The rust fungi (Pucciniales) are the most speciose natural group of plant pathogens, members of which possess the most complex lifecycles in Fungi. How natural selection works on the Pucciniales has been the subject of several hypotheses in mycology. This study uses molecular age estimation using sequence data from multiple loci, and cophylogeny reconciliation analyses to test hypotheses regarding how the aecial and telial stages in the lifecycle of rust fungi may have differentially impacted their diversification. Molecular age estimates show that the timing of diversification in the Pucciniales correlates with the diversification of their gymnosperm and angiosperm hosts. Host reconciliation analyses suggest that systematic relationships of hosts from the aecial stage of the Pucciniales lifecycle better reflect the systematic relationships among the Pucciniales. The results demonstrate the relative importance of this stage on the overall evolution of the Pucciniales and supports hypotheses made by Leppik over half a century ago. This study represents the first evaluation of how different life stages in the Pucciniales shape the evolution of these fungi.

Armillaria is a globally distributed mushroom-forming genus composed primarily of plant pathogens. Species in this genus are prolific producers of rhizomorphs, or vegetative structures, which, when found, are often associated with infection. Because of their importance as plant pathogens, understanding the evolutionary origins of this genus and how it gained a worldwide distribution is of interest. The first gasteroid fungus with close affinities to Armillaria—Guyanagaster necrorhizus—was described from the Neotropical rainforests of Guyana. In this study, we conducted phylogenetic analyses to fully resolve the relationship of G. necrorhizus with Armillaria. Data sets containing Guyanagaster from two collecting localities, along with a global sampling of 21 Armillaria species—including newly collected specimens from Guyana and Africa—at six loci (28S, EF1α, RPB2, TUB, actin-1 and gpd) were used. Three loci—28S, EF1α and RPB2—were analyzed in a partitioned nucleotide data set to infer divergence dates and ancestral range estimations for well-supported, monophyletic lineages. The six-locus phylogenetic analysis resolves Guyanagaster as the earliest diverging lineage in the armillarioid clade. The next lineage to diverge is that composed of species in Armillaria subgenus Desarmillaria. This subgenus is elevated to genus level to accommodate the exannulate mushroom-forming armillarioid species. The final lineage to diverge is that composed of annulate mushroom-forming armillarioid species, in what is now Armillaria sensu stricto. The molecular clock analysis and ancestral range estimation suggest the most recent common ancestor to the armillarioid lineage arose 51 million years ago in Eurasia. A new species, Guyanagaster lucianii sp. nov. from Guyana, is described. The armillarioid lineage evolved in Eurasia during the height of tropical rainforest expansion about 51 million years ago, a time marked by a warm and wet global climate. Species of Guyanagaster and Desarmillaria represent extant taxa of these early diverging lineages. Desarmillaria represents an armillarioid lineage that was likely much more widespread in the past. Guyanagaster likely evolved from a gilled mushroom ancestor and could represent a highly specialized endemic in the Guiana Shield. Armillaria species represent those that evolved after the shift in climate from warm and tropical to cool and arid during the late Eocene. No species in either Desarmillaria or Guyanagaster are known to produce melanized rhizomorphs in nature, whereas almost all Armillaria species are known to produce them. The production of rhizomorphs is an adaptation to harsh environments, and could be a driver of diversification in Armillaria by conferring a competitive advantage to the species that produce them.

Rapid economic development and accelerated urbanization in China has resulted in widespread atmospheric nitrogen (N) deposition. One consequence of N deposition is the alteration of mycorrhizal symbioses that are critical for plant resource acquisition (nitrogen, N, phosphorus, P, water). In this study, we characterized the diversity, composition, and functioning of ectomycorrhizal (ECM) fungal communities in an urban-adjacent Pinus elliottii plantation under ambient N deposition (~24 kg N ha⁻¹ year⁻¹), and following N fertilization (low N, 50 kg N ha⁻¹ year⁻¹; high N, 300 kg N ha⁻¹ year⁻¹). ECM functioning was expressed as the potential activities of extracellular enzymes required for organic N (protease), P (phosphomonoesterase), and recalcitrant polymers (phenol oxidase). Despite high ambient N deposition, ECM community composition shifted under experimental N fertilization, and those changes were linked to disparate levels of soil minerals (P, K) and organic matter (but not N), a decline in acid phosphatase (AP), and an increase in phenol oxidase (PO) potential activities. Based on enzyme stoichiometry, medium-smooth exploration type ECM species invested more in C acquisition (PO) relative to P (AP) following high N fertilization than other exploration types. ECM species with hydrophilic mantles also showed higher enzymatic PO:AP ratios than taxa with hydrophobic mantles. Our findings add to the accumulating evidence that shifts in ECM community composition and taxa specialized in organic C, N, and P degradation could modulate the soil nutrient cycling in forests exposed to chronic elevated N input.

The nuclear ribosomal internal transcribed spacer (nrITS) region has been widely used in fungal diversity studies. Environmental metabarcoding has increased the importance of the fungal DNA barcode in documenting fungal diversity and distribution. The DNA barcode gap is seen as the difference between intra- and inter-specific pairwise distances in a DNA barcode. The current understanding of the barcode gap in macrofungi is limited, inhibiting the development of best practices in applying the nrITS region toward research on fungal diversity. This study examined the barcode gap using 5146 sequences representing 717 species of macrofungi from eleven genera, eight orders and two phyla in datasets assembled by taxonomic experts. Intra- and inter-specific pairwise distances were measured from sequence and phylogenetic data. The results demonstrate that barcode gaps are influenced by differences in intra- and inter-specific variance in pairwise distances. In terms of DNA barcode behavior, variance is greater in the ITS1 than ITS2, and variance is greater in both relative to the combined nrITS region. Due to the difference in variance, the barcode gaps in the ITS2 region are greater than in the ITS1. Additionally, the taxonomic approach of “splitting” taxa into numerous taxonomic units produces greater barcode gaps when compared to “lumping”. The results show variability in the barcode gaps between fungal taxa, demonstrating a need to understand the accuracy of DNA barcoding in quantifying species richness. For taxonomic studies, variability in nrITS sequence data supports the application of multiple molecular markers to corroborate the taxonomic and systematic delineation of species.

Premise: Fungaria are an underutilized resource for understanding fungal biodiversity. The effort and cost of producing DNA barcode sequence data for large numbers of fungal specimens can be prohibitive. This study applies a modified metabarcoding approach that provides a labor‐efficient and cost‐effective solution for sequencing the fungal DNA barcodes of hundreds of specimens at once.Methods: We applied a two‐step PCR approach using nested, barcoded primers to sequence the fungal nrITS2 region of 766 macrofungal specimens using the Illumina platform. The specimens represent a broad taxonomic sampling of the Dikarya. Of these, 382 Lactarius specimens were analyzed to identify molecular operational taxonomic units (MOTUs) using a phylogenetic approach. The raw sequences were trimmed, filtered, assessed, and analyzed using the DADA2 amplicon de‐noising toolkit and Biopython. The sequences were compared to the NCBI and UNITE databases and Sanger nrITS sequences from the same specimens.Results: The taxonomic identities derived from the nrITS2 sequence data were >90% accurate across all specimens sampled. A phylogenetic analysis of the Lactarius sequences identified 20 MOTUs.Discussion: The results demonstrate the capacity of these methods to produce nrITS2 sequences from large numbers of fungarium specimens. This provides an opportunity to more effectively use fungarium collections to advance fungal diversity identification and documentation.