Water levels primarily drive variation in photosynthesis and nutrient use of scrub Red Mangroves in the southeastern Florida Everglades

We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 µmol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades.


INTRODUCTION
1 1 (Pulversette 0, Frtisch GmbH, Idar-Oberstein, Germany). Green and senescent leaf samples were stored in scintillation vials at room temperature and analyzed separately. Leaf TC and TN 3 2 1 content were determined with a Carlo-Erba NA-1500 elemental analyzer (Fisons Instruments   3  2  2 Inc., Danvers, MA, USA). TP was extracted using an acid-digest (HCl) extraction, and and 365.2, US EPA 1983). Leaf C and N bulk isotopic signatures (δ 13 C, δ 15 N) were analyzed on 3 2 5 a Thermo Scientific Delta V Plus CF-IRMS coupled to a Carlo-Erba 1108 elemental analyzer via 3 2 6 a ConFlo IV interface (Thermo Fisher Scientific, Waltham, MA, USA). All C and N analyses 3 2 7 were conducted at the Southeast Environmental Research Center Analysis Laboratory. Using leaf carbon isotope fractionation values, we calculated the concentration of 3 2 9 intracellular CO 2 and plant water use efficiency integrated over the lifespan of the leaf tissue of atmospheric CO 2 of 408 µmol mol -1 for our calculations, which is a conservative estimate for (408 × (1 -c i ÷ 408)) ÷ 1.6, where c i is the value derived from the previous equation. Additionally, the following equation was used to calculate the resorption of N and P using green  Repeated measures analysis of variance (ANOVA) was used to test for differences in 3 4 2 water level, surface water salinity, and porewater salinity among locations (fringe and interior), island habitats (center and edge), and season (wet and dry), as well as for the interaction 3 4 4 between these effects and season, which was used as the repeated measure. For the repeated 3 4 5 measures ANOVA, islands were nested within locations and treated as experimental units. All effects were considered fixed, except for when testing for significant differences in habitat, 3 4 7 which included location as a random effect to account for the nested structure of the sampling 3 4 8 scheme. One-way ANOVAs were used to test for differences in soil surface elevation among locations and habitats and their interaction. Two-way ANOVAs were carried out for all leaf were performed in R v3.5.1 (R Core Team 2018).

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We constructed linear mixed-effects models (with a Gaussian error distribution and included as fixed effects in the models to address questions 1 and 2, respectively, with water 3 5 8 levels and porewater salinity being also included as the continuous covariates to parse out their 3 5 9 marginal effects. We couple inference from these models to leaf nutrient analyses and our 3 6 0 measurements of the hydrological environment to inform about nutrient and water use of R. (wet and dry), habitat (center and edge), porewater salinity, and water level were considered, 3 6 5 including interaction terms for water level and porewater salinity with season. All models 3 6 6 considered random intercept terms for location (i.e., fringe vs. interior), islands, and islands 3 6 7 nested within location. Random slopes were explored but determined not to improve model fits. The best-fit models were determined via stepwise model comparison using AIC based on included a random intercept term for islands, which helped remove variability in the data 3 7 2 because of the sampling design. Random effects for location were insignificant, signifying that 3 7 3 most of the random variance in the gas exchange data was among islands, which we consider 3 7 4 as the experimental unit in all mixed-effects models. The mixed-effect models were fit using were evaluated using model predicting, tabling, and plotting functions from the sjPlot R package 3 7 7 (Lüdecke 2018). All analyses were complete in R v3.5.1 (R Core Team 2018).

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The linear mixed-effects model for A net included fixed effects for island habitat, porewater  Table S4). There was substantial variation in A net rates among leaves (σ 2 of about 6 µmol m -2 s -4 1 5 1 ), and the random variation among islands was about 0.02 µmol m -2 s -1 (see Table S4). All 1 5 about 27 µmol mol -1 greater (19 to 35 µmol mol -1 difference in 95% CI estimates, Figure 2). The 4 5 2 marginal effect of season alone was similar in magnitude to that of habitat; the wet season led 4 5 3 to a decrease in c i of 24 µmol mol -1 (14 to 34 µmol mol -1 difference 95% CI estimates) relative to 4 5 4 the dry season ( Figure 2, Table S6). Water levels, by themselves (again, the marginal effect), 4 5 5 led to a slight decrease in c i but had a positive interaction with season, indicating that the 4 5 6 relative decrease in c i due to increasing water levels was suppressed during the wet season c i , about 9% of which was explained by data from the hydrological environment (Table S6).

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Lastly, we modeled wue using a similar mixed-effects model to that of g sw . In the model the dry season (F 1,12 = 18.88, p < .01, Table 3, Figure 5), with no statistical difference between with c i being about 10 µmol mol -1 greater in island edge habitats relative to their centers ( between season and island habitat (F 1,12 = 0.58, p > .05, Table 3, Figure 5A). Our first research question asked how mangrove-island habitat affects rates of leaf gas 5 1 8 exchange. We can confirm our hypothesis that photosynthetic rates and stomatal conductances 5 1 9 are greater at island centers than edges ( Figure 2). However, contrary to our expectation, 5 2 0 habitat driven-variation in leaf gas exchange rates was roughly equal to seasonal variation, with 5 2 1 no apparent decoupling between A net and g sw (Figure 2). Our second research question asked 5 2 2 whether water levels or salinity exerted a more-substantial effect on mangrove leaf gas affected (i.e., salinities > 35 ppt) and did not vary considerably over time ( Figures S1 & S2).

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Therefore, we conclude that inundation stress is the primary driver of variation in R. mangle leaf 5 2 7 gas exchange rates. Lastly, we predicted that general physiological stress would be lower at 5 2 8 island centers than island edge habitats, leading to increased wue and higher rates of nutrient 5 2 9 resorption at centers relative to edges. Indeed, intrinsic water-use efficiency was greater at 5 3 0 island centers than edge habitats, with results being consistent across gas exchange-measured 5 3 1 wue and isotope-derived WUE. In addition, water levels modulated leaf intrinsic water use habitats, which likely drive variation in leaf gas exchange rates.

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The effect of mangrove-island habitat on leaf gas exchange 5 3 7 Our results showed significant differences in soil elevation of about 30 cm between 5 3 8 mangrove-island habitats (Table 1), which affected R. mangle leaf gas exchange rates (Figures   5  3  9 2, 3, 4). The soil elevation gradient at our study site is driven by differences in mangrove root 5 4 0 biomass and productivity between center and edge island habitats, with higher total root mangrove-island centers than edges, and g sw was >0.1 mol m -2 s -1 (or >37%) higher; these 5 4 5 differences were attributable to mangrove-island habitat alone, after accounting for variation supporting the understanding that R. mangle leaves limit g sw in response to flooding. Limits on 6 8 4 g sw seek to optimize c i for carbon gain without losing unnecessary amounts of water, but our 6 8 5 findings show that g sw can increase with freshwater inputs, resulting in a decrease in c i as A net information Figure S9). Interestingly, the scrub R. mangle leaves of TS/Ph-7 operate with low c i 6 8 8 concentrations (range = 220-260 µmol mol -1 ), which suggests pervasive inundation stress. Such pervasive inundation stress likely leads to water and nutrient (i.e., leaf N and Rubisco)nitrification, denitrification) nutrient cycling processes (Garten 1993). In our study site, patterns including mesophyll and lower level (i.e., cell wall, plasma membrane, cytosol) conductance.

0 1
Additionally, differences in nutrient acquisition and use patterns among scrub R. mangle trees 8 0 2 growing at island edges vs. centers affect leaf-nutrient status and photosynthetic potential. The findings from this study indicate that it is the interaction of inundation stress with Florida Everglades that principally alters tree water and nutrient-use dynamics, which appear to 8 0 6 cascade to affect leaf gas exchange rates through their effects on g sw . Reductions in A net 8 0 7 interact with the salinity of the water that inundates scrub R. mangle trees, in theory, because 8 0 8 g sw rates are low and primarily respond to water loss from leaves rather than carbon gain (see supplemental section on R. mangle CO 2 assimilation and stomatal behavior). In our field 8 1 0 measurements, however, we found that prolonged inundation more than porewater salinity level, such physiological differences in scrub mangrove functioning with habitat and hydrological