Reference-Based Restriction-Site-Associated DNA Sequencing Data Are Useful for Species Delineation in a Recently Diverged Asexually Reproducing Species Complex (Parmeliaceae, Ascomycota).

Cryptic species are common in lichen-forming fungi and have been reported from different genera in the most speciose family, Parmeliaceae. Herein, we address species delimitation in a group of mainly asexually reproducing Parmelina species. The morphologically distinct P. pastillifera was previously found nested within a morphologically circumscribed P. tiliacea based on several loci. However, these studies demonstrated a relatively high genetic diversity within P. tiliacea sensu lato. Here, we revisit the species delimitation in the group by analyzing single-nucleotide polymorphisms (SNPs) through genome-wide assessment using Restriction-Site-Associated sequencing and population genomic methods. Our data support previous studies and provide further insight into the phylogenetic relationships of the four clades found within the complex. Based on the evidence suggesting a lack of gene flow among the clades, we recognize the four clades as distinct species, P. pastillifera and P. tiliacea sensu stricto, and two new species, P. clandestina sp. nov. and P. mediterranea sp. nov.

A New Cryptic Lineage in Parmeliaceae (Ascomycota) with Pharmacological Properties.

We used molecular data to address species delimitation in a species complex of the parmelioid genus Canoparmelia and compare the pharmacological properties of the two clades identified. We used HPLC_DAD_MS chromatography to identify and quantify the secondary substances and used a concatenated data set of three ribosomal markers to infer phylogenetic relationships. Some historical herbarium specimens were also examined. We found two groups that showed distinct pharmacological properties. The phylogenetic study supported the separation of these two groups as distinct lineages, which are here accepted as distinct species: Canoparmelia caroliniana occurring in temperate to tropical ecosystems of a variety of worldwide localities, including America, Macaronesia, south-west Europe and potentially East Africa, whereas the Kenyan populations represent the second group, for which we propose the new species C. kakamegaensis Garrido-Huéscar, Divakar & Kirika. This study highlights the importance of recognizing cryptic species using molecular data, since it can result in detecting lineages with pharmacological properties previously overlooked.

Modulation of Cellular Circadian Rhythms by Secondary Metabolites of Lichens.

Background: Most mammalian cells harbor molecular circadian clocks that synchronize physiological functions with the 24-h day-night cycle. Disruption of circadian rhythms, through genetic or environmental changes, promotes the development of disorders like obesity, cardiovascular diseases, and cancer. At the cellular level, circadian, mitotic, and redox cycles are functionally coupled. Evernic (EA) and usnic acid (UA), two lichen secondary metabolites, show various pharmacological activities including anti-oxidative, anti-inflammatory, and neuroprotective action. All these effects have likewise been associated with a functional circadian clock. Hypothesis/Purpose: To test, if the lichen compounds EA and UA modulate circadian clock function at the cellular level. Methods: We used three different cell lines and two circadian luminescence reporter systems for evaluating dose- and time-dependent effects of EA/UA treatment on cellular clock regulation at high temporal resolution. Output parameters studied were circadian luminescence rhythm period, amplitude, phase, and dampening rate. Results: Both compounds had marked effects on clock rhythm amplitudes and dampening independent of cell type, with UA generally showing a higher efficiency than EA. Only in fibroblast cells, significant effects on clock period were observed for UA treated cells showing shorter and EA treated cells showing longer period lengths. Transient treatment of mouse embryonic fibroblasts at different phases had only minor clock resetting effects for both compounds. Conclusion: Secondary metabolites of lichen alter cellular circadian clocks through amplitude reduction and increased rhythm dampening.

Fungal Taxa Responsible for Mucormycosis/"Black Fungus" among COVID-19 Patients in India.

Mucormycosis is caused by fungi belonging to the order Mucorales. The term "Black Fungus" has been widely applied to human pathogenic Mucorales in India. They mainly infect the sinuses and brain, lungs, stomach and intestines, and skin. While this has been considered a rare disease, thousands of cases have been reported during the second wave of COVID-19 in India, between the months of April and June 2021. Hitherto, more than 45,374 cases and over 4300 deaths have been reported among COVID-19 patients across India from April 2021 to July 21, 2021. Though the mortality rate is estimated to be 50%, it could be above 90% if left untreated. In India, Rhizopus arrhizus has been related to be the most common species to cause human mucormycosis, followed by Apophysomyces variabilis, Rhizopus microsporus, and R. homothallicus. Accurate sample identification of human pathogenic Mucorales species is challenging especially due to the frequent lack of diagnostic morphological features. Traditionally, the culture-based approach has been extensively used to isolate and characterize human pathogenic Mucorales. However, this may not be an appropriate approach to objectively isolate and characterize all species, as the germination and growth of fungal spores are highly dependent on culture media and environmental conditions. Therefore, a robust approach to the accurate and rapid identification of human pathogenic Mucorales species is a prerequisite. The metagenomic approach comprehensively sequences and analyzes all genetic material in a complex biological sample and, consequently, this could be an appropriate approach to objectively characterize human pathogenic Mucorales taxa without the need for in vitro culture. The precise identification of the species will not only be useful for the correct diagnosis of this disease, but also for the development of antifungal drugs specific for each species. Accurate and rapid species identification is desperately needed to save lives in the mucormycosis outbreak among COVID-19 patients in India and neighboring countries.

Biodiversity Patterns and Ecological Preferences of the Photobionts Associated With the Lichen-Forming Genus Parmelia.

The worldwide, ecologically relevant lichen-forming genus Parmelia currently includes 41 accepted species, of which the Parmelia sulcata group (PSULgp) and the Parmelia saxatilis group (PSAXgp) have received considerable attention over recent decades; however, phycobiont diversity is poorly known in Parmelia s. lat. Here, we studied the diversity of Trebouxia microalgae associated with 159 thalli collected from 30 locations, including nine Parmelia spp.: P. barrenoae, P. encryptata, P. ernstiae, P. mayi, P. omphalodes, P. saxatilis, P. serrana, P. submontana, and P. sulcata. The mycobionts were studied by carrying out phylogenetic analyses of the nrITS. Microalgae genetic diversity was examined by using both nrITS and LSU rDNA markers. To evaluate putative species boundaries, three DNA species delimitation analyses were performed on Trebouxia and Parmelia. All analyses clustered the mycobionts into two main groups: PSULgp and PSAXgp. Species delimitation identified 13 fungal and 15 algal species-level lineages. To identify patterns in specificity and selectivity, the diversity and abundance of the phycobionts were identified for each Parmelia species. High specificity of each Parmelia group for a given Trebouxia clade was observed; PSULgp associated only with clade I and PSAXgp with clade S. However, the degree of specificity is different within each group, since the PSAXgp mycobionts were less specific and associated with 12 Trebouxia spp., meanwhile those of PSULgp interacted only with three Trebouxia spp. Variation-partitioning analyses were conducted to detect the relative contributions of climate, geography, and symbiotic partner to phycobiont and mycobiont distribution patterns. Both analyses explained unexpectedly high portions of variability (99 and 98%) and revealed strong correlations between the fungal and algal diversity. Network analysis discriminated seven ecological clusters. Even though climatic conditions explained the largest proportion of the variation among these clusters, they seemed to show indifference relative to climatic parameters. However, the cluster formed by P. saxatilis A/P. saxatilis B/Trebouxia sp. 2/Trebouxia sp. S02/Trebouxia sp. 3A was identified to prefer cold-temperate as well as humid summer environments.

Using target enrichment sequencing to study the higher-level phylogeny of the largest lichen-forming fungi family: Parmeliaceae (Ascomycota).

Parmeliaceae is the largest family of lichen-forming fungi with a worldwide distribution. We used a target enrichment data set and a qualitative selection method for 250 out of 350 genes to infer the phylogeny of the major clades in this family including 81 taxa, with both subfamilies and all seven major clades previously recognized in the subfamily Parmelioideae. The reduced genome-scale data set was analyzed using concatenated-based Bayesian inference and two different Maximum Likelihood analyses, and a coalescent-based species tree method. The resulting topology was strongly supported with the majority of nodes being fully supported in all three concatenated-based analyses. The two subfamilies and each of the seven major clades in Parmelioideae were strongly supported as monophyletic. In addition, most backbone relationships in the topology were recovered with high nodal support. The genus Parmotrema was found to be polyphyletic and consequently, it is suggested to accept the genus Crespoa to accommodate the species previously placed in Parmotrema subgen. Crespoa. This study demonstrates the power of reduced genome-scale data sets to resolve phylogenetic relationships with high support. Due to lower costs, target enrichment methods provide a promising avenue for phylogenetic studies including larger taxonomic/specimen sampling than whole genome data would allow.

Genome-Wide Analysis of Biosynthetic Gene Cluster Reveals Correlated Gene Loss with Absence of Usnic Acid in Lichen-Forming Fungi.

Lichen-forming fungi are known to produce a large number of secondary metabolites. Some metabolites are deposited in the cortical layer of the lichen thallus where they exert important ecological functions, such as UV filtering. The fact that closely related lineages of lichen-forming fungi can differ in cortical chemistry suggests that natural product biosynthesis in lichens can evolve independent from phylogenetic constraints. Usnic acid is one of the major cortical pigments in lichens. Here we used a comparative genomic approach on 46 lichen-forming fungal species of the Lecanoromycetes to elucidate the biosynthetic gene content and evolution of the gene cluster putatively responsible for the biosynthesis of usnic acid. Whole-genome sequences were gathered from taxa belonging to different orders and families of Lecanoromycetes, where Parmeliaceae is the most well-represented taxon, and analyzed with a variety of genomic tools. The highest number of biosynthetic gene clusters was found in Evernia prunastri, Pannoparmelia angustata, and Parmotrema austrosinense, respectively, and lowest in Canoparmelia nairobiensis, Bulbothrix sensibilis, and Hypotrachyna scytodes. We found that all studied species producing usnic acid contain the putative usnic acid biosynthetic gene cluster, whereas the cluster was absent in all genomes of species lacking usnic acid. The absence of the gene cluster was supported by an additional unsuccessful search for ß-ketoacylsynthase, the most conserved domain of the gene cluster, in the genomes of species lacking usnic acid. The domain architecture of this PKS cluster-homologous to the already known usnic acid PKS cluster (MPAS) and CYT450 (MPAO)-varies within the studied species, whereas the gene arrangement is highly similar in closely related taxa. We hypothesize that the ancestor of these lichen-forming fungi contained the putative usnic acid producing PKS cluster and that the gene cluster was lost repeatedly during the evolution of these groups. Our study provides insight into the genomic adaptations to the evolutionary success of these lichen-forming fungal species and sets a baseline for further exploration of biosynthetic gene content and its evolutionary significance.

IMA Genome - F13: Draft genome sequences of Ambrosiella cleistominuta, Cercospora brassicicola, C. citrullina, Physcia stellaris, and Teratosphaeria pseudoeucalypti.

Draft genomes of the fungal species Ambrosiella cleistominuta, Cercospora brassicicola, C. citrullina, Physcia stellaris, and Teratosphaeria pseudoeucalypti are presented. Physcia stellaris is an important lichen forming fungus and Ambrosiella cleistominuta is an ambrosia beetle symbiont. Cercospora brassicicola and C. citrullina are agriculturally relevant plant pathogens that cause leaf-spots in brassicaceous vegetables and cucurbits respectively. Teratosphaeria pseudoeucalypti causes severe leaf blight and defoliation of Eucalyptus trees. These genomes provide a valuable resource for understanding the molecular processes in these economically important fungi.

Lichens of Parmelioid Clade as Promising Multitarget Neuroprotective Agents.

Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are multifactorial disorders which are increasing in incidence and prevalence over the world without existing effective therapies. The search for new multitarget compounds is the latter therapeutic strategy to address these pathological conditions. Lichens have an important and unknown therapeutic value attributed to their unique secondary metabolites. The aim of this study is to evaluate for the first time the in vitro neuroprotective activities and molecular mechanisms underlying methanol extracts of lichens of the parmelioid clade and to characterize major bioactive secondary metabolites responsible for their pharmacological actions. Of the 15 parmelioid lichen species, our results showed that Parmotrema perlatum and Hypotrachyna formosana methanol extracts exhibited high antioxidant activity as evidenced in ORAC, DPPH, and FRAP assays. Then, SH-SY5Y cells were pretreated with methanol extracts (24 h) followed by Fenton reagent exposure (2 h). Pretreatments with these two more antioxidant methanol lichen extracts increased cell viability, reduced intracellular ROS, prevented oxidative stress biomarkers accumulation, and upregulated antioxidant enzyme (CAT, SOD, GR, and GPx) activity compared to Fenton reagent cells. The neuroprotective activity was much higher for H. formosana than for P. perlatum, even equal to or higher than Trolox (reference compound). Moreover, H. formosana extracts inhibited both AChE and BuChE activities in a concentration dependent manner, and P. perlatum only showed concentration dependent activity against AChE. Finally, chemical composition analysis using TLC and HPLC methods revealed that physodic acid, lividic acid, and lichexanthone are major secondary metabolites in H. formosana and stictic acid and constictic acid are in P. perlatum. These results demonstrated that P. perlatum and, specially, H. formosana are promising multitargeted neuroprotective agents due to their antioxidant and AChE and BuChE inhibition activities.

Discovering cryptic species in the Aspiciliella intermutans complex (Megasporaceae, Ascomycota) - First results using gene concatenation and coalescent-based species tree approaches.

Taxonomic identifications in some groups of lichen-forming fungi have been challenge largely due to the scarcity of taxonomically relevant features and limitations of morphological and chemical characters traditionally used to distinguish closely related taxa. Delineating species boundaries in closely related species or species complexes often requires a range of multisource data sets and comprehensive analytical methods. Here we aim to examine species boundaries in a group of saxicolous lichen forming fungi, the Aspiciliella intermutans complex (Megasporaceae), widespread mainly in the Mediterranean. We gathered DNA sequences of the nuclear ribosomal internal transcribed spacer (nuITS), the nuclear large subunit (nuLSU), the mitochondrial small subunit (mtSSU) ribosomal DNA, and the DNA replication licensing factor MCM7 from 80 samples mostly from Iran, Caucasia, Greece and eastern Europe. We used a combination of phylogenetic strategies and a variety of empirical, sequence-based species delimitation approaches to infer species boundaries in this group. The latter included: the automatic barcode gap discovery (ABGD), the multispecies coalescent approach *BEAST and Bayesian Phylogenetics and Phylogeography (BPP) program. Different species delimitation scenarios were compared using Bayes factors species delimitation analysis. Furthermore, morphological, chemical, ecological and geographical features of the sampled specimens were examined. Our study uncovered cryptic species diversity in A. intermutans and showed that morphology-based taxonomy may be unreliable, underestimating species diversity in this group of lichens. We identified a total of six species-level lineages in the A. intermutans complex using inferences from multiple empirical operational criteria. We found little corroboration between morphological and ecological features with our proposed candidate species, while secondary metabolite data do not corroborate tree topology. The present study on the A. intermutans species-complex indicates that the genus Aspiciliella, as currently circumscribed, is more diverse in Eurasia than previously expected.

Draft genome sequences of five Calonectria species from Eucalyptus plantations in China, Celoporthe dispersa, Sporothrix phasma and Alectoria sarmentosa.

Draft genome sequences of five Calonectria species [including Calonectria aciculata, C. crousiana, C. fujianensis, C. honghensis and C. pseudoturangicola], Celoporthe dispersa, Sporothrix phasma and Alectoria sarmentosa are presented. Species of Calonectria are the causal agents of Eucalyptus leaf blight disease, threatening the growth and sustainability of Eucalyptus plantations in China. Celoporthe dispersa is the causal agent of stem canker in native Syzygium cordatum and exotic Tibouchina granulosa in South Africa. Sporothrix phasma was first discovered in the infructescences of Protea laurifolia and Protea neriifolia in South Africa. Alectoria sarmentosa is fruticose lichen belongs to the alectorioid clade of the family Parmeliaceae. The availability of these genome sequences will facilitate future studies on the systematics, population genetics, and genomics of these fungi.

Environment and host identity structure communities of green algal symbionts in lichens.

An understanding of how biotic interactions shape species' distributions is central to predicting host-symbiont responses under climate change. Switches to locally adapted algae have been proposed to be an adaptive strategy of lichen-forming fungi to cope with environmental change. However, it is unclear how lichen photobionts respond to environmental gradients, and whether they play a role in determining the fungal host's upper and lower elevational limits. Deep-coverage Illumina DNA metabarcoding was used to track changes in the community composition of Trebouxia algae associated with two phylogenetically closely related, but ecologically divergent fungal hosts along a steep altitudinal gradient in the Mediterranean region. We detected the presence of multiple Trebouxia species in the majority of thalli. Both altitude and host genetic identity were strong predictors of photobiont community assembly in these two species. The predominantly clonally dispersing fungus showed stronger altitudinal structuring of photobiont communities than the sexually reproducing host. Elevation ranges of the host were not limited by the lack of compatible photobionts. Our study sheds light on the processes guiding the formation and distribution of specific fungal-algal combinations in the lichen symbiosis. The effect of environmental filtering acting on both symbiotic partners appears to shape the distribution of lichens.

Circumscription of the genus Lepra, a recently resurrected genus to accommodate the "Variolaria"-group of Pertusaria sensu lato (Pertusariales, Ascomycota).

Pertusarialean lichens include more than 300 species belonging to several independent phylogenetic lineages. Only some of these phylogenetic clades have been comprehensively sampled for molecular data, and formally described as genera. Here we present a taxonomic treatment of a group of pertusarialean lichens formerly known as "Pertusaria amara-group", "Monomurata-group", or "Variolaria-group", which includes widespread and well-known taxa such as P. amara, P. albescens, or P. ophthalmiza. We generated a 6-locus data set with 79 OTUs representing 75 species. The distinction of the Variolaria clade is supported and consequently, the resurrection of the genus Lepra is followed. Thirty-five new combinations into Lepra are proposed and the new species Lepra austropacifica is described from mangroves in the South Pacific. Lepra is circumscribed to include species with disciform ascomata, a weakly to non-amyloid hymenial gel, strongly amyloid asci without clear apical amyloid structures, containing 1 or 2, single-layered, thin-walled ascospores. Chlorinated xanthones are not present, but thamnolic and picrolichenic acids occur frequently, as well as orcinol depsides. Seventy-one species are accepted in the genus. Although the distinction of the genus from Pertusaria is strongly supported, the relationships of Lepra remain unresolved and the genus is tentatively placed in Pertusariales incertae sedis.

A temporal banding approach for consistent taxonomic ranking above the species level.

Comparable taxonomic ranks within clades can facilitate more consistent classifications and objective comparisons among taxa. Here we use a temporal approach to identify taxonomic ranks. This is an extension of the temporal banding approach including a Temporal Error Score that finds an objective cut-off for each taxonomic rank using information for the current classification. We illustrate this method using a data set of the lichenized fungal family Parmeliaceae. To assess its performance, we simulated the effect of taxon sampling and compared our method with the other temporal banding method. For our sampled phylogeny, 11 of the 12 included families remained intact and 55 genera were confirmed, whereas 32 genera were lumped and 15 genera were split. Taxon sampling impacted the method at the genus level, whereas yielded only insignificant changes at the family level. The other available temporal approach also gives a similar cutoff point to our method. Our approach to identify taxonomic ranks enables taxonomists to revise and propose classifications on an objective basis, changing ranks of clades only when inconsistent with most taxa in a phylogenetic tree. An R script to find the time point with the minimal temporal error is provided.

Panmixia and dispersal from the Mediterranean Basin to Macaronesian Islands of a macrolichen species.

The Mediterranean region, comprising the Mediterranean Basin and the Macaronesian Islands, represents a center of diversification for many organisms. The genetic structure and connectivity of mainland and island microbial populations has been poorly explored, in particular in the case of symbiotic fungi. Here we investigated genetic diversity and spatial structure of the obligate outcrossing lichen-forming fungus Parmelina carporrhizans in the Mediterranean region. Using eight microsatellite and mating-type markers we showed that fungal populations are highly diverse but lack spatial structure. This is likely due to high connectivity and long distance dispersal of fungal spores. Consistent with low levels of linkage disequilibrium and lack of clonality, we detected both mating-type idiomorphs in all populations. Furthermore we showed that the Macaronesian Islands are the result of colonization from the Mediterranean Basin. The unidirectional gene flow, though, seemed not to be sufficient to counterbalance the effects of drift, resulting in comparatively allelic poor peripheral populations. Our study is the first to shed light on the high connectivity and lack of population structure in natural populations of a strictly sexual lichen fungus. Our data further support the view of the Macaronesian Islands as the end of the colonization road for this symbiotic ascomycete.

Fungal-algal association patterns in lichen symbiosis linked to macroclimate.

Both macroclimate and evolutionary events may influence symbiont association and diversity patterns. Here we assess how climatic factors and evolutionary events shape fungal-algal association patterns in the widely distributed lichen-forming fungal genus Protoparmelia. Multilocus phylogenies of fungal and algal partners were generated using 174 specimens. Coalescent-based species delimitation analysis suggested that 23 fungal hosts are associating with 20 algal species. Principal component analysis (PCA) was performed to infer how fungal-algal association patterns varied with climate. Fungi associated with one to three algal partners whereas algae accepted one to five fungal partners. Both fungi and algae were more specific, associating with fewer partners, in the warmer climates. Interaction with more than one partner was more frequent in cooler climates for both the partners. Cophylogenetic analyses suggest congruent fungal-algal phylogenies. Host switch was a more common event in warm climates, whereas failure of the photobiont to diverge with its fungal host was more frequent in cooler climates. We conclude that both environmental factors and evolutionary events drive fungal and algal evolution in Protoparmelia. The processes leading to phylogenetic congruence of fungi and algae are different in different macrohabitats in our study system. Hence, closely related species inhabiting diverse habitats may follow different evolutionary pathways.

Hidden diversity before our eyes: Delimiting and describing cryptic lichen-forming fungal species in camouflage lichens (Parmeliaceae, Ascomycota).

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.

An Integrative Approach for Understanding Diversity in the Punctelia rudecta Species Complex (Parmeliaceae, Ascomycota).

High levels of cryptic diversity have been documented in lichenized fungi, especially in Parmeliaceae, and integrating various lines of evidence, including coalescent-based species delimitation approaches, help establish more robust species circumscriptions. In this study, we used an integrative taxonomic approach to delimit species in the lichen-forming fungal genus Punctelia (Parmeliaceae), with a particular focus on the cosmopolitan species P. rudecta. Nuclear, mitochondrial ribosomal DNA and protein-coding DNA sequences were analyzed in phylogenetic and coalescence-based frameworks. Additionally, morphological, ecological and geographical features of the sampled specimens were evaluated. Five major strongly supported monophyletic clades were recognized in the genus Punctelia, and each clade could be characterized by distinct patterns in medullary chemistry. Punctelia rudecta as currently circumscribed was shown to be polyphyletic. A variety of empirical species delimitation methods provide evidence for a minimum of four geographically isolated species within the nominal taxon Punctelia rudecta, including a newly described saxicolous species, P. guanchica, and three corticolous species. In order to facilitate reliable sample identification for biodiversity, conservation, and air quality bio-monitoring research, these three species have been epitypified, in addition to the description of a new species.

Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi.

We studied the evolutionary history of the Parmeliaceae (Lecanoromycetes, Ascomycota), one of the largest families of lichen-forming fungi with complex and variable morphologies, also including several lichenicolous fungi. We assembled a six-locus data set including nuclear, mitochondrial and low-copy protein-coding genes from 293 operational taxonomic units (OTUs). The lichenicolous lifestyle originated independently three times in lichenized ancestors within Parmeliaceae, and a new generic name is introduced for one of these fungi. In all cases, the independent origins occurred c. 24 million yr ago. Further, we show that the Paleocene, Eocene and Oligocene were key periods when diversification of major lineages within Parmeliaceae occurred, with subsequent radiations occurring primarily during the Oligocene and Miocene. Our phylogenetic hypothesis supports the independent origin of lichenicolous fungi associated with climatic shifts at the Oligocene-Miocene boundary. Moreover, diversification bursts at different times may be crucial factors driving the diversification of Parmeliaceae. Additionally, our study provides novel insight into evolutionary relationships in this large and diverse family of lichen-forming ascomycetes.