1. Author Response:

    Reviewer #1 (Public Review):

    The manuscript by Schrieber et al., explores whether inbreeding affects floral attractiveness to pollinators with additional factors of sex and origin in play, in male and female plants of Silene latifolia. The authors use a combination of spatial sampling, floral volatiles, flower color, and floral rewards coupled with the response of a specialized pollinator to these traits. Their results show that females are more affected by inbreeding and in general inbreeding negatively impacts the "composite nature" of floral traits. The manuscript is well written, the experiments are detailed and quite elaborate. For example., the methodology for flower color estimation is the most detailed effort in this area that I can remember. All the experiments in the manuscript show meticulous planning, with extensive data collection addressing minute details, including the statistics used. However, I do have some concerns that need to be addressed.

    Core strengths: Detailed experimental design, elaborate data collection methods, well-defined methodology that is easy to follow. There is a logical flow for the experiments, and no details are missing in most of the experiemnts.

    Weaknesses: A recent study has addressed some of the questions detailed in the manuscript. So, introduction needs to be tweaked to reflect this.

    Thank you very much for bringing this excellent article to our attention! We adjusted the writing in the introduction and the discussion accordingly. Please consider that this article was first published at the 15th of January 21, while our manuscript was submitted at the 9th of January. Hence, we were not able to account for this study in the first submission. Introduction pp 4-5, ll 48-54: “Although in a few cases inbreeding has been shown to alter single components of flower attractiveness (Ivey and Carr, 2005; Ferrari et al., 2006; Haber et al., 2019), insight into syndrome-wide effects is restricted to a single study. Kariyat et al. (2021) demonstrated that inbred Solanum carolinense L. display reduced flower size, pollen and scent production and receive fewer visits from diurnal generalists. It is necessary to broaden such integrated methodological approaches to other plant-pollinator systems (e.g., nocturnal specialist pollinators) and further floral traits (i.e., flower colour).” Discussion p 19, ll 535-542: “In summary, our research on S. latifolia suggests that in addition to inbreeding disrupting interactions with herbivores by changing plant leaf chemistry (Schrieber et al., 2018) it affects plant interactions with pollinators by altering flower chemistry. Our observations are in line with studies on other plant species (Ivey and Carr, 2005; Kariyat et al., 2012, 2021) and highlight that inbreeding has the potential to reset the equilibrium of species interactions by altering functional traits that have developed in a long history of co-evolution. These threats to antagonistic and symbiotic plant-insect interactions may mutually magnify in reducing plant individual fitness and altering the dynamics of natural plant populations under global change.”

    Some details and controls are missing in floral scent estimation. Flower age, a pesticide treatment of plants that could affect chemistry..needs to be better refined.

    We clarified this issue at different occasions in the methods section. Previous studies (and our study) on S. latifolia have shown no clear differences in the quality of floral scent between sexes. However, one study found higher total emission of VOC in males, while others found no differences. Hence, females produce no specific VOC that are used as oviposition cues but may be differentiated from males by the total amount of emitted VOC and pronounced differences in spatial flower traits. We highlight this at p 6, ll 111-116: “Silene latifolia exhibits various sexual dimorphisms with male plants producing more and smaller flowers that excrete lower volumes of nectar with higher sugar concentrations as compared to females (Gehring et al., 2004; Delph et al., 2010). The quality of floral scent exhibits no clear sex-specific patterns, while male plants have been shown to emit higher or equal total amounts of VOC as compared to females in different studies (Dötterl & Jürgens 2005, Waelti et al. 2009)”.

    Both male and female moths show pronounced behavioural responses to lilac aldehyde isomers and other VOC in the floral scent of S. latifolia (Dötterl et al., 2006). We therefore treated these VOC as typical floral scent compounds. We clarified this at p 7, ll 125-126: “A substantial fraction of floral VOC produced by S. latifolia triggers antennal and behavioural responses in male and female H. bicruris moths (Dötterl et al., 2006).” and p 9, ll 2010-218:” For targeted statistical analyses, we focused on those VOC that evidently mediate communication with H. bicruris according to Dötterl et al. (2006). We analysed the Shannon diversity per plant (calculated with R-package: vegan v.2.5-5, Oksanen et al. 2019) for 20 floral VOC in our data set that were shown to elicit electrophysiological responses in the antennae of H. bicruris (Supplementary File 1). Moreover, we analysed the intensities of three lilac aldehyde isomers, which trigger oriented flight and landing behaviour in both male and female H. bicruris most efficiently when compared to other VOC in the floral scent of S. latifolia. Furthermore, H. bicruris is able to detect the slightest differences in the concentration of these three compounds at very low dosages (Dötterl et al. 2006).”

    We used biological pest control agents in a preventive manner because S. latifolia is often infested by thrips and aphids under greenhouse conditions. The writing in the previous manuscript version was not clear with this regard and we changed the text at p 8, ll 157-161: ” Plants received water and fertilisation (UniversolGelb 12-30-12, Everris-Headquarters, NL) when necessary for the entire experimental period and were prophylactically treated with biological pest control agents under greenhouse conditions to prevent thrips (agent Amblyseius barkeri and Amblyseius cucumeris) and aphid (agent Chrysoperla carnea) infestation (Katz Biotech GmbH, GE) .”

    Indeed, flower size and scent emission can be correlated. Although the question whether differences in scent emission were based on a difference in flower size is an interesting one, it seemed less relevant to us because it is unlikely that our pollinators correct their perception of a scent for the size of a flower (see also p 19, 520-526). We were rather interested in whether scent emission differs between the plant treatments and thus pollinators may chemically perceive such differences. Moreover, we found it problematic to correct our models for flower size by including it as a covariate, which is the reason why we have not assessed this trait during scent collection. In this case, we would have corrected our scent responses for the effects of inbreeding, sex and population origin (i.e., the predictors we are interested in) because all of them determine the size of a flower (Figure 2 c,d). Hence, the inbreeding, sex and origin effects on flower scent would likely vanish. However, it is highly unlikely that the set of genes contributing to sex-, breeding treatment- and origin-based variation in flower size is exactly the same one that determines variation in scent emission per flower, which is basically the assumption underlying the model that includes flower size as a covariate. We critically mentioned the trade-off relationships and our reasoning to not correct for flower size at 9p ll 208-210: “The intensities of VOC were not corrected for flower size because we wanted to capture all variation in scent emission that is relevant for the receiver i.e., the pollinator.”

    While the study is laser-focused on floral traits, as the authors are aware inbreeding affects the total phenotype of the plants including fitness and defense traits. For example, there are quite a few studies that have shown how inbreeding affects the plant defense phenotype. This could be addressed in the introduction and discussion.

    We agree that this aspect is important and therefore addressed it in further detail in the introduction at p 4 ll 34-38: “While it is well established that inbreeding can increase a plant’s susceptibility to herbivores by diminishing morphological and chemical defences (Campbell et al., 2013; Kariyat et al., 2012; Kalske et al., 2014), its effects on plant-pollinator interactions are less well understood. Inbreeding may reduce a plant’s attractiveness to pollinating insects by compromising the complex set of floral traits involved in interspecific communication.” Since other referees suggested to rather tone down than increase the discussion based on floral scent results, we stick to the general feedback relationship among of herbivory and pollination, rather than relating it specifically to volatiles in the discussion at p 19, ll 535-544: “In summary, our research on S. latifolia suggests that in addition to inbreeding disrupting interactions with herbivores by changing plant leaf chemistry (Schrieber et al., 2018) it affects plant interactions with pollinators by altering flower chemistry. Our observations are in line with studies on other plant species (Ivey and Carr, 2005; Kariyat et al., 2012, 2021) and highlight that inbreeding has the potential to reset the equilibrium of species interactions by altering functional traits that have developed in a long history of co-evolution. These threats to antagonistic and symbiotic plant-insect interactions may mutually magnify in reducing plant individual fitness and altering the dynamics of natural plant populations under global change. As such, our study adds to a growing body of literature supporting the need to maintain or restore sufficient genetic diversity in plant populations during conservation programs.”

    Reviewer #2 (Public Review):

    A summary of what the authors were trying to achieve. This interesting and data-rich paper reports the results of several detailed experiments on the pollination biology of the dioceus plant Silene latfolia. The authors uses multiple accessions from several European (native range) and North American (introduced range) populations of S. latifolia to generate an experimental common garden. After one generation of within-population crosses, each cross included either two (half-)siblings or two unrelated individuals, they compared the effects of one-generation of inbreeding on multiple plant traits (height, floral size, floral scent, floral color), controlling for population origin. Thereby, they set out to test the hypothesis that inbreeding reduces plant attractiveness. Furthermore, they ask if the effect is more pronounced in female than male plants, which may be predicted from sexual selection and sex-chromosome-specific expression, and if the effect of inbreeding larger in native European populations than in North American populations, that may have already undergone genetic purging during the bottleneck that inbreeding reduces plant attractiveness. Finally, the authors evaluate to what extent the inbreeding-related trait changes affect floral attractiveness (measured as visitation rates) in field-based bioassays.

    An account of the major strengths and weaknesses of the methods and results. The major strength of this paper is the ambitious and meticulous experimental setup and implementation that allows comparisons of the effect of multiple predictors (i.e. inbreeding treatment, plant origin, plant sex) on the intraspecific variation of floral traits. Previous work has shown direct effects of plant inbreeding on floral traits, but no previous study has taken this wholesale approach in a system where the pollination ecology is well known. In particular, very few studies, if any, has tested the effects of inbreeding on floral scent or color traits. Moreover, I particularly appreciate that the authors go the extra mile and evaluate the biological importance of the inbreeding-induced trait variation in a field bioassay. I also very much appreciate that the authors have taken into account the biological context by using a relevant vision model in the color analyses and by focusing on EAD-active compounds in the floral scent analyses.

    The results are very interesting and shows that the effects of inbreeding on trait variation is both origin- and sex-dependent, but that the strongest effects were not always consistent with the hypothesis that North American plants would have undergone genetic purging during a bottleneck that would make these plants less susceptible to inbreeding effects. The authors made a large collection effort, securing seeds from eight populations from each continent, but then only used population origin and seed family origin as random factors in the models, when testing the overall effect of inbreeding on floral traits. It would have been very interesting with an analysis that partition the variance both in the actual traits under study and in the response to inbreeding to determine whether to what extent there is variation among populations within continents. Not the least, because it is increasingly clear that the ecological outcome of species interactions (mutualistic/antagonistic) in nursery pollination systems often vary among populations (cf. Thompson 2005, The geographic mosaic of coevolution), and some results suggest that this is the case also in Hadena-Silene interactions (e.g. Kephardt et al. 2006, New Phytologist). Furthermore, some plants involved in nursery pollination systems both show evidence of distinct canalization across populations of floral traits of importance for the interaction (e.g. Svensson et al. 2005), whereas others show unexpected and fine-grained variation in floral traits among populations (e.g. Suinyuy et al. 2015, Proceedings B, Thompson et al. 2017 Am. Nat., Friberg et al. 2019, PNAS). Hence, it is possible that the local population history and local variation in the interactions between the plants and their pollinators may be more important predictors for explaining variation in floral trait responses to inbreeding, than the larger-scale continental analyses. Not the least, because North American S. latifolia probably has multiple origins, with subsequent opportunity for admixture in secondary contact.

    Yes, it is necessary to put populations from the same continent into one category, since native and invasive plant populations differ significantly in their evolutionary history (p 5, ll 74-81, http://onlinelibrary.wiley.com/doi/10.1111/j.1365-294X.2012.05751.x). Origin explained sufficient amounts of variation in several traits including flower number, corolla expansion, VOC diversity, lilac aldehyde A intensity, and pollinator visitation rates (see Figures 2-3; and Table 2) and some variation in in the magnitude of inbreeding effects (Figure 2e, f; Figure 3). Even if we would not be interested in differences among native and invasive populations, we would have to include origin as a fixed effect in our models because:

    i) populations within a distribution range are no independent samples,

    ii) origin explains sufficient variation in many responses,

    iii) origin cannot be fitted as a random factor, since it has only two levels (the minimum number of levels for random effect is 4). We agree that it would be very interesting to specifically assess differences in the magnitude of breeding and sex effects among populations within origins. We now discuss this as important future research direction at p 18, ll 500-507: “As such, the precise mechanisms underlying variation in inbreeding effects on different scent traits across population origins of S. latifolia can only be explored based on comprehensive genomic resources, which are currently not available. Future studies should also incorporate field-data on the abundance of specialist pollinators and extend the focus from variation in the magnitude of inbreeding effects among geographic origins to variation among populations within geographic origins and individuals within populations. This would allow a detailed quantification of geographic variation in inbreeding effects and elaborating on the causes and ecological consequences of such variation (Thompson, 2005; Schrieber and Lachmuth, 2017; Thompson et al., 2017)”.

    To empirically address within-origin variation of inbreeding effects with our data, we would have to i) fit correlated random intercepts and slopes for the interaction breeding-sex on the population random factor (models consume min. 22 DF); or ii) include population as a fixed effect in our models (models consume min. 67 DF). We have tried both of these approaches when preparing the revision, but unfortunately it turned out that our study is not designed to address this question. The models for both variants only partially converge (see R-script ll. 1568-1580), and even if they do this does not imply that one can draw solid inference from them. Approach i often results in multiple singular convergence warning messages implying that no variance is explained by population-specific reaction norms to the fixed effects specified in the random effects structure. Approach ii results in odd rank- deficient models (I was seriously worried about type I errors). We simply have too few replicates (5) per population-breeding treatment-sex combination for both approaches. For solid inference we would need 10approach i-40approach ii replicates = 640-2600 individuals. However, our experimental design is sufficient to address the hypothesis we have raised in the introduction as well as general differences in response variables among populations. We now provide information on variance partitioning for all models that include population as a random effect in S9. As you will see, population explains lower amounts of variation in our responses as the fixed effects in 9 out of 12 models. The random effects maternal and paternal genotype (mother&father) explain more variation than the random effect population in 6 of 12 cases. Thus, these data do not make a strong case for an extensive discussion of population-based differences in floral traits and this was also not a question or hypotheses we wanted to address with our study.

    I see no major weaknesses in the study, and but in my detailed response, I have made a few questions and suggestions about the floral scent analyses. In short, the authors have used a technique that is not the standard method used for making quantitative floral scent analyses, and I am curious about how it was made sure that the results obtained from the static headspace sampling using PDMS adsorbents could be used as a quantitative measure. I would suggest the authors to validate the use of this method more thoroughly in the manuscript, and have detailed this comment in my response to the authors.

    Also, and this may seem like a nit-picky comment, I am not convinced that the best way to describe the traits under study is "plant attractiveness", because in the experimental bioassays, most of the traits under study that are affected by the inbreeding treatment, did not result in a reduced pollinator visitation. Most (or all) of these traits may also be involved in other plant functions and important for other interactions, so I suggest potentially using a term like "floral traits" or "(putative) signalling traits".

    We now avoid the term floral attractiveness throughout the manuscript and instead refer to “floral traits”.

    An appraisal of whether the authors achieved their aims, and whether the results support their conclusions: By and large, the authors achieved the aims of this study, and drew conclusions based in these results. One interesting aspect of this work that I think could be discussed a bit deeper is the lack of congruence between the effects of inbreeding on floral traits and the variation in visitation pattern in the bioassay. In fact, the only large effect of inbreeding on a floral trait that may play a role as an explanatory factor is the reduction of emission of lilac aldehyde A in inbred female S. latifolia from North America, which correspond to a reduced visitation rate in this group in the pollinator visitation bioassay. I have made some specific suggestions in my comments to the authors.

    We agree that this aspect required deeper discussion and revised the section at p 19, ll 520-526 accordingly. We believe that the limited spatial vision of H. bicruris in combination with our experimental setup for pollinator observations increased the relative importance of floral scent for pollinator visitation rates (suggested by referee #3).

    A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community: I think that one important aspect of this work that may broaden the impact of this study further is the link between these experiment, and our expectations from the evolution of selfing. Selfing plant species most often conform to the selfing syndrome, presenting smaller, less scented flowers than outcrossing relatives. Traditionally, the selfing syndrome is explained by natural selection against individuals that invest energy into floral signalling, when attracting pollinators is no longer crucial for reproduction. Some studies (for example Andersson, 2012, Am. J. Bot), however, have shown that only one, or a few, generations of inbreeding may reduce floral size as much as quite strong selection for reduced signalling. Here, at least for some populations and sexes, similar results are obtained in this paper regarding several traits (including floral scent), and one way to put this paper in context is by discussing the results in the light of these previous papers.

    We now address this issue at p 16, ll 417-420: “However, our findings highlight that even weak degrees of biparental inbreeding (i.e., one generation sib-mating) can result in a severe reduction of spatial flower trait and scent trait values that is detectable against the background of natural variation among multiple plant populations from a broad geographic region. This observation indirectly supports that the selfing syndrome (i.e., smaller, less scented flowers observed in selfing relative to outcrossing populations of hermaphroditic plant species) may not merely be a result of natural selection against resource investment into floral traits, but also a direct negative consequence of inbreeding (Andersson, 2012).”

    Reviewer #3 (Public Review):

    Schrieber et al. studied the effects of biparental inbreeding in the dioecious plant Silene latifolia, focusing specifically on traits important for floral attractiveness and pollinator attraction. These traits are especially important for dioecious species with separate sexes as they are obligate outcrossers. The authors find that inbreeding mostly decreases floral attractiveness, but that this effect tended to be stronger in the female flowers, which the authors suspect to result from the trade-off with larger investment in the sexual functions in the female plants. The authors then go on to couple the changes in visual and olfactory floral traits to pollinator attraction which allows them to conclude or at least speculate that differences in pollinator behavior are mostly driven by the changes in olfactory traits. The study is robust in its broad and well-balanced sampling of populations, rigorous and in large part meticulously documented experimental designs and linking of the effects on mechanisms to ecological function. The hypothesis are clearly stated and the study is able to address them mostly convincingly. However, some of the aspects of the decisions the authors made and possible caveats need to be addressed and elaborated on.

    A major caveat, in my opinion, is that while the authors find stronger effects of inbreeding on pollinator visitation rates in the plants from the North American (Na) origin, these plants were tested in an environment that was foreign to them, which could have important consequences for the results of this study. This is specifically because the main pollinator Hadena bicruris moth is completely absent from the populations in Na, and yet, was the main pollinator observed in the pollinator attraction experiment. As this pollinator is also a seed predator, the Na populations are released from the selection pressure to avoid attracting the females of this species and thus risking the loss of seeds and fitness. In fact, some of the results suggest that the release from the specialist pollinator and seed predator in Na has led to increase in the attractiveness of the female flowers based on the higher number of flowers visited in the outcrossed females compared to outcrossed males in the plant from the Na origin and the similar, though not statistically significant, pattern in the olfactory cue. While ideally this pollinator attraction experiment should be repeated within the local range of the Na plants, this is of course is not feasible. Instead I suggest the problem should be addressed in the discussion explicitly and its consequences for the interpretation of the results should be considered.

    Indeed, North American populations are tested in their “away”- habitat only and the observed plant performance and pollinator visitation rates can thus provide no direct implications for their “home”-habitat. We state this now more clearly at pp 11-12, ll 283-285. However, our design is appropriate for investigating inbreeding effects on plant-pollinator interactions in multiple plant populations in a common environment. Given the close taxonomic relationship of H. bicruris (main pollinator in Europe) and H. ectypa (main pollinator in North America), the behavioural responses of the former species to variation in the quality of its host plant was considered to overlap sufficiently with responses of the latter species as outlined at pp 11-12, ll 285-291.

    The hypothesis that North American (NA) S. latifolia evolved higher attractiveness to female Hadena moths because H. ectypa is not able to oviposit on female plants in contrast to H. bicruris is indeed a highly interesting one. However, as you have outlined correctly, our study is not designed to elaborate on questions related to adaptive evolutionary differentiation among North American and European plants. Instead of addressing this hypothesis based on our data, we thus take reference to previous studies in the discussion p 17, ll 482-487: “As discussed in detail in previous studies, higher flower numbers in North American S. latifolia plants (Figure 1b) may result from changes in the selective regimes for numerous abiotic factors (Keller et al., 2009) or from the release of seed predation. As opposed to H. bicruris, H. ectypa pollinates North American S. latifolia without incurring costs for seed predation, which may result in the evolution of higher flower numbers, specifically in female plants (Elzinga and Bernasconi, 2009).”

    The incorporation of the VOC data in the actual manuscript was quite limited and I found the reasoning for picking only the three lilac aldehydes (in addition to the Shannon diversity index) for the univariate statistical tests insufficient. How much more efficient was the effect of the lilac aldehydes compared to the other 17 compounds deemed important in the previous study? While the data on this one aldehyde matches the pollinator attraction results, having one compound out of 70 (or out of 20 if only considering the ones identified important for the main pollinator) seems, perhaps, fortuitous lest there is a good reason for focusing on these particular compounds.

    We adapted the text to increase clarity but sticked to our previous choice for the analyses of VOC data.

    i) We now explain our choice of analysing lilac aldehydes with more detail p9, ll 210-218: “For targeted statistical analyses, we focused on those VOC that evidently mediate communication with H. bicruris according to Dötterl et al. (2006). We analysed the Shannon diversity per plant (calculated with R-package: vegan v.2.5-5, Oksanen et al. 2019) for 20 floral VOC in our data set that were shown to elicit electrophysiological responses in the antennae of H. bicruris (Supplementary File 1). Moreover, we analysed the intensities of three lilac aldehyde isomers, which trigger oriented flight and landing behaviour in both male and female H. bicruris most efficiently when compared to other VOC in the floral scent of S. latifolia. Furthermore, H. bicruris is able to detect the slightest differences in the concentration of these three compounds at very low dosages (Dötterl et al. 2006).”

    ii) If one analyses 20 compounds with zero-inflation models (actually two models in one) + 8 floral trait models + 2 pollinator visitation models (zi-models with two component models), one ends up with 52 models investigating complex fixed and random effect structures. To keep type-1 errors as low as possible (see also comment 2.12.b from Referee#2), we approached the more comprehensive VOC data sets with multivariate analyses or Shannon diversity.

    iii) We tested the effect of sexoriginbreeding treatment on the Shannon diversity of 20 active VOC as well as in the random forest analyses with the 20 VOC and 70 VOC dataset and transparently reported the results from all of these analyses in the manuscript. Hence, the incorporation of VOC data was not limited. However, we agree that we have taken too little reference to these results and now changed the text accordingly. Results section p 13 ll 351-354: ”Multivariate statistical analyses of 20 H. bicruris active VOC and all 70 VOC detected in S. latifolia revealed no clear separation of floral headspace VOC patterns for any of the treatments (Figure 2-figure supplement 2). In summary, the combined effects of breeding treatment, sex and range on floral scent were rather week.”

    Sampling time of VOCs is reported ambiguously. Was it from 21:00 to 17:00 the next day or in fact from 9pm to 5AM (instead of 5 pm as reported)? Please be more specific in the text as this is quite important. If sampling tubes were left in place during the daytime, some of the compounds could have evaporated due to heating of the tubes in the summer. It would also be important to mention whether all of the headspace VOCs were sampled on the same day and whether there could be variation in i.e. temperature.

    Thank you very much for identifying this typo! It is from 9 pm to 5 am (p 9, l 186).

    Considering the experimental setup for the pollinator attraction observations and the pooling of the data at the block level (which I think is the right choice) it seems possible the authors were more likely to get a result where pollinator behavior matches the long-distance cue, the VOCs. Short-distance cues such a subtle difference in flower size would perhaps not be distinguished with the current setup. I would be interested to know if the authors agree, and if so, mention this in the discussion.

    Thank you very much for this excellent suggestion! We agree and discuss this aspect in detail at p 19, ll 520-526. Indeed, one would need two different experimental setups to assess the contributions of long and short distance cues. Our setup (large distances among plots) is optimal for long distance cues, while a setup for short distance cues should have all plants in close spatial proximity. However, the latter approach does then not allow to address long-distance cues and to exclude competition/facilitation for pollinators among plants from different treatment groups.

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  2. Reviewer #3 (Public Review):

    Schrieber et al. studied the effects of biparental inbreeding in the dioecious plant Silene latifolia, focusing specifically on traits important for floral attractiveness and pollinator attraction. These traits are especially important for dioecious species with separate sexes as they are obligate outcrossers. The authors find that inbreeding mostly decreases floral attractiveness, but that this effect tended to be stronger in the female flowers, which the authors suspect to result from the trade-off with larger investment in the sexual functions in the female plants. The authors then go on to couple the changes in visual and olfactory floral traits to pollinator attraction which allows them to conclude or at least speculate that differences in pollinator behavior are mostly driven by the changes in olfactory traits. The study is robust in its broad and well-balanced sampling of populations, rigorous and in large part meticulously documented experimental designs and linking of the effects on mechanisms to ecological function. The hypothesis are clearly stated and the study is able to address them mostly convincingly. However, some of the aspects of the decisions the authors made and possible caveats need to be addressed and elaborated on.

    A major caveat, in my opinion, is that while the authors find stronger effects of inbreeding on pollinator visitation rates in the plants from the North American (Na) origin, these plants were tested in an environment that was foreign to them, which could have important consequences for the results of this study. This is specifically because the main pollinator Hadena bicruris moth is completely absent from the populations in Na, and yet, was the main pollinator observed in the pollinator attraction experiment. As this pollinator is also a seed predator, the Na populations are released from the selection pressure to avoid attracting the females of this species and thus risking the loss of seeds and fitness. In fact, some of the results suggest that the release from the specialist pollinator and seed predator in Na has led to increase in the attractiveness of the female flowers based on the higher number of flowers visited in the outcrossed females compared to outcrossed males in the plant from the Na origin and the similar, though not statistically significant, pattern in the olfactory cue. While ideally this pollinator attraction experiment should be repeated within the local range of the Na plants, this is of course is not feasible. Instead I suggest the problem should be addressed in the discussion explicitly and its consequences for the interpretation of the results should be considered.

    The incorporation of the VOC data in the actual manuscript was quite limited and I found the reasoning for picking only the three lilac aldehydes (in addition to the Shannon diversity index) for the univariate statistical tests insufficient. How much more efficient was the effect of the lilac aldehydes compared to the other 17 compounds deemed important in the previous study? While the data on this one aldehyde matches the pollinator attraction results, having one compound out of 70 (or out of 20 if only considering the ones identified important for the main pollinator) seems, perhaps, fortuitous lest there is a good reason for focusing on these particular compounds.

    Sampling time of VOCs is reported ambiguously. Was it from 21:00 to 17:00 the next day or in fact from 9pm to 5AM (instead of 5 pm as reported)? Please be more specific in the text as this is quite important. If sampling tubes were left in place during the daytime, some of the compounds could have evaporated due to heating of the tubes in the summer. It would also be important to mention whether all of the headspace VOCs were sampled on the same day and whether there could be variation in i.e. temperature.

    Considering the experimental setup for the pollinator attraction observations and the pooling of the data at the block level (which I think is the right choice) it seems possible the authors were more likely to get a result where pollinator behavior matches the long-distance cue, the VOCs. Short-distance cues such a subtle difference in flower size would perhaps not be distinguished with the current setup. I would be interested to know if the authors agree, and if so, mention this in the discussion.

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  3. Reviewer #2 (Public Review):

    A summary of what the authors were trying to achieve. This interesting and data-rich paper reports the results of several detailed experiments on the pollination biology of the dioceus plant Silene latfolia. The authors uses multiple accessions from several European (native range) and North American (introduced range) populations of S. latifolia to generate an experimental common garden. After one generation of within-population crosses, each cross included either two (half-)siblings or two unrelated individuals, they compared the effects of one-generation of inbreeding on multiple plant traits (height, floral size, floral scent, floral color), controlling for population origin. Thereby, they set out to test the hypothesis that inbreeding reduces plant attractiveness. Furthermore, they ask if the effect is more pronounced in female than male plants, which may be predicted from sexual selection and sex-chromosome-specific expression, and if the effect of inbreeding larger in native European populations than in North American populations, that may have already undergone genetic purging during the bottleneck that inbreeding reduces plant attractiveness. Finally, the authors evaluate to what extent the inbreeding-related trait changes affect floral attractiveness (measured as visitation rates) in field-based bioassays.

    An account of the major strengths and weaknesses of the methods and results. The major strength of this paper is the ambitious and meticulous experimental setup and implementation that allows comparisons of the effect of multiple predictors (i.e. inbreeding treatment, plant origin, plant sex) on the intraspecific variation of floral traits. Previous work has shown direct effects of plant inbreeding on floral traits, but no previous study has taken this wholesale approach in a system where the pollination ecology is well known. In particular, very few studies, if any, has tested the effects of inbreeding on floral scent or color traits. Moreover, I particularly appreciate that the authors go the extra mile and evaluate the biological importance of the inbreeding-induced trait variation in a field bioassay. I also very much appreciate that the authors have taken into account the biological context by using a relevant vision model in the color analyses and by focusing on EAD-active compounds in the floral scent analyses.

    The results are very interesting and shows that the effects of inbreeding on trait variation is both origin- and sex-dependent, but that the strongest effects were not always consistent with the hypothesis that North American plants would have undergone genetic purging during a bottleneck that would make these plants less susceptible to inbreeding effects. The authors made a large collection effort, securing seeds from eight populations from each continent, but then only used population origin and seed family origin as random factors in the models, when testing the overall effect of inbreeding on floral traits. It would have been very interesting with an analysis that partition the variance both in the actual traits under study and in the response to inbreeding to determine whether to what extent there is variation among populations within continents. Not the least, because it is increasingly clear that the ecological outcome of species interactions (mutualistic/antagonistic) in nursery pollination systems often vary among populations (cf. Thompson 2005, The geographic mosaic of coevolution), and some results suggest that this is the case also in Hadena-Silene interactions (e.g. Kephardt et al. 2006, New Phytologist). Furthermore, some plants involved in nursery pollination systems both show evidence of distinct canalization across populations of floral traits of importance for the interaction (e.g. Svensson et al. 2005), whereas others show unexpected and fine-grained variation in floral traits among populations (e.g. Suinyuy et al. 2015, Proceedings B, Thompson et al. 2017 Am. Nat., Friberg et al. 2019, PNAS). Hence, it is possible that the local population history and local variation in the interactions between the plants and their pollinators may be more important predictors for explaining variation in floral trait responses to inbreeding, than the larger-scale continental analyses. Not the least, because North American S. latifolia probably has multiple origins, with subsequent opportunity for admixture in secondary contact.

    I see no major weaknesses in the study, and but in my detailed response, I have made a few questions and suggestions about the floral scent analyses. In short, the authors have used a technique that is not the standard method used for making quantitative floral scent analyses, and I am curious about how it was made sure that the results obtained from the static headspace sampling using PDMS adsorbents could be used as a quantitative measure. I would suggest the authors to validate the use of this method more thoroughly in the manuscript, and have detailed this comment in my response to the authors.

    Also, and this may seem like a nit-picky comment, I am not convinced that the best way to describe the traits under study is "plant attractiveness", because in the experimental bioassays, most of the traits under study that are affected by the inbreeding treatment, did not result in a reduced pollinator visitation. Most (or all) of these traits may also be involved in other plant functions and important for other interactions, so I suggest potentially using a term like "floral traits" or "(putative) signalling traits".

    An appraisal of whether the authors achieved their aims, and whether the results support their conclusions: By and large, the authors achieved the aims of this study, and drew conclusions based in these results. One interesting aspect of this work that I think could be discussed a bit deeper is the lack of congruence between the effects of inbreeding on floral traits and the variation in visitation pattern in the bioassay. In fact, the only large effect of inbreeding on a floral trait that may play a role as an explanatory factor is the reduction of emission of lilac aldehyde A in inbred female S. latifolia from North America, which correspond to a reduced visitation rate in this group in the pollinator visitation bioassay. I have made some specific suggestions in my comments to the authors.

    A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community: I think that one important aspect of this work that may broaden the impact of this study further is the link between these experiment, and our expectations from the evolution of selfing. Selfing plant species most often conform to the selfing syndrome, presenting smaller, less scented flowers than outcrossing relatives. Traditionally, the selfing syndrome is explained by natural selection against individuals that invest energy into floral signalling, when attracting pollinators is no longer crucial for reproduction. Some studies (for example Andersson, 2012, Am. J. Bot), however, have shown that only one, or a few, generations of inbreeding may reduce floral size as much as quite strong selection for reduced signalling. Here, at least for some populations and sexes, similar results are obtained in this paper regarding several traits (including floral scent), and one way to put this paper in context is by discussing the results in the light of these previous papers.

    Any additional context that would help readers interpret or understand the significance of the work: I would like to reiterate here the potential to utilize the population sampling to make additional conclusions about the geography of trait variation and its importance for the phenotypic response to inbreeding.

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  4. Reviewer #1 (Public Review):

    The manuscript by Schrieber et al., explores whether inbreeding affects floral attractiveness to pollinators with additional factors of sex and origin in play, in male and female plants of Silene latifolia. The authors use a combination of spatial sampling, floral volatiles, flower color, and floral rewards coupled with the response of a specialized pollinator to these traits. Their results show that females are more affected by inbreeding and in general inbreeding negatively impacts the "composite nature" of floral traits. The manuscript is well written, the experiments are detailed and quite elaborate. For example., the methodology for flower color estimation is the most detailed effort in this area that I can remember. All the experiments in the manuscript show meticulous planning, with extensive data collection addressing minute details, including the statistics used. However, I do have some concerns that need to be addressed.

    Core strengths: Detailed experimental design, elaborate data collection methods, well-defined methodology that is easy to follow. There is a logical flow for the experiments, and no details are missing in most of the experiemnts.

    Weaknesses: A recent study has addressed some of the questions detailed in the manuscript. So, introduction needs to be tweaked to reflect this.

    Some details and controls are missing in floral scent estimation. Flower age, a pesticide treatment of plants that could affect chemistry..needs to be better refined. While the study is laser-focused on floral traits, as the authors are aware inbreeding affects the total phenotype of the plants including fitness and defense traits. For example, there are quite a few studies that have shown how inbreeding affects the plant defense phenotype. This could be addressed in the introduction and discussion.

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  5. Evaluation Summary:

    This research is relevant for ecologist and evolutionary biologist in the specific fields of plant conservation, chemical ecology, pollination biology and plant sex evolution. The authors test the interesting hypothesis that traits important for plant-insect interactions are directly affected by inbreeding, which in turn may directly impact the plant-insect interaction. The authors test this prediction in a series of experiments on the plant Silene latifolia, and the results largely support the hypothesis that inbreeding reduces plant attractiveness. In short, the results show that there are indeed strong negative effects of inbreeding on multiple plant/floral traits, but that the effects of these traits do not necessarily translate directly into reduced pollinator visitation rates. The data are of high quality, the sampling of populations was markedly geographically broad and balanced, and the experiments were well implemented, leading to a certain robustness of the results and conclusions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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