The Plot Thickens: Haploid and Triploid-Like Thalli, Hybridization, and Biased Mating Type Ratios in Letharia

The study of the reproductive biology of lichen fungal symbionts has been traditionally challenging due to their complex lifestyles. Against the common belief of haploidy, a recent genomic study found a triploid-like signal in Letharia. Here, we infer the genome organization and reproduction in Letharia by analyzing genomic data from a pure culture and from thalli, and performing a PCR survey of the MAT locus in natural populations. We found that the read count variation in the four Letharia specimens, including the pure culture derived from a single sexual spore of L. lupina, is consistent with haploidy. By contrast, the L. lupina read counts from a thallus' metagenome are triploid-like. Characterization of the mating-type locus revealed a conserved heterothallic configuration across the genus, along with auxiliary genes that we identified. We found that the mating-type distributions are balanced in North America for L. vulpina and L. lupina, suggesting widespread sexual reproduction, but highly skewed in Europe for L. vulpina, consistent with predominant asexuality. Taken together, we propose that Letharia fungi are heterothallic and typically haploid, and provide evidence that triploid-like individuals are hybrids between L. lupina and an unknown Letharia lineage, reconciling classic systematic and genetic studies with recent genomic observations

Drivers of evolutionary change in Podospora anserina

Genomic diversity is shaped by a myriad of forces acting in different directions. Some genes work in concert with the interests of the organism, often shaped by natural selection, while others follow their own interests. The latter genes are considered “selfish”, behaving either neutrally to the host, or causing it harm. In this thesis, I focused on genes that have substantial fitness effects on the fungus Podospora anserina and relatives, but whose effects are very contrasting. In Papers I and II, I explored the evolution of a particular type of selfish genetic elements that cause meiotic drive. Meiotic drivers manipulate the outcome of meiosis to achieve overrepresentation in the progeny, thus increasing their likelihood of invading and propagating in a population. In P. anserina there are multiple meiotic drivers but their genetic basis was previously unknown. In Paper I, we demonstrated that drive is caused by members of the Spok gene family. We discovered two new Spok genes, Spok3 and Spok4, which locate in different chromosomes in different strains. In Paper II, we showed that Spok3 and Spok4 are found on a gigantic (up to 247 Kb long) variant of Enterprise, a Crypton-like transposable element. Enterprise likely mobilize through the action of a putative tyrosine-recombinase that we call Kirc. When carrying the Spoks, this element has double selfish properties: transposition and meiotic drive. In addition, we found that homologs of the Spoks (Paper I) and of Kirc (Paper II) are widespread in fungi but their phylogenies are discordant with that of the species, suggesting that they have undergone horizontal gene transfer. In Papers III and IV, I turned the focus into genes that have an adaptive function. In fungi, the het genes control conspecific self/non-self recognition. Such genes are expected to evolve under frequency-dependent balancing selection. In Paper III, we found evidence of balancing selection acting on some het genes across the P. anserina species complex. Unexpectedly, we also discovered that het genes of the HNWD gene family are duplicated in a transposon-like manner, broadening our understanding of their potential fitness effects. Finally, in Paper IV we show how het genes with pleiotropic effects on sexual recognition lead to the evolution of strong reproductive isolation, and hence speciation. Overall, the results of my thesis highlight the functional intersection between mobile selfish genetic elements and other genes, either selfish or adaptive, and their effects on genome architecture and population structure

Drivers of evolutionary change in Podospora anserina

Genomic diversity is shaped by a myriad of forces acting in different directions. Some genes work in concert with the interests of the organism, often shaped by natural selection, while others follow their own interests. The latter genes are considered “selfish”, behaving either neutrally to the host, or causing it harm. In this thesis, I focused on genes that have substantial fitness effects on the fungus Podospora anserina and relatives, but whose effects are very contrasting. In Papers I and II, I explored the evolution of a particular type of selfish genetic elements that cause meiotic drive. Meiotic drivers manipulate the outcome of meiosis to achieve overrepresentation in the progeny, thus increasing their likelihood of invading and propagating in a population. In P. anserina there are multiple meiotic drivers but their genetic basis was previously unknown. In Paper I, we demonstrated that drive is caused by members of the Spok gene family. We discovered two new Spok genes, Spok3 and Spok4, which locate in different chromosomes in different strains. In Paper II, we showed that Spok3 and Spok4 are found on a gigantic (up to 247 Kb long) variant of Enterprise, a Crypton-like transposable element. Enterprise likely mobilize through the action of a putative tyrosine-recombinase that we call Kirc. When carrying the Spoks, this element has double selfish properties: transposition and meiotic drive. In addition, we found that homologs of the Spoks (Paper I) and of Kirc (Paper II) are widespread in fungi but their phylogenies are discordant with that of the species, suggesting that they have undergone horizontal gene transfer. In Papers III and IV, I turned the focus into genes that have an adaptive function. In fungi, the het genes control conspecific self/non-self recognition. Such genes are expected to evolve under frequency-dependent balancing selection. In Paper III, we found evidence of balancing selection acting on some het genes across the P. anserina species complex. Unexpectedly, we also discovered that het genes of the HNWD gene family are duplicated in a transposon-like manner, broadening our understanding of their potential fitness effects. Finally, in Paper IV we show how het genes with pleiotropic effects on sexual recognition lead to the evolution of strong reproductive isolation, and hence speciation. Overall, the results of my thesis highlight the functional intersection between mobile selfish genetic elements and other genes, either selfish or adaptive, and their effects on genome architecture and population structure

het-B allorecognition in Podospora anserina is determined by pseudo-allelic interaction of genes encoding a HET and lectin fold domain protein and a PII-like protein.

Filamentous fungi display allorecognition genes that trigger regulated cell death (RCD) when strains of unlike genotype fuse. Podospora anserina is one of several model species for the study of this allorecognition process termed heterokaryon or vegetative incompatibility. Incompatibility restricts transmission of mycoviruses between isolates. In P. anserina, genetic analyses have identified nine incompatibility loci, termed het loci. Here we set out to clone the genes controlling het-B incompatibility. het-B displays two incompatible alleles, het-B1 and het-B2. We find that the het-B locus encompasses two adjacent genes, Bh and Bp that exist as highly divergent allelic variants (Bh1/Bh2 and Bp1/Bp2) in the incompatible haplotypes. Bh encodes a protein with an N-terminal HET domain, a cell death inducing domain bearing homology to Toll/interleukin-1 receptor (TIR) domains and a C-terminal domain with a predicted lectin fold. The Bp product is homologous to PII-like proteins, a family of small trimeric proteins acting as sensors of adenine nucleotides in bacteria. We show that although the het-B system appears genetically allelic, incompatibility is in fact determined by the non-allelic Bh1/Bp2 interaction while the reciprocal Bh2/Bp1 interaction plays no role in incompatibility. The highly divergent C-terminal lectin fold domain of BH determines recognition specificity. Population studies and genome analyses indicate that het-B is under balancing selection with trans-species polymorphism, highlighting the evolutionary significance of the two incompatible haplotypes. In addition to emphasizing anew the central role of TIR-like HET domains in fungal RCD, this study identifies novel players in fungal allorecognition and completes the characterization of the entire het gene set in that species

Invasion and maintenance of meiotic drivers in populations of ascomycete fungi

Meiotic drivers (MDs) are selfish genetic elements that are able to become overrepresented among the products of meiosis. This transmission advantage makes it possible for them to spread in a population even when they impose fitness costs on their host organisms. Whether an MD can invade a population, and subsequently reach fixation or coexist in a stable polymorphism, depends on the one hand on the biology of the host organism, including its life cycle, mating system, and population structure, and on the other hand on the specific fitness effects of the driving allele on the host. Here, we present a population genetic model for spore killing, a type of drive specific to fungi. We show how ploidy level, rate of selfing, and efficiency of spore killing affect the invasion probability of a driving allele and the conditions for its stable coexistence with a nondriving allele. Our model can be adapted to different fungal life cycles, and is applied here to two well-studied genera of filamentous ascomycetes known to harbor spore-killing elements, Neurospora and Podospora. We discuss our results in the light of recent empirical findings for these two systems

Functional characterization of the B2 haplotype.

(A) Incompatibility phenotype of a ΔBh2 strain. ΔBh2 strains show a barrage reaction to B1. (B) Schematic summary of transformation efficiencies experiments of three fragments of the B2-locus region (fragments ab, ac, db). No transformants were obtained with fragments db and ab transformed into the B1 strain. (C) Bp2 determines B2-incompatibility. Strains deleted for Bp2 (ΔBp2) show no barrage reaction to B1 and ΔBh1 strains transformed with Bp2 produce a barrage reaction to B1. (PDF)</p

Domain annotation and comparison of BH and BP proteins.

(A) Alignment of the BH1 and BH2 sequences. The diagram above the alignment gives the domain organization of BH and the level of identity between BH1 and BH2 in each domain together with a graphic representation of the sequence identity along the protein sequence. The position of the different domains are also given on the left side of the alignment with the same color-code as in the domain diagram. (B) An AlphaFold model of the BH1 C-terminal lectin fold domain is given together with the structure of the Sclerotium rolfsii SRL lectin (pdb:2OFC). (C) Alignment of the BP1 and BP2 sequences with the polymorphic T-loop and α1-regions colour code as in (D). (D) AlphaFold model of the BP2 trimer. The polymorphic T-loop and α1-regions are given in orange and red respectively.</p

Evidence of balancing selection on <i>het-B</i>.

(A) Allele frequency changes over time for the Wageningen Collection (the Netherlands), with a comparison of total het-B frequencies in three sample groups: a group of 108 strains from the Wageningen area (strains collected between 1991 to 2016 and data extracted from whole-genome sequencing), a second group of 61 strains from the same locality (collected in 2017 and genotyped by PCR) and a smaller collection of phenotyped French strains (for which sampling started in 1937). (B) Alignment of the het-B haplotypes and surrounding regions in P. anserina, along with maximum-likelihood phylogenies of Bh and unrelated genes at the flanks across the P. anserina species complex. Same-sense alignments are represented with gray links, while inverted alignments are in light red. Branch lengths drawn to scale as indicated by their scale bar (nucleotide substitutions per site). Branch support values are nonparametric bootstraps larger than 70%.</p

Incompatibility systems in <i>Podospora</i>, and macro- and microscopic manifestations of <i>het-B</i> incompatibility.

(A) The different genetically defined het systems of P. anserina are shown. Allelic systems are given in blue and non-allelic systems in red. het-V is involved both in allelic and non-allelic interactions and is given in purple. Opposing arrows represent an incompatible interaction. Full arrowheads indicate normal fertility, open arrowheads indicate sexual incompatibility. The arrow direction represents the direction of the cross, with the arrow pointing from the male parent to the female parent in the cross (for example for the V/V1 interaction, the diagram denotes that a male V1 x V female cross shows sexual incompatibility while the opposite cross is fertile). In the s/S interaction, the dashed arrowhead denotes the spore-killing reaction occurring in S male x s female crosses and leading to specific abortion of S spores. B. Barrage reaction (incompatibility) but lack of sexual incompatibility between B1 and B2. The strains of the given genotypes were confronted on corn meal agar and grown for 6 days in the dark (upper panel). The same plate was imaged again after a week under constant illumination (lower panel). Full incompatibility genotypes for relevant loci are as follows: B1: het-B1, het-c2, het-d3, het-e4, het-r, het-V; B2: het-B2, het-c2, het-d3, het-e4, het-r, het-V; E1: het-B1, het-c1, het-d3, het-e1, het-r, het-V; RV1: het-B1, het-c2, het-d3, het-e4, het-R, het-V1. Mating type is designated with—and + symbols. Note that fructifications (perithecia) form at the B1/B2 interface indicating the absence of sexual incompatibility in contrast to the C/E and RV1/rV interactions that show partial or total sterility respectively. C. Microscopic observation of the confrontation zone between a B1 strain expressing GFP and a B2 strain expressing RFP. The presumed anastomosis sites are marked by an arrowhead and lysed cell with an asterisk. In upper and lower right end panels, the confrontation zone between a B1 strain expressing GFP and a B1 strain expressing RFP shows normal heterokaryotic cells expressing both fluorescent markers. Each panel is 140x110 μ in size. (PPTX)</p

<i>Bh1</i> and <i>Bp2</i> control <i>het-B</i> incompatibility.

(A) Strains as given were confronted in all combinations on solid corn meal agar medium and after 5 days at 26°C the confrontation zone was imaged using a binocular lens (image size is 4.3x4.3 mm). Full test plates are given in S3 Fig. Under the strain designation, the constitution at Bh and Bp is recalled. On the top right part of the diagram, the barrage results are repeated graphically (/, for compatibility; //, for incompatibility) to improve readability. Note that ΔBh1 and ΔBp2 are compatible with all testers and that ΔBh2 and ΔBp1 retain the same incompatibility spectrum as the B1 and B2 strains from which they derive. (B) Transformation efficiency of the Bp1 and Bp2 alleles in different het-B genetic backgrounds. Note that Bp2 leads to a strong reduction in transformation efficiency in the B1 background but not in ΔBh1. Results are quadruplicates with standard deviations. (C) Interpretative model of het-B incompatibility interactions, het-B incompatibility is asymmetric and determined solely by the Bh1/Bp2 interaction, the reciprocal Bh2/Bp1 interaction is compatible. Het-B2 incompatibility type is determined by Bp2 and het-B1 incompatibility type by Bh1.</p