Stable Carbon and Oxygen Isotopes Indicate Photophysiology and Calcification Mechanisms of Macroalgae on Little Cayman Reefs

doi:10.21203/rs.3.rs-2743528/v1

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Stable Carbon and Oxygen Isotopes Indicate Photophysiology and Calcification Mechanisms of Macroalgae on Little Cayman Reefs

https://doi.org/10.21203/rs.3.rs-2743528/v1

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Tropical macroalgae serve importance ecological roles on coral reefs but can supplant corals in phase shifts as shown worldwide. Thus, it is important to understand their fundamental photophysiology and calcification mechanisms. We examined organic d¹³C, organic+inorganic d¹⁵N, inorganic CaCO₃ d¹³C and d¹⁸O, and organic %C, %N, C:N of abundant macroalgae on Little Cayman Island (LCI) reefs across irradiance gradients (5-2,000 mmol m^-2 s^-1). We hypothesized macroalgae utilize greater CO₂ in low light as carbon concentration mechanisms (CCMs) for HCO₃^- uptake have higher energetic costs. We proposed that calcification mechanisms are influenced by light and Ci-use strategy in addition to taxonomy. LCI macroalgae had organic d¹³C values (-12‰ to -25‰) indicative of CCMs and exhibited only 1-3‰ ¹³C depletion in irradiances <5 mmol photons m^-2 s^-1. Ochrophyte d¹³C values were enriched in ¹³C at low nitrogen indicative of efficient CCMs. Macroalgae exhibited four clusters of thalli CaCO₃ d¹³C that were distinctly taxon-specific. Rhodophytes exhibited the most negative inorganic d¹³C and d¹⁸O values (-4.1‰ ± 0.7‰; -3.7‰ ± 0.4‰) an indication of high calcifying space pH and rapid calcification, potentially reliant on active ion transport. Relatively higher organic C fractionation when utilizing greater CO₂ for photosynthesis was reflected in depleted thalli carbonates in species/genus with positive d¹³C values. Increasing ocean CO₂ is not likely to enhance dominant LCI macroalgal photosynthesis as these reef macroalgae utilize CCMs across a broad range of irradiances; however, effects on calcification may be taxon specific based on the degree of biotic control elucidated in thalli carbonate isotopes.

reef

macroalgae

light

taxonomy

Rhodophyta

Chlorophyta

Ochrophyta

Tropical macroalgae serve importance functional roles on coral reefs (Fong & Paul, 2011) including as primary producers with 3-D structures which support a high diversity and abundance of fish and invertebrates (Wilson et al., 2022, Fulton et al., 2020, Fulton et al., 2019). In addition, they serve as autochthonous sources of inorganic carbonate sands from which coral reefs accrete (Castro-Sanguino et al., 2020, Littler & Littler, 1984) and as a substrate for larval settlement, particularly crustose coralline algae (Chisholm, 2000). Specific algal species provide chemical cues along with their microbiome promoting settlement and metamorphosis of coral planulae (Gomez-Lemos et al., 2018, Whitman et al., 2020). In addition to providing critical positive functions on tropical reefs, reef macroalgae have been observed to supplant corals in persistent phase shifts from coral-dominated to algal-dominated reefs worldwide (McManus & Polsenberg, 2004, Hughes et al., 2007).

On tropical reefs, herbivory can be important in controlling macroalgal taxonomic assemblages, algal functional forms and biomass leading to high abundance of macroalgae that are either chemically or structurally defended against herbivores and grazed down algal turfs (Hay, 1997, Spadaro & Butler, 2021). However, the ability of herbivores to affect macroalgae can be influenced by geographic variation in species susceptibility to grazing (Mantyka & Bellwood, 2007), nutrient enrichment conditions and other interacting factors (Adam et al., 2015). In the Caribbean region, ochrophytes with high chemical diversity and expression, such as those in the genus Lobophora and Dictyota (Hay, 1988), tend to dominate (Dell et al., 2020, Vieira et al., 2020). Proliferation of some species of the Lobophora genus have been identified as reef macroalgae of concern and referred to as a "Lobophora-syndrome" when the alga becomes epizoic causing coral-algal phase shifts (Vieira, 2019). While Lobophora-coral overgrowth can occur on undisturbed reefs (Vieira et al., 2015), rapid Lobophora expansion primarily occurs at sites with declining coral health and other disturbances (Vieira, 2019). Another dominant group of Caribbean reef macroalgae partially resistant to high grazing pressure are chlorophytes in the Halimeda genus. This group is primarily defended by their CaCO₃ thalli structure making up >95% of their weight. Halimeda also produce chemicals to resist predation and compete with corals and other benthic invertebrates for space (Longo & Hay, 2014). Weakened corals or coral mortality creates space that provides opportunities for reef macroalgae to expand on individual reefs, increase their broader distribution, and occupy space by growing epizotically on corals. As climate change enhances ocean temperature, creating more intense heat waves, and lowers ocean pH, it is projected that corals will become further stressed allowing for opportunistic expansion of macroalgae on reefs (Knowlton & Jackson, 2008, Hoegh-Guldberg et al., 2017). Thus, it is important to understand the basic photophysiology and calcification mechanisms of abundant tropical reef macroalgae and to further our knowledge of how these groups respond to global change (Koch et al., 2013).

As a lower pH ocean shifts the carbonate equilibria toward greater CO₂ availability, macroalgae have the potential to modify their inorganic carbon source (Ci) for photosynthesis due to the dominance of a C3-type photosynthetic pathway with only a few C4-like exceptions (Raven & Hurd, 2012, Koch et al., 2013, Maberly et al., 1992). However, marine macroalgae are recognized to efficiently utilize bicarbonate (HCO₃^-) for photosynthesis by employing carbon concentrating mechanisms (CCMs) (Koch et al., 2013) similar to other marine autotrophs, even those such as seagrasses which exhibit Ci limitation (Beer & Koch, 1996, Maberly et al., 2022). The evolutionary advantage for marine autotrophs to utilize HCO₃^- for photosynthesis is the high concentration of HCO₃^- in seawater accounting for ~90% of the total dissolved inorganic carbon (DIC; ~2,000 mmol kg^-1) pool in contrast to CO₂ which represents <1% of the total DIC (Dickson, 2010). Further, CO₂ has a low diffusive flux rate in seawater (10⁴ slower) compared to air and HCO₃^- dehydration rates to CO₂ without biotic enzymatic enhancement is slow (Israel & Hophy, 2002). CCMs that allow marine macroalgae to utilize HCO₃^- will likely still be favored under elevated atmospheric pCO₂ (~1,000 matm) predicted by 2100 due to HCO₃^- remaining the dominant Ci species (>90%) of total DIC (~2,200 mmol kg^-1) in seawater (Hartin et al., 2016), supported by the conservative CCM trait evolutionarily across marine macroalgae phyla (Stepien et al., 2016). Regardless of CCMs, higher seawater pCO₂ is likely to increase CO₂ as an Ci source for macroalgae as observed across latitudinal and coastal gradients of dissolved CO₂ (Lovelock et al., 2020, Stepien, 2015, Hofmann & Heesch, 2018). In some environments, CO₂-use can be favored as the use of CCMs requires light energy (Raven et al., 2014) to promote enzymatic dehydration of HCO₃^- to CO₂ and/or operate active HCO₃^-/H⁺ transmembrane transport systems. Light was also found to promote facultative HCO₃^- use in tropical macroalgae and enhance maximum photosynthetic rates at saturating irradiances in some species (Zweng et al., 2018). Due to energy requirements, macroalgae that reside in deep reef environments in both temperate and tropical settings have been found to utilize greater amounts of CO₂ compared to species in high irradiance environments (Cornwall et al., 2015, Diaz-Pulido et al., 2016); however, this pattern has not been examined on Caribbean reefs.

Little Cayman Island (LCI) in the western Caribbean, a low-lying carbonate platform with narrow shelves that descend to several thousand-meter basins, possess reefs across a range of irradiances and are some of the most pristine reefs throughout the Caribbean which have recovered from Caribbean-wide bleaching events due to low anthropogenic stress (Manfrino et al., 2013). Little Cayman Island reefs provide a study site to examine reef macroalgae photophysiology across a light gradient in a low-nutrient environment not impacted by high densities of coastal human populations (~200 residents), and within protected areas that support herbivorous fish that consume and partially constrain macroalgae including Lobophora (Dell et al., 2020). Reefs on LCI include a fringing barrier reef that transitions to a fore reef and deeper mesophotic reef wall. The deeper reefs also possess dark crevices within their 3-D structure where macroalgae grow at extremely low irradiance (< 5 mmol photons m^-2 s^-1). Since macroalgae of concern and ecological importance, such as Lobophora and Halimeda, respectively, span irradiance gradients on LCI by several orders of magnitude, identifying their Ci use mechanisms can provide information on the role of irradiance driving shifts in photophysiology and how Ci use might shift under greater CO₂ availability in abundant Caribbean tropical reef macroalgae. To discriminate Ci-use across genus/species, we examined d¹³C stable isotope values in the organic fraction of macroalgal thalli classifying those with values > -11‰ as obligate HCO₃^- users, < -30‰ as obligate CO₂ users and between -11‰ and -30 as facultative HCO₃^-/CO₂ users (Maberly et al., 1992) with some taxonomic and physiological discrimination complexities recognized particularly for species with d¹³C values < -30‰ (Raven et al., 2002). Isolated eukaryote Rubisco discriminates against ¹³C (~30‰), while CCMs minimize this discrimination, thus d¹³C is used as an indicator of the presence of a CCM (Giordano et al., 2005). We also determined the C:N ratios and ¹⁵N values of the thalli to determine any relationship between Ci-use and nitrogen concentration and fractionation, respectively. Macroalgae along the mesophotic wall edge may have unique ¹⁵N values compared to individuals growing in shallow barrier reef sites and potentially affected by local nutrient sources (Duarte et al., 2018, Umezawa et al., 2002, Dailer et al., 2010, Lapointe et al., 2005).

While many studies on macroalgal organic C isotopes relate values to photphysiology and mechanisms of Ci use, fewer studies include inorganic C isotopes (Lee & Carpenter, 2001) even though many tropical species thalli are > 95% inorganic C as CaCO₃. For tropical reefs where calcifying species are abundant, examining the proportion of Ci used for photosynthesis versus calcification can increase our mechanistic understanding of these metabolic processes and potential coupling. While most of the calcifying macroalgae are associated with chlorophytes and rhodophytes, we examined inorganic d¹³C values across all phyla as ochrophyte calcifiers were present (e.g., Padina and Lobophora calcified ruffled form). The d¹⁸O of thalli CaCO₃ was also determined to compare precipitation environments and biotic effects on LCI reef macroalgae to the few studies where tropical d¹³C and d¹⁸O have been determined (Aharon, 1991, Lee & Carpenter, 2001, Keith & Weber, 1965, Stokes et al., 2020, Wefer & Berger, 1981) and calcification models of coral that have been more extensively studied (Chen et al., 2018). Inorganic d¹³C and d¹⁸O values of macroalgal thalli have shown that CaCO₃ precipitation primarily occurs in disequilibrium (Lee & Carpenter, 2001, McConnaughey, 1989). The distance of the d¹³C value from calcification equilibrium has been used to classify calcification mechanisms in marine biogenic carbonates (Chen et al., 2018, McConnaughey, 1989), including metabolic influences (e.g., photosynthesis and respiration). It has also been shown that d¹³C and d¹⁸O values typically exhibit linear covariance and divergence can be indicative of disparate calcification mechanisms.

Herein we establish reef macroalgae organic d¹³C values and thalli d¹³C and d¹⁸O values of macroalgal carbonates from samples collected across a range of irradiances. We hypothesized that under ledges, in crevices and low-light environments on LCI macroalgae utilize greater amounts of CO₂ due to the higher energetic costs of CCMs. We also propose that macroalgal calcification mechanisms are influenced by light availability in addition to taxonomic affinity, particularly in species' dependent on photosynthesis for calcification and utilize Ci-use strategies that depend on high irradiance.

Study Site and Collections

Little Cayman Island (12 x 2 km) is the smallest of three Cayman Islands characterized by low relief and narrow shoals comprised of a fringing barrier reef possessing small lagoons shoreward of the back reef in some locations (Fig. 1a). Abundant macroalgae from three phyla (Ochrophyta, Chlorophyta, Rhodophyta) were collected (July 8-11, 2018) at five reef sites around LCI (Fig. 1a). These sites included: (Fig.1a: 1) a shallow fringing reef and at ~2 m depth (19^o41^'46^" N, 80^o03^'39^"W), (Fig.1a: 2,3) two intermediate depth fore reef sites at ~15 m (19^o40^'17^" N, 80^o06^'12^"W; 19^o39^'18^" N, 80^o06^'13^"W), and (Fig.1a: 4,5) two deeper reefs at the reef wall edge at ~25-30 m (19^o42^'09^" N, 80^o03^'25^"W; 19^o39^'54^" N, 80^o06^'40^"W). These sites and ledges/crevices on the fore (Fig. 1b,c) and wall reefs (Fig. 1. d,e) provided a broad range of irradiances to examine macroalgae thalli stable isotopes and C:N ratios. The macroalgae were randomly collected by 2-3 divers each assigned to a specific light condition associated with open or more cryptic locations depending on the site. Algae were placed into dive bags and transported to the boat where they were kept separated in marked containers in the dark. Following collections, all macroalgae were sorted by site and light environment and identified to genus or species (Littler & Littler, 2000); Lobophora was not identified as L. variegata because genetic analyses suggest greater variability in Caribbean species than previously considered (Vieira et al., 2020). Four replicates of each species, light, and site combination were used for the study. All macroalgae were carefully rinsed in filtered seawater, epiphytes gently removed, rinsed with deionized water and dried at 60°C on LCI. Once macroalgae were dried, they were kept under low humidity with desiccant to be processed at the FAU lab (Boca Raton, FL USA).

During collections, site irradiance (mmol photons m^-2 s^-1) was measured and recorded every minute using a spherical quantum sensor (LI-193) connected to an underwater recorder (RBRsolo3 PAR). Two sensors were deployed at each site, one to measure the benthic surface irradiance (Fig. 1c insert) and the other to place on the edge or within low-irradiance crevices (Fig. 1e insert). Five irradiance levels were recorded amongst sites categorized as: high, intermediate, low, lowlow, and dark that spanned 3 orders of magnitude of irradiance (Table 1).

Processing Samples and Isotope Analyses

Macroalgal samples were ground to a fine powder with a porcelain mortar and pestle in liquid N₂, transferred to Whirl-pak bags and kept in a self-cycling desiccator at low humidity. Thalli were processed to isolate organic and inorganic tissue, thus each individual sample had organic and inorganic fractions that were analyzed. To remove inorganic components of the thalli for organic fraction analyses, a subsample was acidified using a low normality acid (1N HCl) until no effervescence was observed and no carbonate fraction was detected; after which, samples were rinsed twice and dried at 60 ^oC. Samples were also rerun unacidified to establish ¹⁵N values to avoid modification of isotopes with acidification (Strait & Spalding, 2021). To isolate the inorganic thalli fraction, a 0.1 or 0.05 g subsample of the original ground sample was oxidized with hypochlorite (10%). Samples were agitated and left uncovered for 15 min before tubes were capped and tubes transferred to a platform shaker for 4 hr. Subsequently, samples were allowed to sit overnight before they were centrifuged and decanted. Samples were washed three times and subsequently dried at 60 ^oC.

Macroalgal thalli stable isotopes (d¹³C) and total C, N (%) of organic tissue and d¹⁵N of organic+inorganic tissue were analyzed using a Thermo Electron DeltaV Advantage isotope ratio mass spectrometer coupled with a ConFlo II interface linked to a Carlo Erba NA 1500 CNHS Elemental Analyzer (Light Stable Isotope Mass Spectrometry Laboratory, University of Florida). Samples were loaded into tin capsules and placed in a 50-position automated Zero Blank sample carousel on a Carlo Erba NA1500 CNS elemental analyzer. After combustion in a quartz column at 1000 ^oC in an O₂-rich atmosphere, the sample gas was transported in a He carrier stream and passed through a hot reduction column (650 ^oC) consisting of elemental copper to remove O₂. The effluent stream then passed through a chemical (magnesium perchlorate) trap to remove water followed by a 0.7 m GC column at 125 ^oC to separate N₂ from CO₂. The sample gas next passed into a ConFlo II interface and into the inlet of a Thermo Electron Delta V Advantage isotope ratio mass spectrometer running in continuous flow mode where the sample gas was measured relative to laboratory reference N₂ and CO₂ gases. All organic carbon isotopic results are expressed in standard delta notation relative to VPDB. All nitrogen isotopic results are expressed in standard delta notation relative to AIR.

Carbon and oxygen isotopes of thalli carbonates (d¹³C, d¹⁸O) were analyzed using a Kiel III carbonate preparation device coupled with a Finnigan-Mat 252 isotope ratio mass spectrometer. Each batch of samples was measured with eight NBS-19 and two NBS-18 standards. Standard masses were 30 to 50 mg. Samples and standards were loaded in individual glass Kiel vials and placed in a carousel which was inserted in the Kiel oven. Phosphoric acid with a specific gravity of 1.92 (at 25 ^oC) was used. Reaction temperature was 70 ^oC and reaction time was 10 min. All inorganic isotopic results are expressed in standard delta notation relative to VPDB.

Water Column Sampling and Nutrient Analyses

Water column nutrient samples were collected at each site (n = 3) approximately 1-2 m above the reef surface at the end of macroalgal collections. Bottles (60 mL) were rinsed 3 times underwater by using compressed air to empty bottles before final collection. Samples were immediately transported to the lab, filtered (0.45 mm) and kept frozen during travel to the FAU-HBOI lab. Dissolved nutrient samples were analyzed with a SEAL Analytical AA3 Autoanalyzer to determine concentrations of nitrate + nitrite (NO₃^- + NO₂^- ; hereafter NO_x) and soluble reactive phosphorus (SRP). Ammonium (NH₄⁺) was measured with a Turner Designs Aquafluor hand-held fluorometer equipped with mini cuvettes. Method detection limits (MDLs) were 0.070 μM for NH₄⁺, 0.015 μM for NO_x, and 0.009 μM for SRP. NH₄⁺ and NO_x results were combined to determine total dissolved inorganic nitrogen (DIN) concentrations and inorganic N:P ratios.

Statistical Analyses

Significant differences among phyla and site for tissue d¹³C, d¹⁵N isotopic values, %N, %C, N:P ratios, and inorganic nutrients among sites were examined using analysis of variance (ANOVA) following tests for normality (Shapiro-Wilks test) and equal variance (Brown-Forsythe test); regression analysis was used to quantify linear trends (SigmaPlot 15.0, Systat Software, Inc.). ANOVA was used to examine light and phyla effects on d¹³C and d¹⁸O of thalli carbonates. If unequal variance was determined, the ANOVA was conducted on ranked data (Kruskal-Wallis test). Differences among means were established using the Holms-Sidak and Dunn's multiple comparison tests. All significant differences were defined at the P < 0.05 level. The k-means cluster analysis approach was used to establish clusters of d¹³C and d¹⁸O values of thalli carbonates for each species by partitioning x,y data around four centroids using Euclidean distances until convergence (Zaiontz, 2022).

Reef Site Characteristics

Water column nutrient levels were relatively low at reef sites on LCI with total DIN < 1 mM, SRP < 0.3 mM and N:P ratios 3.6 on average (Table 1). No significant differences were found for SRP or N:P among sites. The highest DIN concentration was measured in the water column of the near shore fringing reef (1), but all sites averaged DIN <0.6 mM. The shallow fore reef sites (2, 3) had similar DIN levels to one of the deeper wall edge sites (5), although the lowest DIN level measured was from the reef wall edge (0.3 mM, Table 1).

Organic d¹³C

All species sampled from Ochrophyta, Chlorophyta, and Rhodophyta phyla had organic d¹³C values in accordance with a mixed model of bicarbonate (<-10‰) and CO₂ (>-30‰) use as their Ci source for photosynthesis (Fig. 2). The ochrophytes were significantly more enriched in ¹³C compared to both chlorophytes and rhodophytes (Table 2). This pattern was consistent across reef sites even though they were collected from reefs with contrasting light and depth environments (Table 1). Within genus/species, thalli organic carbon was consistently more enriched in ¹³C along a gradient from darker to lighter conditions across sites (Fig. 2).

Table 1. Site, depth (D) in meters and reef characteristics where macroalgae were collected at 5 sites on Little Cayman Island (Fig. 1a). Reef type/location include fringing reef (FR), fore reef (F), mesophotic wall edge (WE), crevice edge (CE), and crevice (C) with depth (m) and light levels (LL): high (H), intermediate (I), low (L), lowlow (LL), and dark (D). Average irradiance (±SD; mmol photons m^-2 s^-1) and dissolved nutrient (N and P) concentrations (mM) and ratio of total inorganic nitrogen to phosphorus ratios at the sites (mean ± SD), including averages (±SD) of all sites.

Site	Reef	D	LL	Irradiance	NH₄⁺	NO_x	DIN	SRP	N:P
1	FR	2	H	1672 ± 266	0.65 ± 0.08	0.30 ± 0.02	0.95 ± 0.09	0.28 ± 0.16	4.99 ± 3.96
2	F	15	I	644 ± 56	0.48 ± 0.05	0.17 ± 0.04	0.64 ± 0.08	0.24 ± 0.03	2.73 ± 0.33
3	F	17	I	730 ± 135	0.44 ± 0.17	0.27 ± 0.13	0.72 ± 0.29	0.13 ± 0.04	5.51 ± 1.24
4	WE	23	L	409 ± 40	0.25 ± 0.09	0.03 ± 0.03	0.28 ± 0.10	0.10 ± 0.02	2.73 ± 1.07
5	WE	22	L	381 ± 218	0.41 ± 0.06	0.20 ± 0.08	0.61 ± 0.15	0.23 ± 0.07	3.02 ± 1.89
					0.41 ± 0.17	0.17 ± 0.12	0.58 ± 0.28	0.18 ± 0.10	3.62 ± 2.06
4	CE	23	LL	254 ± 31	na	na	na	na	na
2	C	15	D	7 ± 5	na	na	na	na	na
4	C	23	D	6 ± 4	na	na	na	na	na
5	C	22	D	16 ± 7	na	na	na	na	na

na - not applicable

However, this enrichment only accounted for a 1‰ to 3.6‰ shift in d¹³C values. In one genus, Amphirora sp., greater irradiance was associated with a more negative d¹³C signature (-2.4‰), but only two light levels were recorded, and these were both low light (LL, D) and did not span the broader range of light reported for the other genus/species. Overall, the higher d¹³C values were associated with macroalgae growing at the highest light levels, while those from dark sites had lower d¹³C values; this was the case even for the fleshy genus, Lobophora sp. found at all irradiances and in the calcified genus Jania at high light and dark sites (Fig. 2). Incorporating d¹³C data from all species, there was a significant positive relationship between increasing irradiance at reef sites and organic thalli d¹³C values (Fig. 3).

d¹⁵N and %C, %N and CN ratios

The d¹⁵N values across site and phyla were highly variable and the values were low, ranging between -1.70‰ and 1.83‰. The Ochrophyta exhibited more positive d¹⁵N values that were significantly higher than Chlorphyta or Rhodophyta (Table 2). Total N (% wt) was highest in the chlorophytes with greater than 2-fold higher levels than the ochrophytes. The rhodophyte %N was on average higher than the ochrophytes but lower than chlorophytes. The entire range of %C across phyla was only 42-45% on average. Although some of the calcified species were <5% organic tissue by total weight, their %C spanned those of the ochrophytes with primarily fleshy thalli. The phyla dominated by calcifiers had both higher (Chlorophyta) and lower (Rhodophyta) %C in their organic tissue than the phyla dominated by fleshy species (Ochrophyta). The high and low C:N ratio in the ochrophytes and chlorophytes, respectively, was primarily the result of the %N concentrations, while the intermediate C:N ratio in the rhodophytes was also the result of lower %C.

The %N in the organic thalli explained 45% of the variance in d¹³C values, but this trend may have been primarily driven by taxonomic differences as indicated by the grouping of phyla along the trendline (Fig. 4a). The %N vs d¹³C relationship within phyla was greatest in the chlorphytes (R² = 0.29) followed by the rhodophytes (R² = 0.21) and no correspondence in the ochrophytes (R² = 0.04). There was no relationship between d¹³C and d¹⁵N values in the ochrophytes and rhodophytes (Fig. 4b), while the chlorophytes (Halimeda sp., Halimeda tuna, Udotea sp.) enriched in ¹³C were also more enriched in ¹⁵N (Fig. 4c).

Table 2. Stable isotope (d¹³C), nitrogen (%), carbon (%) and C:N of thalli organic tissue, and d¹⁵N of organic+inorganic tissue, by site and phyla (means ± SE) and across phyla (bold). Different letters represent significant differences amongst phyla (P < 0.05).

Reef Site	Ochrophyta	Chlorophyta	Rhodophyta
d¹³C
1	-15.1 ± 0.33	-15.5 ± 0.24	-18.1 ± 0.98
2	-16.7 ± 0.39	-21.1 ± 0.55	nd
3	-17.5 ± 0.40	-21.4 ± 0.49	nd
4	-17.2 ± 0.88	-19.3 ± 0.75	-22.0 ± 0.17
5	-17.1 ± 0.51	-21.8 ± 0.39	-21.5 ± 0.53
X ± SE	-16.5 ± 0.22^a	-20.6 ± 0.30^b	-20.9 ± 0.23^b
d¹⁵N
1	0.59 ± 0.08	0.74 ± 0.17	0.66 ± 0.15
2	0.52 ± 0.17	-0.02 ± 0.39	nd
3	0.66 ± 0.12	0.33 ± 0.30	nd
4	0.28 ± 0.08	-0.09 ± 0.35	-0.63 ± 0.19
5	0.39 ± 0.15	-0.50 ± 0.26	0.12 ± 0.11
X ± SE	0.49 ± 0.09^a	0.09 ± 0.27^b	0.05 ± 0.38^b
%N
1	1.74 ± 0.15	3.15 ± 0.66	2.97 ± 0.14
2	1.65 ± 0.13	5.65 ± 0.14	nd
3	2.01 ± 0.13	5.19 ± 0.21	nd
4	1.45 ± 0.13	4.88 ± 0.27	2.75 ± 0.13
5	1.90 ± 0.13	5.47 ± 0.16	4.22 ± 0.27
X ± SE	1.78 ± 0.07^a	5.17 ± 0.12^b	3.70 ± 0.20^c
%C
1	44.4 ± 0.73	41.8 ± 2.55	40.9 ± 0.72
2	43.0 ± 0.20	45.2 ± 0.66	nd
3	44.5 ± 0.64	40.6 ± 0.97	nd
4	42.3 ± 0.43	47.3 ± 0.58	37.3 ± 1.82
5	42.9 ± 0.22	46.1 ± 0.48	43.1 ± 0.48
X ± SE	43.6 ± 0.27^a	44.8 ± 0.43^b	41.5 ± 0.60^c
C:N
1	30.7 ± 2.84	17.5 ± 2.92	14.0 ± 0.59
2	27.9 ± 1.65	8.1 ± 0.22	nd
3	23.3 ± 1.28	8.0 ± 0.26	nd
4	31.4 ± 3.76	10.3 ± 0.75	13.9 ± 1.06
5	24.1 ± 1.44	8.8 ± 0.38	11.0 ± 0.65
X ± SE	27.3 ± 1.09^a	9.5 ± 0.40^b	12.2 ± 0.50^c

Inorganic d¹³ and d¹⁸O

There were four primary clusters associated with d¹³C and d¹⁸O values of CaCO₃ in LCI reef calcifying macroalgae (Fig. 5). The clusters were highly phyla specific with the ochrophytes indicating the most positive combination of d¹³C and d¹⁸O values, while rhodophytes from three genus/family were associated with the most negative d¹³C and d¹⁸O cluster. The chlorophytes split into two additional groupings: one with a d¹³C and d¹⁸O range from 0.5 to 2.4 and -1.9 to -0.5, the second with a d¹³C and d¹⁸O range from 0.1 to 3.5 and -2.3 to -3.7. One rhodophyte (Peyssonneliaceae-red) was associated with the first chlorophyte grouping, and another rhodophyte species, Galaxaura rugosa, grouped with the ochrophytes.

Macroalgae collected from dark and lowlow light sites had significantly more depleted ¹³C carbonates than those from low to high light. These differences were primarily attributed to more enriched ¹³C carbonates in ochrophytes and depleted ¹³C carbonates in rhodophytes (Fig. 5) that were primarily from lighter and darker sites, respectively. There was no significant light effect on d¹³C values within the chlorophytes and only a 0.3‰ lower d¹³C value from chlorophytes collected in high light compared to dark sites. This modest shift in the inorganic d¹³C across light environments cannot account for the 3.0‰ shift in d¹³C values observed in chlorophyte CaCO₃ that was highly taxon-specific (Fig. 5) or the 1-3‰ d¹³C light effect observed in organic tissue (Fig. 2). In contrast to carbonate d¹³C, no significant light effects were found for d¹⁸O values. Rhodophyte d¹⁸O values were significantly lower than ochrophytes and chlorophytes. Across all phyla and site, there was a linear relationship between d¹³C and d¹⁸O (y = 0.33x-2.18) with an R² = 0.51; this relationship increased to R² = 0.87 when the cluster group 3 was omitted from the relationship (y = 0.40x - 1.89; Fig. 5).

Inorganic and Organic d¹³C

There was a significant (P < 0.01; R = 0.69) correlation between inorganic and organic d¹³C of macroalgae with enriched ¹³C and ¹⁸O values (clusters 1-3; Fig. 5), including species from all three phyla (Fig. 6). The trend was primarily driven by the high enrichment in ochrophytes, but the slope remains significant excluding this phylum (P < 0.01).

Little Cayman Island reef macroalgae had organic d¹³C values indicative of HCO₃^- use even within dark crevices on the upper mesophotic wall reef where irradiance levels are < 4 mmol photons m^-2 s^-1. Fleshy and calcified algal forms from all three phyla had d¹³C values that ranged between -12‰ and -25‰. Similar ranges in d¹³C (-14‰ to -20‰) were found in organic tissue of macroalgae on a subtropical patch reef in the Florida Keys (Zweng et al., 2018) under high irradiance (700 to 1,200 mmol photons m^-2 s^-1) and calcifying rhodophytes and chlorophytes on the great barrier reef (GBR, -13‰ to -19‰). On the GBR only fleshy species within the Chlorophyta and Ochrophyta exclusively utilized CCMs (d¹³C > -10‰) and only three fleshy Rhodophyta species were classified as non-CCM (d¹³C < -30‰) species (Diaz-Pulido et al., 2016). The majority ≥ 75% of GBR marine macroalgae and 84% of the 170 species recently assessed from the Gulf of California (Velázquez-Ochoa et al., 2022) were classified as utilizing Ci uptake strategies for photosynthesis that incorporated both diffusive CO₂ uptake and HCO₃^- use employing CCMs (d¹³C < -10‰ and > -30‰). These data support earlier work combining d¹³C with pH drift experiments that indicate most marine macroalgae utilize a diverse source of Ci for photosynthesis (Maberly et al., 1992). Thus, non-CCM exclusive CO₂ users within marine macroalgae have been considered to be rare (Raven et al., 2002, Maberly et al., 1992), frequently <10% to 20% of the macroalgal community (Hepburn et al., 2011, Stepien, 2015), particularly in tropical species (Stepien, 2015, Zweng et al. 2018, this study). However, temperate reefs of Tasmania, Australia with a high diversity of fleshy species dominated by rhodophytes (~58%) possess a high percentage (60%) of non-CCM species (Cornwall et al., 2015); these results correspond to those observed around south island New Zealand that have shown a high proportion of rhodophytes dependent on CO₂ diffusion (Hepburn et al., 2011). In cold temperate/polar and upwelling systems with higher dissolved CO₂ evolutionary pressure to evolve CCMs would be lessened, but there are also phyla-specific patterns of greater CO₂-diffusive use in the rhodophytes (Giordano et al., 2005) and fleshy species (Cornwall et al., 2015); these trends suggest taxonomy and form are important in defining Ci uptake mechanisms in macroalgae.

We observed a taxonomic trend in d¹³C values with ochrophytes significantly more enriched in ¹³C suggestive of efficient CCMs within this phylum. While the majority of macroalgae on LCI reefs were indicative of a mixed CO₂/HCO₃^- model, one genus (Padina sp.) d¹³C values approached those considered non-CO₂ users, a trait that appears to be highly conserved evolutionarily in the Ochrophyta phylum (Stepien et al., 2016). The presence of only species with CCMs on LCI tropical reefs is likely explained by the fact that all rhodophytes and most chlorophytes were calcifiers. On the GBR, none of the non-CCM macroalgae from either the Chlorophyta or Rhodophyta were calcifiers (Diaz-Pulido et al., 2016) and 26% of the rhodophytes with CCMs were calcifiers on temperate reefs of Tasmania (Cornwall et al., 2015). Crustose coralline algae (CCA) on the GBR across three unique evolutionary lineages were all recently shown to have CCMs with the only distinction being low, moderate and high percentage (35-65%) of diffusive CO₂ usage accompanying HCO₃^- use for photosynthesis (Bergstrom et al., 2020). A survey of rhodoliths, free-living calcifying rhodophytes, across 50^o of latitude (30-80^o N) in the northeast Atlantic, had organic d¹³C values between -26‰ and -3‰ with the majority of species close to -10‰ indicative of HCO₃^- use (Hofmann & Heesch, 2018). Our results, and those discussed above, are supportive of the paradigm that calcifying macroalgae primarily utilize HCO₃^- through efficient CCMs in contrast to fleshy species (Stepien, 2015, Bergstrom et al., 2020, Hofmann & Heesch, 2018, Zweng et al., 2018) consistent with CCM requirements for calcification as well as photosynthesis.

Counter to our initial hypothesis, LCI macroalgae d¹³C values corresponded to efficient CCMs across an irradiance gradient spanning three orders of magnitude within all three phyla and fleshy and calcified forms. Enhanced irradiance contributed to greater HCO₃^- use as indicated by a 1-3‰ enrichment in ¹³C and a linear increase in ¹³C in organic tissue as a function of irradiance across LCI reef sites. A similarly modest ¹³C enrichment of 1.2 ± 2.2‰ was calculated from macroalgae using CCMs (> -30‰) growing in deep versus shallow sites on the GBR (Cornwall et al., 2015), and ¹³C enrichment of ~4‰ in ochrophytes and rhodophytes in a kelp forest of New Zealand at 10-fold greater irradiance at 2 m compared to 10 m depth (Hepburn et al., 2011). The minor shifts observed within genus/species as a function of irradiance are consistent with the idea that irradiance levels influence the proportion of CO₂-use; however, photophysiology with regards to CCMs appears to be fundamentally taxon-specific. This specificity was observed in the CCA groupings of species with Ci-use mechanisms associated with unique evolutionary lineages (Bergstrom et al., 2020) and taxonomy being the dominant factor (genus 46%, species 57%), followed by macroalgal form and structure (35%) with minor attribution to environmental conditions, in a multi-variate model used to explain macroalgal diversity of d¹³C values (-35‰ to -2‰, 170 species) in the Gulf of California (Velázquez-Ochoa et al., 2022). Global meta-analysis also indicate strong taxonomic patterns in Ci-use strategies as clearly observed in unimodal distributions of d¹³C across chlorophytes and ochrophytes and bimodal distribution within rhodophytes (Stepien, 2015).

The low average inorganic nutrient concentrations (0.58 mM DIN , 0.18 mM SRP ), C:N ratios (3.6) and low d¹⁵N values (0.23‰ ± 0.77‰) on LCI reefs indicate the oligotrophic state in which macroalgae were growing (Dailer et al., 2010). Low NO₃^- concentrations (<2 mM) likely accounts for the minimal fraction of d¹⁵N observed (Swart et al., 2014) across all genus/species (-2‰ to +2‰). No significant correlations were found between d¹³C and tissue %N content within phyla, although there were differences among phyla suggesting %N levels were taxon-specific. Ochrophytes had low organic %N simultaneous with ¹³C enrichment, thus this phylum may be able to optimize HCO₃^- use in low N reef environments, a trait that may contribute to its dominance on tropical reefs in addition to a lack of palatability to grazers (Hay, 1997). The higher organic C:N ratio in LCI ochrophytes compared to chlorophytes or rhodophytes supports this conjecture. Chlorophytes had the broadest range in d¹⁵N and the relationship between d¹³C and d¹⁵N was primarily driven by Halimeda tuna and Halimeda sp. with fleshier thalli lower in CaCO₃ (81-92%) exhibiting more positive isotope values (d¹³C -18‰ ± 1.5‰ and d¹⁵N 1.08‰ ± 0.27‰) compared to H. goreaui, H. copiosa, and H. opuntia with relatively higher CaCO₃ (94%) and more negative isotopic values (d¹³C -22‰ ± 1.9‰ and d¹⁵N -0.78‰ ± 0.41‰). Several factors may have contributed to the depleted ¹⁵N and d¹³C values in the dominant Halimeda species (H. goreaui, H. copiosa, and H. opuntia) growing on the fore and mesophotic wall edges at intermediate to low irradiance. (1) These are primarily pendant species suspended in the water column such that they would receive greater seawater exchange and flow which is likely to enhance NO₃^- uptake and CO₂ diffusion compared to fleshy benthic forms. (2) Their smaller utricular cells, compared to H. tuna and the larger fleshy species (Halimeda sp.), shortens the diffusion pathway for seawater (Peach et al., 2017) that may enhance NO₃^- uptake. It is interesting that these same two groups of Halimeda species also differentiated in their CaCO₃ ¹⁸O values.

In comparison to organic d¹³C values, calcified macroalgae on LCI exhibited clustering of thalli CaCO₃ d¹³C values that were distinctly species/genus and phyla-specific, suggesting divergent calcification mechanisms. Isotope values in biogenic carbonates can be associated with unique locations of calcification, in some cases constrained by thalli form, or metabolic effects of photosynthesis/respiration or active ion transport systems. The ochrophytes were enriched in ¹³C by approximately 4‰ ± 1‰ indicating the pool of Ci available for calcification was affected by sequestration of the lighter isotope ¹²C for photosynthesis as calcification and photosynthesis occur together on the thalli surface. This level of enrichment (3.5‰), relative to marine DIC (~1.5‰) corresponds to shallow carbonate shelves in the Bahamas and Shark Bay, Australia that are thought to become more enriched in ¹³C due to ¹²C uptake by photosynthesis during periods of maximum calcification during the day (Geyman & Maloof, 2019). Biotic effects of symbiotic algae in corals also leads to ¹³C enrichment of coral CaCO₃ due to algae sequestering ¹²C for photosynthesis thereby increasing ¹³C in the calcifying fluids (McConnaughey, 2003). Chlorophyte CaCO₃ d¹³C values (1.7‰ ± 0.6‰) were consistent with marine DIC d¹³C and slightly less enriched in ¹³C than those found in ochrophytes. As the majority of species on LCI were of the Halimeda genus the lighter fraction may be the result of greater Ci contribution to calcification from respiratory ¹²C (McConnaughey, 2003) and/or the transport of seawater through diffusive pathways between utricular cells that when compressed create channels for seawater exchange (Borowitzka & Larkum, 2007, Koch et al., 2013). A greater role of photosynthesis by LCI ochrophytes than chlorophytes in calcification is supported by DIC changes primarily attributed to photosynthesis in Padina sp., while to a lesser extent in Halimeda sp. where DIC changes were primarily associated with calcification (Buapet & Sinutok, 2021).

Rhodophytes exhibited the greatest depletion in ¹³C (-4.1‰ ± 0.7‰) with the exception of Peyssonneliacea (red) and Galaxaura rugosa that were more enriched in ¹³C, potentially affected by their primarily aragonite structure (Basso, 2012, Kerkar, 1994) and mechanism of calcification. Aragonite rhodophytes have been shown to be more enriched in ¹³C (Swart, 1983). The more ¹³C depleted rhodophyte carbonates in calcite-forming rhodophytes (Swart, 1983, McConnaughey, 2003) reflect enhanced calcification complexity compared to Halimeda sp. (Borowitzka, 1981). Rhodophytes are primarily characterized by high magnesium calcite cell wall calcification (Borowitzka, 1982, Koch et al., 2013) where the calcification sites are semi-isolated from ambient seawater (Adey, 1998) and pH can rapidly increase (Cornwall et al., 2017). Rhodophytes, including CCA, utilize active transport to control ion flux between the cell and cell wall (Adey, 1998, Borowitzka, 1981) and require calcification/decalcification for growth and reproduction; thus, they likely possess a high degree of biotic control over calcification (Hofmann et al., 2016). In addition to depleted ¹³C carbonates, rhodophytes exhibited depleted ¹⁸O in their carbonates, a similar pattern exhibited in corals and other biogenic carbonates implying comparable calcification mechanisms (McConnaughey, 2003).

The LCI macroalgal carbonate d¹⁸O and d¹³C were highly correlated (R² = 0.87) with a slope of 2.17 (excluding Halimeda sp. and H. tuna) equivalent to slopes reported for rhodophytes (2.13) and similar to slopes for ahermatypic and hermatypic shallow (2.25 and 2.37, respectively) and deep-sea corals (2.5 ± 0.69) (Chen et al., 2018). The divergence in d¹⁸O:d¹³C in less calcified Halimeda sp. and H. tuna (more depleted ¹⁸O) with greater organic:inorganic thalli may be a consequence of calcification driven more by photosynthesis, a condition shown to select for ¹⁶O in biogenic calcification (Adkins et al., 2003). The relationship between d¹⁸O and d¹³C in biogenic carbonates has been attributed to kinetic effects from CO₂ hydration and hydroxylation resulting in depleted ¹⁸O and ¹³C in HCO₃^- and CO₃^2- incorporated into CaCO₃ (McConnaughey, 2003, McConnaughey, 1989). Alternatively, admixtures of cellular CO₂ depleted in ¹³C and seawater Ci diffusion into the calcifying space explains the d¹⁸O:d¹³C relationship in biogenic calcifiers (Adkins et al., 2003). Regardless of the specific mechanism accounting for the d¹⁸O:d¹³C correspondence, depleted ¹⁸O and ¹³C values in LCI rhodophyte carbonates are an indication of high calcifying fluid pH and rapid calcification, which may be indicative of a highly controlled calcification mechanism using active ion transport. Species/genus with positive d¹³C carbonates across phyla had a significant positive correlation between thalli organic d¹³C and inorganic CaCO₃ d¹³C. In addition to the unique phyletic differences in calcification discussed above, these results may reflect the transfer of less fractionated Ci into CaCO₃ of calcified species with more efficient CCMs compared to those relying on greater CO₂ diffusion and establish the importance of photosynthesis/respiration in their mechanisms of calcification and a modest influence of irradiance.

In conclusion, stable isotopes of tropical reef macroalgae on LCI indicate that most genus/species are utilizing CCMs with different phyla varying their reliance on diffusive CO₂. Diffusive CO₂-use is modestly enhanced under low irradiance, but is also more prevalent in chlorophytes and rhodophytes, while the ochrophytes are primarily utilizing HCO₃^-, a positive result given ochrophye propensity to evade herbivory and lead to algal blooms on Caribbean reefs. Increasing ocean CO₂ is thus not likely to significantly enhance photosynthesis if reef macroalgae utilize CCMs to efficiently sequester HCO₃^- even across a broad range of irradiances. Ochrophytes may also be able to engage efficient CCMs at relatively low inorganic nitrogen concentrations explaining their abundance in low nutrient reef environments. Relatively higher organic C fractionation when utilizing greater CO₂ for photosynthesis appears to be reflected in depleted thalli carbonates in species/genus with positive d¹³C values. However, inorganic d¹⁸O and d¹³C values were phyla-specific suggestive of evolutionary importance in defining form and mechanisms of calcification. Specifically, the cell wall calcite forming species were unique in exhibiting highly depleted carbonates not related to organic ¹³C values with evidence of high pH and CO₃^2- concentrations within sites of calcification. Calcification in these rhodophyte genus/species are frequently shown to be highly sensitive to ocean acidification perhaps due to their reliance on active ion transport and the difficulty in sustaining ion balance/flux in seawater high in H⁺ concentrations. More mechanistic studies and assessments of additional reef macroalgal species from various sites are needed to further corroborate the ideas presented herein and advance our understanding of reef macroalgae photophysiology and calcification mechanisms that will determine how macroalgae are likely to respond to global change.

Acknowledgements

Graduate (Regina Zweng) and undergraduate (Steven Olayon) students are thanked for their efforts in collecting/processing samples in the FAU lab and Cayman Island Marine Conservation Board and Department of the Environment for permitting our LCI research. We thank captain Jon Clamp for field support and the staff at LCI for assistance working at the Central Caribbean Research Station.

Funding Statement

This research was funded by the National Science Foundation Ocean Acidification Program-CRI-OA Grant #1416376.

Competing Interests

The authors have no competing interests.

Availability of data and material

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

All authors assisted in data collection, reviewing the manuscript.

Conall McNicholl - processed samples

Carrie Manfrino - coordinated field collection at LCI

Brian Lapointe - analyzed nutrient samples.

Marguerite Koch - coordinated the project, developed hypotheses, and wrote the manuscript

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No competing interests reported.

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Stable Carbon and Oxygen Isotopes Indicate Photophysiology and Calcification Mechanisms of Macroalgae on Little Cayman Reefs

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