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.].

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.

Cophylogenetic patterns in algal symbionts correlate with repeated symbiont switches during diversification and geographic expansion of lichen-forming fungi in the genus Sticta (Ascomycota, Peltigeraceae).

Species in the fungal genus Sticta form symbiotic associations primarily with either green algae or cyanobacteria, but tripartite associations or photosymbiodemes involving both types of photobionts occur in some species. Sticta is known to associate with green algae in the genus Symbiochloris. However, previous studies have shown that algae from other genera, such as Heveochlorella, may also be suitable partners for Sticta. We examined the diversity of green algal partners in the genus Sticta and assessed the patterns of association between the host fungus and its algal symbiont. We used multi-locus sequence data from multiple individuals collected in Australia, Cuba, Madagascar, Mauritius, New Zealand, Reunion and South America to infer phylogenies for fungal and algal partners and performed tests of congruence to assess coevolution between the partners. In addition, event-based methods were implemented to examine which cophylogenetic processes have led to the observed association patterns in Sticta and its green algal symbionts. Our results show that in addition to Symbiochloris, Sticta associates with green algae from the genera Chloroidium, Coccomyxa, Elliptochloris and Heveochlorella, the latter being the most common algal symbiont associated with Sticta in this study. Geography plays a strong role in shaping fungal-algal association patterns in Sticta as mycobionts associate with different algal lineages in different geographic locations. While fungal and algal phylogenies were mostly congruent, event-based methods did not find any evidence for cospeciation between the partners. Instead, the association patterns observed in Sticta and associated algae, were largely explained by other cophylogenetic events such as host-switches, losses of symbiont and failure of the symbiont to diverge with its host. Our results also show that tripartite associations with green algae evolved multiple times in Sticta.

Understanding the evolution of phenotypical characters in the Micarea prasina group (Pilocarpaceae) and descriptions of six new species within the group.

Six new Micarea species are described from Europe. Phylogenetic analyses, based on three loci, i.e. mtSSU rDNA, Mcm7 and ITS rDNA and ancestral state reconstructions, were used to evaluate infra-group divisions and the role of secondary metabolites and selected morphological characters on the taxonomy in the M. prasina group. Two main lineages were found within the group. The Micarea micrococca clade consists of twelve species, including the long-known M. micrococca and the newly described M. microsorediata, M. nigra and M. pauli. Within this clade, most species produce methoxymicareic acid, with the exceptions of M. levicula and M. viridileprosa producing gyrophoric acid. The M. prasina clade includes the newly described M. azorica closely related to M. prasina s.str., M. aeruginoprasina sp. nov. and M. isidioprasina sp. nov. The species within this clade are characterised by the production of micareic acid, with the exception of M. herbarum which lacks any detectable substances and M. subviridescens that produces prasinic acid. Based on our reconstructions, it was concluded that the ancestor of the M. prasina group probably had a thallus consisting of goniocysts, which were lost several times during evolution, while isidia and soredia evolved independently at multiple times. Our research supported the view that the ancestor of M. prasina group did not produce any secondary substances, but they were gained independently in different lineages, such as methoxymicareic acid which is restricted to M. micrococca and allied species or micareic acid present in the M. prasina clade.

Sharpening species boundaries in the Micarea prasina group, with a new circumscription of the type species M. prasina.

Micarea is a lichenized genus in the family Pilocarpaceae (Ascomycota). We studied the phylogeny and reassessed the current taxonomy of the M. prasina group. We focused especially on the taxonomic questions concerning the type species M. prasina and, furthermore, challenges concerning type specimens that are too old for successful DNA barcoding and molecular studies. The phylogeny was reconstructed using nuc rDNA internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS), mitochrondrial rDNA small subunit (mtSSU), and replication licensing factor MCM7 gene from 31 species. Fifty-six new sequences were generated. The data were analyzed using maximum parsimony and maximum likelihood methods. The results revealed four undescribed, well-supported lineages. Three lineages represent new species described here as M. fallax, M. flavoleprosa, and M. pusilla. In addition, our results support the recognition of M. melanobola as a distinct species. Micarea fallax is characterized by a vivid to olive green thallus composed of aggregated granules and whitish or brownish apothecia sometimes with grayish tinge (Sedifolia-gray pigment).Micarea flavoleprosa has a thick, wide-spreading yellowish green, whitish green to olive green sorediate thallus and lacks the Sedifolia-gray pigmentation. The species is mostly anamorphic, developing apothecia rarely. Micarea melanobola is characterized by a pale to dark vivid green granular thallus and darkly pigmented apothecia (Sedifolia-gray). Micarea pusilla is characterized by a whitish green to olive green thinly granular or membranous thallus, numerous and very small whitish apothecia lacking the Sedifolia-gray pigment, and by the production of methoxymicareic acid. Micarea fallax, M. flavoleprosa, and M. melanobola produce micareic acid. The reliability of crystalline granules as a character for species delimitation was investigated and was highly informative for linking the old type specimen of M. prasina to fresh material.

Oligocene origin and drivers of diversification in the genus Sticta (Lobariaceae, Ascomycota).

A major challenge to evolutionary biologists is to understand how biodiversity is distributed through space and time and across the tree of life. Diversification of organisms is influenced by many factors that act at different times and geographic locations but it is still not clear which have a significant impact and how drivers interact. To study diversification, we chose the lichen genus Sticta, by sampling through most of the global range and producing a time tree. We estimate that Sticta originated about 30 million years ago, but biogoegraphic analysis was unclear in estimating the origin of the genus. Furthermore, we investigated the effect of dispersal ability finding that Sticta has a high dispersal rate, as collections from Hawaii showed that divergent lineages colonized the islands at least four times. Symbiont interactions were investigated using BiSSE to understand if green-algal or cyanobacterial symbiont interactions influenced diversification, only to find that the positive results were driven almost completely by Type I error. On the other hand, another BiSSE analysis found that an association with Andean tectonic activity increases the speciation rate of species.

High diversity, high insular endemism and recent origin in the lichen genus Sticta (lichenized Ascomycota, Peltigerales) in Madagascar and the Mascarenes.

Lichen biodiversity and its generative evolutionary processes are practically unknown in the MIOI (Madagascar and Indian Ocean Islands) biodiversity hotspot. We sought to test the hypothesis that lichenized fungi in this region have undergone a rapid radiation, following a single colonization event, giving rise to narrow endemics, as is characteristic of other lineages of plants. We extensively sampled specimens of the lichen genus Sticta in the Mascarene archipelago (mainly Réunion) and in Madagascar, mainly in the northern range (Amber Mt and Marojejy Mt) and produced the fungal ITS barcode sequence for 148 thalli. We further produced a four-loci data matrix for 68 of them, representing the diversity and geographical distribution of ITS haplotypes. We reconstructed the phylogenetic relationships within this group, established species boundaries with morphological context, and estimated the date of the most recent common ancestor. Our inferences resolve a robust clade comprising 31 endemic species of Sticta that arose from the diversification following a single recent (c. 11 Mya) colonization event. All but three species have a very restricted range, endemic to either the Mascarene archipelago or a single massif in Madagascar. The first genus of lichens to be studied with molecular data in this region underwent a recent radiation, exhibits micro-endemism, and thus exemplifies the biodiversity characteristics found in other taxa in Madagascar and the Mascarenes.

Conserved genomic collinearity as a source of broadly applicable, fast evolving, markers to resolve species complexes: A case study using the lichen-forming genus Peltigera section Polydactylon.

Synteny can be maintained for certain genomic regions across broad phylogenetic groups. In these homologous genomic regions, sites that are under relaxed purifying selection, such as intergenic regions, could be used broadly as markers for population genetic and phylogenetic studies on species complexes. To explore the potential of this approach, we found 125 Collinear Orthologous Regions (COR) ranging from 1 to >10kb across nine genomes representing the Lecanoromycetes and Eurotiomycetes (Pezizomycotina, Ascomycota). Twenty-six of these COR were found in all 24 eurotiomycete genomes surveyed for this study. Given the high abundance and availability of fungal genomes we believe this approach could be adopted for other large groups of fungi outside the Pezizomycotina. Asa proof of concept, we selected three Collinear Orthologous Regions (COR1b, COR3, and COR16), based on synteny analyses of several genomes representing three classes of Ascomycota: Eurotiomycetes, Lecanoromycetes, and Lichinomycetes. COR16, for example, was found across these three classes of fungi. Here we compare the resolving power of these three new markers with five loci commonly used in phylogenetic studies of fungi, using section Polydactylon of the cyanolichen-forming genus Peltigera (Lecanoromycetes) - a clade with several challenging species complexes. Sequence data were subjected to three species discovery and two validating methods. COR markers substantially increased phylogenetic resolution and confidence, and highly contributed to species delimitation. The level of phylogenetic signal provided by each of the COR markers was higher than the commonly used fungal barcode ITS. High cryptic diversity was revealed by all methods. As redefined here, most species represent lineages that have relatively narrower, and more homogeneous biogeographical ranges than previously understood. The scabrosoid clade consists of ten species, seven of which are new. For the dolichorhizoid clade, twenty-two new species were discovered for a total of twenty-nine species in this clade.

A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomycota): 1307 fungi representing 1139 infrageneric taxa, 317 genera and 66 families.

The Lecanoromycetes is the largest class of lichenized Fungi, and one of the most species-rich classes in the kingdom. Here we provide a multigene phylogenetic synthesis (using three ribosomal RNA-coding and two protein-coding genes) of the Lecanoromycetes based on 642 newly generated and 3329 publicly available sequences representing 1139 taxa, 317 genera, 66 families, 17 orders and five subclasses (four currently recognized: Acarosporomycetidae, Lecanoromycetidae, Ostropomycetidae, Umbilicariomycetidae; and one provisionarily recognized, 'Candelariomycetidae'). Maximum likelihood phylogenetic analyses on four multigene datasets assembled using a cumulative supermatrix approach with a progressively higher number of species and missing data (5-gene, 5+4-gene, 5+4+3-gene and 5+4+3+2-gene datasets) show that the current classification includes non-monophyletic taxa at various ranks, which need to be recircumscribed and require revisionary treatments based on denser taxon sampling and more loci. Two newly circumscribed orders (Arctomiales and Hymeneliales in the Ostropomycetidae) and three families (Ramboldiaceae and Psilolechiaceae in the Lecanorales, and Strangosporaceae in the Lecanoromycetes inc. sed.) are introduced. The potential resurrection of the families Eigleraceae and Lopadiaceae is considered here to alleviate phylogenetic and classification disparities. An overview of the photobionts associated with the main fungal lineages in the Lecanoromycetes based on available published records is provided. A revised schematic classification at the family level in the phylogenetic context of widely accepted and newly revealed relationships across Lecanoromycetes is included. The cumulative addition of taxa with an increasing amount of missing data (i.e., a cumulative supermatrix approach, starting with taxa for which sequences were available for all five targeted genes and ending with the addition of taxa for which only two genes have been sequenced) revealed relatively stable relationships for many families and orders. However, the increasing number of taxa without the addition of more loci also resulted in an expected substantial loss of phylogenetic resolving power and support (especially for deep phylogenetic relationships), potentially including the misplacements of several taxa. Future phylogenetic analyses should include additional single copy protein-coding markers in order to improve the tree of the Lecanoromycetes. As part of this study, a new module ("Hypha") of the freely available Mesquite software was developed to compare and display the internodal support values derived from this cumulative supermatrix approach.

Do photobiont switch and cephalodia emancipation act as evolutionary drivers in the lichen symbiosis? A case study in the Pannariaceae (Peltigerales).

Lichen symbioses in the Pannariaceae associate an ascomycete and either cyanobacteria alone (usually Nostoc; bipartite thalli) or green algae and cyanobacteria (cyanobacteria being located in dedicated structures called cephalodia; tripartite thalli) as photosynthetic partners (photobionts). In bipartite thalli, cyanobacteria can either be restricted to a well-delimited layer within the thallus ('pannarioid' thalli) or spread over the thallus that becomes gelatinous when wet ('collematoid' thalli). We studied the collematoid genera Kroswia and Physma and an undescribed tripartite species along with representatives of the pannarioid genera Fuscopannaria, Pannaria and Parmeliella. Molecular inferences from 4 loci for the fungus and 1 locus for the photobiont and statistical analyses within a phylogenetic framework support the following: (a) several switches from pannarioid to collematoid thalli occured and are correlated with photobiont switches; the collematoid genus Kroswia is nested within the pannarioid genus Fuscopannaria and the collematoid genus Physma is sister to the pannarioid Parmeliella mariana group; (b) Nostoc associated with collematoid thalli in the Pannariaceae are related to that of the Collemataceae (which contains only collematoid thalli), and never associated with pannarioid thalli; Nostoc associated with pannarioid thalli also associate in other families with similar morphology; (c) ancestors of several lineages in the Pannariaceae developed tripartite thalli, bipartite thalli probably resulting from cephalodia emancipation from tripartite thalli which eventually evolved and diverged, as suggested by the same Nostoc present in the collematoid genus Physma and in the cephalodia of a closely related tripartite species; Photobiont switches and cephalodia emancipation followed by divergence are thus suspected to act as evolutionary drivers in the family Pannariaceae.

A two-gene phylogeny shows the lichen genus Niebla (Lecanorales) is endemic to the New World and does not occur in Macaronesia nor in the Mediterranean basin.

The generic segregates of the widespread fruticose genus Ramalina (mostly based on empirical data on morphology, cortex anatomy and secondary metabolites) are studied using maximum parsimony, maximum likelihood and Bayesian analyses of nuclear LSU and ITS sequences. The species examined include the three species aggregates within Niebla from the western coasts of North America, all species except one assumed to belong to the same genus from Macaronesia and the Mediterranean basin, the type species of Dievernia and Ramalina, and representatives of the genus Fistulariella. The genus Niebla is strongly supported when restricted to species from the New World, and all species referred to it from Macaronesia and the Mediterranean basin belong to Ramalina (R. bourgeana, R. crispatula, R. cupularis, R. hamulosa, R. portosantana, R. rosacea, R. subwebbiana and R. webbii). No support is found for the genera Dievernia and Fistulariella. The internal topology of the large genus Ramalina is unresolved and needs further studies.

Microsatellite primers in the Peltigera dolichorhiza complex (lichenized ascomycete, Peltigerales).

PREMISE OF THE STUDY: Microsatellite primers were developed for the lichen-forming fungus Peltigera dolichorhiza to investigate partitioning of genetic variation in a widespread, morphologically and chemically variable taxon likely to represent a complex of cryptic lineages, including P. neopolydactyla. • METHODS AND RESULTS: Using next generation shotgun sequence reads, 331 primer pairs were designed to amplify microsatellite sequences from an African accession of P. dolichorhiza. Eleven primer pairs representing the longest repeat units identified were tested on 15 P. dolichorhiza accessions from Africa (incl. Réunion), South America, Papua New Guinea, and on two accessions of P. neopolydactyla from North America. The primers amplified di-, tri, tetra-, and pentanucelotide repeats with 3-8 alleles per locus. All individuals represent distinct multiloci genotypes. • CONCLUSIONS: These results indicate the utility of the new microsatellite primers for testing genetic differentiation within the widespread complex of P. dolichorhiza.

A new lineage of lichenized basidiomycetes inferred from a two-gene phylogeny: The Lepidostromataceae with three species from the tropics.

The lichen habit has apparently evolved independently in at least five major clades of mushroom-forming basidiomycetes (Agaricomycetes). Tracing the origin of lichenization in these groups depends on a clearer understanding of the phylogenetic relationships of basidiolichens to other fungi. We describe here a new family of basidiolichens made up of tropical, soil-inhabiting fungi that form lichenized, scale-like squamules and erect, coral-like fruiting structures. These structures are common to two basidiolichen genera, Multiclavula and Lepidostroma. Molecular studies have confirmed the phylogenetic position of Multiclavula species in the Cantharellales, but Lepidostroma species have never been sequenced. We obtained nuclear small and large subunit ribosomal sequences from specimens of L. calocerum collected in Costa Rica and Mexico and also from specimens of two Multiclavula spp. recently described from Rwanda. The phylogenetic placement of these fungi within the Agaricomycetes was investigated using likelihood and Bayesian analyses. Our results indicate that L. calocerum and the Rwandan species form a natural group unrelated to Multiclavula and sister to the Atheliales, members of which are neither lichen-forming nor clavarioid. The independent evolution of morphologically similar forms in so many groups of basidiomycetes is a remarkable example of convergence, indicating similar pathways to lichenization in these fungi.