Audrius Menkis

Archaeorhizomycetes: Unearthing an Ancient Class of Ubiquitous Soil Fungi

Cultivation and cloning allow phylogenetic placement of a prominent fungal lineage. Estimates suggest that only one-tenth of the true fungal diversity has been described. Among numerous fungal lineages known only from environmental DNA sequences, Soil Clone Group 1 is the most ubiquitous. These globally distributed fungi may dominate below-ground fungal communities, but their placement in the fungal tree of life has been uncertain. Here, we report cultures of this group and describe the class, Archaeorhizomycetes, phylogenetically placed within subphylum Taphrinomycotina in the Ascomycota. Archaeorhizomycetes comprises hundreds of cryptically reproducing filamentous species that do not form recognizable mycorrhizal structures and have saprotrophic potential, yet are omnipresent in roots and rhizosphere soil and show ecosystem and host root habitat specificity.

Fungal communities in mycorrhizal roots of conifer seedlings in forest nurseries under different cultivation systems, assessed by morphotyping, direct sequencing and mycelial isolation

Fungi colonising root tips of Pinus sylvestris and Picea abies grown under four different seedling cultivation systems were assessed by morphotyping, direct sequencing and isolation methods. Roots were morphotyped using two approaches: (1) 10% of the whole root system from 30 seedlings of each species and (2) 20 randomly selected tips per plant from 300 seedlings of each species. The first approach yielded 15 morphotypes, the second yielded 27, including 18 new morphotypes. The overall community consisted of 33 morphotypes. The level of mycorrhizal colonisation of roots determined by each approach was about 50%. The cultivation system had a marked effect on the level of mycorrhizal colonisation. In pine, the highest level of colonisation (48%) was observed in bare-root systems, while in spruce, colonisation was highest in polyethylene rolls (71%). Direct internal transcribed spacer ribosomal DNA sequencing and isolation detected a total of 93 fungal taxa, including 27 mycorrhizal. A total of 71 (76.3%) fungi were identified at least to a genus level. The overlap between the two methods was low. Only 13 (13.9%) of taxa were both sequenced and isolated, 47 (50.5%) were detected exclusively by sequencing and 33 (35.5%) exclusively by isolation. All isolated mycorrhizal fungi were also detected by direct sequencing. Characteristic mycorrhizas were Phialophora finlandia, Amphinema byssoides, Rhizopogon rubescens, Suillus luteus and Thelephora terrestris. There was a moderate similarity in mycorrhizal communities between pine and spruce and among different cultivation systems.

Ecology and molecular characterization of dark septate fungi from roots, living stems, coarse and fine woody debris

The aim of the present work was to determine the identity and molecular relationships between 127 strains of dark septate (DS) fungi isolated from healthy root tips, decayed coarse roots, live healthy-looking stems, coarse (stumps, snags and logs) and fine (tree branches and tops) woody debris in temperate-boreal forests in Sweden and Lithuania. Sequence analysis of ITS rDNA was used to identify the fungi. In a neighbour-joining similarity tree, all sequences were grouped into five distinct clusters. Within each of these, ITS rDNA sequence variation consisted of 2-18 nucleotides, corresponding to 1-3% of their total length. The four least variable clusters were supported with high bootstrap values of 86-100%. Comparisons with the sequences in the GenBank database showed that all our strains had a 95-100% homology with identified Phialocephala species, and they were thus assigned to this genus. The representatives of two clusters were identified, as P. fortinii and P. dimorphospora. The representatives of three remaining clusters were defined as Phialocephala sp. 35, Phialocephala sp. 6 and Phialocephala sp. 18. Within each of these clusters, ITS rDNA sequence uniformity was higher than that observed within P. fortinii and P. dimorphospora. Consequently, their clusters were most discrete, supported with bootstrap values of 100%. Genetic variation in the five distinguished Phialocephala species and their possible ecological roles are discussed. Phialocephala sp. 6 was confined to healthy root tips of conifers. P. dimorphospora was only associated with dead woody tissue of P. abies. P. fortinii, Phialocephala sp. 18 and sp. 35 were isolated from both dead and living conifers and Betula pendula. In conclusion, the present study revealed the ability of fungi from the genus Phialocephala to colonise and persist in live and dead trees under strikingly different ecological conditions.

The Mating-Type Chromosome in the Filamentous Ascomycete Neurospora tetrasperma Represents a Model for Early Evolution of Sex Chromosomes

We combined gene divergence data, classical genetics, and phylogenetics to study the evolution of the mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma. In this species, a large non-recombining region of the mating-type chromosome is associated with a unique fungal life cycle where self-fertility is enforced by maintenance of a constant state of heterokaryosis. Sequence divergence between alleles of 35 genes from the two single mating-type component strains (i.e. the homokaryotic mat A or mat a-strains), derived from one N. tetrasperma heterokaryon (mat A+mat a), was analyzed. By this approach we were able to identify the boundaries and size of the non-recombining region, and reveal insight into the history of recombination cessation. The non-recombining region covers almost 7 Mbp, over 75% of the chromosome, and we hypothesize that the evolution of the mating-type chromosome in this lineage involved two successive events. The first event was contemporaneous with the split of N. tetrasperma from a common ancestor with its outcrossing relative N. crassa and suppressed recombination over at least 6.6 Mbp, and the second was confined to a smaller region in which recombination ceased more recently. In spite of the early origin of the first “evolutionary stratum”, genealogies of five genes from strains belonging to an additional N. tetrasperma lineage indicate independent initiations of suppressed recombination in different phylogenetic lineages. This study highlights the shared features between the sex chromosomes found in the animal and plant kingdoms and the fungal mating-type chromosome, despite fungi having no separate sexes. As is often found in sex chromosomes of plants and animals, recombination suppression of the mating-type chromosome of N. tetrasperma involved more than one evolutionary event, covers the majority of the mating-type chromosome and is flanked by distal regions with obligate crossovers.

Phylogenetic and biological species diversity within the Neurospora tetrasperma complex

The objective of this study was to explore the evolutionary history of the morphologically recognized filamentous ascomycete Neurospora tetrasperma, and to reveal the genetic and reproductive relationships among its individuals and populations. We applied both phylogenetic and biological species recognition to a collection of strains representing the geographic and genetic diversity of N. tetrasperma. First, we were able to confirm a monophyletic origin of N. tetrasperma. Furthermore, we found nine phylogenetic species within the morphospecies. When using the traditional broad biological species recognition all investigated strains of N. tetrasperma constituted a single biological species. In contrast, when using a quantitative measurement of the reproductive success, incorporating characters such as viability and fertility of offspring, we found a high congruence between the phylogenetic and biological species recognition. Taken together, phylogenetically and biologically defined groups of individuals exist in N. tetrasperma, and these should be taken into account in future studies of its life history traits.

Root rot, associated fungi and their impact on health condition of declining Fraxinus excelsior stands in Lithuania

Abstract Dieback of Fraxinus excelsior L. associated with Chalara fraxinea is observed in Europe, and in some areas dying trees exhibit symptoms of root and butt rot. Our study was conducted (1) to estimate the impact of the rot on F. excelsior dieback severity; (2) to identify fungi colonizing roots of dieback-affected trees; (3) to check their pathogenicity to F. excelsior; and (4) to estimate sprouting incidence and sprout health condition in relation to presence/absence of rot. The extent of rot was measured in 33 trees with different dieback intensity, 150 fungal isolations were attempted from roots of 50 trees, 26 fungi were tested for pathogenicity to 286 saplings, and sprouting was evaluated for 328 stumps on three clear-felled sites. Root rot was mainly caused by Armillaria cepistipes, and the extent of rot correlated positively with dieback severity although it played a secondary role in tree decline. Four years after tree felling, root rot had a negative impact on sprouting frequency, yet rot did not enter sprouts from stumps, and when experimentally inoculated, fungi associated with root rot in mature ash had no visible impact on tree vigor, showing that after formation of sprouts, rot does not affect the subsequent phytosanitary condition. Sprouts on investigated sites exhibited Chalara dieback symptoms on leaves and bark irrespectively of presence/absence of rot, indicating that vigorous natural regeneration of F. excelsior in dieback-affected areas could not be expected.

Afforestation of abandoned farmland with conifer seedlings inoculated with three ectomycorrhizal fungi—impact on plant performance and ectomycorrhizal community

The aim of a 3-year study was to investigate whether inoculation of Pinus sylvestris L. and Picea abies (L.) Karst. seedlings with mycorrhizas of Cenococcum geophilum Fr., Piceirhiza bicolorata, and Hebeloma crustuliniforme (Bull.) Quel. has any impact on: 1) survival and growth of outplanted seedlings on abandoned agricultural land, and 2) subsequent mycorrhizal community development. For inoculation, the root system of each plant was wrapped in a filter paper containing mycelium, overlaid with damp peat–sand mixture and wrapped in a paper towel. In total, 8,000 pine and 8,000 spruce seedlings were planted on 4-ha of poor sandy soil in randomized blocks. Already after the first year natural mycorrhizal infections prevailed in the inoculated root systems, and introduced mycorrhizas were seldom found. Yet, the seedlings that had been pre-inoculated with C. geophilum and the P. bicolorata during the whole 3-year period showed significantly higher survival and growth as compared to controls. Moreover, the independent colonization of roots by C. geophilum and the P. bicolorata from natural sources was also observed. A diverse mycorrhizal community was detected over two growing seasons in all treatments, showing low impact of inoculation on subsequent fungal community development. A total of 19 additional ectomycorrhizal morphotypes was observed, which clustered into two well-separated groups, according to host tree species (pine and spruce). In conclusion, the results showed limited ability to increase tree survival and growth, and to manipulate the mycorrhizal community even by extensive pre-inoculations, indicating that fungal community formation in root systems is governed mainly by environmental factors.

Conflict between reproductive gene trees and species phylogeny among heterothallic and pseudohomothallic members of the filamentous ascomycete genus Neurospora

In this study, we investigated the genealogies of genes important for sexual identity, i.e. mating-type (mat) and pheromone-receptor (pre) genes, among heterothallic and peudohomothallic taxa of Neurospora. The resulting genealogies were compared with the species phylogeny derived from non-coding sequences. We found conflicting topologies between the reproductive genealogies and the species phylogeny, and these conflicts were supported by both node support analyses and likelihood tests on the relative fit of datasets to alternative phylogenetic hypotheses. We argue that reproductive genes are more permeable to gene flow, i.e. are more often introgressed between species of Neurospora, than other parts of the genome. Certain conflicts between the species phylogeny and both mat genealogies were observed, suggesting that the two mating-type idiomorphs were selectively introgressed into a species from a single ancestral source. Taken together, the results presented here highlight complex evolutionary trajectories of reproductive genes in the fungal kingdom, which may be of importance for reproductive behavior in natural populations.

Large-Scale Introgression Shapes the Evolution of the Mating-Type Chromosomes of the Filamentous Ascomycete Neurospora tetrasperma

The significance of introgression as an evolutionary force shaping natural populations is well established, especially in animal and plant systems. However, the abundance and size of introgression tracts, and to what degree interspecific gene flow is the result of adaptive processes, are largely unknown. In this study, we present medium coverage genomic data from species of the filamentous ascomycete Neurospora, and we use comparative genomics to investigate the introgression landscape at the genomic level in this model genus. We revealed one large introgression tract in each of the three investigated phylogenetic lineages of Neurospora tetrasperma (sizes of 5.6 Mbp, 5.2 Mbp, and 4.1 Mbp, respectively). The tract is located on the chromosome containing the locus conferring sexual identity, the mating-type (mat) chromosome. The region of introgression is confined to the region of suppressed recombination and is found on one of the two mat chromosomes (mat a). We used Bayesian concordance analyses to exclude incomplete lineage sorting as the cause for the observed pattern, and multilocus genealogies from additional species of Neurospora show that the introgression likely originates from two closely related, freely recombining, heterothallic species (N. hispaniola and N. crassa/N. perkinsii). Finally, we investigated patterns of molecular evolution of the mat chromosome in Neurospora, and we show that introgression is correlated with reduced level of molecular degeneration, consistent with a shorter time of recombination suppression. The chromosome specific (mat) and allele specific (mat a) introgression reported herein comprise the largest introgression tracts reported to date from natural populations. Furthermore, our data contradicts theoretical predictions that introgression should be less likely on sex-determining chromosomes. Taken together, the data presented herein advance our general understanding of introgression as a force shaping eukaryotic genomes.

Archaeorhizomyces borealis sp nov and a sequence-based classification of related soil fungal species

The class Archaeorhizomycetes (Taphrinomycotina, Ascomycota) was introduced to accommodate an ancient lineage of soil-inhabiting fungi found in association with plant roots. Based on environmental sequencing data Archaeorhizomycetes may comprise a significant proportion of the total fungal community in soils. Yet the only species described and cultivated in this class is Archaeorhizomyces finlayi. In this paper, we describe a second species from a pure culture, Archaeorhizomyces borealis NS99-600(T) (=CBS138755(ExT)) based on morphological, physiological, and multi-locus molecular characterization. Archaeorhizomyces borealis was isolated from a root tip of a Pinus sylvestris seedling grown in a forest nursery in Lithuania. Analysis of Archaeorhizomycete species from environmental samples shows that it has a Eurasian distribution and is the most commonly observed species. Archaeorhizomyces borealis shows slow growth in culture and forms yellowish creamy colonies, characteristics that distinguish A. borealis from its closest relative A. finlayi. Here we also propose a sequence-based taxonomic classification of Archaeorhizomycetes and predict that approximately 500 species in this class remain to be isolated and described.