The proteins of the ammonium transporter/methylammonium permease/Rhesus factor family (AMT/MEP/Rh family) are responsible for the movement of ammonia or ammonium ions across the cell membrane. Although it has been established that the Rh proteins are distantly related to the other members of the family, the evolutionary history of the AMT/MEP/Rh family remains unclear. Here, we use phylogenetic analysis to infer the evolutionary history of this family of proteins across 191 genomes representing all main lineages of life and to provide a new classification of the proteins in this family. Our phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). We show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: The AMT family as defined here exhibits vertical gene transfer (i.e., standard parent-to-offspring inheritance), whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs). These ancient HGT events include a gene replacement during the early evolution of the fungi, which could be a defining trait for the kingdom Fungi, a gene gain from hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina) that was subsequently lost in most lineages but retained in even distantly related lichenized fungi. This recircumscription of the ammonium transporters/ammonia permeases family into MEP and AMT families informs the debate on the mechanism of transport in these proteins and on the nature of the transported molecule because published crystal structures of proteins from the MEP and Rh clades may not be representative of the AMT clade. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with AMTs and ammonia permeases forming two distinct and possibly monophyletic groups.
Species of the genus Lichenomphalia are mostly restricted to arctic-alpine environments with the exception of Lichenomphalia umbellifera which is also common in northern forests. Although Lichenomphalia species inhabit vast regions in several continents, no information is available on their genetic variation across geographic regions and the underlying population-phylogenetic patterns. We collected samples from arctic and subarctic regions, as well as from newly discovered subantarctic localities for the genus. Phylogenetic, nonparametric permutation methods, and coalescent analyses were used to assess phylogeny and population divergence and to estimate the extent and direction of gene flow among distinct geographic populations. All known species formed monophyletic groups, supporting their morphology-based delimitation. In addition, we found two subantarctic phylogenetic species (Lichenomphalia sp. and Lichenomphalia aff. umbellifera), of which the latter formed a well-supported sister group to L. umbellifera. We found no significant genetic differentiation among conspecific North American and Eurasian populations in Lichenomphalia. We detected high intercontinental gene flow within the northern polar region, suggesting rapid (re)colonisation of suitable habitats in response to climatic fluctuations and preventing pronounced genetic differentiation. On the other hand, our phylogenetic analyses suggest that dispersal between northern circumpolar and subantarctic areas likely happened very rarely and led to the establishment and subsequent divergence of lineages. Due to limited sampling in the Southern Hemisphere, it is currently uncertain whether the northern lineages occur in Gondwanan regions. On the other hand, our results strongly suggest that the southern lineages do not occur in the circumpolar north. Although rare transequatorial dispersal and subsequent isolation may explain the emergence of at least two subantarctic phylogenetic species lineages in Lichenomphalia, more samples from the Southern Hemisphere are needed to better understand the phylogeographic history of the genus. © 2011 The British Mycological Society.
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.
Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli. For this study, comparative analyses were conducted on lichen-associated bacterial communities to test for effects of photobiont-types (i.e. green algal vs. cyanobacterial), mycobiont-types and large-scale spatial distances (from tropical to arctic latitudes). Amplicons of the 16S (SSU) rRNA gene were examined using both Sanger sequencing of cloned fragments and barcoded pyrosequencing. Rhizobiales is typically the most abundant and taxonomically diverse order in lichen microbiomes; however, overall bacterial diversity in lichens is shown to be much higher than previously reported. Members of Acidobacteriaceae, Acetobacteraceae, Brucellaceae and sequence group LAR1 are the most commonly found groups across the phylogenetically and geographically broad array of lichens examined here. Major bacterial community trends are significantly correlated with differences in large-scale geography, photobiont-type and mycobiont-type. The lichen as a microcosm represents a structured, unique microbial habitat with greater ecological complexity and bacterial diversity than previously appreciated and can serve as a model system for studying larger ecological and evolutionary principles. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.
How plants and microbes recognize each other and interact to form long-lasting relationships remains one of the central questions in cellular communication. The symbiosis between the filamentous fungus Cladonia grayi and the single-celled green alga Asterochloris sp. was used to determine fungal and algal genes upregulated in vitro in early lichen development. cDNA libraries of upregulated genes were created with suppression subtractive hybridization in the first two stages of lichen development. Quantitative PCR subsequently was used to verify the expression level of 41 and 33 candidate fungal and algal genes respectively. Induced fungal genes showed significant matches to genes putatively encoding proteins involved in self and non-self recognition, lipid metabolism, and negative regulation of glucose repressible genes, as well as to a putative d-arabitol reductase and two dioxygenases. Upregulated algal genes included a chitinase-like protein, an amino acid metabolism protein, a dynein-related protein and a protein arginine methyltransferase. These results also provided the first evidence that extracellular communication without cellular contact can occur between lichen symbionts. Many genes showing slight variation in expression appear to direct the development of the lichen symbiosis. The results of this study highlight future avenues of investigation into the molecular biology of lichen symbiosis.
Endolichenic fungi live in close association with algal photobionts inside asymptomatic lichen thalli and resemble fungal endophytes of plants in terms of taxonomy, diversity, transmission mode, and evolutionary history. This similarity has led to uncertainty regarding the distinctiveness of endolichenic fungi compared with endophytes. Here, we evaluate whether these fungi represent distinct ecological guilds or a single guild of flexible symbiotrophs capable of colonizing plants or lichens indiscriminately. Culturable fungi were sampled exhaustively from replicate sets of phylogenetically diverse plants and lichens in three microsites in a montane forest in southeastern Arizona (USA). Intensive sampling combined with a small spatial scale permitted us to decouple spatial heterogeneity from host association and to sample communities from living leaves, dead leaves, and lichen thalli to statistical completion. Characterization using data from the nuclear ribosomal internal transcribed spacer and partial large subunit (ITS-LSU rDNA) provided a first estimation of host and substrate use for 960 isolates representing five classes and approximately 16 orders, 32 families, and 65 genera of Pezizomycotina. We found that fungal communities differ at a broad taxonomic level as a function of the phylogenetic placement of their plant or lichen hosts. Endolichenic fungal assemblages differed as a function of lichen taxonomy, rather than substrate, growth form, or photobiont. In plants, fungal communities were structured more by plant lineage than by the living vs. senescent status of the leaf. We found no evidence that endolichenic fungi are saprotrophic fungi that have been "entrapped" by lichen thalli. Instead, our study reveals the distinctiveness of endolichenic communities relative to those in living and dead plant tissues, with one notable exception: we identify, for the first time, an ecologically flexible group of symbionts that occurs both as endolichenic fungi and as endophytes of mosses. © 2010 Springer Science+Business Media, LLC.
The lichen-forming fungal genus Peltigera includes a number of species that are extremely widespread, both geographically and ecologically. However, morphological variability has lead to doubts about the distinctness of some species, and it has been suggested that hybridization is common in nature. We examined species boundaries by looking for evidence of hybridization and gene flow among seven described species collected at five sites in British Columbia, Canada. We found no evidence of gene flow or hybridization between described species, with fixed differences between species for two or more of the three loci examined. Reproductive isolation did not reflect a solely clonal mode of reproduction as there was evidence of ongoing gene flow within species. In addition, we found five undescribed species that were reproductively isolated, although there was evidence of ongoing or historical gene flow between two of the new species. These results indicate that the genus Peltigera is more diverse in western North America than originally perceived, and that morphological variability is due largely to the presence of undescribed species rather than hybridization or intraspecific variation.
Fungi associated with photosynthetic organisms are major determinants of terrestrial biomass, nutrient cycling, and ecosystem productivity from the poles to the equator. Whereas most fungi are known because of their fruit bodies (e.g., saprotrophs), symptoms (e.g., pathogens), or emergent properties as symbionts (e.g., lichens), the majority of fungal diversity is thought to occur among species that rarely manifest their presence with visual cues on their substrate (e.g., the apparently hyperdiverse fungal endophytes associated with foliage of plants). Fungal endophytes are ubiquitous among all lineages of land plants and live within overtly healthy tissues without causing disease, but the evolutionary origins of these highly diverse symbionts have not been explored. Here, we show that a key to understanding both the evolution of endophytism and the diversification of the most species-rich phylum of Fungi (Ascomycota) lies in endophyte-like fungi that can be isolated from the interior of apparently healthy lichens. These "endolichenic" fungi are distinct from lichen mycobionts or any other previously recognized fungal associates of lichens, represent the same major lineages of Ascomycota as do endophytes, largely parallel the high diversity of endophytes from the arctic to the tropics, and preferentially associate with green algal photobionts in lichen thalli. Using phylogenetic analyses that incorporate these newly recovered fungi and ancestral state reconstructions that take into account phylogenetic uncertainty, we show that endolichenism is an incubator for the evolution of endophytism. In turn, endophytism is evolutionarily transient, with endophytic lineages frequently transitioning to and from pathogenicity. Although symbiotrophic lineages frequently give rise to free-living saprotrophs, reversions to symbiosis are rare. Together, these results provide the basis for estimating trophic transition networks in the Ascomycota and provide a first set of hypotheses regarding the evolution of symbiotrophy and saprotrophy in the most species-rich fungal phylum. [Ancestral state reconstruction; Ascomycota; Bayesian analysis; endolichenic fungi; fungal endophytes; lichens; pathogens; phylogeny; saprotrophy; symbiotrophy; trophic transition network.].
The Fungi comprise a diverse kingdom of eukaryotes that are characterized by a typically filamentous but sometimes unicellular vegetative form, and heterotrophic, absorptive nutrition. Their simple morphologies and variable ecological strategies have confounded efforts to elucidate their limits, phylogenetic relationships, and diversity. Here we review progress in developing a phylogenetic classification of Fungi since Darwin's On the Origin of Species. Knowledge of phylogenetic relationships has been driven by the available characters that have ranged from morphological and ultrastructural to biochemical and genomic. With the availability of multiple gene phylogenies a well-corroborated phylogenetic classification has now begun to emerge. In the process some fungus-like heterotrophs have been shown to belong elsewhere, and several groups of enigmatic eukaryotic microbes have been added to the Fungi. © 2009 Elsevier Ltd. All rights reserved.
A comprehensive phylogenetic classification of the kingdom Fungi is proposed, with reference to recent molecular phylogenetic analyses, and with input from diverse members of the fungal taxonomic community. The classification includes 195 taxa, down to the level of order, of which 16 are described or validated here: Dikarya subkingdom nov.; Chytridiomycota, Neocallimastigomycota phyla nov.; Monoblepharidomycetes, Neocallimastigomycetes class. nov.; Eurotiomycetidae, Lecanoromycetidae, Mycocaliciomycetidae subclass. nov.; Acarosporales, Corticiales, Baeomycetales, Candelariales, Gloeophyllales, Melanosporales, Trechisporales, Umbilicariales ords. nov. The clade containing Ascomycota and Basidiomycota is classified as subkingdom Dikarya, reflecting the putative synapomorphy of dikaryotic hyphae. The most dramatic shifts in the classification relative to previous works concern the groups that have traditionally been included in the Chytridiomycota and Zygomycota. The Chytridiomycota is retained in a restricted sense, with Blastocladiomycota and Neocallimastigomycota representing segregate phyla of flagellated Fungi. Taxa traditionally placed in Zygomycota are distributed among Glomeromycota and several subphyla incertae sedis, including Mucoromycotina, Entomophthoromycotina, Kickxellomycotina, and Zoopagomycotina. Microsporidia are included in the Fungi, but no further subdivision of the group is proposed. Several genera of 'basal' Fungi of uncertain position are not placed in any higher taxa, including Basidiobolus, Caulochytrium, Olpidium, and Rozella. © 2007 The British Mycological Society.
We examined endophytic fungi in asymptomatic foliage of loblolly pine (Pinus taeda) in North Carolina, U.S.A., with four goals: (i) to evaluate morphotaxa, BLAST matches and groups based on sequence similarity as functional taxonomic units; (ii) to explore methods to maximize phylogenetic signal for environmental datasets, which typically contain many taxa but few characters; (iii) to compare culturing vs. culture-free methods (environmental PCR of surface sterilized foliage) for estimating endophyte diversity and species composition; and (iv) to investigate the relationships between traditional ecological indices (e.g. Shannon index) and phylogenetic diversity (PD) in estimating endophyte diversity and spatial heterogeneity. Endophytes were recovered in culture from 87 of 90 P. taeda leaves sampled, yielding 439 isolates that represented 24 morphotaxa. Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) for 150 isolates revealed 59 distinct ITS genotypes that represented 24 and 37 unique groups based on 90% and 95% sequence similarity, respectively. By recoding ambiguously aligned regions to extract phylogenetic signal and implementing a conservative phylogenetic backbone constraint, we recovered well supported phylogenies based on ca. 600 bp of the nuclear ribosomal large subunit (LSUrDNA) for 72 Ascomycota and Basidiomycota, 145 cultured endophytes and 33 environmental PCR samples. Comparisons with LSUrDNA-delimited species showed that morphotaxa adequately estimated total species richness but rarely corresponded to biologically meaningful groups. ITS BLAST results were variable in their utility, but ITS genotype groups based on 90% sequence similarity were concordant with LSUrDNA-delimited species. Environmental PCR yielded more genotypes per sampling effort and recovered several distinct clades relative to culturing, but some commonly cultured clades were never found (Sordariomycetes) or were rare relative to their high frequency among cultures (Leotiomycetes). In contrast to traditional indices, PD demonstrated spatial heterogeneity in endophyte assemblages among P. taeda trees and study plots. Our results highlight the need for caution in designating taxonomic units based on gross cultural morphology or ITS BLAST matches, the utility of phylogenetic tools for extracting robust phylogenies from environmental samples, the complementarity of culturing and environmental PCR, the utility of PD relative to traditional ecological indices, and the remarkably high diversity of foliar fungal endophytes in this simplified temperate ecosystem.
Fungal endophytes are found in asymptomatic photosynthetic tissues of all major lineages of land plants. The ubiquity of these cryptic symbionts is clear, but the scale of their diversity, host range, and geographic distributions are unknown. To explore the putative hyperdiversity of tropical leaf endophytes, we compared endophyte communities along a broad latitudinal gradient from the Canadian arctic to the lowland tropical forest of central Panama. Here, we use molecular sequence data from 1403 endophyte strains to show that endophytes increase in incidence, diversity, and host breadth from arctic to tropical sites. Endophyte communities from higher latitudes are characterized by relatively few species from many different classes of Ascomycota, whereas tropical endophyte assemblages are dominated by a small number of classes with a very large number of endophytic species. The most easily cultivated endophytes from tropical plants have wide host ranges, but communities are dominated by a large number of rare species whose host range is unclear. Even when only the most easily cultured species are considered, leaves of tropical trees represent hotspots of fungal species diversity, containing numerous species not yet recovered from other biomes. The challenge remains to recover and identify those elusive and rarely cultured taxa with narrower host ranges, and to elucidate the ecological roles of these little-known symbionts in tropical forests.
The sporadic distribution of nuclear group I introns among different fungal lineages can be explained by vertical inheritance of the introns followed by successive losses, or horizontal transfers from one lineage to another through intron homing or reverse splicing. Homing is mediated by an intron-encoded homing endonuclease (HE) and recent studies suggest that the introns and their associated HE gene (HEG) follow a recurrent cyclical model of invasion, degeneration, loss, and reinvasion. The purpose of this study was to compare this model to the evolution of HEGs found in the group I intron at position S943 of the nuclear ribosomal DNA of the lichen-forming fungus Pleopsidium. Forty-eight S943 introns were found in the 64 Pleopsidium samples from a worldwide screen, 22 of which contained a full-length HEG that encodes a putative 256-amino acid HE, and 2 contained HE pseudogenes. The HEGs are divided into two closely related types (as are the introns that encode them) that differ by 22.6% in their nucleotide sequences. The evolution of the Pleopsidium intron-HEG element shows strong evidence for a cyclical model of evolution. The intron was likely acquired twice in the genus and then transmitted via two or three interspecific horizontal transfers. Close geographical proximity plays an important role in intron-HEG horizontal transfer because most of these mobile elements were found in Europe. Once acquired in a lineage, the intron-HEG element was also vertically transmitted, and occasionally degenerated or was lost.
Degenerate PCR and chromosome-walking approaches were used to identify mating-type (MAT) genes and flanking regions from the homothallic (sexually self-fertile) euascomycete fungus Neosartorya fischeri, a close relative of the opportunistic human pathogen Aspergillus fumigatus. Both putative alpha- and high-mobility-group-domain MAT genes were found within the same genome, providing a functional explanation for self-fertility. However, unlike those in many homothallic euascomycetes (Pezizomycotina), the genes were not found adjacent to each other and were termed MAT1 and MAT2 to recognize the presence of distinct loci. Complete copies of putative APN1 (DNA lyase) and SLA2 (cytoskeleton assembly control) genes were found bordering the MAT1 locus. Partial copies of APN1 and SLA2 were also found bordering the MAT2 locus, but these copies bore the genetic hallmarks of pseudogenes. Genome comparisons revealed synteny over at least 23,300 bp between the N. fischeri MAT1 region and the A. fumigatus MAT locus region, but no such long-range conservation in the N. fischeri MAT2 region was evident. The sequence upstream of MAT2 contained numerous candidate transposase genes. These results demonstrate a novel means involving the segmental translocation of a chromosomal region by which the ability to undergo self-fertilization may be acquired. The results are also discussed in relation to their significance in indicating that heterothallism may be ancestral within the Aspergillus section Fumigati.
The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree.
The Lecanoromycetes includes most of the lichen-forming fungal species (> 13500) and is therefore one of the most diverse class of all Fungi in terms of phenotypic complexity. We report phylogenetic relationships within the Lecanoromycetes resulting from Bayesian and maximum likelihood analyses with complementary posterior probabilities and bootstrap support values based on three combined multilocus datasets using a supermatrix approach. Nine of 10 orders and 43 of 64 families currently recognized in Eriksson's classification of the Lecanoromycetes (Outline of Ascomycota--2006 Myconet 12:1-82) were represented in this sampling. Our analyses strongly support the Acarosporomycetidae and Ostropomycetidae as monophyletic, whereas the delimitation of the largest subclass, the Lecanoromycetidae, remains uncertain. Independent of future delimitation of the Lecanoromycetidae, the Rhizocarpaceae and Umbilicariaceae should be elevated to the ordinal level. This study shows that recent classifications include several nonmonophyletic taxa at different ranks that need to be recircumscribed. Our phylogenies confirm that ascus morphology cannot be applied consistently to shape the classification of lichen-forming fungi. The increasing amount of missing data associated with the progressive addition of taxa resulted in some cases in the expected loss of support, but we also observed an improvement in statistical support for many internodes. We conclude that a phylogenetic synthesis for a chosen taxonomic group should include a comprehensive assessment of phylogenetic confidence based on multiple estimates using different methods and on a progressive taxon sampling with an increasing number of taxa, even if it involves an increasing amount of missing data.
Aspergillus fumigatus is a medically important opportunistic pathogen and a major cause of respiratory allergy . The species has long been considered an asexual organism. However, genome analysis has revealed the presence of genes associated with sexual reproduction, including a MAT-2 high-mobility group mating-type gene and genes for pheromone production and detection (Galagan et al., personal communication; Nierman et al., personal communication; [2, 3]). We now demonstrate that A. fumigatus has other key characteristics of a sexual species. We reveal the existence of isolates containing a complementary MAT-1 α box mating-type gene and show that the MAT locus has an idiomorph structure characteristic of heterothallic (obligate sexual outbreeding) fungi [4, 5]. Analysis of 290 worldwide clinical and environmental isolates with a multiplex-PCR assay revealed the presence of MAT1-1 and MAT1-2 genotypes in similar proportions (43% and 57%, respectively). Further population genetic analyses provided evidence of recombination across a global sampling and within North American and European subpopulations. We also show that mating-type, pheromone-precursor, and pheromone-receptor genes are expressed during mycelial growth. These results indicate that A. fumigatus has a recent evolutionary history of sexual recombination and might have the potential for sexual reproduction. The possible presence of a sexual cycle is highly significant for the population biology and disease management of the species. ©2005 Elsevier Ltd All rights reserved.
Heterocystous cyanobacteria form symbiotic associations with a wide range of plant and fungal hosts. We used a molecular phylogenetic approach to investigate the degree of host specialization of cyanobacteria associated with four closely related species of the lichenized fungus Peltigera, and to compare these strains with other symbiotic cyanobacteria. We conducted phylogenetic analyses on 16S, rbcLX, and trnL sequences from cyanobacteria associated with multiple specimens of each lichen species and from symbionts of other fungi and plants, as well as from free-living strains of Nostoc and related genera of cyanobacteria. The genus Nostoc comprises two divergent lineages, but symbiotic strains occur primarily within a single monophyletic lineage that also includes free-living representatives. Cyanobacteria from the same lichen species were often more closely related to strains from other species or to plant symbionts or free-living strains than to each other. These results indicate that host specialization is low for the genus Nostoc, and suggest that opportunities for coevolution with its partners may be rare. © 2005 British Phycological Society.
Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.
Based on an overview of progress in molecular systematics of the true fungi (Fungi/Eumycota) since 1990, little overlap was found among single-locus data matrices, which explains why no large-scale multilocus phylogenetic analysis had been undertaken to reveal deep relationships among fungi. As part of the project "Assembling the Fungal Tree of Life" (AFTOL), results of four Bayesian analyses are reported with complementary bootstrap assessment of phylogenetic confidence based on (1) a combined two-locus data set (nucSSU and nucLSU rDNA) with 558 species representing all traditionally recognized fungal phyla (Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota) and the Glomeromycota, (2) a combined three-locus data set (nucSSU, nucLSU, and mitSSU rDNA) with 236 species, (3) a combined three-locus data set (nucSSU, nucLSU rDNA, and RPB2) with 157 species, and (4) a combined four-locus data set (nucSSU, nucLSU, mitSSU rDNA, and RPB2) with 103 species. Because of the lack of complementarity among single-locus data sets, the last three analyses included only members of the Ascomycota and Basidiomycota. The four-locus analysis resolved multiple deep relationships within the Ascomycota and Basidiomycota that were not revealed previously or that received only weak support in previous studies. The impact of this newly discovered phylogenetic structure on supraordinal classifications is discussed. Based on these results and reanalysis of subcellular data, current knowledge of the evolution of septal features of fungal hyphae is synthesized, and a preliminary reassessment of ascomal evolution is presented. Based on previously unpublished data and sequences from GenBank, this study provides a phylogenetic synthesis for the Fungi and a framework for future phylogenetic studies on fungi.
Bayesian Markov chain Monte Carlo sampling has become increasingly popular in phylogenetics as a method for both estimating the maximum likelihood topology and for assessing nodal confidence. Despite the growing use of posterior probabilities, the relationship between the Bayesian measure of confidence and the most commonly used confidence measure in phylogenetics, the nonparametric bootstrap proportion, is poorly understood. We used computer simulation to investigate the behavior of three phylogenetic confidence methods: Bayesian posterior probabilities calculated via Markov chain Monte Carlo sampling (BMCMC-PP), maximum likelihood bootstrap proportion (ML-BP), and maximum parsimony bootstrap proportion (MP-BP). We simulated the evolution of DNA sequence on 17-taxon topologies under 18 evolutionary scenarios and examined the performance of these methods in assigning confidence to correct monophyletic and incorrect monophyletic groups, and we examined the effects of increasing character number on support value. BMCMC-PP and ML-BP were often strongly correlated with one another but could provide substantially different estimates of support on short internodes. In contrast, BMCMC-PP correlated poorly with MP-BP across most of the simulation conditions that we examined. For a given threshold value, more correct monophyletic groups were supported by BMCMC-PP than by either ML-BP or MP-BP. When threshold values were chosen that fixed the rate of accepting incorrect monophyletic relationship as true at 5%, all three methods recovered most of the correct relationships on the simulated topologies, although BMCMC-PP and ML-BP performed better than MP-BP. BMCMC-PP was usually a less biased predictor of phylogenetic accuracy than either bootstrapping method. BMCMC-PP provided high support values for correct topological bipartitions with fewer characters than was needed for nonparametric bootstrap.
About one-fifth of all known extant fungal species form obligate symbiotic associations with green algae, cyanobacteria or with both photobionts. These symbioses, known as lichens, are one way for fungi to meet their requirement for carbohydrates. Lichens are widely believed to have arisen independently on several occasions, accounting for the high diversity and mixed occurrence of lichenized and non-lichenized (42 and 58%, respectively) fungal species within the Ascomycota. Depending on the taxonomic classification chosen, 15-18 orders of the Ascomycota include lichen-forming taxa, and 8-11 of these orders (representing about 60% of the Ascomycota species) contain both lichenized and non-lichenized species. Here we report a phylogenetic comparative analysis of the Ascomycota, a phylum that includes greater than 98% of known lichenized fungal species. Using a Bayesian phylogenetic tree sampling methodology combined with a statistical model of trait evolution, we take into account uncertainty about the phylogenetic tree and ancestral state reconstructions. Our results show that lichens evolved earlier than believed, and that gains of lichenization have been infrequent during Ascomycota evolution, but have been followed by multiple independent losses of the lichen symbiosis. As a consequence, major Ascomycota lineages of exclusively non-lichen-forming species are derived from lichen-forming ancestors. These species include taxa with important benefits and detriments to humans, such as Penicillium and Aspergillus.
Phylogenetic analyses of non-protein-coding nucleotide sequences such as ribosomal RNA genes, internal transcribed spacers, and introns are often impeded by regions of the alignments that are ambiguously aligned. These regions are characterized by the presence of gaps and their uncertain positions, no matter which optimization criteria are used. This problem is particularly acute in large-scale phylogenetic studies and when aligning highly diverged sequences. Accommodating these regions, where positional homology is likely to be violated, in phylogenetic analyses has been dealt with very differently by molecular systematists and evolutionists, ranging from the total exclusion of these regions to the inclusion of every position regardless of ambiguity in the alignment. We present a new method that allows the inclusion of ambiguously aligned regions without violating homology. In this three-step procedure, first homologous regions of the alignment containing ambiguously aligned sequences are delimited. Second, each ambiguously aligned region is unequivocally coded as a new character, replacing its respective ambiguous region. Third, each of the coded characters is subjected to a specific step matrix to account for the differential number of changes (summing substitutions and indels) needed to transform one sequence to another. The optimal number of steps included in the step matrix is the one derived from the pairwise alignment with the greatest similarity and the least number of steps. In addition to potentially enhancing phylogenetic resolution and support, by integrating previously nonaccessible characters without violating positional homology, this new approach can improve branch length estimations when using parsimony.
Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.
Differential rates of nucleotide substitutions among taxa are a common observation in molecular phylogenetic studies, yet links between rates of DNA evolution and traits or behaviors of organisms have proved elusive. Likelihood ratio testing is used here for the first time to evaluate specific hypotheses that account for the induction of shifts in rates of DNA evolution. A molecular phylogenetic investigation of mutualist (lichen-forming fungi and fungi associated with liverworts) and nonmutualist fungi revealed four independent transitions to mutualism. We demonstrate a highly significant association between mutualism and increased rates of nucleotide substitutions in nuclear ribosomal DNA, and we demonstrate that a transition to mutualism preceded the rate acceleration of nuclear ribosomal DNA in these lineages. Our results suggest that the increased rate of evolution after the adoption of a mutualist lifestyle is generalized across the genome of these mutualist fungi.
As an initial step toward developing a model system to study requirements for and consequences of transitions to mutualism, the phylogeny of a group of closely related lichenized and nonlichenized basidiomycetes (Omphalina) was reconstructed. The phylogenetic analyses are based on four data sets representing different regions of the nuclear ribosomal repeat unit (ITS1, 5.8S, ITS2, and 25S) obtained from 30 species of Omphalina and related genera. The resulting phylogenetic trees from each of these four data sets, when analyzed separately, were not identical. Testing for the combinability of these four data sets suggested that they could not be combined in their entirety. The removal of ambiguous alignments and saturated sites was sufficient, after reapplying the combinability test on the pruned data sets, to explain the topological discrepancies. In this process, the first of two complementary tests developed by Rodrigo et al. (1993, N.Z. J. Bot. 31:257-268) to assess whether two data sets are the result of the same phylogenetic history was found to be biased, rejecting the combinability of two data sets even when they are samples of the same phylogenetic history. Combining the four pruned data sets yielded phylogenies that suggest the five lichen-forming species of Omphalina form a monophyletic group. The sister group to this symbiotic clade consists mostly of dark brown Omphalina species intermixed with species from the genera Arrhenia and Phaeothellus. The genera Omphalina and Gerronema are shown to be polyphyletic. The lichen-forming species O. ericetorum and the nonmutualistic species O. velutipes, O. epichysium, and O. sphagnicola are the best candidates for experimental work designed to gain a better understanding of mechanisms involved in symbiotic interactions and the role symbiosis has played in the evolution of fungi.