Two new species of Rhizoplaca (Lecanoraceae) from Southwest China.

In this study, two new species, Rhizoplacaadpressa Y. Y. Zhang & Li S. Wang and R.auriculata Y. Y. Zhang, Li S. Wang & Printzen, are described from Southwest China, based on their morphology, phylogeny and chemistry. In phylogeny, the two new species are monophyletic, and sister to each other within Rhizoplacachrysoleuca-complex. Rhizoplacaadpressa is characterized by its placodioid and closely adnate thallus, pale green and heavily pruinose upper surface, narrow (ca. 1 mm) and white free margin on the lower surface of marginal squamules, the absence of a lower cortex, and its basally non-constricted apothecia with orange discs that turn reddish-brown at maturity. Rhizoplacaauriculata is characterized by its squamulose to placodioid thallus, yellowish green and marginally pruinose squamules, wide (1-3 mm) and bluish-black free margin on the lower surface of marginal squamules, the absence of a lower cortex, and its basally constricted apothecia with persistently orange discs. Rhizoplacaadpressa and R.auriculata share the same secondary metabolites of usnic and placodiolic acids.

The phylogeny and taxonomy of Upretia (Caloplacoideae, Teloschistaceae), reveal three new species from Southwestern China.

Several specimens of Upretia from Southwest China are morphologically and phylogenetically distinct from currently recognized species in the genus. These specimens are here accommodated within a new species, Upretiazeorina Li J. Li & Printzen. It is characterized by an areolate to squamulose thallus with brown to blackish brown upper surface, pruinose, zeorine type apothecia, black discs, narrowly bacilliform conidia, and the production of gyrophoric acid. Two other specimens of Upretia from China are distinct from currently accepted species and tentatively referred to as Upretia sp. 1 and Upretia sp. 2. A key to all known species of Upretia is also provided.

Phylogeny, morphology and chemistry reveal two new multispored species in the Lecanorasubfusca group (Lecanoraceae, Ascomycota).

Two new multispored species from China, Lecanoraanhuiensis Li J. Li & Printzen, sp. nov. and Lecanorapseudojaponica Li J. Li & Printzen, sp. nov. are described and illustrated here, based on morphological, chemical and molecular evidence. Lecanoraanhuiensis is characterised by an epruinose, yellowish-brown to deep brown apothecial disc, an epihymenium with fine crystals, an amphithecium with small crystals, 16-spored asci and the presence of zeorin, in addition to atranorin. Lecanorapseudojaponica is characterised by an epruinose, red-brown apothecial disc, an epihymenium without crystals, an amphithecium with small crystals, 8 or 16- spored asci and the presence of zeorin and the stictic acid complex, in addition to atranorin. Phylogenetic reconstructions, based on mtSSU, nrITS and nrLSU suggest that these two species are members of the Lecanorasubfusca group. They are compared with morphologically similar and phylogenetically related species, based on a nrITS dataset. Phylogenetic results show that the multispored taxa of Lecanora are polyphyletic. The number of ascospores per ascus appears to be a taxonomic character of minor importance. Detailed descriptions, discussions and figures for the two new species from China and a key for the multispored species of Lecanora worldwide are provided.

Lichen speciation is sparked by a substrate requirement shift and reproduction mode differentiation.

We show that obligate lignicoles in lichenized Micarea are predominately asexual whereas most facultative lignicoles reproduce sexually. Our phylogenetic analyses (ITS, mtSSU, Mcm7) together with ancestral state reconstruction show that the shift in reproduction mode has evolved independently several times within the group and that facultative and obligate lignicoles are sister species. The analyses support the assumption that the ancestor of these species was a facultative lignicole. We hypothezise that a shift in substrate requirement from bark to wood leads to differentiation in reproduction mode and becomes a driver of speciation. This is the first example of lichenized fungi where reproduction mode is connected to substrate requirement. This is also the first example where such an association is demonstrated to spark lichen speciation. Our main hypothesis is that obligate species on dead wood need to colonize new suitable substrata relatively fast and asexual reproduction is more effective a strategy for successful colonization.

Squamarina (lichenised fungi) species described from China belong to at least three unrelated genera.

New collections of six Squamarina species from type localities in China were studied. The comparison of morphological characteristics and secondary metabolites with those of the type specimens and phylogenetic analyses suggest that S. callichroa and S. pachyphylla belong to Rhizoplaca, S. semisterilis belongs to Lobothallia and S. chondroderma should be retained in Lecanora temporarily. Only two species, S. kansuensis and S. oleosa, remain in Squamarina. The new combinations Lobothallia semisterilis (H. Magn.) Y. Y. Zhang, Rhizoplaca callichroa (Zahlbr.) Y. Y. Zhang and R. pachyphylla (H. Magn.) Y. Y. Zhang are proposed. Detailed descriptions to aid the identification of these species, distributions and phylogenetic trees, based on multiple collections, are presented. The generic concept of Squamarina is recircumscribed in this study.

Repositories for Taxonomic Data: Where We Are and What is Missing.

Natural history collections are leading successful large-scale projects of specimen digitization (images, metadata, DNA barcodes), thereby transforming taxonomy into a big data science. Yet, little effort has been directed towards safeguarding and subsequently mobilizing the considerable amount of original data generated during the process of naming 15,000-20,000 species every year. From the perspective of alpha-taxonomists, we provide a review of the properties and diversity of taxonomic data, assess their volume and use, and establish criteria for optimizing data repositories. We surveyed 4113 alpha-taxonomic studies in representative journals for 2002, 2010, and 2018, and found an increasing yet comparatively limited use of molecular data in species diagnosis and description. In 2018, of the 2661 papers published in specialized taxonomic journals, molecular data were widely used in mycology (94%), regularly in vertebrates (53%), but rarely in botany (15%) and entomology (10%). Images play an important role in taxonomic research on all taxa, with photographs used in >80% and drawings in 58% of the surveyed papers. The use of omics (high-throughput) approaches or 3D documentation is still rare. Improved archiving strategies for metabarcoding consensus reads, genome and transcriptome assemblies, and chemical and metabolomic data could help to mobilize the wealth of high-throughput data for alpha-taxonomy. Because long-term-ideally perpetual-data storage is of particular importance for taxonomy, energy footprint reduction via less storage-demanding formats is a priority if their information content suffices for the purpose of taxonomic studies. Whereas taxonomic assignments are quasifacts for most biological disciplines, they remain hypotheses pertaining to evolutionary relatedness of individuals for alpha-taxonomy. For this reason, an improved reuse of taxonomic data, including machine-learning-based species identification and delimitation pipelines, requires a cyberspecimen approach-linking data via unique specimen identifiers, and thereby making them findable, accessible, interoperable, and reusable for taxonomic research. This poses both qualitative challenges to adapt the existing infrastructure of data centers to a specimen-centered concept and quantitative challenges to host and connect an estimated $ \le $2 million images produced per year by alpha-taxonomic studies, plus many millions of images from digitization campaigns. Of the 30,000-40,000 taxonomists globally, many are thought to be nonprofessionals, and capturing the data for online storage and reuse therefore requires low-complexity submission workflows and cost-free repository use. Expert taxonomists are the main stakeholders able to identify and formalize the needs of the discipline; their expertise is needed to implement the envisioned virtual collections of cyberspecimens. [Big data; cyberspecimen; new species; omics; repositories; specimen identifier; taxonomy; taxonomic data.].

Population genomic analyses of RAD sequences resolves the phylogenetic relationship of the lichen-forming fungal species Usneaantarctica and Usneaaurantiacoatra.

Neuropogonoid species in the lichen-forming fungal genus Usnea exhibit great morphological variation that can be misleading for delimitation of species. We specifically focused on the species delimitation of two closely-related, predominantly Antarctic species differing in the reproductive mode and representing a so-called species pair: the asexual U.antarctica and the sexual U.aurantiacoatra. Previous studies have revealed contradicting results. While multi-locus studies based on DNA sequence data provided evidence that these two taxa might be conspecific, microsatellite data suggested they represent distinct lineages. By using RADseq, we generated thousands of homologous markers to build a robust phylogeny of the two species. Furthermore, we successfully implemented these data in fine-scale population genomic analyses such as DAPC and fineRADstructure. Both Usnea species are readily delimited in phylogenetic inferences and, therefore, the hypothesis that both species are conspecific was rejected. Population genomic analyses also strongly confirmed separated genomes and, additionally, showed different levels of co-ancestry and substructure within each species. Lower co-ancestry in the asexual U.antarctica than in the sexual U.aurantiacoatra may be derived from a wider distributional range of the former species. Our results demonstrate the utility of this RADseq method in tracing population dynamics of lichens in future analyses.

The use of microsatellite markers for species delimitation in Antarctic Usnea subgenus Neuropogon.

Lichens are symbiotic associations consisting of a fungal (mycobiont) and one or more photosynthetic (photobionts) partners and are the dominant component, and most important primary producers, of Antarctic terrestrial ecosystems. The most common lichens in the maritime Antarctic are Usnea antarctica and U. aurantiacoatra, a so-called "species pair" in which U. antarctica shows asexual reproduction and propagation via soredia and U. aurantiacoatra forms ascospores in apothecia. Previous molecular analyses were not able to unambiguously distinguish the two morphotypes as species. Therefore, the goal of this study was to find out whether fast-evolving SSR (single sequence repeat) markers are able to separate morphotypes more clearly and help to clarify their taxonomy. We investigate 190 individuals from five mixed stands of both morphotypes collected in King George Island and Elephant Island (South Shetland Islands, Antarctica). Based on 23 microsatellite markers designed from sequenced genomes, discriminant analysis of principal components (DAPC), Bayesian clustering analysis, and coalescent-based estimation of gene flow show clear evidence for the existence of two different species distinguishable by reproductive mode. We did not detect any statistical association between genetic clusters and three previously reported chemical races of each species.

Fungus-specific SSR markers in the Antarctic lichens Usnea antarctica and U. aurantiacoatra (Parmeliaceae, Ascomycota).

PREMISE OF THE STUDY: Usnea antarctica and U. aurantiacoatra (Parmeliaceae) are common lichens in the maritime Antarctic. These species share the same habitats on King George Island (South Shetland Islands, Antarctica) and are distinguishable based on reproductive strategies. METHODS AND RESULTS: We developed 23 fungus-specific simple sequence repeat (SSR) markers that cross-amplify between the two species. We used a low-coverage genome-skimming approach on one sample of each species to identify SSR repeats in the two species. Primers were designed for 3-4-bp repeats, and only the loci common to both species were selected for further analyses. Seventy-seven samples of the two species were selected to assess fungal specificity, genetic variability, and linkage of the markers. In addition, we tested cross-amplification in other Usnea species. CONCLUSIONS: The 23 newly designed SSR markers are suitable for population genetic and phylogeographic studies of Usnea species.

Characterization of microsatellite loci in the lichen-forming fungus Cetraria aculeata (Parmeliaceae, Ascomycota).

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed for the lichen species Cetraria aculeata (Parmeliaceae) to study fine-scale population diversity and phylogeographic structure. METHODS AND RESULTS: Using Illumina HiSeq and MiSeq, 15 fungus-specific microsatellite markers were developed and tested on 81 specimens from four populations from Spain. The number of alleles ranged from four to 13 alleles per locus with a mean of 7.9, and average gene diversities varied from 0.40 to 0.73 over four populations. The amplification rates of 10 markers (CA01-CA10) in populations of C. aculeata exceeded 85%. The markers also amplified across a range of closely related species, except for locus CA05, which did not amplify in C. australiensis and C. "panamericana," and locus CA10 which did not amplify in C. australiensis. CONCLUSIONS: The identified microsatellite markers will be used to study the genetic diversity and phylogeographic structure in populations of C. aculeata in western Eurasia.

Diagnostics for a troubled backbone: testing topological hypotheses of trapelioid lichenized fungi in a large-scale phylogeny of Ostropomycetidae (Lecanoromycetes).

Trapelioid fungi constitute a widespread group of mostly crust-forming lichen mycobionts that are key to understanding the early evolutionary splits in the Ostropomycetidae, the second-most species-rich subclass of lichenized Ascomycota. The uncertain phylogenetic resolution of the approximately 170 species referred to this group contributes to a poorly resolved backbone for the entire subclass. Based on a data set including 657 newly generated sequences from four ribosomal and four protein-coding gene loci, we tested a series of a priori and new evolutionary hypotheses regarding the relationships of trapelioid clades within Ostropomycetidae. We found strong support for a monophyletic group of nine core trapelioid genera but no statistical support to reject the long-standing hypothesis that trapelioid genera are sister to Baeomycetaceae or Hymeneliaceae. However, we can reject a sister group relationship to Ostropales with high confidence. Our data also shed light on several long-standing questions, recovering Anamylopsoraceae nested within Baeomycetaceae, elucidating two major monophyletic groups within trapelioids (recognized here as Trapeliaceae and Xylographaceae), and rejecting the monophyly of the genus Rimularia. We transfer eleven species of the latter genus to Lambiella and describe the genus Parainoa to accommodate a previously misunderstood species of Trapeliopsis. Past phylogenetic studies in Ostropomycetidae have invoked "divergence order" for drawing taxonomic conclusions on higher level taxa. Our data show that if backbone support is lacking, contrasting solutions may be recovered with different or added data. We accordingly urge caution in concluding evolutionary relationships from unresolved phylogenies.

Pleistocene expansion of the bipolar lichen Cetraria aculeata into the Southern hemisphere.

Many boreal and polar lichens occupy bipolar distributional ranges that frequently extend into high mountains at lower latitudes. Although such disjunctions are more common among lichens than in other groups of organisms, the geographic origin of bipolar lichen taxa, and the way and time frame in which they colonized their ranges have not been studied in detail. We used the predominantly vegetative, widespread lichen Cetraria aculeata as a model species. We surveyed the origin and history of its bipolar pattern using population genetics, phylogenetic and genealogical reconstruction methods. Cetraria aculeata originated in the Northern Hemisphere and dispersed southwards during the Pleistocene. The genetic signal suggests a Pleistocene dispersive burst in which a population size expansion concurred with the acquisition of a South-American range that culminated in the colonization of the Antarctic.

Alphaproteobacterial communities in geographically distant populations of the lichen Cetraria aculeata.

Lichen symbioses were recently shown to include diverse bacterial communities. Although the biogeography of lichen species is fairly well known, the patterns of their bacterial associates are relatively poorly understood. Here we analyse the composition of Alphaproteobacteria in Cetraria aculeata, a common lichen species that occurs at high latitudes and various habitats. Using clone libraries we show that most of the associated Alphaproteobacteria belong to Acetobacteraceae, which have also been found previously in other lichen species of acidic soils and rocks in alpine habitats. The majority of alphaproteobacterial sequences from C. aculeata are very similar to each other and form a single clade. Data from C. aculeata reveal that alphaproteobacterial communities of high latitudes are depauperate and more closely related to each other than to those of extrapolar habitats. This agrees with previous findings for the fungal and algal symbiont in this lichen. Similar to the algal partner, the composition of lichen alphaproteobacterial communities is affected by environmental parameters.

Extreme phenotypic variation in Cetraria aculeata (lichenized Ascomycota): adaptation or incidental modification?

BACKGROUND AND AIMS: Phenotypic variability is a successful strategy in lichens for colonizing different habitats. Vagrancy has been reported as a specific adaptation for lichens living in steppe habitats around the world. Among the facultatively vagrant species, the cosmopolitan Cetraria aculeata apparently forms extremely modified vagrant thalli in steppe habitats of Central Spain. The aim of this study was to investigate whether these changes are phenotypic plasticity (a single genotype producing different phenotypes), by characterizing the anatomical and ultrastructural changes observed in vagrant morphs, and measuring differences in ecophysiological performance. METHODS: Specimens of vagrant and attached populations of C. aculeata were collected on the steppes of Central Spain. The fungal internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPD) and the large sub-unit of the mitochondrial ribosomal DNA (mtLSUm), and the algal ITS and actin were studied within a population genetics framework. Semi-thin and ultrathin sections were analysed by means of optical, scanning electron and transmission electron microscopy. Gas exchange and chlorophyll fluorescence were used to compare the physiological performance of both morphs. KEY RESULTS AND CONCLUSIONS: Vagrant and attached morphs share multilocus haplotypes which may indicate that they belong to the same species in spite of their completely different anatomy. However, differentiation tests suggested that vagrant specimens do not represent a random sub-set of the surrounding population. The morphological differences were related to anatomical and ultrastructural differences. Large intercalary growth rates of thalli after the loss of the basal-apical thallus polarity may be the cause of the increased growth shown by vagrant specimens. The anatomical and morphological changes lead to greater duration of ecophysiological activity in vagrant specimens. Although the anatomical and physiological changes could be chance effects, the genetic differentiation between vagrant and attached sub-populations and the higher biomass of the former show fitness effects and adaptation to dry environmental conditions in steppe habitats.

The delimitation of Antarctic and bipolar species of neuropogonoid Usnea (Ascomycota, Lecanorales): a cohesion approach of species recognition for the Usnea perpusilla complex.

Species of the Neuropogon group in the lichen genus Usnea have their centre of distribution in polar regions of the Southern Hemisphere. Their morphological and chemical variability is poorly understood and several asexual taxa with uncertain relationships to fertile taxa occur in the group. The species concept is controversial. A phylogenetic analysis revealed three related complexes of mainly asexual lineages arranged around three fertile Usnea species: U. aurantiaco-atra, U. trachycarpa and U. perpusilla. In this study a dataset of 80 specimens was used to resolve species circumscriptions in the U. perpusilla complex. We used a phylogenetic and a haplotype network approach based on three gene fragments (ITS, IGS and RPB1) to detect distinct lineages. To support the hypothesis that these lineages represent different species, we tested for correlation of morphological and chemical characters with hierarchical nested haplotype groups, employing statistical tests of contingency tables and analysis of variance (ANOVA). This cohesion species recognition method detected three fertile U. perpusilla lineages. We could also delimit an undescribed fertile species with yellow apothecia and a new asexual species from the High Andes. Interestingly, there is an additional bipolar species, U. lambii, which was formerly confused with U. sphacelata. The fact that U. lambii shows a geographically disjunct distribution pattern, but the genetic distances among specimens are low, points to recent long-distance dispersal.

Local population subdivision in the lichen Cladonia subcervicornis as revealed by mitochondrial cytochrome oxidase subunit 1 intron sequences.

A 753-771 bp long intronic sequence from the mitochondrial cox 1 gene of Cladonia subcervicornis (Cladoniaceae, Lecanorales, Ascomycota) was amplified with newly designed PCR primers. The cox 1 intron sequence, which apparently has not been used for phylogenetic or population genetic research in fungi, displays high infraspecific variation. Sequences were obtained from 124 specimens from four neighboring localities in coastal Hordaland, western Norway. An exact test of population differentiation and population pairwise fixation indices F(ST) show significantly reduced gene flow between the northernmost locality and the other three populations. Although Cladonia subcervicornis frequently produces apothecia, we conclude that dispersal by ascospores over long distances is rather ineffective in this species.