Review Reports - Effect of Line Spacing on Blade Phenotype and Yields of Farmed <i>Alaria marginata</i> from Alaska

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present an experiment conducted on a single Alaska farm to investigate how line spacing affects yield and morphological traits in Alaria marginata. Yield was expressed both as line yield (kg m⁻¹) and areal yield (kg m⁻²). They report no significant differences in yield per area, which they attribute to an increase in individual blade size offset by reduced density.

This work provides valuable insights for future farm design and cultivation practices for A. marginata in Alaska, and it addresses an underexplored aspect of kelp aquaculture. I believe the manuscript has potential for publication after revision. Below, I outline my major and minor suggestions.

Major comments:

Introduction

The introduction would benefit from further discussion on why line spacing is important for kelp farming. Are there related studies in Alaria or other kelp species (e.g., Saccharina or Laminaria) that could provide context? Additionally, consider adding a paragraph on the broader importance of farm design and cultivation settings for optimizing yield.

Materials and Methods

A schematic or photograph of the farm design would help readers better understand the experimental setup. Figure S2 could be refined and moved into this section.

In addition, please clarify the term “kelp seed.” Does this refer to a mixture of gametophytes or another propagation method? Clear terminology will aid reproducibility.

Results

A summary table of all measured traits would make the results more accessible. Figure 3, in particular, requires a clearer explanation. Please expand on the methods underlying this figure and more explicitly integrate it into the results and discussion.

Discussion

The discussion could be strengthened by situating your findings within the context of prior studies on spacing effects in kelp cultivation. Even if work is limited for A. marginata, research on related kelp species would provide useful comparisons.

Minor suggestions:

In the first paragraph of the introduction, species names (e.g., Alaria marginata) should be italicized throughout the manuscript.
In the second paragraph of the introduction, please elaborate on why ribbon kelp (A. marginata) may be particularly well-suited for Alaskan farms.
Add references for all statistical methods used.
In the materials and methods part, Figures 1 and 2: consider adding significance levels directly to the boxplots for clarity.
Figure 3: please revise the caption to provide a more complete description of the figure content and its interpretation.

Author Response

Reviewer 1

Comments and Suggestions for Authors

Major comments:

Introduction

Reply: Thank you for your comment. We have elaborated on the points suggested. Changes were incorporated throughout the introduction. Here we show the most significant onces:

Lines 32-35: While S. latissima has been the focus of most commercial and research efforts, A. marginata is gaining attention for its market potential, particularly in food-grade applications where specific phenotypic traits are valued.

Lines 36- 47: Unlike S. latissima, which thrives in protected coastal environments, A. marginata is naturally found in more exposed, rocky shorelines. Its holdfast and blades are much more robust than those of Alaska S. latissima, offering opportunities for diversification of farm sites and products, particularly in more exposed areas [3]. Despite its commercial potential, limited research exists on the performance of this species as a farmed species within specific farm settings. For example, cultivation line spacing within farm designs may be adjusted based on practical experience or site-specific constraints. However, there are limited systematic studies quantifying how this variable affects key production metrics, such as sporophyte phenotype, yield, biofouling, or tissue nutrient content, under real-world farming conditions [4,5]. Effective seaweed farm design is fundamental to optimizing the growth, quality, and resilience of cultivated species, as it governs the hydrodynamic, optical, and nutritional environment that the thalli experience [6].

Lines 48-56: The arrangement of longline spacing between ropes and the orientation relative to prevailing currents determine light availability, water flow, and nutrient exchange, strongly influencing the photosynthetic performance, tissue nitrogen content, and biomass yield of seaweed [7-8]. Designs that minimize self-shading and mechanical stress while maintaining consistent exposure to currents can enhance productivity compared to designs in which light penetration or nutrient exchange are reduced due to limited water flow [9,6]. Moreover, thoughtful spatial configuration can support uniform growth and biochemical composition, facilitating efficient harvests and reliable product quality for food or bioindustrial uses [10,5].

Lines 57-60: Like many other seaweeds, A. marginata is sensitive to hydrodynamic forces, nutrient availability, and light gradients. Therefore, farm layouts that balance line spacing, depth, and tension are key to achieving sustainable biological performance and operational effectiveness.

Materials and Methods

A schematic or photograph of the farm design would help readers better understand the experimental setup. Figure S2 could be refined and moved into this section.

Reply: We have updated the supplementary figure and moved it to the main narrative as Figure 1.

In addition, please clarify the term “kelp seed.” Does this refer to a mixture of gametophytes or another propagation method? Clear terminology will aid reproducibility.

Reply: We have added the following

Line 101: “Kelp seed, consisting of embryonic sporophytes…”

Results

Reply: Thank you. We have made substantial changes to how we present the results. Changes include, but are not limited to, updating all captions, including more figures as requested by reviewer #2, adding significant letters to boxplots, adding all p-values rather than cutoff values, and descripting outputs much more to improve figure interpretation.

Discussion

Reply: Thank you and acknowledge. We have reworked the entire discussion to include information about new results presented and to further elaborate on the topics provided. In brief, we have included a description of the environmental conditions at the time of the experiments, practical considerations about our overall results, implications and limitations of our data analysis approach, implications for farming, and constraints or further research questions not addressed here. We continued our discussion of density vs. yield trade-offs and added more citations. The new discussion was expanded from 4 to 7 paragraphs in its current version.

Minor suggestions:

In the first paragraph of the introduction, species names (e.g., Alaria marginata) should be italicized throughout the manuscript.

Corrected. We noticed the missing italics too late.

In the second paragraph of the introduction, please elaborate on why ribbon kelp (A. marginata) may be particularly well-suited for Alaskan farms.

Added to lines 36-39: “Unlike S. latissima, which thrives in protected coastal environments, A. marginata is naturally found in more exposed, rocky shorelines. Its holdfast and blades are much more robust than those of Alaska S. latissima, offering opportunities for diversification of farm sites and products, particularly in more exposed areas [3].”

Add references for all statistical methods used.

Reply: We have made substantial updates to the methods, including the statistical analysis, which now include citations.

In the materials and methods part, Figures 1 and 2: consider adding significance levels directly to the boxplots for clarity.

Added.

Figure 3: please revise the caption to provide a more complete description of the figure content and its interpretation.

Updated.

Reviewer 2 Report

Comments and Suggestions for Authors

In general, the information provided in the short communication is interesting and relevant to seaweed farmers, industry, and researchers aiming to identify best practices in a growing aquaculture sector. However, the report is unnecessarily limited in methods, results, and discussion, and there are some inconsistencies in the reported results between Figure 2 and 3 and the discussion of results that should be either explained or corrected. More transparency is needed regarding the data collected and results of statistical testing to help readers draw their own decisions regarding results of the work.

MATERIALS AND METHODS

Comment M1. Deployment orientation and conditions. More information is needed on the orientation of spreader bar arrays at the sea farm and the conditions under which they were deployed. Was biomass production similar to that experienced in other years (e.g., was this year typical or atypical)? Were there external factors (storms or other uncontrollable driving forces) that affected biomass production in the study year? While orientation relative to tidal flows or currents may affect water flow (and nutrient delivery) within the deployment, orientation (north-south vs. east-west) can also affect light availability, especially in more closely spaced lines where shading may occur from A. marginata growing on adjacent lines. Thus, physical orientation can affect photosynthesis and measured differences in biomass production may in part be an artifact of orientation rather than completely attributable to line spacing. Was this controlled in the study?

Comment M2. Exclusion of biomass production on edge lines. The edge lines should be included in the analysis of differences of biomass characteristics between line spacings. The researchers have stated in their methods that they have excluded biomass production on edge lines of the spreader bar system for grow lines in their study. Understandably, edge lines may be more exposed to detrimental mechanical forces in sea farms than interior lines. They may also have greater light exposure and more access to other growth factors than more densely spaced lines in the center of the arrays where growth factors may be more locally depleted. However, their arbitrary exclusion can misleadingly inflate differences between biomass properties and density at different line spacings as actually deployed.

Note that the authors themselves admit that separation is maintained between repeated units of spreader bar arrays (e.g., one spreader bar array is shown in the supplemental information Figure S2) of no more than 20 meters. While not provided by the authors, there is a practical minimum distance between spreader bar arrays that also needs to be maintained. This facilitates boat or shipping lane access along the 50m length between the spreader bar line arrays for deployment, maintenance, and harvesting of A. marginata. Thus, exposed edge lines are a reality - they exist in real deployments at sea farms. Edge lines are exposed and biomass is harvested from edge lines as well as interior lines. Seaweed growers will collect and sell this biomass alongside any interior biomass collected, and thus it is unrealistic to exclude it from the study results.

Comment M3. Phenotypic differences as a function of line position and spacing. Phenotypic differences between A. marginata growth were reported as a function of line spacing (treated as a single population for each line spacing). Within each of these populations from a selected line spacing, did the researchers investigate whether there were phenotypic differences as a function of distance from the edge lines or observe such differences? If so, it would be worthwhile to report and discuss.

Editorial comment. Change “obtained” to “was obtained” in the last sentence of Materials and Methods. Currently, this sentence reads: “Yield per meter (kg m-1) and per area (kg m-2) obtained from the total biomass harvested per treatment.”

RESULTS

General Comment. Transparency. Improve transparency of data and statistical measures throughout. There was an interesting dataset collected for this study. I strongly encourage the authors to share it as supplemental data, which could benefit other researchers. Also, more transparency is needed regarding outcomes of tests for differences (see comment R1 below).

Comment R1. Report p-values of all tests for differences. Reporting the p-values is much more informative than selecting arbitrarily a level of significance that will indicate a “difference” between treatments and making judgement calls. This increased transparency will allow readers to make their own decisions about whether the differences are significant or not and to understand more completely the data. Philosophically, does a p-value of 0.051 really indicate no difference while p=0.049 indicates a difference? This rigid statistical interpretation can mask outcomes and limit interpretation of results.

An example of how this affects outcomes is apparent in paragraph 3 of the results. The authors state that “No significant differences were detected in yield per area (Figure 3), despite the low probability that the 1.83 m (6ft) spacing would yield more than 7 kg per square meter (Figure 2D).” Visually in Figure 2B (corresponds to Figure 2D), the 6 ft spacing had much lower biomass per area relative to the other line spacings. It would be much more informative to know the p-value to this test for differences and allow readers to draw their own conclusions rather than select an arbitrary p-value of p<0.05 and report no difference without transparency or ability for readers to understand the interpretation made.

Comment R2. Apparent discrepancy between Figure 2 and Figure 3. In Figure 2 the difference in biomass yield per meter between the 1 ft and 6 ft spacing (~3kg/m on average) is approximately double that reported between the 1 ft versus 3 ft spacing (~1.5 kg/m on average) of the same figure. However, in Figure 3, the adjusted p-values reporting results of the test for differences suggest that the difference in biomass per meter between the 1 ft. vs 3 ft. spacing (“***” indicating that p<0.001) is more significant than the difference in biomass per meter between the 1 ft vs 6 ft spacing (“*” indicating p<0.05). There does not seem to be enough difference in the overall range or inner / outer quartile ranges of the data between 3ft spacing and 6 ft spacing in Figure 2B to account for this mismatch. Could the authors please either correct any potential typographic errors or provide a more informative discussion about this confusing result? Note that greater data transparency (e.g., raw data provided in the supplemental material and reporting p-values exactly rather than as cutoffs) could help alleviate this type of confusion for readers.

DISCUSSION

Editorial Comment. First and second sentence of discussion. Italicize “Alaria marginata” and “A. marginata”. Please read through and make sure all other instances are properly italicized.

Comment D1. Limited discussion. In paragraph 2, the authors state that “Despite obtaining a lower yield per meter with reduced line spacing, yield per area showed no significant differences across treatments. These results also suggest a density– yield trade-off, where a greater yield per meter does not necessarily result in a greater yield per unit area.” The authors go on to discuss tradeoffs largely as quality differences between biomass produced. This is extremely limited. For example, a very important consideration is the materials use and cost of the production platform, production or purchase of seeded line, and labor for deploying and harvesting A. marginata. While the yield per area may not change between line spacing selection, the economic cost of producing the same biomass quantity outcome with much greater inputs and labor associated with deployment of closely spaced line arrays is significant. Further the increased environmental costs of added materials, fuels usage, and other related costs will also be greater for deployment of closely spaced line arrays with lower resulting biomass per meter. These are practical considerations worthy of discussion.

Comment D2. Limited discussion. In paragraph 3 of the discussion, the authors identify nutritional and biochemical composition, color, and fouling as potentially important qualities of seaweeds that may be affected by line spacing in addition to blade length, width, and thickness and biomass production. Are there nutritional and biochemical properties that are important for A. marginata, and were these nutritional and biochemical properties, color, and fouling also monitored for differences between line spacing in the study? It would be worthwhile to add to the discussion these points specifically if data were collected or at least discuss whether some of these factors exist for A. marginata that were not investigated in this study.

Author Response

Reviewer 2

Comments and Suggestions for Authors

Reply: Thank you for taking the time to review our document. We kept information limited to comply with the requirements of a short communication rather than a full research paper. Nonetheless, we see the value of presenting as much detail as possible. To this end, we have reworked all sections, added more figures, more descriptions, and more discussion, along with more citations and references. More specific comments following your comments.

MATERIALS AND METHODS

Comment M1. Deployment orientation and conditions. More information is needed on the orientation of spreader bar arrays at the sea farm and the conditions under which they were deployed. Was biomass production similar to that experienced in other years (e.g., was this year typical or atypical)? Were there external factors (storms or other uncontrollable driving forces) that affected biomass production in the study year? While orientation relative to tidal flows or currents may affect water flow (and nutrient delivery) within the deployment, orientation (north-south vs. east-west) can also affect light availability, especially in more closely spaced lines where shading may occur from A. marginata growing on adjacent lines. Thus, physical orientation can affect photosynthesis and measured differences in biomass production may in part be an artifact of orientation rather than completely attributable to line spacing. Was this controlled in the study?

Reply: Thanks for your comment. We have made multiple changes to address your concerns. These include describing the study site in more detail, adding a supplementary figure to guide the positioning of the arrays with respect to the features in the experimental site (e.g., shoreline, island), and information on the monitoring of environmental parameters (i.e., nutrients, sea surface temperature, water transparency at the time of the experiment.

A few of these updates read as follows:

Line 68-74: “This study occurred within an 86-acre plot leased by Alaska Ocean Farms, LLC, in Kalsin Bay, Kodiak Island (57.6585800° N, -152.4166224° W; Figure S1). The lease site is situated along the northwestern shoreline of the bay, with Kalsin Island forming its southwestern boundary. Extensive rocky reefs, exposed at low tide to the north and east, characterize the surrounding area. This setting provides natural protection from ocean swells and large seas, offering a sheltered environment ideal for farm operations (Figures S1 and S2).”

Lines 109-120: “Nitrogen, phosphorus, and silicate concentrations, plus sea surface temperature, and water transparency were monitored across the study area to verify that environ-mental conditions remained consistent among the three arrays. Measurements and samples were collected adjacent to each array every two weeks between February and June. Three 30-ml water samples were collected at approximately 2.13 m (7 ft) depth at each sampling station, corresponding to the kelp growth zone. Samples were immediately filtered through 0.45-µm filters and stored at –20 °C until analysis. Water transparency was measured with a Secchi disk to the nearest 0.25 m. HOBO MX2202 Temperature Data Loggers were attached to each array to record temperature (°C) at hourly intervals from seeding through harvest. In situ temperature readings were also taken at the same depth to verify logger accuracy using a YSI EcoSense 300A meter, which was used for calibration checks.”

We have also updated the results section to include environmental details covered in lines 150-164.

Comment M2. Exclusion of biomass production on edge lines. The edge lines should be included in the analysis of differences of biomass characteristics between line spacings. The researchers have stated in their methods that they have excluded biomass production on edge lines of the spreader bar system for grow lines in their study. Understandably, edge lines may be more exposed to detrimental mechanical forces in sea farms than interior lines. They may also have greater light exposure and more access to other growth factors than more densely spaced lines in the center of the arrays where growth factors may be more locally depleted. However, their arbitrary exclusion can misleadingly inflate differences between biomass properties and density at different line spacings as actually deployed.

Reply: Comment acknowledged. On the same token, the decision to exclude biomass production on the edge lines of the spreader-bar system is justified because those edge lines are subject to distinct environmental and mechanical conditions compared to the interior lines and thus may not reflect the “typical” behavior of lines under the intended density-spacing treatment. As you mentioned, edge lines are often more exposed to waves, currents, light, and biomass loading (due to fewer neighbors), which may lead to atypical biomass growth or loss, making them less representative of the interior treatment. By excluding edge lines, we focus comparisons on lines with more uniform neighbors and exposure conditions, which permits a cleaner assessment of how line spacing influences biomass properties in the “bulk” of the array. In other words, exclusion is not arbitrary but rather a methodological decision to reduce extraneous variation and isolate the effect of spacing under comparable conditions. That said, the exclusion should still be transparently reported (which it is in the latest version). With this in mind, we acknowledge some implications for generalizing to full-farm deployments.

Comment M3. Phenotypic differences as a function of line position and spacing. Phenotypic differences between A. marginata growth were reported as a function of line spacing (treated as a single population for each line spacing). Within each of these populations from a selected line spacing, did the researchers investigate whether there were phenotypic differences as a function of distance from the edge lines or observe such differences? If so, it would be worthwhile to report and discuss.

Reply: Good point. We recorded the position of each line within the arrays to ensure that the location of every blade was accurately tracked. We did not conduct statistical comparisons among lines within the same treatment, as increasing line spacing led to a sharp decrease in the number of comparable units, potentially introducing bias. However, we observed that in the 1-ft spacing treatment, blades on center lines appeared visibly smaller than those on the outer lines. This pattern was not evident in any of the other spacing treatments.

Editorial comment. Change “obtained” to “was obtained” in the last sentence of Materials and Methods. Currently, this sentence reads: “Yield per meter (kg m-1) and per area (kg m-2) obtained from the total biomass harvested per treatment.”

Corrected.

RESULTS

Reply: The following three comments are related. Therefore, we crafted a single, comprehensive reply at the end of the last comment for this section.

General Comment. Transparency. Improve transparency of data and statistical measures throughout. There was an interesting dataset collected for this study. I strongly encourage the authors to share it as supplemental data, which could benefit other researchers. Also, more transparency is needed regarding outcomes of tests for differences (see comment R1 below).

Comment R1. Report p-values of all tests for differences. Reporting the p-values is much more informative than selecting arbitrarily a level of significance that will indicate a “difference” between treatments and making judgement calls. This increased transparency will allow readers to make their own decisions about whether the differences are significant or not and to understand more completely the data. Philosophically, does a p-value of 0.051 really indicate no difference while p=0.049 indicates a difference? This rigid statistical interpretation can mask outcomes and limit interpretation of results.

Comment R2. Apparent discrepancy between Figure 2 and Figure 3. In Figure 2 the difference in biomass yield per meter between the 1 ft and 6 ft spacing (~3kg/m on average) is approximately double that reported between the 1 ft versus 3 ft spacing (~1.5 kg/m on average) of the same figure. However, in Figure 3, the adjusted p-values reporting results of the test for differences suggest that the difference in biomass per meter between the 1 ft. vs 3 ft. spacing (“***” indicating that p<0.001) is more significant than the difference in biomass per meter between the 1 ft vs 6 ft spacing (“*” indicating p<0.05). There does not seem to be enough difference in the overall range or inner/outer quartile ranges of the data between 3ft spacing and 6 ft spacing in Figure 2B to account for this mismatch. Could the authors please either correct any potential typographic errors or provide a more informative discussion about this confusing result? Note that greater data transparency (e.g., raw data provided in the supplemental material and reporting p-values exactly rather than as cutoffs) could help alleviate this type of confusion for readers.

Reply: We agree with you that this dataset should be made available. We have a DOI placeholder (see Data availability in line 296) that will be added once our contribution gets recommended for publication. Additionally, we have revised the description of our methods. Lines 124-150 provide more detail on how the data were collected, how they were analyzed, and the rationale for the analysis presented.

As requested, this revised version presents the actual p-values for all main and post hoc comparisons. These are presented in Table 2 for the Kruskal-Wallis test for phenotypic and productivity metrics and Figure 6 for Dunn’s test.

We agree that the lack of significance in yield per meter looks odd, particularly given the boxplot display. Boxplots can suggest differences that are not statistically significant because they emphasize medians and visible gaps. Coupled with that, nonparametric tests, such as the Kruskal–Wallis test, are less sensitive than parametric tests and may fail to detect differences when they exist. With small sample sizes, like in the 6 ft spacing, even moderate differences in medians won’t reach significance. Also, the boxplot may exaggerate the effect because each point (or small sample) defines quartiles sharply. The methods section now includes a brief explanation of how yield was calculated. We have further discussed this topic in lines 254-262 (discussion).

DISCUSSION

Editorial Comment. First and second sentence of discussion. Italicize “Alaria marginata” and “A. marginata”. Please read through and make sure all other instances are properly italicized.

Reply: Oops, thanks for noticing. The text lost formatting when copied and pasted into the template.

Reply: The following three comments are related. Therefore, we crafted a single reply at the end of the last comment.

Comment D1. Limited discussion. In paragraph 2, the authors state that “Despite obtaining a lower yield per meter with reduced line spacing, yield per area showed no significant differences across treatments. These results also suggest a density– yield trade-off, where a greater yield per meter does not necessarily result in a greater yield per unit area.” The authors go on to discuss tradeoffs largely as quality differences between biomass produced. This is extremely limited. For example, a very important consideration is the materials use and cost of the production platform, production or purchase of seeded line, and labor for deploying and harvesting A. marginata. While the yield per area may not change between line spacing selection, the economic cost of producing the same biomass quantity outcome with much greater inputs and labor associated with deployment of closely spaced line arrays is significant. Further the increased environmental costs of added materials, fuels usage, and other related costs will also be greater for deployment of closely spaced line arrays with lower resulting biomass per meter. These are practical considerations worthy of discussion.

Comment D2. Limited discussion. In paragraph 3 of the discussion, the authors identify nutritional and biochemical composition, color, and fouling as potentially important qualities of seaweeds that may be affected by line spacing in addition to blade length, width, and thickness and biomass production. Are there nutritional and biochemical properties that are important for A. marginata, and were these nutritional and biochemical properties, color, and fouling also monitored for differences between line spacing in the study? It would be worthwhile to add to the discussion these points specifically if data were collected or at least discuss whether some of these factors exist for A. marginata that were not investigated in this study.

Reply: Thanks for for thorough comments. We intended to keep the discussion as succinct as possible to fit this contribution as a short communication rather than a research paper. We have considered all your points to rework the discussion. This section was completely rewritten to accommodate the new results presented and elaborate on the points requested. In brief, the discussion now addresses the following topics: environmental conditions at the time of the experiments, density–yield trade-offs, economics of line spacing, limitations arising from the analysis used, remaining knowledge gaps, and overall limitations of the study.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for their revisions, and still believe the data set and study objectives are of strong interest. I still have concerns regarding the methodology / data analysis, particularly around the exclusion of edge lines, and its impact on the conclusions drawn from the research work, that should be addressed before publication.

General comment on exclusion of edge lines:

Both biomass quality and quantity are relevant economically; both may be affected by positioning of biomass within a spreader bar array whether grown on more sheltered inner lines or exposed outer lines. The authors mirror my comments in the first review regarding potential detrimental effects of mechanical action at exposed outer lines on biomass by stating that even under expected seasonal conditions, mechanical damage can occur to seaweeds on the outermost (edge) lines in a spreader bar array (Page 5, lines 151-154). However, this exposure may also reduce detrimental effects of dense line spacings experienced by seaweed grown on inner lines such as light or nutrient shading. Further, they may provide a buffer for other detrimental forces to remaining biomass. Regardless, biomass is grown on these edge lines and it is relevant to the activity at hand. Growers will harvest and sell biomass from all lines, including the outermost lines, rather than simply disposing of it. Considering that all biomass is relevant and that edge line effects may not be unidirectional, the argument to exclude edge lines because they are not representative of biomass grown under different line spacing conditions is confusing and unsubstantiated, as is the argument that it masks the effects of increased line density. For example, at 1.83m spacing, the inner line only represents 33% of the total growing line length in the array. How can biomass from edge lines be excluded as non-representative when it comprises as much as 67% of the biomass produced at this line spacing? Reluctance to report this data and include it in the analysis leaves the reader unclear to what extent this exclusion introduces bias or even the direction of the bias. If the data is available, it should be reported upon and discussed. It is relevant to the analysis and towards the stated objectives of the study. If it is not available, that too should be reported and its potential impact on the conclusions discussed.

Typographical error in Figure 1. Please correct the line spacing shown in the third array. It should read “1.23 m (4 ft)”

Methods, Page 4, lines 126 – 128, Sampling strategy: The authors report that “For each treatment, 69 subsamples, consisting of 0.5m sections from each of the non-edge lines, were randomly collected to obtain phenotypic measurements.” Assuming this is true, for each treatment an equivalent of 69 x 0.5m = 34.5 m of line would have been sampled. However, for the 1.83 m line spacing, excluding edge lines leaves only one 10m line per array and 3 arrays (10m x 3 = 30 m line). Thus, even if the entire population were sampled, it would fall short of the reported line length subsampled. The authors should report whether they used a smaller length interval for subsamples for the 1.83 m line spacing to total 69 subsamples or whether they used 60 subsamples for this line length and sampled the entire population (= 30m total line length / 0.5 m per subsample).

Methods, Page 4, lines 131-132, Yield per meter line (kg/m): While the authors report that only non-edge lines were used for phenotypic measurements (see lines 126-128), it is not clear whether biomass measurements were made on all lines (including edge lines) or whether edge lines were excluded. Please clarify.

Methods, Page 4, lines 132-134, Yield per area (kg/m^2): It is understood that the growing area for each treatment and array was 3.7m x 10m = 37 m^2, and this growing area was used in all yield per area calculations. However, it is not clear whether yield per area for each line spacing treatment was calculated based upon (i) actual biomass measurements of all lines (biomass on the edge lines included in the calculation); (ii) actual biomass measured on non-edge lines only (biomass on edge lines excluded from the calculation); or (iii) actual biomass on non-edge lines plus estimated biomass on edge lines (e.g. assumed equivalent to non-edge lines or other rationale). Please clarify.

Methods, Page 4, lines 134-136, Imputation of missing biomass values: Provide the imputation function and procedure used to estimate missing biomass values and the extent of missing biomass values per treatment. Report the criteria used to determine whether biomass needed to be corrected for mechanical damage using the imputation procedure. How did the authors decide whether a correction was needed and then apply the rationale equally across all treatments such that it did not bias results?

Results, Page 7, lines 195-197, Yield per area (kg/m^2): Figure 5 reports yield per area for each line spacing investigated in the study. However, these reported results are not an appropriate representation of actual conditions because of the method of calculation of yield per area used. Inspection of Figure 5 reveals that the biomass per area was estimated as biomass measured on non-edge lines only divided by the full growing area of 37 m^2 for each treatment block in the array. This is an inaccurate measurement of yield per area because it ignores biomass on the outer (edge) lines produced within the 37 m^2 area of interest. This error disproportionately affects larger line spacings as compared to closer line spacings because the edge lines represent a larger fraction of the line length available for biomass growth at more distant line spacings than closer spacings. This leads to errors in the conclusions drawn by the authors regarding differences in biomass yield per area between treatments (line spacings).

For example, at 6 ft (1.83 m) line spacing, Figure 5A reports a median of approximately 5.3 kg/m biomass growth. While there are 3 growth lines in this 37 m^2 plot (1 inner and 2 edge lines), biomass from only one line (the inner line) was used in areal estimates of biomass growth (5.3 kg/m x 10m x 1 line / 37 m^2 area = 1.4 kg/m^2 – see Figure 5B which reports approximately this biomass yield per square meter). In reality, there are three lines at that spacing within the 37 m^2 growing area. Assuming no edge effects on biomass quantity per m line (because the authors provide no data to understand the actual biomass growth and properties on the edge lines), the yield for that line spacing would more appropriately be as much as three times the reported value (5.3 kg/m x 10m x 3 lines / 37 m^2 area = 4.3 kg/m^2). Even if detrimental effects of sea forces on the edge lines reduced biomass quantity by half on edge lines, the biomass yield would still be double that reported (5.3 kg/m x 10m x 1 line + 2.65 kg/m x 10m x 2 lines)/ 37 m^2 area = 2.9 kg/m^2). In other words, neglecting edge lines introduces up to 67% underestimate of total biomass per area for the 6 ft spacing on each spreader bar array. The effect of neglecting biomass grown on edge lines is exacerbated for large line spacings over close line spacings, and this biases the discussion and conclusions drawn by the authors. For example, for the 1 ft (0.31 m) spacing, neglecting the edge lines produced as much as 15.4% underestimate of total biomass per area (2 lines / 13 lines), much less than the 67% error for the 6 ft line spacing. This should be corrected and the tests for differences, results, and discussion modified appropriately.

Results, Page 7, lines 197-198, Yield per area maximums: The authors report that all treatments besides 6 ft (1.83 m) line spacing could achieve 15 kg biomass per square meter or more, referring the reader to Figure 5D. The axes of Figure 5D and 5B represent yield of biomass per area only up to 7 or 8 kg per square meter, and none of these reported values realistically approach 15kg biomass per square meter. More data or explanation is needed to substantiate this claim.

Author Response

Reviewer #2

Reply: Once again, thank you for your thoughtful feedback. Following an in-depth discussion, we have implemented several revisions. Most notably, we removed all references to yield per area, as your comments highlighted important concerns that led us to recognize that our attempt to fill data gaps could bias results and inadvertently misrepresent system performance for this metric. While we initially sought to present outcomes using criteria we believed were appropriate, your points clarified that this approach was not sufficiently robust for this analysis.

Regarding edge lines, we acknowledge the differing perspectives between the authors and Reviewer #2 (excluding yield per area). We have revised the manuscript to articulate our rationale more clearly and to state that edge-line data were not collected a priori as part of our established sampling design agreement. As a result of these revisions, the Discussion section has been substantially updated to reflect these clarifications and adjustments.

Thank you for being so descriptive while providing comments. It indeed guided our revisions and made the process more straightforward.

General comment on exclusion of edge lines:

Reply: Thank you for this thoughtful and detailed comment. We fully understand and agree with your point that biomass from edge lines is economically relevant, harvested in practice, and certainly influences overall yield per area. As you note, edge-line effects are not unidirectional. While exposure may enhance light or nutrient availability in some cases, mechanical stress can reduce biomass quality or quantity in others. This bidirectionality is precisely why we were cautious about including edge lines in a strict comparison of line-spacing treatments. Unlike inner lines, edge lines are not flanked by seeded lines on both sides and therefore do not experience the same density-dependent competitive environment we sought to isolate. Our narrative now includes a line clarifying that edge line data was not collected. The narrative also includes an explanation of why.

That said, we acknowledge the importance of your point regarding representativeness and yield per area, mainly where edge lines constitute a large proportion of total line length. Despite our efforts to present data and results clearly, we do not have sufficient edge-line biomass data to adequately address yield per area. Therefore, to avoid any extrapolation and confounding the interpretation of spacing-specific effects, we have removed yield per area from this manuscript and focused the analysis solely on lines that experienced comparable conditions. Nonetheless, given the relevance of yield per area, we still discuss that this variable would provide a more comprehensive assessment of production.

Typographical error in Figure 1. Please correct the line spacing shown in the third array. It should read “1.23 m (4 ft)”

Reply: Corrected

Reply: Thank you for your careful revision. The current description is inaccurate. We have updated this section to: “ For each treatment, we randomly collected 0.5 m sections, totaling 66 subsamples, to obtain phenotypic measurements. Edge lines were not considered.”

Reply: Following careful consideration of your comments above, we have removed all “yield per area” components.

Reply: When initially calculated, we first used only the biomass from non-edge lines. We have removed yield per area from the analysis.

Reply: The former imputation approach was removed from the manuscript because it was used to fill missing biomass data to estimate yield per area.

Reply: Agree. We attempted to fill gaps utilizing an imputation function to account for missing data. However, the results could have misrepresented the actual yield per area.

Reply: We have removed any mention of yield per area.