Stefan Ekman

The Limitations of Ancestral State Reconstruction and the Evolution of the Ascus in the Lecanorales (Lichenized Ascomycota)

Ancestral state reconstructions of morphological or ecological traits on molecular phylogenies are becoming increasingly frequent. They rely on constancy of character state change rates over trees, a correlation between neutral genetic change and phenotypic change, as well as on adequate likelihood models and (for Bayesian methods) prior distributions. This investigation explored the outcomes of a variety of methods for reconstructing discrete ancestral state in the ascus apex of the Lecanorales, a group containing the majority of lichen-forming ascomycetes. Evolution of this character complex has been highly controversial in lichen systematics for more than two decades. The phylogeny was estimated using Bayesian Markov chain Monte Carlo inference on DNA sequence alignments of three genes (small subunit of the mitochondrial rDNA, large subunit of the nuclear rDNA, and largest subunit of RNA polymerase II). We designed a novel method for assessing the suitable number of discrete gamma categories, which relies on the effect on phylogeny estimates rather than on likelihoods. Ancestral state reconstructions were performed using maximum parsimony and maximum likelihood on a posterior tree sample as well as two fully Bayesian methods. Resulting reconstructions were often strikingly different depending on the method used; different methods often assign high confidence to different states at a given node. The two fully Bayesian methods disagree about the most probable reconstruction in about half of the nodes, even when similar likelihood models and similar priors are used. We suggest that similar studies should use several methods, awaiting an improved understanding of the statistical properties of the methods. A Lecanora-type ascus may have been ancestral in the Lecanorales. State transformations counts, obtained using stochastic mapping, indicate that the number of state changes is 12 to 24, which is considerably greater than the minimum three changes needed to explain the four observed ascus apex types. Apparently, the ascus in the Lecanorales is far more apt to change than has been recognized. Phylogeny corresponds well with morphology, although it partly contradicts currently used delimitations of the Crocyniaceae, Haematommataceae, Lecanoraceae, Megalariaceae, Mycoblastaceae, Pilocarpaceae, Psoraceae, Ramalinaceae, Scoliciosporaceae, and Squamarinaceae.

Phylogeography of Cavernularia hultenii: evidence of slow genetic drift in a widely disjunct lichen

Population structure and history is poorly known in most lichenized ascomycetes. Many species display large‐scale infraspecific disjunctions, which have been explained alternately by range fragmentation in species of high age and widespread long‐distance dispersal. Using the lichen Cavernularia hultenii, which is widely disjunct across North America and Europe, Pleistocene and Holocene population history was inferred. The internal transcribed spacer (ITS) and part of the the intergenic spacer (IGS) region of the nuclear ribosomal DNA were sequenced in 300 individuals representing 62 populations across the range of the species. While four ancestral haplotypes are found in all areas, none of the observed tip haplotypes is present in more than one of the three part ranges. Although this is evidence for a past fragmentation event, nested clade analysis (NCA) remains equivocal in the choice between allopatric fragmentation and long‐distance dispersal. Mismatch distributions indicate exponential population growth, probably during postglacial invasion of C. hultenii into formerly glaciated areas of western North America. The presence of one southern and at least one northern glacial refugium in South Central Alaska is inferred. Evidence for another refugium in the Queen Charlotte Islands or Alexander Archipelago is inconclusive because of sparse sampling. However, a range expansion was not confirmed unambiguously by NCA. The limited power of NCA to infer past range fragmentations and expansions is due apparently to the shallow haplotype network and widespread ancestral haplotypes. This can be explained by slow genetic drift causing incomplete removal of ancestral haplotypes from the postfragmentation and postexpansion areas.

Phylogenetic relationships of Lecanoromycetes (Ascomycota) as revealed by analyses of mtSSU and nLSU rDNA sequence data

The phylogeny of Lecanoromycetes (Ascomycota, Fungi) is investigated utilizing parsimony and Bayesian Markov Chain Monte Carlo analyses, of combined nLSU rDNA and mtSSU rDNA sequence datasets. The results suggest that Acarosporaceae, Candelariaceae, Phlyctis and Pycnora are not members of the monophyletic Lecanorales, and that Timdalia and Pleopsidium are members of a monophyletic Acarosporaceae. Pycnora, Candelariaceae and Acarosporaceae form a monophyletic group. Umbilicariaceae, Hypocenomyce scalaris, H. friesii, Ophioparmaceae, Boreoplaca, Elixia and Fuscidea form either a basal paraphyletic assemblage in Lecanoromycetes, or a monophyletic group which is the sister-group to Lecanorales and the rest of Lecanoromycetes (excluding Acarosporaceae). The Acarosporaceae forms a group with Pycnora and Candelariaceae, which may be outside the Lecanoromycetes. Chaetothyriales, Verrucariales, Eurotiales, Lichinales and Mycocaliciales form a monophyletic group, but with low support. We briefly discuss incongruence between datasets from different genetic markers, comparing the differences between the separate parsimony analyses, where the ILD test indicated a very significant incongruence. The phylogenetic significance of ascus-types that have influenced most recent Ascomycota classifications heavily is also discussed, and we finally point out risks with formalizing classifications too early.

Most species of Lepraria and Leproloma form a monophyletic group closely related to Stereocaulon

The phylogenetic position of members of the entirely asexually reproducing lichen-forming genera Lepraria and Leproloma was investigated using sequence data from the ITS1-5.8S-ITS2 and small subunit (SSU) nuclear ribosomal DNA. Phylogenetic reconstructions were carried out using a likelihood-based Bayesian Markov chain Monte Carlo (MCMC) tree sampling technique and the unweighted least squares optimality criterion, the latter based on maximum likelihood distances obtained via an alignment-free distance estimation technique. The results indicate that most species currently referred to Lepraria and Leproloma form a single monophyletic group. This monophyletic group is the sister group to Stereocaulon and Muhria and belongs in the Stereocaulaceae (Lecanorales, Lecanoromycetes, Ascomycota ). This indicates that an ancestor of Lepraria switched from a sexual to an asexual mode of dispersal. Subsequent speciation must have taken place in the absence of sexual processes, which contradicts the view of asexual taxa as ‘evolutionary dead ends’. Leproloma is polyphyletic and nested within Lepraria. Lepraria flavescens is a Lecanora probably belonging in subgenus Glaucomaria. Lepraria lesdainii and L. obtusatica are unrelated to each other and to other species currently referred to Lepraria or Leproloma. Leprocaulon and Crocynia are distantly related to the core group of Lepraria and Leproloma.

Molecular phylogeny of the Bacidiaceae (Lecanorales, lichenized Ascomycota)

The phylogeny of the family Bacidiaceae (Lecanorales, Ascomycota) was investigated using 65 nuclear ITS1-5,8S-ITS2 ribosomal DNA sequences, 63 of which were newly determined. After exclusion of ambiguous alignment, the data set contained 285 variable characters, 212 of which were parsimony-informative. Phylogenetic estimations were performed with maximum parsimony (unweighted and weighted) and maximum likelihood optimality criteria. Four different phylogenetic hypotheses were tested using a parametric bootstrap approach to simulate the expected null distribution of the difference between the globally optimal tree and the best (constrained) tree agreeing with the null hypotheses under unweighted and weighted parsimony, and maximum likelihood: (1) the genus Bacidia is monophyletic; (2) the genus Bacidina is monophyletic; (3) the genus Toninia is monophyletic; and (4) the family Ramalinaceae is monophyletic and distinct from a monophyletic Bacidiaceae. The monophyly of Bacidia, Toninia, and the Ramalinaceae was rejected under all circumstances. Hence, Bacidiaceae is likely to be a younger synonym of Ramalinaceae. The monophyly of Bacidina was not rejected under any optimality criterion. Furthermore, the data set suggests that the Bacidia beckhausii and B. sabuletorum groups are unrelated to Bacidia s. str., that Megalaria is monophyletic, and that Lecania auct. is polyphyletic.

Genetic variation and population differentiation in the lichen-forming ascomycete Xanthoria parietina on the island Storfosna, central Norway.

Genetic diversity and fine‐scale population structure in the lichen‐forming ascomycete Xanthoria parietina was investigated using sequence variation in part of the intergenic spacer (IGS) and the complete internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA. Sampling included 213 and 225 individuals, respectively, from seven populations in two different habitats, bark and rock, on the island Storfosna off the central west coast of Norway. Both markers revealed significant variation and a total of 10 IGS and 16 ITS haplotypes were found. There were no signs of significant positive spatial autocorrelation at any spatial size class down to 10% of transect length, nor did we find significant deviations from neutrality or signs of historical population expansion. Analysis of molecular variance (amova) indicated that most of the genetic variance observed was within populations, but when populations were grouped according to habitat, more than a quarter of the variance was explained among groups. Pairwise comparisons of populations (FST, exact tests of population differentiation) revealed significant differentiation between populations in different habitats (on bark or rock), but not between populations in the same habitat. Haplotype networks show that internal and presumably old haplotypes are shared between habitats, whereas terminal haplotypes tend to be unique to a habitat, mostly bark. We interpret the observed pattern to mean that there is no evidence of restricted gene flow between populations in the same habitat at the present spatial scale (interpopulation distances one or a few kilometres). On the other hand, differentiation between habitats is considerable, which we attribute to restricted gene flow between habitats (habitat isolation). Evidence suggests that the observed differentiation did not evolve locally. Estimates of divergence time between populations in the respective habitats indicate that an ancestral population started to diverge at least 34 000 years ago but probably much further back in time.

Phylogeny and character evolution in the jelly fungi (Tremellomycetes, Basidiomycota, Fungi)

The Tremellomycetes (Agaricomycotina, Basidiomycota, Fungi) are a nutritionally heterogeneous group comprising saprotrophs, animal parasites, and fungicolous species (fungal-inhabiting, including lichen-inhabiting). The relationships of many species, particularly those with a lichenicolous habit, have never been investigated by molecular methods. We present a phylogeny of the Tremellomycetes based on three nuclear DNA ribosomal markers (nSSU, 5.8S and nLSU), representing all main taxonomic groups and life forms, including lichenicolous taxa. The Cystofilobasidiales, Filobasidiales, Holtermanniales, and Tremellales (including the Trichosporonales) are recovered as monophyletic, but this is not the case for the Tremellomycetes. We suggest, however, that the Cystofilobasidiales tentatively continue to be included in the Tremellomycetes. As currently circumscribed, the Filobasidiaceae, Sirobasidiaceae, Syzygosporaceae and Tremellaceae are non-monophyletic. Cuniculitremaceae, Sirobasidiaceae and Tetragoniomycetaceae are nested within Tremellaceae. The lichenicolous species currently included within the Tremellomycetes belong in this group, distributed across the Filobasidiales and Tremellales. Lichen-inhabiting taxa do not form a monophyletic group; they are distributed in several clades and sometimes intermixed with taxa of other nutritional habits. Character state reconstruction indicates that two morphological traits claimed to characterize groups in the Tremellomycetes (the basidium habit and basidium septation) are highly homoplastic. Comparative phylogenetic methods suggest that the transitions between single and catenulate basidia in the Tremellales are consistent with a punctuational model of evolution whereas basidium septation is likely to have evolved under a graduational model in the clade comprising the Holtermanniales, Filobasidiales, and Tremellales.

Slippery when wet: Phylogeny and character evolution in the gelatinous cyanobacterial lichens (Peltigerales, Ascomycetes)

Many lichen fungi form symbioses with filamentous Nostoc cyanobacteria, which cause the lichen to swell and become extremely gelatinous when moist. Within the Lecanoromycetes, such gelatinous lichens are today mainly classified in the Collemataceae (Peltigerales, Ascomycota). We performed Bayesian MCMC, maximum likelihood, and maximum parsimony analyses of three independent markers (mtSSU rDNA, nuLSU rDNA, and RPB1), to improve our understanding of the phylogeny and classification in the Peltigerales, as well as the evolution of morphological characters that have been used for classification purposes in this group. The Collemataceae and the non-gelatinous Pannariaceae are paraphyletic but can be re-circumscribed as monophyletic if Leciophysma, Physma, Ramalodium and Staurolemma are transferred to the Pannariaceae. The gelatinous taxa transferred to the Pannariaceae deviate from other Collemataceae in having simple ascospores, and several also have a ring-shaped exciple as in other Pannariaceae, rather than the disc-shaped exciple found in the typical Collemataceae. Both Collema and Leptogium are non-monophyletic. The re-circumscribed Collemataceae shares a distinct ascus type with the sister group Placynthiaceae and the Coccocarpiaceae, whereas Pannariaceae includes a variety of structures. All Pannariaceae have one-celled ascospores, whereas all Collemataceae have two- or multi-celled spores. Reconstructions of the number of character state transformations in exciple structure, thallus gelatinosity, and ascus apex structure indicate that the number of transformations is distinctly higher than the minimum possible. Most state transformations in the exciple took place from a ring-shaped to a disc-shaped exciple. Depending on the reconstruction method, most or all transformations in thallus structure took place from a non-gelatinous to a gelatinous thallus. Gains and losses of internal structures in the ascus apex account for all or a vast majority of the number of transformations in the ascus, whereas direct transformations between asci with internal structures appear to have been rare.

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 FST 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.

Contributions to an epiphytic lichen flora of northwest North America: I. Eight new species from British Columbia inland rain forests

Abstract Recent surveys of the inland rain forests of British Columbia and adjacent regions have brought to light an unexpectedly rich epiphytic lichen flora, including several species apparently new to science. In the first of a series of papers, we describe eight species discovered during these surveys as new: Absconditella amabilis T. Sprib. (Ostropales), Bacidina contecta S. Ekman & T. Sprib., Biatora aureolepra T. Sprib. & Tønsberg, Biatora ligni-mollis T. Sprib. & Printzen (all Lecanorales), Collema coniophilum Goward (Peltigerales), Pertusaria diluta C. Björk, G. Thor & T. Wheeler (Pertusariales), Schaereria brunnea C. Björk, T. Sprib. & T. Wheeler (Ostropomycetidae incertae sedis) and Scoliciosporum abietinum T. Sprib. (Lecanorales). We also call attention to a ninth species, Bacidina sp. A, a poorly known and possibly undescribed colonizer of moribund cyanolichens. A majority of the above species appear to be confined to old-growth forests, while two (Biatora ligni-mollis and Schaereria brunnea) are currently known only from “antique” forests older than about 500 years. Many additional undescribed epiphytic lichens are known from inland rain forests, underscoring the need for further baseline biodiversity research in light of its ongoing disappearance as a result of resource extraction. In addition to the eight new species, we report Absconditella celata as new to North America, Absconditella lignicola as new to Canada and Montana, Bacidina chloroticula as new to British Columbia and Gyalideopsis piceicola as new to Montana.