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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.

The phylogeny of Lecidella species is studied, based on a 7-locus data set using ML and Bayesian analyses. Phylogenetic relationships among 43 individuals representing 11 Lecidella species, mainly from mainland China, were included in the analyses and phenotypical characters studied and mapped onto the phylogeny. The Lecidella species fall into three major clades, which are proposed here as three informal groups-Lecidella stigmatea group, L. elaeochroma group and L. enteroleucella group, each of them strongly supported. Our phylogenetic analyses support traditional species delimitation based on morphological and chemical traits in most but not all cases. Individuals considered as belonging to the same species based on phenotypic characters were found to be paraphyletic, indicating that cryptic species might be hidden under these names (e.g. L. carpathica and L. effugiens). Potentially undescribed species were found within the phenotypically circumscribed species L. elaeochroma and L. stigmatea. Additional sampling across a broader taxonomic and geographic scale will be crucial to fully resolving the taxonomy in this cosmopolitan genus.

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.

The inclusion of molecular data is increasingly an integral part of studies assessing species boundaries. Analyses based on predefined groups may obscure patterns of differentiation, and population assignment tests provide an alternative for identifying population structure and barriers to gene flow. In this study, we apply population assignment tests implemented in the programs STRUCTURE and BAPS to single nucleotide polymorphisms from DNA sequence data generated for three previous studies of the lichenized fungal genus Letharia. Previous molecular work employing a gene genealogical approach circumscribed six species-level lineages within the genus, four putative lineages within the nominal taxon L. columbiana (Nutt.) J.W. Thomson and two sorediate lineages. We show that Bayesian clustering implemented in the program STRUCTURE was generally able to recover the same six putative Letharia lineages. Population assignments were largely consistent across a range of scenarios, including: extensive amounts of missing data, the exclusion of SNPs from variable markers, and inferences based on SNPs from as few as three gene regions. While our study provided additional evidence corroborating the six candidate Letharia species, the equivalence of these genetic clusters with species-level lineages is uncertain due, in part, to limited phylogenetic signal. Furthermore, both the BAPS analysis and the ad hoc ΔK statistic from results of the STRUCTURE analysis suggest that population structure can possibly be captured with fewer genetic groups. Our findings also suggest that uneven sampling across taxa may be responsible for the contrasting inferences of population substructure. Our results consistently supported two distinct sorediate groups, 'L. lupina' and L. vulpina, and subtle morphological differences support this distinction. Similarly, the putative apotheciate species 'L. lucida' was also consistently supported as a distinct genetic cluster. However, additional studies will be required to elucidate the relationships of other L. columbiana s.l. populations with the two sorediate genetic clusters.

Pleistocene climatic fluctuations influenced patterns of genetic variation and promoted speciation across a wide range of species groups. Lichens are commonly found in habitats that were directly impacted by glacial cycles; however, the role of Pleistocene climate in driving speciation in most lichen symbionts remains unclear. This uncertainty is due in part to limitations in our ability to accurately recognize independently evolving lichen-forming fungal lineages and a lack of relevant fossil calibrations. Using a coalescent-based species tree approach, we estimated divergence times for two sister clades in the genus Xanthoparmelia (Parmeliaceae) restricted to western North America. We assessed the influence of two different species circumscription scenarios and various locus-specific rates of molecular evolution on divergence estimates. Species circumscriptions were validated using the program BP&P. although speciation was generally supported in both scenarios, divergence times differed between traditional species circumscriptions and those based on genetic data, with more recent estimates resulting from the former. Similarly, rates of evolution for different loci resulted in variable divergence time estimates. However, our results unambiguously indicate that diversification in the sampled Xanthoparmelia clades occurred during the Pleistocene. Our study highlights the potential impact of ambiguous species circumscriptions and uncertain rates of molecular evolution on estimating divergence times within a multilocus species tree framework.

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.