Emmanuël Sérusiaux

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.

Lichen-symbiotic cyanobacteria associated with Peltigera have an alternative vanadium-dependent nitrogen fixation system

In past decades, environmental nitrogen fixation has been attributed almost exclusively to the action of enzymes in the well-studied molybdenum-dependent nitrogen fixation system. However, recent evidence has shown that nitrogen fixation by alternative pathways may be more frequent than previously suspected. In this study, the nitrogen fixation systems employed by lichen-symbiotic cyanobacteria were examined to determine whether their diazotrophy can be attributed, in part, to an alternative pathway. The mining of metagenomic data (generated through pyrosequencing) and PCR assays were used to determine which nitrogen-fixation systems are present in cyanobacteria from the genus Nostoc associated with four samples from different geographical regions, representing different lichen-forming fungal species in the genus Peltigera. A metatranscriptomic sequence library from an additional specimen was examined to determine which genes associated with N2 fixation are transcriptionally expressed. Results indicated that both the standard molybdenum-dependent system and an alternative vanadium-dependent system are present and actively transcribed in the lichen symbiosis. This study shows for the first time that an alternative system is utilized by cyanobacteria associated with fungi. The ability of lichen-associated cyanobacteria to switch between pathways could allow them to colonize a wider array of environments, including habitats characterized by low temperature and trace metal (e.g. molybdenum) availability. We discuss the implications of these findings for environmental studies that incorporate acetylene-reduction assay data.

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.

Macroevolution of Specificity in Cyanolichens of the Genus Peltigera Section Polydactylon (Lecanoromycetes, Ascomycota).

Abstract Patterns of specificity among symbiotic partners are key to a comprehensive understanding of the evolution of symbiotic systems. Specificity of mutualistic partners, within a widespread monophyletic group for which all species are sampled has rarely been explored. Here, we assess the level of specificity between the cosmopolitan lichen‐forming fungus (mycobiont) from the genus Peltigera, section Polydactylon, and its cyanobacterial partner Nostoc (cyanobiont). The mycobiont and cyanobiont phylogenies are inferred from five nuclear loci and the rbcLX region, respectively. These sequences were obtained from 206 lichen thalli, representing ca. 40 closely related Peltigera species sampled worldwide, doubling the number of known species in this group. We found a broad spectrum of specificity for both partners ranging from strict specialists to generalists. Overall, mycobionts are more specialized than cyanobionts by associating mostly with one or a few Nostoc phylogroups, whereas most cyanobionts associate frequently with several Peltigera species. Specialist mycobionts are older than generalists, supporting the hypothesis that specialization of mycobionts to one or few cyanobionts, is favored through time in geographic areas where species have been established for long periods of time. The relatively recent colonization of a new geographic area (Central and South America) by members of section Polydactylon is associated with a switch to a generalist pattern of association and an increased diversification rate by the fungal partner, suggesting that switches to generalism are rare events that are advantageous in new environments. We detected higher genetic diversity in generalist mycobionts. We also found that Peltigera species specialized on a single Nostoc phylogroup have narrower geographical distributions compared with generalist species.

Dismantling the treasured flagship lichen Sticta fuliginosa (Peltigerales) into four species in Western Europe

In the framework of a worldwide project on the phylogeny of the lichen genus Sticta, dedicated sampling was performed in four regions of Western Europe, roughly along an east–west line between N 48°02′ E 07°01′ and N 52°01′ W 09°30′, ranging from France/Vosges to Ireland/Kerry. Five clearly distinct ITS haplotypes were detected for isidia-producing species where only two were expected. Subtle anatomical and morphological characters, together with a strongly supported 4-loci molecular phylogeny, permit distinguishing, besides the easily recognized S. canariensis and S. limbata: 1) the two well-known S. fuliginosa and S. sylvatica, whose type collections have been carefully reassessed; the former is widespread in both hemispheres, while the latter is correctly identified only from continental Europe and the Andes in Colombia. The barcode ITS of S. fuliginosa differs by a single substitution from S. limbata (with a single exception), and the 4-loci phylogenetic tree does not resolve them as distinct lineages, most probably highlighting a very recent divergence and incomplete lineage sorting. 2) Three species that were formely included in S. fuliginosa: the resurrected S. ciliata Taylor, belonging to a complex group yet to be disentangled and occurring in the Neotropics, Africa, Macaronesia, and Western Europe, and two species described as new to science, S. fuliginoides, found in continental Europe, the Canary Islands, eastern North America, and Colombia, and S. atlantica only known from Ireland and the Azores archipelago. Molecular inferences demonstrate active divergence and dispersion within S. ciliata that may require recognition of further species. Fresh material can be identified with a morphological and anatomical preliminary key provided here. We propose that the taxonomy of all lichen species be urgently reviewed in the light of molecular data in an evolutionary context, particularly those used as bioindicators of environmental change and woodland management.

Isalonactis, a new genus of Roccellaceae (Arthoniales), from southern Madagascar

The new genus and species Isalonactis madagascariensis is characterized by a crustose, non- corticate, often sorediate thallus containing psoromic acid, tiny white pruinose ascomata with a thalline margin, an inconspicuous excipulum, a pale brown hypothecium, 3-septate hyaline ascospores and curved filiform conidia. Phylogenetic analyses using nuLSU and RPB2 sequences place Isalonactis in the Roccellaceae, close to the genera Lecanactis and Chiodecton. The new species was collected on sheltered siliceous rocks in the dry landscape of the Isalo Massif (S Madagascar). Dermatiscum thunbergii is newly recorded from Madagascar.

Unveiling a surprising diversity in the lichen genus Micarea ( Pilocarpaceae ) in Réunion (Mascarenes archipelago, Indian Ocean)

Detailed anatomical and chemical studies conducted on recent collections made in almost all suitable habitats on Reunion, a small remote tropical island in the Indian Ocean, yielded a surprising diversity in the widespread lichen genus Micarea ( Pilocarpaceae , Lecanorales ). Twenty-one species are recognized, including 13 described here as new to science. They are: Micarea alectorialica, M. bebourensis, M. borbonica, M. boryana, M. cilaoensis, M. hyalinoxanthonica, M. isidiosa, M. melanoprasina, M. pseudocoppinsii, M. pseudolignaria, M. sublithinella, M. takamakae and M. tenuispora . Notes on local ecology and important biogeographical features are also given and a key to the species is provided. Isidiiform areolae are reported for the first time in the genus ( M. isidiosa and M. tenuispora ), as well as the production of protolichesterinic and confluentic acids ( M. sublithinella and M. takamakae , respectively). Two groups within the genus are species-rich on the island: the M. peliocarpa group with possibly 5 species, including 3 new to science, and the M. prasina group with 4 species, including 2 new to science. Micarea levicula is reported here for the first time since its description, and the status of the material that can be referred to M. micrococca s. lat. needs further study.

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.

Hypotrachyna penduliloba and Remototrachyna pandani, two new species in the hyperdiverse lichen family Parmeliaceae from Réunion in the Mascarene Archipelago

Extensive exploration of lichen diversity in the tiny and remote tropical island of Réunion (Mascarene Archipelago, Indian Ocean) yielded two new species in the hyperdiverse lichen family Parmeliaceae. Morphological, anatomical and chemical characters and molecular inferences from 3 loci (ITS, nuLSU and mtSSU) strongly support their assignment to Hypotrachyna (subgenus Longilobae) and Remototrachyna. Hypotrachyna producta is further assigned to H. subgenus Longilobae and is newly reported for Réunion as well as Remototrachyna costaricensis.

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.