Phylogenetic studies in Porinaceae

Baloch, E. & Grube, M.
Institute for Plant Science, Karl-Franzens-University Graz, Holteigasse 6, A-8010 Graz, Austria

Porinaceae (Trichotheliales) are a diverse pyrenocarpous lichen family with cosmopolitan distribution and highest diversity in the tropics and humid subtropics. Representatives of this lichen family are among the most common lichens on living leaves. We present some relationships within foliicolous Porinaceae and clarify the systematic position of this family in the system of ascomycetes. The phylogenetic questions were investigated using mtSSU rDNA sequences.
The analysis applying a Bayesian approach suggests that the pyrenocarpous Porinaceae are close to Graphidaceae or Gyalectaceae. Together with these families the Porinaceae share a special type of hemiangiocarpous ontogeny of the ascomata. This ontogenetic type gives apparently rise to both perithecia and apothecia. The closed fruitbodies of the Porinaceae are likely a result of neotenic ontogeny and are correlated with special hymenial characters: rather thin walled narrow asci, and a different consistency of the hymenial gels. Phylogenetic relationships within Porinaceae were investigated using a dataset that contains epiphyllous species of Porina as well as some species of Trichothelium. The results show that Porina in today’s circumscription is not monophyletic. The Porina nitidula group represented by P. repanda and P. subnitidula are closely related to Trichothelium. Morphological and especially chemical characters underline the phylogenetic results. Taxa of the Porina rufula group form a distinct monophyletic group. P. mirabilis and Phyllophiale alba are confirmed as synonyms and closely related to P. radiata.


The lichen genus Buellia De Not in the Greater Sonoran Desert Region: saxicolous species with one-septate ascospores

Bungartz, F. (1) & Nash III, T. H. (2)
(1) Botanische Staatssammlung München, Menzinger Straße 67, D-80638 München, Germany; (2) Arizona State University, School of Life Sciences, PO Box 87 4601, Tempe, AZ 85287

31 saxicolous species of Buellia s.l. with one-septate ascospores were examined from the Sonoran Desert Region. They are distinguished by morphology, anatomy, spore ultrastructure, secondary chemistry, ecology and distribution. All species are traditionally included within the Physciaceae. Recent molecular studies suggests that calicioid lichens are closely related to Buellia s.l. A comparison confirms that spore wall ultrastructure is identical in both groups. Iodine staining of the ascus in the light microscope corresponds well with ascus ultrastructure. The Bacidia-type is variable but common to all species. The prototunicate ascus of calicioid Physciaceae may have evolved by reduction from this type. Spore ontogeny of Buellia s.l. is very dynamic. Four stages can be distinguished: immature, premature, mature and overmature. During these stages wall differentiation, septum formation and pigmentation develop independently. Wall thickenings result from endospore differentiation, are most obvious in premature and mature spores, but become reduced with age. Exciple anatomy of Buellia s.l. is diagnostic and corresponds to some degree with pigmentation. Only B. mamillana has a thalline exciple, which soon becomes excluded. Other species lack a distinct thalline margin. Inconspicuous species show some degree of substrate penetration but only Buellia sequax and B. vilis are chasmolithic. Pycnidia of all species are similar and a general transfer of taxa with filiform conidia into Amandinea is not supported. Amandinea needs to be revised to include only species closely related to the type. A phylogenetic analysis demonstrates that most generic segregates of Buellia s.l. are not well supported. Groups like Diplotomma s.str. or Hafellia may be recognized but even these are not strongly supported. The low support is inherent to the small amount of characters. As a consequence, genera should only be accepted if classical concepts are re-evaluated with molecular tools.


Testing "species pair" concepts: An analysis of processes driving the evolution of Porpidia flavocoerulescens/P. melinodes

Buschbom, J. (1, 2) & Mueller, G.(2)
(1) University of Chicago, Committee on Evolutionary Biology, USA; (2) Field Museum of Natural History, Botany Department, USA

The evolutionary history and processes underlying the contrasting reproductive modes in the crustose, epilithic lichen-forming genus Porpidia (Porpidiaceae, Ascomycota) were investigated using an integrated phylogenetic and population-genetic approach. Embedded in a phylogenetic framework of tree and ancestral character state reconstructions at the genus-level, the study focused on the "species pair" P. flavocoerulescens (sexual) and P. melinodes (vegetative). The population-level processes that shape the phylogenetic patterns observed within the "species pair" were analyzed through a combination of pattern-orientated (hypothesis testing and visualizations based on tree and network reconstructions) and classical population-genetic approaches.
Stringent tests employing maximum likelihood and Bayesian approaches rejected previously proposed "species pair" hypotheses by Poelt (1963), Robinson (1975) and Tehler (1982). A detailed analysis of DNA-sequence variability revealed closely linked gene loci in combination with high levels of conflict within each of the gene fragments, as well as between observed, deeply divided genetic lineages. Such a pattern is neither congruent with concepts of exclusively asexual organisms nor with a process of cryptic speciation.
The observed results suggest that conflicting reproductive and nutritional requirements imposed by an obligate symbiotic lifestyle produce recurring selective sweeps within predominantly vegetatively reproducing lineages and are thus the main forces that shape the evolution within the investigated "species pair".


Phylogenetic analyses of the lichen family Umbilicariaceae based on material from Siberia

Davydov, E. A. (1), Persoh, D (2) & Rambold, G. (2)
(1) Altai State University, Barnaul, Russia; (2) University of Bayreuth, Bayreuth,

The Umbilicariaceae represent a distinctive and apparently isolated family of lichenized Ascomycetes. Within the Umbilicariaceae, genera, subgenera, and sections were distinguished based on different morphological characters (e.g. apothecium structure, number and types of spores, special morphological structures of the thallus). The classification of the family has been revised several times, and its relation to the Lecanoromycetes remained unclear.
The aim of this study was to investigate phylogenetic relationships among Siberian species of Umbilicariaceae by using morphological, chemical, and ribosomal DNA data. The major part of the work is based on fresh material from the Altai Mountains. To obtain a representative taxon selection DNA was also extracted from herbarium specimens collected in other regions. At least 2 specimens were included for each taxon. More then one hundred double stranded 5.8S/ITS nrDNA sequences were obtained from 35 described species of Umbilicaria and 8 of Lasallia, representing almost all known Siberian species, as well as representatives from the Russian Far East, Europe, and China.
Resulting phylogenetic trees from maximum parsimony and maximum likelihood analyses are comparatively discussed. Furthermore, the distribution of different morphological and anatomical characters used in traditional classification is discussed against the background of the ITS nrDNA phylogeny.


Phylogeny of pyrenocarpous lichens: implications for the evolution of ascomata

Del Prado, R. & Lumbsch, H.T.
Field Museum of Natural History, Dept. of Botany, 1400 S. Lake Shore Drive, Chicago, IL 60605 USA

The phylogenetic relationships of some groups of lichen-forming ascomycetes with perithecioid ascomata, such as Pyrenulales, Trichotheliales, Verrucariales, Arthopyreniaceae, Thelenellaceae, and the non-lichenized Chaetothyriales were investigated using sequence data of the nuclear LSU rDNA and the mitochondrial SSU rDNA. The combined dataset was analyzed using Bayesian, distance, and parsimony approaches. The molecular data allowed a better understanding of the phylogeny of lichen-forming pyrenomycetes and the evolution of their traits.
The lichenized pyrenomycetes basically fall into three groups: 1) the Arthopyreniaceae with bitunicate asci and strictly ascolocular ascoma development belong to the Dothideomycetes s. str., 2) some families, such as Thelenellaceae and Trichoteliales with unitunicate asci and ascohymenial ascoma development are derived independently within the usually discocarpous Lecanoromycetes, and 3) taxa with bitunicate asci, but ascohymenial development, including Pyrenulales, Verrucariales, and numerous lichenicolous fungi, belong to Chaetothyriomycetes. The last group is closely related to the non-lichenized Chaetothyriales. This raises the question of the type of ascoma development in this order.
The placement of pyrenocarpous taxa in the usually discocarpous Lecanoromycetes appears surprising. In morphologically based classifications, lichen-forming discomycetes were believed to be closely related to non-lichenized discomycetes, especially the Heliotales. However, the ascoma types in Lecanoromycetes and the developmental types occurring within this class are extremely variable.
In addition to the molecular studies, the ontogeny of ascoma types was also studied in these groups to reach a better understanding of the evolution of the fruiting bodies in lichen-forming fungi and related non-lichenized taxa.


Molecular phylogeny of the genus Stereocaulon

Högnabba, F.
Department of Biological and Environmental Sciences, P.O.Box 65, FIN-00014
University of Helsinki, Finland

Species typically included in the lichen genus Stereocaulon have a dimorphic thallus consisting of a crustose primary thallus and a fruticose secondary thallus. In the currently valid infrageneric classification of the genus, three subgenera, a number of sections and subsections are recognized based on anatomical, morphological and chemical characters. However, the traditional view of Stereocaulon has changed in recent years as five crustose species completely lacking the secondary thallus have been transferred to the genus based on similarities in anatomy, morphology and chemistry. This transfer was confirmed in a molecular study where representatives of the crustose Stereocaulon where nested within the genus together with the monotypic crustose genus Muhria, which shares many features with Stereocaulon.
In the present study the phylogenetic relationships of Stereocaulon, including the crustose species and Muhria, have been examined by using direct optimization of DNA sequences from the ITS 1-5.8s-ITS 2 rDNA gene cluster and the protein-coding ß-tubulin gene. The results support the inclusion of the crustose Stereocaulon and Muhria while the current infrageneric classification is not supported as such. Further, the species concept for some species need to be re-examined as they do not form monophyletic entities in the phylogeny.


A phylogenetic study of Siphula

Kantvilas, G. (1) & Grube, M. (2)
(1) Tasmanian Herbarium, Private Bag 4, Hobart, Tasmania 7001, Australia; (2) Institute of Plant Sciences, Karl-Franzens University Graz, Holteigasse 6, 8010 Graz, Austria

The genus Siphula comprises c. 25 species of strictly sterile fruticose or foliose lichens. The results of a phylogenetic investigation are presented using several loci of nuclear and mitochondrial ribosomal DNA. The ITS molecular data support the existing infra-generic classification that was based on correlations between chemistry and morphology, and the results also show that the genus in its traditional circumscription is not monophyletic. Instead there are two main lineages within the phylogeny of ascomycetes. The first lineage consists of strictly cool to cold temperate Southern Hemisphere species (including S. complanata, S. elixii, S. foliacea, S. fragilis, S. georginae and S. jamesii). This group is characterised by a foliose to fruticose, generally very fragile, typically grey-brown to brown thallus, either lacking lichen substances or containing depsidones or dibenzofuranes. The relationships of this group with other ascomycete lineages is discussed. The second lineage includes the type species S. ceratites as well as several others (S. coriacea, S. decumbens, S. gracilis, S. fastigiata and S. pickeringii). These taxa typically have a rather chalky, fruticose thallus and contain depsides and/or chromones. Several species of this group are geographically widespread from temperate to montane, tropical areas, but local endemics are also known. The variation within the groups is fairly low in the mitochondrial datasets, while nuclear LSU rDNA provides additional information on the relationships between species. Relationships within the S. ceratites lineage as a whole, and its relationships with the genus Icmadophila remain to be analysed in detail.


Chemical variation in the Ramalina siliquosa chemotype complex: revisiting an old question with new methods

LaGreca, S.
The Natural History Museum, London, United Kingdom

Many morphologically-defined lichen species comprise multiple, morphologically indistinguishable chemical races, or chemotypes. The aim of the present study is to provide a more detailed picture of chemotype variation in lichenized fungi using analyses of molecular phylogenetic data. My study group, the Ramalina siliquosa complex, offers the best-documented example of this variation. A review of traditional taxonomic studies of these lichens shows that there has been little agreement regarding the status or relationships of the chemotypes. In recent years, molecular-genetic studies of the Ramalina siliquosa complex have been attempted, but with similarly mixed results. A study by Mattsson of a cliff in Sweden, utilizing isozymes, showed that each of the chemotypes yielded a different isozyme pattern, and thus should be accepted as distinct taxonomic entities. Culberson et al. came to a similar conclusion using chemical analyses of spore progeny from thalli collected on a cliff in Wales, except that two of the chemotypes emerged as a clear polymorphism. Most recently, I have completed a pilot molecular study of the genus Ramalina using nuclear rDNA ITS and RPB2 sequences, including twelve samples from the R. siliquosa complex selected from throughout the range of the complex. These samples sort into two distinct groups, each comprising three chemotypes, which supports the two-species taxonomy advocated by Sheard, Purvis et al., and others. Surprisingly, these two groups are not sister to each other. Future work includes testing the monophyly of the R. siliquosa complex further, and determine its exact relationships within the genus. An expanded analysis of the R. siliquosa group, including more samples and more genes, is also planned.


New insights in the phylogenetic relationships of lichenized Pezizomycotina

Persoh, D.
Universität Bayreuth, Lehrstuhl für Pflanzensystematik, Universitätsstraße 30 - NW I, D-95440 Bayreuth, Germany

Within the last decade, many molecular studies in mycology focussed on the relationships among lichenized Ascomycetes. Unfortunately, published phylogenies were often contradictory with respect to the taxa at higher taxonomic levels. To achieve a more convincing overall picture of phylogenetic relationships among these groups, we set up an alignment of all publicly available SSU nrDNA sequences of the Pezizomycotina and added sequences representing hitherto neglected or under-represented groups. Sequences not matching certain quality criteria (sequence
length, number of ambiguous bases, and agreement with secondary structure information) were discarded. The resulting alignment of 750 taxa was analysed with eight different phylogenetic algorithms, representing the three optimality criteria Distance, Parsimony, and Likelihood. For the publication of the extensive phylogenetic trees a new online database was established (www.mycophylogeny.net). The resulting tree topologies are comparatively discussed with respect to length, likelihood, overall resolution, and agreement with the current classification system of the Pezizomycotina. Furthermore, the impact of ambiguous sequences on the tree topology is addressed. Finally, the phylogenetic position of selected groups with closer relationship to Lecanorales (i.e., Acarosporaceae, Lichinales, Mycocaliciaceae, Umbilicariaceae) is discussed against the background of the phylogenetic tree best revealing the overall results of the various analyses.


Phylogenetic methods and the delimitation and detection of lichen species

Printzen, C. (1) & Ekman, S. (2)
(1) Forschungsinstitut Senckenberg, Abteilung Botanik/Paläobotanik, Senckenberganlage 25, D-60325 Franfurt am Main, Germany; (2) Universitetet i Bergen, Institutt for Biologi, Postboks 7800, N-5020 Bergen, Norway

The delimitation of lichen species is a controversial issue. One example for this is the question whether closely related asexual and sexual individuals represent species pairs or morphotypes of a single species. In the past, some authors concluded that presumed species pairs in fact represent single species. Conversely, results based on genealogical concordance among phylogenetic trees indicated the presence of additional „cryptic species". In order to explore whether phylogenetic methods are appropriate for the detection and delimitation of lichen species, we simulated datasets under different models of population history and different recombination and mutation rates. We investigated for how long recombination in an ancestral species remains detectable after cessation of gene flow among populations and tested whether high bootstrap values of single branches indicate the absence of recombination among clades in a multigene phylogeny. Traces of ancestral recombination persist in populations long after actual cessation of recombination. Hence, the detection of recombination is a poor predictor of ongoing gene flow. Decreasing mutation rate and increasing recombination rate cause a decline in the proportion of branches with high bootstrap support. The influence of mutation rate is more pronounced than that of recombination rate. Therefore, a high bootstrap value does not necessarily indicate reproductive isolation of the respective clade, but rather a high number of uncontradicted characters that support this clade. As an alternative to phylogenetic methods and a phylogenetic species concept we illustrate the cohesion species concept and methods based on haplotype networks in the case of Cavernularia hultenii and C. lophyrea. These species do not form monophyletic groups in a phylogenetic analysis but the association of morphological differences with genetical differences is statistically supported.


Molecular phylogeny and character evolution in Graphidaceae (Ostropomycetidae)

Staiger, B. (1) & Grube, M. (2)
(1) Institute of Botany, University of Regensburg, Universitätsstrasse 31, D-93040 Regensburg, Germany; (2) Institute of Plant Sciences, Karl-Franzens-University Graz, Holteigasse 6, A-8010 Graz, Austria

The generic concepts in Graphidaceae based on spore characters ("spore genera") were introduced by Müller Arg. and – although being considered as artificial – persisted for more than 100 years. Only recently, extensive phenotypic studies by the first author lead to a revision of this "old-fashioned" spore-based system: New generic concepts included ascocarp morphology, ascospore type and secondary chemistry, and both several old generic names were reintroduced and two new genera were described. A molecular evaluation of the recent intrafamiliar classification of Graphidaceae is presented here. We analysed mtSSU and nLSU rDNA sequence data to investigate the monophyly of the newly proposed genera and to trace character evolution. Thereby special attention was drawn to morphological characters such as excipular structure, carbonization, paraphysis-type and spore-type being considered as important features in respect of generic delimitation. Species of the genus Fissurina (including species with well-developed excipular labia) form a well supported clade basal to all other graphids. The families Thelotremataceae and Graphidaceae as presently circumscribed are not monophyletic. Both datasets (mtSSU & nLSU rDNA) as well as the combined dataset does not resolve Graphis as monophyletic genus – two distinct and well supported clades are present. Albeit we confirm heterogeneity within Graphis with phenotypic data, the distant position of Graphis species is surprising. Glyphis as defined by Staiger is monophyletic in the present study. This genus includes species with ascocarpia embedded in stromata, species without stromata and round "divergent" ascocarps (distinctly open discs) as well as species with lirelline Graphis-like, "convergent" ascocarps and narrow, slit-like discs. In contrast, although showing uniform ascocarp morphology, Hemithecium as recently delimited is not monophyletic: One sample (belonging to the subgenus Leucogramma with brownish-coloured ascospores) belongs to a larger clade of brown-spored taxa; H. implicatum with hyaline spores (belonging to the subgenus Hemithecium) clusters inside one of the Graphis-clades suggesting that the uncarbonized exciple is a homoplasious character in Graphidaceae.


Phylogenetic relationships of the Lecanoromycetes utilizing combined mtSSU rDNA and nLSU rDNA sequence datasets

Wiklund, E. (1), Lumbsch, T. (2), Ekman, S. (3), Crewe, A. (1) & Wedin, M. (1)
(1) Dept. of Ecology and Environmental Science, Umeå University, SE-901 87 Umeå, Sweden; (2) Field Museum of Natural History, Dept. of Botany, 1400 S. Lake Shore Drive, Chicago, IL 60605 USA; (3) Universitetet i Bergen, Institutt for Biologi, Postboks 7800,N-5020 Bergen, Norway

Lichenization is one of the most important ways of life among ascomycetes, and most lichenized ascomycetes are currently classified in the Lecanoromycetes, a group where the delimitation and ranking of subgroups vary considerably between recent classifications. We have investigated the phylogenetic relationships within the Lecanoromycetes and closely related groups, using maximum parsimony, parsimony jackknifing and Bayesian (MCMC) analyses based on mtSSU rDNA and nLSU rDNA sequence datasets. Here, we will discuss some of the well supported groupings and a selection of relevant morphological characters. In particular, the 'Umbilicariaceae'-group, which was highlighted as in need of further studies in earlier investigations, and the 'Acarosporaceae'-group, will be examined.

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