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Lichens are well-known examples of complex symbiotic associations between organisms from different Kingdoms. Microfungi in particular, establish diverse associations with the hosting lichen thallus, as species-specific parasites or transient co-inhabitants. The whole community of lichen-associated fungi constitute the 'lichen mycobiome' comprising both ascomycetes and basidiomycetes, including filamentous and yeast taxa. Metabarcoding results and microscopy analyses show that in some thalli, basidiomycetes are frequent lichen-associated fungi but still only a few species could be axenically isolated and morphologically characterized. Within a broad project aiming at characterizing the mycobiome diversity by culture-dependent and independent approaches in two lichen species selected as reference models - Rhizoplaca melanophthalma and Tephromela atra, we succeed in isolating and culturing 76 new strains of basidiomycetous yeasts. The lichen thalli were collected in different mountain regions worldwide and at relatively high elevation. The yeast strains were isolated on different growth media and were studied for their morphological and genetic diversity. Nuclear internal transcribed spacer (ITS) and ribosomal large subunit (LSU) sequence analyses identified them to belong to ten families within the orders Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Tremellomycetes and Ustilaginomycetes. The yeasts here detected showed patterns of host-preference in a few cases and they are potentially related to the ecological conditions., Competing Interests: Declaration of competing interest None., (Copyright © 2022 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Advancements in molecular genetics have revealed that hybridization may be common among plants, animals, and fungi, playing a role in evolutionary dynamics and speciation. While hybridization has been well-documented in pathogenic fungi, the effects of these processes on speciation in fungal lineages with different life histories and ecological niches are largely unexplored. Here we investigated the potential influence of hybridization on the emergence of morphologically and reproductively distinct asexual lichens. We focused on vagrant forms (growing obligately unattached to substrates) within a clade of rock-dwelling, sexually reproducing species in the Rhizoplaca melanophthalma (Lecanoraceae, Ascomycota) species complex. We used phylogenomic data from both mitochondrial and nuclear genomes to infer evolutionary relationships and potential patterns of introgression. We observed multiple instances of discordance between the mitochondrial and nuclear trees, including the clade comprising the asexual vagrant species R. arbuscula, R. haydenii, R. idahoensis, and a closely related rock-dwelling lineage. Despite well-supported phylogenies, we recovered strong evidence of a reticulated evolutionary history using a network approach that incorporates both incomplete lineage sorting and hybridization. These data suggest that the rock-dwelling western North American subalpine endemic R. shushanii is potentially the result of a hybrid speciation event, and introgression may have also played a role in other taxa, including vagrant species R. arbuscula, R. haydenii and R. idahoensis. We discuss the potential roles of hybridization in terms of generating asexuality and novel morphological traits in lichens. Furthermore, our results highlight the need for additional study of reticulate phylogenies when investigating species boundaries and evolutionary history, even in cases with well-supported topologies inferred from genome-scale data.

Background: Regions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. In spite of the prevalent use of ribosomal markers in fungal research, central features of nuclear ribosomal DNA (nrDNA) evolution are poorly characterized for fungi in general, including lichenized fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. However, little is known about intragenomic variation in the nrDNA in symbiotic fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing., Results: We estimated copy numbers for the nrDNA operon, ranging from nine to 48 copies for members of this complex, and found low levels of intragenomic variation in the standard barcode region (ITS). Monophyly of currently described species in this complex was supported in phylogenetic inferences based on the ITS, 28S, intergenic spacer region, and some intronic regions, independently; however, a phylogenetic inference based on the 18S provided much lower resolution. Phylogenetic analysis of concatenated ITS and intergenic spacer sequence data generated from 496 specimens collected worldwide revealed previously unrecognized lineages in the nrDNA phylogeny., Conclusions: The results from our study support the general assumption that the ITS region of the nrDNA is an effective barcoding marker for fungi. For the R. melanophthalma group, the limited amount of potential intragenomic variability in the ITS region did not correspond to fixed diagnostic nucleotide position characters separating taxa within this species complex. Previously unrecognized lineages inferred from ITS sequence data may represent undescribed species-level lineages or reflect uncharacterized aspects of nrDNA evolution in the R. melanophthalma species complex.

Molecular data provide unprecedented insight into diversity of lichenized fungi, although morphologically cryptic species-level lineages circumscribed from sequence data often remain undescribed even in well-studies groups. Using diagnostic characters from DNA sequence data and support from the multispecies coalescent model, we formally describe a total of eleven new species and resurrect two others in the hyperdiverse lichen-forming fungal family Parmeliaceae. These include: four in the genus Melanelixia - M. ahtii sp. nov., M. epilosa comb. nov., M. hawksworthii sp. nov., and M. robertsoniorum sp. nov.; six in Melanohalea - M. austroamericana sp. nov., M. beringiana sp. nov., M. clari sp. nov., M. columbiana sp. nov., M. davidii sp. nov., and M. tahltan sp. nov.; and three species in Montanelia - M. occultipanniformis sp. nov., M. saximontana comb. nov., and M. secwepemc sp. nov. Morphological, ecological and geographical features were revised to corroborate species descriptions. These species can consistently be distinguished by differences in nucleotide position characters in the fungal barcoding marker (ITS) and high speciation probabilities. This study helps close the "taxonomic gap" between molecular species delimitation studies and formal taxonomy by incorporating statistical evaluation of lineage independence, diagnostic differences in DNA data, and additional consideration of differences in morphology and species distributions., (Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Renewed interests in macroevolutionary dynamics have led to the proliferation of studies on diversification processes in large taxonomic groups, such as angiosperms, mammals, and birds. However, such a study has yet to be conducted in lichenized fungi--an extremely successful and diverse group of fungi. Analysing the most comprehensive time-calibrated phylogenies with a new analytical method, we illustrated drastically different diversification dynamics between two hyper-diverse families of lichenized fungi, Graphidaceae and Parmeliaceae, which represent more than a fourth of the total species diversity of lichenized fungi. Despite adopting a similar nutrition mode and having a similar number of species, Graphidaceae exhibited a lower speciation rate, while Parmeliaceae showed a sharp increase in speciation rate that corresponded with the aridification during the Oligocene-Miocene transition, suggesting their adaptive radiation into a novel arid habitat.

Species recognition in lichen-forming fungi has been a challenge because of unsettled species concepts, few taxonomically relevant traits, and limitations of traditionally used morphological and chemical characters for identifying closely related species. Here we analyze species diversity in the cosmopolitan genus Protoparmelia s.l. The ~25 described species in this group occur across diverse habitats from the boreal-arctic/alpine to the tropics, but their relationship to each other remains unexplored. In this study, we inferred the phylogeny of 18 species currently assigned to this genus based on 160 specimens and six markers: mtSSU, nuLSU, ITS, RPB1, MCM7, and TSR1. We assessed the circumscription of species-level lineages in Protoparmelia s. str. using two coalescent-based species delimitation methods--BP&P and spedeSTEM. Our results suggest the presence of a tropical and an extra-tropical lineage, and eleven previously unrecognized distinct species-level lineages in Protoparmelia s. str. Several cryptic lineages were discovered as compared to phenotype-based species delimitation. Many of the putative species are supported by geographic evidence.

Parmelioid lichens form a species-rich group of predominantly foliose and fruticose lichenized fungi encompassing a broad range of morphological and chemical diversity. Using a multilocus approach, we reconstructed a phylogeny including 323 OTUs of parmelioid lichens and employed ancestral character reconstruction methods to understand the phenotypical evolution within this speciose group of lichen-forming fungi. Specifically, we were interested in the evolution of growth form, epicortex structure, and cortical chemistry. Since previous studies have shown that results may differ depending on the reconstruction method used, here we employed both maximum-parsimony and maximum-likelihood approaches to reconstruct ancestral character states. We have also implemented binary and multistate coding of characters and performed parallel analyses with both coding types to assess for potential coding-based biases. We reconstructed the ancestral states for nine well-supported major clades in the parmelioid group, two higher-level sister groups and the ancestral character state for all parmelioid lichens. We found that different methods for coding phenotypical characters and different ancestral character state reconstruction methods mostly resulted in identical reconstructions but yield conflicting inferences of ancestral states, in some cases. However, we found support for the ancestor of parmelioid lichens having been a foliose lichen with a non-pored epicortex and pseudocyphellae. Our data suggest that some traits exhibit patterns of evolution consistent with adaptive radiation.

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

Premise of the Study: In spite of the recent advances in generic and species circumscriptions and in recognizing species diversity in lichen-forming fungi, the timing of speciation and the factors that promote diversification in lichens remain largely unexplored. We used brown parmelioids as a model to assess the timing of divergence and explore the impact of geological and climatic events on lineage divergence and diversification in lichenized fungi. Additionally, to clarify the phylogenetic position of the species currently placed in Melanelia disjuncta group, we evaluated the taxonomic status and phylogenetic relationships within Parmeliaceae. •, Methods: Phylogenetic relationships and divergence time estimates were inferred from a four-loci data set. Alternative hypotheses were tested using Shimodaira-Hasegawa and expected likelihood weights tests. •, Key Results: The M. disjuncta group forms a strongly supported, monophyletic lineage independent from Melanelia s.s. The M. disjuncta clade arose ca. 23.1 million years ago (Ma). Our results suggest that most of the lineages within the clade diversified during the Miocene (17.6 to 11.2 Ma). The split of other brown parmelioids, such as Emodomelanelia-Melanelixia occurred ca. 41.70 Ma, and the radiation of Melanelixia began during the Eocene-Oligocene transition (ca. 33.75 Ma). •, Conclusions: Montanelia is described here as a new genus to accommodate species of the Melanelia disjuncta group. Further, the study indicates that the current species delimitation within the newly described genus requires revision. We provide evidence of lineage divergence of Montanelia at the Oligocene-Miocene boundary. Our results indicate that the diversification during Miocene would have happened during major mountain uplifts.

Premise of the Study: Diversification in neotropical regions has been attributed to both Tertiary geological events and Pleistocene climatic fluctuations. However, the timing and processes driving speciation in these regions remain unexplored in many important groups. Here, we address the timing of diversification in the neotropical lichenized fungal genus Oropogon (Ascomycota) and assess traditional species boundaries., Methods: We analyzed sequence data from three loci to assess phenotypically circumscribed Oropogon species from the Oaxacan Highlands, Mexico. We provide a comparison of dated divergence estimates between concatenated gene trees and a calibrated multilocus species-tree using substitution rates for two DNA regions. We also compare estimates from a data set excluding ambiguously aligned regions and a data set including the hyper-variable regions in two ribosomal markers., Key Results: Phylogenetic reconstructions were characterized by well-supported monophyletic clades corresponding to traditionally circumscribed species, with the exception of a single taxon. Divergence estimates indicate that most diversification of the sampled Oropogon species occurred throughout the Oligocene and Miocene, although diversification of a single closely related clade appears to have occurred during the late Pliocene and into the Pleistocene. Divergence estimates calculated from a data set with ambiguously aligned regions removed were much more recent than those from the full data set., Conclusions: Overall, our analyses place the majority of divergence events of Oropogon species from the Oaxacan Highlands within the Neogene and provide strong evidence that climatic changes during the Pleistocene were not a major factor driving speciation in the lichenized genus Oropogon in neotropical highlands.

Background: Factors promoting diversification in lichen symbioses remain largely unexplored. While Pleistocene events have been important for driving diversification and affecting distributions in many groups, recent estimates suggest that major radiations within some genera in the largest clade of macrolichens (Parmeliaceae, Ascomycota) vastly predate the Pleistocene. To better understand the temporal placement and sequence of diversification events in lichens, we estimated divergence times in a common lichen-forming fungal genus, Melanohalea, in the Northern Hemisphere. Divergence times were estimated using both concatenated gene tree and coalescent-based multilocus species tree approaches to assess the temporal context of major radiation events within Melanohalea. In order to complement our understanding of processes impacting genetic differentiation, we also evaluated the effects of Pleistocene glacial cycles on population demographics of distinct Melanohalea lineages, differing in reproductive strategies., Results: We found that divergence estimates, from both concatenated gene tree and coalescent-based multilocus species tree approaches, suggest that diversification within Melanohalea occurred predominantly during the Miocene and Pliocene, although estimated of divergence times differed by up to 8.3 million years between the two methods. These results indicate that, in some cases, taxonomically diagnostic characters may be maintained among divergent lineages for millions of years. In other cases, similar phenotypic characters among non-sister taxa, including reproductive strategies, suggest the potential for convergent evolution due to similar selective pressures among distinct lineages. Our analyses provide evidence of population expansions predating the last glacial maximum in the sampled lineages. These results suggest that Pleistocene glaciations were not inherently unfavorable or restrictive for some Melanohalea species, albeit with apparently different demographic histories between sexually and vegetatively reproducing lineages., Conclusions: Our results contribute to the understanding of how major changes during the Miocene and Pliocene have been important in promoting diversification within common lichen-forming fungi in the northern Hemisphere. Additionally, we provide evidence that glacial oscillations have influenced current population structure of broadly distributed lichenized fungal species throughout the Holarctic.

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

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

Premise of the Study: Accurate species delimitation is important for understanding the diversification of biota and has critical implications for ecological and conservation studies. However, a growing body of evidence indicates that morphology-based species circumspection in lichenized fungi misrepresents fungal diversity. The foliose lichen genus Xanthoparmelia includes over 800 species displaying a complex array of morphological and secondary metabolite diversity., Methods: We used a multifaceted approach, applying phylogenetic, population genetic, and genealogical analyses to delimit species in a single well-supported monophyletic clade containing 10 morphologically and chemically diverse Xanthoparmelia species in western North America. Sequence data from four ribosomal and two low-copy, protein-coding markers, along with chemical and morphological data were used to assess species diversity., Key Results: We found that traditionally circumscribed species are not supported by molecular data. Rather, all sampled taxa were better represented by three polymorphic population clusters. Our results suggest that secondary metabolite variation may have limited utility in diagnosing lineages within this group, while identified populations clusters did not reflect major phylogeographic or ecological patterns., Conclusions: In contrast to studies revealing previously undiscovered fungal lineages masked within lichen species circumscribed by traditional morphological and chemical concepts, the present study suggests that species diversity has been overestimated in the species-rich genus Xanthoparmelia.