Species boundaries in the messy middle-A genome-scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi.

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.

Prioritizing natural product compounds using 1D-TOCSY NMR spectroscopy.

Natural product (NP) secondary metabolites are designed evolutionarily to have biological effects in other organisms for defense and the mediation of ecological interactions. Their structural complexity and diversity complement biological systems, allowing them to display unique bioactivities. Although more than half of all pharmaceuticals stem from NPs, pharmaceutical companies have reduced NP-based drug discovery programs due to various time and cost-consuming pitfalls; the re-isolation of already known, bioactive compounds being one of the most common. Dereplication methods minimize cost and speed up the discovery of new, bioactive leads by quickly identifying known small molecules. Liquid chromatography coupled mass spectrometry (LC-MS) is the most widely utilized dereplication technique because of its sensitivity and the open-source availability of MS libraries. However, single-ionization techniques are not able to detect all metabolites in a biological sample. Even more concerning, bioactive isomers cannot be differentiated by their mass alone. In response to these issues, complementary dereplication tools are needed to assist MS. Total correlation spectroscopy (TOCSY) is an NMR experiment that illustrates the connection between all coupled protons in a spin system. Most molecules contain several spin systems, and together, these networks form a unique fingerprint that can be utilized to quickly differentiate and dereplicate known compounds, even those with identical masses. In addition, these fingerprints can be used to identify possible new compounds in a crude NP-extract that are structurally related to known small molecules. From a sample of the U.S. endemic lichen Niebla homalea, five non-cytotoxic, new triterpenoids and three known triterpenoids were isolated in our laboratory. As our goal is to discover both new and cytotoxic compounds, we developed a one-dimensional TOCSY-based dereplication method to quickly identify these non-bioactive triterpenoids. After prioritizing triterpenoid-free fractions that showed antiproliferative activity in various cancer cell lines, the new compound 11 was isolated from another Niebla species.

Corrigendum: Spjut R, Simon A, Guissard M, Magain N, Sérusiaux E (2020) The fruticose genera in the Ramalinaceae (Ascomycota, Lecanoromycetes): their diversity and evolutionary history. MycoKeys 73: 1-68. https://doi.org/10.3897/mycokeys.73.47287.

[This corrects the article DOI: 10.3897/mycokeys.73.47287.].

Specialized metabolites of the United States lichen Niebla homalea and their antiproliferative activities.

Three undescribed stictanes, nieblastictanes A-C, two flavicanes, nieblaflavicanes A and B, together with three already reported stictanes, along with the known compounds (+)-usnic acid, sekikaic acid, divaricatic acid, and divaricatinic acid methyl ester were isolated from an ethyl acetate extract of the western North American lichen Niebla homalea. The structures of the new and known compounds were established by spectroscopic methods including nuclear magnetic resonance spectroscopy, mass spectrometry and electronic circular dichroism. Among the compounds isolated, usnic acid exhibited moderately potent antiproliferative activities against the A2780 ovarian (IC50 3.8 µM) and MCF-7 breast cancer (IC50 6.8 µM) cell lines. A plausible mode of formation of the chlorine-containing compound nieblastictane C is provided and the contribution of the isolated compounds to the chemotaxonomy of United States lichen species of the genus Niebla is also discussed.

The fruticose genera in the Ramalinaceae (Ascomycota, Lecanoromycetes): their diversity and evolutionary history.

We present phylogenetic analyses of the fruticose Ramalinaceae based on extensive collections from many parts of the world, with a special focus on the Vizcaíno deserts in north-western Mexico and the coastal desert in Namibia. We generate a four-locus DNA sequence dataset for accessions of Ramalina and two additional loci for Niebla and Vermilacinia. Four genera are strongly supported: the subcosmopolitan Ramalina, the new genus Namibialina endemic to SW Africa, and a duo formed by Niebla and Vermilacinia, endemic to the New World except the sorediate V. zebrina that disjunctly occurs in Namibia. The latter three genera are restricted to coastal desert and chaparral where vegetation depends on moisture from ocean fog. Ramalina is subcosmopolitan and much more diverse in its ecology. We show that Ramalina and its sister genus Namibialina diverged from each other at c. 48 Myrs, whereas Vermilacinia and Niebla split at c. 30 Myrs. The phylogeny of the fruticose genera remains unresolved to their ancestral crustose genera. Species delimitation within Namibialina and Ramalina is rather straightforward. The phylogeny and taxonomy of Vermilacinia are fully resolved, except for the two youngest clades of corticolous taxa, and support current taxonomy, including four new taxa described here. Secondary metabolite variation in Niebla generally coincides with major clades which are comprised of species complexes with still unresolved phylogenetic relationships. A micro-endemism pattern of allopatric species is strongly suspected for both genera, except for the corticolous taxa within Vermilacinia. Both Niebla and saxicolous Vermilacinia have chemotypes unique to species clades that are largely endemic to the Vizcaíno deserts. The following new taxa are described: Namibialina gen. nov. with N. melanothrix (comb. nov.) as type species, a single new species of Ramalina (R. krogiae) and four new species of Vermilacinia (V. breviloba, V. lacunosa, V. pustulata and V. reticulata). The new combination V. granulans is introduced. Two epithets are re-introduced for European Ramalina species: R. crispans (= R. peruviana auct. eur.) and R. rosacea (= R. bourgeana auct. p.p). A lectotype is designated for Vermilacinia procera. A key to saxicolous species of Vermilacinia is presented.

α-Pyrone and Sterol Constituents of Penicillium aurantiacobrunneum, a Fungal Associate of the Lichen Niebla homalea.

Four new metabolites, 4-epi-citreoviridin (1), auransterol (3), and two analogues (2 and 4) of paxisterol (6), together with two known metabolites (15R*,20S*)-dihydroxyepisterol (5) and (6), were isolated from cultures of the fungal associate, Penicillium aurantiacobrunneum, of the lichen Niebla homalea, endemic to California and Baja California. The structures of all compounds were determined by comprehensive spectroscopic and spectrometric methods, as well as single-crystal X-ray diffraction for the determination of the absolute configuration of 3. Compound 1 showed selective cytotoxicity toward MCF-7 breast and A2780 ovarian cells with IC50 values of 4.2 and 5.7 µM, respectively.