The fungus Neurospora comprises a novel model for testing hypotheses involving the role of sex and reproduction in eukaryotic genome evolution. Its variation in reproductive mode, lack of sex-specific genotypes, availability of phylogenetic species, and young sex-regulating chromosomes make research in this genus complementary to animal and plant models.
The impact of sex and reproductive traits on genome evolution is the subject of much theoretical and empirical attention. Central hypotheses involve the relationships between genome evolution and reproductive mode, reproductive- and sex-biased gene expression, speciation, as well as the origin of sex chromosomes 1–6. Empirical testing in multicellular eukaryotes has mostly focused on animal systems, and certain plants. Here, we highlight the traits of the filamentous fungus Neurospora that make it a novel and highly complementary model system for testing major hypotheses in genome evolution of multicellular eukaryotes.
Neurospora's advantages as a model
Neurospora is commonly known as the orange bread mold. The fungus first gained notoriety as a result of infestation in bread bakeries in the 1830s 7. Later, in the 1920s, it was domesticated for research 7. The most well-studied Neurospora species, Neurospora crassa, was used in experiments that lead to the 1958 Nobel Prize for the “one-gene, one enzyme hypothesis” 7, 8. Today, Neurospora is the multicellular eukaryotic model organism of choice for hundreds of laboratories studying cytology, genetics, and biochemistry. Its attractive traits have been well documented (e.g. 7) and include: a simple morphology and reproductive biology, a rapid life cycle (<10 days), an ease of growth and inter-strain crossings, a haploid life cycle, which allows recessive traits to be exposed in offspring, a low cost of maintenance, and the absence of ethical concerns in experimentation. In addition, extensive genomic data and tools are available for Neurospora 9 (http://www.broadinstitute.org/annotation/genome/neurospora; http://genome.jgi-psf.org), and thousands of mutants and wild-type strains are accessible at the Fungal Genetics Stock Center (FGSC, http://www.fgsc.net).
The knowledge base for Neurospora's reproductive biology is conducive to testing hypotheses in genome evolution. Its main reproductive features are as follows. It is typically anisogamous (produces dissimilar gametes) and is a morphological hermaphrodite. The sexual organs emerge from the vegetative tissue (the mycelium) following environmental cues; thus it does not have sequestered germlines. Sexual reproduction is regulated by the biallelic mating type (mat) locus (mat a and A alleles, known as “idiomorphs”) located on the mat chromosomes 10, 11, and certain species also produce asexual propagules 10, 11. Taken together, Neurospora's well-studied biology, combined with its genomics resources cited above, make it an attractive platform to test hypotheses on genome evolution.
Reproductive mode alters genome evolution
Two central hypotheses regarding mating systems and genome evolution are that: (i) inbreeding reduces the effective population size, and impairs purifying and positive selection as compared to outbreeding, leading to the accumulation of deleterious mutations 1, 12, and (ii) that selfers are evolutionary dead ends, i.e. selfers frequently go extinct due to low genetic diversity 12 and high levels of deleterious mutations 1. The dead-end theory also posits that the transition from outcrossing to selfing is irreversible 1. Empirical studies to date have provided both supporting and contradictory evidence for these theories 1, and additional studies are needed. Here, we underscore the traits of Neurospora that make it suited to testing these hypotheses.
Neurospora contains 49 established taxa. These include taxa that are sexually outcrossing (self-incompatible, or heterothallic) and those that are highly or strictly selfing (self-incompatible, or homothallic) 11. Taxa with intermediate mating systems (pseudohomothallic) also exist (see http://www.fgsc.net/neurospora/sectionb2.htm for details). Selfing lifestyles have evolved at least six independent times from outcrossing in Neurospora, and data suggest that these switches are likely irreversible 11, 13. Thus, the genus provides a means to test hypotheses about reproductive modes using replicated and phylogenetically independent samples, which is essential for discerning cause–effect relationships 14. One addressable question is: Do genes in selfers evolve faster than in outbreeders? Indeed, data from Neurospora (7 genes and 43 taxa) indicate that genes evolve more rapidly at the protein level in selfers than outcrossers, consistent with relaxed selection 11. But, other genes have not shown this effect 15. Additional relevant questions include: Do outcrossers present evidence of more positive selection events than selfers? Do selfers exhibit reduced selection on codon usage at the species level, or, at the population level? Recent data of segregation frequencies of mutations at polymorphic sites suggest that N. crassa exhibits selection on these traits at the population level 16; further studies are needed to reveal whether selective pressures vary with mating systems. Notably, Neurospora may also be utilized to test theories about mating systems and mutation rates 1. In fact, recent data based on synonymous substitution rates suggest mutation rates are lower in selfers, perhaps evolving as a mechanism to mitigate their reduced selective efficiency 11. Taken together, Neurospora provides a framework to address major questions about reproductive mode and genome evolution.
Sex biases drive gene evolution
Sex-biases in mutation and selection are believed to be major factors driving genome evolution. For instance, DNA carried in the male germline tends to have higher mutation rates than in female counterparts in a wide range of animals and certain plants 2, 17. With respect to selection, genes with male-biased expression typically exhibit higher rates of protein evolution and reduced selection for codon bias than female-biased or unbiased genes 4. Male-specific genes, e.g. spermatogenesis genes, evolve especially rapidly, possibly resulting from sperm competition and positive selection 3–5. Minimal data are available on this subject from fungi.
Outcrossing Neurospora species draws on a unique combination of traits that makes this approach complementary to animals and plants for the study of sex-biased evolution. These traits include: (i) the presence of sex-regulating (mat) loci or chromosomes; (ii) the existence of male and female sexual organs in a single organism, and (iii) the lack of sex-specific genotypes, i.e. the male or the female organs can carry the mat a or A chromosome. The latter trait is highly significant given that in most dioecious animal/plant models, sex is linked to sex-specific genotypes, e.g. Y is male-specific in X/Y systems; that makes it challenging to distinguish between the effects of sex and sex-specific genotypes on mutation and selection. Accordingly, one question that can be addressed in Neurospora is: Do sex-biased mutation rates or sex-biased selection occur in organisms that have sex-regulating chromosomes but lack sex-specific genotypes? Such studies may allow the elucidation of the precise roles of sex per se versus sex-specific genotypes on mutation-biases and selection biases. It may also reveal whether the lack of separate germlines inhibits sex-effects in Neurospora. Another addressable question is: Do sex-biased genes have a non-random distribution among the sex chromosomes and autosomes, as has been inferred in animals 2, 4? In other words, does such a phenomenon occur even when the sex chromosomes are not sex-specific? Such data may help reveal the association between sex-biases and sex chromosome evolution 2, 4. In sum, Neurospora provides complementary systems to address questions about sex-driven evolution.
Sex and reproductive genes mediate reproductive isolation
The rapid evolution of reproductive genes, including sex-biased and sex-specific genes discussed above, has been postulated to give rise to reproductive isolation 3–5, 18, 19. Specific cases of rapidly evolving reproductive proteins, e.g. mating and fertilization proteins, including those under positive selection, have been identified in animals, plants, and in Neurospora 3–5, 18, 19. At present, however, the precise role of rapidly evolving reproductive proteins in speciation remains unknown. Advancing this topic requires further genome-wide and gene functionality studies among closely related taxa 3, 5, 18. We assert that Neurospora provides systems to test these relationships.
Neurospora contains two fitting systems for such research. Specifically, the morphospecies Neurospora tetrasperma and N. discreta consist of complexes of at least nine and eight distinct phylogenetic species, respectively, defined based on DNA sequence analysis 20, 21. The phylogenetic species of each complex exhibit some level of pre- and post-zygotic reproductive isolation, which can be readily experimentally quantified in these organisms 20, 21. Comparative analyses among N. tetrasperma and among N. discreta lineages could expose key genes involved in reproductive isolation. Examples of questions include: What are the reproductive genes and gene regions exhibiting rapid evolution among lineages? Which genes are associated with male and female stages of fertilization 3, 5? Do they experience positive selection? Such analyses may allow the identification of genes experiencing sexual selection and sexual antagonism 3, 5. Additional questions that may reveal the precise reproductive stages driving reproductive isolation include: Do genes associated with pre-fertilization (e.g. sexual attraction) versus post-fertilization (e.g. meiosis, sporogenesis) evolve differently? Do pairs of lineages that exhibit higher levels of reproductive isolation have more rapidly evolving reproductive genes? Addressing these questions may help reveal whether rapid protein evolution is a cause or a consequence of speciation 3. It may also expose genes driving reproductive isolation; candidate genes may be readily studied on a molecular level using derived or available mutant strains (FGSC).
How do young sex chromosomes evolve?
The origin and divergence of sex chromosomes is a central issue in genome evolution 6, 22–24. Heterogametic sex chromosomes of many model species are highly degenerated due to extended periods of recombination suppression. For example, Y in mammalian X/Y and W in bird Z/W systems originated more than 100 mya 22, and retain minimal information about their early stages of divergence. Thus, young sex chromosome systems are needed for research 6. Two main young systems studied to date include the Y chromosome in the dioecious plant taxon Silene (e.g. Silene latifolia ∼5 mya) and the neo-Y chromosomes in Drosophila (e.g. Drosophila miranda, 1 mya) 22. We argue that the N. tetrasperma mat chromosomes, in which recombination ceased relatively recently, provide a useful model to study early stages of sex chromosome evolution 10, 23.
Distinct advantages for using N. tetrasperma in the study of sex chromosome evolution include the fact that the region of recombination suppression is on both mat chromosomes and is very young and large (<5.8 mya, >75% of the chromosomes 23). Furthermore, as discussed above, sex is unlinked to the mat chromosomes and the morphospecies contains nine closely related phylogenetic lineages. Fundamental questions can be addressed in this system including: What are the evolutionary forces driving sex chromosome evolution; postulated factors include genetic drift, selective sweeps, background selection, and/or Muller's Ratchet 2, 6, 24? What are the earliest symptoms of gene degeneration? Recent studies indicate that these regions exhibit losses of preferred codons and gains in allele-specific non-synonymous substitutions 25, 26. Furthermore, the absence of linkage between sex and the N. tetrasperma mat chromosomes allows one to address novel questions. For example: How do the sex-chromosomes degenerate in the absence of differential pressures from sex-specific biases in mutation or selection, sex-specific genotypes, and without differences in the effective population size (Ne) among chromosomes? This is important as sex- and Ne- effects are believed to contribute to genomic changes in X/Y and W/Z systems 2, 24; Neurospora may reveal how the chromosomes evolve in the absence of such effects. Sexual antagonism has been hypothesized to drive expansion of recombination suppression in sex chromosomes of dioecious species 27. Thus, another significant question is: What is the role of sexual antagonism in recombination suppression in a taxon without separate sexes? Such data may contribute towards the verification/nullification of this hypothesis and reveal whether it can occur without separate sexes.
The fact that N. tetrasperma is comprised of phylogenetic lineages 21 is highly useful for studying sex chromosome evolution. For example, comparative analysis among lineages has revealed that the segment of recombination suppression has expanded and/or shrunk numerous times 21, has undergone gene conversions 28, and contains a large introgression from other Neurospora taxa 29. Such analyses cannot be conducted in a single species. Future questions include: Do the sex chromosomes evolve differently from autosomes, e.g. changes in proteins, codon usage, and transpositions? Are sex chromosomes hotspots for adaptive mutations 2? Do genes with specific functions evolve more rapidly among lineages? Such research may help ascertain the genes and genome changes associated with the evolution of young sex chromosomes.
Neurospora's extensive variation in reproductive mode, lack of sex-specific genotypes, young sex-regulating chromosomes, and complexes of closely related phylogenetic species make it well positioned to test evolutionary genomics concepts in a manner that is novel and highly complementary to animal and plant models.
We are grateful to Dr. A. Moore and an anonymous reviewer for valuable comments on our manuscript. We acknowledge research funding from the Erik Philip-Sörensens Stiftelse (CAW), the Lars Hiertas Foundation (CAW), the Helge Ax:son Jonssons. Stifttelse (CAW), and the Swedish Research Council (HJ). The DNA data used in the background of our graphical abstract were extracted from the mat-A1 sequence of Neurospora crassa. The complete DNA sequence is available from the Neurospora crassa database.