Diploid Nuclei Occur throughout the Life Cycles of Pucciniales Fungi

ABSTRACT Within Eukaryotes, fungi are the typical representatives of haplontic life cycles. Basidiomycota fungi are dikaryotic in extensive parts of their life cycle, but diploid nuclei are known to form only in basidia. Among Basidiomycota, the Pucciniales are notorious for presenting the most complex life cycles, with high host specialization, and for their expanded genomes. Using cytogenomic (flow cytometry and cell sorting on propidium iodide-stained nuclei) and cytogenetic (FISH with rDNA probe) approaches, we report the widespread occurrence of replicating haploid and diploid nuclei (i.e., 1C, 2C and a small proportion of 4C nuclei) in diverse life cycle stages (pycnial, aecial, uredinial, and telial) of all 35 Pucciniales species analyzed, but not in sister taxa. These results suggest that the Pucciniales life cycle is distinct from any cycle known, i.e., neither haplontic, diplontic nor haplodiplontic, corroborating patchy and disregarded previous evidence. However, the biological basis and significance of this phenomenon remain undisclosed. IMPORTANCE Within Eukaryotes, fungi are the typical representatives of haplontic life cycles, contrasting with plants and animals. As such, fungi thus contain haploid nuclei throughout their life cycles, with sexual reproduction generating a single diploid cell upon karyogamy that immediately undergoes meiosis, thus resuming the haploid cycle. In this work, using cytogenetic and cytogenomic tools, we demonstrate that a vast group of fungi presents diploid nuclei throughout their life cycles, along with haploid nuclei, and that both types of nuclei replicate. Moreover, haploid nuclei are absent from urediniospores. The phenomenon appears to be transversal to the organisms in the order Pucciniales (rust fungi) and it does not occur in neighboring taxa, but a biological explanation or function for it remains elusive.

The manuscript "Diploid nuclei occur throughout the life cycles of Pucciniales fungi" describes the analysis of Picciniales fungi nuclei through cytogenetic and FISH analysis. This study is important in understanding the Picciniales species and why they are so different to other closely related fungal species. However, it is unclear how the methodologies were benchmarked and validated and more detail in the materials and methods is required. Referring to methodologies from other papers without specifically providing information with what was done is not adequate.
This reviewer is not convinced that genome sizes can be determined from cytometry, and some sort of validation is required via genome sequencing or other methodologies.
It also seems assumptions are made about nuclear DNA content and signal, as well as signal from the FISH. I would like to see other validations for these experiments or at least these limitations to be addressed and discussed.
It is unclear why certain lifecycles for certain species are chosen, and need to be discussed.
The gating strategy for cytometry looks like for some fungal species cut off the population right down the middle. Why is this? No data given to back this up.
Multiple species are analysed and mean data is shown in several tables -this should include standard errors/deviations. Reviewer #3 (Comments for the Author): The paper by Talhinhas at al explores ploidy diversity in multiple species of Puccinales and close relatives.
The main findings of the paper are that Puccinales have a unique nuclear biology, whereby haplontic, diplontic nor haplodiplontic states can co-exist in the same cell.
The results are exciting, and compelling. I also feel can possibly apply to other fungi that have uncoventional cellular biology.
The paper is very well writtes, and I have one minor comments that relates to the speculation that Puccinales represent the only MAJOR EXCEPTION that challenges text-book pre-conceptions.
It would be interesting for the authors to perhaps slightly expand their views outside the Dikarya and see if similar patterns exist elsewhere in the Mycota?
For example, the statement that "karyogamy is immediately followed by meiosis, and thus diploid nuclei should occur only in rust fungi basidia textbook assumptions, is also challenged by recent work on Arbuscular Mycorrhizal Heterokayons.
Indeed, Sperschneider et al 2023 (BiorXiv) have reported that strains containing TWO parental haplotypes physically separate among thousands of nuclei originated from two parental strains but HAVE YET TO UNDERGO meiosis; a situation (at least partially) similar to that reported here. This is just one example worth mentioning, but it's possible that some literature search may find more; further increasing the impact of this work.

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Thank you for submitting your paper to Microbiology Spectrum.

Dear Dr. Michael Klutstein
We would like to thank you for considering our work for possible publication in Microbiology Spectrum and would also like to acknowledge the constructive comments raised by reviewers. Bellow are point-by-point responses to those questions, marked in red for clarity. DOIs for all references cited in our responses are given in the end of this letter. Changes to the manuscript file are also highlighted in red.
We hope these modifications and responses are found satisfactory and that you can consider the manuscript suitable for publication.

Best wishes Pedro Talhinhas
Reviewer comments: Reviewer #2 (Comments for the Author): The manuscript "Diploid nuclei occur throughout the life cycles of Pucciniales fungi" describes the analysis of Picciniales fungi nuclei through cytogenetic and FISH analysis. This study is important in understanding the Picciniales species and why they are so different to other closely related fungal species. However, it is unclear how the methodologies were benchmarked and validated and more detail in the materials and methods is required. Referring to methodologies from other papers without specifically providing information with what was done is not adequate.
R: It was our intention to validate the results obtained by FCM, and we have chosen FISH for this. Because n2C samples are indistinguishable from 2nC using FCM, FISH was used to distinguish both populations. A clear correlation between singlet or doublet FISH signals with the nonreplicated or replicated loci, respectively, has been established a long time ago (Selig et al. 1992;Kitsberg et al. 1993;Boggs & Chinault 1997;Singh et al. 2003;Dutta et al. 2009; please refer to the end of this response letter for the DOIs of these references), and has been accepted and used since then. Moreover, the simultaneous use of FCM sorted nuclei and further validation of stages of cell-cycle by FISH is also a common technique (for example Lavoie et al. 2004;Huang et al. 2009) to assess G0/G1 or G2 populations. Furthermore, FISH is a standard technique to assess nuclear ploidy level since the number of individual signals is directly correlated with the ploidy level. In this way, this is a technique used in national laboratories worldwide to diagnose chromosomes abnormalities that can cause disease. We have added to material and methods section information about the FISH methodology.
This reviewer is not convinced that genome sizes can be determined from cytometry, and some sort of validation is required via genome sequencing or other methodologies. R: Flow cytometry is the standard technique for genome size measurement in eukaryotes. This has been demonstrated and reviewed exhaustively (e.g., Genome size measurement by FCM can in fact be employed to measure the quality and the completeness of genome sequencing initiatives (as an example, see Figure  2 and the discussion of the article by Hill et al. 2021, but also Pflug et al. 2020 andWyngaard et al. 2022 for examples from animals). The main focus of the present work, however, was not the measurement of fungal genomes, but the analysis of ploidy level in rust nuclei (still, some fungal genome sizes were newly determined in this study). Flow cytometry of nuclear particles is also a standard technique for ploidy and nuclear cycle analyses (Galbraith et al 1983;DeLaat et al. 1987;Schutte et al. 1995;Johnston et al. 2005;Suda & Travnicek 2006;Ramsey & Ramsey 2014;Fomicheva & Domblides 2023). Therefore, it is clear that the techniques used in this study are solidly validated for the purposes for which they were employed.
It also seems assumptions are made about nuclear DNA content and signal, as well as signal from the FISH. I would like to see other validations for these experiments or at least these limitations to be addressed and discussed. R: Please see the answer about methodologies.
It is unclear why certain lifecycles for certain species are chosen, and need to be discussed. R: We have analysed as many rust species as possible. Similarly, we have analysed as many lifecycle stages as possible. Not all lifecycle stages were found for all species. For instance, we have analysed the aecial stage of Gymnosporangium confusum on Crataegus monogyna, but we were unable to find the uredinial/telial stage on Juniperus spp., in spite of multiple surveys. We were also not able to analyse the pycnial stage of this fungus in spite of several attempts, as it turn out to be very ephemeral, yield very limited amount of biomass (insufficient for analysis) and very soon giving rise to the aecial structures. We did attempt performing detailed dissection of in planta fungal structures using Laser Capture Microdissection, but this yielded no reliable FCM results. A clarification on the sampling strategy was added to the Materials and Methods section.
The gating strategy for cytometry looks like for some fungal species cut off the population right down the middle. Why is this? No data given to back this up. R: In most cases, to avoid as much as possible the background noise due to cell wall debris autofluorescence, a gating strategy (following Talhinhas et al. 2021) was employed aiming to maximize the read of positive events and to minimize that of debris, while attempting to maintain consistency among the different samples. This information was added to the text.
Multiple species are analysed and mean data is shown in several tables -this should include standard errors/deviations. R: Standard deviation values were included next to average values corresponding to multiple species (Table 2). The main text was also adjusted to encompass the interpretation of these values.

Reviewer #3 (Comments for the Author):
The paper by Talhinhas at al explores ploidy diversity in multiple species of Puccinales and close relatives. The main findings of the paper are that Puccinales have a unique nuclear biology, whereby haplontic, diplontic nor haplodiplontic states can co-exist in the same cell. The results are exciting, and compelling. I also feel can possibly apply to other fungi that have uncoventional cellular biology. The paper is very well writtes, and I have one minor comments that relates to the speculation that Puccinales represent the only MAJOR EXCEPTION that challenges text-book pre-conceptions. It would be interesting for the authors to perhaps slightly expand their views outside the Dikarya and see if similar patterns exist elsewhere in the Mycota? For example, the statement that "karyogamy is immediately followed by meiosis, and thus diploid nuclei should occur only in rust fungi basidia textbook assumptions, is also challenged by recent work on Arbuscular Mycorrhizal Heterokayons. Indeed, Sperschneider et al 2023 (BiorXiv) have reported that strains containing TWO parental haplotypes physically separate among thousands of nuclei originated from two parental strains but HAVE YET TO UNDERGO meiosis; a situation (at least partially) similar to that reported here. This is just one example worth mentioning, but it's possible that some literature search may find more; further increasing the impact of this work. R: Thank you for your enthusiasm regarding our results. The results described by Sperschneider et al. (2023) are compelling and we have included a reference to that study in our discussion in order to expand the interpretation of the results outside the Dikarya, as suggested by the reviewer. Still, we would like to keep our discussion mainly centred in the Pucciniales and their sister taxa, so we have not expanded much on AMF. We believe that the strength of our results rely on the analysis of a phylogenetically diverse set of organisms, for all of which the phenomenon is documented, and on the fact that we were able to show that the nuclear ploidy level varies along the life cycle of these fungi. congratulations! the reviewers were satisfied with your revision, and your manuscript is accepted.
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