PONE-D-22-23499Pollination service provided by honey bees to buzz-pollinated crops in the NeotropicsPLOS ONE

Dear Dr. Quezada-Euan,

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a very interesting study investigating the contribution of native and introduced bees on the pollination of two crops associated with buzz pollination. The study is extremely valuable in its contribution to the pollinator visitation, and crop yield consequences of visits by different pollinator guilds and will make a very valuable contribution to the literature. Below I provide some major and minor suggestions.

Major suggestions

1) Data. The data provided in the Supplemental Information is not of sufficient quality to allow replicating the analyses of the paper. You must include all necessary data, with a clear explanation of column names, units and how they were calculated. The objective of providing the data for your study should be to allow others to replicate your analysis and enable subsequent investigations, and now the Excel file is poorly organised and described. Maybe the necessary information is there, but without a README file is hard to tell what is what. https://journals.plos.org/plosone/s/data-availability

2) Introduction. Please provide a brief overview of native bee diversity in the study region. This will be very useful when trying to understand what level of diversity is encompassed in the term you use (native bees) throughout the rest of the paper. The honeybee vs. native bee seems a simplistic comparison and you need to give more context here.

3) Details of methods, sample sizes and analyses are missing in some parts. See comments below for specific suggestions.

Minor suggestions

4) Abstract. Consider removing the acronyms in the abstract.

5) Line 46. “Few studies…” Perhaps, but it might be a good idea to add other references as a single citation here may give a bias idea of the numerous studies of pollination services in the tropics.

6) Line 60. Here you should mention that honeybees are introduced in the Americas. Is not obvious to all readers.

7) Line 64. What do you mean by “natural” abundance?

8) Suggested change: “The Solanaceae is…”

9) Line 78. Introduce authority and year for eggplant as you do for achiote in line 85

10) Line 79. Remove “thus” as self-compatibility does not imply self-pollination

11) Line 89. Which region is the major producer of achiote?

12) Line 94. AHB. I am not a fan of acronyms. Could you simply define and use “honeybees” throughout the paper?

13) Line 94. No question has been formulated. Rephrase.

14) Line 116-117. More details needed. How many observers/cameras? Provide a standardised unit for observations (person-hours, etc.).

15) Line 117. How was it determined if a bee buzz pollinated. Details needed as this is essential for the functional classification done in the next paragraph.

16) Line 119. Why was this initital analysis done? What was the sample size? It may be best to formally analyse this and report in the results with sample size details, etc.

17) Lines 121-122. How were these determinations done? Are voucher specimens collected and stored? How many species per genus? The information on determination appears briefly later. Consider re-organising.

18) Line 147. Was time nested in day? More details of statistical model are needed. Consider providing the full model used, and make sure it matches the results (eg. lines 233-236). You need to also explain whether the model assumptions are met.

19) Line 152. “Both functional groups” I think you mean the three functional groups? Clarify.

20) Line 162. Why 2-3 visits? Was visit number included in the statistical model, and if not why not?

21) Line 193. Not sure what the AHB2, AHB3,… mean.

22) Line 198-200. Explain what the Friedman test is.

23) Line 34-347. Please rephrase. Most genera in Solanaceae are not buzz pollinated. Within Solanum, not all species have anthers fused into a cone (the majority do not).

24) Line 414. In this sentence, consider including in the discussion a more nuanced evaluation of the benefit of honeybees for “sustainable” pollination (as you state at the end of the paragraph). To the extent that honeybees impact native bees, it might not be very sustainable in a broader context.

Reviewer #2: The study provides some very interesting information about how non-native, feral honey bee colonies affect buzz-pollinated crops. Your analyses support your conclusions that 1) honey bees have become the dominant visitors to these crops, 2) that despite their lack of buzz pollination behavior honey bees seem to pollinate eggplant effectively by visiting multiple times, and 3) that honey bees may be decreasing pollination success for annato by competing with more effective native pollinators. These conclusions contribute interesting and useful information for anyone interested in agriculture or bee conservation in the tropics, and should be published. It was especially interesting to see how multiple visits by honey bees seem to compensate for their inability to vibrate the anthers of eggplant flowers but there is no similar effect in annato flowers. The experiments allowing 1, 2, or 3 honey bee visits and counting or estimating seed production are impressive. I imagine that took a lot of careful work.

My main suggestion would be to double-check your bee abundance data for the 3 guilds and to make sure that you provide all data for figure 5. Table 1 does not match the data in your eggplant and annato data files. I have some suggestions below to add or clarify information at particular lines and figure/table captions.

You do not need to do this, but I would highly recommend providing your R code and providing your data in separate (ideally csv) files that can be read directly from your R code. This would help make your study more reproducible and easier to interpret. It would also help you to double-check your results (or clarify what data you included in each summary/analysis).

Introduction-

Good introduction, sets up the importance/questions well

Line 44-45: I know you understand that many crops are self-compatible or wind pollinated, but the way you say this, it makes it sound like all crops need visits from pollinators.

Materials and Methods-

Overall thorough description of your methods, but there are a few places that need clarification

Lines 116-117: Did you pause walking the transects each time you saw a bee and record it?

Line 154: When I hear the word proximate, I usually think of close in spatial location, but here you mean close in time, correct?

Line 198: So the data violated the assumption of normality and you used a non-parametric test instead?

Line 218: I'm assuming the dotted line is this function? It looks like a good fit, but it would be helpful to include a little more detail about how you decided that this was the best fit

Lines 219-228: It was helpful that you included a description of the formula here

Results-

Overall thorough and clear, but there seems to be a problem in one of the tables and the captions need more detail

Line 296: So am I correct that your point here is that even though the increase in seed production with unlimited access to the pollinators was statistically indistinguishable from the seed production produced by hand pollination, it's still possible that a different community of pollinators (maybe one with more native bee species) could have done a better job of providing pollination services to the eggplants?

Line 303: Do you mean 38% less than the maximum possible?

Table 1:

There seems to be a few errors in the eggplant column and many errors in the annato column when I compare them to your data files. Please double-check these!

What does EE refer to?

Picky comment- I would recommend presenting times of day in the format 8:00 or 16:00 rather than 8h and 16h because at first I thought those were durations

Figure 1:

This figure contains interesting information about the diversity of pollinators you found, but I wonder if it wouldn't be better to present pie charts because it would better convey the relative proportions. But that's personal preference.

Figure 2:

It would help to put in the caption what the dotted line represents (maybe even including the equation you present in the text) because saying correlation makes me expect a straight line here (clearly you didn't just look for a linear correlation, but it would help to clarify that).

Figure 3:

This figure is very clear and contains a lot of information about the variation and trends

Figure 4:

This figure is very helpful, providing context for the methods and discussion. Very nice photos of the anthers!

Figure 5:

Why are all the y values between 0 and 3? Was that the range for both species or did you normalize somehow? I don't see where those numbers are in your eggplant data file.

Table 2:

The percentages of the hand-pollinated results are very helpful. I think the letters to indicate significant differences would be good to add to the figure.

Table 3:

It would helpful to switch the NB and PLUS rows so they match the order in the figure

S1 Video:

Very interesting behaviors! You don't need to do this, but I wonder if it would make the video more likely to be used by others to either slow the it down or use video stabilization so it is easier to focus on the bee's behavior rather the flower moving in the wind (I know ffmpeg has a way to do this, although I haven't used it: https://www.paulirish.com/2021/video-stabilization-with-ffmpeg-and-vidstab/). If you do either of those things, please include a description of what you did in the video caption

S2 Video:

This looks like it was slowed down. It would be helpful to indicate how you did that (what % change, when in video)

S3 Video:

The cloud of pollen is quite striking. I'm not sure that you need the arrow, but if you keep it, I think it would be good to say in the caption that it is pointing to the cloud of pollen

Discussion-

It was helpful that you related your findings to previous literature on the pollination of buzz-pollinated crops and talked about potential reasons for the difference in efficiency of honey bees across the 2 crops. Your conclusions about supporting diverse native pollinators to maximize pollination services across crops seems reasonable.

Line 402-403: How are you calculating AHB contribution here? Is this because native non-sonicating bees were also poor pollinators per trip?

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Reviewer #1: No

Reviewer #2: No

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Pollination service provided by honey bees to buzz-pollinated crops in the Neotropics

PONE-D-22-23499R1

Dear Dr. Quezada-Euan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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