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The occurrence of cryptic species is well documented in fungi but the extent of their diversity is not fully understood. This study assessed the fungal diversity within a part of the Larry F. Grand Mycological Herbarium (NCSLG), a small, well-maintained collection at North Carolina State University, with a focus on the powdery mildew fungi ( Erysiphaceae ). Erysiphaceae were selected due to their economic impact as plant pathogens and availability of extensive DNA sequence data for multiple barcode loci. Our research objectives included determining the number of phylogenetic species compared with those identified morphologically, and to identify undescribed species. We generated sequence data for 220 of the 299 powdery mildew specimens (73% success rate) in the herbarium, which represented 60 species in 10 genera, collected from 134 host plant species. Our analyses revealed that ~83% (183/220) of the sequenced specimens had identifications that were incorrect and/or outdated based on current genus/species concepts. Additionally, four new species are described: Erysiphe amphicarpaeicola , E. ulmi-alatae , E. quercus-virginianae , and Takamatsuella grandii . A specimen deposited at NCSLG is designated as an epitype for Phyllactinia liriodendri , and a species of Phyllactinia identified on Carpinus caroliniana , as well as multiple species infecting Quercus spp., likely represent additional undescribed species that require more data. This research highlights the critical role of herbarium collections in uncovering fungal biodiversity, and underscores the importance of preserving these valuable resources, particularly with the growing trend to discard herbaria due to financial and space constraints.

The fungal mitochondrial small subunit (mtSSU) ribosomal DNA is one of the most commonly used loci for phylogenetic analysis of lichen-forming fungi, but their primer specificity to mycobionts has not been evaluated. The current study aimed to design mycobiont-specific mtSSU primers and highlights their utility with an example from the saxicolous lichen-forming fungal genus Melanelia Essl. in Iceland. The study found a 12.5% success rate (3 out of 24 specimens with good-quality mycobiont mtSSU sequences) using universal primers (i.e. mrSSU1 and mrSSU3R), not including off-target amplification of environmental fungi, e.g. Cladophialophora carrionii and Lichenothelia convexa . New mycobiont-specific primers (mt-SSU-581-5’ and mt-SSU-1345-3’) were designed by targeting mycobiont-specific nucleotide sites in comparison with environmental fungal sequences, and assessed for mycobiont primer specificity using in silico PCR. The new mycobiont-specific mtSSU primers had a success rate of 91.7% (22 out of 24 specimens with good-quality mycobiont mtSSU sequences) on the studied Melanelia specimens. Additional testing confirmed the specificity and yielded amplicons from 79 specimens of other Parmeliaceae mycobiont lineages. This study highlights the effectiveness of designing mycobiont-specific primers for studies on lichen identification, barcoding and phylogenetics.

Historical and recent material of Bacidia akompsa (basionym: Biatora akompsa) was studied to determine the taxonomic position of this crustose lichen, prompted by the appearance of the provisional name “Lecanactis akompsa (Tuck.) ined.” in multiple online lichen databases and checklists. Initial morphological assessment of verified material confirmed B. akompsa as a member of Arthoniales but distinct from Lecanactis by having epruinose ascomata with a thin exciple open below the hymenium; a pale, K/I+ violet to deep blue hypothecium; and ascospores with a gelatinous sheath. Molecular phylogenetic analysis of specimens recently collected near the type locality revealed B. akompsa to be nested within the genus Pentagenella in Opegraphaceae. The same placement was found using phenotype-based phylogenetic binning with 37 morphological, anatomical and chemical characters. The new combination Pentagenella akompsa is proposed and the species represents the first North American member of this genus, otherwise known only from Chile and Peru. Therefore, our results reveal the genus Pentagenella to have a remarkably disjunct distribution between North and South America. Several specimens previously labeled as Bacidia, Biatora, or Lecanactis akompsa were found to be misidentifications, narrowing the range of P. akompsa to coastal California, where it is found on two coastal conifer species that are (like P. akompsa itself) narrowly endemic and of conservation concern.

Peltigera globulata Miadl. & Magain, a new species in the P. ponojensis/monticola species complex of section Peltigera, is formally described. This clade was previously given the interim designation Peltigera sp. 17. It is found in sun-exposed and xeric habitats at high altitudes in Peru and Ecuador. Peltigera globulata can be easily recognized by its irregularly globulated margins covered mostly by thick, white pruina, somewhat resembling the sorediate thallus margins of P. soredians, another South American species from section Peltigera. The hypervariable region of ITS1 (ITS1-HR), which is in general highly variable among species of section Peltigera, does not have diagnostic value for species identification within the P. ponojensis/monticola complex. Nevertheless, no significant level of gene flow was detected among eight lineages representing a clade of putative species (including P. globulata) within this complex. ITS sequences from the holotype specimens of P. monticola Vitik. (collected in 1979) and P. soredians Vitik. (collected in 1981) and lectotype specimens of P. antarctica C. W. Dodge (collected in 1941) and P. aubertii C. W. Dodge (collected in 1952) were successfully obtained through Sanger and Illumina metagenomic sequencing. BLAST results of these sequences revealed that the type specimen of P. monticola falls within the P. monticola/ponojensis 7 clade, which represents P. monticola s. str., and confirmed that the type specimen of P. aubertii falls within a clade identified previously as P. aubertii based on morphology. The ITS sequence from the type specimen of P. soredians, which superficially resembles P. globulata, confirms its placement in the P. rufescens clade. Finally, we discovered that the name P. antarctica was erroneously applied to a lineage in the P. ponojensis/monticola clade. The ITS sequence from the type specimen of P. antarctica represents a lineage within the P. rufescens clade, which is sister to the P. ponojensis/monticola clade.

Historical and recent material of Bacidia akompsa (basionym: Biatora akompsa) was studied to determine the taxonomic position of this crustose lichen, prompted by the appearance of the provisional name ‘‘Lecanactis akompsa (Tuck.) ined.’’ in multiple online lichen databases and checklists. Initial morphological assessment of verified material confirmed B. akompsa as a member of Arthoniales but distinct from Lecanactis by having epruinose ascomata with a thin exciple open below the hymenium; a pale, K/Iþ violet to deep blue hypothecium; and ascospores with a gelatinous sheath. Molecular phylogenetic analysis of specimens recently collected near the type locality revealed B. akompsa to be nested within the genus Pentagenella in Opegraphaceae. The same placement was found using phenotype-based phylogenetic binning with 37 morphological, anatomical and chemical characters. The new combination Pentagenella akompsa is proposed and the species represents the first North American member of this genus, otherwise known only from Chile and Peru. Therefore, our results reveal the genus Pentagenella to have a remarkably disjunct distribution between North and South America. Several specimens previously labeled as Bacidia, Biatora, or Lecanactis akompsa were found to be misidentifications, narrowing the range of P. akompsa to coastal California, where it is found on two coastal conifer species that are (like P. akompsa itself) narrowly endemic and of conservation concern.

Lichens of the Ramalina siliquosa complex dominate seashore cliffs in Europe and South-East Asia, but their taxonomy has been vigorously debated for over a century. On many cliffs, they exhibit a bewildering zonation of chemotypes that resembles the classic zonation of organisms that occupy the littoral zone below. Do the chemotypes represent separate species, or infraspecific variation? To better understand the systematics of this group, sequences from four genetic loci (ITS, IGS, RPB1 and RPB2 ) were obtained for 59 samples from Denmark, France, Iceland, Norway, UK, Japan and Korea, including all major chemotypes. Maximum likelihood analysis of these sequences, together with sequences from 36 other Ramalina species, reveals that the complex comprises two distinct phylogenetic lineages, each including multiple chemotypes. These two putative species-level lineages correspond to the currently accepted taxa R. cuspidata and R. siliquosa . There is no evidence that these two taxa are phylogenetic sister species. Consequently, the explanation of this chemotype complex as an example of ‘sibling speciation’ is rejected. Specimens traditionally called ‘ R . siliquosa ’ from South-East Asia form a third clade, identified here as R . semicuspidata , with an additional, divaricatic acid chemotype. Other results include a robustly supported clade of Ramalina species that produce medullary depsides and depsidones; this clade includes another well-supported clade of south-eastern United States coastal plain and tropical Ramalina species. By contrast, large, strap-shaped Ramalina species that lack medullary depsides and depsidones occur in separate lineages. In addition, close relationships between the following groups of species are indicated: R. farinacea with R. subfarinacea ; R . fraxinea with R. leptocarpha , R. menziesii and R. subleptocarpha ; R. sinensis with R. unifolia . Furthermore, a new, variolaric acid-only chemotype is reported for R . farinacea , and a new, acid-deficient chemotype is reported for a more broadly circumscribed R. culbersoniorum .

Two members of the Lecanora varia group (sensu Eigler), L. strobilina and L. symmicta, can be difficult to distinguish in northeastern North America. Morphological and chemical investigation of 277 specimens recognizes two species in northeastern North America, both fitting European concepts of L. strobilina and L. symmicta. The only reliable character for separating them is the size of the exciple (consistently larger in L. symmicta). Within each of these two species, previously unknown chemical variation is revealed—L. strobilina comprises three distinct chemotypes, while L. symmicta includes two. An isotype of L. symmictera is shown to contain the thiophanic acid chemosyndrome by HPLC, and hence the name L. symmictera cannot be applied to the xanthone-deficient chemotype of L. symmicta. Chemistry is not correlated with morphology or geographic distribution in either L. strobilina or L. symmicta, so we regard the chemotypes of these two species as intraspecific variation.

The nature and significance of morphologically indistinguishable chemical races have been active areas of lichenological research for decades. This study uses a phylogenetic analysis of rDNA sequences to investigate whether the morphologically indistinguishable chemotypes of Ramalina americana are distinct species. Cladistic analysis of ITS sequences from 19 R. americana individuals (representing eight of the nine described chemotypes) and four outgroup Ramalina species reveals that R. americana comprises at least two cryptic, phylogenetic species. One, R. americana, is largely acid-deficient and occurs in the northern half of the range of the complex (and at high elevations in the Southern Appalachian Mountains); the other, R. culbersoniorum sp. nov., encompasses most of the chemical diversity and occurs in the southern half of the range. There is no meaningful resolution within either species. Among the outgroup Ramalina species, R. intermedia appears to be sister to the northern species, while R. fastigiata, the presumed sister group to the complex, forms an unresolved trichotomy with both the northern and southern species. Ramalina sinensis, often assumed to be closely related to the complex, is only distantly related. The results of this study agree with previous studies of other lichens in that each chemotype does not represent a separate species, nor are they all conspecific. Rather, the complex can be divided into two species, each consisting of multiple chemical races.