Global Biodiversity Patterns of the Photobionts Associated with the Genus Cladonia (Lecanorales, Ascomycota).

The diversity of lichen photobionts is not fully known. We studied here the diversity of the photobionts associated with Cladonia, a sub-cosmopolitan genus ecologically important, whose photobionts belong to the green algae genus Asterochloris. The genetic diversity of Asterochloris was screened by using the ITS rDNA and actin type I regions in 223 specimens and 135 species of Cladonia collected all over the world. These data, added to those available in GenBank, were compiled in a dataset of altogether 545 Asterochloris sequences occurring in 172 species of Cladonia. A high diversity of Asterochloris associated with Cladonia was found. The commonest photobiont lineages associated with this genus are A. glomerata, A. italiana, and A. mediterranea. Analyses of partitioned variation were carried out in order to elucidate the relative influence on the photobiont genetic variation of the following factors: mycobiont identity, geographic distribution, climate, and mycobiont phylogeny. The mycobiont identity and climate were found to be the main drivers for the genetic variation of Asterochloris. The geographical distribution of the different Asterochloris lineages was described. Some lineages showed a clear dominance in one or several climatic regions. In addition, the specificity and the selectivity were studied for 18 species of Cladonia. Potentially specialist and generalist species of Cladonia were identified. A correlation was found between the sexual reproduction frequency of the host and the frequency of certain Asterochloris OTUs. Some Asterochloris lineages co-occur with higher frequency than randomly expected in the Cladonia species.

A spectral analysis of common boreal ground lichen species.

Lichens dominate a significant part of the Earth's land surface, and are valuable bioindicators of various environmental changes. In the northern hemisphere, the largest lichen biomass is in the woodlands and heathlands of the boreal zone and in tundra. Despite the global coverage of lichens, there has been only limited research on their spectral properties in the context of remote sensing of the environment. In this paper, we report spectral properties of 12 common boreal lichen species. Measurements of reflectance spectra were made in laboratory conditions with a standard spectrometer (350-2500 nm) and a novel mobile hyperspectral camera (400-1000 nm) which was used in a multiangular setting. Our results show that interspecific differences in reflectance spectra were the most pronounced in the ultraviolet and visible spectral range, and that dry samples always had higher reflectance than fresh (moist) samples in the shortwave infrared region. All study species had higher reflectance in the backward scattering direction compared to nadir or forward scattering directions. Our results also reveal, for the first time, that there is large intraspecific variation in reflectance of lichen species. This emphasizes the importance of measuring several replicates of each species when analyzing lichen spectra. In addition, we used the data in a spectral clustering analysis to study the spectral similarity between samples and species, and how these similarities could be linked to different physical traits or phylogenetic closeness of the species. Overall, our results suggest that spectra of some lichen species with large ground coverage can be used for species identification from high spatial resolution remote sensing imagery. On the other hand, for lichen species growing as small assemblages, mobile hyperspectral cameras may offer a solution for in-situ species identification. The spectral library collected in this study is available in the SPECCHIO Spectral Information System.

Phylogeny of the family Cladoniaceae (Lecanoromycetes, Ascomycota) based on sequences of multiple loci.

Cladoniaceae is a family of lichenized fungi that belongs to the Lecanorales, Ascomycota. The family is distributed widely, although several genera are restricted to the Southern Hemisphere. The circumscriptions of the genera and species in the family have traditionally been based on thallus morphology, the type of vegetative propagules and the secondary metabolites. However, numerous species are highly variable phenotypically, making their delimitation problematic. In the present study a new phylogeny of Cladoniaceae is constructed using five loci (ITS rDNA, IGS rDNA, RPB2, RPB1, EF-1a) from a worldwide sample of 643 specimens representing 304 species. Cladoniaceae was resolved as a monophyletic group. The circumscription of the genera and the relationships among them are discussed. Pycnothelia, Carassea and Metus are closely related, forming a sister clade to the larger genus Cladonia. Cladia in its recent wide sense turned out to be paraphyletic, including species that have been recognized in Thysanothecium and Notocladonia. Cladonia was resolved as monophyletic, with C. wainioi as the earliest diverging lineage. Eleven major clades were resolved in Cladonia. No synapomorphies were found for most of them. We propose the new genera Pulchrocladia and Rexia, as segregates of Cladia, five new combinations, and the resurrection of the genus Heterodea.

Genetic variation and factors affecting the genetic structure of the lichenicolous fungus Heterocephalacria bachmannii (Filobasidiales, Basidiomycota).

Heterocephalacria bachmannii is a lichenicolous fungus that takes as hosts numerous lichen species of the genus Cladonia. In the present study we analyze whether the geographical distance, the host species or the host secondary metabolites determine the genetic structure of this parasite. To address the question, populations mainly from the Southern Europe, Southern Finland and the Azores were sampled. The specimens were collected from 20 different host species representing ten chemotypes. Three loci, ITS rDNA, LSU rDNA and mtSSU, were sequenced. The genetic structure was assessed by AMOVA, redundance analyses and Bayesian clustering methods. The results indicated that the host species and the host secondary metabolites are the most influential factors over the genetic structure of this lichenicolous fungus. In addition, the genetic structure of H. bachmannii was compared with that of one of its hosts, Cladonia rangiformis. The population structure of parasite and host were discordant. The contents in phenolic compounds and fatty acids of C. rangiformis were quantified in order to test whether it had some influence on the genetic structure of the species. But no correlation was found with the genetic clusters of H. bachmannii.

Three common bryophilous fungi with meristematic anamorphs and phylogenetic alliance to Teratosphaeriaceae, Capnodiales.

Bryophilous ascomycetes are an overlooked and poorly known fungal group. In this study, the extreme and small-sized niche of Polytrichum piliferum hyaline leaf tips was screened for the presence of these fungi in Finland. Three closely related species were found. Bryochiton perpusillus and Bryochiton monascus were identified from several samples, and DNA isolations revealed a third closely related species, Bryochiton sp. In addition, melanised hyphae, typical to the Bryochiton species, were present in all the samples. According to phylogenetic analyses consisting of combined small subunit (SSU), large subunit (LSU), and 5.8S rDNA sequences, and internal transcribed spacer (ITS) rDNA sequences, the species showed affinity with Teratosphaeriaceae within Capnodiales, and especially with black, meristematic species often inhabiting rock substrate in extreme environments. The connection was supported by meristematic growth of the Bryochiton species in culture. Bryochiton is the second sexual genus associated within the family Teratosphaeriaceae, and B. perpusillus, and B. monascus constitute examples of teleomorphs within a group of meristematic anamorphs. These findings emphasize the multiform diversity underlying poorly researched fungal groups, such as the bryophilous fungi.

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.

Multilocus approach to species recognition in the Cladonia humilis complex (Cladoniaceae, Ascomycota).

PREMISE OF THE STUDY: The members of the Cladonia humilis complex are characterized by a well-developed primary thallus and broadly scyphose podetia. In the present study, this complex was phylogenetically analyzed to test the boundaries between the species and to determine the usefulness of the phenotypic characters to distinguish them. The species C. conista, C. cyathomorpha, C. hammeri, C. humilis, C. kurokawae, C. nashii, C. pulvinella, and C. subconistea were examined. METHODS: Four DNA loci were sequenced and analyzed to test the monophyly of the species. For the phylogenetic reconstructions, maximum parsimony, maximum likelihood, and Bayesian methods were employed. The genealogical sorting index was used to quantify the exclusive ancestry of the nonmonophyletic species on the tree. KEY RESULTS: The performed phylogenetic analyses showed that the C. humilis complex is not monophyletic. Cladonia nashii is not closely related to the remaining taxa within the complex. Seven monophyletic lineages were identified, most of which comprise specimens belonging to more than one chemotype. Cladonia hammeri and C. pulvinella are conspecific, and this taxon is not present in Europe. CONCLUSIONS: This study suggests that morphological characters and secondary metabolites have less taxonomical value than thought in the Cladonia humilis complex. Use of multilocus phylogeny is recommended to delimit species.

Pleistocene speciation in North American lichenized fungi and the impact of alternative species circumscriptions and rates of molecular evolution on divergence estimates.

Pleistocene climatic fluctuations influenced patterns of genetic variation and promoted speciation across a wide range of species groups. Lichens are commonly found in habitats that were directly impacted by glacial cycles; however, the role of Pleistocene climate in driving speciation in most lichen symbionts remains unclear. This uncertainty is due in part to limitations in our ability to accurately recognize independently evolving lichen-forming fungal lineages and a lack of relevant fossil calibrations. Using a coalescent-based species tree approach, we estimated divergence times for two sister clades in the genus Xanthoparmelia (Parmeliaceae) restricted to western North America. We assessed the influence of two different species circumscription scenarios and various locus-specific rates of molecular evolution on divergence estimates. Species circumscriptions were validated using the program BP&P. although speciation was generally supported in both scenarios, divergence times differed between traditional species circumscriptions and those based on genetic data, with more recent estimates resulting from the former. Similarly, rates of evolution for different loci resulted in variable divergence time estimates. However, our results unambiguously indicate that diversification in the sampled Xanthoparmelia clades occurred during the Pleistocene. Our study highlights the potential impact of ambiguous species circumscriptions and uncertain rates of molecular evolution on estimating divergence times within a multilocus species tree framework.

Implementing a cumulative supermatrix approach for a comprehensive phylogenetic study of the Teloschistales (Pezizomycotina, Ascomycota).

The resolution of the phylogenetic relationships within the order Teloschistales (Ascomycota, lichen-forming-fungi), with nearly 2000 known species and outstanding phenotypic diversity, has been hindered by the limitation in the resolving power that single-locus or two-locus phylogenetic studies have provided to date. In this context, an extensive taxon sampling within the Teloschistales with more loci (especially nuclear protein-coding genes) was needed to confront the current taxonomic delimitations and to understand evolutionary trends within this order. Comprehensive maximum likelihood and bayesian analyses were performed based on seven loci using a cumulative supermatrix approach, including protein-coding genes RPB1 and RPB2 in addition to nuclear and mitochondrial ribosomal RNA-coding genes. We included 167 taxa representing 12 of the 15 genera recognized within the currently accepted Teloschistineae, 22 of the 43 genera within the Physciineae, 49 genera of the closely related orders Lecanorales, Lecideales, and Peltigerales, and the dubiously placed family Brigantiaeaceae and genus Sipmaniella. Although the progressive addition of taxa (cumulative supermatrix approach) with increasing amounts of missing data did not dramatically affect the loss of support and resolution, the monophyly of the Teloschistales in the current sense was inconsistent, depending on the loci-taxa combination analyzed. Therefore, we propose a new, but provisional, classification for the re-circumscribed orders Caliciales and Teloschistales (previously referred to as Physciineae and Teloschistineae, respectively). We report here that the family Brigantiaeaceae, previously regarded as incertae sedis within the subclass Lecanoromycetidae, and Sipmaniella, are members of the Teloschistales in a strict sense. Within this order, one lineage led to the diversification of the mostly epiphytic crustose Brigantiaeaceae and Letrouitiaceae, with a circumpacific center of diversity and found mostly in the tropics. The other main lineage led to another epiphytic crustose family, mostly tropical, and with an Australasian center of diversity--the Megalosporaceae--which is sister to the mainly rock-inhabiting, cosmopolitan, and species rich Teloschistaceae, with a diversity of growth habits ranging from crustose to fruticose. Our results confirm the use of a cumulative supermatrix approach as a viable method to generate comprehensive phylogenies summarizing relationships of taxa with multi-locus to single locus data.

Expansion of the Stictidaceae by the addition of the saxicolous lichen-forming genus Ingvariella.

The monotypic, lichen-forming genus Ingvariella originally was segregated from Diploschistes and placed within the Thelotremataceae (Ostropales) based on aspects of exciple morphology. However, the I+ hymenium and amyloid ascus wall suggest affinities to families other than the Thelotremataceae. To assess the identity of Ingvariella and to investigate its placement within the Ostropales, we inferred phylogenetic relationships of I. bispora by comparison of mtSSU rDNA and nuLSU rDNA sequences for 59 species encompassing a broad array of ostropalean fungi by means of Bayesian, maximum likelihood and weighted maximum parsimony methods. Here we report that Ingvariella is a member of the Stictidaceae, sister to the mainly saprotrophic genus Cryptodiscus. The inclusion of the first saxicolous lichen-forming fungus within this family expands the broad ecological diversity of the Stictidaceae, where saprotrophic fungi, corticicolous lichen-forming fungi and lichenized and non-lichenized conspecific taxa have been described previously. We also present new insights into the relationships among other families within the Ostropales.

Multiple origins of symbioses between ascomycetes and bryophytes suggested by a five-gene phylogeny.

Numerous species of microscopic fungi inhabit mosses and hepatics. They are severely overlooked and their identity and nutritional strategies are mostly unknown. Most of these bryosymbiotic fungi belong to the Ascomycota. Their fruit-bodies are extremely small, often reduced and simply structured, which is why they cannot be reliably identified and classified by their morphological and anatomical characters. A phylogenetic hypothesis of bryosymbiotic ascomycetes is presented. New sequences of 78 samples, including 61 bryosymbionts, were produced, the total amount of terminals being 206. Of these, 202 are Ascomycetes. Sequences from the following five gene loci were used: rDNA SSU, rDNA LSU, RPB2, mitochondrial rDNA SSU, and rDNA 5.8S. The program TNT was used for tree search and support value estimation. We show that bryosymbiotic fungi occur in numerous lineages, one of which represents a newly discovered lineage among the Ascomycota and exhibits a tripartite association with cyanobacteria and sphagna. A new genus Trizodia is proposed for this basal clade. Our results demonstrate that even highly specialized life strategies can be adopted multiple times during evolution, and that in many cases bryosymbionts appear to have evolved from saprobic ancestors. © The Willi Hennig Society 2009.

Joergensenia, a new genus to accommodate Psoroma cephalodinum (lichenized Ascomycota).

The taxonomy of the genus Psoroma (lichenized Ascomycota) is currently under revision, as it has long been recognized as heterogeneous. The aim of the present study was to reconstruct the phylogeny of Psoroma and related genera. The ITS region of nu-rDNA and the mtSSU rDNA from 22 collections of Psoroma and Pannaria were amplified. Sequences from GenBank were also used. For the phylogenetic analysis, direct optimization was implemented, using the program POY, and standard MP using PAUP. Both analyses resulted in similar trees. Two main clades were obtained, one including Santesoniella and most Psoroma species and the other including Pannaria and two Psoroma species, indicating that Psoroma is polyphyletic. Based on its phylogenetic position and ascus type, P. implexum is transferred to the genus Pannaria, and the new combination P. implexa is proposed. Psoroma cephalodinum clusters with Parmeliella and Degelia as a basal group within the Pannariaceae. The ascus apex of P. cephalodinum has a strong amyloid reaction revealing a compact cap-shaped plug. This differs from most of the ascus apical structures observed in the family. Both ascus internal structure and phylogenetic position suggest that P. cephalodinum is distantly related to Psoroma or Pannaria, and thus a new genus Joergensenia is proposed.

High cyanobiont selectivity of epiphytic lichens in old growth boreal forest of Finland.

Here, cyanobiont selectivity of epiphytic lichen species was examined in an old growth forest area in Finland. Samples of the eight lichen species were collected from the same aspen (Populus tremula) and adjacent aspens in the same stand. The cyanobionts of these samples were compared with free and symbiotic Nostoc obtained from other habitats and geographic regions. Our results, based on the phylogenetic analysis of a partial small subunit of the ribosomal DNA (16S rDNA) and the rbcLX gene complex did not show any correlation with the geographic origin of the samples at any spatial scale. Instead, there was a correlation between the cyanobionts and the alleged taxonomy of their mycobionts. The results indicate that the lichen species examined are highly selective towards their cyanobiont partners. Only Lobaria pulmonaria proved to be more flexible, being able to associate with a wide range of Nostoc. A same Nostoc strain was found to form associations with taxonomically unrelated lichens indicating that the cyanobiont-mycobiont associations as a whole were not highly specific in the examined species.

New insights into classification and evolution of the Lecanoromycetes (Pezizomycotina, Ascomycota) from phylogenetic analyses of three ribosomal RNA- and two protein-coding genes.

The Lecanoromycetes includes most of the lichen-forming fungal species (> 13500) and is therefore one of the most diverse class of all Fungi in terms of phenotypic complexity. We report phylogenetic relationships within the Lecanoromycetes resulting from Bayesian and maximum likelihood analyses with complementary posterior probabilities and bootstrap support values based on three combined multilocus datasets using a supermatrix approach. Nine of 10 orders and 43 of 64 families currently recognized in Eriksson's classification of the Lecanoromycetes (Outline of Ascomycota--2006 Myconet 12:1-82) were represented in this sampling. Our analyses strongly support the Acarosporomycetidae and Ostropomycetidae as monophyletic, whereas the delimitation of the largest subclass, the Lecanoromycetidae, remains uncertain. Independent of future delimitation of the Lecanoromycetidae, the Rhizocarpaceae and Umbilicariaceae should be elevated to the ordinal level. This study shows that recent classifications include several nonmonophyletic taxa at different ranks that need to be recircumscribed. Our phylogenies confirm that ascus morphology cannot be applied consistently to shape the classification of lichen-forming fungi. The increasing amount of missing data associated with the progressive addition of taxa resulted in some cases in the expected loss of support, but we also observed an improvement in statistical support for many internodes. We conclude that a phylogenetic synthesis for a chosen taxonomic group should include a comprehensive assessment of phylogenetic confidence based on multiple estimates using different methods and on a progressive taxon sampling with an increasing number of taxa, even if it involves an increasing amount of missing data.

Phylogeny of the Polytrichales (Bryophyta) based on simultaneous analysis of molecular and morphological data.

Phylogenetic analyses of Polytrichales were conducted using morphology and sequence data from the chloroplast genes rbcL and rps4 plus the trnL-F gene region, part of the mitochondrial nad5 and the nuclear-encoded 18S rDNA. Our analyses included 46 species representing all genera of Polytrichales. Phylogenetic trees were constructed with simultaneous parsimony analyses of all sequences plus morphology and separate combinations of sequence data only. Results lend support for recognition of Polytrichales as a monophyletic entity. Oedipodium griffithianum appears as a sister taxon to Polytrichales or as a sister taxon of all mosses excluding Sphagnales and Andreaeles. Within Polytrichales, Alophosia and Atrichopsis, species without the adaxial lamellae (in Atrichopsis present but poorly developed on male gametophyte) otherwise typical of the group are sister to the remaining species followed by a clade including Bartramiopsis and Lyellia, species with adaxial lamellae covering only the central portion of the leaves. Six taxa with an exclusively Southern Hemisphere distribution form a grade between the basal lineages and a clade including genera that are mostly confined to the Northern Hemisphere.

Phylogeny of bipolar Cladonia arbuscula and Cladonia mitis (Lecanorales, Euascomycetes).

Phylogenetic relationships and levels of geographic differentiation of two closely related bipolar taxa, Cladonia arbuscula and Cladonia mitis, were cladistically examined with ITS regions, SSU rDNA introns, partial beta-tubulin, and partial glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes. In the combined analysis of the four genes, C. arbuscula was paraphyletic, while C. mitis, nested within C. arbuscula, formed a strongly supported monophyletic group. C. arbuscula samples were divided into three separate clades: "arbuscula I," appearing as basal to the other ingroup taxa, "arbuscula II," and "arbuscula III" (the latter represented by only one specimen), which were not correlated with any morphological trait. Only C. mitis specimens formed a morphologically and chemically distinct group. None of the main clades was correlated with geographic origin. The separate analyses were poorly resolved, and in most cases samples from "arbuscula I," "arbuscula II," and "arbuscula III" clades were intermixed. An incongruence test revealed conflict among the four gene regions in almost all cases. Only ITS regions and introns were not significantly incongruent, suggesting lack of recombination within the ribosomal DNA locus. Incomplete lineage sorting and recombination were considered to be the main reasons accounting for the incongruencies. The high proportion of shared polymorphisms between the "arbuscula I" and "arbuscula II" clades, especially found from the beta-tubulin gene and from the ITS regions, and the lack of corroborating morphological characters both indicate a short history of reproductive isolation among the groups. The lack of genetic differentiation among the northern and southern samples within the main clades indicates a relatively recent gene flow, which may have resulted from migrations during the Pleistocene glaciations or from more recent long-distance dispersal.

Phylogeny of the Genus Cladonia s.lat. (Cladoniaceae, Ascomycetes) Inferred from Molecular, Morphological, and Chemical Data.

Phylogenetic relationships within the genus Cladonia, including Cladina (Cladoniaceae, Lecanoromycetes), were reconstructed based upon simultaneous analyses of DNA sequences and morphological and chemical data. We used sequences from the internal transcribed spacer 1 (ITS1), the 5.8 rDNA gene, and the internal transcribed spacer 2 (ITS2) of the nuclear rDNA gene cluster, and partial sequences from the protein-coding ß-tubulin gene. The analyses included 235 specimens of 168 taxa representing all currently recognized sections of Cladonia and Cladina and the outgroup genera Cladia, Pycnothelia, and Ramalea. Analyses were performed using optimization alignment with three different parameter values. The results of all analyses support the inclusion of Cladina in Cladonia. The current sectional division of Cladonia was not supported, and a new provisional classification for the genus is proposed.