Kelps may compensate for low nitrate availability by using regenerated forms of nitrogen, including urea and ammonium

Nitrate, the form of nitrogen often associated with kelp growth, is typically low in summer during periods of high macroalgal growth. More ephemeral, regenerated forms of nitrogen, such as ammonium and urea, are much less studied as sources of nitrogen for kelps, despite the relatively high concentrations of regenerated nitrogen found in the Southern California Bight, where kelps are common. To assess how nitrogen uptake by kelps varies by species and nitrogen form in southern California, USA, we measured uptake rates of nitrate, ammonium, and urea by Macrocystis pyrifera and Eisenia arborea individuals from four regions characterized by differences in nitrogen availability—Orange County, San Pedro, eastern Santa Catalina Island, and western Santa Catalina Island—during the summers of 2021 and 2022. Seawater samples collected at each location showed that overall nitrogen availability was low, but ammonium and urea were often more abundant than nitrate. We also quantified the internal %nitrogen of each kelp blade collected, which was positively associated with ambient environmental nitrogen concentrations at the time of collection. We observed that both kelp species readily took up nitrate, ammonium, and urea, with M. pyrifera taking up nitrate and ammonium more efficiently than E. arborea. Urea uptake efficiency for both species increased as internal percent nitrogen decreased. Our results indicate that lesser‐studied, more ephemeral forms of nitrogen can readily be taken up by these kelps, with possible upregulation of urea uptake as nitrogen availability declines.


INTRODUCTION
Kelps provide vital ecosystem services globally, including enhancing primary production (Mann, 1973), increasing biodiversity (Graham, 2004), regulating water flow and coastal erosion (Gaylord et al., 2012), and supporting fisheries (Bertocci et al., 2015).As the climate changes, kelp populations are facing multiple stressors, resulting in declines around the world (Krumhansl et al., 2016;Smale et al., 2019).In southern California, USA, kelp populations have experienced long-term declines (Tegner et al., 1996), with recent studies highlighting associated substantial structural and functional changes in kelp forest communities (Beas-Luna et al., 2020).Amid declining kelp populations and subsequent shifts in community dynamics, understanding factors that influence kelp growth and productivity is essential for managing and restoring these habitats.
Nutrient supply is a major factor affecting algal growth and productivity, and nitrogen (N) is the most common limiting nutrient affecting growth in coastal habitats (Elser et al., 2007).Work on nutrients and kelp growth has historically focused on nitrate (Gerard, 1982b;Sánchez-Barredo et al., 2011;Wheeler & North, 1981), which is brought into shallow coastal waters through upwelling and seasonal overturn, but evidence is emerging of the importance of other, regenerated forms of nitrogen, including ammonium and urea, as nitrogen sources for kelp.Ammonium can be readily taken up and assimilated by Macrocystis pyrifera (Haines & Wheeler, 1978) and can sustain M. pyrifera growth when nitrate concentrations are low (Brzezinski et al., 2013).Research also indicates that M. pyrifera can take up urea during periods of low nitrogen availability (Smith et al., 2018(Smith et al., , 2021)).The use of urea as a source of nitrogen for macroalgae is of particular interest due to its year-round availability associated with local-scale inputs from marine consumers (Regnault, 1987) and coastal nitrogen loading (Kudela et al., 2008).Furthermore, urea provides two atoms of N per molecule, although the energetic cost of uptake and assimilation in macroalgae may be higher than for inorganic N forms (Phillips & Hurd, 2004).
In southern California, nitrate concentrations are low during summer months when upwelling is infrequent, but Macrocystis pyrifera maintains growth, with natural population growth rates saturating at relatively low nitrate concentrations (Wheeler & North, 1981;Zimmerman & Kremer, 1984).Although nitrate availability in southern California varies seasonally, oceanographic time series have shown that nitrate concentrations have significantly declined over multiple years while ammonium concentrations have increased (Martiny et al., 2016).With chronically low nitrate concentrations in the summer and declining nitrate concentrations in coastal waters overall, there remains a clear gap in our understanding of how kelps use alternative forms of nitrogen and of the potential for those forms to mitigate stress associated with low nitrogen availability.Previous studies have focused on nitrate uptake and assimilation, assuming that concentrations of ammonium and other regenerated forms of nitrogen were too low and/or ephemeral to substantially affect kelp growth in southern California (Wheeler & North, 1981).However, in addition to relatively high concentrations of ammonium and organic nitrogen associated with wastewater outfalls and terrestrial runoff along southern California's highly urbanized coastline (Howard et al., 2014), high concentrations of these alternative, regenerated nitrogen forms can occur due to pulses from consumer waste, sediment efflux, and riverine input in close proximity to kelp beds (Bray et al., 1986(Bray et al., , 1988;;Mayer et al., 1998).Additionally, N can quickly cycle among these different forms in coastal waters due to microbial activity (Hutchins & Capone, 2022;Jetten, 2008;Klawonn et al., 2019), suggesting that standing concentrations may underestimate the importance of ammonium and organic nitrogen.There is a clear need to understand the ability of macroalgae to use not only nitrate but also alternative forms of nitrogen such as ammonium and urea.Here, we fill this knowledge gap by evaluating uptake of nitrate, ammonium, and urea by two kelp species: Macrocystis pyrifera and Eisenia arborea.
Macrocystis pyrifera and Eisenia arborea are southern California kelp species that frequently occur along the same shorelines but occupy separate niches.Eisenia arborea inhabits the low intertidal and shallow subtidal zones, whereas M. pyrifera is located at greater depths, those of up to 25 m (Graham et al., 2010).Although nutrient uptake, storage, and limitation in M. pyrifera have been investigated for decades, E. aborea's nutrient dynamics remain understudied, despite its ability to survive in warm, nutrient-poor waters (Hernández-Carmona et al., 2001;Matson & Edwards, 2006), conditions that can induce stress in other kelp species.Although E. arborea populations persist in stressful conditions, studies of its nitrogen use and storage have focused on nitrate (Sánchez-Barredo et al., 2011), and E. arborea's ability to take up and assimilate regenerated forms of nitrogen remains unknown.It has been hypothesized that M. pyrifera can tolerate nutrientpoor surface waters in part because individuals grow from the bottom to the surface, potentially spanning the thermocline/nutricline, although nutrient input from episodic thermocline motion has not been shown to sustain maximal growth (Gerard, 1982b;Zimmerman & Kremer, 1984).In contrast, E. arborea does not have access to a vertical gradient in nutrient availability, as it lives in well-mixed shallow waters.Given E. arborea's proximity to the shore, it could also encounter pulses of regenerated nitrogen forms (i.e., ammonium, urea) associated with freshwater runoff (Kudela et al., 2008), consumer waste (Aquilino et al., 2009), and increased microbial activity (Pfister et al., 2014).
In this study, we sought to understand the importance of regenerated nitrogen for Macrocystis pryifera and Eisenia arborea in southern California during low-nutrient conditions by measuring the uptake rates of different nitrogen forms.Specifically, we aimed to answer the following questions: (1) Does uptake efficiency of nitrate, ammonium, and urea by M. pyrifera and E. arborea change with environmental N availability?(2) What forms of N are contributing the most to the nitrogen taken up by each species across various sites in southern California?We predicted that uptake efficiency of regenerated forms of N would increase as N availability declined.We predicted that this pattern would be especially apparent for urea, as it may be more energetically costly to assimilate.In the San Pedro and east Catalina sampling sites, M. pyrifera and E. arborea did not coexist during sampling events and were therefore collected from separate sites as close as possible to each other (i.e., ~2 km) within the region (Figure 1).We collected seawater samples and blades from the target species of kelp twice from every site, yielding a total of four sampling events per region, with the exception of White Point in the San Pedro region, which could only be sampled once due to permit constraints, and Shaw's Cove in Orange County, which had one extra sampling event for methodological comparisons (Table S1 in the Supporting Information).At each sampling event, we collected six blades from four individuals of the target species of kelp.Blades collected from M. pyrifera were randomly selected from the top 3 m of adult plants.Blades were cut from the stipe, stored in individual mesh bags, and placed into a cooler with seawater from the collection site for transport to the laboratory.Each blade was then tagged with an individual blade ID and weighed before being placed into a flowing seawater system to recover from wounding for at least 24 h.Blades were kept in flowing seawater prior to use in nitrogen (N) uptake trials and subjected to 12:12 h light:dark cycles using two Luxx Clone LED Lights (Hawthorne Gardening, Vancouver, Washington, USA) in Catalina trials and four T5 10,000 K high-output fluorescent lamps in mainland trials.Blades were kept in laboratory seawater systems for no longer than 60 h prior to uptake trials.Laboratory tanks were maintained at 16°C and a salinity of 30-35 to reflect typical conditions in local waters.After 24-60 h of recovery in laboratory seawater systems, each blade was removed and transferred to an individual chamber to experimentally measure the nitrogen uptake rate of that blade.After use in a singular trial, each blade was frozen and then dried for internal N analysis as described below.

Ambient nitrogen (N) availability and internal N content
To quantify ambient environmental N availability, 500 mL of seawater were collected in triplicate at each collection event (n = 4 per region, Table S1), kept on ice for transport, and filtered using GF/F filters within 2 h of collection.Filtered seawater samples were aliquoted into 50-mL tubes and frozen for later nitrate (NO 3 − ), ammonium (NH 4 + ), and urea quantification.Nitrate concentrations were quantified with a QuickChem FIA 8500 autoanalyzer (Lachat Instruments, Loveland, Colorado, USA; detection limit: 0.014 μmol • L −1 NO 3 − ).Ammonium and urea concentrations were quantified spectrophotometrically using methods adapted from Solórzano (1969)  To quantify internal N concentrations, all kelp blades were immediately frozen after uptake trials were completed.Blades were then dried in an oven at 65°C to constant mass, ground to a fine powder using a mixer mill, and analyzed for %N (Thermo Flash 2000 Elemental Analyzer, CE Elantech, Inc., Lakewood, New Jersey, USA).

Nitrogen (N) uptake experiments
To measure the form-specific N uptake rates of Macrocystis pyrifera and Eisenia arborea in different regions of southern California, N uptake was measured at four different initial concentrations of either nitrate, ammonium, or urea.Nitrogen uptake of kelps was measured over 1 h by placing blades into eight chambers containing artificial seawater with target concentrations of 2, 10, 20, and 30 μM of the nitrogen form, adapting methods from Bracken et al. (2011) and Benes and Bracken (2016).Two blades from each kelp individual were used in each trial.During the trials, high water flow was maintained by using magnetic stir bars and stir plates, which provided sufficient velocities to maximize uptake (Hurd et al., 1996).Chambers were kept at ~15°C by placing them in a circulating chilled water bath.All trials took place outside in natural sunlight (>1000 μmol photons • m −2 • s −1 ).In 2021, 1-L chambers were used, but these were replaced with 3-L chambers in 2022 to increase the volume and amount of N in chambers.There were no differences in uptake efficiency associated with chamber volume based on uptake trials performed using blades collected from the same individuals at Shaw's Cove (linear model: initial concentration x method interaction: p > 0.05 in all cases; Figure S1 in the Supporting Information; see analytical details below).Kelp blades were rinsed with deionized water to remove epiphytes and residual N and placed into individual chambers.The target form of N was then added to each chamber to achieve the desired initial N concentrations.After a 1-min mixing period, water (10 mL for ammonium and nitrate; 35 mL for urea) was collected from each chamber every 15 min for 2 h (n = 9 samples per chamber) in 2021 and 1 h (n = 5 samples per chamber) for 2022.The duration of the trials was shortened after 2021 because our data from those trials revealed that uptake remained relatively linear for 60 min, allowing accurate calculation of uptake rates over that time interval.Therefore, no time points after 60 min were used in further analyses.Water samples taken from chambers during uptake experiments on Santa Catalina Island were immediately frozen for later analysis, while experimental samples from mainland trials were immediately analyzed due to laboratory proximity.Nitrogen concentrations of each experimental sample were quantified using the methods described above for environmental N concentrations.

Data analyses
Micromolar urea concentrations were multiplied by 2 to calculate the nitrogen (N) concentration from urea, as urea contains two amino groups (CO[NH 2 ] 2 ).To determine if some forms of N contributed more than others to the total observed N availability, the percentage of the total environmental N from ammonium, urea, and nitrate was calculated for each sampling event to assess whether N availability varied by form and site, alone and interactively.Differences in environmental N concentrations between sites and regions were assessed with one-way ANOVAs examining N concentrations and [N from urea] from each site.To assess if internal %N was associated with environmental nitrogen concentrations, separate linear models were fit on a loglog scale for each form of N independently, as well as for total environmental N.These analyses evaluated whether environmental N concentrations were associated with the average internal %N for each kelp from a collection event.As we had greater sampling depth for blade internal %N, internal %N was used as a proxy for each blade's recent N history in subsequent models.
The rate of mass-specific rate of N uptake for each chamber was calculated using the following equation: The relationship between dry-mass-specific N uptake rate (μmol N • h −1 • g −1 ) and initial concentration (μM N) was fit to a linear model to assess the efficiency of uptake.Uptake model fits (linear models versus Michaelis-Menten models) were compared using data from all sites for each kelp species and N form as well as within each site to account for variation by site.Sitespecific models showed significant linear relationships between mass-specific uptake rates and initial N concentrations (p < 0.05), whereas no Michaelis-Menten models were significant.Models using all data from each kelp species and N form significantly fit linear models, but few significantly fit only V max parameters with no significant K of saturating kinetics (Methods S1 in the Supporting Information).Overall, linear uptake models provided better fits for these relationships compared with traditional Michaelis-Menten models of uptake kinetics in every case, as saturating rates of N uptake were not reached despite experimental concentrations of N far exceeding environmental concentrations.
To assess whether kelp species and nitrogen availability at the time of collection were related to the efficiency of N uptake (slope of the relationship between initial N concentrations and N uptake rates), general linear models were fit examining the main and interactive effects of initial N concentration, species, and internal %N on N uptake rate for each form of N studied.Kelp species and %N were added into the linear models sequentially and tested against the simple linear regression between uptake rate and initial N concentration to determine the most parsimonious model for each form of N. Percent nitrogen was treated as a continuous variable in the models but was visualized as a factor with the levels high, medium, and low %N.High, medium, and low %N groups were calculated by quartiles, with low being the bottom quartile of %N values, medium being 25%-27% of %N values, and high being the top quartile of %N values in the dataset.To test if experimental N addition significantly altered %N, a linear model was used to test the interactive effects of N form and experimental initial N concentration on %N.There was no significant effect of experimental N concentration on %N for any form of N (linear model, F 2,376 = 0.705, p = 0.505).Differences between methods used in 2021 and 2022 were investigated within each species using a similar model structure looking for significant effects of method on the slope between N uptake rates and initial N concentration.To compare expected rates of uptake for each form of N, theoretical site-and species-specific uptake rates for each N form were calculated by inputting each observed environmental N concentration into the associated site and species-specific linear uptake model.These expected uptake rates for each form of N were then pooled to assess what form of N contributed the most to overall N uptake.To assess if one form contributed more to the overall N uptake of the kelps studied, we ran a two-way ANOVA, testing if the calculated uptake rates varied by N form and kelp species.

RESULTS
There were significant positive linear relationships between N uptake rate and initial N concentration for all forms of nitrogen studied (linear regression, p < 0.001, R 2 > 0.15; Table S2 in the Supporting Information), and both Macrocystis pyrifera and Eisenia arborea took up all forms of N studied.The most parsimonious model examining N uptake from urea as a function of initial N concentration included the interactive effect of internal %N (linear model, F 1,124 = 7.04, p < 0.001), but not kelp species.Urea uptake efficiency (i.e., the slope of the relationship between initial concentration and uptake) increased as total internal percent N decreased (linear model, F 1,124 = 7.04, p = 0.008, Figure 2).This pattern was consistent overall (i.e., there was no "species × initial concentration" interaction), but it was primarily associated with M. pyrifera rather than E. arborea based on within-species patterns.There was no difference in the efficiency of urea uptake between M. pyrifera and E. arborea.Neither ammonium nor nitrate uptake efficiency varied significantly with blade internal %N.However, ammonium uptake efficiency differed between kelp species (linear model, F 1,126 = 21.10,p < 0.001, Figure 3), with M. pyrifera exhibiting more efficient uptake.Similarly, nitrate uptake by M. pyrifera was more efficient than uptake by E. arborea (linear model, F 1,120 = 217.08,p < 0.001, Figure 3).There was a significant positive, saturating (linear on a log-log plot) relationship between internal %N and total environmental N concentrations (linear model, F 1,14 = 8.137, p = 0.013, R 2 = 0.322, Figure S2 in the Supporting Information).We observed positive, saturating relationships between %N and concentrations of urea (linear model, F 1,14 = 14.48, p = 0.002, R 2 = 0.475) and ammonium (linear model, F 1,14 = 6.096, p = 0.027, R 2 = 0.254), but Total environmental nitrogen concentrations-the sum of nitrogen from nitrate, ammonium, and urea in seawater collections-at collection sites averaged 5.14 ± 0.702 μM N and ranged from 1.14 to 12.64 μM N. Overall, ammonium contributed more to the total observed N availability than urea and nitrate, regardless of site (linear model, F 2,27 = 5.27, p = 0.012, Figure 1b).
There was a significant difference in total environmental N concentration between regions (ANOVA, i 3,11 = 3.74, p = 0.045), driven by higher total N concentrations in Orange County (7.63 ± 1.69 μM N, n = 4) and San Pedro (6.35 ± 0.503 μM N, n = 3) compared to east (3.54 ± 0.826 μM N, n = 4) and west Catalina Island (1.58 ± 0.789 μM N).When environmental N concentrations were examined within each N form, we determined significant differences between regions in urea concentrations (ANOVA, F 3,11 = 3.69, p = 0.047), with San Pedro (2.53 ± 0.788 μM N from urea, n = 3) having higher urea concentrations than west Catalina (0.859 ± 0.216 μM N from urea, n = 4).Although there was a trend of higher ammonium concentrations on the mainland, these differences were statistically insignificant (p > 0.05).There were also no significant differences between nitrate concentrations of our study regions (mean = 1.15 ± 0.318 NO 3 − , n = 15) with all samples being less than 2 μM NO 3 − except one sample from Orange County with 4.95 μM NO 3 − .Based on the calculated rate of uptake by kelps at each site associated with observed environmental N concentrations, ammonium was taken up more than nitrate and urea (ANOVA, F 2,18 = 5.58, p = 0.013, Figure 4), with no significant differences between kelp species.East and west Catalina Macrocystis pyrifera and east Catalina Eisenia arborea took up urea more than nitrate, whereas kelps from other sites took up more nitrate than urea, though the differences in the calculated uptake rates of N from urea and nitrate were frequently very small (Figure 4).

D ISCUSSION
We had predicted that kelps' use of regenerated forms of nitrogen (N)-ammonium and urea-would increase as N availability declined.This prediction was supported, as urea uptake efficiency increased as internal nitrogen content decreased for both species of kelp, with no differences in uptake between species.This apparent upregulation of N uptake with decreasing N availability was not seen for ammonium or nitrate, but Macrocystis pyrifera took up both ammonium and nitrate more efficiently than Eisenia arborea, a pattern not seen in urea trials.Based on calculated uptake rates and observed environmental N concentrations, regenerated forms of N, like ammonium and urea, contribute substantially to the N taken up by kelps in southern California during seasonally low-N conditions.Moreover, our results suggest that these kelps increase urea uptake as overall N concentrations decline.
Decreased N availability, as approximated by kelp blade internal percent N, was associated with increased efficiency of urea uptake, indicating possible upregulation of urea uptake as N stress increases.Although the presence and concentration of one form of N is known to impact the uptake of another N form, this had primarily been shown for nitrate and ammonium F I G U R E 3 Efficiency of (a) ammonium, (b) urea, and (c) nitrate by the kelps Macrocystis pyrifera and Eisenia arborea.Significant linear relationships between nitrogen uptake rate and initial nitrogen concentration for each species illustrate the efficiency of uptake for each nitrogen form.P-values indicate whether nitrogen uptake efficiency differed between M. pyrifera and E. arborea; "g" is grams dry mass.(Rees et 2007), and these patterns are not universal, particularly in kelps (Ahn et al. 1998).However, Phillips and Hurd (2004) showed that ammonium and nitrate were preferred to urea in intertidal seaweeds.Similarly, increased ammonium availability was related to decreased urea uptake in green and red macroalgae (Ross et al., 2018;Tyler et al., 2005), but the role of urea and the regulation of its uptake and breakdown have been better studied in phytoplankton and bacteria than in macroalgal lineages.Eukaryotic phytoplankton, particularly diatoms, reared in low-N conditions can exhibit reduced urea uptake rates when ammonium and nitrate are more available (Lomas, 2004;Lund, 1987;Solomon et al., 2010).The impacts of ammonium presence and concentration on urea uptake across various algal lineages highlight the potential of ammonium availability as an environmental cue that regulates urea uptake.We used internal percent N as a measure of recent nitrogen history but also saw a significant positive relationship between percent N and water-column ammonium concentrations at the time of collection, highlighting the potential for reduction of urea uptake efficiency associated with higher water-column ammonium concentrations.
Although potential mechanisms of urea uptake regulation remain speculative in macroalgae, the main mechanism of urea uptake regulation in phytoplankton is modification of transporter activity, during which ammonium represses urea active transport (Berg et al., 2008;Solomon et al., 2010).There is also evidence that seaweed-associated bacteria contribute to urea use in macroalgae, with antibiotic-treated algae exhibiting substantially reduced urea-degrading enzyme activity (Bekheet & Syrett, 1977).The metagenomes of kelp-associated bacteria contain urease and other hydrolase metabolisms (Miranda et al., 2022), and host-associated bacteria have shown to increase ammonium availability to kelp by cleaving carbonnitrogen bonds (Hochroth & Pfister, 2024).The observed reduction in the uptake efficiency of urea with increasing nitrogen availability may be associated not only with regulation of uptake by the kelp but also by kelp-associated microbes.Although much of the classic macroalgal nutrient dynamic literature has neglected the contribution of kelp-associated microbes to kelps' physiological processes, recognition of the importance of macroalgal microbiomes in a variety of functions is growing (Egan et al., 2013;Miranda et al., 2022;Tarquinio et al., 2018).Specifically, bacterial ureases on kelp blades could have generated ammonium in this study, altering the concentration of both urea and ammonium.Because the natural microbiome was left intact on our experimental blades, the differences we documented in N uptake may be attributable to site differences at the holobiont level.Although metagenomic studies have indicated the functional capacity of macroalgal microbiomes in N cycling, experimental decoupling of the microbiome and the kelp is still needed to fully understand the role of the microbiome in kelps' N uptake.
Macroalgal internal N content is primarily associated with stored N reserves and amino acids and varies with nutrient conditions recently experienced by the algae (Lyngby, 1990;Wheeler & North, 1981).In Macrocystis pyrifera, %N declined when individuals were exposed to low-N environments, as they depleted N reserves to maintain growth.The internal N concentrations seen in those low-N individuals were similar to those observed in our study (Gerard, 1982a).Likewise, the %N values we measured in field-collected Eisenia arborea were consistent with those experimentally starved nitrate (Sánchez-Barredo et al., 2011).Although we saw significant positive relationships between ambient N availability and internal %N, blades from certain locations exhibited higher %N than expected given the low N concentrations observed, particularly M. pyrifera collected from east Catalina Island (Figure S2e).Blades present near the top of a mature frond are actively growing and may therefore have increased %N values due to increased protein synthesis (Gerard, 1982a).As the blades used in this study were collected from this portion of the thallus, it is possible that a recent pulse of N availability led to an increase in N reserves and growth rates, leading to increased internal %N.However, these blades also exhibited some of the highest rates of urea uptake, supporting the idea that mechanisms of urea uptake regulation are more likely associated with ambient ammonium concentrations than internal N content.
There were also differences between the ammonium and nitrate uptake rates of Macrocystis pyrifera and Eisenia arborea, with M. pyrifera exhibiting more efficient uptake of these forms.Morphologically, M. pyrfiera blades are thinner compared to the thick, leathery blades of E. arborea, likely related to increased wave exposure on the shallow, subtidal, moderately waveexposed shores where E. arborea occurs (Roberson & Coyer, 2004).This difference in blade morphology likely translates to higher surface-area-to-volume ratios in M. pyrifera blades, enhancing uptake rates.
Although urea uptake rates were relatively high, observed environmental concentrations of urea may limit the uptake of urea at our study locations, particularly in comparison to ammonium along the California coast.Ammonium contributed the most to our estimates of total N uptake, especially along the mainland; urea contributed relatively similar amounts of N to ammonium at Catalina sites.However, regenerated forms of nitrogen (ammonium and urea) have increased relative to nitrate along the highly urbanized southern California coast (Howard et al., 2014;Martiny et al., 2016), and kelp populations exposed to more freshwater runoff may experience even higher urea concentrations.Although these regenerated N forms are associated with anthropogenic sources, they are also produced by consumers within kelp beds (Bray et al., 1986(Bray et al., , 1988)).By providing habitat for consumers, canopy-forming kelps may enhance N regeneration by animals, facilitating kelp growth during periods of low nitrate availability.The importance of ammonium is also likely underestimated, as ammonium fluctuates rapidly in coastal waters because it is generated and used rapidly (Klawonn et al., 2019).
We have shown that regenerated forms of N contribute to the N taken up by kelps during seasonally low-N conditions, with urea contributing relatively more to total N uptake up at more isolated sites further from densely populated areas.As kelp populations continue to face multiple stressors, including decreased nutrient concentrations and increased ocean stratification, understanding nutrient uptake dynamics becomes increasingly important for the protection and management of these populations.As coastal nitrate concentrations decline and anthropogenic N inputs-including regenerated forms such as ammonium and urea-increase, elucidating the use of these N forms, not only by kelps but also by their associated microbiomes and the phytoplankton assemblages within kelp beds, will give us a better understanding of how these communities function from a microscopic-to-macroscopic scale.
and Goeyens et al. (1998), respectively.F I G U R E 1 Sampling sites and the average available nitrogen available at each site.(a) Location of sampling sites in southern California for two species of kelp, organized into four regions.The inset in the map shows the study location in the context of the broader region.(b) Average total nitrogen concentration of three forms of nitrogen collected from each sampling site.Sites are abbreviated as follows: Little Harbor (LH), Big Fisherman's Cove (BF), Bird Rock (BR), Shaw's Cove (SC), Point Fermin (PF), White Point (WP).[Color figure can be viewed at wileyonlinelibrary.com]

F
Relationships between internal %nitrogen and efficiency of nitrogen uptake for (a) ammonium, (b) urea, and (c) nitrate.Significant linear relationships between nitrogen uptake rate and initial nitrogen concentration for high, medium, or low values of %N illustrate the efficiency of uptake for each form of nitrogen.Pvalues indicate whether N uptake was related to internal %nitrogen for each form of nitrogen; "g" is grams dry mass.[Color figure can be viewed at wileyonlinelibrary.com] not concentrations of nitrate (linear model, F 1,14 = 0.22, p = 0.642).

F
I G U R E 4 Calculated rates of nitrogen uptake.(a) Expected nitrogen uptake rates based on average environmental nitrogen concentrations and uptake equations for each species, region, and site: Little Harbor (LH), Big Fisherman's Cove (BF), Bird Rock (BR), Shaw's Cove (SC), Point Fermin (PF), White Point (WP).(b) Overall average expected nitrogen uptake rates.[Color figure can be viewed at wileyonlinelibrary.com]