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In this study we demonstrate the utility of whole genome shotgun (WGS) metagenomics in study organisms with small genomes to improve upon amplicon-based estimates of biodiversity and microbial diversity in environmental samples for the purpose of understanding ecological and evolutionary processes. We generated a database of full-length and near-full-length ribosomal DNA sequence complexes from 273 lichenized fungal species and used this database to facilitate fungal species identification in the southern Appalachian Mountains using low coverage WGS at higher resolution and without the biases of amplicon-based approaches. Using this new database and methods herein developed, we detected between 2.8 and 11 times as many species from lichen fungal propagules by aligning reads from WGS-sequenced environmental samples compared to a traditional amplicon-based approach. We then conducted complete taxonomic diversity inventories of the lichens in each one-hectare plot to assess overlap between standing taxonomic diversity and diversity detected based on propagules present in environmental samples (i.e., the “potential” of diversity). From the environmental samples, we detected 94 species not observed in organism-level sampling in these ecosystems with high confidence using both WGS and amplicon-based methods. This study highlights the utility of WGS sequence-based approaches in detecting hidden species diversity and demonstrates that amplicon-based methods likely miss important components of fungal diversity. We suggest that the adoption of this method will not only improve understanding of biotic constraints on the distributions of biodiversity but will also help to inform important environmental policy.

A total of 171 species of lichens and allied fungi are reported from the spruce-fir forests of Mount Mitchell State Park, in the Black Mountains of North Carolina, based on both historical and modern records. Comparison of the modern baseline with the historical macrolichen baseline generated in the 1970s revealed potential losses of high-elevation southern Appalachian endemics (2 species), cyanolichens (5 species), species typical of exposed rock outcrops (1 species), and widespread species typical of hardwood substrates at high elevations (8 species). In addition to a checklist and summary of lichen biodiversity, dichotomous keys are provided that include all reported species.

Recent lichenological investigations of Fox Hills Formation sandstone outcrops in Colorado resulted in the discovery of three populations that represent an undescribed member of the Lecanora dispersa group (=Myriolecis). This new species is different from all others in the group in its production of usnic acid, which yields apothecia that are yellowish-green in color in fresh field material. The new species, here formally described as Lecanora lendemeri, is further characterized by its relatively large ascospores, endolithic thallus, presence of POL+ granules, and apparent restriction to this sandstone formation. We conducted molecular phylogenetic analyses to place the new species into the context of other members of Lecanora using new shotgun sequence data generated for this study in tandem with previously published rDNA data, and found that the new species is resolved as nested within the L. dispersa group, which was a strongly supported clade in our analysis. Using IUCN criteria including a known occurrence of only three populations, the largest of which is under conservation threat, we herein formally rank this new species as Endangered. This discovery comes on the heels of several other recent lichen discoveries on Fox Hills Sandstone, all species that are, so far as known, restricted to this rock type, suggesting that substrate endemism may be a common element of the biotic communities of the Fox Hills Formation. From the results of this and prior studies, it is clear that sandstone outcrops serve as important, yet still incompletely documented, habitats for cryptogamic diversity. This discovery further highlights the significance of conservation areas, even tiny units (e.g., 40 ha or less) that represent mere islands in a sea of urban development, such as in the Front Range of Colorado.

Hydrogen/deuterium exchange (HDX) using matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry is a sensitive, salt-tolerant and high-throughput method useful to probe protein conformation and molecular interactions. However, a drawback of the MALDI HDX technique is that sample preparation methods can typically result in higher levels of artificial deuterium in-exchange and/or hydrogen back-exchange just prior to or during mass analysis; this may impair data reproducibility and impede structural and kinetic data interpretation. While methods to minimize effects of back-exchange during protein analyte deposition on MALDI plates have been reported, this study presents a readily available, highly sensitive protein control set to facilitate rapid MALDI HDX protocol workup. The Ca2+-induced solvent accessible surface area (ASA) changes of calmodulin (CaM) and S100 proteins were employed to monitor and optimize HDX protocol efficiency. Under non-stringent room temperature conditions, the Ca2+-induced deuterium exchange of CaM, ΔDca2+, MH+ shifts −17 Da to −24 Da, while S100 ΔDca2+ MH+ shifts +8 Da to +12 Da. By comparing the divergent CaM and S100 Ca2+-induced deuterium mass shift differences, HDX sample workup and MALDI plate spotting conditions can easily be monitored.

Recent lichenological investigations of Fox Hills Formation sandstone outcrops in Colorado yielded discovery of three populations that represent an undescribed member of the Lecanora dispersa group. This new species is different from all others in the group in its production of usnic acid, which yields apothecia that are brilliant lime green in color in fresh, field material. The new species, here formally described as Lecanora lendemerii, is further characterized by its relatively large spores, endolithic thallus, presence of POL+ granules, and apparent geographical restriction to this type of sandstone formation. We conducted molecular phylogenetic analyses to place the new species into the context of other members of Lecanora using new shotgun sequence data generated for this study in tandem with previously published rDNA data and found that the new species is resolved as nested within L. dispersa clade, which was a strongly supported clade in our analysis. Using IUCN criteria including a known occurrence of only three populations, the largest of which is under conservation threat, we herein formally rank this new species as Endangered. This discovery comes on the heels of several other recent lichen discoveries on Fox Hills sandstone, all species of which are so far as known restricted to this rock type, suggesting edaphic endemism may be a common element of the biotic communities of the Fox Hills Formation. From the results of this and prior studies, it is clear that sandstone outcrops serve as important, yet still incompletely documented, habitats for cryptogamic diversity. This discovery further highlights the significance of conservation areas, even tiny units (e.g., 100 acres or less) that represent mere islands in a sea of urban development, such as in the Front Range of Colorado.

Microbial communities have increasingly been broadly investigated using sequence-based methods, leading to improved understanding of these important contributors to ecological and evolutionary processes. In this study we demonstrate the utility of whole genome shotgun (WGS) metagenomics in study organisms with small genomes to improve upon amplicon-based estimates of biodiversity and microbial diversity in environmental samples. We generated a database of full-length and near-full-length ribosomal DNA sequence complexes from 273 lichenized fungal species and used this database to compare WGS versus amplicon-based approaches to detect diversity in environmental samples collected in two, one-hectare plots in the southern Appalachian Mountains. This database was established to facilitate fungal species identification using low coverage WGS at higher resolution and without the biases of amplicon-based approaches. Using this new database and methods herein developed, we detected between 2.8 and 11 times as many species from lichen fungal propagules by aligning reads from WGS-sequenced environmental samples compared to a traditional amplicon-based approach applied to the same samples. We then conducted complete taxonomic diversity inventories of the lichens in each one-hectare plot to assess overlap between standing taxonomic diversity (TD) and diversity detected based on propagules present in environmental samples (i.e., the "potential" of diversity, or PoD). From the environmental samples, we detected 94 species not observed in organism-level sampling in these ecosystems with high confidence using both WGS and amplicon-based methods. To our knowledge, no studies to date have applied this method to macro-eukaryotes. This study highlights the utility of WGS sequence-based approaches in detecting hidden species diversity and demonstrates that amplicon-based methods likely miss important components of fungal diversity. We suggest that the adoption of this method, which relies on quantification of TD vs. PoD, will not only improve understanding of biotic constraints on the distributions of biodiversity but will also help to inform important environmental policy.

Microbial communities have increasingly been broadly investigated using sequence-based methods, leading to improved understanding of these important contributors to ecological and evolutionary processes. In this study we demonstrate the utility of whole genome shotgun (WGS) metagenomics in study organisms with small genomes to improve upon amplicon-based estimates of biodiversity and microbial diversity in environmental samples. We generated a database of full-length and near-full-length ribosomal DNA sequence complexes from 273 lichenized fungal species and used this database to compare WGS versus amplicon-based approaches to detect diversity in environmental samples collected in two, one-hectare plots in the southern Appalachian Mountains. This database was established to facilitate fungal species identification using low coverage WGS at higher resolution and without the biases of amplicon-based approaches. Using this new database and methods herein developed, we detected between 2.8 and 11 times as many species from lichen fungal propagules by aligning reads from WGS-sequenced environmental samples compared to a traditional amplicon-based approach applied to the same samples. We then conducted complete taxonomic diversity inventories of the lichens in each one-hectare plot to assess overlap between standing taxonomic diversity (TD) and diversity detected based on propagules present in environmental samples (i.e., the "potential" of diversity, or PoD). From the environmental samples, we detected 94 species not observed in organism-level sampling in these ecosystems with high confidence using both WGS and amplicon-based methods. To our knowledge, no studies to date have applied this method to macro-eukaryotes. This study highlights the utility of WGS sequence-based approaches in detecting hidden species diversity and demonstrates that amplicon-based methods likely miss important components of fungal diversity. We suggest that the adoption of this method, which relies on quantification of TD vs. PoD, will not only improve understanding of biotic constraints on the distributions of biodiversity but will also help to inform important environmental policy.

Recent lichenological investigations of Fox Hills Formation sandstone outcrops in Colorado yielded discovery of three populations that represent an undescribed member of the Lecanora dispersa group. This new species is different from all others in the group in its production of usnic acid, which yields apothecia that are brilliant lime green in color in fresh, field material. The new species, here formally described as Lecanora lendemerii, is further characterized by its relatively large spores, endolithic thallus, presence of POL+ granules, and apparent geographical restriction to this type of sandstone formation. We conducted molecular phylogenetic analyses to place the new species into the context of other members of Lecanora using new shotgun sequence data generated for this study in tandem with previously published rDNA data and found that the new species is resolved as nested within L. dispersa clade, which was a strongly supported clade in our analysis. Using IUCN criteria including a known occurrence of only three populations, the largest of which is under conservation threat, we herein formally rank this new species as Endangered. This discovery comes on the heels of several other recent lichen discoveries on Fox Hills sandstone, all species of which are so far as known restricted to this rock type, suggesting edaphic endemism may be a common element of the biotic communities of the Fox Hills Formation. From the results of this and prior studies, it is clear that sandstone outcrops serve as important, yet still incompletely documented, habitats for cryptogamic diversity. This discovery further highlights the significance of conservation areas, even tiny units (e.g., 100 acres or less) that represent mere islands in a sea of urban development, such as in the Front Range of Colorado.