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Evolutionary histories are now being inferred from unprecedented, genome-scale datasets for a broad range of organismal groups. While phylogenomic data has helped in resolving a number of difficult, long-standing questions, constructing appropriate datasets from genomes is not straightforward, particularly in non-model groups. Here we explore the utility of phylogenomic data to infer robust phylogenies for a lineage of closely related lichen-forming fungal species. We assembled multiple, distinct nuclear phylogenomic datasets, ranging from ca. 25 Kb to 16.8 Mb and inferred topologies using both concatenated gene tree approaches and species tree methods based on the multispecies coalescent model. In spite of evidence for rampant incongruence among individual loci, these genome-scale datasets provide a consistent, well-supported phylogenetic hypothesis using both concatenation and multispecies coalescent approaches (ASTRAL-II and SVDquartets). However, the popular full hierarchical coalescent approach implemented in *BEAST provided inconsistent inferences, both in terms of nodal support and topology, with smaller subsets of the phylogenomic data. While comparable, well-supported topologies can be accurately inferred with only a small fraction of the overall genome, consistent results across a variety of datasets and methodological approaches provide reassurance that phylogenomic data can effectively be used to provide robust phylogenies for closely related lichen-forming fungal lineages.

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.

DNA barcoding approaches provide powerful tools for characterizing fungal diversity. However, DNA barcoding is limited by poor representation of species-level diversity in fungal sequence databases. Can the development of custom, regionally focused DNA reference libraries improve species-level identification rates for lichen-forming fungi? To explore this question, we created a regional ITS database for lichen-forming fungi (LFF) in the Intermountain West of the United States. The custom database comprised over 4800 sequences and represented over 600 formally described and provisional species. Lichen communities were sampled at 11 sites throughout the Intermountain West, and LFF diversity was characterized using high-throughput ITS2 amplicon sequencing. We compared the species-level identification success rates from our bulk community samples using our regional ITS database and the widely used UNITE database. The custom regional database resulted in significantly higher species-level assignments (72.3%) of candidate species than the UNITE database (28.3–34.2%). Within each site, identification of candidate species ranged from 72.3–82.1% using the custom database; and 31.5–55.4% using the UNITE database. These results highlight that developing regional databases may accelerate a wide range of LFF research by improving our ability to characterize species-level diversity using DNA barcoding. [ABSTRACT FROM AUTHOR]

Subalpine habitats in sky islands in the Southwestern USA are currently facing large-scale transformations. Lichens have widely been used as bioindicators of environmental change. On the Colorado Plateau, fruticose lichens occur in patchy, disconnected populations, including unique lichen-draped conifer sites in subalpine forests in the La Sal Mountains in southeastern Utah. Here, we document the distribution and fungal diversity within these lichen communities. We find that lichen-draped conifer sites in the La Sal Mountains are restricted to only three known, small areas in Picea englemannii forests above 3000 m above sea level, two of which have recently been impacted by wildfire. We document 30 different species of lichen-forming fungi in these communities, several which represent the first reports from the Colorado Plateau. We also characterize mycobiont haplotype diversity for the fruticose lichens Evernia divaricata, Ramalina sinensis, and multiple Usnea species. We also report a range of diverse fungi associated with these lichens, including genetic clusters representing 22 orders spanning seven classes of Ascomycetes and fewer clusters representing Basidiomycetes. Our results provide a baseline for ongoing monitoring and help to raise awareness of unique lichen communities and other biodiversity in the region. [ABSTRACT FROM AUTHOR]

Robust species delimitations provide a foundation for investigating speciation, phylogeography, and conservation. Here we attempted to elucidate species boundaries in the cosmopolitan lichen-forming fungal taxon Lecanora polytropa. This nominal taxon is morphologically variable, with distinct populations occurring on all seven continents. To delimit candidate species, we compiled ITS sequence data from populations worldwide. For a subset of the samples, we also generated alignments for 1209 single-copy nuclear genes and an alignment spanning most of the mitochondrial genome to assess concordance among the ITS, nuclear, and mitochondrial inferences. Species partitions were empirically delimited from the ITS alignment using ASAP and bPTP. We also inferred a phylogeny for the L. polytropa clade using a four-marker dataset. ASAP species delimitations revealed up to 103 species in the L. polytropa clade, with 75 corresponding to the nominal taxon L. polytropa. Inferences from phylogenomic alignments generally supported that these represent evolutionarily independent lineages or species. Less than 10% of the candidate species were comprised of specimens from multiple continents. High levels of candidate species were recovered at local scales but generally with limited overlap across regions. Lecanora polytropa likely ranks as one of the largest species complexes of lichen-forming fungi known to date. [ABSTRACT FROM AUTHOR]

Species delimitation among closely related species is challenging because traditional phenotype‐based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high‐throughput sequencing, it has become increasingly cost‐effective to acquire genome‐scale data which can resolve previously ambiguous species boundaries. As the availability of genome‐scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro‐endemism in a lineage of lichen‐forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single‐locus and genome‐scale data consisting of (a) single‐locus species delimitation analysis using ASAP, (b) maximum likelihood‐based phylogenetic tree inference, (c) genome‐scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross‐validate results between genome‐scale and single‐locus datasets, differently sampled subsets of genomic data and to control for population‐level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro‐endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories. [ABSTRACT FROM AUTHOR]

Lichens are classic models of symbiosis, and one of the most frequent nutritional modes among fungi. The ecologically and geographically widespread lichen-forming algal (LFA) genus Trebouxia is one of the best-studied groups of LFA and associates with over 7000 fungal species. Despite its importance, little is known about its diversification. We synthesized twenty years of publicly available data by characterizing the ecological preferences of this group and testing for time-variant shifts in climatic regimes over a distribution of trees. We found evidence for limited shifts among regimes, but that disparate lineages convergently evolved similar ecological tolerances. Early Trebouxia lineages were largely forest specialists or habitat generalists that occupied a regime whose extant members occur in moderate climates. Trebouxia then convergently diversified in non-forested habitats and expanded into regimes whose modern representatives occupy wet-warm and cool-dry climates. We rejected models in which climatic diversification slowed through time, suggesting climatic diversification is inconsistent with that expected under an adaptive radiation. In addition, we found that climatic and vegetative regime shifts broadly coincided with the evolution of biomes and associated or similar taxa. Together, our work illustrates how this keystone symbiont from an iconic symbiosis evolved to occupy diverse habitats across the globe. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Taxonomic ranks above the species level are inherently arbitrary. However, there is a growing number of publications aimed at more consistent classifications with comparable ranks among taxa. For this study, we use a recently developed temporal approach that utilizes time-calibrated chronograms to identify and define temporal bands for comparable ordinal and family ranks in Lecanoromycetes, the most diverse lineage of lichen-forming fungi. A multi-locus dataset consisting of 539 taxa in the two major subclasses of Lecanoromycetes, Lecanoromycetidae and Ostropomycetidae, was used to address the circumscription of families and orders. Based on the temporal banding approach, clades that share a common ancestor between 176 and 194 Mya and a time window of 111–135 Mya correspond to order-level and family-level, respectively. Most currently accepted orders and families were supported in their current circumscription, but some new taxa are described. Here we propose a revised, temporally based classification for the two subclasses. Specifically, three new orders are proposed: Sporastatiales, Schaereriales and Thelenellales. Arctomiales, Hymeneliales and Trapeliales are synonymized with Baeomycetales. Varicellariaceae are proposed as a new family, and Diploschistaceae and Thelotremataceae are resurrected. Squamarinaceae and Stereocaulaceae are synonymized with Cladoniaceae, Carbonicolaceae are synonymized with Lecanoraceae, Letrouitiaceae are synonymized with Brigantiaeaceae, Lobariaceae and Nephromataceae are synonymized with Peltigeraceae, Thrombiaceae are synonymized with Protothelenellaceae, and Miltideaceae are synonymized with Agyriaceae. This study represents an important step towards more consistent, comparable deeper-level taxonomic rankings in the most diverse lineages of lichen-forming fungi. [ABSTRACT FROM AUTHOR]

Accurate species delimitation is important as species are a fundamental unit in ecological, evolutionary and conservation biology research. In lichenized fungi, species delimitation has been difficult due to a lack of taxonomically important characteristics and due to the limits of traditional, morphology-based species concepts. In this study we reassess the current taxonomy of the Parmotrema perforatum group, which recognizes six closely related species divided into three species pairs, each pair comprising one apotheciate (sexual) and one sorediate (asexual) species. Each pair is further characterized by a distinct combination of secondary metabolites. It was hypothesized that the three apotheciate species are reproductively isolated sibling species and that each sorediate species evolved once from the chemically identical apotheciate species. In this study, species boundaries were re-examined using an integrative approach incorporating morphological, chemical and molecular sequence data to delimit species boundaries. Phylogenetic trees were inferred from a seven-locus DNA sequence dataset using concatenated gene tree and coalescent-based species-tree inference methods. Furthermore, we employed a multi-species coalescent method to validate candidate species. Micromorphological measurements of conidia were found to be congruent with phylogenetic clusters. Each approach that we applied to the P. perforatum group consistently recovered four of the currently circumscribed species ( P. perforatum, P. hypotropum, P. subrigidum and P. louisianae), whereas P. preperforatum and P. hypoleucinum were consistently combined and are thus interpreted as conspecific. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Brown parmelioid lichens comprise a number of distinct genera in one of the most species-rich families of lichen-forming fungi, Parmeliaceae (Ascomycota). In spite of their superficial similarity, a number of studies of brown parmelioids have provided important insight into diversification in lichen-forming fungi with cosmopolitan distributions. In this study we assess species diversity, biogeography and diversification of the genus Montanelia , which includes alpine to temperate saxicolous species. We sampled each of the five known species, four of which are known from broad, intercontinental distributions. In order to identify potential biogeographical patterns, each broadly distributed species was represented by individuals collected across their intercontinental distributions. Molecular sequence data were generated for six loci, including three nuclear protein-coding markers (MCM7, RPB1, and RPB2), two nuclear ribosomal markers (ITS and nrLSU), and a fragment of the mitochondrial small subunit. We used three sequence-based species delimitations methods to validate traditional, phenotype-based species and circumscribe previously unrecognized species-level lineages in Montanelia . Relationships among putative lineages and divergence times were estimated within a coalescent-based multi-locus species tree framework. Based on the results of the species delimitation analyses, we propose that the genus Montanelia is likely comprised of six to nine species-level lineages, including previously unrecognized species-level diversity in the nominal taxa M. panniformis and M. tominii . In contrast, molecular sequence data suggest that M. predisjuncta may be conspecific with the widespread taxon M. disjuncta in spite of distinct morphological differences . The rate-based age estimation of the most recent common ancestor of Montanelia (ca. 23.1 Ma) was similar to previous estimates based on the fossil record. Furthermore, our data suggest that diversification in Montanelia occurred largely during the Neogene. At least three Montanelia species are broadly distributed throughout Asia, Europe, and North America with no evidence of phylogeographic substructure. In contrast to broadly distributed Montanelia species, our study suggests Pleistocene-dominated diversification and complex biogeographic history in the M. tominii group. Our analyses provide additional insight for understanding diversification and uncovering cryptic diversity in cosmopolitan species of lichen-forming fungi. [ABSTRACT FROM AUTHOR]

The largest family of lichen-forming fungi, the Parmeliaceae, is reviewed. It includes 79 genera in current use and ca 2726 species, a large majority of which belong to one of five main clades: the parmelioid, cetrarioid, usneoid, alectorioid and hypogymnioid. However, 21 genera are positioned outside these clades, and four genera in current use still lack DNA- data. The family has been shown to be monophyletic and the generic classification is relatively well-settled compared with other lecanoralean families. Each clade and its genera are presented here with the latest results from phylogenetic analyses and current taxonomy. In addition, a historical outline of the family and its most prominent researchers is provided. [ABSTRACT FROM AUTHOR]

In show caves, artificial lighting is intended to illuminate striking cave formations for visitors. However, artificial lighting also promotes the growth of novel and diverse biofilm communities, termed lampenflora, that obtain their energy from these artificial light sources. Lampenflora, which generally consist of cyanobacteria, algae, diatoms, and bryophytes, discolor formations and introduce novel ecological interactions in cave ecosystems. The source of lampenflora community members and patterns of diversity have generally been understudied mainly due to technological limitations. In this study, we investigate whether members of lampenflora communities in an iconic show cave—Lehman Caves—in Great Basin National Park (GRBA) in the western United States also occur in nearby unlit and rarely visited caves. Using a high-throughput environmental DNA metabarcoding approach targeting three loci—the ITS2 (fungi), a fragment of the 16S (bacteria), and a fragment of 23S (photosynthetic bacteria and eukaryotes)—we characterized diversity of lampenflora communities occurring near artificial light sources in Lehman Caves and rock surfaces near the entrances of seven nearby "wild" caves. Most caves supported diverse and distinct microbial-dominated communities, with little overlap in community members among caves. The lampenflora communities in the show cave were distinct, and generally less diverse, from those occurring in nearby unlit caves. Our results suggest an unidentified source for a significant proportion of lampenflora community members in Lehman Caves, with the majority of community members not found in nearby wild caves. Whether the unique members of the lampenflora communities in Lehman Caves are related to distinct abiotic conditions, increased human visitation, or other factors remains unknown. These results provide a valuable framework for future research exploring lampenflora community assemblies in show caves, in addition to a broad perspective into the range of microbial and lampenflora community members in GRBA. By more fully characterizing these communities, we can better monitor the establishment of lampenflora and design effective strategies for their management and removal. [ABSTRACT FROM AUTHOR]

• Comprehensive molecular phylogeny of the Holarctic clade of lichen-forming fungi genus Xanthoparmelia. • Despite reconstructing relatively consistent phylogenetic trees, rampant phylogenetic conflict was revealed. • Widespread hybridization was detected among species in the most diverse genus of lichen-forming fungi using three independent tests for hybridization. While advances in sequencing technologies have been invaluable for understanding evolutionary relationships, increasingly large genomic data sets may result in conflicting evolutionary signals that are often caused by biological processes, including hybridization. Hybridization has been detected in a variety of organisms, influencing evolutionary processes such as generating reproductive barriers and mixing standing genetic variation. Here, we investigate the potential role of hybridization in the diversification of the most speciose genus of lichen-forming fungi, Xanthoparmelia. As Xanthoparmelia is projected to have gone through recent, rapid diversification, this genus is particularly suitable for investigating and interpreting the origins of phylogenomic conflict. Focusing on a clade of Xanthoparmelia largely restricted to the Holarctic region, we used a genome skimming approach to generate 962 single-copy gene regions representing over 2 Mbp of the mycobiont genome. From this genome-scale dataset, we inferred evolutionary relationships using both concatenation and coalescent-based species tree approaches. We also used three independent tests for hybridization. Although different species tree reconstruction methods recovered largely consistent and well-supported trees, there was widespread incongruence among individual gene trees. Despite challenges in differentiating hybridization from ILS in situations of recent rapid radiations, our genome-wide analyses detected multiple potential hybridization events in the Holarctic clade, suggesting one possible source of trait variability in this hyperdiverse genus. This study highlights the value in using a pluralistic approach for characterizing genome-scale conflict, even in groups with well-resolved phylogenies, while highlighting current challenges in detecting the specific impacts of hybridization. [ABSTRACT FROM AUTHOR]

• The vast majority of species-level lineages in Trebouxia lack formal description. • Four major Trebouxia lineages are recovered in our multi-locus phylogeny. • Integrative taxonomy will be essential for these important lichen photobionts. • Other under-sampled regions likely harbor additional species-level diversity. Lichens provide valuable systems for studying symbiotic interactions. In lichens, these interactions are frequently described in terms of availability, selectivity and specificity of the mycobionts and photobionts towards one another. The lichen-forming, green algal genus Trebouxia Puymaly is among the most widespread photobiont, associating with a broad range of lichen-forming fungi. To date, 29 species have been described, but studies consistently indicate that the vast majority of species-level lineages still lack formal description, and new, previously unrecognized lineages are frequently reported. To reappraise the diversity and the evolutionary relationships of species-level lineages in Trebouxia , we assembled DNA sequence data from over 1600 specimens, compiled from a range of sequences from previously published studies, axenic algal cultures, and lichens collected from poorly sampled regions. From these samples, we selected representatives of the currently known genetic diversity in the lichenized Trebouxia and inferred a phylogeny from multi-locus sequence data (ITS, rbcL , cox 2). We demonstrate that the current formally described species woefully underrepresent overall species-level diversity in this important lichen-forming algal genus. We anticipate that an integrative taxonomic approach, incorporating morphological and physiological data from axenic cultures with genetic data, will be required to establish a robust, comprehensive taxonomy for Trebouxia. The data presented here provide an important impetus and reference dataset for more reliably characterizing diversity in lichenized algae and in using lichens to investigate the evolution of symbioses and holobionts. [ABSTRACT FROM AUTHOR]