No abstract

We present an exhaustive analysis of the ITS barcoding marker in the genus Usnea s.lat., separated into Dolichousnea, Eumitria, and Usnea including the subgenus Neuropogon, analyzing 1,751 accessions. We found only a few low-quality accessions, whereas information on voucher specimens and accuracy and precision of identifications was of subpar quality for many accessions. We provide an updated voucher table, alignment and phylogenetic tree to facilitate DNA barcoding of Usnea, either locally or through curated databases such as UNITE. Taxonomic and geographic coverage was moderate: while Dolichousnea and subgenus Neuropogon were well-represented among ITS data, sampling for Eumitria and Usnea s.str. was sparse and biased towards certain lineages and geographic regions, such as Antarctica, Europe, and South America. North America, Africa, Asia and Oceania were undersampled. A peculiar situation arose with New Zealand, represented by a large amount of ITS accessions from across both major islands, but most of them left unidentified. The species pair Usnea antarctica vs. U. aurantiacoatra was the most sampled clade, including numerous ITS accessions from taxonomic and ecological studies. However, published analyses of highly resolved microsatellite and RADseq markers showed that ITS was not able to properly resolve the two species present in this complex. While lack of resolution appears to be an issue with ITS in recently evolving species complexes, we did not find evidence for gene duplication (paralogs) or hybridization for this marker. Comparison with other markers demonstrated that particularly IGS and RPB1 are useful to complement ITS-based phylogenies. Both IGS and RPB1 provided better backbone resolution and support than ITS; while IGS also showed better resolution and support at species level, RPB1 was less resolved and delineated for larger species complexes. The nuLSU was of limited use, providing neither resolution nor backbone support. The other three commonly employed protein-coding markers, TUB2, RPB2, and MCM7, showed variable evidence of possible gene duplication and paralog formation, particularly in the MCM7, and these markers should be used with care, especially in multimarker coalescence approaches. A substantial challenge was provided by difficult morphospecies that did not form coherent clades with ITS or other markers, suggesting various levels of cryptic speciation, the most notorious example being the U. cornuta complex. In these cases, the available data suggest that multimarker approaches using ITS, IGS and RPB1 help to assess distinct lineages. Overall, ITS was found to be a good first approximation to assess species delimitation and recognition in Usnea s.lat., as long as the data are carefully analyzed, and reference sequences are critically assessed and not taken at face value. In difficult groups, we recommend IGS as a secondary barcode marker, with the option to employ more resource-intensive approaches, such as RADseq, in species complexes involving so-called species pairs or other cases of disparate morphology not reflected in the ITS or IGS. Attempts should be made to close taxonomic and geographic gaps especially for the latter two markers, in particular in Eumitria and Usnea s.str. and in the highly diverse areas of North America and Central America, Africa, Asia, and Oceania.

Seventeen corticolous shrubby apotheciate Usnea species without vegetative propagules are reported from Brazil, including five species that are new to science: Usnea aurantiaca-parvula A. Gerlach & P. Clerc (characterized by an orange medulla and lageniform spinulose fibrils), U. cirrosa Motyka, U. cladocarpa Fée (syn. nov.: U. ramillosa Motyka), U. concinna Stirton (lectotype designated here, syn. nov. U. radiata Stirton, U. florida var. scabrosa Zahlbr.), U. cristatula Motyka, U. erinacea Vain., U. fleigiae A. Gerlach & P. Clerc (characterized by large spores and a thin, lax medulla), U. grandispora A. Gerlach & P. Clerc (characterized by large spores, a black base and protocetraric or salazinic acids in the medulla), U. kalbiana P. Clerc & A. Gerlach (characterized by a vitreous cortex and annular cracks in the basal part), U. lunaria Motyka, U. meridionalis Zahlbr. (syn. nov.: U. michauxii I. I. Tav.), Usnea cf. moreliana Motyka, U. parvula Motyka, U. steineri Zahlbr, U. subelegans (Vain.) B. de Lesd. (lectotype designated here), U. subparvula A. Gerlach & P. Clerc (characterized by spinulose fibrils and protocetraric acid in the medulla) and one as yet unidentified species (named Usnea sp. 1). Usnea cirrosa, U. cristatula and U. erinacea are new records for Brazil. A full description with morphological, anatomical (CMA and ascospores) and chemical features (TLC), as well as geographical distribution, is provided for each species along with an identification key to all species reported. Molecular data from the ITS rDNA, RPB1 and Mcm7 markers are present for most taxa, except for U. concinna, U. cristatula, U. kalbiana, U. lunaria, U. cf. moreliana and U. subelegans.

Presence and identity of secondary metabolites are two of the main components of lichen taxonomy. Aromatic compounds formed via the acetyl-polymalonate pathway are the most studied lichen substances. In addition, compounds derived from the mevalonic acid pathway (e.g. terpenes and steroids) are sometimes detected in the medulla. However, their identity and value as diagnostic characters in the genus Usnea are yet poorly understood despite the fact that they were mentioned in several taxonomical papers. We conclude that i) aside from the previously recognized polyphenolic compounds, carbohydrates and steroids are also detected in the medulla of some Usnea species; ii) the use of sulfuric anisaldehyde reagent greatly improves the detection of terpenes, carbohydrates and steroids compared with the sulfuric acid reagent routinely used in thin layer chromatography; iii) among carbohydrates, we detected arabitol and sucrose in the medulla; iv) steroids and terpenes remain unidentified and deserve further investigations.

This study is a reassessment of the Usnea cornuta aggregate diversity mainly in Brazil. Based on previous multi-locus species delimitation analyses using the multispecies coalescent model (MSC), we carried out a posteriori morphological and anatomical studies. The following corticolous new species are described: Usnea arianae with norstictic or fatty or lobaric acids, convex and efflorescent soralia and a lax medulla; U. flabelliformis with protocetraric acid, fan-shaped main branches and a high medulla/cortex ratio; U. rubropallens with protocetraric acid and a faint orange cortical/subcortical pigment; U. stipitata with constictic acid, stipitate soralia and a high medulla/cortex ratio; and U. tenuicorticata with protocetraric acid, a thin cortex and a high medulla/cortex ratio. The newly described species were found to occur so far only in the Americas, except U. arianae which shows an amphi-atlantic distribution in Europe and on the American continent. Seven species already described belonging to the U. cornuta aggr. in Brazil are further treated here: Usnea boomiana with caperatic acid and large, concave soralia, new to South America; U. brasiliensis with protocetraric acid, minute and irregular soralia; U. cornuta with mainly salazinic acid and minute soralia fusing into consoralia; U. macaronesica (syn. nov.: U. subglabrata) with barbatic acid, large excavate soralia and a lax medulla; Usnea subpectinata, a so far european species resurrected from the U. cornuta synonymy, with stictic acid and numerous isidiofibrils, new to South America; and U. trachyclada with thamnolic acid and K+ bright yellow stipitate soralia. Full descriptions with morphological, anatomical and chemical features, geographical distributions, and illustrations are provided for each species along with an identification key.