Cross-feeding affects the target of resistance evolution to an antifungal drug.

Pathogenic fungi are a cause of growing concern. Developing an efficient and safe antifungal is challenging because of the similar biological properties of fungal and host cells. Consequently, there is an urgent need to better understand the mechanisms underlying antifungal resistance to prolong the efficacy of current molecules. A major step in this direction would be to be able to predict or even prevent the acquisition of resistance. We leverage the power of experimental evolution to quantify the diversity of paths to resistance to the antifungal 5-fluorocytosine (5-FC), commercially known as flucytosine. We generated hundreds of independent 5-FC resistant mutants derived from two genetic backgrounds from wild isolates of Saccharomyces cerevisiae. Through automated pin-spotting, whole-genome and amplicon sequencing, we identified the most likely causes of resistance for most strains. Approximately a third of all resistant mutants evolved resistance through a pleiotropic drug response, a potentially novel mechanism in response to 5-FC, marked by cross-resistance to fluconazole. These cross-resistant mutants are characterized by a loss of respiration and a strong tradeoff in drug-free media. For the majority of the remaining two thirds, resistance was acquired through loss-of-function mutations in FUR1, which encodes an important enzyme in the metabolism of 5-FC. We describe conditions in which mutations affecting this particular step of the metabolic pathway are favored over known resistance mutations affecting a step upstream, such as the well-known target cytosine deaminase encoded by FCY1. This observation suggests that ecological interactions may dictate the identity of resistance hotspots.

Complex patterns shape immune genes diversity during invasion of common raccoon in Europe - Selection in action despite genetic drift.

Rapid adaptation is common in invasive populations and is crucial to their long-term success. The primary target of selection in the invasive species' new range is standing genetic variation. Therefore, genetic drift and natural selection acting on existing variation are key evolutionary processes through which invaders will evolve over a short timescale. In this study, we used the case of the raccoon Procyon lotor invasion in Europe to identify the forces shaping the diversity of immune genes during invasion. The genes involved in the defence against infection should be under intense selection pressure in the invasive range where novel pathogens are expected to occur. To disentangle the selective and demographic processes shaping the adaptive immune diversity of its invasive and expanding populations, we have developed species-specific single-nucleotide polymorphism markers located in the coding regions of targeted immune-related genes. We characterised the genetic diversity of 110 functionally important immune genes in two invasive and one native raccoon genetic clusters, each presenting a different demographic history. Despite the strong effect of demographic processes in the invasive clusters, we detected a subset of genes exhibiting the diversity pattern suggestive of selection. The most likely process shaping the variation in those genes was balancing selection. The selected genes belong to toll-like receptors and cytokine-related genes. Our results suggest that the prevalence of selection depends on the level of diversity, that is - less genetically diverse invasive population from the Czech Republic displayed fewer signs of selection. Our results highlight the role of standing genetic variation in adapting to new environment. Understanding the evolutionary mechanisms behind invasion success would enable predicting how populations may respond to environmental change.

Independent Evolution Has Led to Distinct Genomic Signatures in Dutch Elm Disease-Causing Fungi and Other Vascular Wilts-Causing Fungal Pathogens.

Vascular wilts are important diseases caused by plant pathogenic fungi that result in the rapid death of their plant hosts. This is due to a systemic defense mechanism whereby the plant induces the compartmentalization of the infected vascular system in order to reduce the propagation of the fungus. The ascomycete class Sordariomycetes contains several species that cause vascular wilts in diverse plant hosts, and they can be classified into four taxonomic orders. The genetic mechanisms of pathogenesis have already been investigated in Fusarium and Verticillium species, but they have not yet been compared with other well-known wilt-causing species, especially fungi causing oak wilt or Dutch elm disease (DED). Here we analyzed 20 whole genome assemblies of wilt-causing fungi together with 56 other species using phylogenetic approaches to trace expansions and contractions of orthologous gene families and gene classes related to pathogenicity. We found that the wilt-causing pathogens evolved seven times, experiencing the largest fold changes in different classes of genes almost every time. However, some similarities exist across groups of wilt pathogens, particularly in Microascales and Ophiostomatales, and these include the common gains and losses of genes that make up secondary metabolite clusters (SMC). DED pathogens do not experience large-scale gene expansions, with most of the gene classes, except for some SMC families, reducing in number. We also found that gene family expansions in the most recent common ancestors of wilt pathogen groups are enriched for carbohydrate metabolic processes. Our study shows that wilt-causing species evolve primarily through distinct changes in their repertoires of pathogenicity-related genes and that there is the potential importance of carbohydrate metabolism genes for regulating osmosis in those pathogens that penetrate the plant vascular system.

Heterogeneous Mutation Rates and Spectra in Yeast Hybrids.

Mutation rates and spectra vary between species and among populations. Hybridization can contribute to this variation, but its role remains poorly understood. Estimating mutation rates requires controlled conditions where the effect of natural selection can be minimized. One way to achieve this is through mutation accumulation experiments coupled with genome sequencing. Here, we investigate 400 mutation accumulation lines initiated from 11 genotypes spanning intralineage, interlineage, and interspecific crosses of the yeasts Saccharomyces paradoxus and S. cerevisiae and propagated for 770 generations. We find significant differences in mutation rates and spectra among crosses, which are not related to the level of divergence of parental strains but are specific to some genotype combinations. Differences in number of generations and departures from neutrality play a minor role, whereas polyploidy and loss of heterozygosity impact mutation rates in some of the hybrid crosses in an opposite way.

The Genome Sequence of the Jean-Talon Strain, an Archeological Beer Yeast from Québec, Reveals Traces of Adaptation to Specific Brewing Conditions.

The genome sequences of archeological Saccharomyces cerevisiae isolates can reveal insights about the history of human baking, brewing and winemaking activities. A yeast strain called Jean-Talon was recently isolated from the vaults of the Intendant's Palace of Nouvelle France on a historical site in Québec City. This site was occupied by breweries from the end of the 17th century until the middle of the 20th century when poisoning caused by cobalt added to the beer led to a shutdown of brewing activities. We sequenced the genome of the Jean-Talon strain and reanalyzed the genomes of hundreds of strains to determine how it relates to other domesticated and wild strains. The Jean-Talon strain is most closely related to industrial beer strains from the beer and bakery genetic groups from the United Kingdom and Belgium. It has numerous aneuploidies and Copy Number Variants (CNVs), including the main gene conferring cobalt resistance in yeast. The Jean-Talon strain has indeed higher tolerance to cobalt compared to other yeast strains, consistent with adaptation to the most recent brewing activities on the site. We conclude from this that the Jean-Talon strain most likely derives from recent brewing activities and not from the original breweries of Nouvelle France on the site.

Similarities in biological processes can be used to bridge ecology and molecular biology.

Much of the research in biology aims to understand the origin of diversity. Naturally, ecological diversity was the first object of study, but we now have the necessary tools to probe diversity at molecular scales. The inherent differences in how we study diversity at different scales caused the disciplines of biology to be organized around these levels, from molecular biology to ecology. Here, we illustrate that there are key properties of each scale that emerge from the interactions of simpler components and that these properties are often shared across different levels of organization. This means that ideas from one level of organization can be an inspiration for novel hypotheses to study phenomena at another level. We illustrate this concept with examples of events at the molecular level that have analogs at the organismal or ecological level and vice versa. Through these examples, we illustrate that biological processes at different organization levels are governed by general rules. The study of the same phenomena at different scales could enrich our work through a multidisciplinary approach, which should be a staple in the training of future scientists.

Hybridization and introgression drive genome evolution of Dutch elm disease pathogens.

Hybridization and the resulting introgression can drive the success of invasive species via the rapid acquisition of adaptive traits. The Dutch elm disease pandemics in the past 100 years were caused by three fungal lineages with permeable reproductive barriers: Ophiostoma ulmi, Ophiostoma novo-ulmi subspecies novo-ulmi and Ophiostoma novo-ulmi subspecies americana. Using whole-genome sequences and growth phenotyping of a worldwide collection of isolates, we show that introgression has been the main driver of genomic diversity and that it impacted fitness-related traits. Introgressions contain genes involved in host-pathogen interactions and reproduction. Introgressed isolates have enhanced growth rate at high temperature and produce different necrosis sizes on an in vivo model for pathogenicity. In addition, lineages diverge in many pathogenicity-associated genes and exhibit differential mycelial growth in the presence of a proxy of a host defence compound, implying an important role of host trees in the molecular and functional differentiation of these pathogens.

Differential introgression across newt hybrid zones: Evidence from replicated transects.

Genomic heterogeneity of divergence between hybridizing species may reflect heterogeneity of introgression, but also processes unrelated to hybridization. Heterogeneous introgression and its repeatability can be directly tested in natural hybrid zones by examining multiple transects. Here, we studied hybrid zones between the European newts Lissotriton montandoni and two lineages of Lissotriton vulgaris, with replicate transects within each zone. Over 1,000 nuclear genes located on a linkage map and mitochondrial DNA were investigated using geographical and genomic clines. Overall, the five transects were all similar, showing hallmarks of strong reproductive isolation: bimodal distribution of genotypes in central populations and narrow allele frequency clines. However, the extent of introgression differed between the zones, possibly as a consequence of their different ages, as suggested by the analysis of heterozygosity runs in diagnostic markers. In three transects genomic signatures of small-scale (~2 km) zone movements were detected. We found limited overlap of cline outliers between transects, and only weak evidence of stronger differentiation of introgression between zones than between transects within zones. Introgression was heterogeneous across linkage groups, with patterns of heterogeneity similar between transects and zones. Predefined candidates for increased or reduced introgression exhibited only a subtle tendency in the expected direction, suggesting that interspecific differentiation is not a reliable indicator for the strength of introgression. These hierarchically sampled hybrid zones of apparently different ages show how introgression unfolds with time and offer an excellent opportunity to dissect the dynamics of hybridization and architecture of reproductive isolation at advanced stages of speciation.

Author Correction: Hybridization is a recurrent evolutionary stimulus in wild yeast speciation.

The original version of the Supplementary Information associated with this Article contained errors in Supplementary Figures 2, 12, 20 and 22. The HTML has been updated to include a corrected version of the Supplementary Information; the original incorrect versions of these Figures can be found as Supplementary Information associated with this Correction.

Hybridization is a recurrent evolutionary stimulus in wild yeast speciation.

Hybridization can result in reproductively isolated and phenotypically distinct lineages that evolve as independent hybrid species. How frequently hybridization leads to speciation remains largely unknown. Here we examine the potential recurrence of hybrid speciation in the wild yeast Saccharomyces paradoxus in North America, which comprises two endemic lineages SpB and SpC, and an incipient hybrid species, SpC*. Using whole-genome sequences from more than 300 strains, we uncover the hybrid origin of another group, SpD, that emerged from hybridization between SpC* and one of its parental species, the widespread SpB. We show that SpD has the potential to evolve as a novel hybrid species, because it displays phenotypic novelties that include an intermediate transcriptome profile, and partial reproductive isolation with its most abundant sympatric parental species, SpB. Our findings show that repetitive cycles of divergence and hybridization quickly generate diversity and reproductive isolation, providing the raw material for speciation by hybridization.

Balancing selection and introgression of newt immune-response genes.

The importance of interspecific introgression as a source of adaptive variation is increasingly recognized. Theory predicts that beneficial genetic variants cross species boundaries easily even when interspecific hybridization is rare and gene flow is strongly constrained throughout the genome. However, it remains unclear whether certain classes of genes are particularly prone to adaptive introgression. Genes affected by balancing selection (BS) may constitute such a class, because forms of BS that favour novel, initially rare alleles, should facilitate introgression. We tested this hypothesis in hybridizing newts by comparing 13 genes with signatures of BS, in particular an excess of common non-synonymous polymorphisms, to the genomic background (154 genes). Parapatric hybridizing taxa were less differentiated in BS candidate genes than more closely related allopatric lineages, while the opposite was observed in the control genes. Coalescent and forward simulations that explored neutral and BS scenarios under isolation and migration showed that processes other than differential gene flow are unlikely to account for this pattern. We conclude that BS, probably involving a form of novel allele advantage, promotes introgression. This mechanism may be a source of adaptively relevant variation in hybridizing species over prolonged periods.

Linkage Map of Lissotriton Newts Provides Insight into the Genetic Basis of Reproductive Isolation.

Linkage maps are widely used to investigate structure, function, and evolution of genomes. In speciation research, maps facilitate the study of the genetic architecture of reproductive isolation by allowing identification of genomic regions underlying reduced fitness of hybrids. Here we present a linkage map for European newts of the Lissotriton vulgaris species complex, constructed using two families of F2 L. montandoni × L. vulgaris hybrids. The map consists of 1146 protein-coding genes on 12 linkage groups, equal to the haploid chromosome number, with a total length of 1484 cM (1.29 cM per marker). It is notably shorter than two other maps available for salamanders, but the differences in map length are consistent with cytogenetic estimates of the number of chiasmata per chromosomal arm. Thus, large salamander genomes do not necessarily translate into long linkage maps, as previously suggested. Consequently, salamanders are an excellent model to study evolutionary consequences of recombination rate variation in taxa with large genomes and a similar number of chromosomes. A complex pattern of transmission ratio distortion (TRD) was detected: TRD occurred mostly in one family, in one breeding season, and was clustered in two genomic segments. This is consistent with environment-dependent mortality of individuals carrying L. montandoni alleles in these two segments and suggests a role of TRD blocks in reproductive isolation. The reported linkage map will empower studies on the genomic architecture of divergence and interactions between the genomes of hybridizing newts.

Selective Landscapes in newt Immune Genes Inferred from Patterns of Nucleotide Variation.

Host-pathogen interactions may result in either directional selection or in pressure for the maintenance of polymorphism at the molecular level. Hence signatures of both positive and balancing selection are expected in immune genes. Because both overall selective pressure and specific targets may differ between species, large-scale population genomic studies are useful in detecting functionally important immune genes and comparing selective landscapes between taxa. Such studies are of particular interest in amphibians, a group threatened worldwide by emerging infectious diseases. Here, we present an analysis of polymorphism and divergence of 634 immune genes in two lineages of Lissotriton newts: L. montandoni and L. vulgaris graecus Variation in newt immune genes has been shaped predominantly by widespread purifying selection and strong evolutionary constraint, implying long-term importance of these genes for functioning of the immune system. The two evolutionary lineages differ in the overall strength of purifying selection which can partially be explained by demographic history but may also signal differences in long-term pathogen pressure. The prevalent constraint notwithstanding, 23 putative targets of positive selection and 11 putative targets of balancing selection were identified. The latter were detected by composite tests involving the demographic model and further validated in independent population samples. Putative targets of balancing selection encode proteins which may interact closely with pathogens but include also regulators of immune response. The identified candidates will be useful for testing whether genes affected by balancing selection are more prone to interspecific introgression than other genes in the genome.

Para-allopatry in hybridizing fire-bellied toads (Bombina bombina and B. variegata): Inference from transcriptome-wide coalescence analyses.

Ancient origins, profound ecological divergence, and extensive hybridization make the fire-bellied toads Bombina bombina and B. variegata (Anura: Bombinatoridae) an intriguing test case of ecological speciation. Previous modeling has proposed that the narrow Bombina hybrid zones represent strong barriers to neutral introgression. We test this prediction by inferring the rate of gene exchange between pure populations on either side of the intensively studied Kraków transect. We developed a method to extract high confidence sets of orthologous genes from de novo transcriptome assemblies, fitted a range of divergence models to these data and assessed their relative support with analytic likelihood calculations. There was clear evidence for postdivergence gene flow, but, as expected, no perceptible signal of recent introgression via the nearby hybrid zone. The analysis of two additional Bombina taxa (B. v. scabra and B. orientalis) validated our parameter estimates against a larger set of prior expectations. Despite substantial cumulative introgression over millions of years, adaptive divergence of the hybridizing taxa is essentially unaffected by their lack of reproductive isolation. Extended distribution ranges also buffer them against small-scale environmental perturbations that have been shown to reverse the speciation process in other, more recent ecotypes.

Detecting balancing selection in genomes: limits and prospects.

In spite of the long-term interest in the process of balancing selection, its frequency in genomes and evolutionary significance remain unclear due to challenges related to its detection. Current statistical approaches based on patterns of variation observed in molecular data suffer from low power and a high incidence of false positives. This raises the question whether balancing selection is rare or is simply difficult to detect. We discuss genetic signatures produced by this mode of selection and review the current approaches used for their identification in genomes. Advantages and disadvantages of the available methods are presented, and areas where improvement is possible are identified. Increased specificity and reduced rate of false positives may be achieved by using a demographic model, applying combinations of tests, appropriate sampling scheme and taking into account intralocus variation in selection pressures. We emphasize novel solutions, recently developed model-based approaches and good practices that should be implemented in future studies looking for signals of balancing selection. We also draw attention of the readers to the results of recent theoretical studies, which suggest that balancing selection may be ubiquitous but transient, leaving few signatures detectable by existing methods. Testing this new theory may require the development of novel high-throughput methods extending beyond genomic scans.

Constraint and adaptation in newt toll-like receptor genes.

Acute die-offs of amphibian populations worldwide have been linked to the emergence of viral and fungal diseases. Inter and intraspecific immunogenetic differences may influence the outcome of infection. Toll-like receptors (TLRs) are an essential component of innate immunity and also prime acquired defenses. We report the first comprehensive assessment of TLR gene variation for urodele amphibians. The Lissotriton newt TLR repertoire includes representatives of 13 families and is compositionally most similar to that of the anuran Xenopus. Both ancient and recent gene duplications have occurred in urodeles, bringing the total number of TLR genes to at least 21. Purifying selection has predominated the evolution of newt TLRs in both long (∼70 Ma) and medium (∼18 Ma) timescales. However, we find evidence for both purifying and positive selection acting on TLRs in two recently diverged (2-5 Ma) allopatric evolutionary lineages (Lissotriton montandoni and L. vulgaris graecus). Overall, both forms of selection have been stronger in L. v. graecus, while constraint on most TLR genes in L. montandoni appears relaxed. The differences in selection regimes are unlikely to be biased by demographic effects because these were controlled by means of a historical demographic model derived from an independent data set of 62 loci. We infer that TLR genes undergo distinct trajectories of adaptive evolution in closely related amphibian lineages, highlight the potential of TLRs to capture the signatures of different assemblages of pathogenic microorganisms, and suggest differences between lineages in the relative roles of innate and acquired immunity.

Nuclear and mitochondrial phylogeography of the European fire-bellied toads Bombina bombina and Bombina variegata supports their independent histories.

Exact location and number of glacial refugia still remain unclear for many European cold-blooded terrestrial vertebrates. We performed a fine-scaled multilocus phylogeographic analysis of two Bombina species combining mitochondrial variation of 950 toads from 385 sites and nuclear genes (Rag-1, Ncx-1) from a subset of samples to reconstruct their colonization and contemporary variation patterns. We identified the lowlands northwest of the Black Sea and the Carpathians to be important refugial areas for B. bombina and B. variegata, respectively. This result emphasizes the importance of Central European refugia for ectothermic terrestrial species, far north of the Mediterranean areas regarded as exclusive glacial refugia for the animals. Additional refugia for B. variegata have been located in the southern Apennines and Balkans. In contrast, no evidence for the importance of other east European plains as refugial regions has been found. The distribution of mtDNA and Ncx-1 variation suggests the presence of local refugia near the Black Sea for B. bombina; however, coalescent simulations did not allow to distinguish whether one or two refugia were present in the region. Strong genetic drift apparently accompanied postglacial expansions reducing diversity in the colonization areas. Extended sampling, coupled with the multilocus isolation with migration analysis, revealed a limited and geographically restricted gene flow from the Balkan to Carpathian populations of B. variegata. However, despite proximity of inferred B. bombina and B. variegata refugia, gene exchange between them was not detected.

Hybrid speciation in sparrows II: a role for sex chromosomes?

Homoploid hybrid speciation in animals is poorly understood, mainly because of the scarcity of well-documented cases. Here, we present the results of a multilocus sequence analysis on the house sparrow (Passer domesticus), Spanish sparrow (P. hispaniolensis) and their proposed hybrid descendant, the Italian sparrow (P. italiae). The Italian sparrow is shown to be genetically intermediate between the house sparrow and Spanish sparrow, exhibiting genealogical discordance and a mosaic pattern of alleles derived from either of the putative parental species. The average variation on the Z chromosome was significantly reduced compared with autosomal variation in the putative parental species, the house sparrow and Spanish sparrow. Additionally, divergence between the two species was elevated on the Z chromosome relative to the autosomes. This pattern of variation and divergence is consistent with reduced introgression of Z-linked genes and/or a faster-Z effect (increased rate of adaptive divergence on the Z). F(ST) -outlier tests were consistent with the faster-Z hypothesis: two of five Z-linked loci (CHD1Z and PLAA) were identified as candidates for being subject to positive, divergent selection in the putative parental species. Interestingly, the two latter genes showed a mosaic pattern in the (hybrid) Italian sparrow; that is, the Italian sparrow was found to be fixed for Spanish sparrow alleles at CHD1Z and to mainly have house sparrow alleles at PLAA. Preliminary evidence presented in this study thus suggests that sex chromosomes may play a significant role in this case of homoploid hybrid speciation.