RPB1 and RPB2) and three ribosomal RNA-coding (nucSSU, nucLSU, and mitSSU) genes individually and in various combinations were investigated based on maximum likelihood bootstrap analyses on lichen-forming fungi from the class Lecanoromycetes (Ascomycota). Our results indicate that the optimal loci (single and combined) to use for molecular systematics of lichen-forming Ascomycota are protein-coding genes (RPB1 and RPB2). RPB1 and RPB2 genes individually were phylogenetically more efficient than all two- and three-locus combinations of ribosomal loci. The 3rd codon position of each of these two loci provided the most characters in support of phylogenetic relationships within the Lecanoromycetes. Of the three ribosomal loci we used in this study, mitSSU contributed the most to phylogenetic analyses when combined with RPB1 and RPB2. Except for the mitSSU, ribosomal genes were the most difficult to recover because they often contain many introns, resulting in PCR bias toward numerous and intronless co-extracted contaminant fungi (mainly Dothideomycetes, Chaetothyriomycetes, and Sordariomycetes in the Ascomycota, and members of the Basidiomycota), which inhabit lichen thalli. Maximum likelihood analysis on the combined five-locus data set for 82 members of the Lecanoromycetes provided a well resolved and well supported tree compared to existing phylogenies. We confirmed the monophyly of three recognized subclasses in the Lecanoromycetes, the Acarosporomycetidae, Ostropomycetidae, and Lecanoromycetideae; the latter delimited as monophyletic for the first time, with the exclusion of the family Umbilicariaceae and Hypocenomyce scalaris. The genus Candelariella (formerly in the Candelariaceae, currently a member of the Lecanoraceae) represents the first evolutionary split within the Lecanoromycetes, before the divergence of the Acarosporomycetidae. This study provides a foundation necessary to guide the selection of loci for future multilocus phylogenetic studies on lichen-forming and allied ascomycetes.

Although associated with all plants, fungal endophytes (microfungi that live within healthy plant tissues) represent an unknown proportion of fungal diversity. While there is a growing appreciation of their ecological importance and human uses, little is known about their host specificity, geographic structure, or phylogenetic relationships. We surveyed endophytic Ascomycota from healthy photosynthetic tissues of three plant species (Huperzia selago, Picea mariana, and Dryas integrifolia, representing lycophytes, conifers, and angiosperms, respectively) in northern and southern boreal forest (Québec, Canada) and arctic tundra (Nunavut, Canada). Endophytes were recovered from all plant species surveyed, and were present in <1-41% of 2 mm2 tissue segments examined per host species. Sequence data from the nuclear ribosomal internal transcribed spacer region (ITS) were obtained for 280 of 558 isolates. Species-accumulation curves based on ITS genotypes remained non-asymptotic, and bootstrap analyses indicated that a large number of genotypes remain to be found. The majority of genotypes were recovered from only a single host species, and only 6% of genotypes were shared between boreal and arctic communities. Two independent Bayesian analyses and a neighbor-joining bootstrapping analysis of combined data from the nuclear large and small ribosomal subunits (LSUrDNA, SSUrDNA; 2.4 kb) showed that boreal and arctic endophytes represent Dothideomycetes, Sordariomycetes, Chaetothyriomycetidae, Leotiomycetes, and Pezizomycetes. Many well-supported phylotypes contained only endophytes despite exhaustive sampling of available sequences of Ascomycota. Together, these data demonstrate greater than expected diversity of endophytes at high-latitude sites and provide a framework for assessing the evolution of these poorly known but ubiquitous symbionts of living plants.

The highly divergent characters of morphology, ecology, and biology in the Helotiales make it one of the most problematic groups in traditional classification and molecular phylogeny. Sequences of three rDNA regions, SSU, LSU, and 5.8S rDNA, were generated for 50 helotialean fungi, representing 11 out of 13 families in the current classification. Data sets with different compositions were assembled, and parsimony and Bayesian analyses were performed. The phylogenetic distribution of lifestyle and ecological factors was assessed. Plant endophytism is distributed across multiple clades in the Leotiomycetes. Our results suggest that (1) the inclusion of LSU rDNA and a wider taxon sampling greatly improves resolution of the Helotiales phylogeny, however, the usefulness of rDNA in resolving the deep relationships within the Leotiomycetes is limited; (2) a new class Geoglossomycetes, including Geoglossum, Trichoglossum, and Sarcoleotia, is the basal lineage of the Leotiomyceta; (3) the Leotiomycetes, including the Helotiales, Erysiphales, Cyttariales, Rhytismatales, and Myxotrichaceae, is monophyletic; and (4) nine clades can be recognized within the Helotiales.

Parmelioid lichens comprise about 1500 species and have a worldwide distribution. Numerous species are widely distributed and well known, including important bioindicators for atmospheric pollution. The phylogeny and classification of parmelioid lichens has been a matter of debate for several decades. Previous studies using molecular data have helped to establish hypotheses of the phylogeny of certain clades within this group. In this study, we infer the phylogeny of major clades of parmelioid lichens using DNA sequence data from two nuclear loci and one mitochondrial locus from 145 specimens (117 species) that represent the morphological and chemical diversity in these taxa. Parmelioid lichens are not monophyletic; however, a core group is strongly supported as monophyletic, excluding Arctoparmelia and Melanelia s. str., and including Parmeliopsis and Parmelaria. Within this group, seven well-supported clades are found, but the relationships among them remain unresolved. Stochastic mapping on a MC/MCMC tree sampling was employed to infer the evolution of two morphological and two chemical traits believed to be important for the evolutionary success of these lichens, and have also been used as major characters for classification. The results suggest that these characters have been gained and lost multiple times during the diversification of parmelioid lichens.

The dothideomycetous fungus Leptosphaeria maculans comprises a complex of species differing in specificity and pathogenicity on Brassica napus. Twenty-eight isolates were investigated and compared to 20 other species of the Pleosporales order. Sequences of the mating type MAT1-2 (23), fragments of actin (48) and beta-tubulin (45) genes were determined and used for phylogenetic analyses inferred by maximum parsimony, distance, maximum likelihood, and Bayesian approaches. These different approaches using single genes essentially confirmed findings using concatanated sequences. L. maculans formed a monophyletic group separate from Leptosphaeria biglobosa. The L. biglobosa clade encompasses five sub-clades; this finding is consistent with classification made previously on the basis of internal-transcribed sequences of the ribosomal DNA repeat. The propensity for purifying and neutral evolution of the three genes was determined using sliding window analysis, a technique not previously applied to genes of filamentous fungi. For members of the L. maculans species complex, this approach showed that in comparison to actin and beta-tubulin, exonic sequences of MAT1-2 were more diverse and appeared to evolve at a faster rate. However, different regions of MAT1-2 displayed different degrees of sequence conservation. The more conserved upstream region (including the High Mobility Group domain) may be better suited for interspecies differentiation, while the more diverse downstream region is more appropriate for intraspecies comparisons.

To provide a robust phylogeny of Pezizaceae, partial sequences from two nuclear protein-coding genes, RPB2 (encoding the second largest subunit of RNA polymerase II) and beta-tubulin, were obtained from 69 and 72 specimens, respectively, to analyze with nuclear ribosomal large subunit RNA gene sequences (LSU). The three-gene data set includes 32 species of Peziza, and 27 species from nine additional epigeous and six hypogeous (truffle) pezizaceous genera. Analyses of the combined LSU, RPB2, and beta-tubulin data set using parsimony, maximum likelihood, and Bayesian approaches identify 14 fine-scale lineages within Pezizaceae. Species of Peziza occur in eight of the lineages, spread among other genera of the family, confirming the non-monophyly of the genus. Although parsimony analyses of the three-gene data set produced a nearly completely resolved strict consensus tree, with increased confidence, relationships between the lineages are still resolved with mostly weak bootstrap support. Bayesian analyses of the three-gene data, however, show support for several more inclusive clades, mostly congruent with Bayesian analyses of RPB2. No strongly supported incongruence was found among phylogenies derived from the separate LSU, RPB2, and beta-tubulin data sets. The RPB2 region appeared to be the most informative single gene region based on resolution and clade support, and accounts for the greatest number of potentially parsimony informative characters within the combined data set, followed by the LSU and the beta-tubulin region. The results indicate that third codon positions in beta-tubulin are saturated, especially for sites that provide information about the deeper relationships. Nevertheless, almost all phylogenetic signal in beta-tubulin is due to third positions changes, with almost no signal in first and second codons, and contribute phylogenetic information at the "fine-scale" level within the Pezizaceae. The Pezizaceae is supported as monophyletic in analyses of the three-gene data set, but its sister-group relationships is not resolved with support. The results advocate the use of RPB2 as a marker for ascomycete phylogenetics at the inter-generic level, whereas the beta-tubulin gene appears less useful.

Ascospore characters have commonly been used for distinguishing ascomycete taxa, while ascomal wall characters have received little attention. Although taxa in the Sordariales possess a wide range of variation in their ascomal walls and ascospores, genera have traditionally been delimited based on differences in their ascospore morphology. Phylogenetic relationships of multiple representatives from each of several genera representing the range in ascomal wall and ascospore morphologies in the Sordariales were estimated using partial nuclear DNA sequences from the 28S ribosomal large subunit (LSU), beta-tubulin, and ribosomal polymerase II subunit 2 (RPB2) genes. These genes also were compared for their utility in predicting phylogenetic relationships in this group of fungi. Maximum parsimony and Bayesian analyses conducted on separate and combined data sets indicate that ascospore morphology is extremely homoplastic and not useful for delimiting genera. Genera represented by more than one species were paraphyletic or polyphyletic in nearly all analyses; 17 species of Cercophora segregated into at least nine different clades, while six species of Podospora occurred in five clades in the LSU tree. However, taxa with similar ascomal wall morphologies clustered in five well-supported clades suggesting that ascomal wall morphology is a better indicator of generic relationships in certain clades in the Sordariales. The RPB2 gene possessed over twice the number of parsimony-informative characters than either the LSU or beta-tubulin gene and consequently, provided the most support for the greatest number of clades.

The Pertusariaceae is a diverse, cosmopolitan group of crustose lichen-forming fungi. It is particularly rich in secondary metabolites, containing a variety of compounds from different substance classes, such as xanthones, depsides, depsidones, and depsones. Morphology and chemistry-based studies have provided conflicting views of relationships among taxa, and phylogenetic relationships among genera in the Pertusariaceae are still unsettled. To evaluate these relationships, we generated a phylogeny based on nucleotide sequences of the nuclear large subunit (nu LSU) and the mitochondrial small subunit (mt SSU) rRNA genes. For a subset of taxa, we additionally sequenced the mitochondrial large subunit (mt LSU) rDNA. We studied a total of 49 taxa from the family Pertusariaceae and the enigmatic genus Loxosporopsis including four Agyrialean taxa used as outgroups. The alignments were analyzed using a Bayesian approach with Markov Chain Monte Carlo tree sampling (B/MCMC), and maximum parsimony methods. In the resulting phylogenetic estimates the genus Pertusaria in its current circumscription is polyphyletic comprising three major clades: the Pertusaria s.str.-group, the Variolaria-group and the Varicellaria-group. Loxosporopsis is closely related to Pertusaria s.str. We re-analyzed morphological, anatomical, and chemical features in the light of the molecular study and found novel character combinations to describe monophyletic entities. The taxonomic significance of particular secondary chemical constituents in the Pertusariaceae is corroborated.

Despite the recent progress in molecular phylogenetics, many of the deepest relationships among the main lineages of the largest fungal phylum, Ascomycota, remain unresolved. To increase both resolution and support on a large-scale phylogeny of lichenized and non-lichenized ascomycetes, we combined the protein coding-gene RPB2 with the traditionally used nuclear ribosomal genes SSU and LSU. Our analyses resulted in the naming of the new subclasses Acarosporomycetidae and Ostropomycetidae, and the new class Lichinomycetes, as well as the establishment of the phylogenetic placement and novel circumscription of the lichen-forming fungi family Acarosporaceae. The delimitation of this family has been problematic over the past century, because its main diagnostic feature, true polyspory (numerous spores issued from multiple post-meiosis mitoses) with over 100 spores per ascus, is probably not restricted to the Acarosporaceae. This observation was confirmed by our reconstruction of the origin and evolution of this form of true polyspory using maximum likelihood as the optimality criterion. The various phylogenetic analyses carried out on our data sets allowed us to conclude that: (1) the inclusion of phylogenetic signal from ambiguously aligned regions into the maximum parsimony analyses proved advantageous in reconstructing phylogeny; however, when more data become available, Bayesian analysis using different models of evolution is likely to be more efficient; (2) neighbor-joining bootstrap proportions seem to be more appropriate in detecting topological conflict between data partitions of large-scale phylogenies than posterior probabilities; and (3) Bayesian bootstrap proportion provides a compromise between posterior probability outcomes (i.e., higher accuracy, but with a higher number of significantly supported wrong internodes) vs. maximum likelihood bootstrap proportion outcomes (i.e., lower accuracy, with a lower number of significantly supported wrong internodes).

There has been considerable disagreement regarding the relationships among Pestalotiopsis species and their delimitations. A molecular phylogenetic analysis was conducted on 32 species of Pestalotiopsis in order to evaluate the utility of morphological characters currently used in their taxonomy. Phylogenetic relationships were inferred from nucleotide sequences in the ITS regions and 5.8S gene of the rDNA under four optimality criteria: maximum parsimony, weighted parsimony, maximum likelihood, and neighbor joining. Phylogenies estimated from all analyses yielded trees of essentially similar topology and revealed 3 major groups that correspond with morphology-based classification systems. Molecular data indicated that the genus contains two distinct lineages based on pigmentation of median cells and four distinct groupings based on morphology of apical appendages. The analyses did not support reliability of other phenotypic characters of this genus, such as spore dimensions. Characters with particular phylogenetic significance are discussed in relation to the taxonomy of Pestalotiopsis.

To understand the evolution of host-parasite relationships in the genus Golovinomyces (Ascomycete: Erysiphaceae), which are obligate parasitic fungi of plants, we investigated the phylogenetic relationships of the genus based on 60 internal transcribed spacer (ITS) and 41 28S rDNA sequences. Five major groups, each represented by isolates from a single tribe of the Asteraceae, were identified in the taxa analyzed in this study. Host plants of four groups were strictly restricted to the Asteraceae. The fifth group, the Lactuceae group, is a large group composed of isolates collected from the tribe Lactuceae of the Asteraceae and all other plant families, which suggests a close affinity between Golovinomyces and the Asteraceae in the early stages of their evolution. Tree topology comparisons between the asteraceous hosts and their parasites suggest that Golovinomyces diverged along with the phylogeny of host tribes Carsueae, Astereae, Heliantheae, and Lactuceae of the Asteraceae. However, a conflict of branching order between the tribe Anthemideae and their parasites suggests that host-jumping has occurred in the tribe Anthemideae. Consequently, we suggest that there are two different phases in the evolutionary history of the host-parasite relationships of Golovinomyces. One phase is divergence in accord with the phylogeny of their hosts, which occurred within the Asteraceae. The another phase is host-jumping, which occurred from the Asteraceae to other families and within the Asteraceae.

Phylogenetic relationships and levels of geographic differentiation of two closely related bipolar taxa, Cladonia arbuscula and Cladonia mitis, were cladistically examined with ITS regions, SSU rDNA introns, partial beta-tubulin, and partial glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes. In the combined analysis of the four genes, C. arbuscula was paraphyletic, while C. mitis, nested within C. arbuscula, formed a strongly supported monophyletic group. C. arbuscula samples were divided into three separate clades: "arbuscula I," appearing as basal to the other ingroup taxa, "arbuscula II," and "arbuscula III" (the latter represented by only one specimen), which were not correlated with any morphological trait. Only C. mitis specimens formed a morphologically and chemically distinct group. None of the main clades was correlated with geographic origin. The separate analyses were poorly resolved, and in most cases samples from "arbuscula I," "arbuscula II," and "arbuscula III" clades were intermixed. An incongruence test revealed conflict among the four gene regions in almost all cases. Only ITS regions and introns were not significantly incongruent, suggesting lack of recombination within the ribosomal DNA locus. Incomplete lineage sorting and recombination were considered to be the main reasons accounting for the incongruencies. The high proportion of shared polymorphisms between the "arbuscula I" and "arbuscula II" clades, especially found from the beta-tubulin gene and from the ITS regions, and the lack of corroborating morphological characters both indicate a short history of reproductive isolation among the groups. The lack of genetic differentiation among the northern and southern samples within the main clades indicates a relatively recent gene flow, which may have resulted from migrations during the Pleistocene glaciations or from more recent long-distance dispersal., (Copyright 2002 Elsevier Science (USA))

The taxonomy of the coelomycetous fungus Pestalotiopsis and other closely related genera based on morphological characters has been equivocal. To gain insight in the phylogenetic relationships of Pestalotiopsis and its allies, part of the large subunit (28S) ribosomal DNA region was examined and compared with existing morphological information. Phylogenetic analyses were conducted using parsimony, distance, and likelihood criteria. Results of the analyses showed that Bartalinia, Pestalotiopsis, Seimatosporium, and Seiridium represent distinct monophyletic groups with high bootstrap support. However, Truncatella species are paraphyletic with Bartalinia, sharing a common ancestor. Pestalotia species sequenced clustered together with Pestalotiopsis. These genera should be recognized as distinct natural groups except for Monochaetia and Discosia, which need to be further resolved. Tree topologies are generally in concordance with previous morphological hypotheses, most notably the placement of all Pestalotia species, except the type P. pezizoides, in Pestalotiopsis. Well-supported clades corresponding to groupings based on conidial morphology were resolved and the relative importance of morphological characters for generic delimitation is discussed. Molecular data also provide further evidence to support the association of these coelomycetes with the Amphisphaeriaceae.

Despite various morphological and anatomical similarities, the two orders Gyalectales (lichenized ascomycetes) and Ostropales (lichenized and non-lichenized ascomycetes) have been considered to be distantly related to each other and their position within the Ascomycota was unsettled. To estimate relationships within these groups and their respective phylogenenetic placement within the Ascomycota, we analyzed DNA sequences from the nuclear small and large subunit ribosomal RNA genes using Maximum Parsimony, Maximum Likelihood, and Bayesian statistics with Markov chain Monte Carlo algorithms. Support for internal branches estimated with bootstrap was compared to Bayesian posterior probabilities. We report here that the Ostropales, in their current circumscription, are paraphyletic, and that the Ostropales s.l. include the Gyalectales and Trapeliaceae. The Unitunicate Ascohymenials are redelineated to include the Ostropales s.l., as defined here, and the Baeomycetaceae. Dimerella and Coenogonium are congeneric, and Petractis thelotremella and P. hypoleuca are reunited with members of the genus Gyalecta. In addition to requiring less computational time, Bayesian inference of phylogeny recovered the same topology as a conventional heuristic search using Maximum Likelihood as the optimization criterion and seems superior to bootstrapping in estimating support for short internal branches.

Molecular phylogenies for the fungi in the Ascomycota rely heavily on 18S rRNA gene sequences but this gene alone does not answer all questions about relationships. Particularly problematical are the relationships among the first ascomycetes to diverge, the Archiascomycetes, and the branching order among the basal filamentous ascomycetes, the Euascomycetes. Would more data resolve branching order? We used the jackknife and bootstrapping resampling approach that constitutes the "pattern of resolved nodes" method to address the relationship between number of variable sites in a DNA sequence alignment and support for taxonomic clusters. We graphed the effect of increasing sizes of subsamples of the 18S rRNA gene sequences on bootstrap support for nodes in the Ascomycota tree. Nodes responded differently to increasing data. Some nodes, those uniting the filamentous ascomycetes for example, would still have been well supported with only two thirds of the 18S rRNA gene. Other nodes, like the one uniting the Archiascomycetes as a monophyletic group, would require about double the number of variable sites available in the 18S gene for 95% neighbor-joining bootstrap support. Of the several groups emerging at the base of the filamentous ascomycetes, the Pezizales receive the most support as the first to diverge. Our analysis suggests that we would also need almost three times as much sequence data as that provided by the 18S gene to confirm the basal position for the Pezizales and more than seven times as much data to resolve the next group to diverge. If more data from other genes show the same pattern, the lack of resolution for the filamentous ascomycetes may indicate rapid radiation within this clade., (Copyright 2000 Academic Press.)

The ontogeny of the ascostroma, in particular the centrum structures, has always been regarded as an important criterion in the subdivision of the Loculoascomycetideae (ascomycetous fungi). However, the use of pseudoparaphysis type, cellular or trabeculate, to classify taxa at the ordinal level has been contentious due to the lack of information about their evolution. To determine the phylogenetic significance of the pseudoparaphysis and its variants, DNA sequences of the 18S nuclear rRNA genes from representatives of the orders Pleosporales and Melanommatales were obtained and analyzed. Species with pseudoparaphyses formed a monophyletic group with high statistical confidence. The monophyly of a distinct lineage of species with cellular pseudoparaphyses (the order Pleosporales) is rejected. Likewise, monophyly of a distinct lineage of species with trabeculate pseudoparaphyses (the order Melanommatales) is rejected also. The Pleosporales and Melanommatales are, therefore, not natural orders. The Lophiostomataceae, Phaeosphaeriaceae, and Melanommataceae are most probably polyphyletic, as is the genus Massarina., (Copyright 2000 Academic Press.)

Extensive morphological convergence or divergence, a common occurrence in fungi, tends to obscure recognition of phylogenetic relationships among Pezizales, widespread filamentous Ascomycetes with either enclosed underground (hypogeous) or exposed (epigeous) fruit bodies, that often establish mutualistic interactions with arboreous plants. Focusing on hypogeous Pezizales commonly known as truffles, we sequenced the 18S rDNA from nine species belonging to three different families (Tuberaceae, Terfeziaceae, and Balsamiaceae). A data set consisting of 1700 secondary structure-aligned sites, including 24 homologous sequences from the GenBank DNA database and using three reconstruction methods, was employed to infer phylogenies in an interval ranging from the subordinal to the subgeneric level. As revealed by the 18S phylogenetic scheme, Balsamiaceae represent a monophyletic clade, comprising the hypogeous taxa Balsamia and Barssia, nested within Helvellaceae. Similarly, the terfeziacean genera Pachyphloeus and Terfezia constitute together with Cazia a distinct hypogeous clade nested within Pezizaceae. The lack of clustering between Terfezia arenaria and Terfezia terfezioides strongly supports the reassignment of the latter taxon to the original monotypic genus Mattirolomyces. Within Tuberaceae, which are sister to the highly evolved Helvellaceae, the genus Tuber cannot be considered monophyletic if Choiromyces is recognized. The paraphyly of Tuber and other relationships that were not supported by high bootstrap values, nor corroborated by morphological evidence, were supported by a parallel analysis of the faster evolving internal transcribed spacer (ITS) rDNA. Distinct episodes of fruit body morphology shifts are discernable in the 18S rDNA phylogenetic tree. In all cases, the shift from an epigeous to a hypogeous form is the most parsimonious interpretation of character transformation, without any instance of character reversal., (Copyright 1999 Academic Press.)

Four Roccella species, R. canariensis, R. fimbriata, R. montagnei, and R. tuberculata, were found to possess sequence insertions in up to four locations in the first half of the SSU rDNA. Insertions from one of these positions have been classified as group I introns, while the others may represent degenerative forms of group I introns or messenger RNA introns. Two of the insertion-containing taxa, R. canariensis and R. tuberculata, differ only in their dispersal strategy: R. canariensis is sexual, producing only fruiting bodies and R. tuberculata is sterile, producing only vegetative propagules, i.e., soredia. Because insertions occurred in specimens of both taxa, they were used to examine the phylogenetic relationships between and within the two species. The sequence insertions from each of the four positions were aligned and cladistically analyzed separately. Internal transcribed spacers (ITS) were additionally sequenced to study the phylogeny of all R. canariensis and R. tuberculata specimens. Three other Roccella species (R. babingtonii, R. fimbriata, and R. montagnei) and Dirina catalinariae were used as outgroups in this parsimony analysis. Sequence insertions were found to be potentially useful in phylogenetic studies, although due to the sequence dissimilarity, homology relations were difficult to establish above the species level and in some cases even within the species. The phylogenies obtained from the insertion matrices were totally consistent with the ITS data and the insertions were concluded to have been inherited. When the insertion and ITS data were combined for total evidence, R. canariensis and R. tuberculata did not form distinct lineages in the phylogenetic tree, but appeared mixed in well-supported groups containing both sorediate and fertile specimens., (Copyright 1999 Academic Press.)

The phylogenetic relationship of the asexual mycorrhizal fungus Cenococcum geophilam Fr. among sexual ascomycetes was examined by phylogenetic analysis of nucleotide sequence data from the nuclear small subunit (18S) ribosomal RNA genie region. A specific focus of this study was to test the hypothesis that the genus Elaphomyces is the closest sexual relative of C. geophilum. Thus nucleotide sequence data of five C. geophilum isolates, three Elaphomyces species, and 44 additional genera of ascomycetes were included in the phylogenetic analyses. The percentage of similarity among the 18S rDNA sequences of the C. geophilum isolates examined was 99.8 to 100%, indicating that C. geophilum is monophyletic. Percent similarity of nucleotide sequence among the three Elaphomyces species was also high and ranged from 99.4 to 99.5%. DNA parsimony and distance analysis of the sequence data separated these 2 genera on distant clades when sequence from 44 additional genera of ascomycetes was included. Parsimony and distance analyses positioned C. geophilum as a basal, intermediate lineage between the two Loculoascomycete orders, the Pleosporales and the Dothidiales, and strongly supported Elaphomyces to be of Plectomycete origin. Among the sexual Ascomycetes examined, which included representative taxa from four classes of filamentous Ascomycetes (Plectomycetes, Pyrenomycetes, Discomycetes, and Loculoascomycetes), no close sexual relative to C. geophilum was identified. At least four independent lineages of mycorrhizal fungi were identified among the ascomycetes examined.

Nucleotide sequences of the small subunit ribosomal RNA (18S) gene were used to investigate evolutionary relationships within the Fungi. The inferred tree topologies are in general agreement with traditional classifications in the following ways: (1) the Chytridiomycota and Zygomycota appear to be basal groups within the Fungi. (2) The Ascomycota and Basidiomycota are a derived monophyletic group. (3) Relationships within the Ascomycota are concordant with traditional orders and divide the hemi- and euascomycetes into distinct lineages. (4) The Basidiomycota is divided between the holobasidiomycetes and phragmobasidiomycetes. Conflicts with traditional classification were limited to weakly supported branches of the tree. Strongly supported relationships were robust to minor changes in alignment, method of analysis, and various weighting schemes. Weighting, either of transversions or by site, did not convincingly improve the status of poorly supported portions of the tree. The rate of variation at particular sites does not appear to be independent of lineage, suggesting that covariation of sites may be an important phenomenon in these genes.