Dry-Season Fog Water Utilization by Epiphytes in a Subtropical Montane Cloud Forest of Southwest China

Round 1

Reviewer 1 Report

The manuscript can be accepted.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Water

Liu et al. 2021—Manuscript # 1437822

Overall statement: 

This manuscript reports on the water use efficiency, as measured by stable isotope techniques, of four groups of epiphytes in southwestern China. It is very interesting and important research, because these data are scarcely available for most epiphytic species. I commend the authors for having such a broad taxonomic scope (including lichens, bryophytes, ferns, and angiosperms), and am sure these data will be useful to many an epiphyte researcher. However, prior to consideration for publication in Water, I suggest a few clarifications:

1. Introduction needs to prepare the reader for differences among epiphyte taxonomic groups. This is a great part of the study which, frankly, is undersold!
2. Justify why epiphytes were measured during dry season into the methods (or introduction) — easy argument but necessary somewhere
3. Consider putting some mean quantitative measurements into a table rather than text in the results. 
4. Add a general paragraph introducing the broad topic again in the discussion; consider decreasing emphasis on water differences per se and highlight epiphyte-epiphyte group differences

I also have several suggestions for minor changes, such as grammar, wording, and clarity (see in-line comments below).

In-line comments and section-specific recommendations:

Abstract:

General comments:

Abstract is well-structured and clear. The important take-home messages are highlighted and give an accurate depiction of the results to come in the paper. Can you add one sentence to interpret why groups differed as such? I was surprised that seed plants and ferns did not groups together (using more humus water because of their true roots) and that lichens and bryophytes differed in strategy so much (without true roots).

In-line comments

28-29 “group-specific and interspecific variations” is vague. Can you specify what you are talking about here biologically, rather than statistically?

Introduction:

General comments:

Two general comments:

-One element that is needed for the narrative is how/why we would expect the different epiphyte groups (bryophytes, lichens, angiosperms, and ferns) to partition water differently. At the very least, mention roots versus no true roots. 

-The paragraph on stable isotopes (72-92) is slightly wandering and may not be necessary. Move all the methodological arguments to the methods section or remove. A simple discussion of why we expect epiphytes to have higher WUE than ground-rooted plants is important; this paragraph should be focused on that, rather than defending the methods.

In-line comments

40-43 Break into two sentences. “Epiphytes, which grow on living or residual host trees, are one of the most diverse groups in the forest ecosystem. These structurally dependent plants play an important role in maintaining biodiversity and biomass and promoting the processes of nutrient and water cycling.”

53-54 I don’t think we actually have good evidence that epiphytes are competing with their hosts for water and nutrients (that’s why they are generally considered commensalists, not parasites). This statement needs some modification or to be deleted. If you have some papers that show indirect competition, be very clear.  Epiphytes don’t need to be competing with their host trees to be important to canopy dynamics.

76-78 Sentence not necessary especially if these are not epiphytes?

79 remove “but by now”

82 remove “besides” and replace with another transition such as “likewise” or “similarly”

84-85 unnecessary sentence. You can just cite a few other studies that have used stable isotopes for this in the beginning or in the methods section. See comment above.

87-88 Yes, this should be the focus of the paragraph. Why do we expect this difference between epiphytic and ground-rooted plants? And how would we expect this to be different among the different epiphyte groups?

93 add “predicted by future climate models” after drought

101-103 Exactly the expectation you need to justify! I think the previous  paragraph should be replaced/modified with a discussion of why you expect the water use patterns of epiphytes to differ among groups of epiphytes. Differences in roots? Stomata? Photosynthetic pathway? A few useful citations:

Horwath, Aline B., et al. "Bryophyte stable isotope composition, diversity and biomass define tropical montane cloud forest extent." Proceedings of the Royal Society B 286.1895 (2019): 20182284.

Longton, R. E. "Physiological ecology of mosses." The mosses of North America (1980): 77-113.

Ah-Peng, Claudine, et al. "The role of epiphytic bryophytes in interception, storage, and the regulated release of atmospheric moisture in a tropical montane cloud forest." Journal of Hydrology 548 (2017): 665-673.

Proctor, M. C. F. "Climatic responses and limits of bryophytes: comparisons and contrasts with vascular plants." Z. Tuba, NG Slack, and LR Stark. Cambridge University Press, New York, New York, USA (2011): 35-54.

Methods

General comments

Somewhere you should discuss why the dry season in particular was chosen for the data collection period.

In-line comments

133 Roots for bryophytes too?

149 Remove And

154 Remove immediately

234-243 This level of detail seems unnecessary, but I will defer to the subject editor and other reviewers.

Results

In-line comments

264 Replace “remarkably” with some quantitative measure of how much different the ratios were (X% higher..)

270 Replace “the figure also showed” with “we also found”

266-281 These are very useful measurements that might be easier to access for future researchers if put into a table/chart.

Figure 5 Please increase the font size on the y-axis and/or expand to be thicker so that the groups are legible

320 Replace period after Figure 6 with a comma to join into 1 sentence

321-334 Again, some of these numbers in a table would be useful

Figure 6 Resize so that the bars are much bigger and the letters from statistical tests have more space between tick marks and error bars

Discussion

General comments:

-The first paragraph seems like it should have been in the methods or introduction section. Better to start with the more general idea of why the study was conducted—go back to why it is important to know from which source the epiphytes are getting water. Then you can get into the subsection on the specifics of why the water differed. These are not as interesting as results as the epiphyte WUE data, so these first two paragraphs could be shortened.

-I wanted a little bit more depth in interpreting why the different species and different groups had different water source ratios—could you tell the readers more about the natural history of the groups/species?

In-line comments

406-408 Is lack of availability the only reasons why lichens could only acquire atmospheric water? Lichens grow in areas with humus in other regions (e.g., Oregon & Washington State in the USA).

433 specify: “some methodological concerns, such as…”

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Thank you for giving me the opportunity to read and review the manuscript “Dry-season fog water utilization by epiphytes in a subtropical montane cloud forest of Southwest China” by Liu et al.

This study focus on the differentiation of water utilization in epiphytes and confirms the importance of fog water for epiphytes during the dry season. As mentioned by the authors and several other studies, epiphytes are very sensitive to changes in environmental conditions and can be good biological indicators of global climate change. Therefore, this study provides an interesting insight into the impact of climate change on plant water use.

Here are a few questions to ask about the manuscript.

P3 L121-: How was ‘Foggy days’ evaluated and measured?

P3 L121-: The product name of evaporation pan was shown, but I could not find the company name or anything else.

P3 L131-: It was a little difficult to understand the epiphyte samples used for the isotope analysis.

1. Were the 12 epiphytic plants collected daily, in the morning, from 10 to 23 January 2019, in the sampling design described in L146-151?

2.For δ13C analysis, 5 - 10 individual plants were randomly selected on the dates of plant tissues collection. Is there any reason why you did not use a mixture of all samples? While fog and rain water were collected during those events, did the randomly selected samples include samples collected on the same day as those events?

3. For the δ2H and δ18O analyses, you say that water was extracted for all plant samples, but how many samples of each epiphyte were used in the analysis in total?

Figure. 2: Figure 2 shows the mean values for each species of epiphyte, and the small legend behind it may indicate the samples used in the analysis. What samples are the results of the data shown in these small legends?

Figure. 4 and 5: Why are there more than 4 results for each species of epiphyte sample in Figure 2, but the results shown in Figures 4 and 5 are limited to 4 samples of each species? Is it not possible that an arbitrary four were selected? If the results of the analysis would change if all the data were used, it might affect the conclusion.

I hope you find at least some of my comments useful -Thanks.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The manuscript has been improved by revisions made by the authors and my questions were answered. Thank you for sharing your interesting research results.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

General comments:

By using stable isotope (δ²H, δ¹⁸O, and δ¹³C) techniques, Liu et al. investigated the potential water sources (i.e., fog water, humus and rainwater) and the intrinsic water use efficiency ( ) of four epiphyte groups (i.e., epiphytic lichens, epiphytic bryophytes, epiphytic ferns, and epiphytic seed plants) in the dry season. This study revealed that the epiphytes greatly relied on fog water, although differences in water absorption proportion were detected among different groups and species. The  of epiphytic lichens was significantly higher than the other epiphyte groups. For the first time, the authors qualified water sources of different epiphyte groups and highlighted the importance of fog water for epiphytes during the dry season. This study provides valuable data on water balance of epiphyte communities under future climate change.

The manuscript is generally well written and has a nice logical progression to the organization. The methodology used in this work seems to be reasonable, but some clarifications may be needed to make it clearer. The discussion section as a whole is good, even though some parts need to be improved. More comments and suggestions were added directly to the manuscript. Please see the attachment for the more details. The information originated from this study may be useful for better understanding of the maintaining mechanisms of biodiversity and the hydrological cycles in the montane forest ecosystem. Thus, I consider the overall objectives and the data of this study is a valuable contribution to the study of the water use strategies of epiphytes and fall into the scope of the Water.

Specific comments:

Line 47-49: The paper you cited here (Song et al. 2012) focused on non-vascular epiphytes, which is not good to show the abundance of epiphytes in SMCF. I would recommend you to cite the following literatures focused on the species biomass and diversity of epiphytes in the study region, which have more comprehensive information of epiphytes.

Xu, H.-Q.; Liu, W.-Y. Species diversity and distribution of epiphytes in the montane moist evergreen broad-leaved forest in AilaoMountain, Yunnan. Biodiversity Science 2005, 137-147.

Ma, W.-Z. The composition and biomass of epiphytic materials and their relationships with ecological factors in Xujiaba Region from Ailao Mountain, Yunnan. Graduate School of the Chinese Academy of Sciences, Beijing. 2009.

Line 80-81: Please provide the reference basis to support this inference.

Line 114: How do you define the neighboring trees? You’d better to provide the specific distances or range.

Line 114-115: Why do you choose different parts (non - photosynthetic tissues, shoots, and thalli) for epiphytes samplings? Will these affect the water relations and isotope measurements of the epiphytes? Please clarify.

Line 122: What is the basis of the fog sampling time?

Line 127: How do you define the ‘mature leaves’ of epiphytes. It is not clear to me on whether the growth period of epiphytes is consistent with the host trees. Please explain it more clearly.

Line 152: The MixSIAR model has been widely used in the investigations of plants water use sources. The cited references can be inserted as followed:

Wu et al., Identifying water sources used by alpine riparian plants in a restoration zone on the Qinghai-Tibet Plateau: Evidence from stable isotopes. 2019. Science of The Total Environment.

Wang et al., Seasonal variation in water uptake patterns of three plant species based on stable isotopes in the semi-arid Loess Plateau. 2017. Science of The Total Environment.

Line 160: You should tell if all data have been subjected to normality and homoscedasticity tests before further statistical analysis.

Line 176: The direction of unit names on the right side of the y-axis should be consistent with the left side.

Line 192: The Figure 2 cannot differentiate the average epiphytes and water sources well， especially epiphytic seed plants. You may need to adjust the size or color transparency of the icons to display the results better.

Line 208-210: You’d better to provide the specific δ2H and δ18O values of four species in epiphytic ferns.

Line 235-237: Different from other three epiphyte groups, you only describes the utilization of fog water of two epiphytic ferns. In this way, you cannot directly draw the conclusion in the second paragraph of the 4.2:

The average contribution proportions of fog water to epiphytic bryophytes, epiphytic ferns, and epiphytic seed plants were 32.7%, 38.8%, and 63.7%, respectively.

I will suggest you to show the contributions of fog water to four epiphytic ferns (AI, Asplenium indicum; LL, Lepisorus loriformis; HP, Hymenophyllum polyanthos; LC, Loxogramme chinensis.).

Line 309-310: We can not directly see the water sources of epiphytes were different among groups from this paragraph. I will suggest you to be more specific to discuss the water absorption (e.i., fog water and humus) of epiphyte groups during the dry season.

Line 316-317. table 1 change to Table 1.

Line 375: If possible, you can take a picture of the fog water on the fog collector. It will help you to describe the collection of fog water better.

Reviewer 2 Report

I reviewed the manuscript entitled “Dry-season fog water utilization by epiphytes in a subtropical montane cloud forest of Southwest China” by Liu et al. While I have some minor issues, the authors provided some solid work. However, they have to be careful not to “overstate” the novelty of their work and I do have some questions regarding their assessment of WUE, especially with respect to all species being C3 plants (which is not mentioned in the text but needed according to their own text). I do feel that the authors can address these points in a revised version of their manuscript.

Major points:

1. Line 44-46 you have also mentioned this in your abstract line 16-18. I do not completely agree. I understand that you want to push you work forward as novel, but a lot of research has been performed on the water use strategies of epiphytes. For example:

• Feild T.S. and Dawson T.E. 1998. Water sources used by Didymopanax pittieri at different life stages in a tropical cloud forest
• Pan et al. 2021. Leaf trichomes of Dendrobium species (epiphytic orchids) in relation to foliar water uptake, leaf surface wettability, and water balance

Please include those references where needed and rephrase your novelty regarding “unexplored” mechanisms.

2. Line 106-110: are all these species C3? Can you confirm this for all species? Your whole C13 method depends on this being true… (see line 72-74)

Minor points:

Introduction

Line 36 either it has a tendency or a significant increase, one excludes the other, so pick the right one

Line 46 spelling: “unexplored”

Line 64 “invariant to water taken up by the roots” is unclear, please rephrase

Line 65-66 this sentence implies that epiphytes use soil and groundwater which is not true, please rephrase/correct

Materials and Methods

Move figure 1 between line 104 and 105. That was it is positioned better and you can put the figure legend on the same page as the figure

Line 112 Your number of host trees does not determine your number of replicates. Your number of individuals does maybe put (n = …) besides every species?

Line 152-165 add any statistical assumptions you made to allow the use of these methods, e.g. did you assume normality? For all tests or a few of them?

Line 165 please include the linear model you used (the equation)

Results

Line 167-179 you could move your meteorological conditions to your materials and methods

Line 173-174 I don’t know which data you have available, but PAR would be more useful

Line 186-187 this could be the result of an enrichment in O18 or a depletion of H2. I don’t think you can tell based on your data, but an enrichment of O18 looks most probable so I don’t think this is correct.

Line 226-245 you indicated before that humus and rainwater are not significantly different, can you explain in your text how the MixSIAR model solves this?

Line 248-249 your figure does show a significant difference between epiphytic bryophytes and epiphytic seed plants…

Line 252 they are correlated, that does not mean one affects the other. Remember the difference between correlation and causation and write as such.

Line 253-256 only HM was different from the rest, that does not really allow this order you’re provide. Clearly state that only HM was significantly different from the other bryophytes and rephrase

Table 1 is hard to interpret without the equation for the model (see comment line 165)

Discussion

Line 280-284 optional comment: I would expand on the topic of foliar water uptake as this has a large impact on your research

Line 294-296 check my comment on line 186-187

Line 297-300 You have not defined how you sampled lichens in your materials and methods, so I assume you took the whole lichens. As such fractionation because of evaporation is very probable. This is confirmed by the position of lichen on figure 2. Lichen can only use fog water or rainwater, so they have to be on a line between these two sources in this plot. That is not the case, probably because of fractionation. Add this information

Line 303 differentiated how? This sentence has no real meaning

Line 307-308 and rainwater, as you can see in figure 5

Line 314 how can a difference in water acquisition have an effect on WUE, explain

Line 343-344 I think I know what you mean, but this is not completely clear, please rephrase

Line 363-365 this is a bold claim, I’m not sure this is true

Reviewer 3 Report

The manuscript entitled "Dry-season fog water utilization by epiphytes in a subtropical montane cloud forest of Southwest China" shows the underline importance of cloud forests worldwide. The manuscript is written with a proper structure and shows the results in an easy and understandable way. Despite the extensive and arduous fieldwork done by the authors, the current version is not suitable for publication for the following reasons:

1. The authors did not carry out an extensive literature review to grasp the state of the art of the main topics addressed in the manuscript (i.e., water use by epiphytes and stable isotopes research). Cloud forests have been widely studied in the Neotropics and there is a wide understanding of the epiphyte's roles and interactions with the water cycle (e.g., Cardelus 2009, Liu 2014, Mendieta-Leiva 2020, Yoder 2000). Also, it is a big gap of context to justify the implementation of stable isotope techniques in this study. The authors should try to address questions such as: What has been done before in SMCF in China and other regions (e.g., cloud forests in Costa Rica, Guatemala, Mexico, Africa, Ecuador)? How stable isotopes (i.e., oxygen, hydrogen, and carbon) have been used in cloud forests? among other questions.
2. There is a large number of concerns related to the Materials and Methods section that does not allow to replicate the sampling procedures carried out by the authors. Some of these concerns are:
   - 2.1. It is clear that the sampling procedure was carried out during the dry season of 2019. However, the authors do not state how long took the sampling period (days), nor the timing among samples. This is really important in terms of isotopic analysis considering the type of species that are under analysis. It is widely known the strong effect that evaporation and mixing processes can have on isotopic signatures. Also, considering that these plant species rely on the absorption of fog or rain or hummus water or the mixing of all of them, it becomes more important to know the timing between individual samples of source waters and target waters (temporal distribution of water and tissue samples collected on site). This is different than the analysis of carbon isotopes that are more linked to the carbon fixed to plant tissues for a longer period of time.
   - 2.2. It is not possible to establish a reliable LMWL based on 5 samples collected only during a period of 15 days. Any assumption done based on this LMWL will mislead the main findings.
   - 2.3. How did the authors ensure that the water sampled from lichens does not correspond to liquid water on the surface of them?
   - 2.4. Do the fog and water samples correspond to cumulative samples over different days, or do they were collected daily?
   - 2.5. How did the authors calculate the evaporation?
   - 2.6. Here, I will stress a really important issue about the field sampling procedures with respect to the physical location of the trees and rain/fog samplers. As epiphytes are located on tree canopy tops, they are strongly influenced by tree interception capacity. So, the main question here is if the fog sampler was located within the forest canopy at the same height and within the forest canopy. Then, where did the sampled plants were situated within the trees (e.g., canopy top, within the canopy, on the main stem, close to stem base). This is really important because the water mixing during the rainfall interception processes will affect strongly the outcome of the samples.
   - 2.7. Do the samples were filtered or not?
3. Considering the sampling timing, it is not clear if the Bayesian analysis procedure did consider the time as one of the variables ingested to be considered. 
4. The authors stated in the objectives section the analysis of the "intrinsic water use efficiency" (Line 86), however, it is not evidence in the methodology, results, or discussion about this.
5. The authors did considerer fog, rain, and humus as independent sources of water. However, the water retained in the canopy hummus is a mixing of these two sources (i.e., fog and rain), and this water (i.e., hummus) has also been affected by the evaporation processes of previous days. The fact that the authors did not account for this issue made the analysis, discussion, and conclusions unreliables.

References

Cardelús, 2009. https://link.springer.com/article/10.1007/s11258-009-9651-y

Liu, 2014. https://doi.org/10.1002/eco.1475

Mendieta-Leiva, 2020. https://link.springer.com/chapter/10.1007/978-3-030-29702-2_9

Yoder, 2000. https://doi.org/10.1016/S0168-9452(00)00246-6