Recombination suppression on sex chromosomes often extends in a stepwise manner, generating evolutionary strata of differentiation between sex chromosomes. Sexual antagonism is a widely accepted explanation for evolutionary strata, postulating that sets of genes beneficial in only one sex are successively linked to the sex-determining locus. The anther-smut fungus Microbotryum lychnidis-dioicae has mating-type chromosomes with evolutionary strata, only some of which link mating-type genes. Male and female roles are non-existent in this fungus, but mating-type antagonistic selection can also generate evolutionary strata, although the life cycle of the fungus suggests it should be restricted to few traits. Here, we tested the hypothesis that mating-type antagonism may have triggered recombination suppression beyond mating-type genes in Microbotryum by searching for footprints of antagonistic selection in evolutionary strata not linking mating-type loci. We found that these evolutionary strata (i) were not enriched in genes upregulated in the haploid phase, where cells are of alternative mating types, (ii) carried no gene differentially expressed between mating types, and (iii) carried no genes displaying footprints of specialization in terms of protein sequences (dN/dS) between mating types after recommended filtering. Without filtering, eleven genes showed signs of positive selection in the strata not linking mating-type genes, which constituted an enrichment compared to autosomes, but their functions were not obviously involved in antagonistic selection. Thus, we found no strong evidence that antagonistic selection has contributed to extending recombination suppression beyond mating-type genes. Alternative hypotheses should therefore be explored to improve our understanding of the sex-related chromosome evolution.
The genus Rhizopogon are ectomycorrhizal (ECM) fungi that are globally distributed with host trees of the family Pinaceae. Rhizopogon species have a very high degree of ECM host specificity and many species will associate with only a single host tree genus or species. There are currently five recognized subgenera of Rhizopogon and these subgenera possess a range of host specificity from obligate association with a single host genus to generalist association with several host genera. Despite the clear ecological significance of these fungi in both natural and managed forests, very little is known regarding their population structure and reproductive strategies. A few studies have performed landscape scale comparisons of closely related species in natural stands of Pseudotsuga and Pinus host trees. Recent studies have also investigated the organization and diversity of mating-type loci from individuals of the sister species R. vinicolor and R. vesiculosus in Rhizopogon subgenus Villosuli. In this study we expand the study of Rhizopogon reproductive strategies. To do so we have characterized the mating-type loci of 6 additional individuals of the sister species R. vinicolor and R. vesiculosus and another 8 individuals from species spanning all five Rhizopogon subgenera. Our results show shared structural traits and content between the B-locus mating-type pheromone receptors of many species of Rhizopogon, suggesting that Rhizopogon species have developed similar reproductive strategies in response to similar selective pressures.
Gemmamyces piceae causes bud blight disease of Picea species in Europe and China. It was first discovered in Alaska in 2016 and by 2017 Gemmamyces bud blight was recorded at over 31 locations in South Central and Interior Alaska. The pathogen is of concern because 90% of forest land in Alaska is boreal forest dominated by Picea species and the fungus causes reduced growth, dieback and in Europe even mortality. Two isolates were subjected to whole-genome sequencing with 150 bp paired-end reads yielding 15 Gb data at >100X coverage. The isolate with more sequence data was used for genome assembly. Reads were quality trimmed using Trimmomatic and de novo assembly was performed using ABySS with a k-mer pair span of 64. Simple Sequence Repeats (SSRs) were mined using Msat Commander. A total of 528 candidate SSR loci were identified for which suitable primer sequence were designed. Motifs sought were tri-, tetra-, penta-, and hexa-nucleotide repeats, with shorter repeats being more abundant than longer repeats (239 were tri-nucleotide repeats and 61 were hexa-nucleotide repeats). One candidate primer for each locus was designed for indirect fluorescent labeling by addition of a short CAG-sequence that was complementary to a third primer. On the alternate primer, a GTTT sequence was added to create a poly(A) tail that reduces stutter peak formation. A selection of candidate primers will be evaluated for ability to detect polymorphism within populations from Alaska and the Czech Republic for subsequent applications to characterize the population structure and migration patterns.
Changes to developmental gene expression drive morphological evolution across eukaryotic
lineages. The cis-regulatory elements and transcription factors that control differential genee expressionplay an important role in the early stages of development from determining celli identityto influencing body plans. The regulators of development have been studied acrosse eukaryotesfrom Hox genes in animals to MADS-box genes in plants. In fungi, the mating type locus (MAT) harbors genes involved in sexual development. The bipolar mating system inM Mucoromycotina(Phylum: Mucoromycota) is exemplified by a single MAT locus which encodes idiomorphicalleles. MAT alleles designated plus (+) have a High Mobility Group-domaine encodinggene SexP and minus (-) alleles a SexM gene. Evidence from a subset of Mucoromycotina suggests the mating type locus is syntenically conserved, where the HMG-domain genes are flanked by a triose phosphate transporter and RNA helicase gene. Our analysis of 144 Mucoromycotina genomes finds that gene content and MAT locus order are conserved. We find that MAT loci are more compact in species with observed sexual reproduction than those where it has not been seen, suggesting that relaxed selection and drift may be occurring. In some species MAT loci have gained additional genes within the locus in a mating-type specific manner. We generated mating-type specific phylogenies for species with known mating pairs and suggest that SexP locus has an ancient origin as the topology is consistent with the species phylogeny in contrast to SexM.
Puccinia hordei is a heteroecious macrocyclic rust fungus that causes leaf rust of barley. The uredinial and telial stages of P. hordei are found on Hordeum spp. and the pycnial and aecial stages are found on Ornithogalum spp. Uromyces scillarium is a microcyclic rust fungus, producing only telia and basidia on Ornithogalum spp. Telia of P. hordei were induced to germinate and used to infect O. eigii plants in a screen house. It was observed that P. hordei infections near natural occurring infections of U. scillarium produced only aecia. Aeciospores were collected from these “near” aecial cups and used to infect barley. Teliospores derived from the “near” aecia had morphological characteristics (teliospore area and percentage of mesospores) that were intermediate between P. hordei and U. scillarium. P. hordei infections of O. eigii “far” from U. scillarium infections formed normal pycnia and aecia and were used as controls. DNA sequence analysis of a 1.5kb segment of the gene encoding the elongation factor 1-alpha (EF1a) was performed. Collections of P. hordei and U. scillarium each had distinct EF1a sequences while the collections that originated from the “near” infections contained both EF1a sequence types (EF1a-Ph, EF1a-Us). Selected isolates originating from the “near” aecia were selfed. The resulting progeny exhibited two distinct classes: “hybrid” type with intermediate teliospore morphology and both EF1a sequence types (EF1a-Ph, EF1a-Us); “Ph” type with teliospore morphology and only the Ph-EF1a sequence type. These results indicate that natural hybridization occurs between P. hordei and U. scillarium.
Calonectria pseuodonaviculata (Cps) is currently the most devastating pathogen of boxwood (Buxus spp.) worldwide. Cps can also infect other members of the Buxaceae, namely, pachysandra (Pachysandra procumbens, P. terminalis) and sweet box (Sarcococca spp.). In the U.S. the disease was detected for the first time in North Carolina and Connecticut in 2012 and is now present in 28 states. A previous study reported that Cps residing in the U.S. prior to 2014 had a clonal population structure, and only the MAT1-2 mating type was present. However, the genetic structure of contemporary populations of this pathogen remains unknown. In the present study, we set out to estimate genetic diversity and gene flow in U.S. populations of Cps, and to assess the importance of sexual reproduction. We used Illumina technology to sequence 11 SSR loci and the MAT1 locus from 176 isolates of Cps, sampled from 2014 to 2019 across 18 states. Preliminary data (10% of isolates examined) showed the prevalence of a single SSR genotype, which was also the most common genotype in the U.S. prior to 2014. A new SSR genotype was identified from a Cps isolate from Pennsylvania, which differed from the predominant genotype by a single SSR allele.
Macrophomina phaseolina is an important pathogen of crops worldwide. Charcoal rot caused by M. phaseolina can significantly reduce yield and seed quality in soybean and dry bean mainly in tropical and subtropical regions. Little information is available on the population structure of M. phaseolina. Genomes of 96 M. phaseolina isolates from 13 states across the US, Puerto Rico, and Colombia, isolated from various hosts were sequenced to determine whether populations are structured by host or geographic origin. Sequencing was performed on an Illumina HiSeq 4000 with 150 bp paired-end reads to an average depth of 23X. Minimum spanning network analysis (MSN) based on Provesti’s genetic distance indicated isolates exhibit a clonal genetic structure, which can be supported by the biology of M. phaseolina since a sexual stage has not been identified. Interestingly, MSN and discriminant analysis of principal components revealed clustering of Puerto Rican and Colombian isolates from dry bean, while isolates from the US regardless dry bean or soybean were assigned to a separate cluster and exhibited lower diversity. The clustering and the higher genetic diversity in isolates from Puerto Rico and Colombia indicated the possibility of population differentiation. To investigate temperature adaptation, mycelial growth at 15ºC and 35ºC was measured and some isolates from the states of Michigan, Wisconsin and Minnesota grew faster at 15ºC than isolates from southern states. Genome-wide differentiation between proposed populations is being investigated, as well as identification of candidate genes that are associated to cold adaptation.
The smut fungus Sporisorium ellisii (G. Winter)M. Piepenbr. on its common U.S. host, Andropogon virginicus (Broomsedge) has received little attention beyond taxonomic placement in its current genus and mycogeographic citations. This obligate parasite, once successfully infective, produces sori which replace the inflorescences of its host as is typical of most smut fungi. It does not, however, generate sporidia upon the germination of teliospores on PDA, water agar or host extract-spiked agar, but instead produces a slow-growing, limited mycelium. In culture on PDA, the mycelium darkens and ceases growth after about 10 days at 25oC.
Three populations of this parasite were located in New Jersey, Pennsylvania and Ohio (the latter, focal). From infected plants collected along multiple, parallel transect lines 50m long, spores were collected, cold-stored for >2 months, surface-sterilized with dilute bleach and cultured on PDA with streptomycin (0.06g/l). Mycelia grown from single spore cultures were inoculated into PD broth and within a week, homogenized in lysis buffer and chloroform-isoamyl extracted. The genomic DNA from ~20 isolates/population was then run against 18 ISSR primers and 10 ScoT primers that reliably produced clear bands. The ISSR primers yielded 154 useful bands (ave. of 8.6 bands/primer) ranging from 400-2000 bps. The ScoT primer work is ongoing. For the ISSR primers, the PIC/primer values ranged from about 0.1 – 0.4. Via AMOVA, within population variance is 79% and among-population variance is 21%. A UPGMA dendrogram and correlation of genetic distance with geographic distance (via Mantel tests) will be presented.
Mushroom forming fungi (Agaricomycetes) is one of the biggest classes (>20,000 species) in the fungal kingdom with diverse morphologies and various ecological roles, yet its macro-evolutionary history is poorly known. To examine its evolution, we inferred a multigene phylogeny of the Agaricomycetes consisting of 5,284 species and three loci (nrLSU, RPB2 and ef1-a), combined with a phylogenomic backbone of 104 species and chronograms were estimated by applying temporal information of eight fungal fossils. First, we estimated the character state independent diversification rates using BAMM. We found an acceleration in diversification of species that started in the early Jurassic, coinciding with the spread of coniferous forest and emergence of pileate-stipitate fruiting bodies. Signs for a mass extinction were detected (BAMM and TESS analyses) in the late Jurassic, but not at the Cretaceous-Tertiary boundary, when a mass extinction event affected wide range of animal and plants species. The broad macro-evolutionary pattern mentioned above consists of several (>85) lineage-specific shifts in diversification rate suggesting a complex evolutionary history of mushroom forming fungi. This pattern could be shaped by several key morphological innovations; therefore, we inferred the character state dependent diversification rates related with fruiting bodies and found that the presence of a cap, increased hymenophore surface and the presence of veil tissues could have spurred lineage diversification.