Rapid promoter evolution of male accessory gland genes is accompanied by divergent expression in closely related Drosophila species

  1. David W. J. McQuarrie
  2. Frannie H. S. Stephens
  3. Alexander D. Ferguson
  4. Roland Arnold
  5. Alberto Civetta
  6. Matthias Soller

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Abstract

Seminal fluid proteins (Sfps) are essential for reproductive success and evolve fast, possibly driven by post-copulatory sexual selection (PCSS) originating from sperm competition and cryptic female choice. Counterintuitively, however, the coding region only in few Sfps evolves adaptively. Hence, additional genomic and functional factors must play a role in Sfp evolution independent of the protein coding region. To shed light on drivers of Sfp evolution we focus on those Sfps predominantly expressed in male accessory glands, because this allows examination of their evolution in the tissue which produces the majority of Sfps. Moreover, accessory glands develop normally in hybrids in contrast to testis allowing to control for changes in cellular environment arising during speciation. Here, we discover that Acp promoters contain hot spots for rapid evolution from accumulation of sequence changes and insertions/deletions (indels). We further show that changes in promoter sequences are accompanied by gene expression divergence among closely related Drosophila species. We then validate these observations in Drosophila hybrids to show that species-specific expression divergence of Acps with rapidly evolving promoters are maintained in hybrids for some Acps, while others show dominance of one allele, a phenomenon termed transvection. These results indicate that cis -regulatory evolution, rather than genome background variation, drives Acp expression changes and promotes their rapid evolution.

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    Reply to the reviewers

    1. Response to reviewers

    We would like to thank the reviewers for carefully reading our manuscript and for their valuable comments in support for the publication of our investigation of rapid promoter evolution of accessory gland genes between Drosophila species and hybrids. We are glad to read that the reviewers find our work interesting and that it provides valuable insights into the regulation and divergence of genes through their promoters. We are encouraged by their acknowledgement of the overall quality of the work and the importance of our analyses in advancing the understanding of cis-regulatory changes in species divergence.

    2. Point-by-point description of the revisions

    Reviewer #

    Reviewer Comment

    Author Response/Revision

    Reviewer 1

    The authors test the hypothesis that promoters of genes involved in insect accessory glands evolved more rapidly than other genes in the genome. They test this using a number of computational and experimental approaches, looking at different species within the Drosophila melanogaster complex. The authors find an increased amount of sequence divergence in promoters of accessory gland proteins. They show that the expression levels of these proteins are more variable among species than randomly selected proteins. Finally, they show that within interspecific hybrids, each copy of the gene maintains its species-specific expression level.

    We thank Reviewer 1 for their detailed review and positive feedback on our manuscript, and for their helpful suggestions. We have now fully addressed the points raised by Reviewer 1 and have provided the suggested clarifications and revisions to improve the flow, readability, and presentation of the data, which we believe have improved the manuscript significantly.

    The work is done with expected standards of controls and analyses. The claims are supported by the analysis. My main criticism of the manuscript has to do not with the experiments or conclusion themselves but with the presentation. The manuscript is just not very well written, and following the logic of the arguments and results is challenging.

    The problem begins with the Abstract, which is representative of the general problems with the manuscript. The Abstract begins with general statements about the evolution of seminal fluid proteins, but then jumps to accessory glands and hybrids, without clarifying what taxon is being studied, and what hybrids they are talking about. Then, the acronym Acp is introduced without explanation. The last two sentences of the Abstract are very cumbersome and one has to reread them to understand how they link to the beginning of the Abstract.

    More generally, if this reviewer is to be seen as an "average reader" of the paper, I really struggled through reading it, and did not understand many of the arguments or rationale until the second read-through, after I had already read the bottom line. The paragraph spanning lines 71-83 is another case in point. It is composed of a series of very strongly worded sentences, almost all starting with a modifier (unexpectedly, interestingly, moreover), and supported by citations, but the logical flow doesn't work. Again, reading the paragraph after I knew where the paper was going was clearer, but on a first read, it was just a list of disjointed statements.

    Since most of the citations are from the authors' own work, I suspect they are assuming too much prior understanding on the part of the reader. I am sure that if the authors read through the manuscript again, trying to look through the eyes of an external reader, they will easily be able to improve the flow and readability of the text.

    We thank the reviewer for their detailed feedback and are glad that they acknowledge our work fully supports the claims of our manuscript. We also appreciate their helpful suggestions for improving the readability of the manuscript and have done our best to re-write the abstract and main text where indicated. In particular, the paragraph between lines 71-83 have been rewritten and we have taken care to write to non-expert readers.

    1. In the analysis of expression level differences, it is not clear what specific stage / tissue the levels taken from the literature refer to. Could it be that the source of the data is from a stage or tissue where seminar fluid proteins will be expressed with higher variability in general (not just inter-specifically) and this could be skewing the results? Please add more information on the original source of the data and provide support for their validity for this type of comparison.

    These were taken from publicly available adult male *Drosophila *datasets, listed in the data availability statement and throughout the manuscript. We have provided more detail on the tissue used for analysis of Acp gene expression levels.

    1. The sentence spanning lines 155-157 needs more context.

    We have added more context to lines 155-157.

    1. Line 203-204: What are multi-choice enhancers?

    We replaced the sentence with "... such as rapidly evolving enhancers or nested epistasis enhancer networks"

    1. Figure 1: The terminology the authors use, comparing the gene of interest to "Genome" is very confusing. They are not comparing to the entire genome but to all genes in the genome, which is not the same.

    We have changed the word "genome" to "all genes in the genome" on the reviewer's suggestion.

    1. Figure 2: Changes between X vs. Y is redundant (either changes between X and Y or changes in X vs. Y).

    We assume that the reviewer is referring to Fig. 2B, which does not measure changes between X and Y, but changes in distribution between Acps and the control group. We have explained this in the figure legend.

    The manuscript addresses a general question in evolutionary biology - do control regions diverge more quickly protein coding regions. The answer is that yes, they do, but this is actually not very surprising. The work is probably thus of more interest to people interested in the copulatory proteins or in the evolution of mating systems, than to people interested in broader evolutionary questions.

    We appreciate this reviewer's recognition of the significance of our work and would like to point out that there are very few studies looking at promoter evolution as detailed in the introduction. Of particular relevance, our study using Acp genes allows us to directly test the impact of promoter mutations on the expression by comparing two alleles in male accessory glands of Drosophila hybrids. Male accessory glands consist of only two secretory cell types allowing us to study evolution of gene expression in a single cell type (Acps are either expressed in main cells or secondary cells). Amid this unique experimental set up we can conclude that promoter mutations can act dominant, in contrast to mutations in protein coding regions, which are generally recessive. Thus, our study is unique in pointing out a largely overseen aspect of gene evolution.

    Reviewer 2

    This manuscript explores promoter evolution of genes encoding seminal fluid proteins expressed in the male accessory gland of Drosophila and finds cis-regulatory changes underlie expression differences between species. Although these genes evolve rapidly it appears that the coding regions rarely show signs of positive selection inferring that changes in their expression and hence promoter sequences can underlie the evolution of their roles within and among species.

    We thank Reviewer 2 for their thorough review, positive feedback on the importance of our work, and suggestions for improving the manuscript. We have addressed all points raised by the reviewer, including analysis of Acp coding region evolution, additional analyses of hybrid expression data, and improved the clarity of the text.

    Figure 1 illustrates evidence that the promoter regions of these gene have accumulated more changes than other sampled genes from the Drosophila genome. While this convinces that the region upstream of the transcription start site has diverged considerably in sequence (grey line compared to black line), Figure 1A also suggests the "Genespan" region which includes the 5'UTR but presumably also part of the coding region is also highly diverged. It would be useful to see how the pattern extends into the coding region further to compare further to the promoter region (although Fig 1H does illustrate this more convincingly).

    The reviewer raises an interesting point, and certainly all parts of genes evolve. Fig. 1A shows the evolutionary rates of Acps compared to the genome average from phyloP27way scores calculated from 27 insect species. Since these species are quite distant it is unsurprising that they show divergence in coding regions as well as promoter regions. In fact, we addressed whether promoter regions evolve fast in closely related *Drosophila *species in Fig. 1H compared to coding regions. We have included an additional analysis of coding region evolution in Figure 1B.

    Figure 2 presents evidence for significant changes in (presumably levels of) expression of male accessory gland protein (AcP) genes and ribosomal proteins genes between pairs of species, which is reflected in the skew of expression compared to randomly selected genes.

    Correct, we have rephrased the statement for clarity.

    Figure 3 shows detailed analysis for 3 selected AcP genes with significantly diverged expression. The authors claim this shows 'substitution' hotspots in the promoter regions of all 3 genes but this could be better illustrated by extending the plots in B-D further upstream and downstream to compare to these regions.

    We picked the 300-nucleotide promoter region for this analysis as it accumulated significant changes as shown in Fig. 1E-H, and extending the G plots (Fig. 3B-D) to regions with lower numbers of sequence changes would not substantially change the conclusion. Specifically, this analysis identifies sequence change hotspots within fast-evolving promoter regions, rather than comparing promoter regions to other genomic regions, as we previously addressed. The plot is based on a cumulative distribution function and the significant positive slope in the upstream region where promoters are located identifies a hotspot for accumulation of substitutions. There could be other hotspots, but the point being made is that significant hotspots consistently appear in the promoter region of these three genes.

    Figure 4 shows the results of expression analysis in parental lines of each pair of species and F1 hybrids. However the results are very difficult to follow in the figure and in the relevant text. While the schemes in A, C. E and G are helpful, the gel images are not the best quality and interpretations confusing. An additional scheme is needed to illustrate hypothetical outcomes of trans change, cis change and transvection to help interpret the gels. On line 169 (presumably referring to panels D and F although C and D are cited on the next line) the authors claim that Obp56f and CG11598 'were more expressed in D. melanogaster compared to D. simulans' but in the gel image the D. sim band is stronger for both genes (like D. sechellia) compared to the D. mel band. The authors also claim that the patterns of expression seen in the F1s are dominant for one allele and that this must be because of transvection. I agree this experiment is evidence for cis-regulatory change. However the interpretation that it is caused by transvection needs more explanation/justification and how do the authors rule out that it is not a cis X trans interaction between the species promoter differences and differences in the transcription factors of each species in the F1? Also my understanding is that transvection is relatively rare and yet the authors claim this is the explanation for 2/4 genes tested.

    We appreciate the reviewer's comments on Figure 4 and the opportunity to improve its clarity. To address these concerns, we have carefully checked the figure citations and corrected any inconsistencies.

    The reviewer raises an important point about our interpretation of transvection. We have expanded our discussion of this result to consider why transvection is a plausible explanation for the observed dominance patterns and also consider cis x *trans *interactions between species-specific promoters and transcription factor binding. While rare, transvection likely has more relevance in hybrid regulatory contexts involving homologous chromosome pairing which we discuss this in the revised text.

    Line 112 states that the melanogaster subgroup contains 5 species - this is incorrect - while this study looked at 5 species there are more species in this subgroup such as mauritiana and santomea.

    We have corrected the statement about the number of species in the melanogaster subgroup.

    Lines 131-134 could explain better what the conservation scores and their groupings mean and the rationale for this approach.

    We have clarified what the conservation scores and their groupings mean and the rationale for this approach.

    Line 162 - the meaning of the sentence starting on this line is unclear - it sounds very circular.

    We have rephrased the statement for more clarity.

    Line 168 should cite Fig 4 H instead of F.

    We have amended citation of Fig 4F to H.

    Reviewer 3

    In this study, McQuarrie et al. investigate the evolution of promoters of genes encoding accessory gland proteins (Acps) in species within the D. melanogaster subgroup. Using computational analyses and available genomic and transcriptomic datasets, they demonstrate that promoter regions of Acp genes are highly diverse compared to the promoters of other genes in the genome. They further show that this diversification correlates with changes in gene expression levels between closely related species. Complementing these computational analyses, the authors conduct experiments to test whether differences in expression levels of four Acp genes with highly diverged promoter regions are maintained in hybrids of closely related species. They find that while two Acp genes maintain their expression level differences in hybrids, the other two exhibit dominance of one allele. The authors attribute these findings to transvection. Based on their data, they conclude that rapid evolution of Acp gene promoters, rather than changes in trans, drives changes in Acp gene expression that contribute to speciation.

    We thank Reviewer 3 for their thorough review and suggestions. We further thank the reviewer for acknowledging the importance of our findings and for pointing out that it contributes to our understanding of speciation. We have thoroughly addressed all comments from the reviewer and significantly revised the manuscript. We believe that this has greatly improved the manuscript.

    Unfortunately, the presented data are not sufficient to fully support the conclusions. While many of the concerns can be addressed by revising the text to moderate the claims and acknowledge the methodological limitations, some key experiments require repetition with more controls, biological replicates, and statistical analyses to validate the findings.

    Specifically, some of the main conclusions heavily rely on the RT-PCR experiments presented in Figure 4, which analyze the expression of four Acp genes in hybrid flies. The authors use PCR and RFLP to distinguish species-specific alleles but draw quantitative conclusions from what is essentially a qualitative experiment. There are several issues with this approach. First, the experiment includes only two biological replicates per sample, which is inadequate for robust statistical analysis. Second, the authors did not measure the intensity of the gel fragments, making it impossible to quantify allele-specific expression accurately. Third, no control genes were used as standards to ensure the comparability of samples.

    The gold standard for quantifying allele-specific expression is using real-time PCR methods such as TaqMan assays, which allow precise SNP genotyping. To address this major limitation, the authors should ideally repeat the experiments using allele-specific real-time PCR assays. This would provide a reliable and quantitative measurement of allele-specific expression.

    If the authors cannot implement real-time PCR, an alternative (though less rigorous) approach would be to continue using their current method with the following adjustments:

    • Include a housekeeping gene in the analysis as an internal control (this would require identifying a region distinguishable by RFLP in the control).

    • Quantify the intensity of the PCR products on the gel relative to the internal standard, ensuring proper normalization.

    • Increase the sample size to allow for robust statistical analysis.

    These experiments could be conducted relatively quickly and would significantly enhance the validity of the study's conclusions.

    We thank the reviewer for their detailed suggestions for improving the conclusions in Fig. 4. Indeed, incorporating a housekeeping gene as a control supports our results for qualitative analysis of gene expression in hybrids assessing each allele individually (Fig 4), and improves interpretation for non-experts. We have also quantified differential gene expression in hybrids between species alleles and the log2 fold change from D. melanogaster. In addition, we have included an additional analysis in the new Fig. 5 which analyses RNA-seq expression changes in *D. melanogaster *x *D. simulans *hybrid male accessory glands. We believe these additions have significantly improved the manuscript and its conclusions.

    While the following comments are not necessarily minor, they can be addressed through revisions to the text without requiring additional experimental work. Some comments are more conceptual in nature, while others concern the interpretation and presentation of the experimental results. They are provided in no particular order.

    1. A key limitation of this study is the use of RNA-seq datasets from whole adult flies for interspecies gene expression comparisons. Whole-body RNA-seq inherently averages gene expression across all tissues, potentially masking tissue-specific expression differences. While Acp genes are likely restricted to accessory glands, the non-Acp genes and the random gene sets used in the analysis may have broader expression profiles. As a result, their expression might be conserved in certain tissues while diverging in others- an aspect that whole-body RNA-seq cannot capture. The authors should acknowledge that tissue-specific RNA-seq analyses could provide a more precise understanding of expression divergence and potentially reveal reduced conservation when considering specific tissues independently.

    We have added a section discussing the limitations in gene expression analysis in the discussion. In addition, we have included an additional Figure analysing gene expression in hybrid male accessory glands (Fig. 5).

    1. The statement in line 128, "Consistent with this model," does not accurately reflect the findings presented in Figures 2A and B. Specifically, the data in Figure 2A show that Acp gene expression divergence is significantly different from the divergence of non-Acp genes or a random sample only in the comparison between D. melanogaster and D. simulans. However, when these species are compared to D. yakuba, Acp gene expression divergence aligns with the divergence patterns of non-Acp genes or random samples. In contrast, Figure 2B shows that the distribution of expression changes is skewed for Acp genes compared to random control samples when D. melanogaster or D. simulans are compared to D. yakuba. However, this skew is absent when the two D. melanogaster and D. simulans are compared. Therefore, the statement in line 128 should be revised to accurately reflect these nuanced results and the trends shown in Figure 2A and B.

    We have updated the statement for clarity. Here, the percentage of Acps showing significant gene expression changes is greater between more closely related species, but the distribution of expression changes increases between more distantly related species.

    1. The statement in lines 136-138, "Acps were enriched for significant expression changes in the faster evolving group across all species," while accurate, overlooks a key observation. This trend was also observed in other groups, including those with slower evolving promoters, in some of the species' comparisons. Therefore, the enrichment is not unique to Acps with rapidly evolving promoters, and this should be explicitly acknowledged in the text.

    This is a valid point, and we have updated this statement as suggested.

    1. It would be helpful for the authors to explain the meaning of the d score at the beginning of the paragraph starting in line 131, to ensure clarity for readers unfamiliar with this metric.

    This scoring method is described in the methods sections, and we have now included reference to thorough explanation of how d was calculated at the indicated section.

    1. In Figure 2C-E - the title of the Y-axis does not match the text. If it represents the percentage of genes with significant expression changes, as in Figure 2A, the discrepancies between the percentages in this figure and those in Figure 2A need to be addressed.

    We have updated the method used to categorise significant changes in gene expression in the text and the figure legend for clarity.

    1. The experiment in Figure 3 needs a better explanation in the text. What is the analysis presented in Figure 3B-D. How many species were compared?

    We have added additional details in the results section and an explanation of how sequence change hotspots were calculated in the results section is available.

    1. The concept of transvection should be omitted from this manuscript. First, the definition provided by the authors is inaccurate. Second, even if additional experiments were to convincingly show that one allele in hybrid animals is dominant over the other, there are alternative explanations for this phenomenon that do not involve transvection. The authors may propose transvection as a potential model in the discussion, but they should do so cautiously and explicitly acknowledge the possibility of other mechanisms.

    We have updated the text to more conservatively discuss transvection, moving this to the discussion section with additional possibilities discussed.

    1. The statement at the end of the introduction is overly strong and would benefit from more cautious phrasing. For instance, it could be reworded as: "These findings suggest that promoter changes, rather than genomic background, play a significant role in driving expression changes, indicating that promoter evolution may contribute to the rise of new species."

    We have reworded this line following the reviewer's suggestion.

    1. Line 32 of the abstract: The term "Acp" is introduced without explaining what it stands for. Please define it as "Accessory gland proteins (Acp)" when it first appears.

    We have updated the manuscript to define Acp where it is first mentioned.

    1. Line 61: The phrase "...through relaxed,..." is unclear. Specify what is relaxed (e.g., "relaxed selective pressures").

    We have included description of relaxed selective pressures.

    1. The sentence in lines 74-76, starting in "Interestingly,...." Needs revision for clarity.

    We have removed the word interestingly.

    1. Line 112: Revise "we focused on the melanogaster subgroup which is made up of five species" to: "we focused on the melanogaster subgroup, which includes five species."

    We have made this change in the text.

    1. In line 144 use the phrase "promoter conservation" instead of "promoter evolution"

    We have updated the phrasing.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this study, McQuarrie et al. investigate the evolution of promoters of genes encoding accessory gland proteins (Acps) in species within the D. melanogaster subgroup. Using computational analyses and available genomic and transcriptomic datasets, they demonstrate that promoter regions of Acp genes are highly diverse compared to the promoters of other genes in the genome. They further show that this diversification correlates with changes in gene expression levels between closely related species. Complementing these computational analyses, the authors conduct experiments to test whether differences in expression levels of four Acp genes with highly diverged promoter regions are maintained in hybrids of closely related species. They find that while two Acp genes maintain their expression level differences in hybrids, the other two exhibit dominance of one allele. The authors attribute these findings to transvection. Based on their data, they conclude that rapid evolution of Acp gene promoters, rather than changes in trans, drives changes in Acp gene expression that contribute to speciation.

    Major comments:

    Unfortunately, the presented data are not sufficient to fully support the conclusions. While many of the concerns can be addressed by revising the text to moderate the claims and acknowledge the methodological limitations, some key experiments require repetition with more controls, biological replicates, and statistical analyses to validate the findings.

    Specifically, some of the main conclusions heavily rely on the RT-PCR experiments presented in Figure 4, which analyze the expression of four Acp genes in hybrid flies. The authors use PCR and RFLP to distinguish species-specific alleles but draw quantitative conclusions from what is essentially a qualitative experiment. There are several issues with this approach. First, the experiment includes only two biological replicates per sample, which is inadequate for robust statistical analysis. Second, the authors did not measure the intensity of the gel fragments, making it impossible to quantify allele-specific expression accurately. Third, no control genes were used as standards to ensure the comparability of samples.

    The gold standard for quantifying allele-specific expression is using real-time PCR methods such as TaqMan assays, which allow precise SNP genotyping. To address this major limitation, the authors should ideally repeat the experiments using allele-specific real-time PCR assays. This would provide a reliable and quantitative measurement of allele-specific expression.

    If the authors cannot implement real-time PCR, an alternative (though less rigorous) approach would be to continue using their current method with the following adjustments:

    • Include a housekeeping gene in the analysis as an internal control (this would require identifying a region distinguishable by RFLP in the control).
    • Quantify the intensity of the PCR products on the gel relative to the internal standard, ensuring proper normalization.
    • Increase the sample size to allow for robust statistical analysis. These experiments could be conducted relatively quickly and would significantly enhance the validity of the study's conclusions.

    Minor comments

    While the following comments are not necessarily minor, they can be addressed through revisions to the text without requiring additional experimental work. Some comments are more conceptual in nature, while others concern the interpretation and presentation of the experimental results. They are provided in no particular order.

    1. A key limitation of this study is the use of RNA-seq datasets from whole adult flies for interspecies gene expression comparisons. Whole-body RNA-seq inherently averages gene expression across all tissues, potentially masking tissue-specific expression differences. While Acp genes are likely restricted to accessory glands, the non-Acp genes and the random gene sets used in the analysis may have broader expression profiles. As a result, their expression might be conserved in certain tissues while diverging in others- an aspect that whole-body RNA-seq cannot capture. The authors should acknowledge that tissue-specific RNA-seq analyses could provide a more precise understanding of expression divergence and potentially reveal reduced conservation when considering specific tissues independently.
    2. The statement in line 128, "Consistent with this model," does not accurately reflect the findings presented in Figures 2A and B. Specifically, the data in Figure 2A show that Acp gene expression divergence is significantly different from the divergence of non-Acp genes or a random sample only in the comparison between D. melanogaster and D. simulans. However, when these species are compared to D. yakuba, Acp gene expression divergence aligns with the divergence patterns of non-Acp genes or random samples. In contrast, Figure 2B shows that the distribution of expression changes is skewed for Acp genes compared to random control samples when D. melanogaster or D. simulans are compared to D. yakuba. However, this skew is absent when the two D. melanogaster and D. simulans are compared. Therefore, the statement in line 128 should be revised to accurately reflect these nuanced results and the trends shown in Figure 2A and B.
    3. The statement in lines 136-138, "Acps were enriched for significant expression changes in the faster evolving group across all species," while accurate, overlooks a key observation. This trend was also observed in other groups, including those with slower evolving promoters, in some of the species' comparisons. Therefore, the enrichment is not unique to Acps with rapidly evolving promoters, and this should be explicitly acknowledged in the text.
    4. It would be helpful for the authors to explain the meaning of the d score at the beginning of the paragraph starting in line 131, to ensure clarity for readers unfamiliar with this metric.
    5. In Figure 2C-E - the title of the Y-axis does not match the text. If it represents the percentage of genes with significant expression changes, as in Figure 2A, the discrepancies between the percentages in this figure and those in Figure 2A need to be addressed.
    6. The experiment in Figure 3 needs a better explanation in the text. What is the analysis presented in Figure 3B-D. How many species were compared?
    7. The concept of transvection should be omitted from this manuscript. First, the definition provided by the authors is inaccurate. Second, even if additional experiments were to convincingly show that one allele in hybrid animals is dominant over the other, there are alternative explanations for this phenomenon that do not involve transvection. The authors may propose transvection as a potential model in the discussion, but they should do so cautiously and explicitly acknowledge the possibility of other mechanisms.
    8. The statement at the end of the introduction is overly strong and would benefit from more cautious phrasing. For instance, it could be reworded as: "These findings suggest that promoter changes, rather than genomic background, play a significant role in driving expression changes, indicating that promoter evolution may contribute to the rise of new species."

    Text edits:

    Throughout the manuscripts there are incomplete sentences and sentences that are not clear. Below is a list of corrections:

    1. Line 32 of the abstract: The term "Acp" is introduced without explaining what it stands for. Please define it as "Accessory gland proteins (Acp)" when it first appears.
    2. Line 61: The phrase "...through relaxed,..." is unclear. Specify what is relaxed (e.g., "relaxed selective pressures").
    3. The sentence in lines 74-76, starting in "Interestingly,...." Needs revision for clarity.
    4. Line 112: Revise "we focused on the melanogaster subgroup which is made up of five species" to: "we focused on the melanogaster subgroup, which includes five species."
    5. In line 144 use the phrase "promoter conservation" instead of "promoter evolution"

    Significance

    This study addresses an important question in evolutionary biology: how seminal fluid proteins achieve rapid evolution despite showing limited adaptive changes in their coding regions. By focusing on accessory gland proteins (Acps) and examining their promoter regions, the authors suggest promoter-driven evolution as a potential mechanism for rapid seminal fluid protein diversification. While this hypothesis is intriguing and can contribute to our understanding of speciation, more rigorous analysis and experimental validation would be needed to support the conclusions. The revised manuscript can be of interest to fly geneticists and to scientists in the fields of gene regulation and evolution.

    Keywords for my expertise: Enhancers, transcriptional regulation, development, evolution, Drosophila.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This manuscript explores promoter evolution of genes encoding seminal fluid proteins expressed in the male accessory gland of Drosophila and finds cis-regulatory changes underlie expression differences between species. Although these genes evolve rapidly it appears that the coding regions rarely show signs of positive selection inferring that changes in their expression and hence promoter sequences can underlie the evolution of their roles within and among species.

    Major comments

    Figure 1 illustrates evidence that the promoter regions of these gene have accumulated more changes than other sampled genes from the Drosophila genome. While this convinces that the region upstream of the transcription start site has diverged considerably in sequence (grey line compared to black line), Figure 1A also suggests the "Genespan" region which includes the 5'UTR but presumably also part of the coding region is also highly diverged. It would be useful to see how the pattern extends into the coding region further to compare further to the promoter region (although Fig 1H does illustrate this more convincingly).

    Figure 2 presents evidence for significant changes in (presumably levels of) expression of male accessory gland protein (AcP) genes and ribosomal proteins genes between pairs of species, which is reflected in the skew of expression compared to randomly selected genes.

    Figure 3 shows detailed analysis for 3 selected AcP genes with significantly diverged expression. The authors claim this shows 'substitution' hotspots in the promoter regions of all 3 genes but this could be better illustrated by extending the plots in B-D further upstream and downstream to compare to these regions.

    Figure 4 shows the results of expression analysis in parental lines of each pair of species and F1 hybrids. However the results are very difficult to follow in the figure and in the relevant text. While the schemes in A, C. E and G are helpful, the gel images are not the best quality and interpretations confusing. An additional scheme is needed to illustrate hypothetical outcomes of trans change, cis change and transvection to help interpret the gels. On line 169 (presumably referring to panels D and F although C and D are cited on the next line) the authors claim that Obp56f and CG11598 'were more expressed in D. melanogaster compared to D. simulans' but in the gel image the D. sim band is stronger for both genes (like D. sechellia) compared to the D. mel band. The authors also claim that the patterns of expression seen in the F1s are dominant for one allele and that this must be because of transvection. I agree this experiment is evidence for cis-regulatory change. However the interpretation that it is caused by transvection needs more explanation/justification and how do the authors rule out that it is not a cis X trans interaction between the species promoter differences and differences in the transcription factors of each species in the F1? Also my understanding is that transvection is relatively rare and yet the authors claim this is the explanation for 2/4 genes tested.

    Minor comments

    Line 112 states that the melanogaster subgroup contains 5 species - this is incorrect - while this study looked at 5 species there are more species in this subgroup such as mauritiana and santomea.

    Lines 131-134 could explain better what the conservation scores and their groupings mean and the rationale for this approach.

    Line 162 - the meaning of the sentence starting on this line is unclear - it sounds very circular.

    Line 168 should cite Fig 4 H instead of F.

    Significance

    This paper is generally well written although some sections would benefit from more explanation. The paper demonstrates cis-regulatory changes between the promoters of orthologs of male accessory gland genes underlie expression differences but that the species differences are not always reflected in hybrids, which the authors interpret as being caused by transvection although there could be other explanations. Overall this provides new insights into the regulation and divergence of these interesting genes. The paper does not explore the consequences of these changes in gene expression although this is discussed to some extent in the Discussion section.

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    Referee #1

    Evidence, reproducibility and clarity

    The authors test the hypothesis that promoters of genes involved in insect accessory glands evolved more rapidly than other genes in the genome. They test this using a number of computational and experimental approaches, looking at different species within the Drosophila melanogaster complex. The authors find an increased amount of sequence divergence in promoters of accessory gland proteins. They show that the expression levels of these proteins are more variable among species than randomly selected proteins. Finally, they show that within interspecific hybrids, each copy of the gene maintains its species-specific expression level.

    The work is done with expected standards of controls and analyses. The claims are supported by the analysis. My main criticism of the manuscript has to do not with the experiments or conclusion themselves but with the presentation. The manuscript is just not very well written, and following the logic of the arguments and results is challenging. The problem begins with the Abstract, which is representative of the general problems with the manuscript. The Abstract begins with general statements about the evolution of seminal fluid proteins, but then jumps to accessory glands and hybrids, without clarifying what taxon is being studied, and what hybrids they are talking about. Then, the acronym Acp is introduced without explanation. The last two sentences of the Abstract are very cumbersome and one has to reread them to understand how they link to the beginning of the Abstract.

    More generally, if this reviewer is to be seen as an "average reader" of the paper, I really struggled through reading it, and did not understand many of the arguments or rationale until the second read-through, after I had already read the bottom line. The paragraph spanning lines 71-83 is another case in point. It is composed of a series of very strongly worded sentences, almost all starting with a modifier (unexpectedly, interestingly, moreover), and supported by citations, but the logical flow doesn't work. Again, reading the paragraph after I knew where the paper was going was clearer, but on a first read, it was just a list of disjointed statements.

    Since most of the citations are from the authors' own work, I suspect they are assuming too much prior understanding on the part of the reader. I am sure that if the authors read through the manuscript again, trying to look through the eyes of an external reader, they will easily be able to improve the flow and readability of the text.

    More specific comments:

    1. In the analysis of expression level differences, it is not clear what specific stage / tissue the levels taken from the literature refer to. Could it be that the source of the data is from a stage or tissue where seminar fluid proteins will be expressed with higher variability in general (not just inter-specifically) and this could be skewing the results? Please add more information on the original source of the data and provide support for their validity for this type of comparison.
    2. The sentence spanning lines 155-157 needs more context.
    3. Line 203-204: What are multi-choice enhancers?
    4. Figure 1: The terminology the authors use, comparing the gene of interest to "Genome" is very confusing. They are not comparing to the entire genome but to all genes in the genome, which is not the same.
    5. Figure 2: Changes between X vs. Y is redundant (either changes between X and Y or changes in X vs. Y).

    Significance

    The manuscript addresses a general question in evolutionary biology - do control regions diverge more quickly protein coding regions. The answer is that yes, they do, but this is actually not very surprising. The work is probably thus of more interest to people interested in the copulatory proteins or in the evolution of mating systems, than to people interested in broader evolutionary questions.

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    Reply to the reviewers

    1. Response to reviewers

    We would like to thank the reviewers for carefully reading our manuscript and for their valuable comments in support for the publication of our investigation of rapid promoter evolution of accessory gland genes between Drosophila species and hybrids. We are glad to read that the reviewers find our work interesting and that it provides valuable insights into the regulation and divergence of genes through their promoters. We are encouraged by their acknowledgement of the overall quality of the work and the importance of our analyses in advancing the understanding of cis-regulatory changes in species divergence.

    2. Point-by-point description of the revisions

    Reviewer #

    Reviewer Comment

    Author Response/Revision

    Reviewer 1

    The authors test the hypothesis that promoters of genes involved in insect accessory glands evolved more rapidly than other genes in the genome. They test this using a number of computational and experimental approaches, looking at different species within the Drosophila melanogaster complex. The authors find an increased amount of sequence divergence in promoters of accessory gland proteins. They show that the expression levels of these proteins are more variable among species than randomly selected proteins. Finally, they show that within interspecific hybrids, each copy of the gene maintains its species-specific expression level.

    We thank Reviewer 1 for their detailed review and positive feedback on our manuscript, and for their helpful suggestions. We have now fully addressed the points raised by Reviewer 1 and have provided the suggested clarifications and revisions to improve the flow, readability, and presentation of the data, which we believe have improved the manuscript significantly.

    The work is done with expected standards of controls and analyses. The claims are supported by the analysis. My main criticism of the manuscript has to do not with the experiments or conclusion themselves but with the presentation. The manuscript is just not very well written, and following the logic of the arguments and results is challenging.

    The problem begins with the Abstract, which is representative of the general problems with the manuscript. The Abstract begins with general statements about the evolution of seminal fluid proteins, but then jumps to accessory glands and hybrids, without clarifying what taxon is being studied, and what hybrids they are talking about. Then, the acronym Acp is introduced without explanation. The last two sentences of the Abstract are very cumbersome and one has to reread them to understand how they link to the beginning of the Abstract.

    More generally, if this reviewer is to be seen as an "average reader" of the paper, I really struggled through reading it, and did not understand many of the arguments or rationale until the second read-through, after I had already read the bottom line. The paragraph spanning lines 71-83 is another case in point. It is composed of a series of very strongly worded sentences, almost all starting with a modifier (unexpectedly, interestingly, moreover), and supported by citations, but the logical flow doesn't work. Again, reading the paragraph after I knew where the paper was going was clearer, but on a first read, it was just a list of disjointed statements.

    Since most of the citations are from the authors' own work, I suspect they are assuming too much prior understanding on the part of the reader. I am sure that if the authors read through the manuscript again, trying to look through the eyes of an external reader, they will easily be able to improve the flow and readability of the text.

    We thank the reviewer for their detailed feedback and are glad that they acknowledge our work fully supports the claims of our manuscript. We also appreciate their helpful suggestions for improving the readability of the manuscript and have done our best to re-write the abstract and main text where indicated. In particular, the paragraph between lines 71-83 have been rewritten and we have taken care to write to non-expert readers.

    1. In the analysis of expression level differences, it is not clear what specific stage / tissue the levels taken from the literature refer to. Could it be that the source of the data is from a stage or tissue where seminar fluid proteins will be expressed with higher variability in general (not just inter-specifically) and this could be skewing the results? Please add more information on the original source of the data and provide support for their validity for this type of comparison.

    These were taken from publicly available adult male *Drosophila *datasets, listed in the data availability statement and throughout the manuscript. We have provided more detail on the tissue used for analysis of Acp gene expression levels.

    1. The sentence spanning lines 155-157 needs more context.

    We have added more context to lines 155-157.

    1. Line 203-204: What are multi-choice enhancers?

    We replaced the sentence with "... such as rapidly evolving enhancers or nested epistasis enhancer networks"

    1. Figure 1: The terminology the authors use, comparing the gene of interest to "Genome" is very confusing. They are not comparing to the entire genome but to all genes in the genome, which is not the same.

    We have changed the word "genome" to "all genes in the genome" on the reviewer's suggestion.

    1. Figure 2: Changes between X vs. Y is redundant (either changes between X and Y or changes in X vs. Y).

    We assume that the reviewer is referring to Fig. 2B, which does not measure changes between X and Y, but changes in distribution between Acps and the control group. We have explained this in the figure legend.

    The manuscript addresses a general question in evolutionary biology - do control regions diverge more quickly protein coding regions. The answer is that yes, they do, but this is actually not very surprising. The work is probably thus of more interest to people interested in the copulatory proteins or in the evolution of mating systems, than to people interested in broader evolutionary questions.

    We appreciate this reviewer's recognition of the significance of our work and would like to point out that there are very few studies looking at promoter evolution as detailed in the introduction. Of particular relevance, our study using Acp genes allows us to directly test the impact of promoter mutations on the expression by comparing two alleles in male accessory glands of Drosophila hybrids. Male accessory glands consist of only two secretory cell types allowing us to study evolution of gene expression in a single cell type (Acps are either expressed in main cells or secondary cells). Amid this unique experimental set up we can conclude that promoter mutations can act dominant, in contrast to mutations in protein coding regions, which are generally recessive. Thus, our study is unique in pointing out a largely overseen aspect of gene evolution.

    Reviewer 2

    This manuscript explores promoter evolution of genes encoding seminal fluid proteins expressed in the male accessory gland of Drosophila and finds cis-regulatory changes underlie expression differences between species. Although these genes evolve rapidly it appears that the coding regions rarely show signs of positive selection inferring that changes in their expression and hence promoter sequences can underlie the evolution of their roles within and among species.

    We thank Reviewer 2 for their thorough review, positive feedback on the importance of our work, and suggestions for improving the manuscript. We have addressed all points raised by the reviewer, including analysis of Acp coding region evolution, additional analyses of hybrid expression data, and improved the clarity of the text.

    Figure 1 illustrates evidence that the promoter regions of these gene have accumulated more changes than other sampled genes from the Drosophila genome. While this convinces that the region upstream of the transcription start site has diverged considerably in sequence (grey line compared to black line), Figure 1A also suggests the "Genespan" region which includes the 5'UTR but presumably also part of the coding region is also highly diverged. It would be useful to see how the pattern extends into the coding region further to compare further to the promoter region (although Fig 1H does illustrate this more convincingly).

    The reviewer raises an interesting point, and certainly all parts of genes evolve. Fig. 1A shows the evolutionary rates of Acps compared to the genome average from phyloP27way scores calculated from 27 insect species. Since these species are quite distant it is unsurprising that they show divergence in coding regions as well as promoter regions. In fact, we addressed whether promoter regions evolve fast in closely related *Drosophila *species in Fig. 1H compared to coding regions. We have included an additional analysis of coding region evolution in Figure 1B.

    Figure 2 presents evidence for significant changes in (presumably levels of) expression of male accessory gland protein (AcP) genes and ribosomal proteins genes between pairs of species, which is reflected in the skew of expression compared to randomly selected genes.

    Correct, we have rephrased the statement for clarity.

    Figure 3 shows detailed analysis for 3 selected AcP genes with significantly diverged expression. The authors claim this shows 'substitution' hotspots in the promoter regions of all 3 genes but this could be better illustrated by extending the plots in B-D further upstream and downstream to compare to these regions.

    We picked the 300-nucleotide promoter region for this analysis as it accumulated significant changes as shown in Fig. 1E-H, and extending the G plots (Fig. 3B-D) to regions with lower numbers of sequence changes would not substantially change the conclusion. Specifically, this analysis identifies sequence change hotspots within fast-evolving promoter regions, rather than comparing promoter regions to other genomic regions, as we previously addressed. The plot is based on a cumulative distribution function and the significant positive slope in the upstream region where promoters are located identifies a hotspot for accumulation of substitutions. There could be other hotspots, but the point being made is that significant hotspots consistently appear in the promoter region of these three genes.

    Figure 4 shows the results of expression analysis in parental lines of each pair of species and F1 hybrids. However the results are very difficult to follow in the figure and in the relevant text. While the schemes in A, C. E and G are helpful, the gel images are not the best quality and interpretations confusing. An additional scheme is needed to illustrate hypothetical outcomes of trans change, cis change and transvection to help interpret the gels. On line 169 (presumably referring to panels D and F although C and D are cited on the next line) the authors claim that Obp56f and CG11598 'were more expressed in D. melanogaster compared to D. simulans' but in the gel image the D. sim band is stronger for both genes (like D. sechellia) compared to the D. mel band. The authors also claim that the patterns of expression seen in the F1s are dominant for one allele and that this must be because of transvection. I agree this experiment is evidence for cis-regulatory change. However the interpretation that it is caused by transvection needs more explanation/justification and how do the authors rule out that it is not a cis X trans interaction between the species promoter differences and differences in the transcription factors of each species in the F1? Also my understanding is that transvection is relatively rare and yet the authors claim this is the explanation for 2/4 genes tested.

    We appreciate the reviewer's comments on Figure 4 and the opportunity to improve its clarity. To address these concerns, we have carefully checked the figure citations and corrected any inconsistencies.

    The reviewer raises an important point about our interpretation of transvection. We have expanded our discussion of this result to consider why transvection is a plausible explanation for the observed dominance patterns and also consider cis x *trans *interactions between species-specific promoters and transcription factor binding. While rare, transvection likely has more relevance in hybrid regulatory contexts involving homologous chromosome pairing which we discuss this in the revised text.

    Line 112 states that the melanogaster subgroup contains 5 species - this is incorrect - while this study looked at 5 species there are more species in this subgroup such as mauritiana and santomea.

    We have corrected the statement about the number of species in the melanogaster subgroup.

    Lines 131-134 could explain better what the conservation scores and their groupings mean and the rationale for this approach.

    We have clarified what the conservation scores and their groupings mean and the rationale for this approach.

    Line 162 - the meaning of the sentence starting on this line is unclear - it sounds very circular.

    We have rephrased the statement for more clarity.

    Line 168 should cite Fig 4 H instead of F.

    We have amended citation of Fig 4F to H.

    Reviewer 3

    In this study, McQuarrie et al. investigate the evolution of promoters of genes encoding accessory gland proteins (Acps) in species within the D. melanogaster subgroup. Using computational analyses and available genomic and transcriptomic datasets, they demonstrate that promoter regions of Acp genes are highly diverse compared to the promoters of other genes in the genome. They further show that this diversification correlates with changes in gene expression levels between closely related species. Complementing these computational analyses, the authors conduct experiments to test whether differences in expression levels of four Acp genes with highly diverged promoter regions are maintained in hybrids of closely related species. They find that while two Acp genes maintain their expression level differences in hybrids, the other two exhibit dominance of one allele. The authors attribute these findings to transvection. Based on their data, they conclude that rapid evolution of Acp gene promoters, rather than changes in trans, drives changes in Acp gene expression that contribute to speciation.

    We thank Reviewer 3 for their thorough review and suggestions. We further thank the reviewer for acknowledging the importance of our findings and for pointing out that it contributes to our understanding of speciation. We have thoroughly addressed all comments from the reviewer and significantly revised the manuscript. We believe that this has greatly improved the manuscript.

    Unfortunately, the presented data are not sufficient to fully support the conclusions. While many of the concerns can be addressed by revising the text to moderate the claims and acknowledge the methodological limitations, some key experiments require repetition with more controls, biological replicates, and statistical analyses to validate the findings.

    Specifically, some of the main conclusions heavily rely on the RT-PCR experiments presented in Figure 4, which analyze the expression of four Acp genes in hybrid flies. The authors use PCR and RFLP to distinguish species-specific alleles but draw quantitative conclusions from what is essentially a qualitative experiment. There are several issues with this approach. First, the experiment includes only two biological replicates per sample, which is inadequate for robust statistical analysis. Second, the authors did not measure the intensity of the gel fragments, making it impossible to quantify allele-specific expression accurately. Third, no control genes were used as standards to ensure the comparability of samples.

    The gold standard for quantifying allele-specific expression is using real-time PCR methods such as TaqMan assays, which allow precise SNP genotyping. To address this major limitation, the authors should ideally repeat the experiments using allele-specific real-time PCR assays. This would provide a reliable and quantitative measurement of allele-specific expression.

    If the authors cannot implement real-time PCR, an alternative (though less rigorous) approach would be to continue using their current method with the following adjustments:

    • Include a housekeeping gene in the analysis as an internal control (this would require identifying a region distinguishable by RFLP in the control).

    • Quantify the intensity of the PCR products on the gel relative to the internal standard, ensuring proper normalization.

    • Increase the sample size to allow for robust statistical analysis.

    These experiments could be conducted relatively quickly and would significantly enhance the validity of the study's conclusions.

    We thank the reviewer for their detailed suggestions for improving the conclusions in Fig. 4. Indeed, incorporating a housekeeping gene as a control supports our results for qualitative analysis of gene expression in hybrids assessing each allele individually (Fig 4), and improves interpretation for non-experts. We have also included an additional analysis in the new Fig. 5 which analyses RNA-seq expression changes in *D. melanogaster *x *D. simulans *hybrid male accessory glands. We believe these additions have significantly improved the manuscript and its conclusions.

    While the following comments are not necessarily minor, they can be addressed through revisions to the text without requiring additional experimental work. Some comments are more conceptual in nature, while others concern the interpretation and presentation of the experimental results. They are provided in no particular order.

    1. A key limitation of this study is the use of RNA-seq datasets from whole adult flies for interspecies gene expression comparisons. Whole-body RNA-seq inherently averages gene expression across all tissues, potentially masking tissue-specific expression differences. While Acp genes are likely restricted to accessory glands, the non-Acp genes and the random gene sets used in the analysis may have broader expression profiles. As a result, their expression might be conserved in certain tissues while diverging in others- an aspect that whole-body RNA-seq cannot capture. The authors should acknowledge that tissue-specific RNA-seq analyses could provide a more precise understanding of expression divergence and potentially reveal reduced conservation when considering specific tissues independently.

    We have added a section discussing the limitations in gene expression analysis in the discussion. In addition, we have included an additional Figure analysing gene expression in hybrid male accessory glands (Fig. 5).

    1. The statement in line 128, "Consistent with this model," does not accurately reflect the findings presented in Figures 2A and B. Specifically, the data in Figure 2A show that Acp gene expression divergence is significantly different from the divergence of non-Acp genes or a random sample only in the comparison between D. melanogaster and D. simulans. However, when these species are compared to D. yakuba, Acp gene expression divergence aligns with the divergence patterns of non-Acp genes or random samples. In contrast, Figure 2B shows that the distribution of expression changes is skewed for Acp genes compared to random control samples when D. melanogaster or D. simulans are compared to D. yakuba. However, this skew is absent when the two D. melanogaster and D. simulans are compared. Therefore, the statement in line 128 should be revised to accurately reflect these nuanced results and the trends shown in Figure 2A and B.

    We have updated the statement for clarity. Here, the percentage of Acps showing significant gene expression changes is greater between more closely related species, but the distribution of expression changes increases between more distantly related species.

    1. The statement in lines 136-138, "Acps were enriched for significant expression changes in the faster evolving group across all species," while accurate, overlooks a key observation. This trend was also observed in other groups, including those with slower evolving promoters, in some of the species' comparisons. Therefore, the enrichment is not unique to Acps with rapidly evolving promoters, and this should be explicitly acknowledged in the text.

    This is a valid point, and we have updated this statement as suggested.

    1. It would be helpful for the authors to explain the meaning of the d score at the beginning of the paragraph starting in line 131, to ensure clarity for readers unfamiliar with this metric.

    This scoring method is described in the methods sections, and we have now included reference to thorough explanation of how d was calculated at the indicated section.

    1. In Figure 2C-E - the title of the Y-axis does not match the text. If it represents the percentage of genes with significant expression changes, as in Figure 2A, the discrepancies between the percentages in this figure and those in Figure 2A need to be addressed.

    We have updated the method used to categorise significant changes in gene expression in the text and the figure legend for clarity.

    1. The experiment in Figure 3 needs a better explanation in the text. What is the analysis presented in Figure 3B-D. How many species were compared?

    We have added additional details in the results section and an explanation of how sequence change hotspots were calculated in the results section is available.

    1. The concept of transvection should be omitted from this manuscript. First, the definition provided by the authors is inaccurate. Second, even if additional experiments were to convincingly show that one allele in hybrid animals is dominant over the other, there are alternative explanations for this phenomenon that do not involve transvection. The authors may propose transvection as a potential model in the discussion, but they should do so cautiously and explicitly acknowledge the possibility of other mechanisms.

    We have updated the text to more conservatively discuss transvection, moving this to the discussion section with additional possibilities discussed.

    1. The statement at the end of the introduction is overly strong and would benefit from more cautious phrasing. For instance, it could be reworded as: "These findings suggest that promoter changes, rather than genomic background, play a significant role in driving expression changes, indicating that promoter evolution may contribute to the rise of new species."

    We have reworded this line following the reviewer's suggestion.

    1. Line 32 of the abstract: The term "Acp" is introduced without explaining what it stands for. Please define it as "Accessory gland proteins (Acp)" when it first appears.

    We have updated the manuscript to define Acp where it is first mentioned.

    1. Line 61: The phrase "...through relaxed,..." is unclear. Specify what is relaxed (e.g., "relaxed selective pressures").

    We have included description of relaxed selective pressures.

    1. The sentence in lines 74-76, starting in "Interestingly,...." Needs revision for clarity.

    We have removed the word interestingly.

    1. Line 112: Revise "we focused on the melanogaster subgroup which is made up of five species" to: "we focused on the melanogaster subgroup, which includes five species."

    We have made this change in the text.

    1. In line 144 use the phrase "promoter conservation" instead of "promoter evolution"

    We have updated the phrasing.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this study, McQuarrie et al. investigate the evolution of promoters of genes encoding accessory gland proteins (Acps) in species within the D. melanogaster subgroup. Using computational analyses and available genomic and transcriptomic datasets, they demonstrate that promoter regions of Acp genes are highly diverse compared to the promoters of other genes in the genome. They further show that this diversification correlates with changes in gene expression levels between closely related species. Complementing these computational analyses, the authors conduct experiments to test whether differences in expression levels of four Acp genes with highly diverged promoter regions are maintained in hybrids of closely related species. They find that while two Acp genes maintain their expression level differences in hybrids, the other two exhibit dominance of one allele. The authors attribute these findings to transvection. Based on their data, they conclude that rapid evolution of Acp gene promoters, rather than changes in trans, drives changes in Acp gene expression that contribute to speciation.

    Major comments:

    Unfortunately, the presented data are not sufficient to fully support the conclusions. While many of the concerns can be addressed by revising the text to moderate the claims and acknowledge the methodological limitations, some key experiments require repetition with more controls, biological replicates, and statistical analyses to validate the findings.

    Specifically, some of the main conclusions heavily rely on the RT-PCR experiments presented in Figure 4, which analyze the expression of four Acp genes in hybrid flies. The authors use PCR and RFLP to distinguish species-specific alleles but draw quantitative conclusions from what is essentially a qualitative experiment. There are several issues with this approach. First, the experiment includes only two biological replicates per sample, which is inadequate for robust statistical analysis. Second, the authors did not measure the intensity of the gel fragments, making it impossible to quantify allele-specific expression accurately. Third, no control genes were used as standards to ensure the comparability of samples.

    The gold standard for quantifying allele-specific expression is using real-time PCR methods such as TaqMan assays, which allow precise SNP genotyping. To address this major limitation, the authors should ideally repeat the experiments using allele-specific real-time PCR assays. This would provide a reliable and quantitative measurement of allele-specific expression.

    If the authors cannot implement real-time PCR, an alternative (though less rigorous) approach would be to continue using their current method with the following adjustments:

    • Include a housekeeping gene in the analysis as an internal control (this would require identifying a region distinguishable by RFLP in the control).
    • Quantify the intensity of the PCR products on the gel relative to the internal standard, ensuring proper normalization.
    • Increase the sample size to allow for robust statistical analysis. These experiments could be conducted relatively quickly and would significantly enhance the validity of the study's conclusions.

    Minor comments

    While the following comments are not necessarily minor, they can be addressed through revisions to the text without requiring additional experimental work. Some comments are more conceptual in nature, while others concern the interpretation and presentation of the experimental results. They are provided in no particular order.

    1. A key limitation of this study is the use of RNA-seq datasets from whole adult flies for interspecies gene expression comparisons. Whole-body RNA-seq inherently averages gene expression across all tissues, potentially masking tissue-specific expression differences. While Acp genes are likely restricted to accessory glands, the non-Acp genes and the random gene sets used in the analysis may have broader expression profiles. As a result, their expression might be conserved in certain tissues while diverging in others- an aspect that whole-body RNA-seq cannot capture. The authors should acknowledge that tissue-specific RNA-seq analyses could provide a more precise understanding of expression divergence and potentially reveal reduced conservation when considering specific tissues independently.
    2. The statement in line 128, "Consistent with this model," does not accurately reflect the findings presented in Figures 2A and B. Specifically, the data in Figure 2A show that Acp gene expression divergence is significantly different from the divergence of non-Acp genes or a random sample only in the comparison between D. melanogaster and D. simulans. However, when these species are compared to D. yakuba, Acp gene expression divergence aligns with the divergence patterns of non-Acp genes or random samples. In contrast, Figure 2B shows that the distribution of expression changes is skewed for Acp genes compared to random control samples when D. melanogaster or D. simulans are compared to D. yakuba. However, this skew is absent when the two D. melanogaster and D. simulans are compared. Therefore, the statement in line 128 should be revised to accurately reflect these nuanced results and the trends shown in Figure 2A and B.
    3. The statement in lines 136-138, "Acps were enriched for significant expression changes in the faster evolving group across all species," while accurate, overlooks a key observation. This trend was also observed in other groups, including those with slower evolving promoters, in some of the species' comparisons. Therefore, the enrichment is not unique to Acps with rapidly evolving promoters, and this should be explicitly acknowledged in the text.
    4. It would be helpful for the authors to explain the meaning of the d score at the beginning of the paragraph starting in line 131, to ensure clarity for readers unfamiliar with this metric.
    5. In Figure 2C-E - the title of the Y-axis does not match the text. If it represents the percentage of genes with significant expression changes, as in Figure 2A, the discrepancies between the percentages in this figure and those in Figure 2A need to be addressed.
    6. The experiment in Figure 3 needs a better explanation in the text. What is the analysis presented in Figure 3B-D. How many species were compared?
    7. The concept of transvection should be omitted from this manuscript. First, the definition provided by the authors is inaccurate. Second, even if additional experiments were to convincingly show that one allele in hybrid animals is dominant over the other, there are alternative explanations for this phenomenon that do not involve transvection. The authors may propose transvection as a potential model in the discussion, but they should do so cautiously and explicitly acknowledge the possibility of other mechanisms.
    8. The statement at the end of the introduction is overly strong and would benefit from more cautious phrasing. For instance, it could be reworded as: "These findings suggest that promoter changes, rather than genomic background, play a significant role in driving expression changes, indicating that promoter evolution may contribute to the rise of new species."

    Text edits:

    Throughout the manuscripts there are incomplete sentences and sentences that are not clear. Below is a list of corrections:

    1. Line 32 of the abstract: The term "Acp" is introduced without explaining what it stands for. Please define it as "Accessory gland proteins (Acp)" when it first appears.
    2. Line 61: The phrase "...through relaxed,..." is unclear. Specify what is relaxed (e.g., "relaxed selective pressures").
    3. The sentence in lines 74-76, starting in "Interestingly,...." Needs revision for clarity.
    4. Line 112: Revise "we focused on the melanogaster subgroup which is made up of five species" to: "we focused on the melanogaster subgroup, which includes five species."
    5. In line 144 use the phrase "promoter conservation" instead of "promoter evolution"

    Significance

    This study addresses an important question in evolutionary biology: how seminal fluid proteins achieve rapid evolution despite showing limited adaptive changes in their coding regions. By focusing on accessory gland proteins (Acps) and examining their promoter regions, the authors suggest promoter-driven evolution as a potential mechanism for rapid seminal fluid protein diversification. While this hypothesis is intriguing and can contribute to our understanding of speciation, more rigorous analysis and experimental validation would be needed to support the conclusions. The revised manuscript can be of interest to fly geneticists and to scientists in the fields of gene regulation and evolution.

    Keywords for my expertise: Enhancers, transcriptional regulation, development, evolution, Drosophila.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This manuscript explores promoter evolution of genes encoding seminal fluid proteins expressed in the male accessory gland of Drosophila and finds cis-regulatory changes underlie expression differences between species. Although these genes evolve rapidly it appears that the coding regions rarely show signs of positive selection inferring that changes in their expression and hence promoter sequences can underlie the evolution of their roles within and among species.

    Major comments

    Figure 1 illustrates evidence that the promoter regions of these gene have accumulated more changes than other sampled genes from the Drosophila genome. While this convinces that the region upstream of the transcription start site has diverged considerably in sequence (grey line compared to black line), Figure 1A also suggests the "Genespan" region which includes the 5'UTR but presumably also part of the coding region is also highly diverged. It would be useful to see how the pattern extends into the coding region further to compare further to the promoter region (although Fig 1H does illustrate this more convincingly).

    Figure 2 presents evidence for significant changes in (presumably levels of) expression of male accessory gland protein (AcP) genes and ribosomal proteins genes between pairs of species, which is reflected in the skew of expression compared to randomly selected genes.

    Figure 3 shows detailed analysis for 3 selected AcP genes with significantly diverged expression. The authors claim this shows 'substitution' hotspots in the promoter regions of all 3 genes but this could be better illustrated by extending the plots in B-D further upstream and downstream to compare to these regions.

    Figure 4 shows the results of expression analysis in parental lines of each pair of species and F1 hybrids. However the results are very difficult to follow in the figure and in the relevant text. While the schemes in A, C. E and G are helpful, the gel images are not the best quality and interpretations confusing. An additional scheme is needed to illustrate hypothetical outcomes of trans change, cis change and transvection to help interpret the gels. On line 169 (presumably referring to panels D and F although C and D are cited on the next line) the authors claim that Obp56f and CG11598 'were more expressed in D. melanogaster compared to D. simulans' but in the gel image the D. sim band is stronger for both genes (like D. sechellia) compared to the D. mel band. The authors also claim that the patterns of expression seen in the F1s are dominant for one allele and that this must be because of transvection. I agree this experiment is evidence for cis-regulatory change. However the interpretation that it is caused by transvection needs more explanation/justification and how do the authors rule out that it is not a cis X trans interaction between the species promoter differences and differences in the transcription factors of each species in the F1? Also my understanding is that transvection is relatively rare and yet the authors claim this is the explanation for 2/4 genes tested.

    Minor comments

    Line 112 states that the melanogaster subgroup contains 5 species - this is incorrect - while this study looked at 5 species there are more species in this subgroup such as mauritiana and santomea.

    Lines 131-134 could explain better what the conservation scores and their groupings mean and the rationale for this approach.

    Line 162 - the meaning of the sentence starting on this line is unclear - it sounds very circular.

    Line 168 should cite Fig 4 H instead of F.

    Significance

    This paper is generally well written although some sections would benefit from more explanation. The paper demonstrates cis-regulatory changes between the promoters of orthologs of male accessory gland genes underlie expression differences but that the species differences are not always reflected in hybrids, which the authors interpret as being caused by transvection although there could be other explanations. Overall this provides new insights into the regulation and divergence of these interesting genes. The paper does not explore the consequences of these changes in gene expression although this is discussed to some extent in the Discussion section.

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    Referee #1

    Evidence, reproducibility and clarity

    The authors test the hypothesis that promoters of genes involved in insect accessory glands evolved more rapidly than other genes in the genome. They test this using a number of computational and experimental approaches, looking at different species within the Drosophila melanogaster complex. The authors find an increased amount of sequence divergence in promoters of accessory gland proteins. They show that the expression levels of these proteins are more variable among species than randomly selected proteins. Finally, they show that within interspecific hybrids, each copy of the gene maintains its species-specific expression level.

    The work is done with expected standards of controls and analyses. The claims are supported by the analysis. My main criticism of the manuscript has to do not with the experiments or conclusion themselves but with the presentation. The manuscript is just not very well written, and following the logic of the arguments and results is challenging. The problem begins with the Abstract, which is representative of the general problems with the manuscript. The Abstract begins with general statements about the evolution of seminal fluid proteins, but then jumps to accessory glands and hybrids, without clarifying what taxon is being studied, and what hybrids they are talking about. Then, the acronym Acp is introduced without explanation. The last two sentences of the Abstract are very cumbersome and one has to reread them to understand how they link to the beginning of the Abstract.

    More generally, if this reviewer is to be seen as an "average reader" of the paper, I really struggled through reading it, and did not understand many of the arguments or rationale until the second read-through, after I had already read the bottom line. The paragraph spanning lines 71-83 is another case in point. It is composed of a series of very strongly worded sentences, almost all starting with a modifier (unexpectedly, interestingly, moreover), and supported by citations, but the logical flow doesn't work. Again, reading the paragraph after I knew where the paper was going was clearer, but on a first read, it was just a list of disjointed statements.

    Since most of the citations are from the authors' own work, I suspect they are assuming too much prior understanding on the part of the reader. I am sure that if the authors read through the manuscript again, trying to look through the eyes of an external reader, they will easily be able to improve the flow and readability of the text.

    More specific comments:

    1. In the analysis of expression level differences, it is not clear what specific stage / tissue the levels taken from the literature refer to. Could it be that the source of the data is from a stage or tissue where seminar fluid proteins will be expressed with higher variability in general (not just inter-specifically) and this could be skewing the results? Please add more information on the original source of the data and provide support for their validity for this type of comparison.
    2. The sentence spanning lines 155-157 needs more context.
    3. Line 203-204: What are multi-choice enhancers?
    4. Figure 1: The terminology the authors use, comparing the gene of interest to "Genome" is very confusing. They are not comparing to the entire genome but to all genes in the genome, which is not the same.
    5. Figure 2: Changes between X vs. Y is redundant (either changes between X and Y or changes in X vs. Y).

    Significance

    The manuscript addresses a general question in evolutionary biology - do control regions diverge more quickly protein coding regions. The answer is that yes, they do, but this is actually not very surprising. The work is probably thus of more interest to people interested in the copulatory proteins or in the evolution of mating systems, than to people interested in broader evolutionary questions.

  9. Version published to 10.1101/2024.10.14.618274 on bioRxiv