Accelerated rates of floral evolution at the upper size limit for flowers.

Evolutionary theory explains phenotypic change as the result of natural selection, with constraint limiting the direction, magnitude, and rate of response [1]. Constraint is particularly likely to govern evolutionary change when a trait is at perceived upper or lower limits. Macroevolutionary rates of floral-size change are unknown for any angiosperm family, but it is predicted that rates should be diminished near the upper size limit of flowers, as has been shown for mammal body mass [2]. Our molecular results show that rates of floral-size evolution have been extremely rapid in the endoholoparasite Rafflesia, which contains the world's largest flowers [3]. These data provide the first estimates of macroevolutionary rates of floral-size change and indicate that in this lineage, floral diameter increased by an average of 20 cm (and up to 90 cm)/million years. In contrast to our expectations, it appears that the magnitude and rate of floral-size increase is greater for lineages with larger flowered ancestors. This study suggests that constraints on rates of floral-size evolution may not be limiting in Rafflesia, reinforcing results of artificial- and natural-selection studies in other plants that demonstrated the potential for rapid size changes [4-6].

High diversity of root associated fungi in both alpine and arctic Dryas octopetala.

BACKGROUND: Dryas octopetala is a widespread dwarf shrub in alpine and arctic regions that forms ectomycorrhizal (ECM) symbiotic relationships with fungi. In this study we investigated the fungal communities associated with roots of D. octopetala in alpine sites in Norway and in the High Arctic on Svalbard, where we aimed to reveal whether the fungal diversity and species composition varied across the Alpine and Arctic regions. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was used to identify the fungal communities from bulk root samples obtained from 24 plants. RESULTS: A total of 137 operational taxonomic units (OTUs) were detected (using 97% similarity cut off during sequence clustering) and well-known ECM genera such as Cenococcum, Cortinarius, Hebeloma, Inocybe and Tomentella occurred frequently. There was no decrease in fungal diversity with increasing latitude. The overall spatial heterogeneity was high, but a weak geographical structuring of the composition of OTUs in the root systems was observed. Calculated species accumulation curves did not level off. CONCLUSIONS: This study indicates that the diversity of fungi associated with D. octopetala does not decrease in high latitude arctic regions, which contrasts observations made in a wide spectrum of other organism groups. A high degree of patchiness was observed across root systems, but the fungal communities were nevertheless weakly spatially structured. Non-asymptotical species accumulation curves and the occurrence of a high number of singletons indicated that only a small fraction of the fungal diversity was detected.

Elucidating the evolutionary history of the Southeast Asian, holoparasitic, giant-flowered Rafflesiaceae: pliocene vicariance, morphological convergence and character displacement.

The aim of the present study is to elucidate the evolutionary history of the enigmatic holoparasitic Rafflesiaceae. More specifically, floral morphological evolution is interpreted in a molecular phylogenetic context, the biogeographic history of the family is investigated, and the possibility of character displacement to have been operating in this family is assessed. Parsimony and Bayesian methods are used to estimate phylogeny and divergence times among Rafflesiaceae species based on nuclear and mitochondrial DNA sequence data from Barkman et al. (2008) as well as new sequence data from additional samples and an additional genetic marker, the plastid 16S. Ancestral areas are inferred using dispersal-vicariance analysis (DIVA) as well a more recently developed parametric likelihood method (LAGRANGE), now including an update that allows for estimation over the posterior distribution of dated trees. Our extended molecular phylogeny of Rafflesiaceae implies a general lack of morphological synapomorphies as well as a high level of morphological homoplasy. In particular, a high level of floral morphological homoplasy is detected among Rafflesia species suggestive of similar patterns of pollinator-based selection in different geographic areas, and multiple instances of divergent floral size evolution is consistent with a model of character displacement. Initial diversification of Rafflesiaceae during the Late Cretaceous was followed by a long period of no-net diversification, likely due to extinctions caused by a Late Eocene to Miocene dramatic reduction in rainforest cover. A Late Miocene to Early Pliocene rise in sea-level probably caused the vicariant diversification observed between areas of endemism. The most recent species divergences are concordant with Pleistocene changes in climate and sea-levels, but apparently with no successful inter-area migrations, supportive of savannah, rather than rainforest, covered landbridges. An explosive increase in net diversification rate, most pronounced in Rafflesia, may be explained by Mid-Miocene to Pliocene rainforest-favorable conditions as well as natural selection promoting character displacement for Rafflesia flower size.

Piceirhiza bicolorata- the ectomycorrhizal expression of the Hymenoscyphus ericae aggregate?

The mycobionts of Piceirhiza bicolorata, a distinct ectomycorrhizal morphotype of conifers and hardwoods, have been identified by internal transcribed spacer 1 (ITS1) nuclear ribosomal DNA (rDNA) sequence comparison of the fungi involved. Samples of Piceirhiza bicolorata were obtained from seedlings of Picea abies, Pinus sylvestris, Betula pubescens, Populus tremula, Quercus robur and Salix phylicifolia. In an initial screening, the fungus amplified with universal ITS primers from ectomycorrhizal root samples of P. bicolorata shared approx. 95% ITS1 sequence identity with the ericoid mycorrhizal fungus Hymenoscyphus ericae. A total of 77 out of 88 (= 87.5%) DNA samples (i.e. 52/56 root samples and 25/32 axenic culture isolates) of P. bicolorata were successfully amplified with a taxon-selective primer designed for exclusive amplification of H. ericae-like strains. Forty-seven amplicons were sequenced, yielding 15 different ITS1 genotypes that differed by 1-14 nucleotide character state changes. An inferred ITS1 phylogeny (maximum parsimony) showed that a single major evolutionary lineage of P. bicolorata embraced the historically important H. ericae isolates in a 100% bootstrap-supported clade. The 15 P. bicolorata genotypes were positioned in four subclades, roughly corresponding to morphological groups of P. bicolorata isolates observed in axenic culture. Culture isolates of H. ericae and P. bicolorata share some common morphological features including slow, dense growth and formation of short aerial hyphal aggregates. Our results suggest that members of the H. ericae aggregate participate in the formation of the distinct ectomycorrhizal morphotype P. bicolorata. This opposes the widely accepted discrimination of ericoid and ectomycorrhizal mycobionts of the boreal forest ecosystem. The high prevalence of the P. bicolorata morphotype on pioneer seedlings of P. sylvestris, B. pubescens and S. phylicifolia at a copper mine spoil was remarkable. Hypotheses of possible nutrient mobilization and detoxification potentials of the fungal associates of P. bicolorata are discussed. We hypothesize that ericoid and ectomycorrhizal plants may share mycobionts of the H. ericae aggregate.

Molecular diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Helotiales in burnt and metal polluted habitats.

• The diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Helotiales are reported here based on ITS1-5.8S-ITS2 (internal transcribed spacer, ITS) nrDNA sequences. • Mycobionts were obtained from roots of ericoid plants and grasses and from Piceirhiza bicolorata ectomycorrhizas (pbECM) on conifers and hardwoods, predominantly in burnt and metal-polluted habitats. The mycobionts were sequenced through the ITS and compared with sequences of known helotialean taxa. • We recognized 132 fungal ITS-sequences with affinity to the Helotiales, of which 75% (54 different ITS-genotypes) grouped within the Hymenoscyphus ericae aggregate including Phialophora finlandia. This aggregate showed stronger affinity to members of the Hyaloscyphaceae and Dermateaceae than to Hymenoscyphus fructigenus (genus-type species; Helotiaceae). Most of the pbECM mycobionts grouped with P. finlandia, although some grouped with H. ericae. Two genotypes co-occurred in ericoid and ectomycorrhizal roots. • The H. ericae aggregate may be referable to a generic unit, and includes a diverse group of closely related, more or less darkly pigmented, root-associated ascomycetes where the borders between intra- and interspecific ITS-sequence variation, as well as different mycorrhizal and nonmycorrhizal root-symbioses, remains unclear.

Regional and local population structure of the pioneer wood-decay fungus Trichaptum abietinum.

The population structure of 11 Fennoscandian geographic populations of the pioneer wood-decay basidiomycete Trichaptum abietinum was assessed with PCR-RFLPs, intersequence simple repeats (ISSRs) and mating studies. The three codominant PCR-RFLP markers (1) internal transcribed spacer 2 (nrDNA), (2) glyceraldehyde-3-phosphate dehydrogenase and (3) translation elongation factor 1α showed that genotype distributions in most cases (94%) agreed with Hardy-Weinberg expectations and that random association of alleles occurred across loci. The molecular data suggest that T. abietinum is a highly outcrossing fungus that regularly proliferates and spreads by sexual spores. Interstock mating reactions suggest a high number of mating factors among individuals and that biological barriers to gene flow are nonexistent in the region. The three PCR-RFLP loci gave an overall F(ST) = 0.03, indicating a low level of genetic differentiation and presumably high gene flow among the geographic populations. The ISSR markers revealed no systematic substructuring and the among-population variance component was low (6.1%) in AMOVA. However, all PCR-RFLP and most ISSR markers (7/12) showed significant deviation from the null hypothesis of an even distribution of allele frequencies across the 11 geographic populations. Allele frequencies varied in an apparently random manner, suggesting that genetic drift might be an important structuring factor in T. abietinum. The spatial small-scale distribution of heterokaryons on three selected substrate units (logs) showed that most isolates represented discrete individuals and that a number of genets (19) may occupy a single log. The small-scale genotype distributions (within logs) were in agreement with panmictic Hardy-Weinberg expectations.

Kohninia linnaeicola, a new genus and species of the Sclerotiniaceae pathogenic to Linnaea borealis.

A new genus and species is described to accommodate a newly discovered fungus pathogenic to Linnaea borealis. The fungus forms true sclerotia on stems and leaves of its host and apothecia arise singly or gregariously on the surface of ripe sclerotia. The new fungus was collected together with a stromatic conidiomal fungus that occurred on the same host. A putative teleomorph-anamorph connection of the observed taxa was ruled out by sequence comparison of the nuclear ribosomal internal transcribed spacer DNA sequences (ITS rDNA). Based on morphology and pathogenicity, the new fungus belongs in the family Sclerotiniaceae, Helotiales, Ascomycota. A phylogenetic analysis of ITS rDNA sequences from 26 taxa of the family Sclerotiniaceae was performed to conclude on the systematic position of the new fungus. The small tuberoid sclerotia, brownish subsessile to substipitate apothecia, four-spored asci, ellipsoid to isthmoid ascospores, inability to grow on PDA culture media and a number of ITS rDNA sequence autapomorphies characterize and distinguish the fungus from other taxa of the Sclerotiniaceae.

Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi.

The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3-5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.

Identification of singlet oxygen photosensitizes in lambs drinking water in an alveld risk area in West Norway.

Alveld is a hepatogenous photosensitivity disorder in lambs. Although alveld has been known in Norway for more than 100years, there are still questions related to the cause of the disease. Phytoporphyrin has long been incriminated as the photosensitizer in hepatogenous photosensitivity diseases but previous findings suggest that the photosensitizing mechanism in alveld is more complex, possibly involving other co-factors. The current work investigates the presence of non-hepatogenous photosensitizers originating in lamb's drinking water from various sources. In addition samples of two of the predominent cyanobacteria found in a representative biofilm (i.e. aggregates of microbes) were identified and isolated in axenic (i.e. pure) cultures. Information from the absorption-, fluorescence emission-, and -excitation spectra and the action spectrum for the formation of singlet oxygen was combined in order to identify the chromophores responsible for the formation of singlet oxygen, e.g. phycocyanins from the cyanobacteria. The highest level of singlet oxygen formation was detected in lotic (i.e. flowing) water in the period consistent with the outbreak of the alveld disease in the area. Meteorological data indicate a warm and wet May with a high radiation exposure leading up to a colder and wet June with an even higher solar irradiance. The seasonal variation in the amount of photosensitizers in lamb's drinking water combined meteorological data can be important to predict the outbreak of alveld.

Impaired secondary oxidant deactivation capacity and enhanced oxidative stress in serum from alveld affected lambs.

Alveld is a hepatogenous photosensitivity disorder in lambs. The aim of the study was to investigate if alveld affected lambs had a reduced capacity to handle oxidative stress induced from either endogenous and/or exogenous photosensitizers. Serum samples from alveld lambs (n=33) were compared to serum samples from control lambs (n=31) and exposed to a controlled amount of singlet oxygen ((1)O2). The sera from alveld lambs were found to have an impaired ability to deactivate reactive oxygen species (ROS) compared to control sera. A higher degree of initial hemolysis and a higher concentration of the exogenous photosensitizer phytoporphyrin (PP) were detected in alveld sera compared to the controls. The action spectrum for the formation of (1)O2 indicated that PP as well as the endogenous compound protoporphyrin IX (PP IX) may act as in vivo photosensitizers. A relatively high level of iron was detected in pooled serum from alveld lambs that showed a high degree of hemolysis. It was concluded that alveld photosensitivity is likely to be initiated by a photodynamic reaction involving PP and possibly also PP IX followed by a light-independent reaction involving hemoglobin-related products and catalysis by the Fenton reaction.

Detection of singlet oxygen in blood serum samples of clinically healthy lambs and lambs suffering from alveld disease.

Alveld is a disease in lambs of domestic sheep (Ovis aries L.), characterized by a combination of photosensitivity and liver damage. Generation of singlet oxygen play a major role in phototoxicity reactions. The compound phylloerythrin (phytoporphyrin) is so far assumed to be the main photodynamic agent in hepatogenous photosensitivity diseases in sheep. Phylloerythrin is a potent photosensitizer and an efficient source of singlet oxygen. The compound accumulates in the peripheral circualtion upon liver damage. Liver dysfunction is also likely to cause an increase in the blood level of bilirubin. Formation of singlet oxygen by bilirubin is reported. In the present work the photosensitizing potential of serum has been measured and related to the bilirubin- and phylloerythrin levels in lambs suffering from alveld and in clinically healthy controls. The singlet oxygen level of the serum was taken as a measure of the photosensitizing potential. The observed singlet oxygen values in serum from alveld lambs were significantly higher than the corresponding values observed in clinically healthy control lambs. This indicates that the serum of the alveld lambs contains an elevated concentration of photosensitizer. The singlet oxygen level was not correlated to the concentration of bilirubin or phylloerythrin. The results indicate that the photosensitizing mechanism is quite complex and may involve other sensitizer(s) than phylloerythrin.

Accelerated nrDNA evolution and profound AT bias in the medicinal fungus Cordyceps sinensis.

Cordyceps sinensis is a reputed medicinal fungus growing parasitically on buried larvae of ghost moths in Asian high-altitude grassland ecosystems. We have analysed the intraspecific ITS nrDNA (ITS1, 5.8S gene, ITS2) variation among 71 sequences of C. sinensis available in EMBL/Genbank. The ITS sequences, submitted to Bayesian ML analyses, were distributed into five groups, referred to as A-E. Nine of the sequences (groups D and E) grouped with distantly related hypocrealean/clavicipitalean taxa and are interpreted as sequences erroneously accessioned under wrong taxon names. The remaining 62 sequences constituted three highly supported clades (groups A-C), that may represent cryptic (phylogenetic) species currently ascribed to C. sinensis. A remarkably high sequence divergence occurred in the 5.8S gene between the three groups. Sequences of groups B and C showed accelerated substitution rates and high AT nucleotide bias. We hypothesize that the accelerated evolution and AT bias have been caused by a shift in life historical attributes or ecology. We also suggest that the recorded differences in medicinal effects among C. sinensis populations may be attributed to the existence of genetically differentiated chemotypes in this morphotaxon.

Multiple gene genealogies and AFLPs suggest cryptic speciation and long-distance dispersal in the basidiomycete Serpula himantioides (Boletales).

Serpula himantioides (Boletales, Basidiomycota) produces thin resupinate basidiocarps on dead coniferous wood worldwide and causes damage in buildings as well. In this study, we present evidence for the existence of at least three phylogenetically defined cryptic species (referred to as Sib I-III) within the morphospecies S. himantioides, a conclusion based on analyses of sequence data from four DNA regions and amplified fragment length polymorphisms (AFLPS). A low degree of shared sequence polymorphisms was observed among the three lineages indicating a long-lasting separation. The AFLPs revealed two additional subgroups within Sib III. Results from mating studies were consistent with the molecular data. In Sib III, no correspondence between genetic and geographical distance was observed among isolates worldwide, presumably reflecting recent dispersal events. Our results indicate that at least two of the lineages (Sib II and Sib III) have wide sympatric distributions. A population genetic analysis of Sib III isolates, scoring sequence polymorphisms as codominant SNP markers, indicates that panmictic conditions exist in the Sib III group. This study supports the view that cryptic speciation is a common phenomenon in basidiomycete fungi and that phylogenetic species recognition can be a powerful inference to detect cryptic species. Furthermore, this study shows that AFLP data are a valuable supplement to DNA sequence data in that they may detect a finer level of genetic variation.

Genetics of self/nonself recognition in Serpula lacrymans.

This study provides an analysis of the vegetative incompatibility system in Serpula lacrymans (Basidiomycota), a genetic system used to recognize nonself in fungi. Seventy-five worldwide isolates could be grouped into eight vegetative compatibility (VC) types, some of them distributed on different continents. Mating studies combined with vegetative incompatibility analyses revealed that the vegetative incompatibility response between isolates mainly could be explained by two biallelic vegetative incompatibility (vic) loci. The frequency distributions of the interpreted vic alleles do not seem to support the idea of frequency-dependent or balancing selection acting on the vic loci. We find little genetic variation at the vic loci and in one of the loci there was a significant heterozyote deficiency among strains in the overall material. The results may be explained by a recent worldwide dispersal of a few S. lacrymans isolates and, correspondingly, only a few vic alleles are being maintained in these populations.

Towards a phylogenetic classification of Cordyceps: ITS nrDNA sequence data confirm divergent lineages and paraphyly.

The ascomycetous genus Cordyceps accommodates endoparasitic species that attack arthropods or other fungi. Analyses of ITS nrDNA sequence data of 72 taxa from the teleomorph genera Cordyceps, Claviceps, Epichloë, and the anamorph genera Akanthomyces, Beauveria, Metarhizium, Hirsutella, Hymenostilbe, Paecilomyces, Polycephalomyces, and Tolypocladium assigned the taxa to four main evolutionary lineages not reflected in the current classification of Cordyceps. Ten subclades were recognized from separate analyses of data subsets. Judged from the ITS phylogenies, Cordyceps spp. with branched stromata were highly supported as a divergent lineage. Host specificity was found to be of limited phylogenetic significance, and several host shifts are suggested to have occurred during the evolution of Cordyceps. Similar ascospore morphology was not reflected in the phyletic groups, and closely related taxa showed large interspecific variation with respect to the number of segments in which the ascospores are divided. However, combinations of selected characters were found to delimitate some lineages, e.g. all Cordyceps spp. that attack hosts in the insect orders Coleoptera and Lepidoptera, and with non-immersed perithecia and clavate to acicular, brightly yellowish to reddish stromata, constituted a separate clade. Furthermore, all Cordyceps spp. with perithecia obliquely immersed in the stroma were recognized as a distinct monophyletic group. This clade is additionally characterized by the formation of anamorphs ascribable to the genus Hymenostilbe. The mycogenous Cordyceps spp. grouped in a separate subclade, interspersed by two cicadaen parasites and all Tolypocladium spp. except T. parasiticum. Tolypocladium and Beauveria were found to be polyphyletic. The included Claviceps and Epichloë taxa appeared to be derived within Cordyceps, thus making Cordyceps paraphyletic as suggested in other studies.

Genetic structure of Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota).

The genetic structure of five Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota) was investigated using codominant PCR-RFLP, allele specific amplification (ASA) markers, inter simple sequence repeat (ISSR) markers and mating studies. Sequence analyses of a subset of single spore isolates revealed sequence variation in four target sequences; internal transcribed spacer (ITS) and intergenic spacer (IGS1) of the nuclear ribosomal DNA, the translation elongation factor 1 alpha (efa) gene and the super oxide dismutase (sod) gene. No sequence variation was found in amplified portions of the mitochondrial large and small rRNA genes. Genotype distributions were mostly (90%) in accordance with Hardy-Weinberg expectations, and the nrDNA markers (ITS/IGS1), efa and sod were in most cases (87%) in linkage equilibrium, indicating an outcrossing reproductive mode, panmictic conditions and large population sizes of the fungus. Mating tests confirmed that F. rosea exhibits an outcrossing bipolar heterothallic mating system. Mating allele richness was high in two investigated populations. Phylogenetic analyses of ITS and IGS1 sequences from the five geographic populations revealed some geographic sub-structuring of the ITS sequences, but no sub-structuring of IGS1. The nrDNA (ITS/IGS1), efa and sod markers gave a low overall FST (0.013). The ISSR markers gave no clustering of the populations in UPGMA, and the between-population variance component was very low in AMOVA (0.4%), indicating a high level of gene flow.

Molecular phylogenetics suggest a North American link between the anthropogenic dry rot fungus Serpula lacrymans and its wild relative S. himantioides.

The dry rot fungus Serpula lacrymans causes damages in wooden buildings and constructions in temperate regions worldwide. In this study, the global phylogeography of S. lacrymans and its wild relative S. himantioides has been investigated to clarify genealogical relationships and determine the origin and spread of the building strains. Internal transcribed spacer (ITS) nrDNA and parts of the beta-tubulin (tub) and the translation elongation factor (efa) 1a genes were sequenced, and phylogenetic relationships inferred. Some analyses suggest that S. lacrymans may have originated from an ancient S. himantioides lineage, but most results support that S. lacrymans and S. himantioides are monophyletic sister species. Phylogenetic analysis of the ITS data revealed two subgroups within S. lacrymans corresponding to two earlier described varieties; one group occurring frequently in houses worldwide ('Domesticus'), and one group represented by individuals from forests in Northern California ('Shastensis'). A few collections from nature were included in the Domesticus group as well, among other specimens from two newly discovered localities in Far East Russia and Siberia. In the Domesticus group little sequence variation occurs, suggesting a recent worldwide dispersal, possibly linked to human activity. Phylogenetic analyses indicate that the Domesticus group may have originated from an ancient lineage related closely to the Shastensis group. A remarkable shift in morphology and habitat preferences has occurred during the evolution of the Domesticus lineage, linked to the transition from nature to human-made habitats.

Morphology and ultrastructure of Neolecta species.

Several independent molecular phylogenetic analyses have indicated that the genus Neolecta has a unique position within the Ascomycota. It is the only taxon outside the core-group of filamentous, ascoma-forming ascomycetes that also has the ability to form ascomata. Light and electron microscope studies indicate that hymenial structure and development in Neolecta spp. are unique. Ascogenous hyphae in N. vitellina branched repeatedly and successively to produce asci. Non-ascogenous hyphae were multinucleate, often with nuclei in pairs. Nuclear pairing was particularily prominent in the ascogenous hyphae. A basal septum delimited the dikaryotic ascus. Ascosporogenesis was initiated by nuclear fusion followed by a meiotic and mitotic division to form eight nuclei. The ascus apex was thin with an annular subapical thickening. Ascospores were forcibly released through a 'split' in the ascus apex. Woronin bodies were frequently associated with hyphal septa. Attempts to culture N. vitellina and to obtain molecular information from the type species, N. flarovirescens, were unsuccessful. However, N. flavovirescens showed several microscopic characters that indicated close relationships with the two other species in the genus, N. vitellina and N. irregularis. The position of Neolecta spp. within the Ascomycota is discussed.