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  1. Author Response

    Reviewer #1 (Public Review):

    There are various ways in which homothallism (self-fertility) has arisen in the fungal kingdom from supposed heterothallic (obligate outbreeding) ancestors. Understanding the genetic basis of homothallism is important from both a fundamental basis, as it provides intriguing evolutionary insights, and also from a practical viewpoint as it impacts on variation and sporulation of a species - of particular importance for pathogenic and species of economic importance. In the present study, the authors describe an investigation of the genetic basis of homothallism in Cryptococcus depauperatus, a fungus closely related to Cryptococcus species causing serious human lung disease. The authors use a combination of genome analysis and experimental gene expression and manipulation work to show that C. depauperatus has a novel form of homothallism never reported before from fungi. This involves loss of the homeodomain genes which normally control mating in basidiomycete fungi, and instead signalling by a cognate pheromone and pheromone receptor and pathway seems sufficient to achieve self-fertility and induction of the sexual cycle. This is a very interesting and significant finding, adding to knowledge in the fungal kingdom and beyond as to the evolution of sexual breeding systems in nature. Overall my conclusion is that the authors' claims and conclusions are justified by their data and the work presented has a large number of strengths, although there are some minor weaknesses and need to qualify one assertion as follows.

    Strengths

    (1) The work has been conducted to a very high and thorough standard and is very well written and illustrated throughout. The authors base their findings on a combination of genome analysis and experimental gene expression and manipulation work, together with additional work (e.g. microscopy and CHEF gel studies) where required. Results arising are then all subject to suitable statistical analysis.

    (2) Regarding the genomics part of the work particular credit is given for aspects such as the very high standard of bioinformatic analysis (e.g. use of both nanopore and Illumina sequencing methodologies and care taken in contig assembly) and presentation of data in figures; the thorough phylogenetic analysis involving over 4,000 protein-encoding genes in a concatenated study to show species relationships; careful checking of a range of mating genes to show mixed evolutionary origins of the de novo mating locus and other regions of the genome (e.g. via analysis of MYO2, STE12, STE11, STE20 origins from an a- or alpha-ancestor).

    (3) Regarding the experimental part of the work particular credit is given for aspects such as the de novo development of a gene transformation and selection system in C. depauperatus (based on Agrobacterium-mediated transformation); the use of a heterologous C. neoformans system to confirm the bioactivity of the putative MAT-2 alpha-type pheromone from C. depauperatus; very clever use of recessive drug resistance markers to select putative recombinant progeny and then use UV-induced markers to show recombination in intra-strain pairings; and analysis of expression of putative 'sex' genes during the sporulation cycle.

    (4) Novelty of the findings. Previous examples from the fungal kingdom have shown the evolution of homothallism by mechanisms such as the incorporation of complementary mating-type (MAT) genes into the same genome, mating-type switching, and unisexual mating. This is the very first study to describe a situation where the homeodomain genes that normally control sexual development in basidiomycete fungi have been lost, and sexual development is instead achieved by activation of a cognate pheromone and pheromone receptor system. To add further to the novelty is the fact that only one complementary pheromone precursor (a MAT-2 alpha-type) and pheromone receptor (a STE3 a-type) pair of genes were found, whereas normally in basidiomycete fungi and beyond a set of two complementary pheromone precursor and pheromone receptor genes are normally found in the same genome (i.e. an additional MAT-1 a-type pheromone precursor and STE2 alpha-type receptor gene).

    We thank the reviewer for highlighting the loss of the homeodomain genes as one of the main findings of our work. Indeed, these genes are invariably present and define a second compatibility checkpoint in basidiomycetes. However, we would like to note that whereas a set of two complementary pheromone precursor and pheromone receptor genes are normally found in the same genome in ascomycete fungi controlling cell-cell communication and fusion, in basidiomycetes, only one non-complementary pair of pheromone and receptors is typically found.

    (5) The work contains an appropriate balance of reporting and yet also some speculation, such as the model at the end suggesting possible evolutionary routes.

    (6) The work is very well referenced throughout. The work also has a very extensive set of supporting data included as supplementary files to support the assertions made.

    We would like to thank the reviewer for taking the time and effort necessary to review the manuscript. We are grateful for the reviewer’s view that this manuscript is well-written, exciting, and of high relevance for both colleagues studying fungi as well as those studying mating systems in eukaryotes, which is a highly active field of research attracting broad interest. We sincerely appreciate all the thoughtful comments and suggestions, which helped us to improve the quality of the manuscript. In our response, we submitted both track-changed and finalized edited copies of the manuscript. Line numbers refer to the revised untracked manuscript file.

    Weaknesses

    (1) The work only involves analysis of two isolates of C. depauperatus, whereas analysis of a wider range of isolates might have revealed additional insights. But to be fair to the authors, the species C. depauperatus has only been reported very rarely and the two isolates examined appear to be the only publicly available accessible isolates. The authors also concede themselves that additional isolates would ideally be examined in the future to see if the proposed models stand.

    We agree with the reviewer’s assessment that this work would benefit from the analysis of additional C. depauperatus isolates. However, as indicated in the manuscript and acknowledged by the reviewer, there are only two isolates presently available for study. Importantly, in a recent survey for mycoparasites of the coffee leaf rust (Guterres et al. 2021) two isolates were found that are apparently closely related to C. depauperatus based on morphological and molecular evidence (analysis of the 28S rDNA sequences). Unfortunately, attempts by the authors at isolating these strains in pure culture after prolonged storage (over 6 months), have repeatedly failed. We concur that increased sampling efforts will be required in the future to better understand the diversity, evolution, and biology of C. depauperatus.

    (2) The authors provide evidence for meiotic recombination based on a very low number of markers - just three UV-induced markers and two drug resistance markers. And recombination is only shown conclusively in a very limited number of progeny (as shown in Figure 8C). Based on this very limited dataset they then produce some centimorgan mapping data and compare this rate to kb/cM data from other Cryptococcus species. However, this is at best preliminary, pilot data and should be cautioned as such, and ideally, many more markers would be used. Though to be fair to the authors they only had one intra-strain 'cross' to work with so were very limited in the markers available, and even within this limited dataset, there was good evidence for some meiotic recombination.

    We concur with the reviewer that recombination frequency determined from only two UV-induced markers along the same chromosome should be seen as preliminary. Indeed, we recognize this limitation in our study by indicating that the genetic distances are likely underestimated as multiple cross-over events between distant marks would skew these estimates (lines 623-627). Nevertheless, we consider that the data presented provides ample evidence that sexual reproduction in C. depauperatus involves a meiotic cycle with genetic exchange through recombination. It is currently unclear why the recovered viable strains did not accumulate additional mutations following UV mutagenesis, and future studies will be necessary to understand if this might be associated with changes in the DNA repair machinery.

    (3) Some minor errors and clarifications are required at various points in the manuscript.

    We thank the reviewer for their careful reading of the manuscript and for pointing out typographical errors, and the sections that would benefit from some clarification. We have carefully addressed all of these points in the revised manuscript.

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  2. Evaluation Summary:

    There are various ways in which self-fertility has arisen in the fungal kingdom. This study describes a novel form of self-fertility that evolved in a species closely related to the Cryptococcus species causing serious human lung disease, in which sexual development is achieved by self signaling of a cognate pheromone and pheromone receptor pair. Through a combination of high-quality genomic analysis and experimental gene expression and manipulation work, the study adds to our understanding of the evolution and flexibility of fungal breeding systems. This work will be of interest to colleagues studying fungi as well as mating systems in any eukaryote.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    There are various ways in which homothallism (self-fertility) has arisen in the fungal kingdom from supposed heterothallic (obligate outbreeding) ancestors. Understanding the genetic basis of homothallism is important from both a fundamental basis, as it provides intriguing evolutionary insights, and also from a practical viewpoint as it impacts on variation and sporulation of a species - of particular importance for pathogenic and species of economic importance. In the present study, the authors describe an investigation of the genetic basis of homothallism in Cryptococcus depauperatus, a fungus closely related to Cryptococcus species causing serious human lung disease. The authors use a combination of genome analysis and experimental gene expression and manipulation work to show that C. depauperatus has a novel form of homothallism never reported before from fungi. This involves loss of the homeodomain genes which normally control mating in basidiomycete fungi, and instead signalling by a cognate pheromone and pheromone receptor and pathway seems sufficient to achieve self-fertility and induction of the sexual cycle. This is a very interesting and significant finding, adding to knowledge in the fungal kingdom and beyond as to the evolution of sexual breeding systems in nature. Overall my conclusion is that the authors' claims and conclusions are justified by their data and the work presented has a large number of strengths, although there are some minor weaknesses and need to qualify one assertion as follows.

    Strengths
    (1) The work has been conducted to a very high and thorough standard and is very well written and illustrated throughout. The authors base their findings on a combination of genome analysis and experimental gene expression and manipulation work, together with additional work (e.g. microscopy and CHEF gel studies) where required. Results arising are then all subject to suitable statistical analysis.
    (2) Regarding the genomics part of the work particular credit is given for aspects such as the very high standard of bioinformatic analysis (e.g. use of both nanopore and Illumina sequencing methodologies and care taken in contig assembly) and presentation of data in figures; the thorough phylogenetic analysis involving over 4,000 protein-encoding genes in a concatenated study to show species relationships; careful checking of a range of mating genes to show mixed evolutionary origins of the de novo mating locus and other regions of the genome (e.g. via analysis of MYO2, STE12, STE11, STE20 origins from an a- or alpha-ancestor).
    (3) Regarding the experimental part of the work particular credit is given for aspects such as the de novo development of a gene transformation and selection system in C. depauperatus (based on Agrobacterium-mediated transformation); the use of a heterologous C. neoformans system to confirm the bioactivity of the putative MAT-2 alpha-type pheromone from C. depauperatus; very clever use of recessive drug resistance markers to select putative recombinant progeny and then use UV-induced markers to show recombination in intra-strain pairings; and analysis of expression of putative 'sex' genes during the sporulation cycle.
    (4) Novelty of the findings. Previous examples from the fungal kingdom have shown the evolution of homothallism by mechanisms such as the incorporation of complementary mating-type (MAT) genes into the same genome, mating-type switching, and unisexual mating. This is the very first study to describe a situation where the homeodomain genes that normally control sexual development in basidiomycete fungi have been lost, and sexual development is instead achieved by activation of a cognate pheromone and pheromone receptor system. To add further to the novelty is the fact that only one complementary pheromone precursor (a MAT-2 alpha-type) and pheromone receptor (a STE3 a-type) pair of genes were found, whereas normally in basidiomycete fungi and beyond a set of two complementary pheromone precursor and pheromone receptor genes are normally found in the same genome (i.e. an additional MAT-1 a-type pheromone precursor and STE2 alpha-type receptor gene).
    (5) The work contains an appropriate balance of reporting and yet also some speculation, such as the model at the end suggesting possible evolutionary routes.
    (6) The work is very well referenced throughout. The work also has a very extensive set of supporting data included as supplementary files to support the assertions made.

    Weaknesses
    (1) The work only involves analysis of two isolates of C. depauperatus, whereas analysis of a wider range of isolates might have revealed additional insights. But to be fair to the authors, the species C. depauperatus has only been reported very rarely and the two isolates examined appear to be the only publicly available accessible isolates. The authors also concede themselves that additional isolates would ideally be examined in the future to see if the proposed models stand.
    (2) The authors provide evidence for meiotic recombination based on a very low number of markers - just three UV-induced markers and two drug resistance markers. And recombination is only shown conclusively in a very limited number of progeny (as shown in Figure 8C). Based on this very limited dataset they then produce some centimorgan mapping data and compare this rate to kb/cM data from other Cryptococcus species. However, this is at best preliminary, pilot data and should be cautioned as such, and ideally, many more markers would be used. Though to be fair to the authors they only had one intra-strain 'cross' to work with so were very limited in the markers available, and even within this limited dataset, there was good evidence for some meiotic recombination.
    (3) Some minor errors and clarifications are required at various points in the manuscript.

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  4. Reviewer #2 (Public Review):

    The manuscript makes large steps forward in our understanding of the life cycle of C. depauperatus, and includes the development of a transformation method for this species. The experimental work has been carried out to a very high standard and the Figures are very clear. The Introduction is also comprehensive and clear. I congratulate the authors on this excellent work.

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