Nutrient and arsenic biogeochemistry of Sargassum in the western Atlantic

Domain: earth_science
Published: 2023-10-05
Editorial outcome: accept

Abstract
The oceanographic ecology of pelagic Sargassum, and the means by which these floating macroalgae thrive in the nutrient-poor waters of the open ocean, have been studied for decades. Beginning in 2011, the Great Atlantic Sargassum Belt (GASB) emerged, with Sargassum proliferating in the tropical Atlantic and Caribbean where it had not previously been abundant. Here we show that the nutritional status of Sargassum in the GASB is distinct, with higher nitrogen and phosphorus content than populations residing in its Sargasso Sea habitat. Moreover, we find that variations in arsenic content of Sargassum reflect phosphorus limitation, following a hyperbolic relationship predicted from Michaelis-Menten nutrient uptake kinetics. Although the sources of nutrients fueling the GASB are not yet clear, our results suggest that nitrogen and phosphorus content of Sargassum, together with its isotopic composition, can be used to identify those sources, whether they be atmospheric, oceanic, or riverine in origin.

Introduction
The floating macroalgae Sargassum spp. serves as a habitat for more than 200 types of organisms, including 10 endemic species1–3, and provides habitat for fish nursery, spawning, and foraging4,5. Neonate and juvenile sea turtles use Sargassum habitat for feeding as well as protection from predators6,7, and several species of seabirds forage over Sargassum8,9. In contrast to these traditionally beneficial ecological impacts, the Great Atlantic Sargassum Belt10 (GASB) has caused unprecedented inundations of Sargassum on Caribbean and Florida coastlines with deleterious effects on near-shore seagrass and coral reef ecosystems11–14, and declines in turtle hatchling survival15. The GASB has also presented challenges to regional economies, particularly those that rely heavily on tourism16,17. Sargassum decay on coastal margins can cause respiratory and other human health issues18, and the presence of arsenic in Sargassum tissue19–21 puts significant constraints on the utilization of the biomass that washes ashore22–24.

A complex set of interconnected hypotheses has been offered to explain the dynamics of the GASB10,25,26. Anomalous wind patterns in 2009-2010 are thought to have introduced Sargassum from its habitat in the Sargasso Sea into the eastern North Atlantic where it was subsequently entrained into the equatorial current system27. Since that time, Sargassum has increased dramatically in the tropical Atlantic, begging the question of nutrient supply. A variety of nutrient sources have been suggested10,27–29, including upwelling, vertical mixing, discharge from the Amazon and Congo rivers, and atmospheric deposition. However, the causes of the GASB and the mechanisms controlling its seasonal to interannual variability remain unknown. An underlying question is: is this massive abundance of Sargassum a result of higher nutrient availability in the GASB?

In this work we show that GASB Sargassum populations are enriched in both nitrogen and phosphorus content relative to its Sargasso Sea habitat, clearly identifying nutrient supply as a primary driver of this phenomenon. In addition, we demonstrate that Sargassum’s arsenic content varies with the degree of phosphorus limitation, linking our observations with a theoretical prediction based on nutrient uptake kinetics. Our results show that ascertaining the nutrient sources and their regulation is essential to understand the underlying causes of this basin-scale phenomenon—and only then will society have a conceptual basis on which to design potential strategies to mitigate the consequences of the GASB.

Results
Sargassum distributions and nutritional status
In order to address this question, Sargassum tissue samples were collected in spring 2021 along with hydrographic and nutrient measurements on two hydrographic sections in the western Atlantic (Methods) that intersected the western portion of the GASB (Fig. 1). The samples were separated into two species S. fluitans and S. natans based on their morphology30. S. fluitans and S. natans co-occurred in the southern portions of both A20 and A22. S. natans was more prevalent north of 24°N on A22, and north of 27°N on A20. Both species were present at 32°N on A20 as well as the farthest north sample, collected just south of the Gulf Stream on A22.

Sargassum elemental composition was similar between species (Fig. 2, Supplementary Table 1), a finding that is consistent with earlier studies29. Our data reveal three distinct regimes within the sampling domain. In the Sargasso Sea, carbon content tends to be relatively high (particularly in the eastern transect A20), whereas nitrogen and phosphorus content tend to be low. Nutritional status in the GASB was much better, with the nitrogen and phosphorus content of Sargassum in the Caribbean and western tropical Atlantic significantly higher than in the Sargasso Sea (Supplementary Fig. 1, Supplementary Table 2). Interestingly, nitrogen and phosphorus content were highest in the northern Sargasso Sea.

These trends are also evident in elemental ratios (Supplementary Fig. 2). As expected, C:N was high in the Sargasso Sea, driven by higher carbon content and lower nitrogen content (Fig. 2). These ratios are higher in A20 than in A22, particularly in S. natans. This zonal gradient with C:N increasing to the east is driven by both increasing carbon content and decreasing nitrogen content. C:P is also high in the Sargasso Sea, although the zonal gradient evident in C:N is not clear in C:P. N:P shows a pronounced maximum in the Sargasso Sea region of A22, which is driven by a combination of increased N content and decreased P content. Little meridional trend in N:P is evident in A20, and in aggregate N:P in the Sargasso Sea differs less from the surrounding regions than does C:N and C:P (Supplementary Fig. 1).

Based on the seasonal analysis conducted on prior datasets29, the springtime samples described herein would be expected to be at their seasonal maxima of nitrogen and phosphorus content, and minima of their C:N and C:P ratios.

Nutrient sources
Because Sargassum is subject to both surface currents and wind, hydrodynamic transport plays a key role in its dynamics31–33. In order to assess the recent history of our Sargassum samples, passive particles were inserted into a numerical ocean model hindcast at each collection site and tracked backward in time (Fig. 3; see Methods). The southernmost sample of A20 appears to have come from the southeast, whereas samples in the 10–30°N latitude band come primarily from the east. Exceptions to that pattern include an eddy-like flows from 25°N to 28°N on A22. Samples collected north of 30°N on A20 appear to have come from the north, consistent with a Gulf Stream origin. The northernmost sample on A22 was clearly under the influence of the Gulf Stream, although its point of entry into the Gulf Stream varied widely within the ensemble—ranging from the continental shelf north of Cape Hatteras to the South Atlantic Bight, with some in the core of the Gulf Stream as far south as 30°N.

This information, together with δ15N values of the Sargassum tissue, facilitates some inferences about nitrogen sources. Specifically, Sargassum utilizing riverine nitrogen tend to have enriched (positive) δ15N values, whereas those fueled by near-surface oceanic and atmospheric sources have lower (negative to slightly positive) δ15N values29. Nitrogen fixation typically results in a fractionation of −2‰34. Patterns in δ15N from our survey reveal spatial coherence, with no systematic difference between S. fluitans and S. natans (Fig. 2). Depleted δ15N values in the Sargasso Sea, Caribbean, and western tropical Atlantic could result from nitrogen fixation by epiphytic cyanobacteria35,36 or perhaps atmospheric deposition37. The highest δ15N values occurred in the northern Sargasso Sea, with values of approximately +2‰ potentially reflecting riverine sources along the U.S. east coast (as indicated by spatial connectivity depicted in Fig. 3), or as distant as the Gulf of Mexico which has been identified as a source region for Sargassum in the western Atlantic38. For example, a coherent plume emanating from the Mississippi River in the summer of 2004 was entrained into the Loop Current, reaching the Straits of Florida in approximately 25 days, continuing northward in the Gulf Stream to be found in the South Atlantic Bight another 25 days after that39. Our northern Sargasso Sea samples were collected another 600 nm farther downstream, so assuming a flow of 4 kt, they could have been influenced by Mississippi River water discharged as recently as 150 days before sampling. With end-member δ15N values of +6–8‰ in the northern Gulf of Mexico Sargassum29, dilution to +2‰ would require several doublings of the population using oceanic sources of nitrogen—which is certainly possible based on observed growth rates36,40,41. Alternatively, enrichment of δ15N in samples from the northern Sargasso Sea could be caused by upwelling and/or vertical mixing, as values of +2‰ are characteristic of nitrate in the upper thermocline in that region42,43.

Enriched δ15N values were also found in the southernmost samples of the western tropical Atlantic (Fig. 2), under the direct influence of the Amazon River plume (as evidenced by the salinity distribution in Fig. 1) which has been implicated as a nutrient source for the GASB10,27–29,44. There was one station in the Caribbean interior (17°N, 67°W) where similarly high δ15N values were measured, but the corresponding salinity (Fig. 1) does not bespeak riverine influence. Particle backtracking calculations suggest source waters to the east-southeast (Fig. 3), and given the cross-isohaline transport enabled by wind drag, Amazon River influence is plausible. However, it is noteworthy that neither the elemental composition (Fig. 2) nor nutrient ratios (Supplementary Fig. 2) in the high-δ15N samples of the tropical Atlantic and Caribbean Seas show corresponding anomalies.

Hydrographic data from sections A20 and A22 provide environmental information with which to interpret the Sargassum observations. The nitracline and phosphocline are deepest in the warm and salty waters of the Sargasso Sea, and shallower in the warmer and fresher waters of the Caribbean and western tropical Atlantic (Supplementary Fig. 3). Both nutriclines are shallower in the colder and fresher waters of the northern Sargasso Sea. Near-surface nutrient concentrations are generally very low except for the northernmost portions of each transect where the nutriclines outcrop. Nitrate and phosphate are at or below the limit of detection in the surface waters of the Sargasso Sea (Supplementary Fig. 4). Nitrate is enhanced in the Caribbean, and less so in the western tropical Atlantic, where surface phosphate is elevated. The presence of detectable nitrate and phosphate in these areas is consistent with the enhanced nutritional status of Sargassum in the GASB.

Arsenic biogeochemistry
The fact that Sargassum bioaccumulates arsenic has generated considerable interest, particularly because the associated toxicity puts practical limitations on the valorization of the biomass inundating coastal communities of the GASB. Our survey revealed regional scale variations in arsenic content (Fig. 2). Arsenic content was lowest in the GASB (Caribbean and Western Tropical Atlantic samples) and highest in the Sargasso Sea, particularly in the eastern transect A20. Other stations in A20 show modest enrichment, such as in the southernmost samples at 9°N, 52°W and the Northern Sargasso Sea samples at 31–33°N, 52°W. There are no systematic differences in arsenic content between S. fluitans and S. natans (Supplementary Table 1), although interspecies variability is apparent at some stations (Fig. 2).

The high arsenic content of Sargassum in the subtropical gyre is accompanied by the lowest phosphorus content, which is consistent with the depression of the phosphocline (Supplementary Fig. 3) and surface dissolved phosphate concentrations generally below the limit of detection (Supplementary Fig. 4). Thus, the arsenic to phosphorus ratio of Sargassum of the subtropical gyre stands out as uniquely high in that region (Fig. 2), where Sargassum is known to be phosphorus limited45.

Uptake of arsenate (AsO43−) by aquatic plants occurs as a byproduct of phosphorus uptake, owing to its chemical similarity with the phosphate ion (PO43−)46,47. Using the standard Michaelis-Menten form, the uptake of dissolved phosphate and arsenate can be expressed as:

ρP = μP [PO43−] / (kP + [PO43−]) and ρAs = μAs [AsO43−] / (kAs + [AsO43−]) (1)

For low concentrations of phosphate and arsenate, uptake is approximately linear in concentration:

ρP ≈ μP [PO43−] / kP and ρAs ≈ μAs [AsO43−] / kAs (2)

The ratio of As to P uptake is therefore:

ρAs / ρP = (μAs / μP) * (kP / kAs) * ([AsO43−] / [PO43−]) (3)

Surface arsenate concentrations in the tropical and subtropical Atlantic are relatively uniform in comparison with phosphate, and up to an order of magnitude lower48. Moreover, surface concentrations of arsenate and phosphate are uncorrelated49, so the ratio of As to P uptake can be simplified to:

ρAs / ρP ∼ 1 / [PO43−] (4)

Assuming that the tissue content of these two constituents reflects their proportionate uptake, one would expect that the ratio of arsenic to phosphorus in Sargassum would be a hyperbolic function of phosphorus content:

%As / %P ∼ 1 / %P (5)

This prediction is qualitatively consistent with the present data as well as prior observations dating back to the 1980s (Fig. 4), although the dependence on phosphorus content varies as %P−1.3 rather than %P−1.0 predicted by the theory. The reason for this supra-hyperbolic dependence is not known, but it is statistically reliable with a p-value < 0.001 (Supplementary Note). This is a clear demonstration of arsenic content as a diagnostic of phosphorus limitation in a natural population of marine algae and is consistent with laboratory studies50,51, biogeochemical proxies based on dissolved As speciation in the ocean49, and other recent field data52.

It could be argued that the As:P versus P relationship in Fig. 4 is a result of the intrinsic association among variables: for two random variables a and b, plotting the ratio a:b versus b will take on a hyperbolic form. However, the elemental composition of Sargassum is not random. For example, the As:C ratio does not show a hyperbolic dependence on carbon content (Supplementary Fig. 5). Interestingly, As:N varies hyperbolically with nitrogen content, which we attribute to covariation in nitrogen and phosphorus content. In contrast, As content is positively correlated with carbon content.

Notwithstanding our theoretical prediction of the hyperbolic relationship of As:P and P content, one might also expect a negative correlation between As and P content as a symptom of phosphorus stress in Sargassum. Observations are also consistent with this expectation (Supplementary Fig. 6). A similar negative correlation is observed between arsenic and nitrogen content, which we again attribute to the correlation between nitrogen and phosphorus content.

Methods
Sampling was conducted on R/V Thomas G. Thompson voyages TN389 (16 March–16 April 2021) and TN390 (20 April–16 May 2021), occupying GO-SHIP lines A2054 and A2255, respectively.

Hydrography
Hydrographic profiles and water samples were collected with a standard Conductivity, Temperature, Depth (CTD) rosette system with Niskin bottles. Nutrient analyses were carried out at sea using a Seal Analytical continuous-flow AutoAnalyzer 3, consistent with the methods described in the GO-SHIP repeat hydrography manual56.

Sargassum collection and identification
Sargassum spp. samples were collected with a dip net and sorted into the species and morphotypes, S. natans I and S. fluitans III per Parr30. Recent literature has indicated the increasing presence of a previously rare form S. natans VIII on the basis of both morphology26 and genetics57,58. Based on morphological similarity, any S. natans VIII in our samples would have been classified as S. fluitans III.

Sargassum elemental analysis and isotopic composition
Samples for each morphotype were separated into up to three replicates as quantities allowed (6 to 10 thalli/species), rinsed briefly (3 to 5 s) in deionized water, cleaned of macroscopic epizoa and epiphytes, dried in a laboratory oven at 65 to 70 °C for 48 h, and powdered with a mortar and pestle53. The dried Sargassum samples were split in half and stored in plastic screw top vials. One half was used for arsenic analysis (see below), and the other half was shipped to the University of Georgia’s Center for Applied Isotope Studies Stable Isotope Ecology Laboratory (UGA-SIEL; https://cais.uga.edu/facilities/stable-isotope-ecology-laboratory/) for analysis of δ15N as well as %C and %N on a Thermo Delta V IRMS coupled to a Carlo Erba NA 1500 CHN-Combustion Analyzer via a Thermo Conflo Interface. National Institute of Standards and Technology reference materials 8549, 8558, 8568, and 8569 were used to routinely calibrate working standards prepared in the laboratory. QA/QC results were incorporated into the raw data reports received by UGA-SIEL. The other part of this half sample was analyzed at UGA-SIEL for %P, where approximately 2 mg of dried tissue was weighed into crucibles, ashed at 500 °C for four hours, and extracted with 0.2 mL of Aqua Regia acid59,60. The acid extracts were then diluted 41:1 with deionized water for TP (as PO4-P) analysis on an Alpkem 300 series analyzer.

Sargassum arsenic content
Arsenic content of Sargassum tissue was measured by the University of Missouri Soil and Plant Testing Laboratory (MU SPTL) using an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES; Agilent 5800; λ = 188.980 nm). Subsamples of rinsed, dried, and powdered Sargassum tissue from the GO-SHIP A20/A22 expeditions were digested at MU SPTL in 2022 according to EPA method 3052. In brief, the powdered samples were digested with a combination of HNO3 acid at 175 °C for 15 min using a microwave-accelerated digestion system (CEM MARS Xpress). Samples collected prior to the 2021 surveys were retrieved from the B. Lapointe archives (1980–2018) at the Harbor Branch Oceanographic Institute, Florida Atlantic University. Prior to sending to the MU SPTL for ICP-OES analysis, this subset was digested at the National High Magnetic Field Laboratory at Florida State University (P. Morton) in 2020 following a two-step process. First, ~0.1 g aliquots of the powdered sample were carefully weighed into 15-mL Teflon digestion beakers (Savillex), to which 3 mL of concentrated HNO3 (Fisher Optima) were added. The beakers were tightly capped and left overnight (~12 h) on a hot plate at 150 °C. The beakers were then uncapped, and the digested sample was taken to dryness (150 °C, 2–4 h). The sample residue was then digested a second time (capped, 150 °C, overnight) using 3 mL concentrated HNO3 (Fisher Optima) and 200 µL of concentrated HF (Fisher Optima). The samples were taken to dryness again (uncapped, 150 °C), and the residue dissolved in 3.0 mL of 0.32 M HNO3 (Fisher Optima). All samples were analyzed at MU SPTL with ICAP-OES at a 1/100 dilution to bring the As concentrations into a working range of the matrix-matched (0.16 M HNO3) external standard calibration curve. Triplicate independent digestions and analyses of four Sargassum tissue samples were used to determine the representative reproducibility of the sample processing and instrumental analysis methods. For more details see https://extension.missouri.edu/programs/soil-and-plant-testing-laboratory/spl-researchers.

Backtracking of source waters
At each station where Sargassum was collected, the source waters were assessed by tracking particles backward in time for 60 days using a Lagrangian algorithm61. Surface currents were specified from the OSCAR 1/3° resolution analysis, described at https://www.esr.org/research/oscar/oscar-surface-currents/ and available at https://podaac.jpl.nasa.gov/dataset/OSCAR_L4_OC_third-deg. A total of 100 particles were released at each location where Sargassum was found, with random walk diffusion applied each time step Δt with gaussian perturbations σ defined by σ2 = 4DΔt. The horizontal diffusivity D was chosen to be 4000 m2 s−1 based on estimates for this region derived from Argo float observations62. In addition to being affected by surface currents, wind also influences Sargassum transport31,32. Windage factors ranging from 0.5% and 3% produce the most accurate simulations of Sargassum trajectories63,64, and a mid-point value of 2% was used here. Wind forcing was specified using daily NCEP/NCAR reanalysis65 and validated with shipboard meteorological measurements made on R/V Thomas G. Thompson during voyages TN389 and TN390. Trajectories for each virtual particle were plotted in Fig. 3 using R and The Source61. Transparency was used to visually indicate relative variance in particle trajectories, and the mean path for each station was overlaid. The script used to run the model and generate the plot is available at https://gist.github.com/tbrycekelly/830f5278269ba46df15f883c1c3cccc5.

Satellite observations
Pelagic Sargassum distributions (https://optics.marine.usf.edu/projects/saws.html) were derived from MODIS measurements using a floating algal index10,66,67. MODIS data were obtained from the U.S. National Aeronautics and Space Administration (NASA) Goddard Space Flight Center (https://oceancolor.gsfc.nasa.gov). Surface salinity distributions were obtained from SMOS Earth Explorer mission and the data were accessed from https://www.catds.fr/Products/Products-over-Ocean/CEC-Lops-SSS-SMOS-SMAP-OI-L4.

Data availability
The Sargassum tissue data generated in this study have been deposited in the Figshare database under accession code https://doi.org/10.6084/m9.figshare.2268450768. Shipboard hydrographic data: https://cchdo.ucsd.edu/cruise/325020210316 (TN38954). https://cchdo.ucsd.edu/cruise/325020210420 (TN39055). Surface currents used for particle tracking: https://podaac.jpl.nasa.gov/dataset/OSCAR_L4_OC_third-deg69. Satellite-based surface salinity: https://www.catds.fr/Products/Products-over-Ocean/CEC-Lops-SSS-SMOS-SMAP-OI-L470. Ocean color data for computation of satellite-based Sargassum distributions: https://oceancolor.gsfc.nasa.gov71.

Code availability
Particle tracking code: https://doi.org/10.5281/zenodo.346852461.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "McGillicuddy, Jr., D.J., P.L. Morton, R. A. Brewton, C. Hu, T. B. Kelly, A. R. Solow, B. E. Lapointe.\n2023. Nutrient and arsenic biogeochemistry of Sargassum in the western Atlantic.\nReview for Nature Communications\nGeneral\nThis paper could be recommended for publication if the nutrient fingerprinting could be extended\nmore definitively to the central Atlantic Sargassum. The abstract for this paper is excellent in\nterms of defining the status of the nutrient problem and intriguing for the promise of a defining\nfingerprint for source nutrients in the central Atlantic that support the excessive Sargassum growth\nthere. Data presented is used to point out differences in Sargassum nutrient content between the\nnorth Atlantic-Sargasso Sea region and the western central Atlantic. In the results section as\npointed out below, it is sometimes difficult for the reader to understand which data points are\nbeing referred to in survey transects A20. It would enhance the paper to make this clear. There is\na comparatively long explanation of how Arsenic and As:P indicates P limitation which may not be\nnecessary to the paper? It is disappointing that the Arsenic fingerprinting presented, applies\nmostly to the SS and does not apparently apply to the new GASB where the source of nutrients\nneeds to be defined. The authors suggests that a more comprehensive sampling of the entire\nbreadth of the GASB might provide such a fingerprint although not based on the Arsenic which the\npaper emphasizes. The scale of such an effort is daunting to the authors.\nSpecific\nP2 Line 83 omit beginning of sentence: North of 24oN, as redundant; is the first A22 supposed to\nbe A20 in this sentence?\nP4 line 139 one station…, Sentence 143 not sure what is meant by this sentence Or maybe I am\nlooking at the wrong point? Please designate location.\nP4 Line 164 Do not see Arsenic enrichment in southern most samples (or sample?) on A20? It may\nbe useful to show limit of subtropical gyre on figures.\nP4 Line 164-165 Arsenic in fluitans and natans appears somewhat different on A20 in the\nsubtropical gyre?\nP4 Line 172- 216 not sure what this material adds to the already stated P limitation in paragraph\nabove, lines 166-171?\nP5 line 201 arsenic content as a diagnostic of phosphorus limitation, what does this mean for\nnutrient sources with respect to the subtropical gyre? Deep nutri-cline, lack of Saharan dust or an\nisolated water mass??\nP6 Line 234 if …. There is an unimportant if and no useful fingerprint for the central Atlantic in the\nconclusion. Sargassum in the subtropical gyre and the SS has perhaps, always been high in\nArsenic and P limited (Fig 4) but biomass is and always has been low in the subtropical gyre and\nSS? So historically and in the future not much Sargassum with high Arsenic washes up on the\nbeaches. The Fig 2 data show a lack of evidence that Sargassum is P limited or Arsenic is high in\nthe GASB where Sargassum biomass is now very high?? So Arsenic fingerprinting is not an\nindicator for nutrient sources for the central Atlantic problem location.", "reviewer_2": "Key results\nThis study examines the carbon, nitrogen, and phosphorus content, N stable isotopic signatures,\nas well as arsenic content of Sargassum spp. along two latitudinal gradients from the Sargasso\nSea to the Caribbean and West Tropical Atlantic (WTA). The methods of using tissue stoichiometry\nand N isotopes to identify potential limitation and sources of nutrients are not new, however, the\nuse of the As/P method is more interesting and novel. Overall, the data from the intensive\nsampling of Sargassum along two latitudinal transects is quite compelling, as it shows the regional\ndifferences in nutrient limitation and sources of nutrients across these regions. These data suggest\nnutrient levels are higher in the Northern Sargasso Sea and the Caribbean and WTA, with higher\nnutrient limitation in the Sargasso Sea. The relationship between As/P and % P also strongly\nconfirms phosphorous limitation in certain regions. Although the d15N data do show variation\nacross regions, I find the linking of these differences to riverine sources only speculative. There is\nno evidence that the higher signatures are due to riverine sources and that they are reaching the\nopen ocean. Higher values could also be due to upwelling. Additionally the negative signatures in\nthe Sargasso Sea could be due to N-fixation.\nThe manuscript is generally well-written and easy to follow. The methods are well described and\nreferences relevant. Overall, I think this study is significant to the field to start to understand what\nnutrients are important to sustain the largest algal bloom worldwide.\nSpecific comments:\nLine 96-check reference to fig. 2. C:N data are not represented in this figure. Perhaps you mean\nExtended Data Fig. 1?\nLine 126-what does the riverine source have to do with Fig. 3? Please explain better.\nLine 132-136- This is highly speculative, although feasible, it seems like there could be other\nsources as well, such as upwelling.\nLine 138-I do not follow the connection with the Amazon River plume and Fig. 1.\nFig. 1- It is difficult to see velocity vectors in Fig 1 a. Also do you talk about them in the text?\nFig. 2 What do the 2000 and 3000 stand for in the graphs? Include in figure legend.\nFig. 3 please add A22 and A20 to the graph\nI would suggest reduction of figures in the Extended data section as a few are redundant. I would\nalso recommend more thorough descriptions of some of the figure legends. Are you displaying\nmeans +/- standard deviation or standard error?\nExtended Table 1-(mean, standard deviation?)\nExtended data Fig. 1- redudant to Table 1. I would use one or the other.", "reviewer_3": "Review of the article “Nutrient and arsenic biogeochemistry of Sargassum in the western Atlantic”.\nThis is a very interesting study that highly contributes to the biological and physiological\nknowledge of Sargassum. The inundations of Sargassum on Caribbean and Florida is causing\nseveral negative ecological, social and economic impacts, hence knowledge of the species biology\nis urgently needed. The authors make a great work collecting many biological samples along the\nspecies distribution, as well as seawater samples. The study showed that tissue samples from\nCaribbean waters showed greater nitrogen (N) and phosphate (P) concentrations than those from\nthe Sargasso Sea, which is positively correlated with nutrient seawater concentrations. These\nresults suggest that invasion of this species might be highly related with eutrophication. Moreover,\nand the most interested part of the study, is the relation found between P and Arsenic content.\nThey suggested that uptake of arsenic might occurred under P limitations. Higher arsenic content\nin the tissue make the species very toxic, and therefore is inedible. Overall the manuscript is very\nwell written, and very easy to follow. I surely suggest the paper acceptance after few minors’\nchanges.\nExtended figures:\nPlease check your axis; it will be better if you use the same amount of decimals in all your graphs.\nAlso, in the extended figure 4, you should keep the same maximum value in your Y axis, as you\nare comparing your stations results. This will highlight the differences between stations."}, "author_response": "Response to reviews\nWe are grateful for the constructive comments provided by the three referees. We have\nincorporated nearly all of the reviewers’ suggestions into the revised version. A detailed\nnarrative of our responses to each of their comments (in bold) follows. We note that additional\nchanges have been made to bring the manuscript into compliance with Nature Communications\nformatting policies. Specifically, we (1) shortened the abstract to 150 words and removed\nreferences therein; (2) added a final paragraph to the introduction containing a brief summary of\nthe major results and conclusions of the study; (3) moved the Methods and Data Availability\nsections to just after the Discussion; and (4) made a number of other miscellaneous changes in\naccordance with the guidelines. For convenience, we have provided both a “clean” and a change-\ntracked version of the revised manuscript.\nReviewer #1\nGeneral\nThis paper could be recommended for publication if the nutrient fingerprinting could be\nextended more definitively to the central Atlantic Sargassum. The abstract for this paper is\nexcellent in terms of defining the status of the nutrient problem and intriguing for the promise of\na defining fingerprint for source nutrients in the central Atlantic that support the excessive\nSargassum growth there. Data presented is used to point out differences in Sargassum nutrient\ncontent between the north Atlantic-Sargasso Sea region and the western central Atlantic. In the\nresults section as pointed out below, it is sometimes difficult for the reader to understand which\ndata points are being referred to in survey transects A20. It would enhance the paper to make this\nclear. There is a comparatively long explanation of how Arsenic and As:P indicates P limitation\nwhich may not be necessary to the paper? It is disappointing that the Arsenic fingerprinting\npresented, applies mostly to the SS and does not apparently apply to the new GASB where the\nsource of nutrients needs to be defined. The authors suggests that a more comprehensive\nsampling of the entire breadth of the GASB might provide such a fingerprint although not based\non the Arsenic which the paper emphasizes. The scale of such an effort is daunting to the\nauthors.\nWe thank the referee for this positive assessment and constructive suggestions. We have\nmodified the results section to include geographic coordinates of the specific stations that\nare called out in the text. We have clarified the fact that the fingerprinting idea pertains to\nnitrogen and phosphorus, not to arsenic—which is an indicator of phosphorus stress that\ndoes not depend on the source. We stand by the novelty of the arsenic biogeochemistry\npresented herein, consisting of a theoretical prediction tested with our observations—which\nhas important implications for management if the nutrient source(s) turn out to be\nanthropogenic. Details of the revisions are described below.\nSpecific\nP2 Line 83 omit beginning of sentence: North of 24oN, as redundant; is the first A22 supposed to\nbe A20 in this sentence?\nP4 line 139 one station…, Sentence 143 not sure what is meant by this sentence Or maybe I am\nlooking at the wrong point? Please designate location.\nStation position now indicated in the text: 17° N, 67° W.\nP4 Line 164 Do not see Arsenic enrichment in southern most samples (or sample?) on A20? It\nmay be useful to show limit of subtropical gyre on figures.\nWe have clarified that the lowest Arsenic concentrations are in the GASB (Caribbean and\nWestern Tropical Atlantic samples), and the modest enrichment on A20 to which we refer\nis the southernmost station at 9° N, 52°W. We have also noted modest enrichment of\nArsenic in the Northern Sargasso Sea samples on A20 (31-33° N, 52°W).\nP4 Line 164-165 Arsenic in fluitans and natans appears somewhat different on A20 in the\nsubtropical gyre?\nWe have rephrased the statement to acknowledge interspecies variability, and point to\nSupplementary Table 1 to support the statement that there are no systematic differences:\n“There are no systematic differences in arsenic content between S. fluitans and S. natans\n(Supplementary Table 1), although interspecies variability is apparent at some stations\n(Fig. 2).”\nP4 Line 172- 216 not sure what this material adds to the already stated P limitation in paragraph\nabove, lines 166-171?\nThis material develops a theoretical prediction for how the As:P ratio varies as a function\nof P content, which to our knowledge has not been described before. Moreover, we test the\ntheoretical prediction with our data in Figure 4. This is a truly novel aspect of the paper\nand thus have chosen to keep this material intact.\nnutrient sources with respect to the subtropical gyre? Deep nutri-cline, lack of Saharan dust or an\nisolated water mass??\nThis issue is discussed in the second paragraph of this section:\n“The high arsenic content of Sargassum in the subtropical gyre is accompanied by the\nlowest phosphorus content, which is consistent with the depression of the phosphocline\n(Supplementary Fig. 3) and surface dissolved phosphate concentrations generally below the\nlimit of detection (Supplementary Fig. 4). Thus, the arsenic to phosphorus ratio of\nSargassum of the subtropical gyre stands out as uniquely high in that region (Fig. 2), where\nSargassum is known to be phosphorus limited42.”\nthe conclusion. Sargassum in the subtropical gyre and the SS has perhaps, always been high in\nArsenic and P limited (Fig 4) but biomass is and always has been low in the subtropical gyre and\nSS? So historically and in the future not much Sargassum with high Arsenic washes up on the\nbeaches. The Fig 2 data show a lack of evidence that Sargassum is P limited or Arsenic is high in\nthe GASB where Sargassum biomass is now very high?? So Arsenic fingerprinting is not an\nindicator for nutrient sources for the central Atlantic problem location.\nWe agree with the referee that arsenic is not an indicator of nutrient source(s)—arsenic\ncontent is an indicator of phosphorus limitation, regardless of what the phosphorus\nsource(s) may be. The fingerprinting of the nutrient sources we suggest has to do with\nnitrogen and phosphorus, not arsenic. We have clarified in both the abstract and in the\nconclusions that fingerprinting pertains to nitrogen and phosphorus content.\nThe referee is correct that “Sargassum in the subtropical gyre and the SS has perhaps,\nalways been high in Arsenic and P limited (Fig 4) but biomass is and always has been low\nin the subtropical gyre and SS…” As for phosphorus limitation of Sargasso Sea\npopulations, the evidence for that is presented in the second paragraph of the section on\nArsenic Biogeochemistry (see above) which references both the present data\n(Supplementary Figs. 3 and 4) as well as a prior study [ref. 42, Lapointe (1986)].\nThe referee is also correct that arsenic is relatively low in the GASB where biomass is high,\nand we have emphasized that point on the new line 161: “Arsenic content was lowest in the\nGASB (Caribbean and Western Tropical Atlantic samples) and highest in the Sargasso\nSea, particularly in the eastern transect A20.”\nThe main point of the sentence starting on line 234 is that a reduction in phosphorus supply\nto the GASB (relative to nitrogen) could increase the arsenic content—which would have\nimportant implications for management purposes in case the nutrient sources turn out to\nbe anthropogenic. We have clarified the sentence accordingly and added prose with the\nadditional point concerning management.\nReviewer #2:\nKey results\nThis study examines the carbon, nitrogen, and phosphorus content, N stable isotopic signatures,\nas well as arsenic content of Sargassum spp. along two latitudinal gradients from the Sargasso\nSea to the Caribbean and West Tropical Atlantic (WTA). The methods of using tissue\nstoichiometry and N isotopes to identify potential limitation and sources of nutrients are not new,\nhowever, the use of the As/P method is more interesting and novel. Overall, the data from the\nintensive sampling of Sargassum along two latitudinal transects is quite compelling, as it shows\nthe regional differences in nutrient limitation and sources of nutrients across these regions. These\ndata suggest nutrient levels are higher in the Northern Sargasso Sea and the Caribbean and WTA,\nwith higher nutrient limitation in the Sargasso Sea. The relationship between As/P and % P also\nstrongly confirms phosphorous limitation in certain regions. Although the d15N data do show\nvariation across regions, I find the linking of these differences to riverine sources only\n4\nspeculative. There is no evidence that the higher signatures are due to riverine sources and that\nthey are reaching the open ocean. Higher values could also be due to upwelling. Additionally the\nnegative signatures in the Sargasso Sea could be due to N-fixation.\nThe manuscript is generally well-written and easy to follow. The methods are well described and\nreferences relevant. Overall, I think this study is significant to the field to start to understand\nwhat nutrients are important to sustain the largest algal bloom worldwide.\nWe thank the referee for this positive assessment. As per the referee’s wise advice, we have\nbolstered the discussion of \"15N and nitrogen sources to include upwelling and nitrogen\nfixation, adding relevant references.\nSpecific comments:\nLine 96-check reference to fig. 2. C:N data are not represented in this figure. Perhaps you mean\nExtended Data Fig. 1?\nThe sentence is correct as is. The C:N ratio is presented in Supplementary Fig. 2\nreferenced in the preceding sentence; the C and N content used to explain that are shown in\nFig. 2.\nLine 126-what does the riverine source have to do with Fig. 3? Please explain better.\nThe figure reference has been clarified: “as indicated by spatial connectivity depicted in\nFig. 3.”\nLine 132-136- This is highly speculative, although feasible, it seems like there could be other\nsources as well, such as upwelling.\nIndeed, we have added a sentence stating upwelling as another possible source:\n“Alternatively, enrichment of \"15N in samples from the northern Sargasso Sea could reflect\nupwelling and/or vertical mixing, as values of 2 ‰ are characteristic of nitrate in the upper\nthermocline in that region.”\nLine 138-I do not follow the connection with the Amazon River plume and Fig. 1.\nThe figure reference has been revised to “as evidenced by the salinity distribution in Fig.\n1.”\nFig. 1- It is difficult to see velocity vectors in Fig 1 a. Also do you talk about them in the text?\nThe vectors are now plotted in black instead of light blue to improve clarity. Yes, they are\nreferred to in the text (e.g., Gulf Stream, eddies, etc.).\nFig. 2 What do the 2000 and 3000 stand for in the graphs? Include in figure legend.\nFig. 2 and Fig. 1.\nFig. 3 please add A22 and A20 to the graph\nThis has been done.\nI would suggest reduction of figures in the Extended data section as a few are redundant. I would\nalso recommend more thorough descriptions of some of the figure legends. Are you displaying\nmeans +/- standard deviation or standard error?\nWe could not identify any redundancies other than those noted below. The +/- values are\n95% confidence intervals, and that information has been added to the relevant captions.\nAdditional information has been added to the figure captions, including identification of\nthe 2000 m and 3000 m isobaths in Supplementary Fig. 2.\nExtended Table 1-(mean, standard deviation?)\nThese are the means and 95% confidence intervals, and that information has been added to\nthe caption for Supplementary Table 1 and Supplementary Fig. 1.\nExtended data Fig. 1- redudant to Table 1. I would use one or the other.\nWhile there is some overlap in the information, Supplementary Fig. 1 actually has more\nquantities plotted than are reported in Supplementary Table 1, specifically As:C, As:N,\nand As:P. Moreover, we feel that both presentations are valuable, with the figure\nproviding graphical comparison among the quantities and the table providing the actual\nnumbers which are helpful for reference. For both of these reasons we have chosen to\nretain both the figure and the table.\nReviewer #3 (Remarks to the Author):\nReview of the article “Nutrient and arsenic biogeochemistry of Sargassum in the western\nAtlantic”. This is a very interesting study that highly contributes to the biological and\nphysiological knowledge of Sargassum. The inundations of Sargassum on Caribbean and Florida\nis causing several negative ecological, social and economic impacts, hence knowledge of the\nspecies biology is urgently needed. The authors make a great work collecting many biological\nsamples along the species distribution, as well as seawater samples. The study showed that tissue\nsamples from Caribbean waters showed greater nitrogen (N) and phosphate (P) concentrations\nthan those from the Sargasso Sea, which is positively correlated with nutrient seawater\nconcentrations. These results suggest that invasion of this species might be highly related with\neutrophication. Moreover, and the most interested part of the study, is the relation found between\nP and Arsenic content. They suggested that uptake of arsenic might occurred under P limitations.\nHigher arsenic content in the tissue make the species very toxic, and therefore is inedible.\nOverall the manuscript is very well written, and very easy to follow. I surely suggest the paper\nacceptance after few minors’ changes.\nWe thank the referee for this positive assessment.\nExtended figures:\nPlease check your axis; it will be better if you use the same amount of decimals in all your\ngraphs. Also, in the extended figure 4, you should keep the same maximum value in your Y axis,\nas you are comparing your stations results. This will highlight the differences between stations.\nAs per the referee’s suggestion, we have revised Supplementary Fig. 4 so that the y-axis\nlimits are the same in both columns.\nAs for the decimal places, it is not practical to use the same number in all cases given the\nwide variation in precision of the measurements, from arsenic content to temperature. In\nall cases we have chosen an appropriate number of significant figures to report, keeping\nthem consistent among the plots to facilitate comparison.\n6"}]

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