The oldest fossil evidence of animal parasitism by fungi supports a Cretaceous diversification of fungal-arthropod symbioses

Paleoophiocordyceps coccophagus, a fungal parasite of a scale insect from the Early Cretaceous (Upper Albian), is reported and described here. This fossil not only provides the oldest fossil evidence of animal parasitism by fungi but also contains morphological features similar to asexual states of Hirsutella and Hymenostilbe of the extant genus Ophiocordyceps (Ophiocordycipitaceae, Hypocreales, Sordariomycetes, Pezizomycotina, Ascomycota). Because species of Hypocreales collectively exhibit a broad range of nutritional modes and symbioses involving plants, animals and other fungi, we conducted ancestral host reconstruction coupled with phylogenetic dating analyses calibrated with P.coccophagus. These results support a plant-based ancestral nutritional mode for Hypocreales, which then diversified ecologically through a dynamic process of intra- and interkingdom host shifts involving fungal, higher plant and animal hosts. This is especially evident in the families Cordycipitaceae, Clavicipitaceae and Ophiocordycipitaceae, which are characterized by a high occurrence of insect pathogens. The ancestral ecologies of Clavicipitaceae and Ophiocordycipitaceae are inferred to be animal pathogens, a trait inherited from a common ancestor, whereas the ancestral host affiliation of Cordycipitaceae was not resolved. Phylogenetic dating supports both a Jurassic origin of fungal-animal symbioses within Hypocreales and parallel diversification of all three insect pathogenic families during the Cretaceous, concurrent with the diversification of insects and angiosperms.     

Clarification of the host substrate of Ascopolyporus and description of Ascopolyporus philodendrus sp. nov

During a recent collection trip to Barro Colorado Island, Panama, two species belonging to genus Ascopolyporus (Clavicipitaceae, Hypocreales) were collected. Species of Ascopolyporus are epibionts of their bamboo (Poaceae) host and long thought to be biotrophs of their plant hosts. However, based on morphological observations and phylogenetic evidence using large subunit ribosomal DNA data, we propose that genus Ascopolyporus is likely composed of pathogens of scale insects (Coccoideae, Homoptera). Phylogenetic analyses included Ascopolyporus spp. in a clade containing only entomopathogenic clavicipitaceous species (100% posterior probability), and the scale insect pathogen Hyperdermium bertonii was found to share the most recent common ancestor with the Ascopolyporus clade (98% posterior probability). In addition remnants of the scale insect were observed to be embedded within stromata during early stages of stroma development. Ascopolyporus philodendrus sp. nov. was described and distinguished from the type species of the genus, A. polychrous, based on perithecial size, ascus size, plant host substrate and phylogenetic evidence. Furthermore subfamily Clavicipitoideae (Clavicipitaceae) was included and well supported in a single clade (100% posterior probability).     

New species of Septobasidium from southern Costa Rica and the southeastern United States

New species are described in Septobasidium, a genus of urediniomycete parasites on scale insects. One new species, S. gomezii, is described from Costa Rica, and another, S. meredithiae, is described from Louisiana. S. gomezii is most similar to S. septobasidioides, but macroscopic and microscopic differences support it being a distinct species. S. meredithiae is similar to S. alni and S. castaneum but differs from these species in several macroscopic and microscopic characters, especially when the species are observed on the same host tree and insect species. Another species collected only once in Costa Rica is listed with observations but it is not formally described here. This Septobasidium species shares some key characteristics with S. ramorum but combines a dense, compact, nearly black thallus and pigmented probasidia-like structures with spindle-shaped haustoria. Implications for taxonomy, morphological evolution and host specificity in Septobasidium are discussed.     

Regiocrella, a new entomopathogenic genus with a pycnidial anamorph and its phylogenetic placement in the Clavicipitaceae

A new genus, Regiocrella, is described with two species, R. camerunensis and R. sinensis, based on specimens collected in Cameroon and China. Both species are parasitic on scale insects (Coccidae, Homoptera). Morphological and molecular evidence place the new genus in the Clavicipitaceae (Hypocreales), despite its combination of characters that are atypical of that family; Regiocrella is characterized by having perithecia partly immersed in a subiculum, noncapitate asci, unicellular fusiform ascospores and pycnidial-acervular conidiomata. The two new species, R. camerunensis and R. sinensis, are distinguished based on ascospore and perithecium size. Morphological characters were evaluated and compared to other genera in the Clavicipitaceae, especially those parasitic on scale insects or with pycnidial-acervular anamorphs or synanamorphs (i.e. Aschersonia, Ephelis or Sphacelia): Atkinsonella, Balansia, Claviceps, Epichl?e, Hypocrella, Myriogenospora and Neoclaviceps. The phylogenetic relationships of Regiocrella were examined with three gene loci: large subunit nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha (TEF), and RNA polymerase II subunit 1 (RPB1). The results of this study confirm that Regiocrella is distinct from other genera in the Clavicipitaceae and that its two species form a monophyletic group. Regiocrella is shown to be closely related to the scale insect pathogen Hypocrella and the plant-associated genera Balansia, Claviceps, Epichl?e, Myriogenospora and Neoclaviceps. This study also provides insights into the evolution of pycnidial-acervular conidiomata and scale insect parasitism within the Clavicipitaceae. Plant-associated genera form a monophyletic group correlated with Clavicipitaceae subfamily Clavicipitoideae sensu Diehl. We also demonstrate that scale insect parasites have multiple evolutionary origins within the family and genera with pycnidial-acervular anamorphs or synanamorphs have a single origin.     

Origins of social parasitism: the importance of divergence ages in phylogenetic studies

Phylogenetic studies on insect social parasites have found very close host-parasite relationships, and these have often been interpreted as providing evidence for sympatric speciation. However, such phylogenetic inferences are problematic because events occurring after the origin of parasitism, such as extinction, host switching and subsequent speciation, or an incomplete sampling of taxa, could all confound the interpretation of phylogenetic relationships. Using a tribe of bees where social parasitism has repeatedly evolved over a wide time-scale, we show the problems associated with phylogenetic inference of sympatric speciation. Host-parasite relationships of more ancient species appear to support sympatric speciation, whereas in a case where parasitism has evolved very recently, sympatric speciation can be ruled out. However, in this latter case, a single extinction event would have lead to relationships that support sympatric speciation, indicating the importance of considering divergence ages when analysing the modes of social parasite evolution.     

Phylogeny and biogeography of the flowering plant genus Styrax (Styracaceae) based on chloroplast DNA restriction sites and DNA sequences of the internal transcribed spacer region

Phylogenetic relationships within the flowering plant genus Styrax were investigated with DNA sequence data from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and with chloroplast DNA restriction site data from the genes trnK, rpoC1, and rpoC2. The data sets from each genome were analyzed separately and in combination with parsimony methods. The results strongly support the monophyly of each of the four series of the genus but provide little phylogenetic resolution among them. Reticulate evolution may at least partly explain discordance between the molecular phylogenetic estimates and a prior morphological estimate within series Cyrta. The historical biogeography of the genus was inferred with unweighted parsimony character optimization of trees recovered from a combined ITS and morphological data set, after a series of combinability tests for data set congruence was conducted. The results are consistent with the fossil record in supporting a Eurasian origin of Styrax. The nested phylogenetic position of the South American members of the genus within those from southern North America and Eurasia suggests that the boreotropics hypothesis best explains the amphi-Pacific tropical disjunct distribution occurring within section Valvatae. The pattern of relationship recovered among the species of section Styrax ((western North America + western Eurasia) (eastern North America + eastern Eurasia)) is rare among north-temperate Tertiary forest relicts. The monophyly of the group of species from western North America and western Eurasia provides qualified support for the Madrean-Tethyan hypothesis, which posits a Tertiary floristic connection among the semiarid regions in which these taxa occur. A single vicariance event between eastern Asia and eastern North America accounts for the pattern of relationship among intercontinental disjuncts in series Cyrta.     

A molecular phylogenetic study of armoured scale insects (Hemiptera: Diaspididae)

Abstract.  Armoured scale insects are economically important parasites of woody plants and grasses. They are promising subjects for the evolutionary study of physiology (no complete gut), genetics (chimerism, paternal genome elimination, frequent parthenogenesis) and coevolution (with host plants, parasitoids, Septobasidium fungi, endosymbiotic bacteria). Little phylogenetic work has been accomplished with armoured scales, and uncertainty surrounds their classification. Here, we report the phylogenetic results of Bayesian and parsimony analyses of 705 base pairs of Elongation Factor 1α and 660 base pairs of 28S from eighty-nine species of armoured scale insects, representing forty-seven genera and five tribes in the subfamilies Diaspidinae and Aspidiotinae, together with two outgroups. 28S was aligned based on a secondary structural model. Our results broadly corroborate the major features of the existing classification, although we do not find perfect monophyly of any of the traditionally recognized subfamilies or tribes. The subfamily Aspidiotinae is paraphyletic with respect to the subfamily Diaspidinae. Diaspidinae consists of two main clades that only roughly correspond to the tribes Lepidosaphidini and Diaspidini. Diaspidini is nearly monophyletic, except that it includes a single aspidiotine species. Other members of the tribe Aspidiotini form a clade, except that the clade includes a single species of Leucaspidini and excludes Maskellia and Pseudaonidia . Our results weakly support the hypothesis that the most recent common ancestor of the Diaspididae had adult females that were permanently enclosed within the derm of the second instar (the pupillarial habit) and had diploid adult males that eliminated their paternal genomes during spermatogenesis (late paternal genome elimination).     

An overview of the higher level classification of Pucciniomycotina based on combined analyses of nuclear large and small subunit rDNA sequences

In this study we provide a phylogenetically based introduction to the classes and orders of Pucciniomycotina (= Urediniomycetes), one of three subphyla of Basidiomycota. More than 8000 species of Pucciniomycotina have been described including putative saprotrophs and parasites of plants, animals and fungi. The overwhelming majority of these (approximately 90%) belong to a single order of obligate plant pathogens, the Pucciniales (= Uredinales), or rust fungi. We have assembled a dataset of previously published and newly generated sequence data from two nuclear rDNA genes (large subunit and small subunit) including exemplars from all known major groups in order to test hypotheses about evolutionary relationships among the Pucciniomycotina. The utility of combining nuc-lsu sequences spanning the entire D1-D3 region with complete nuc-ssu sequences for resolution and support of nodes is discussed. Our study confirms Pucciniomycotina as a monophyletic group of Basidiomycota. In total our results support eight major clades ranked as classes (Agaricostilbomycetes, Atractiellomycetes, Classiculomycetes, Cryptomycocolacomycetes, Cystobasidiomycetes, Microbotryomycetes, Mixiomycetes and Pucciniomycetes) and 18 orders.     

Host jumping onto close relatives and across kingdoms by Tyrannicordyceps (Clavicipitaceae) gen. nov. and Ustilaginoidea_(Clavicipitaceae)

? Premise of study: This research seeks to advance understanding of conditions allowing movement of fungal pathogens among hosts. The family Clavicipitaceae contains fungal pathogens exploiting hosts across three kingdoms of life in a pattern that features multiple interkingdom host shifts among plants, animals, and fungi. The tribe Ustilaginoideae potentially represents a third origin of plant pathogenesis, although these species remain understudied. Fungal pathogens that cause ergot are linked morphologically with Clavicipitaceae, but are not yet included in phylogenetic studies. The placement of Ustilaginoideae and ergot pathogens will allow differentiation between the host habitat and host relatedness hypotheses as mechanisms of phylogenetic diversification of Clavicipitaceae. ? Methods: A multigene data set was assembled for Clavicipitaceae to test phylogenetic placement and ancestral character-state reconstructions for Ustilaginoidea virens and U. dichromonae as well as the ergot mycoparasite Cordyceps fratricida. Microscopic morphological observations of sexual and asexual states were also performed. ? Key results: Phylogenetic placement of U. virens and U. dichromonae represents a third acquisition of the plant pathogenic lifestyle in Clavicipitaceae. Cordyceps fratricida was also placed in Clavicipitaceae and recognized as a new genus Tyrannicordyceps. Ancestral character state reconstructions indicate initially infecting hemipteran insect hosts facilitates subsequent changes to a plant pathogenic lifestyle. The ancestor of T. fratricida is inferred to have jumped from grasses to pathogens of grasses. ? Conclusions: The host habitat hypothesis best explains the dynamic evolution of host affiliations seen in Clavicipitaceae and throughout Hypocreales. Co-occurrence in the same habitat has allowed for host shifts from animals to plants, and from plants to fungi.     

How do obligate parasites evolve? A multi-gene phylogenetic analysis of downy mildews

Plant parasitism has independently evolved as a nutrition strategy in both true fungi and Oomycetes (stramenopiles). A large number of species within phytopathogenic Oomycetes, the so-called downy mildews, are defined as obligate biotrophs since they have not, to date, been cultured on any artificial medium. Other genera like Phytophthora and Pythium can in general be cultured on standard or non-standard agar media. Within all three groups there are many important plant pathogens responsible for severe economic losses as well as damage to natural ecosystems. Although they are important model systems to elucidate the evolution of obligate parasites, the phylogenetic relationships between these genera have not been clearly resolved. Based on the most comprehensive sampling of downy mildew genera to date and a representative sample of Phytophthora subgroups, we inferred the phylogenetic relationships from a multi-gene dataset containing both coding and non-coding nuclear and mitochondrial loci. Phylogenetic analyses were conducted under several optimality criteria and the results were largely consistent between all the methods applied. Strong support is achieved for monophyly of a clade comprising both the genus Phytophthora and the obligate biotrophic species. The facultatively parasitic genus Phytophthora is shown to be at least partly paraphyletic. Monophyly of a cluster nested within Phytophthora containing all obligate parasites is strongly supported. Within the obligate biotrophic downy mildews, four morphologically or ecologically well-defined subgroups receive statistical support: (1) A cluster containing all species with brownish-violet conidiosporangia, i.e., the genera Peronospora and Pseudoperonospora; (2) a clade comprising the genera with vesicular to pyriform haustoria (Basidiophora, Benua, Bremia, Paraperonospora, Plasmopara, Plasmoverna, Protobremia); (3) a group containing species included in Hyaloperonospora and Perofascia which almost exclusively infect Brassicaceae; (4) a clade including the grass parasites Viennotia oplismeni and Graminivora graminicola. Phylogenetic relationships between these four clades are not clearly resolved, and neither is the position of Sclerospora graminicola within the downy mildews. Character analysis indicates an evolutionary scenario of gradually increasing adaptation to plant parasitism in Peronosporales and that at least the most important of these adaptive steps occurred only once, including major host shifts within downy mildews.     

Assessing the diversity of AM fungi in arid gypsophilous plant communities

In the present study, we used PCR-Single-Stranded Conformation Polymorphism (SSCP) techniques to analyse arbuscular mycorrhizal fungi (AMF) communities in four sites within a 10 km² gypsum area in Southern Spain. Four common plant species from these ecosystems were selected. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, SSCP analysis, sequencing and phylogenetic analyses. A total of 1443 SSU rRNA sequences were analysed, for 21 AM fungal types: 19 belonged to the genus Glomus , 1 to the genus Diversispora and 1 to the Scutellospora. Four sequence groups were identified, which showed high similarity to sequences of known glomalean species or isolates: Glo G18 to Glomus constrictum , Glo G1 to Glomus intraradices , Glo G16 to Glomus clarum , Scut to Scutellospora dipurpurescens and Div to one new genus in the family Diversisporaceae identified recently as Otospora bareai . There were three sequence groups that received strong support in the phylogenetic analysis, and did not seem to be related to any sequences of AM fungi in culture or previously found in the database; thus, they could be novel taxa within the genus Glomus : Glo G4, Glo G2 and Glo G14. We have detected the presence of both generalist and potential specialist AMF in gypsum ecosystems. The AMF communities were different in the plant studied suggesting some degree of preference in the interactions between these symbionts.     

Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.     

Community structure of arbuscular mycorrhizal fungi in undisturbed vegetation revealed by analyses of LSU rDNA sequences

Arbuscular mycorrhizal fungi (AMF) form a mutualistic symbiosis with plant roots and are found in most ecosystems. In this study the community structure of AMF in a clade of the genus Glomus was examined in undisturbed costal grassland using LSU rDNA sequences amplified from roots of Hieracium pilosella. Roots were sampled from May to November along eight 30-m transects, 30-120 m apart. Phylogenetic analysis of the sequences revealed 11 phylogenetic clusters within the clade of Glomus. The phylogenetic clusters were patchily distributed within the area; time had no influence on the distribution pattern. The dominant cluster covered up to 10 m along the transect, whereas other clusters formed what can be interpreted as small individual mycelia. Four of the phylogenetic clusters included known species; the other clusters, including the dominant sequence types, were unknown. The dominant phylogenetic cluster enclosed nine haplotypes, and analyses of genetic diversity of this phylogenetic cluster showed that the total diversity could be found within single root fragments, suggesting that the multiple sequences were derived from a single individual.     

Remarkable nutritional diversity of basidiomycetes in the Corticiales, including a new foliicolous species of Marchandiomyces (anamorphic Basidiomycota, Corticiaceae) from Australia

Fungi in the basidiomycete order Corticiales are remarkably diverse nutritionally, including a variety of saprotrophs, plant and fungal pathogens, and lichen-forming fungi. Tracing the origin of this diversity depends on a clearer understanding of the phylogenetic relationships of fungi in the order. One of its core members is the genus Marchandiomyces, originally established for lichen pathogens that form orange or coral bulbils. We describe here a new species in the genus, M. marsonii sp. nov., which is unusual in its appearance, habit, and geographic provenance. It is foliicolous on leaves of Pandanus (screw pines, Pandanaceae) and produces flattened, coral bulbils resembling apothecia of the ascomycete genus Orbilia. It is also the first member of the genus to be collected from Australia. An isolate of the new fungus and several additional cultures of related plant pathogenic fungi were obtained and investigated phylogenetically using parsimony, likelihood, and Bayesian analyses of nuclear small and large subunit ribosomal sequences. Our phylogeny makes clear that Marchandiomyces species and their close relatives contribute significantly to the ecological diversity of the Corticiales and that this diversity is derived mainly from lignicolous ancestors.     

Community composition, host range and genetic structure of the fungal entomopathogen Beauveria in adjoining agricultural and seminatural habitats

Although intensively investigated for biological control of insect pests, little is known about the ecology of the fungal entomopathogenic genus Beauveria in natural or agricultural habitats. In this study, we used molecular phylogenetic and genotypic information to infer species diversity, reproductive potential and genetic structure of Beauveria occurring within a single arable field and bordering hedgerow in Denmark. Isolates were sampled from cultivated field and hedgerow soils, from insects harbouring latent fungal infections, and from the phylloplanes of three plant species common in the hedgerow flora. A nuclear phylogeny of this local Beauveria assemblage resolved seven phylogenetic species, including (i) five phylogenetic species within Beauveria bassiana sensu stricto ; (ii) Clade C, a taxonomically uncharacterized species that is morphologically indistinguishable but phylogenetically distant from B. bassiana s.s. ; and (iii) Beauveria brongniartii. All seven species were present throughout the hedgerow habitat, including as infections in insects. Significantly, only B . bassiana s.s. phylogenetic species Eu_1 was isolated from tilled soils. Mating type polymerase chain reaction assays demonstrated that all five B. bassiana s.s. phylogenetic species possess bipolar outcrossing mating systems. Of these, only the Eu_1 population contained two mating types; however, a 31:2 skew in MAT1:MAT2 mating types suggests a low frequency of sexual reproduction in this population. The four remaining B. bassiana s.s. phylogenetic species were fixed for single mating types and these populations are evidently clonal. Multilocus microsatellite genotyping revealed polymorphism in all five phylogenetic species of B. bassiana s.s. ; however, all show evidence of clonal genetic structure.     

Effects of variation among plant species on the interaction between a herbivore and its parasitoid

Abstract.  1. Previous studies have demonstrated that phenotypic traits of plants have the potential to affect interactions between herbivores and their natural enemies. Consequently, the impact of natural enemies on herbivore vital rates and population dynamics may vary among plant species. This study was designed to investigate the potential for density-dependent parasitism of an aphid herbivore feeding on six different host plant species.
2. Population densities of the aphid Aphis nerii B de F (Homoptera: Aphididae) and its parasitoid Lysiphlebus testaceipes Cresson (Hymenoptera: Braconidae) were recorded within a single growing season on six different species of milkweed in the genus Asclepias L. (Asclepiadaceae). Asclepias species are known to vary in their quality as food for herbivores. Although data on plant quality were not available in this study, population data were analysed to determine the effects of different Asclepias species on rates of parasitism and aphid population growth.
3. Parasitism rates of A. nerii varied among Asclepias species but were temporally density dependent over at least some range of aphid density on all plant species. Aphid population growth rates also varied among Asclepias species, and declined with an increase in the maximum parasitism rates among plant species; however, in no case was density-dependent parasitism sufficient to prevent exponential population growth of aphids within the growing season. The results serve to emphasise that, if natural enemies are to regulate herbivore populations, density-dependent mortality is a necessary, but not sufficient, condition for regulation.     

Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA.

Marram grass (Ammophila arenaria L.), a sand-stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes. To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 18S rRNA (rDNA). A nested PCR strategy was employed to amplify a 569-bp region of the 18S rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands. PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species. DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species. The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data. DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis. Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences. The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys.     

Phylogenetic comparison of protein-coding versus ribosomal RNA-coding sequence data: a case study of the Lecanoromycetes (Ascomycota)

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.     

Bezerromycetales and Wiesneriomycetales ord. nov. (class Dothideomycetes), with two novel genera to accommodate endophytic fungi from Brazilian cactus

During a survey of endophytic fungi from the cactus Tacinga inamoena in a Brazilian tropical dry forest (Caatinga) some undescribed ascomycetous fungi were isolated. These fungi are characterized by superficial and immersed, globose to subglobose, smooth or hairy ascomata, bitunicate asci, and muriformly septate, ellipsoidal ascospores. Multigene phylogenetic analyses using sequences from partial ITS, SSU and LSU nrDNA and the translation elongation factor 1-alpha gene (tef1) demonstrated a monophyletic clade accommodating these endophytic fungi in the class Dothideomycetes, closely related to the order Tubeufiales. Based on morphological features and phylogenetic analyses, these fungi could not be placed in the order Tubeufiales, in the new order Wiesneriomycetales, or any other known genus in the class Dothideomycetes. Thus, two new genera (Bezerromyces, with B. brasiliensis and B. pernambucoensis, and Xiliomyces with X. brasiliensis), a new family (Bezerromycetaceae) and a new order (Bezerromycetales) are introduced to accommodate these novel taxa. Our phylogenetic analyses also demonstrated that the clade accommodating Wiesneriomycetaceae represents a new order, here introduced as Wiesneriomycetales.     

Intrageneric phylogeny of Capsella (Brassicaceae) and the origin of the tetraploid C. bursa-pastoris based on chloroplast and nuclear DNA sequences

Polyploidization, often accompanied by hybridization, has been of major importance in flowering plant evolution. Here we investigate the importance of these processes for the evolution of the tetraploid crucifer Capsella bursa-pastoris using DNA sequences from two chloroplast loci as well as from three nuclear low-copy genes. The near-absence of variation at the C. bursa-pastoris chloroplast markers suggests a single and recent origin of the tetraploid. However, despite supporting a single phylogeny, chloroplast data indicate that neither of the extant Capsella diploids is the maternal parent of the tetraploid. Combined with data from the three nuclear loci, our results do not lend support to previous hypotheses on the origin of C. bursa-pastoris as an allopolyploid between the diploids C. grandiflora and C. rubella or an autopolyploid of C. grandiflora. Nevertheless, for each locus, some of the C. bursa-pastoris accessions harbored C. rubella alleles, indicating that C. rubella contributed to the gene pool of C. bursa-pastoris, either through allopolyploid speciation or, more likely, through hybridization and introgression. To our knowledge, this study is the first of a wild, nonmodel plant genus that uses a combination of chloroplast and multiple low-copy nuclear loci for phylogenetic inference of polyploid evolution.