Martin Kemler

Anther smuts of Caryophyllaceae: molecular characters indicate host-dependent species delimitation +

Phylogenetic relationships of Microbotryum species (Urediniomycetes, Basidiomycota) inhabiting anthers of Caryophyllaceae were investigated by molecular analyses using internal transcribed spacer (ITS) sequences and collections from different host plants. The data show that the current taxonomy of Microbotryum on Caryophyllaceae is only partly satisfactory. Microbotryum violaceum is confirmed to be a paraphyletic grouping and is split up in monophyletic groups. Microbotryum silenesinflatae and M. violaceo-verrucosum appear as polyphyletic. Host data are in good agreement with molecular results. Two new species, Microbotryum chloranthae-verrucosum and M. saponariae, are described based on morphological, ecological, and molecular characteristics. An emended circumscription of Microbotryum dianthorum is given. The name Ustilago major (= Microbotryum major) is lectotypified.

Implications of molecular characters for the phylogeny of the Microbotryaceae (Basidiomycota: Urediniomycetes)

BackgroundAnther smuts of the basidiomycetous genus Microbotryum on Caryophyllaceae are important model organisms for many biological disciplines. Members of Microbotryum are most commonly found parasitizing the anthers of host plants in the family Caryophyllaceae, however they can also be found on the anthers of members of the Dipsacaceae, Lamiaceae, Lentibulariaceae, and Portulacaceae. Additionally, some members of Microbotryum can be found infecting other organs of mainly Polygonaceae hosts. Based on ITS nrDNA sequences of members of almost all genera in Microbotryaceae, this study aims to resolve the phylogeny of the anther smuts and their relationship to the other members of the family of plant parasites. A multiple analysis strategy was used to correct for the effects of different equally possible ITS sequence alignments on the phylogenetic outcome, which appears to have been neglected in previous studies.ResultsThe genera of Microbotryaceae were not clearly resolved, but alignment-independent moderate bootstrap support was achieved for a clade containing the majority of the Microbotryum species. The anther parasites appeared in two different well-supported lineages whose interrelationship remained unresolved. Whereas bootstrap support values for some clades were highly vulnerable to alignment conditions, other clades were more robustly supported. The differences in support between the different alignments were much larger than between the phylogenetic optimality criteria applied (maximum parsimony and maximum likelihood).ConclusionThe study confirmed, based on a larger dataset than previous work, that the anther smuts on Caryophyllaceae are monophyletic and that there exists a native North American group that diverged from the European clade before the radiation of the European species. Also a second group of anther smuts was revealed, containing parasites on Dipsacaceae, Lamiaceae, and Lentibulariaceae. At least the majority of the parasites of Asteraceae appeared as a monophylum, but delimitations of some species in this group should be reconsidered. Parasitism on Polygonaceae is likely to be the ancestral state for the Microbotryaceae on Eudicot hosts.

Anther smuts of Caryophyllaceae: molecular analyses reveal further new species.

Recent collections of Microbotryum (Pucciniomycotina, Basidiomycota) specimens inhabiting anthers of different caryophyllaceous host plants were analysed using LM and electron microscopy, as well as molecular phylogenetic analyses using rDNA (ITS and LSU) sequences. The phylogenetic relationships of caryophyllaceous anther parasites are discussed. Three new species, Microbotryum adenopetalae, M. minuartiae, and M. silenes-acaulis, are described based on morphological, ecological, and molecular characteristics. New host plants are reported for Microbotryum dianthorum (Dianthus jacquemontii and Petrorhagia saxifraga) and M. lychnidis-dioicae (Cucubalus baccifer and Silene zawadskii). For Microbotryum violaceum, a neotype is selected.

Ion Torrent PGM as Tool for Fungal Community Analysis: A Case Study of Endophytes in Eucalyptus grandis Reveals High Taxonomic Diversity

The Kingdom Fungi adds substantially to the diversity of life, but due to their cryptic morphology and lifestyle, tremendous diversity, paucity of formally described specimens, and the difficulty in isolating environmental strains into culture, fungal communities are difficult to characterize. This is especially true for endophytic communities of fungi living in healthy plant tissue. The developments in next generation sequencing technologies are, however, starting to reveal the true extent of fungal diversity. One of the promising new technologies, namely semiconductor sequencing, has thus far not been used in fungal diversity assessments. In this study we sequenced the internal transcribed spacer 1 (ITS1) nuclear encoded ribosomal RNA of the endophytic community of the economically important tree, Eucalyptus grandis, from South Africa using the Ion Torrent Personal Genome Machine (PGM). We determined the impact of various analysis parameters on the interpretation of the results, namely different sequence quality parameter settings, different sequence similarity cutoffs for clustering and filtering of databases for removal of sequences with incomplete taxonomy. Sequence similarity cutoff values only had a marginal effect on the identified family numbers, whereas different sequence quality filters had a large effect (89 vs. 48 families between least and most stringent filters). Database filtering had a small, but statistically significant, effect on the assignment of sequences to reference sequences. The community was dominated by Ascomycota, and particularly by families in the Dothidiomycetes that harbor well-known plant pathogens. The study demonstrates that semiconductor sequencing is an ideal strategy for environmental sequencing of fungal communities. It also highlights some potential pitfalls in subsequent data analyses when using a technology with relatively short read lengths.

Species Accumulation Curves and Incidence-Based Species Richness Estimators to Appraise the Diversity of Cultivable Yeasts from Beech Forest Soils

Background Yeast-like fungi inhabit soils throughout all climatic zones in a great abundance. While recent estimations predicted a plethora of prokaryotic taxa in one gram of soil, similar data are lacking for fungi, especially yeasts. Methodology/Principal Findings We assessed the diversity of soil yeasts in different forests of central Germany using cultivation-based techniques with subsequent identification based on rDNA sequence data. Based on experiments using various pre-cultivation sample treatment and different cultivation media we obtained the highest number of yeasts by analysing mixed soil samples with a single nutrient-rich medium. Additionally, several species richness estimators were applied to incidence-based data of 165 samples. All of them predicted a similar range of yeast diversity, namely 14 to 16 species. Randomized species richness curves reached saturation in all applied estimators, thus indicating that the majority of species is detected after approximately 30 to 50 samples analysed. Conclusions/Significance In this study we demonstrate that robust species identification as well as mathematical approaches are essential to reliably estimate the sampling effort needed to describe soil yeast communities. This approach has great potential for optimisation of cultivation techniques and allows high throughput analysis in the future.

Hidden diversity in the non-caryophyllaceous plant-parasitic members of Microbotryum (Pucciniomycotina: Microbotryales)

Abstract Members of the fungal genus Microbotryum are well‐known parasites on eudicotyledonous plant hosts. However, recent studies focused exclusively on Microbotryum species being parasites in the anthers of Caryophyllaceae in which strong host‐specificity was confirmed by molecular analyses. Consequently, species numbers have risen considerably as multi‐host parasites were split up in so‐called cryptic species. We subjected three non‐caryophyllaceous Microbotryum groups to molecular phylogenetic analyses to see whether we would confirm multi‐host morphospecies or if host‐specific cryptic species in these selected groups could be revealed as well (i.e. a group of non‐caryophyllaceous anther smuts, parasites on different Fallopia species, and parasites on Polygonum bistorta and Polygonum vi‐viparum). We applied a multiple analysis strategy to correct for varying alignment effects on a two‐locus dataset (ITS and LSU rDNA). The results obtained by the different approaches are uniform; high host‐specificity exists in the non‐ caryophyllaceous anther smuts, but overlapping host ranges occur in the parasites of Fallopia species. Results for the parasites of Polygonum are similar, with Microbotryum bistortarum being separated into three lineages and M. marginale forming a lineage on P. bistorta which apparently is conspecific with M. bistortarum p.p. Our study shows that phylogenetic patterns within Microbotryum are much more complicated than deduced from morphological observations alone. Even though Microbotryum species are highly host‐specific, it is impossible to identify species based solely on host taxa affiliation. Species status is reinstated for the anther smut on Salvia pratensis.

Microbotryum heliospermae, a new anther smut fungus parasitic on Heliosperma pusillum in the mountains of the European Alpine System.

The members of the smut genus Microbotryum are pathogens of a wide range of host plant species from nine dicotyledonous families. Within the genus, the species sporulating in anthers of Caryophyllaceae form a monophyletic group that in recent years attracted much interest in various biological studies. The phylogenetic framework developed for species delimitation within Microbotryum revealed that high level host-specificity is a major feature of most caryophyllaceous anther smuts. However, the great number of anther smut specimens on diverse host plant species reported worldwide has still not been included in phylogenetic analyses due to the inaccessibility of recently collected specimens, and thus many species remain still undiscovered. In this study, anther smut specimens on Heliosperma pusillum originating from all main mountain ranges of the European Alpine System were examined using partial rDNA sequence and/or morphological analyses. The investigation revealed that all specimens are morphologically uniform and phylogenetically represent a monophyletic lineage, sister to Microbotryum lagerheimii complex on Atocion rupestre/Silene lacera/Silene vulgaris/Viscaria vulgaris. This lineage cannot be attributed to any of the previously described species, and therefore the smut in anthers of H. pusillum is described and illustrated here as a new species, Microbotryum heliospermae. The species is known from subalpine zone of the Alps, the Carpathians, the Dinaric Alps, and the Pyrenees, inhabiting host plants growing in open spring communities or semihumid mountain meadows.

Botryosphaeria dothidea : a latent pathogen of global importance to woody plant health

Botryosphaeria dothidea is the type species of Botryosphaeria (Botryosphaeriaceae, Botryosphaeriales). Fungi residing in this order are amongst the most widespread and important canker and dieback pathogens of trees worldwide, with B. dothidea one of the most common species on a large number of hosts. Its taxonomic circumscription has undergone substantial change in the past decade, making it difficult to interpret the large volume of literature linked to the name B. dothidea. This pathogen profile synthesizes the current understanding of B. dothidea pertaining to its distribution, host associations and role as a pathogen in managed and natural woody environments. The prolonged latent infection or endophytic phase is of particular importance, as it implies that the fungus can easily pass undetected by quarantine systems in traded living plants, fruits and other plant parts. Infections typically become obvious only under conditions of host stress, when disease symptoms develop. This study also considers the knowledge emerging from the recently sequenced B. dothidea genome, elucidating previously unknown aspects of the species, including mating and host infection strategies. Despite more than 150 years of research on B. dothidea, there is clearly much to be learned regarding this global tree pathogen. This is increasingly important given the stresses imposed on various woody hosts as a result of climate change. TAXONOMY Botryosphaeria dothidea (Moug. ex Fr) Ces. & De Not, 1863. Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Botryosphaeriales, Family Botryosphaeriaceae, Genus Botryosphaeria, Species dothidea. HOST RANGE Confirmed on more than 24 host genera, including woody plants, such as Acacia (= Vachellia), Eucalyptus, Vitis and Pistachio. DISEASE SYMPTOMS Associated with twig, branch and stem cankers, tip and branch dieback, fruit rot, blue stain and plant death. USEFUL WEBSITES The Botryosphaeria site for detailed morphological descriptions (http://www.crem.fct.unl.pt/botryosphaeria_site/); Systematic Mycology and Microbiology Laboratory Fungal Database for all literature and associated hosts (https://nt.ars-grin.gov/fungaldatabases/); TreeBASE link for the combined ITS and TEF-1α tree (http://purl.org/phylo/treebase/phylows/study/TB2:S18906); DOE Joint Genome Institute, JGI Mycocosm for the Botryosphaeria dothidea genome (http://genome.jgi.doe.gov/Botdo1_1/Botdo1_1.home.html).

Contrasting phylogenetic patterns of anther smuts (Pucciniomycotina: Microbotryum) reflect phylogenetic patterns of their caryophyllaceous hosts

Anther smuts in the genus Microbotryum often show very high host specificity toward their caryophyllaceous hosts, but some of the larger host groups such as Dianthus are crucially undersampled for these parasites so that the question of host specificity cannot be answered conclusively. In this study we sequenced the internal transcribed spacer (ITS) region of members of the Microbotryum dianthorum species complex as well as their Dianthus hosts. We compared phylogenetic trees of these parasites including sequences of anther smuts from other Caryophyllaceae, mainly Silene, with phylogenies of Caryophyllaceae that are known to harbor anther smuts. Additionally we tested whether observed patterns in parasites are due to shared ancestry or if geographic separation is a factor that should be taken into consideration in delimitating species. Parasites on Dianthus showed mainly an arbitrary distribution on Dianthus hosts, whereas parasites on other Caryophyllaceae formed well-supported monophyletic clades that corresponded to restricted host groups. The same pattern was observed in the Caryophyllaceae studied: morphologically described Dianthus species did not correspond well with monophyletic clades based on molecular data, whereas other Caryophyllaceae mainly did. We suggest that these different patterns primarily result from different breeding systems and speciation times between different host groups as well as difficulties in species delimitations in the genus Dianthus.

Microbotryum silenes-saxifragae sp. nov. sporulating in the anthers of Silene saxifraga in southern European mountains.

Currently, the monophyletic lineage of anther smuts on Caryophyllaceae includes 22 species classified in the genus Microbotryum. They are model organisms studied in many disciplines of fungal biology. A molecular phylogenetic approach was used to resolve species boundaries within the caryophyllaceous anther smuts, as species delimitation based solely on phenotypic characters was problematic. Several cryptic species were found amongst the anther smuts on Caryophyllaceae, although some morphologically distinct species were discernible, and most species were characterized by high host-specificity. In this study, anther smut specimens infecting Silene saxifraga were analysed using rDNA sequences (ITS and LSU) and morphology to resolve their specific status and to discuss their phylogenetic position within the lineage of caryophyllaceous anther smuts. The molecular phylogeny revealed that all specimens form a monophyletic lineage that is supported by the morphological trait of reticulate spores with tuberculate interspaces (observed in certain spores). This lineage cannot be attributed to any of the previously described species, and the anther smut on Silene saxifraga is described and illustrated here as a new species, Microbotryum silenes-saxifragae. This species clusters in a clade that includes Microbotryum species, which infect both closely and distantly related host plants growing in diverse ecological habitats. It appears possible that host shifts combined with changes to ecological host niches drove the evolution of Microbotryum species within this clade.