Comparative Mitogenomics Reveals Intron Dynamics and Mitochondrial Gene Expression Shifts in Domesticated and Wild Pleurotus ostreatus

Round 1

Reviewer 1 Report

This is an interesting study that demonstrates the changes that occur in fungi when they have undergone consistent reculturing under laboratory conditions. Overall the work is well-performed and interpreted, but some additional discussion of the results in connection to the conditions would strengthen the manuscript. 

• Line 93-94: are the Standard Culture conditions under which the strains were maintained the same as MESM at 24 degrees? I assume the authors did not also maintain mycelial plugs at -80 degrees that could be used to check strain adaptation? Would they be available from another lab?
• Line 185-191. The authors should provide a bit more detail about how the identity percentages were calculated. Were only the conserved regions included in this calculation? Considering the size difference with the commercial strain, this cannot be taking all sequences into account.
• Lines 209-210. Were all 11 ORFs from the commercial strain present in the other two strains? Can the authors make predictions on whether the loss of the other ORFs would be inconsequential under laboratory conditions?
• It would be helpful if teh authors would use consistent colors for the conditions and strains across all figures. E.g., in Fig S5A a nice set of colors is used with different main colors per strain and different shades for the different temperatures. However, these same samples do not maintain these colors in Fig. S5B and most of the other figures, which makes it not intuitive to compare the data in different figures.
• Figure S2. The legend does not seem to match what is displayed in the figure or if it does, it is not very clear. 
• Section 3.4.2. The comparison of the carbon sources requires more discussion and interpretation. BAsed on Fig. 4, the sucrose and glycerol condition are highly similar and different from SC. This is probably not surprising as the media composition is highly different between SC and the other two. SC has malt extract (typically consisting of 52% maltose, 20% glucose, 15% dextrin, 6% other carbohydrates, and 5% protein), while the other two conditions have a basic salt medium with ammonium as a nitrogen source and a specific carbon source). Even though there was a strong difference in growth rate between sucrose and glycerol (was this only based on diameter or was mycelial density also considered?), the expression seems to be mostly determined by the medium difference, possibly the nitrogen source or the concentration of other elements. The authors should consider this and add relevant discussion about the importance of these medium components for Pleurotus.
• Discussion (as well as other text): The authors commonly use the term domestication in their manuscript, but I would question whether this is the correct term here. There is no question that there are domesticated pleurotus strains that are being used for mushroom production. However, based on the description in this manuscript the authors did not use one of these strains but rather a progeny that has had years of adaptation to laboratory (malt extract) conditions. In mushroom production, typically crude substrates (e.g. wood shavings) are used, which require a larger arsenal of enzymes and metabolic pathways of the fungus to use as a carbon source. It has been demonstrated for several fungi that prolonged re-culturing on easy digestable substrates reduced the ability to utilize complex substrates. Could this also explain part of the differences shown in this study? E.g., could longer periods of adaptation have resulted in more reduced metabolism, not due to domestication as such, but rather by adaptation to a specific growth condition? 

Author Response

Reviewer 1

Detailed comments Reviewer 1

• Line 93-94: are the Standard Culture conditions under which the strains were maintained the same as MESM at 24 degrees? I assume the authors did not also maintain mycelial plugs at -80 degrees that could be used to check strain adaptation? Would they be available from another lab?

We thank the reviewer for this comment. We would like to clarify that the Standard Condition (SC) for maintaining P. ostreatus strains in our laboratory is in MESM at 24ºC. The strains are maintained through regular subculturing on fresh medium several times per year. Maintaining basidiomycete strains at -80ºC is problematic. We are working on that. The strains are available to other laboratories upon request. In addition, strain dkN001 is deposited in the Spanish Type Culture Collection (CECT).

• Line 185-191. The authors should provide a bit more detail about how the identity percentages were calculated. Were only the conserved regions included in this calculation? Considering the size difference with the commercial strain, this cannot be taking all sequences into account.

We agree with this comment. To clarify how the identity percentages were calculated, we have included the following text in lines 200-204 of Results section 3.1:

“Sequence identity percentages were calculated using BLAST alignments between the mitogenomes; therefore, only the regions that could be aligned were considered in the calculation, meaning that the reported values reflect similarity in conserved regions rather than across the entire genome. Given the differences in genome size among strains, the overall genome-wide identity would be lower.”

• Lines 209-210. Were all 11 ORFs from the commercial strain present in the other two strains? Can the authors make predictions on whether the loss of the other ORFs would be inconsequential under laboratory conditions?

All ORFs identified in the commercial strain dkN001 were also present in the wild-type strains; however, some were only partially conserved. For example, orf188 from dkN001 exhibits complete sequence homology with a segment of orf377 found in the wild strain dkN009 and in the dkF515 strain adapted to laboratory culture conditions. Importantly, orf188 was located at one end of the genomic inversion observed in the commercial strain relative to the wild-type strains, suggesting a partial loss of this sequence during the inversion process. The strain dkN001 lacks some ORFs present in the wild isolates. These orfs correspond to homing endonucleases present in the intron of cox1 gene. Some of these introns are lost in dkN001. The effect of the loss of these ORFs on gene expression is discussed later in the paper (Lines 573-584).

• It would be helpful if the authors would use consistent colors for the conditions and strains across all figures. E.g., in Fig S5A a nice set of colors is used with different main colors per strain and different shades for the different temperatures. However, these same samples do not maintain these colors in Fig. S5B and most of the other figures, which makes it not intuitive to compare the data in different figures.

We fully agree with this suggestion. We have revised the figure colours to ensure consistency, thereby facilitating easier comparison.

• Figure S2. The legend does not seem to match what is displayed in the figure or if it does, it is not very clear.

Thank you for your comment. We have revised the legend of Figure S2 to improve clarity. The new legend of Figure S2 is:

Figure S2: Structural alignments of the cox1 sequences from dkN001, dkF515, and dkN009 based on analyses with the Mauve and MFannot tools. DNA homologous regions among strains identified with the Mauve tool are shown in the same colour and appear just below the bar indicating the gene length in base pairs. Arrows indicate exon and intron regions in the cox1 sequences and are drawn to scale. Exons are represented as coloured blocks and the same colour denoting sequence homology. For example, exon 1 in dkF515 and dkN009 contains two regions (red and green). These two regions are separated in two exons in dkN001 by the presence of an intron.  Black lines connecting the blocks represent intronic regions of the cox1 gene.

• Section 3.4.2. The comparison of the carbon sources requires more discussion and interpretation. BAsed on Fig. 4, the sucrose and glycerol condition are highly similar and different from SC. This is probably not surprising as the media composition is highly different between SC and the other two. SC has malt extract (typically consisting of 52% maltose, 20% glucose, 15% dextrin, 6% other carbohydrates, and 5% protein), while the other two conditions have a basic salt medium with ammonium as a nitrogen source and a specific carbon source). Even though there was a strong difference in growth rate between sucrose and glycerol (was this only based on diameter or was mycelial density also considered?), the expression seems to be mostly determined by the medium difference, possibly the nitrogen source or the concentration of other elements. The authors should consider this and add relevant discussion about the importance of these medium components for Pleurotus.

We thank the reviewer for this comment. We want to indicate that the main objective of this study was to compare how three P. ostreatus strains with different origins responded to different culture conditions, and to seek for a correlation between these responses and their culture stories.

We agree that the similarity expression patterns observed between the strains grown in minimal solid medium with sucrose or with glycerol conditions, and their clear distinction from the SC medium, is likely influenced not only by the carbon source but also by the overall medium composition.

To clarify one of the questions of this reviewer, we want to indicate that the strong differences in growth rate between sucrose and glycerol was only based on linear growth.

In this context, the differential expression of mitochondrial nad5 and nad6 genes (complex I) in the wild strain dkN009, may reflect adjustments in respiratory metabolism associated with the availability of defined versus complex nutrients. Both sucrose and glycerol require active oxidative metabolism and NADH-dependent respiration, which may explain the similar expression profiles of Complex I genes, whereas growth on SC medium may reduce the reliance on mitochondrial respiration due to the presence of readily assimilable organic nutrients.

We have proceeded to include, in lines 446-452 of Results section 3.4.2, the following paragraph:

”In addition, opposite expression patterns were observed when strains were grown in SC compared to MSM supplemented with sucrose or with glycerol. For instance, the cox1 gene exhibited higher expression levels in the laboratory-adapted strains dkN001 and dkF515 when grown in minimal medium, whereas lower expression was detected in the wild isolate dkN009 under the same conditions. In contrast, under SC conditions, the highest cox1 expression was observed in dkN009 compared with the other strains (figure 4).”

• Discussion (as well as other text): The authors commonly use the term domestication in their manuscript, but I would question whether this is the correct term here. There is no question that there are domesticated Pleurotus strains that are being used for mushroom production. However, based on the description in this manuscript the authors did not use one of these strains but rather a progeny that has had years of adaptation to laboratory (malt extract) conditions. In mushroom production, typically crude substrates (e.g. wood shavings) are used, which require a larger arsenal of enzymes and metabolic pathways of the fungus to use as a carbon source. It has been demonstrated for several fungi that prolonged re-culturing on easy digestable substrates reduced the ability to utilize complex substrates. Could this also explain part of the differences shown in this study? E.g., could longer periods of adaptation have resulted in more reduced metabolism, not due to domestication as such, but rather by adaptation to a specific growth condition?

We would like to point out that in this study we used the commercial strain dkN001 (and not its progeny). This strain has been maintained through long-term subculturing on fresh medium, therefore, it is also adapted to laboratory culture conditions and bred for mushroom production (domesticated and adapted strain).

In the Discussion section, we have addressed and clarified both the concepts of domestication and adaptation, also as response to referee #2, between lines 639 and 650, including the following paragraph:

“Several studies support the distinction between the concepts of domestication and adaptation to laboratory conditions. In S. cerevisiae, for example, strains maintained in laboratory conditions over thousands of generations show predictable adaptations in growth rate and metabolic regulation that are not necessarily associated with deliberate selection for industrial traits (Johnson et al., 2021). Conversely, domesticated yeasts used in brewing, baking or winemaking exhibit signatures of selection on metabolic pathways linked to fermentation performance, stress tolerance, and flavour compound production, reflecting human-mediated selection rather than neutral laboratory adaptation (Gallone et al., 2016). In summary, while domestication refers to human-driven selection for desirable commercial traits, adaptation broadly refers to selection processes occurring in a neutral or stable environment, highlighting the importance of distinguishing between these two concepts in fungal biology.”

 

Author Response File: Author Response.pdf

Reviewer 2 Report

This study presents a well-designed and clearly articulated comparative analysis of mitochondrial genomes and gene expression in three Pleurotus ostreatus strains with differing domestication histories. The topic is relevant to fungal evolutionary biology, domestication studies, and mitochondrial genetics. The manuscript is generally well-written, the methods are sound, and the conclusions are largely supported by the data. The integration of structural genomics with expression profiling under stress conditions is a particular strength. The work is suitable for publication in Journal of Fungi after addressing the points below.

Major Comments

1.The study is built on three strains. While their selection (commercial/long-term lab-adapted/recent wild) is logical for the research question, the small sample size limits the generalizability of conclusions about "domestication" as a broad process. The authors should explicitly acknowledge this limitation in the Discussion and clarify that their findings suggest a pattern consistent with domestication-driven changes, which requires validation in larger strain collections.

2.The manuscript clearly documents intron loss and genome streamlining in the domesticated strain (dkN001). However, the direct functional consequenceof these structural changes (beyond correlation with expression shifts) is not deeply explored. For instance, does intron loss in rnl or cox1 affect splicing efficiency, mRNA stability, or respiratory complex assembly/function? Some speculation or reference to known mechanisms in other fungi would strengthen the discussion in Sections 4.1 and 4.2.

3.The expression data in Figures 3, 4, S4, S6, S8, S9 are central to the paper's argument. Error bars are mentioned (biological replicates) but statistical tests (e.g., ANOVA with post-hoc tests) should be explicitly stated in the figure legends and Methods (Section 2.4).

4. The description of expression patterns in Sections 3.4.1 and 3.4.2 is sometimes qualitative (e.g., "markedly elevated," "reduced expression"). A more quantitative summary (e.g., fold-changes for key contrasts) would be helpful.
5. Interpretation of "Domestication" vs. "Laboratory Adaptation":
   The authors interchangeably use "domestication" and "long-term laboratory maintenance." Strain dkF515 is a wild isolate but lab-adapted, while dkN001 is a commercial cultivar. It would be valuable to discuss more explicitly whether the observed changes (especially in gene expression) are driven primarily by duration under controlled conditions(convergent evolution) or by the specific history of commercial selection (dkN001). This is touched upon in the final paragraph but could be sharpened.

 

 

Minor Comments & Suggestions

1. Abstract:

Consider specifying the approximate sequence identity percentages earlier (e.g., ">99% between wild isolates, ~95% with the commercial strain") to immediately highlight genetic divergence.

2. Introduction:

The transition from general mitochondrial biology to the specific P. ostreatus model could be slightly smoother. A sentence explicitly stating why P. ostreatus is a good model for studying mitochondrial domestication would help.

3. Methods (Section 2.2):

Clarify the RNA extraction: was it performed on mycelia grown under all tested conditions (SC, different temps, carbon sources)? This is implied but should be stated for reproducibility.

4. Results (Section 3.1):

When first presenting genome sizes, consider adding a brief parenthetical note on the % difference (e.g., "dkN001 was ~10.6% smaller than dkN009") to emphasize the scale of size reduction.

5 Figure 1: Ensure the inversion in dkN001 is clearly visually distinguishable from the wild-type arrangement.

6 Page 5, Section 2.4: "2 µL of 3 µM forward and reverse primers" – specify if this is 2 µL of a primer mix or 2 µL each.

7.Page 9, Figure 2 caption: "in P. ostreatus strains dkF515, dkN001 and dkN009." -> "in P. ostreatus strains dkN001, dkF515, and dkN009." (Match order in figure).

8. Page 12, Caption for Figure 3: Specify the culture medium used (presumably MESM/SC?).

9 Throughout: Ensure consistent formatting of gene names in italics (e.g., cox1, rnl).

Author Response

Major Comments

1.The study is built on three strains. While their selection (commercial/long-term lab-adapted/recent wild) is logical for the research question, the small sample size limits the generalizability of conclusions about "domestication" as a broad process. The authors should explicitly acknowledge this limitation in the Discussion and clarify that their findings suggest a pattern consistent with domestication-driven changes, which requires validation in larger strain collections.

We agree with this comment and we have proceeded to include this sentence at the end of penultimate paragraph of Discussion section (lines 670-672):

“Further validation in larger strain collections will be necessary to confirm the consistency of differential expression patterns of PCGs associated with domestication-driven changes.”

2.The manuscript clearly documents intron loss and genome streamlining in the domesticated strain (dkN001). However, the direct functional consequence of these structural changes (beyond correlation with expression shifts) is not deeply explored. For instance, does intron loss in rnl or cox1 affect splicing efficiency, mRNA stability, or respiratory complex assembly/function? Some speculation or reference to known mechanisms in other fungi would strengthen the discussion in Sections 4.1 and 4.2.

We thank the reviewer for highlighting the need to discuss potential functional consequences of structural mitochondrial changes beyond their correlation with expression shifts. Mitochondrial introns — particularly group I and group II introns found in genes such as cox1, cob and rnl — are not merely neutral sequence elements; their presence and removal can affect several layers of gene expression and organelle function.

We have proceeded to include, in lines 575-584 of the Discussion section the following paragraph:

“Mitochondrial introns located in genes such as cox1, cob and rnl, are not merely neutral elements but can influence splicing efficiency, transcript processing, and mRNA stability, thereby affecting mitochondrial transcriptional homeostasis and respiratory function, as it was observed in the nde1 (complex I) gene expression in P. ostreatus (Garde et al., 2025). Intron removal has been shown to disrupt normal expression of cox1 and other respiratory genes by affecting mRNA stability and transcript abundance. Intron-less mitochondrial genomes can accumulate excess mature transcripts altering the stoichiometry of respiratory complexes and leading to physiological stress, as demonstrated in S. cerevisiae (Rudan et al., 2018). It is important to note that the effects of mitochondrial (prokaryotic-type) intron processing differ from those observed in nuclear intron processing.“

3.The expression data in Figures 3, 4, S4, S6, S8, S9 are central to the paper's argument. Error bars are mentioned (biological replicates) but statistical tests (e.g., ANOVA with post-hoc tests) should be explicitly stated in the figure legends and Methods (Section 2.4).

We appreciate this comment and we have proceeded to include this sentence at the end of Section 2.4 (lines 165-169):

“Data were analysed using IBM SPSS Statistics version 27.0 (IBM Corp., released 2020; Armonk, NY, USA). One-way ANOVA was applied to determine significant differences, followed by Scheffé’s post hoc test to compare mean values for intra-strain and inter-strain differences under different culture conditions”. In addition, we have indicated the statistical test used in the legends of Figures 3, 4, S4, S6, S8, and S9.

4. The description of expression patterns in Sections 3.4.1 and 3.4.2 is sometimes qualitative (e.g., "markedly elevated," "reduced expression"). A more quantitative summary (e.g., fold-changes for key contrasts) would be helpful.

We agree with this comment and have now included quantitative fold-change values for the most relevant gene expression comparisons.

5.Interpretation of "Domestication" vs. "Laboratory Adaptation": The authors interchangeably use "domestication" and "long-term laboratory maintenance." Strain dkF515 is a wild isolate but lab-adapted, while dkN001 is a commercial cultivar. It would be valuable to discuss more explicitly whether the observed changes (especially in gene expression) are driven primarily by duration under controlled conditions (convergent evolution) or by the specific history of commercial selection (dkN001). This is touched upon in the final paragraph but could be sharpened.

We thank the reviewer for pointing out the need to clarify our use of the terms “domestication” and “long-term laboratory maintenance”, and for suggesting a more explicit discussion of the potential drivers underlying the observed genomic and gene expression changes.

We agree that these concepts are related but distinct. Domestication in edible basidiomycete strains refers typically refers to directed selection imposed by humans with the purpose to obtain new strains with desirable traits, resulting in genetic differentiation from wild progenitors. An example is the dkN001 strain.

In contrast, laboratory adaptation as it was observed in dkF515 strain reflects the effects of neutral or adaptive changes that occur under prolonged culture condition under controlled environment without deliberate artificial selection. The latter is often driven by relaxed selection on traits required in wild or variable environments, and by genetic drift or specific laboratory practices.

We have proceeded to include, in lines 639-650 of the Discussion section, the following paragraph:

“Several studies support the distinction between the concepts of domestication and adaptation to laboratory conditions. In S. cerevisiae, for example, strains maintained in laboratory conditions over thousands of generations show predictable adaptations in growth rate and metabolic regulation that are not necessarily associated with deliberate selection for industrial traits (Johnson et al., 2021). Conversely, domesticated yeasts used in brewing, baking or winemaking exhibit signatures of selection on metabolic pathways linked to fermentation performance, stress tolerance, and flavour compound production, reflecting human-mediated selection rather than neutral laboratory adaptation (Gallone et al., 2016). In summary, while domestication refers to human-driven selection for desirable commercial traits, adaptation broadly refers to selection processes occurring in a neutral or stable environment, highlighting the importance of distinguishing between these two concepts in fungal biology.”

 

Minor Comments & Suggestions

1. Abstract: Consider specifying the approximate sequence identity percentages earlier (e.g., ">99% between wild isolates, ~95% with the commercial strain") to immediately highlight genetic divergence.

In response to this suggestion, we have uploaded this sentence in the abstract (lines 19-21).

2. Introduction: The transition from general mitochondrial biology to the specific ostreatus model could be slightly smoother. A sentence explicitly stating why P. ostreatus is a good model for studying mitochondrial domestication would help.

Thank you for that suggestion, we have incorporated, in lines 66-70 of the Introduction section, this sentence to explain why P. ostreatus is a good model for this study: “Pleurotus ostreatus, commonly known as the oyster mushroom, is an excellent model for studying mitochondrial domestication because it combines a well-characterized genome, the availability of both wild and commercially cultivated strains, and a long history of laboratory maintenance, allowing the disentangling of evolutionary changes driven by natural variation, artificial selection, and controlled growth conditions.”

3. Methods (Section 2.2): Clarify the RNA extraction: was it performed on mycelia grown under all tested conditions (SC, different temps, carbon sources)? This is implied but should be stated for reproducibility.

We have included this sentence at the beginning of section 2.2 (lines 115-116):

“Mycelia obtained in all conditions (MESM at 15ºC; MESM at 24ºC; MEMS at 32ºC; MSM supplemented with saccharose, and MSM supplemented with glycerol) were harvested….”

4. Results (Section 3.1): When first presenting genome sizes, consider adding a brief parenthetical note on the % difference (e.g., "dkN001 was ~10.6% smaller than dkN009") to emphasize the scale of size reduction.

We have added this comment in the text of Section 3.1 (lines 191-192).

5. Figure 1: Ensure the inversion in dkN001 is clearly visually distinguishable from the wild-type arrangement.

The inversion is indicated in the legend of Figure 1: “…., and blocks below the center line represent inversions“ (line 225).

6. Page 5, Section 2.4: "2 µL of 3 µM forward and reverse primers" – specify if this is 2 µL of a primer mix or 2 µL each.

We have clarified this sentence by adding (lines 159-160):

“... 2 μL of each primer (3 μM, forward and reverse)”.

7. Page 9, Figure 2 caption: "in P. ostreatus strains dkF515, dkN001 and dkN009." -> "in P. ostreatus strains dkN001, dkF515, and dkN009." (Match order in figure).

Thank you for this correction. We have changed the order of names to match that shown in the Figure 2.

8. Page 12, Caption for Figure 3: Specify the culture medium used (presumably MESM/SC?).

To observe the effects of temperature on growth rate and mitochondrial genes transcription levels, all strains were grown in MESM at different temperatures: 24ºC (the Standard Condition for P. ostreatus in our laboratory), 15ºC, and 32ºC. We have included this clarification in the legend of Figure 3 (line 416).

9. Throughout: Ensure consistent formatting of gene names in italics (e.g.,  cox1, rnl).

We have revised the gene names to check their format.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors sufficiently dealt with my previous comments, so I have no further comments.

see above