PONE-D-21-40078Illuminating Protist Diversity in Pitcher Plants and Bromeliad TanksPLOS ONE

Dear Dr. Sleith,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

 The three reviewers have provide detailed comments and recommendations for improvements to the manuscript. Please consider the reviews carefully and pay particular attention to reviewer 3's issue with succession - this needs to be addressed. 

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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 straightforward report of two small-scale studies analyzing the composition of protist communities in pitcher plant tanks, both in different species and different timepoints. It addresses a novel question, executed in a generally technically sound manner, readable, and is a good fit for PLoS ONE. All of my suggestions could feasibly be addressed without additional wetlab experimentation.

One addition that could improve the paper is some additional information within the main text figures on the identity of the OTUs of interest. The introduction indicates that ciliates are dominant in other studies of bromeliad protist communities using different methods, and that bromeliads harbor rare species, but universal primers miss a lot of diversity that SAR primers could capture- three interesting hypotheses. There isn’t much detail given on the taxonomic resolution of these primers, but the final results paragraph and previous Sisson study cited indicates that the sequences are identifiable to some more specific taxonomic level. This information is not necessary for all the OTUs, but would be helpful to add to the dominant OTU highlighted in Fig. 4 and the specialized OTUs shown in figure 5, to put those initial questions into context. Furthermore, several recent studies of protists living on other types of aerial plant tissues have indicated the presence of a large diversity of Cercozoa esp. dominated by Sarcomonads (Ploch et al. 2016, Flues et al. 2018, Walden et al. 2021, Sun et al. 2021), but a few Ciliophora (Colpodea) have been reported to be universal too (Muller and Muller 1970). Some additional OTU identity could help contextualize which patterns you are observing are common to other plant structures vs. novel or specialized to bromeliads. Given the acidity, one would expect the residents to be very different from that of other plants, and the paragraph starting on line 278 indicates this could be true! Paper dois below:

10.1111/jeu.12314

https://doi.org/10.1111/jeu.12503

https://doi.org/10.1111/1462-2920.15385

https://doi.org/10.2307/2423721

https://doi.org/10.1093/femsec/fiab081

The introduction talks a lot about pitcher plant microbiomes, but could benefit from a little more general information on the pitcher plant environments being studied, to be accessible to researchers not in this specific area. Are the tanks thought to be rain-fed or does the water mainly seep in from the surrounding bog? What type of plant exudates feed the microbiome (i.e., sugars, lipids, insecticidal enzymes, acidity?). When one plant has multiple pitchers, do they usually emerge at the same time or do they have different ages? Is the type and amount of insect prey known to affect the microbiome? Finally, what are the phenotypic differences among the species being studied here (volume, habitat, shape, prey range, lids vs. no lids)? This information would help rationalize your hypotheses about species effects and seasonality.

Also in the introduction, there are some references to there being more studies of bacteria and animal communities than of microbial eukaryotes (line 113), meaning protists, but fungal studies are only mentioned in the discussion. Fungi are technically microbial eukaryotes, so please clarify when you are talking about only protist communities.

Line 126- the genetic diversity of the microbial community was similar to the host plant? Unclear phrasing.

-Line 145 “differentiate active community members from cysts” makes it sound like both parts of the community were measured and compared, it would be better rephrased to say the method primarily targets active protists.

Methods:

-Please provide more information about the sequencing beyond that it was MiSeq. Did you sequence paired end reads? 2x300 or 2x150 length? How many total reads? Were reads filtered for quality or length before entering them in the pipeline? Even if some of the QC is in the cited link, it’s best to say briefly what was done.

-line 201 – meaning of “OTU libraries” is unclear.

In figure 3A it would be helpful to indicate somewhere in the figure or legend that open symbols are background samples. Circles indicating confidence intervals and p-values would greatly help demonstrate the finding that Day 53 communities are significantly different.

I really do not understand what Figure 3B is showing. PCA is typically used to differentiate different sample types, but these are all from the same treatment. If the point is that most OTUs are alveolate at this timepoint, isn’t that already shown in figure 4, and what are the principal components for?

Figure 5 is discussed before Figure 3. Figures should be in the order they are discussed in the text.

There are some places in the results where the verb tense suddenly changes from past to present tense (such as line 268). Please check for verb tense consistency.

I would suggest limiting the discussion to be no longer than the length of the results text, which should be enough to synthesize how the results match initial expectations stated in the introduction and extend upon previous work. The recent Walden et al. paper on phenology of protists in aerial plant tissues may be good to mention for context. Also it seems that the findings here (seasonal successional shifts, origin from surrounding environmental microbiota, and specialized/reduced diversity from surrounding microbiota) align well with other emerging paradigms from the leaf and floral ecology field, which may be worth a mention (good overview by Britt Koskella here: https://doi.org/10.1016/j.cub.2020.07.037)

Reviewer #2: In their manuscript “Illuminating Protist Diversity in Pitcher Plants and Bromeliad Tanks”, the authors present a study that used a molecular approach to characterizing eukaryotic microbes (specifically SAR protists) in pitcher plants (Sarracenia and Nepenthes) and tank bromeliads. In the study, the authors sampled inquiline communities from their respective hosts over varying spatial and temporal scales. They found that host plant species affects SAR community structure and that there is a large amount of variability between communities within the same host plant. They also found that Sarracenia pitcher plants from the same bog are more diverse and heterogenous earlier in the growing season than later in the growing season. I enjoyed reading this manuscript immensely. The methods are rigorous and stand out from previous work that characterizes protists in phytotelma primarily based on morphology. Overall, I believe that the manuscript has merit for publication following a round of revisions from the authors. I have made some specific suggestions below.

Thank you for a fun read,

Erica Holdridge

NSF Postdoctoral Research Fellow

Boise State University

1. Although the authors have provided a nice table (Table 1), I still have a hard time understanding the sampling structure of their study. This could use quite a bit of clarification in the text of the manuscript. In particular, there are 3 samples from each bromeliad species taken across 2 dates. Are these from 3 different plants? How many were sampled on each date? etc. The same questions apply for the Nepenthes sampled. There were also 17 Sarracenia samples from Hawley Bog separately from the temporal study. Were these from a separate part of the bog than the Sarracenia that were used in the temporal study?

2. I can’t help but wonder if your Sarracenia samples are so variable (e.g. Figure 2B) because (a) you simply have more samples taken over a wider range of time points and (b) they’re taken from the field rather than a greenhouse.

3. Lines 341-346: Are Nepenthes at Lyman Conservatory grown in hanging baskets? If so, this may be one reason why bromeliads and Nepenthes from the same greenhouse have distinct communities (although I do believe that most of it does have to do with host identity).

4. Do you notice more mosquitoes (particularly W. smithii) in Hawley bog later in the growing season? It’s possible that they are aiding in dispersal and could explain some of the homogeneity you see later in the growing season. You do mention “animal movement” on lines 371-372, which may be a reference to this idea.

Minor comments:

• lines 77-78: “Much less is known about freshwater habitats” to “Much less is known about SAR in freshwater habitats”

• lines 92-93: “host available” to “host-available”

• line 163: “was collected” to “were collected”

• line 203: “SWARM v2 d = 1” to “SWARM v2 with the parameter d = 1”

• line 212: “weighted and unweighted (for relative abundance)” to “weighted (relative abundance) and unweighted (presence/absence)”

• lines 231-232: How many OTUs exactly?

• lines 253-255: The first sentence of this section is a fragment.

• line 267: You discuss Figure 5 before Figure 4; they should probably be reordered so they are organized by the order in which they are discussed

• line 286: OTU8 seems to be most abundant in the background bog water (Figure 4) – what is the taxonomic assignment of that OUT?

• line 342: This is the first place where you clarify that you mean Nepenthes when you say “tropical pitcher plants”. I know that this is the common name but other readers may not, so this should be clarified earlier in the manuscript.

• line 355: This would be a great place to site Aaron Ellison and Nick Gotelli’s new book “Scaling Ecology with a Model System” (2021)

Reviewer #3: The manuscript by Sleigh and Katz investigates the eukaryotic diversity within the inquiline communities of pitcher plants by using a taxon-focused approach. The technique they used is very timely and needed because eukaryotic diversity is understudied in general and especially in pitcher plant systems. When eukaryotic diversity is studied, it is usually with broad primers that can underestimate our knowledge about protist diversity because of the overabundance of insect DNA that can also be captured. Overall, the manuscript was very well written and there was a very indepth literature search. My main comments are that more information is needed in the methods section, and I am not fully comfortable with considering this a study that addresses Succession. With most of the inquiline community removed at each sampling point, this study seems to address more the resilience of the community after a disturbance, and the role of stochastic events and dispersal, instead of internal biotic interactions, on protist diversity throughout time, than a proper assessment of Succession. The study did definitely investigate the difference in protist composition across the different locations in the bog, but their question about Succession may need to be re-thought.

Main comments in Method Section:

In the Methods section of Study 2, additional information is needed in order for a better understanding of what was done. Specifically,

1) Were new pitchers selected on each of the rosettes at the beginning of the field season? This would allow for an accurate assessment of early succession.

2) It is unclear in the Methods if the three pitchers were sampled on each of the three sampling dates, or if one pitcher represented a specific sampling date and the replicate was on the other two rosettes in the section (i.e., Pitcher 1 = May 6, Pitcher 2 = May 29, Pitcher 3 = June 27). This information is stated in the Results section (Line 253-255) and should be moved up to the Methods for clarity.

3) For a better assessment of the methods and results, it would also be helpful to know how much volume was in each pitcher during each sampling time and the distance between each plant in Forest edge versus open bog, for example.

Main comment regarding Succession:

On Line 179-181, the authors state that a maximum of 25mL of fluid was removed and fluid was concentrated onto filters. If each pitcher was sampled three times (the three time periods), removing up to 25mL on each time period would be an exhaustive sampling, which would not capture protist succession through time if the community was removed almost completely during each sampling and needed to reset. To follow succession properly, it would be better to either exhaustively remove the volume of one pitcher on each of 3 replicate rosettes. This would represent one time period, replicated 3 times within 1 section of the bog, or, to take a smaller volume from each pitcher during the three sampling events to prevent a disturbance.

My concern about the large volume that was removed each time continues in Lines 361-365 of the Discussion. These follow-up questions are indeed important, but I just cannot see how biotic interactions will play a role in the authors’ sampling method if they removed most of the volume in the leaf each time. The internal biotic interactions were basically reset at each time point, making it instead a study that addressed the role of stochastic events and dispersal at different time points throughout the growing season, and a study that tests the resilience of the community after such a large disturbance each time. (I am happy to see though that the authors recognize this potentially large perturbation of removing almost all the volume during each sampling point (Line 374-375) and their results do indeed match what is found with microscopic counts of protists in the Sarracenia system, with flagellates dominating at the beginning of succession and ciliates being more abundant later in the season).

Additional comments:

- Why were OTUs used instead of ASVs?

- Figure 2. It would be helpful to visualize which of the Sarracenia samples came from which site.

- Figure 3A. Labelling the Date as a circle is confusing with the Forest Edge circle. Additionally, it is confused what “Pool Edge” means.

- Table 1. Minor detail, but signify that the dates are Month/Day.

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Reviewer #1: No

Reviewer #2: Yes: Erica Holdridge

Reviewer #3: No

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PONE-D-21-40078R1Illuminating Protist Diversity in Pitcher Plants and Bromeliad TanksPLOS ONE

Dear Dr. Sleith,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

 In the revised (2nd revision) manuscript, please address the continued concerns of reviewer three. There remains a need for clarification on the age of the pitcher plants sampled and the possible sampling from pitchers that lasted over the winter from an earlier season. If there is a reason that the pitcher plants in this study are maturing earlier than expected by the third reviewer, please make that clear. Please address the question regarding dipteran larvae remaining in the pitcher plants.

Please submit your revised manuscript by May 28 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Theodore Raymond Muth

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. 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: I feel that the authors responded satisfactorily to the previous comments and that the manuscript meets PLoS ONE review criteria.

Reviewer #2: The authors have revised their manuscript, “Illuminating Protist Diversity in Pitcher Plants and Bromeliad Tanks”, following comments from myself and two other reviewers. The revised manuscript adequately addresses all of my previous comments and I do not have any additional comments. Great job handling all of the (sometimes conflicting) comments and thanks for a fun read!

Reviewer #3: Thank you very much for the revisions to the manuscript. I commend the authors for the work they did. Overall, I find the study interesting and timely. Eukaryotic diversity is understudied in general, and especially in pitcher plant systems. I still, however, have concerns about the methods in Study 2. My concerns do not mean that there is a problem with the data, but rather in the question that is being addressed (Succession). My reasons for this are detailed below:

1) Standardization of pitcher leaf age:

Sampling in the beginning of May in Massachusetts seems very early in the growing season for Sarracenia purpurea at this latitude. It is thus surprising that there were new leaves/pitchers available on the rosettes (especially 3 new leaves per rosette). More surprising is that the new leaves that were used in the study were open and filled with large volumes of water (as shown in the supplemental data), unless several large rainstorms occurred just before sampling. I just can’t imagine that the new pitchers (from that season) would be large enough to hold such a large volume at this time period.

Not all pitchers die back each year. Instead, they overwinter, with the snow protecting them. There will, of course, be some old pitchers from several seasons in the past that have died back (the pitchers closer to the ground), but the middle of the rosette will have pitchers from last season that have survived the winter as well as the new pitchers that are growing in the current season. It can sometimes be difficult to determine last season’s pitchers from new growth pitchers if the new growth pitchers are ~4 weeks or older. I am therefore uncertain if the authors used pitchers from that season or overwintered pitchers. For the study to examine Succession, the age and opening time of the pitchers needed to be better standardized. This can be done by selecting very new (fresh, flexible green leaves that had just opened) pitchers. The first sampling event should have then been done several weeks after they opened, so that there was time for the pitchers to fill with water.

Study 2 still informs us about the inquiline community that inhabits pitchers, but without the initial standardization, it acts more of a snapshot at each point in the season, and not succession.

Apologies if my phenology is incorrect. Addition of field site information (flowering time, new leaf production, precipitation) should be added to the manuscript.

2) Dipteran larvae in the inquiline community:

The large volume of water that was removed from each pitcher during sampling would also remove any of the dipteran larvae that are essential for dynamics of the inquiline community. Some (the midge and some of the mosquito larvae) could be down at the bottom of the leaf near the decomposing insects, so it is likely that those ones could remain in the community after each sampling. It is unfortunate that the authors did not record midge and mosquito larvae during the sampling so that the authors have an idea of how many were lost from the community during each sampling. Were there any mosquito larvae stuck on the filter after the inquiline community was filtered?

Along those lines, a better reference than Rango 1999 for seasonal Wyeomyia dynamics would be to use the Harvard Forest Data Archives. These archives contain data from the authors’ field site and/or sites in the region and have information about the seasonal dynamics of the inquiline mosquito larvae in the system. https://harvardforest1.fas.harvard.edu/exist/apps/datasets/showData.html?id=hf193

It very well might be that the mosquito larvae densities do not peak until mid-summer, and therefore are not a part of the May-June sampling that occurred in this study. In the lower latitude part of Sarracenia purpurea’s distribution (e.g., Florida) the mosquito larvae are the most abundant as soon as the leaf opens. If that is the case in this study site as well, then the mosquito density would be greatly impacted. If overwintered leaves were indeed used for this study, there could be mosquito larvae present because they were in diapause in the leaves during the winter.

3) Disturbance by removing large volumes of water during sampling: a large volume was taken from each community during each sampling time period in Study 2. The authors do replace the volume they removed with the supernatant, but this is still a large, open niche space. I agree that there will be some microbes that remain at the bottom of the leaf during each sampling time period, but the community is undergoing a great disturbance during each sampling period, with diversity potentially being diluted each time.

In summary, I do think this study has accomplished a lot of things and is interesting, it is just that there are some potential issues in the methods which will affect the focus of the paper as it currently is written. In general, I suggest that the authors be careful with the word “succession” in this study. Instead, I suggest that the authors use “seasonal” dynamics or something along those lines.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Erica Holdridge

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Illuminating Protist Diversity in Pitcher Plants and Bromeliad Tanks

PONE-D-21-40078R2

Dear Dr. Sleith,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Theodore Raymond Muth

Academic Editor

PLOS ONE

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