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The northern North American Cordillera is a globally significant center of endemism. In western North America, imperiled arid steppe habitats support a number of unique species, including several endemic lichens. However, processes driving diversification and endemism in this region remain unclear. In this study, we investigate diversity and phylogeography of the threatened wanderlust lichens (mycobiont = Rhizoplaca species) which occur unattached on calcareous soils in steppe habitats. Wanderlust lichens comprise three species of lichen-forming fungi (LFF) - Rhizoplaca arbuscula, R. haydenii, and R. idahoensis (endangered, IUCN Red List) - which occur in fragmented populations in Idaho and Wyoming, with more limited populations in southern Montana and northern Utah. These lichens reproduce almost exclusively via large, asexual vegetative propagules. Here, our aims were to (i) assess the evolutionary origin of this group and identify phylogeographic structure, (ii) infer ancestral geographic distributions for lineages within this clade, and (iii) use species distribution modeling to better understand the distribution of contemporary populations. Using a genome-skimming approach, we generated a 19.1Mb alignment, spanning ca. half of the complete LFF genome, from specimens collected throughout the entire range of wanderlust lichens. Based on this phylogeny, we investigated phylogeographic patterns using RASP. Finally, we used MaxEnt to estimate species distribution models for R. arbuscula and R. haydenii. We inferred a highly structured topology, with clades corresponding to distinct geographic regions and morphologies collected throughout the group's distribution. We found that R. robusta, a sexually reproducing taxon, is clearly nested within the vagrant Rhizoplaca clade. Phylogeographic analyses suggest that both dispersal and vicariance played significant roles throughout the evolutionary history of the vagrant Rhizoplaca clade, with most of the dispersal events originating from the Salmon Basin in eastern Idaho - the center of diversity for this group. Despite the fact that wanderlust lichens are dispersal limited due to large, unspecialized vegetative propagules, we inferred multiple dispersal events crossing the Continental Divide. Comparing herbarium records with species distribution models suggests that wanderlust lichens don't fully occupy the areas of highest distribution probability. In fact, documented records often occur in areas predicted to be only marginally suitable. These data suggest a potential mismatch between contemporary habitats outside of the center of diversity in eastern Idaho with the most suitable habitat, adding to the vulnerability of this imperiled complex of endemic lichens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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.

Thirty years after its designation as a federally endangered species, the Florida Perforate Cladonia (FPC) remains imperiled in isolated populations in the Florida scrub in the southeastern USA. For threatened and endangered species, such as FPC, reference genomes provide critical insight into genomic diversity, local adaptations, landscape-level genetics, and phylogenomics. Using high-throughput sequencing, we assemble the first draft nuclear and mitochondrial genomes for the FPC mycobiont- Cladonia perforata . We also assess genetic diversity within and among populations in southeastern Florida using genome-scale data and investigate diversity across the entire nuclear ribosomal cistron, including the standard DNA barcoding marker for fungi. The draft nuclear genome spanned 33.6 Mb, and the complete, circular mitochondrial genome was 59 Kb. We also generated the first chloroplast genome, to our knowledge, for the photobiont genus associated with FPC, an undescribed Asterochloris species. We inferred the presence of multiple, distinct mycobiont parental genotypes (genets) occurring at local scales in southeastern Florida, and strikingly, no genets were shared among even the closest sample sites. All sampled thalli shared identical mitochondrial genomes, while the nuclear ribosomal cistron showed limited variability-highlighting the genetic resolution provided by nuclear genome-scale datasets. The genomic resources generated here provide critical resources for informed conservation efforts for the FPC.

In this study, two new species, Peltula confusa Q.X. Yang & X.L. Wei, sp. nov., growing in a dry microenvironment within a semi-humid area, and Peltula subpatellata Q.X. Yang & X.L. Wei, sp. nov., occurring in arid and semi-arid regions, are described. In addition, two species, P. polyspora (Tuck.) Wetmore and P. obscuratula (Nyl.) Poelt ex Egea, are recorded for the first time in China. All four species are described based on morphological, anatomical and molecular data. Peltula confusa is characterized by a lighter, brighter, and more yellowish upper surface than other species of this genus, with discs concentrated in the central part of squamules, and a thick lower cortex. Peltula subpatellata can be distinguished from P. patellata (Bagl.) Swinscow & Krog by its non-thickened and sometimes darkened margins and sometimes the presence of peltate squamules. Phylogenetic analysis based on DNA sequences of four loci (ITS, nrSSU, nrLSU, and RPB2) demonstrates the placement of these species within Peltula , and supports current species delimitations. We suggest that the growth substrate should be considered as an additional characteristic for species delimitation.

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Lichens are well-known examples of complex symbiotic associations between organisms from different Kingdoms. Microfungi in particular, establish diverse associations with the hosting lichen thallus, as species-specific parasites or transient co-inhabitants. The whole community of lichen-associated fungi constitute the 'lichen mycobiome' comprising both ascomycetes and basidiomycetes, including filamentous and yeast taxa. Metabarcoding results and microscopy analyses show that in some thalli, basidiomycetes are frequent lichen-associated fungi but still only a few species could be axenically isolated and morphologically characterized. Within a broad project aiming at characterizing the mycobiome diversity by culture-dependent and independent approaches in two lichen species selected as reference models - Rhizoplaca melanophthalma and Tephromela atra, we succeed in isolating and culturing 76 new strains of basidiomycetous yeasts. The lichen thalli were collected in different mountain regions worldwide and at relatively high elevation. The yeast strains were isolated on different growth media and were studied for their morphological and genetic diversity. Nuclear internal transcribed spacer (ITS) and ribosomal large subunit (LSU) sequence analyses identified them to belong to ten families within the orders Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Tremellomycetes and Ustilaginomycetes. The yeasts here detected showed patterns of host-preference in a few cases and they are potentially related to the ecological conditions., Competing Interests: Declaration of competing interest None., (Copyright © 2022 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

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.

Fungal-algal relationships-both across evolutionary and ecological scales-are finely modulated by the presence of the symbionts in the environments and by the degree of selectivity and specificity that either symbiont develop reciprocally. In lichens, the green algal genus Trebouxia Puymaly is one of the most frequently recovered chlorobionts. Trebouxia species-level lineages have been recognized on the basis of their morphological and phylogenetic diversity, while their ecological preferences and distribution are still only partially unknown. We selected two cosmopolitan species complexes of lichen-forming fungi as reference models, i.e., Rhizoplaca melanophthalma and Tephromela atra , to investigate the diversity of their associated Trebouxia spp. in montane habitats across their distributional range worldwide. The greatest diversity of Trebouxia species-level lineages was recovered in the altitudinal range 1,000-2,500 m a.s.l. A total of 10 distinct Trebouxia species-level lineages were found to associate with either mycobiont, for which new photobionts are reported. One previously unrecognized Trebouxia species-level lineage was identified and is here provisionally named Trebouxia "A52." Analyses of cell morphology and ultrastructure were performed on axenically isolated strains to fully characterize the new Trebouxia "A52" and three other previously recognized lineages, i.e., Trebouxia "A02," T. vagua "A04," and T. vagua "A10," which were successfully isolated in culture during this study. The species-level diversity of Trebouxia associating with the two lichen-forming fungi in extreme habitats helps elucidate the evolutionary pathways that this lichen photobiont genus traversed to occupy varied climatic and vegetative regimes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Carolis, Cometto, Moya, Barreno, Grube, Tretiach, Leavitt and Muggia.)

Lichen associations are overwhelmingly supported by carbon produced by photosynthetic algal symbionts. These algae have diversified to occupy nearly all climates and continents; however, we have a limited understanding of how their climatic niches have evolved through time. Here we extend previous work and ask whether phylogenetic signal in, and the evolution of, climatic niche, varies across climatic variables, phylogenetic scales, and among algal lineages in Trebouxia- the most common genus of lichen-forming algae. Our analyses reveal heterogeneous levels of phylogenetic signal across variables, and that contrasting models of evolution underlie the evolution of climatic niche divergence. Together these analyses demonstrate the variable processes responsible for shaping climatic tolerance in Trebouxia , and provide a framework within which to better understand potential responses to climate change-associated perturbations. Such predictions reveal a disturbing trend in which the pace at which modern climate change is proceeding will vastly exceed the rate at which Trebouxia climatic niches have previously evolved., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nelsen, Leavitt, Heller, Muggia and Lumbsch.)

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., Competing Interests: The authors declare that they have no conflict of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)