Phylogenetic relationships of Gomphillaceae and Asterothyriaceae: evidence from a combined Bayesian analysis of nuclear and mitochondrial sequences

The phylogeny and systematic position of Gomphillaceae was reconstructed using a combined Bayesian analysis of nuclear LSU rDNA and mitochondrial SSU rDNA sequences. Twenty-four partial sequences of 12 taxa (11 Gomphillaceae and one Asterothyriaceae) plus two new sequences of Stictis radiata (Ostropales outgroup) were generated and aligned with the corresponding sequences retrieved from GenBank, resulting in an alignment of 82 taxa that was analyzed using a Bayesian approach with Markov chain Monte Carlo (B/MCMC) methods. Our results confirm Gomphillaceae sensu Vezda and Poelt plus Asterothyriaceae to be a monophyletic group, with an unresolved relationship between the two families. Placement of Gomphillaceae and Asterothyriaceae within Ostropales sensu Kauff and Lutzoni, as sister of Thelotremataceae, also is strongly supported. Alternative hypotheses placing Gomphillaceae in Lecanorales (Cladoniaceae), Agyriales (Baeomycetaceae) or within bitunicate Ascomycota (Arthoniomycetes, Chaetothyriomycetes, Dothideomycetes) were rejected with our dataset. After recent synonymization of Dimerella with Coenogonium (Ostropales: Coenogoniaceae), we propose the new combination Coenogonium pineti (one of our Ostropales outgroup taxa in this analysis).     

Ascoma morphology is homoplaseous and phylogenetically misleading in some pyrenocarpous lichens

The phylogenetic relationships of many lichen-forming perithecioid ascomycetes are unknown. We generated nuLSU and mtSSU rDNA sequences of members of seven families of pyrenocarpous lichens and used a Bayesian framework to infer a phylogenetic estimate. Members of the perithecioid Protothelenellaceae, Thelenellaceae and Thrombiaceae surprisingly cluster within the mainly discocarpous Lecanoromycetes, while Strigulaceae, Verrucariaceae and Pyrenulaceae are related to the ascolocular Chaetothyriomycetes. Micromorphological studies of the ascomata showed that the two main groups of pyrenocarpous lichen-forming fungi differ in their ascus types. The Strigulaceae, Verrucariaceae and Pyrenulaceae have apically and laterally thick-walled asci, whereas the Thelenellaceae, Protothelenellaceae and Thrombiaceae have only apically thickened asci. The latter two show ring-shaped amyloid apical structures. Based on morphological and molecular evidence we propose to reduce Thrombiaceae to synonymy with Protothelenellaceae.     

Positions of multiple insertions in SSU rDNA of lichen-forming fungi

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.      

SSU rDNA phylogeny of cladoniiform lichens

To examine phylogenetic relationships among the "cladoniiform" lichenized fungi, i.e., the families Cladoniaceae, Baeomycetaceae, Icmadophilaceae, Stereocaulaceae, and Siphulaceae, and to provide evidence for the anticipated independent origins of podetia and pseudopodetia, we conducted phylogenetic analyses of SSU (small subunit) rDNA sequences from 39 lichen-forming fungi. These fungi represent all of the major growth forms of lichen associations, fruticose (including "cladoniiform"), foliose, and crustose. Our analysis suggests that lichen-forming fungi with a "cladoniiform" morphology arose multiple times within the ascomycetes. Additionally, each of the other thallus growth forms, crustose, foliose, and fruticose, have originated multiple times. It also seems to be clear that neither all podetiate nor all pseudopodetiate taxa form a monophyletic group. Therefore the term "podetium" should be restricted to homologous structures that are most probably limited to the genera Cladonia, Cladina, Pycnothelia, and allies. The "pseudopodetia" of Stereocaulon (Stereocaulaceae) and Cladia (Cladiaceae) may represent different states of the same homologous character. Our phylogenetic hypothesis supports the monophyletic origin of the order Lecanorales sensu stricto, including representatives of five suborders Cladoniineae, Lecanorineae, Teloschistineae, Agyriineae and Peltigerineae, but excluding representatives of the suborders Acarosporineae (Acarospora schleicheri and Megaspora verrucosa), Pertusariineae (Pertusaria trachythallina), and Umbilicarineae. The suborder Cladoniineae and the family Cladoniaceae both appear to be polyphyletic assemblages.     

Phylogenetic affiliations of members of the heterogeneous lichen-forming fungi of the genus Lecidea sensu Zahlbruckner (Lecanoromycetes, Ascomycota)

The genus Lecidea Ach. sensu lato (sensu Zahlbruckner) includes almost 1200 species, out of which only 100 species represent Lecidea sensu stricto (sensu Hertel). The systematic position of the remaining species is mostly unsettled but anticipated to represent several unrelated lineages within Lecanoromycetes. This study attempts to elucidate the phylogenetic placement of members of this heterogeneous group of lichen-forming fungi and to improve the classification and phylogeny of Lecanoromycetes. Twenty-five taxa of Lecidea sensu lato and 22 putatively allied species were studied in a broad selection of 268 taxa, representing 48 families of Lecanoromycetes. Six loci, including four ribosomal and two protein-coding genes for 315- and 209-OTU datasets were subjected to maximum likelihood and Bayesian analyses. The resulting well supported phylogenetic relationships within Lecanoromycetes are in agreement with published phylogenies, but the addition of new taxa revealed putative rearrangements of several families (e.g. Catillariaceae, Lecanoraceae, Lecideaceae, Megalariaceae, Pilocarpaceae and Ramalinaceae). As expected, species of Lecidea sensu lato and putatively related taxa are scattered within Lecanoromycetidae and beyond, with several species nested in Lecanoraceae and Pilocarpaceae and others placed outside currently recognized families in Lecanorales and orders in Lecanoromycetidae. The phylogenetic affiliations of Schaereria and Strangospora are outside Lecanoromycetidae, probably with Ostropomycetidae. All species referred to as Lecidea sensu stricto based on morphology (including the type species, Lecidea fuscoatra [L.] Ach.) form, with Porpidia species, a monophyletic group with high posterior probability outside Lecanorales, Peltigerales and Teloschistales, in Lecanoromycetidae, supporting the recognition of order Lecideales Vain. in this subclass. The genus name Lecidea must be redefined to apply only to Lecidea sensu stricto and to include at least some members of the genus Porpidia. Based on morphological and chemical similarities, as well as the phylogenetic relationship of Lecidea pullata sister to Frutidella caesioatra, the new combination Frutidella pullata is proposed here.     

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.     

Evolution of host-parasite relationships of Golovinomyces (Ascomycete: Erysiphaceae) inferred from nuclear rDNA sequences

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

Evolutionary dynamics of multiple group I introns in nuclear ribosomal RNA genes of endoparasitic fungi of the genus Cordyceps

A large number of group I introns were discovered in coding regions of small and large subunits of nuclear ribosomal RNA genes (SSU rDNA and LSU rDNA) in ascomycetous fungi of the genus CORDYCEPS: From 28 representatives of the genus, we identified in total 69 group I introns which were inserted at any of four specific sites in SSU rDNA and four specific sites in LSU rDNA. These group I introns reached sizes of up to 510 bp, occurred in up to eight sites in the same organism, and belonged to either subgroup IB3 or subgroup IC1 based on their sequence and structure. Introns inserted at the same site were closely related to each other among Cordyceps fungi, whereas introns inserted at different sites were phylogenetically distinct even in the same species. Mapped on the host phylogeny, the group I introns were generally not restricted to a particular lineage, but, rather, widely and sporadically distributed among distinct lineages. When the phylogenetic relationships of introns inserted at the same site were compared with the phylogeny of their hosts, the topologies were generally significantly congruent to each other. From these results, the evolutionary dynamics of multiple group I introns in Cordyceps fungi was inferred as follows: (1) most of the group I introns were already present at the eight sites in SSU and LSU rDNAs of the ancestor of the genus Cordyceps; (2) the introns have principally been immobile and vertically transmitted throughout speciation and diversification of Cordyceps fungi, which resulted in the phylogenetic congruence between the introns at the same site and their hosts; (3) in the course of vertical transmission, the introns have repeatedly been lost in a number of lineages independently, which has led to the present sporadic phylogenetic distribution of the introns; and (4) a few acquisitions of new introns, presumably through horizontal transmission, were identified in the evolutionary history of the genus Cordyceps, while no transpositions were detected. Losses of group I introns in SSU rDNA have occurred at least 27 times in the evolutionary course of the 28 Cordyceps members.     

The potential saprotrophic capacity of foliar endophytic fungi from Quercus gambelii

Endophytic fungi occur in living tissues of terrestrial plants. Many of these fungi are primarily biotrophic, but the trophic range of endophytic fungi as a group may not be fully appreciated. In this study, our goals were (1) for the Class 3 foliar endophytic fungi isolated from Quercus gambelii, determine their potential saprotrophic capacity, which we define as the difference in growth rate in culture on Quercus gambelii leaf litter medium and control medium lacking leaf litter and (2) quantify sources of variation among isolates of these endophytic fungi in potential saprotrophic capacity, including variation due to microsite within host trees (leaves receiving full sun vs. shade) and variation within and among fungal genera. We found that 48 of the 49 tested endophytic fungal isolates have significant potential saprotrophic capacity. Contrary to expectation, the amount of solar radiation available to the leaf from which the fungi were isolated had no significant impact on potential saprotrophic capacity and there was more variability in potential saprotrophic capacity among isolates within a genus than among genera. Our results suggest that some Class 3 endophytic fungi may have the potential to function as saprotrophic fungi within plant litter, but this remains to be seen for these Quercus gambelii isolates under more natural circumstances.     

Supraordinal phylogenetic relationships of Lecanoromycetes based on a Bayesian analysis of combined nuclear and mitochondrial sequences

Phylogenetic relationships of lichen-forming discomycetes and their relatives in the class Lecanoromycetes were examined by using nuclear large subunit and mitochondrial small subunit ribosomal DNA sequences. Ninety-eight partial sequences of 53 ascomycetes were generated and aligned with the corresponding sequences retrieved from GenBank resulting in an alignment of 100 taxa that was analyzed using a Bayesian approach with Markov chain Monte Carlo (B/MCMC) methods. The analysis revealed the monophyly of the Lecanoromycetes with two major clades: one clade including the monophyletic orders Graphidales and Ostropales and the paraphyletic Gyalectales, the other clade including the monophyletic Lecanorales (incl. Caliciales, Peltigerales, and Teloschistales) and a clade containing the polyphyletic Agyriales, a yet undescribed order Umbilicariales (including Elixiaceae and Umbilicariaceae), and Pertusariales. The monophyly of the Pertusariales was not resolved. Testing of alternative hypotheses revealed that a placement of Chaetothyriomycetes and Eurotiomycetes within Lecanoromycetes and the monophyly of Agyriales s. lat. (incl. Elixiaceae and Schaereriaceae) and Ostropales s. lat. (incl. Graphidales) can be rejected, while monophyly of Gyalectales and the Pertusariales and placement of Umbilicariales on the Lecanorales branch cannot be rejected with the current data set.     

Molecular phylogeny of truffles (Pezizales: Terfeziaceae, Tuberaceae) derived from nuclear rDNA sequence analysis.

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

Molecular phylogeny of Zygomycota based on EF-1alpha and RPB1 sequences: limitations and utility of alternative markers to rDNA

Earlier molecular phylogenetic analyses based on nuclear small subunit ribosomal DNA (nSSU rDNA) suggest that the Zygomycota are polyphyletic within the Chytridiomycota. However, these analyses failed to resolve almost all interordinal relationships among basal fungi (Chytridiomycota and Zygomycota), due to lack of sufficient characters within the nSSU rDNA. To further elucidate the higher-level phylogeny of Zygomycota, we have sequenced partial RPB1 (DNA dependent RNA polymerase II largest subunit) and EF-1alpha (translation elongation factor 1 alpha) genes from 10 and 3 zygomycete fungi, respectively. Independent molecular phylogenetic analyses were performed based on each sequence by distance and maximum likelihood methods. Although deep phylogenetic relationships among basal fungi still remain poorly resolved using either gene, the RPB1-based phylogeny identified a novel monophyletic clade consisting of the Dimargaritales, Harpellales, and Kickxellales. This result suggests that regularly formed septa (cross walls that divide hyphae into segments) with a lenticular cavity are plesiomorphic for this clade, and indicates the importance of septal pore ultrastructure in zygomycete phylogeny. In addition, a peculiar mucoralean genus Mortierella, which was considered to be distantly related to the other Mucorales based on previous nSSU rDNA analyses, was resolved as the basal most divergence within the Mucorales, consistent with traditional phenotypic-based taxonomy. Although the taxa included in our analysis are restricted, the monophyly of each order suggested by nSSU rDNA phylogeny is supported by the present RPB1-based analysis. These results support the potential use of RPB1 as an alternative marker for fungal phylogenetic studies. Conversely, the overall fungal phylogeny based on EF-1alpha sequence is poorly resolved. A comparison of numbers of observed substitutions versus inferred substitutions within EF-1alpha indicates that this gene is much more saturated than RPB1. This result suggests that the EF-1alpha gene is unsuitable for resolving higher-level phylogenetic relationships within the Fungi.     

The causal agents of witches' broom and frosty pod rot of cacao (chocolate, Theobroma cacao) form a new lineage of Marasmiaceae

The two most devastating diseases of cacao (Theobroma cacao)--the source of chocolate--in tropical America are caused by the fungi Crinipellis perniciosa (witches' broom disease) and Moniliophthora roreri (frosty pod rot or moniliasis disease). Despite the agricultural, socio-economic and environmental impact of these fungi, most aspects of their life cycles are unknown, and the phylogenetic relationships of M. roreri have yet to be conclusively established. In this paper, extensive phylogenetic analyses of five nuclear gene regions (28S rDNA, 18S rDNA, ITS, RPB1, and EF1-alpha) confirm that C. perniciosa and M. roreri are sister taxa that belong in the Marasmiaceae (euagarics). Furthermore, these taxa form part of a separate and distinct lineage within the family. This lineage includes the biotrophic fungi Moniliophthora perniciosa comb. nov. and M. roreri, as well as one undescribed endophytic species. The sister genera to Moniliophthora are Marasmius, Crinipellis and Chaetocalathus, which consist mainly of saprotrophic litter fungi.     

Revival of saprotrophic and mycorrhizal basidiomycete cultures after 20 years in cold storage in sterile water

Vegetatively colonized agar cores of 69 basidiomycete fungus isolates (48 species in 30 genera and 17 families) were stored at 5 degrees C in tubes of sterile distilled water without manipulation for 20 years. These were represented by 34 isolates of saprotrophic fungi (29 species in 19 genera) and 35 isolates of mycorrhizal fungi (19 species in 11 genera). Viability was evaluated based on revived growth on agar media at room temperature. Fifty-seven of the 69 isolates (82.6%) grew vigorously when revived after storage for 20 years; of the 34 saprotrophic fungus isolates, 30 revived (88.2%); of the 35 mycorrhizal fungus isolates, 27 revived (77.1%). Thirteen isolates of Laccaria were all viable after 20 years, indicating cold storage in sterile water to be a good method for maintaining this important genus of mycorrhizal fungi. In general, however, mycorrhizal fungus species demonstrated lower viability than saprotrophic fungi.     

The obligately lichenicolous genus Lichenoconium represents a novel lineage in the Dothideomycetes

Lichenicolous fungi are obligately lichen-associated organisms that have evolved many times throughout the Ascomycota and Basidiomycota. Approximately 20% of lichenicolous ascomycetes are recognized only from asexual (anamorphic) characteristics, so the phylogenetic position of many groups has never been resolved. Here we present the first molecular phylogeny of Lichenoconium, a genus of strictly asexual, obligately lichenicolous species with broad geographic distributions and diverse host ecologies. We obtained nuclear and mitochondrial rDNA sequences from fungal cultures isolated from four species in the genus, including a new species, Lichenoconium aeruginosum sp. nov., collected in France, Luxembourg and Netherlands. Our multilocus phylogeny supports the monophyly of fungi in the genus Lichenoconium, and places the genus in the Dothideomycetes, an ascomycete class made up mainly of saprobes and plant-associated endophytes and pathogens. There are only a few recognized groups of lichen-formers in the Dothideomycetes, but Lichenoconium is not supported as being closely related to any of these, nor to any other recognized order within the Dothideomycetes. Given that Lichenoconium is but one of over 100 genera of anamorphic lichenicolous fungi, most of which have never been studied phylogenetically, we suggest that asexual lichenicolous fungi may represent novel and evolutionarily significant phylogenetic groups in the Kingdom Fungi.     

Patterns of Group I Intron Presence in Nuclear SSU rDNA of the Lichen Family Parmeliaceae

Group I introns are commonly reported within nuclear SSU ribosomal DNA of eukaryotic micro-organisms, especially in lichen-forming fungi. We have studied the primary and secondary structure of 70 new nuclear SSU rDNA group I introns of Parmeliaceae (Ascomycota: Lecanorales) and compared them with those available in databases, covering more than 60 species. The analyzed samples of Parmeliaceae fell into two groups, one having an intron at the 1506 site and another lacking this one but having another at the 1516 or 1521 position. Introns at the 1521 position seem to be transposed from 1516 sites. Introns at the 1516 position were similar in structure to ones previously reported at this site and known from other lecanoralean fungi, while those at the 1506 position showed structural differences and no similar introns are known from related fungi. The study of the distribution of group I introns within a large monophyletic ensemble of fungi has revealed an unexpected correlation between intron types and ecological and geographical parameters. The introns at the 1516 position occurred in mainly arctic, boreal, and temperate lichens, while those at position 1506 were present in mainly tropical and subtropical to oceanic mild-temperate taxa. Further, the 1516 introns occurred in genera with few distributed species that could represent older taxa, while the 1506 ones were mainly in species-rich genera that could be of recent speciation, as many species have wide distribution areas. The transition between two different environments has been accompanied by a change in introns gained and lost. [Reviewing Editor: Dr. Debashish Bhattacharya]