1, Raphael Ritson-Williams1,2, Brian N Popp3, Ruth D Gates1 · 4 Christopher B Wall1, Raphael Ritson-Williams1,2, Brian N Popp3, Ruth D Gates1 5 6 *corresponding author: [email protected]

Spatial variation in the biochemical and isotopic composition of corals during bleaching and 1

recovery 2

3

Christopher B Wall1*, Raphael Ritson-Williams1,2, Brian N Popp3, Ruth D Gates1 4

5

*corresponding author: [email protected] 6

7

1University of Hawai‘i at Mānoa, Hawai‘i Institute of Marine Biology, PO Box 1346, Kāne‘ohe, 8

HI 96744, USA 9

2California Academy of Sciences, Invertebrate Zoology Department, 55 Concourse Dr, San 10

Francisco, CA 94118, USA 11

3University of Hawai‘i at Mānoa, Department of Geology and Geophysics, 1680 East-West Rd, 12

POST 701, Honolulu, HI 96822, USA 13

14

author emails: 15

[email protected], [email protected], [email protected], [email protected] 16

17

keywords: energy reserves, thermal stress, heterotrophy 18

19

running header: Biology of bleached and recovering corals 20

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted September 11, 2018. ; https://doi.org/10.1101/414086doi: bioRxiv preprint

https://doi.org/10.1101/414086

Abstract 21

Ocean warming and the increased prevalence of coral bleaching events threaten coral reefs. 22

However, the biology of corals during and following bleaching events under field conditions is 23

poorly understood. We examined bleaching and post-bleaching recovery in Montipora capitata 24

and Porites compressa corals that either bleached or did not bleach during a 2014 bleaching 25

event at three reef locations in Kāne‘ohe Bay, O‘ahu. We measured changes in chlorophylls, 26

biomass, and nutritional plasticity using stable isotopes (δ13C, δ15N). Coral traits showed 27

significant variation among bleaching conditions, reef sites, time periods, and their interactions. 28

Bleached colonies of both species had lower chlorophyll and total biomass. While M. capitata 29

chlorophyll and biomass recovered three months later, P. compressa chlorophyll recovery was 30

location-dependent and total biomass of previously bleached colonies remained low. Biomass 31

energy reserves were not affected by bleaching, instead M. capitata proteins and P. compressa 32

biomass energy declined over time, and P. compressa lipid biomass was site-specific. Stable 33

isotope analyses of host and symbiont tissues did not indicate increased heterotrophic nutrition in 34

bleached colonies of either species, during or after thermal stress. Instead, mass balance 35

calculations revealed variance in δ13C values was best explained by augmented biomass 36

composition, whereas δ15N values reflected spatial and temporal variability in nitrogen sources in 37

addition to bleaching effects on symbiont nitrogen demand. These results emphasize total 38

biomass quantity may change substantially during bleaching and recovery. Consequently, there 39

is a need to consider the influence of biomass composition in the interpretation of isotopic values 40

in corals. 41

42


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Introduction 43

Scleractinian corals in association with Symbiodinium spp. symbionts are important primary 44

producers on coral reefs, which through biogenic processes create the complex calcium 45

carbonate framework of the reef milieu. The coral-Symbiodinium symbiosis can be disturbed 46

under environmental stress, which through a variety of cellular mechanisms, leads to the 47

reduction of symbiotic algal cells in coral tissue (i.e., coral bleaching) (Weis et al. 2008). 48

Depending on the severity or duration of stress, bleaching causes coral mortality, although some 49

corals survive and recover their symbionts post-bleaching (Fitt et al. 1993; Cunning et al. 2016). 50

The strength and frequency of bleaching events has increased over the last three decades from a 51

combination of progressive seawater warming (Heron et al. 2016) and climatic events (i.e., 52

ENSO) (Hughes et al. 2017). It is therefore critical to advance an understanding of the 53

environmental conditions and biological mechanisms that underpin the physiological resilience 54

of corals to thermal stress. 55

56

Coral resistance to and recovery from bleaching stress has been related to associations with 57

thermally tolerant Symbiodinium spp. (Sampayo et al. 2008), replete tissue biomass (Thornhill et 58

al. 2011) or high-quality biomass (i.e., lipid content), and the capacity to maintain positive 59

energy budgets through nutritional plasticity (Anthony et al. 2009). Coral nutrition is largely 60

supported by fixed-carbon derived from Symbiodinium, however, particle feeding, plankton 61

capture, and the uptake of dissolved nutrients (collectively, ‘heterotrophy’) can account for < 15 62

– 50 % of energy demands (Porter 1976; Houlbrèque and Ferrier-Pagès 2009) and > 100 % of 63

respiratory carbon demand in bleached corals (Grottoli et al. 2006; Palardy et al. 2008; Levas et 64

al. 2016). Facultative shifts from autotrophic to heterotrophic nutrition are often linked to 65


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reduced Symbiodinium photosynthesis in response to periodic light attenuation (i.e., turbidity) 66

and/or environmental stress (Houlbrèque and Ferrier-Pagès 2009). As such, nutritional plasticity 67

is an important acclimatization mechanism shaping corals’ physiological niche (Anthony and 68

Fabricius 2000) and supporting the resilience of reef-building corals to changing environments 69

and resource availability (Grottoli et al. 2006; Ferrier-Pagès et al. 2010; Connolly et al. 2012; 70

Hughes and Grottoli 2013). 71

72

Thermal stress and bleaching can increase coral feeding on zooplankton (Grottoli et al. 2006; 73

Ferrier-Pagès et al. 2010; Hughes and Grottoli 2013; Levas et al. 2013) and suspended particles 74

(Anthony and Fabricius 2000), and stimulate the uptake of diazotroph-derived nitrogen (Bednarz 75

et al. 2017) and dissolved organic carbon by corals (Levas et al. 2016). Periods of stress or 76

resource limitation do not facilitate shifts towards heterotrophic nutrition in all corals (Anthony 77

and Fabricius 2000; Schoepf et al. 2015), instead catabolism of energy-rich biomass (i.e., 78

proteins, lipids, carbohydrates) supports energetic demands (Fitt et al. 1993; Grottoli et al. 2006; 79

Schoepf et al. 2015). Considering the limited size of biomass reserves, corals capable of 80

increasing the acquisition of heterotrophic energy may experience a fitness advantage during 81

times of stress and symbiosis disruption, as well as increased rates of physiological recovery 82

(Rodrigues and Grottoli 2007; Connolly et al. 2012; Grottoli et al. 2014). 83

84

Elevated temperature effects on corals are also mediated by co-occurring environmental factors, 85

including: ultraviolet (UV) (Shick et al. 1996) and photosynthetically active radiation (PAR) 86

(Coles and Jokiel 1977), the concentration (Vega-Thurber et al. 2014) and stoichiometry of 87

dissolved nutrients (e.g., nitrogen, phosphorous) (Wiedenmann et al. 2012), as well as water 88


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motion (Nakamura and van Woesik 2001). For instance, elevated light levels and chronic 89

nutrient loading can exacerbate thermal stress (Coles and Jokiel 1977; Vega-Thurber et al. 2014), 90

while high water motion and seawater turbidity can reduce bleaching severity and mortality 91

(Nakamura and van Woesik 2001; Anthony et al. 2007). Heterotrophic feeding preceding and 92

following thermal stress can reduce bleaching severity and coral mortality (Anthony et al. 2009; 93

Ferrier-Pagès et al. 2010) and replenish lipid biomass (Baumann et al. 2014). Additionally, 94

heterotrophy and dissolved nutrients promote post-bleaching recovery (Connolly et al. 2012) and 95

support Symbiodinium repopulation (Marubini and Davies 1996). Spatiotemporal variation in 96

abiotic conditions that affect coral performance and resource availability/demand, therefore, can 97

influence coral holobiont response trajectories and outcomes to physiological stress 98

(Hoogenboom et al. 2011; Connolly et al. 2012; Scheufen et al. 2017). Considering reef corals 99

may experience bleaching effects > 12 months following initial thermal stress and well beyond 100

the return of normal tissue pigmentation (Fitt et al. 1993; Baumann et al. 2014; Grottoli et al. 101

2014; Levitan et al. 2014; Schoepf et al. 2015), it is important to consider the environmental 102

effects and physiological mechanism(s) that facilitate or stymie post-bleaching recovery. 103

104

The occurrence of large-scale coral bleaching episodes has been historically rare in the Main 105

Hawaiian Islands, being limited to 1996 (Bahr et al. 2017). However, coastal seawater in 106

Hawai‘i is warming (0.02 °C y-1) (Bahr et al. 2015) and the frequency and severity of global 107

bleaching events is increasing (Hughes et al. 2017). From September – October 2014, the 108

Hawaiian Island archipelago experienced a protracted period of elevated sea surface warming. 109

Degree heating weeks (DHW) for the Main Hawaiian Islands began to accumulate on 15 110

September, peaking at 7 DHW on 20 October, and declining below < 7 DHW after 08 December 111


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(NOAA Coral Reef Watch 2018). Water temperatures (29 – 30.5 °C) (Bahr et al. 2015) 112

exceeded O‘ahu summertime maximum temperatures by ≤ 3.0 °C (Bahr et al. 2017) and resulted 113

in a rare coral bleaching event spanning the archipelago (Bahr et al. 2017; Couch et al. 2017) 114

with extensive bleaching within Kāne‘ohe Bay, O‘ahu (62 – 100 % of corals; Bahr et al. 2015). 115

This event provided a rare opportunity to track the biology of bleaching resistant and susceptible 116

corals during and after thermal stress under natural field conditions, with the potential to inform 117

trajectories of bleaching recovery among reef habitats. 118

119

In this study different bleaching phenotypes (bleached vs. non-bleached) of two dominant 120

Kāne‘ohe Bay coral species (Montipora capitata and Porites compressa) (Fig. 1) previously 121

shown to increase heterotrophy (M. capitata) and catabolize tissues (P. compressa) during 122

bleaching (Grottoli et al. 2006; Rodrigues and Grottoli 2007). Corals were collected during peak 123

bleaching and three months following thermal stress (Fig. S1a) from three patch reefs within an 124

environmental gradient of decreasing oceanic influence (Lowe et al. 2009) and terrigenous 125

nutrient perturbations (Smith et al. 1981), which allowed an examination of the spatial variance 126

and environmental influence (temperature, light, sedimentation, dissolved nutrients) on corals 127

after thermal stress. We tested (1) whether photopigments, coral biomass (total biomass, 128

proteins, lipids, carbohydrates, energy content), and contributions of heterotrophic nutrition 129

(δ13C and δ15N values) differed among time periods, bleaching conditions, or reef sites, and (2) 130

whether environmental conditions influenced bleaching severity and mechanisms of 131

physiological recovery. 132

133

Materials and Methods 134


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Site description 135

Corals were collected from three patch reefs (Fig. 1a): one in northern (Reef 44: 21°28’36.4” N, 136

157°50’01.0” W), central (Reef 25: 21°27’40.3” N, 157°49’20.1” W), and southern (Hawai‘i 137

Institute of Marine Biology (HIMB): 21°26’06.0” N, 157°47’27.9” W) Kāne‘ohe Bay, Oʻahu, 138

Hawaiʻi (see Cunning et al. 2016 for more detail). Reef sites were identified for their location 139

within the longitudinal axis of Kāne‘ohe Bay, which spans a north-south hydrodynamic gradient 140

of seawater residence times (north: < 2 d; south: 30 – 60 d)) and oceanic influence (high in north, 141

low in south) (Lowe et al. 2009). 142

143

Environmental data 144

Dissolved inorganic nutrients in seawater were measured on samples collected (ca. 100 ml) from 145

surface waters (< 1 m) at each reef site at a period of once every two weeks from 11 November 146

2014 to 27 January 2015. In total, seven seawater samples were analyzed for each reef site over 147

the study period. Additional samples were also collected to analyze seawater nitrate δ15N using 148

the bacterial denitrifier method (see Supporting Information). Seawater was filtered (0.7 µm) 149

and stored in 0.1 N HCl-washed bottles and frozen at -20 °C until analyzed. Dissolved inorganic 150

nutrients – ammonium (NH4+), nitrate + nitrite (NO3

- + NO2-) (i.e., N+N), phosphate (PO4

3-), and 151

silicate (Si(OH)4) – were measured by University of Hawai‘i at Mānoa SOEST Laboratory for 152

Analytical Biogeochemistry using a Seal Analytical AA3 HR nutrient autoanalyzer and 153

expressed as µmol L-1. Photosynthetically active radiation (PAR) and temperature data were 154

continuously recorded at 15 min intervals at 2 m depth at each reef site using cross-calibrated 155

Odyssey PAR loggers (Dataflow Systems Limited, Christchurch, New Zealand) and Hobo 156

Pendant UA-002-08 loggers (Onset Computer Corp., Bourne, MA) (see Supporting Information). 157


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PAR and temperature loggers at Reef 25 experienced mechanical errors; therefore, only data 158

from Reef 44 and HIMB are presented. Instantaneous PAR values were used to calculate the 159

daily light integral (DLI) for each site (mol photons m-2 d-1). Rates of sedimentation at the three 160

sites were measured using sediment traps collected each month, and expressed as mg sediment-1 161

d-1 (see Supporting Information). 162

163

Coral collection and tissue analysis 164

During peak bleaching in October 2014, five adjacent pairs of Montipora capitata (Dana, 1846) 165

and five pair of Porites compressa (Dana, 1846) exhibiting different bleaching conditions – 166

tissue paling (bleached) and fully pigmented (non-bleached) (Fig. 1b-c) – were collected. 167

Colonies were identified and tagged (depth: <1 – 3 m) with cattle tags and zip ties, and fragments 168

(4 cm in length) from each coral colony pair (5 pairs per species) were collected from each of the 169

three reefs during bleaching (24 October 2014) and ca. 3 month following peak seawater 170

temperatures (14 January 2015) (Fig. S1). Fragments were immediately frozen in liquid nitrogen 171

and stored at -80 °C until processing. 172

173

All biomass assays were performed on the holobiont tissues (host + symbionts), following 174

established procedures (Wall et al. 2017). Additional information methodology information can 175

be found in the Supporting Information. Coral tissues were removed from skeletons using an 176

airbrush filled with filtered seawater (0.2 µm). The tissue slurry was briefly homogenized and 177

stored on ice. Chlorophyll (a+c2) was used as a metric of bleaching (Grottoli et al. 2006) and 178

symbiont densities (symbiont:host cell ratio) were measured previously (Cunning et al. 2016). 179

Symbiodinium chlorophyll was extracted in 100 % acetone and measured by spectrophotometry 180


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(Jeffrey and Humphrey 1975). Pigment concentrations were normalized to skeletal surface area 181

(cm2) determined by the wax-dipping technique (Stimson and Kinzie 1991). 182

183

Total tissue biomass was determined from the difference of dry (60 °C) and combusted (4 h, 450 184

°C) masses of an aliquot of tissue extract and expressed as the ash-free dry weight (AFDW) of 185

biomass cm-2. Total protein (soluble + insoluble) was measured spectrophotometrically 186

following the Pierce BCA Protein Assay Kit (Pierce Biotechnology, Waltham, MA) using a 187

bovine serum albumin standard curve (Smith et al. 1985). Tissue lipids were quantified on 188

lyophilized tissue slurry in a 2:1 chloroform:methanol solution followed by 0.88 % KCl and 189

100 % chloroform wash. The lipid extract was evaporated in pre-combusted (450 °C, 4h) 190

aluminum pans, and measured to nearest 0.0001 g (Wall et al. 2017). Carbohydrates were 191

measured by the phenol-sulfuric acid method using glucose as a standard (Dubois et al. 1965). 192

Finally, changes in tissue biomass reserves were assessed energetically using compound-specific 193

enthalpies of combustion (Gnaiger and Bitterlich 1984). Proteins, lipids, carbohydrates, and 194

biomass kilojoules (i.e., energy content) were normalized to g AFDW of the tissue slurry (see 195

Supporting Information). 196

197

Stable isotope analysis 198

Skeletal carbonates were filtered from the tissue slurry (Maier et al. 2010) and host and symbiont 199

tissues were separated by centrifugation (2000 g × 3 min) with filtered seawater (0.2 µm) rinses 200

(Muscatine et al. 1989). Tissues were filtered onto pre-combusted 25 mm GF/F filters (450 °C, 201

4h), dried overnight (60°C), and packed in tin capsules. Carbon (δ13C) and nitrogen (δ15N) 202

isotopic values and molar ratios of carbon:nitrogen (C:N) for coral host (δ13CH, δ15NH, C:NH) and 203


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algal symbiont (δ13CS, δ15NS, C:NS) tissues were determined with a Costech elemental 204

combustion system coupled to a Thermo-Finnigan Delta Plus XP Isotope Ratio Mass-205

Spectrometer. Analytical precision of δ13C and δ15N values of samples was < 0.2 ‰ determined 206

by analysis of laboratory reference material run before and after every 10 samples (see 207

Supporting Information). Isotopic data are reported in delta values (δ) using the conventional 208

permil (‰) notation and expressed relative to Vienna Pee-Dee Belemnite (V-PBD) and 209

atmospheric N2 standards (air) for carbon and nitrogen, respectively, using the following 210

equation: 211

δ13C or δ15N = [(Rsample/Rstandard) – 1] × 1000 212

where R is the ratio of 13C:12C or 15N:14N in the sample and its respective standard. The relative 213

differences in isotopic values in the host and symbiont for carbon (δ13CH-S) and nitrogen (δ15NH-214

S) were calculated to evaluate changes in the proportion of heterotrophic carbon to coral host 215

nutrition (i.e., δ13CH-S) and changes in trophic enrichment among host and symbiont (i.e., δ15NH-216

S) (Rodrigues and Grottoli 2006; Reynaud et al. 2009). 217

218

An isotope mass balance was modeled to measure changes in tissue biomass composition on 219

holobiont (host + symbiont) δ13C values during bleaching recovery, following Hayes (2001). 220

First, the isotopic composition of the holobiont (δ13CHolobiont) was modeled for each time period: 221

δ13CHolobiont = (mH * δ13CH) + (mS * δ13CS) 222

where m is the estimated proportion of host (mH) and Symbiodinium (mS) tissues in holobiont 223

biomass (g AFDW), and δ13C (defined above) are isotopic values of tissues. Second, the 224

measured proportion of biomass compounds (i.e., % of proteins, lipids, carbohydrates) and 225

δ13CHolobiont were used to estimate compound-specific isotopic values (δ13CCompound) for each 226


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compound. The influence of changes in biomass composition on δ13CHolobiont values (i.e., 227

observed-δ13CHolobiont) during bleaching recovery δ13CCompound estimates were applied to the same 228

colonies in January 2015 to determine expected-δ13CHolobiont. The relationship between observed 229

and expected δ13CHolobiont was evaluated using a linear regression (see Supporting Information). 230

231

Statistical analysis 232

A matrix of all biological response variables for M. capitata and P. compressa was first analyzed 233

using a permutational multivariate analysis of variance (PERMANOVA) with periods (October 234

2014, January 2015), sites (Reef 44, Reef 25, HIMB), and colony-level physiological condition 235

observed in October 2014 (i.e., bleached or non-bleached) as main effects. δ13C values were 236

incorporated into the data matrix by transforming to absolute values (i.e., |δ13C|). Sum of squares 237

were partitioned according to Bray-Curtis dissimilarity matrix and sequential tests were applied 238

on 1000 model permutations using adonis2 in package vegan (Oksanen et al. 2017), with 239

pairwise comparisons over an additional 1000 permutations in RVAideMemoire. Results of 240

PERMANOVA were applied to distinguish the hierarchy of main effects between coral species 241

and to holistically evaluate post-bleaching recovery. Multivariate relationship between periods, 242

sites, and bleaching condition were visualized for each species separately using nonmetric 243

multidimensional scaling (NMDS) plots with ellipses representing standard errors of point means. 244

NMDS plots were used to visualize differences among reefs and bleaching condition (i.e., site × 245

condition), and among bleached and non-bleached corals across all sites with vectors 246

representing significant biological responses (p ≤ 0.05). 247

248


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Environmental data (temperatures, light, dissolved nutrients, sedimentation) from each reef were 249

analyzed using a linear mixed effect model using lmer in package lme4 (Bates et al. 2015). Reef 250

site was treated as a fixed effect and date of sample collection as a random effect. Biological 251

response variables for individual species were analyzed using three-way linear mixed effect 252

models in lme4 with period, site, and condition as fixed effects and coral colony and colony-pairs 253

as random effects. Model selection was performed on candidate models using a combination of 254

AIC and likelihood ratio tests. Where significant interactions were observed, pairwise post hoc 255

slice-tests of main effects by least-square means were performed in package lsmeans (Lenth 256

2016). Analysis of variance tables for all environmental and biological metrics were generated 257

using type II sum of squares with Satterthwaite approximation of degrees freedom using 258

lmerTest (Kuznetsova et al. 2017). Temperature, light, and sedimentation data from these reefs 259

are publically available (Ritson-Williams and Gates, 2016a; 2016b). Data and R code to 260

reproduce tables, figures, and analyses are archived at Zenodo (xxx). 261

262

Results 263

Environmental data 264

Kāne‘ohe Bay reef flats sustained a maximum seawater temperatures of ca. 31 °C (Bahr et al. 265

2015). Peak seawater warming at HIMB spanned 15 – 24 September 2014 with temperatures 266

ranging from 29.8 – 30.2 °C (NOAA 2017) (Fig. S1a). From October 2014 to January 2015 267

daily maximum seawater temperatures were 0.01 °C different among sites (p <0.001) (Table S1, 268

Fig. S1c), however, this difference is below the accuracy of the temperature loggers (0.53 °C; 269

Onset Computer Corp) and should be interpreted with caution. Seawater temperatures at both 270

Reef 44 and HIMB declined from peaks in mid-October (≤ 29.2 °C), and daily mean (p = 0.192) 271


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and minimum (p = 0.687) seawater temperatures were comparable (Fig. S1d). Light values 272

integrated over a 24 h day (i.e. DLI mol photons m-2 d-1) was 4.5 mol photons m-2 d-1 greater at 273

HIMB compared to Reef 44 (p < 0.001) (Fig. S1b, Table S1) 274

275

Dissolved inorganic nutrients differed among the three reefs (Fig. 2a-d, Table S1). Phosphate (p 276

= 0.020) was lowest at Reef 25 and highest at Reef 44, with intermediate values at HIMB. 277

Ammonium concentrations were equivalent among reefs (p = 0.161), but nitrate + nitrite 278

concentrations were greater at Reef 44 compared to other reefs (p = 0.002). While, silicate (p = 279

0.724) and short-term sedimentation rates (p = 0.161) (Fig. 2e) did not differ among sites, silicate 280

tended to be higher at Reef 44 and an extended monitoring of sedimentation rates (2015 January 281

– 2016 January) show annual sedimentation rates at Reef 44 and HIMB are approximately two-282

fold greater than that of Reef 25 (p = 0.041) (Fig. 2f). δ15N values for nitrate ranged from 3.8 to 283

4.9 ‰ (Table S2), however, low [nitrate + nitrite] precluded isotopic reduced sample sizes for 284

δ15N nitrate analysis (n = 1 – 2 samples per site). 285

286

Coral physiology 287

Multivariate analysis of sixteen response variables in M. capitata and P. compressa revealed 288

significant changes in corals among time periods (<0.001), between bleached and non-bleached 289

corals (p ≤ 0.004) and the interaction of period × condition (p ≤ 0.029) (Table S3). Reef sites 290

significantly influenced M. capitata (p = 0.006), especially during October 2014 (Fig. 3a), 291

whereas P. compressa colonies were less influenced by site (p = 0.099) and instead 292

predominantly influenced by bleaching condition (Fig. 4a). NMDS plots showed differences in 293

bleached and non-bleached colonies of both species during October 2014 (post-hoc: p ≤ 0.008) 294


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where bleaching resulted in a negative correlation with chlorophyll concentration (chl) and 295

biomass in both species (Fig. 3b, 4b) and lower host and symbiont C:N in P. compressa (Fig. 4b). 296

By January 2015, the physiological condition of previously bleached M. capitata (post-hoc: p = 297

0.337) and P. compressa colonies (post-hoc: p = 0.125) were indistinguishable from non-298

bleached conspecifics, indicating a convergence of physiological properties in corals across 299

bleaching histories and a rapid physiological recovery from bleaching (Fig. 3c-d, Fig. 4c-d). A 300

summary of significant effects for all response variables can be found in Table 1. 301

302

Montipora capitata chlorophyll and tissue biomass quantity (Fig. 5a-b) and composition (Fig. 303

6a-d) were similar across the three sites (p ≥ 0.222), but total chlorophyll (p = 0.041) and tissue 304

biomass (p = 0.011) were affected by the interaction of period × condition (Table S4). In 305

October bleached M. capitata had 63 % less chlorophyll and 30 % less tissue biomass than non-306

bleached phenotypes (Fig. 5a-b). By January, however, M. capitata chlorophyll and tissue 307

biomass were equivalent among bleached and non-bleached corals, having increased 255 % and 308

95 % in bleached phenotypes and 54 % and 37 % in non-bleached phenotypes, respectively, 309

from October 2014 levels (Fig. 5a-b). Over the recovery period, M. capitata protein biomass (g 310

gdw-1) declined by 20 % (p = 0.010) but did not differ among sites (p = 0.461) or between 311

bleached and non-bleached colonies (p = 0.267) (Fig. 6a, Table S4). M. capitata tissue lipids, 312

carbohydrates and energy content did not differ among periods (p ≥ 0.073), sites (p ≥ 0.093) or 313

between bleached and non-bleached colonies (p ≥ 0.267) (Fig. 6b-d), although carbohydrate 314

biomass tended to be higher in January 2015 relative to October 2014. 315

316

317


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Porites compressa chlorophyll content differed according to period × condition (p <0.001) and 318

site × condition (p = 0.008) interactions (Fig. 5c, Fig. S5). In October, chlorophyll in bleached P. 319

compresa was reduced by 84 % (Reef 44), 78 % (Reef 25), and 92 % (HIMB) relative to non-320

bleached corals. By January, chlorophyll was equivalent between all P. compressa at Reef 25 321

and 44, but chlorophyll recovery was suppressed in colonies at HIMB, with previously bleached 322

corals having 25 % less chlorophyll than corals that did not bleach. P. compressa total biomass 323

was on average 19 % higher in non-bleached relative to bleached colonies (p = 0.025) but did not 324

differ among periods or sites (p ≥ 0.173) (Fig. 5d). 325

326

Porites compressa protein biomass was affected by period × condition (p = 0.011) (Fig. 6e, 327

Table S5). In October, bleached colonies had 20 % more protein biomass than non-bleached 328

corals, however, previously bleached colonies in January had 20 % less protein biomass relative 329

to colonies that did not bleach. Tissue lipids and energy content did not differ among bleached 330

and non-bleached P. compressa (p ≥ 0.179) but the period × site interaction (p ≤ 0.008). At the 331

time of bleaching, P. compressa lipids and biomass energy content was equivalent among reefs, 332

being 0.386 – 0.440 g lipids gdw-1 and 19 – 20 kJ gdw-1, respectively (Fig. 6f,h). However, three 333

months post-bleaching, tissue lipids and energy content had declined by ca. 27 % and 18 %, 334

respectively, in Reef 44 and Reef 25 P. compressa but were unchanged in corals from HIMB; 335

carbohydrate biomass showed no significant changes during the study (p ≥ 0.114) (Fig. 6g). 336

337

Tissue isotopic analysis 338

The carbon isotopic composition of M. capitata host (δ13CH) tissues was on average 0.7 ‰ 339

higher in bleached relative to non-bleached colonies (p = 0.022), while mean symbiont δ13C 340


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(δ13CS) was lower (0.7 ‰) during bleaching and increased during post-bleaching recovery (p = 341

0.001). Host and symbiont δ13C did not differ among sites (p ≥ 0.073) (Table 1, Table S6), 342

although M. capitata δ13CH and δ13CS values tended to be lower at HIMB and increased along a 343

northern gradient (Fig. 7a). The relative difference in M. capitata host and symbiont δ13C values 344

(δ13CH-S) – a metric for greater proportion of autotrophic (positive values) and heterotrophic 345

(negative values) derived carbon – changed over time, with higher δ13CH-S values in October and 346

a decline in δ13CH-S values in January (p = 0.001) (Fig. 7c) and slightly higher δ13CH-S values 347

(0.3 ‰) in bleached relative to non-bleached corals (p = 0.050). Nitrogen isotopic composition 348

of M. capitata host (δ15NH) and Symbiodinium (δ15NS) tissues did not differ among time periods 349

(p ≥ 0.381) or among bleaching conditions (p ≥ 0.541) (Fig. 7e-f). However, δ15NH values (mean 350

± SE) were spatially dependent (p = 0.042), being highest at HIMB (5.4 ± 0.1 ‰) and lowest at 351

Reef 25 (4.4 ± 0.1 ‰) but not statistically different at Reef 44 compared to other locations (4.6 ± 352

0.1 ‰) (Fig. 7d). δ15NH-S values (p ≥ 0.066) showed no statistically significant effects (Fig. 7e-f). 353

M. capitata C:NH was affected by period × condition (p = 0.046) with no differences in C:NH 354

among bleached and non-bleached corals in October, but a slightly larger increase in C:NH in 355

bleached (9 %) relative to non-bleached colonies (4 %) in January (Fig. S2a). C:NS (p ≥ 0.064) 356

was unaffected across the study (Fig. S2b, Table S6). 357

358

P. compressa host carbon isotopic composition was affected by the interaction of period × site × 359

condition (p = 0.032) (Table 1, Table S7). δ13CH values were comparable among all corals and 360

sites in October during bleaching. During January recovery, however, previously bleached 361

colonies at HIMB were on average enriched in 13C by 2 ‰ relative non-bleached colonies, while 362

bleached colonies at Reef 25 and Reef 44 did not differ from each other (Fig. 7g). δ13CS values 363


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were affected by the period × condition interaction (p = 0.048). Coral condition did not affect P. 364

compressa δ13C in October, but in January δ13CS values in previously bleached corals were 1 ‰ 365

higher relative non-bleached colonies, although largely driven by δ13CS in HIMB colonies (Fig. 366

7h). P. compressa δ13CH-S values did not differ over the study (p ≥ 0.136) (Fig. 7i). P. 367

compressa δ15NH values differed among periods (p = 0.014), sites (p <0.001), and was affected 368

by the period × condition interaction (p = 0.033), although this effect was not significant in post-369

hoc tests (p ≥ 0.078). Overall, mean δ15NH values were lower (0.4 ‰) in October compared to 370

January and higher (1 ‰) in colonies from HIMB relative to other sites (Fig. 7j). The nitrogen 371

isotopic composition of Symbiodinium differed among reef sites (p = 0.024) with Symbiodinium 372

becoming progressively 15N-enriched (1.2 ‰) from northern Reef 44 to southern HIMB (Fig. 7k). 373

δ15NS values were also higher (1.1 ‰) in bleached corals relative to non-bleached corals in 374

October, but not January (p = 0.009). This corresponded to lower P. compressa δ15NH-S values 375

(p = 0.001) for bleached colonies relative non-bleached corals (p = 0.001) during October alone 376

(Fig. 7l). P. compressa C:NH was higher in bleached relative to non-bleached colonies in 377

October and January (p <0.001) (Table S7). While C:NH was affected by period × site (p = 378

0.004), in post-hoc tests C:NH did not differ among sites within each period (Fig. S2c). C:NS 379

showed no significant effects (p ≥ 0.085) (Fig. S2d). 380

381

Mass balance calculations between total biomass and constituent compounds (i.e., proteins, 382

lipids, carbohydrates) (Hayes 2001) produced estimates for compound-specific δ13C values (i.e., 383

δ13CCompound) from coral holobiont δ13C values (i.e., δ13CHolobiont) in bleached and non-bleached 384

corals at the time of thermal stress (Fig. S3). During bleaching recovery the relationship 385

between the expected-δ13CHolobiont (calculated from δ13CCompound values and measured compound 386


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proportions) and the observed-δ13CHolobiont values was significant in both M. capitata (R2 = 0.88, 387

p <0.001) and P. compressa (R2 = 0.56, p <0.001) (Fig. 8). Thus, a significant proportion (56 – 388

88 %) of the observed variance in δ13CHolobiont values in both species during bleaching recovery 389

can be explained by changes in the relative abundance of carbohydrates, lipids and protein in 390

tissues and not changes in nutritional modes. 391

392

Discussion 393

A half-century of assessing the causes and consequences of coral bleaching has advanced our 394

understanding coral bleaching mechanisms (Weis et al. 2008) and the impact of environmental 395

and biological factors that influence bleaching sensitivity and resilience. However, few studies 396

have monitored changes in coral physiology and nutritional plasticity during and after large scale 397

bleaching events (Fitt et al. 1993; Edmunds et al. 2003; Rodrigues et al. 2008; Grottoli et al. 398

2011) or evaluated local environmental effects on physiological conditions that shape bleaching 399

recovery (Cunning et al. 2016). We observe rapid post-bleaching recovery Montipora capitata 400

and Porites compressa from three reefs spanning 6.3 km along Kāne‘ohe Bay. With the 401

exception of chlorophyll and total biomass at the time of bleaching, spatial and/or temporal 402

effects influenced coral physiology and tissue isotopic values at a level equivalent to, or greater 403

than, differences between bleached and non-bleached corals. However, spatial effects were not 404

equivalent in each coral species, indicating sensitivity to local conditions determines both 405

trajectories of bleaching as well as post-bleaching recovery. Individual and interactive effects of 406

site were most abundant for isotopic values in both species, whereas site effects on coral 407

physiology (chlorophylls, lipids, energy content) were limited to P. compressa alone. These 408

results confirm significant variance in the bleaching and recovery responses of two coral species 409


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at small spatial scales and emphasize environmental influences before, during, and after thermal 410

stress are integral in shaping physiological outcomes for corals, as well as the mechanisms of 411

physiological resilience. 412

413

Environmental context, bleaching, and recovery 414

Bleaching and subsequent recovery can be influenced by local environmental factors, such as 415

light, salinity, water motion, and dissolved nutrients in addition to thermal stress (Coles and 416

Jokiel 1977; Nakamura and van Woesik 2001; Anthony et al. 2007; Wiedenmann et al. 2012). In 417

addition, seawater cooling, including the influence of hurricanes, can benefit corals during and 418

after periods of thermal stress (Manzello et al. 2007). In the present study, cooling 419

corresponding with the passage of Hurricane Ana by the Hawaiian Islands (ca. 17 – 23 October 420

2014; NOAA 2018) days before our sampling (24 October 2014) may have mitigated further 421

physiological stress (Fig. S1a). Seawater temperatures among the three locations were similar. 422

However, Reef 44 in northern Kāne‘ohe Bay (Fig. 1a) had 27 % less light (Fig. S1), higher 423

[N+N], and a trend for higher ammonium and silicate concentrations and rates of sedimentation 424

(Fig. 2). Kāne‘ohe Bay encompasses several distinct flow regimes [northern (< 1 d) to southern 425

(> 30 d) (Lowe et al. 2009)] and is exposed to diverse nutrient inputs (runoff from watersheds, 426

streams, groundwater) (Drupp et al. 2001; Dulai et al. 2016). In Hawai‘i, subterranean 427

groundwater discharge (SGD) inputs can be 2 – 5-fold greater than coastal drainage (i.e., 428

watershed, streams) (Garrison et al. 2003; Dulai et al. 2016) and is a major source of silicate and 429

dissolved inorganic nitrogen (DIN) fluxes, whereas streams and runoff are dissolved inorganic 430

phosphorous (DIP) sources (Dulai et al. 2016). Nutrient enrichment can harm corals by reducing 431

growth (Silbiger et al. 2018) and increasing coral bleaching severity disease (Vega-Thurber et al. 432


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2014). Conversely, moderate nutrient enrichment and stochastic nutrient perturbations may 433

benefit corals post-bleaching by stimulating Symbiodinium growth (Sawall et al. 2014) and 434

plankton biomass (Selph et al. 2018) to the benefit of coral energy acquisition. Background 435

nutrient concentrations reported here (N:P range: 0.6 – 10.5) are below those reported in cases 436

where nutrient enrichment produced negative effects (i.e., bleaching, tissue loss) on corals [N:P 437

of 255:1 (Rosset et al. 2017), 22:1 and 43:1 (Wiedenmann et al. 2012)]. Therefore, the spatial 438

distribution of PAR (Cunning et al. 2016) and dissolved nutrients may explain some site-specific 439

differences in the biology of bleached and non-bleached corals during and after thermal stress 440

but do not appear to have interacted with accumulated heat stress to exacerbate bleaching 441

responses or impair post-bleaching recovery. 442

443

Physiological impacts of bleaching and recovery 444

Three months after a regional bleaching event the bleached corals had regained 445

photopigmentation and were indistinguishable from non-bleached conspecifics, with the 446

exception of moderately lower chlorophyll in bleached P. compressa at HIMB. Bleaching 447

recovery can be affected by the magnitude and/or duration of thermal stress (Bahr et al. 2017; 448

Claar et al. 2018), as well as the capacity for cellular and genetic properties of Symbiodinium and 449

host genotypes to mitigate cellular damage during bleaching (Weis et al. 2008; Kenkel et al. 450

2013). Interactions of host genotype and environmental history (Kenkel and Matz 2016) may be 451

particularly important in thermotolerance and influence site-specific recovery trajectories in P. 452

compressa, which hosts only clade C15 symbionts (LaJeunesse et al. 2004). Alternatively, 453

flexible symbiont partnerships in M. capitata among bleaching phenotypes [this study: bleached, 454

C-dominated; non-bleached, D- or C-dominated (Cunning et al. 2016)] and habitats (Innis et al. 455


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2018) indicate thermal stress responses and recovery outcomes in this species might be expected 456

to be a function of host and symbiont combinations and environmental conditions (Cunning et al. 457

2016). 458

459

Energy inputs available to corals during the recovery process, such as tissue biomass (Anthony et 460

al 2009; Thornhill et al. 2011), heterotrophy (Grottoli et al. 2006; Connolly et al. 2012) and 461

dissolved nutrients (Sawall et al. 2014), can provide an energetic context to support holobiont 462

function and symbiont growth (Palardy et al. 2008; Hughes and Grottoli 2013). However, rapid 463

recovery rates observed here over short periods do not negate possible long-term effects of 464

bleaching. For instance, in many corals bleaching can reduce long-term reproduction capacity 465

(Levitan et al. 2014), alter tissue biochemistry (Rodrigues and Grottoli 2007; Baumann et al. 466

2014), and affects gene expression (Pinzón et al. 2015) several months and up to a year after the 467

onset of thermal stress (Rodrigues and Grottoli 2007; Schoepf et al. 2015; Thomas and Palumbi 468

2017). Moreover, effects of repeat bleaching events can be complex and multiplicative, reducing 469

coral physiological resilience long-term (Grottoli et al. 2014). Therefore, it is important to 470

recognize short-term recovery of pigmentation and biomass (Fig. 5) as one part of the bleaching 471

condition, while acknowledging the uncertainty in long-term effects of bleaching on coral 472

biology after Symbiodinium repopulation. 473

474

Coral host biomass quantity (i.e., total biomass), quality (i.e., % lipids) and thickness are 475

important determinants for environmental stress resilience and post-bleaching survival (Loya et 476

al. 2001; Anthony et al. 2009; Thornhill et al. 2011). In the present study, bleached colonies of 477

both species had between 25 – 30 % less biomass than non-bleached corals, and during post-478


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bleaching recovery, changes in tissue biomass were species-specific and dependent on bleaching 479

history. Bleached M. capitata recovered biomass quickly after bleaching (< 3 months) (Fig. 5). 480

In contrast, biomass in bleached P. compressa colonies remained low (17 % less than non-481

bleached colonies) at both time periods. These results agree with laboratory experiments, where 482

bleaching reduced M. capitata and P. compressa biomass quickly, but rates of biomass recovery 483

are species-specific and much slower in P. compressa (4 – 6 months post-bleaching) compared 484

to M. capitata (1.5 months) (Grottoli et al. 2006; Rodrigues and Grotolli 2007). Declining 485

biomass (i.e., g AFDW cm-2) in bleached corals (Porter et al. 1989) can reflected a combination 486

of tissue catabolism (Rodrigues and Grottoli 2007) and/or cellular detachment (Gates et al. 1992) 487

resulting in 34 – 50 % decline in tissue biomass (Fitt et al. 1993; Grottoli et al. 2006). Low 488

tissue biomass in bleached corals might also be due to lower biomass in bleaching phenotypes 489

prior to the onset of bleaching which may also influence the susceptibility to thermal stress and 490

mortality (Thornhill et al. 2011). 491

492

In October, M. capitata and P. compresa biomass composition did not differ between bleached 493

and non-bleached corals. Three months later, M. capitata proteins were 20 % lower relative to 494

October (Fig. 6). Over the same period, P. compressa biomass energy (kJ gdw-1) fell by 12 %, 495

and tissue lipids at Reef 25 and Reef 44 fell by 20 % (Fig. 6). Host C:N, however, did differ 496

between bleached and non-bleached colonies during (P. compressa) and following bleaching 497

(both species) (Fig. S2). Lower C:NH in bleached P. compressa in October indicates a general 498

decline in biomass carbon relative to protein (Bodin et al. 2007), whereas higher C:NH in 499

previously bleached colonies of both species during recovery from bleaching suggest an 500


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increased breakdown and/or decreased acquisition of nitrogen (M. capitata) and carbon (P. 501

compressa) in these species. 502

503

Differential biomass utilization among species can relate to metabolic demand. Higher 504

metabolic rates, and a lower photosynthesis:respiration ratio in P. compressa relative to M. 505

capitata (Coles and Jokiel 1977) may determine the differential metabolism of high-energy lipids 506

(P. compressa) or proteins (M. capitata) based on energy requirements (Rodrigues and Grottoli 507

2007). Energetic investments in tissue biomass are also size-dependent (Anthony et al. 2002) 508

and tissue biomass (and its composition) can change along the surface of coral tissue (Oku et al. 509

2002). As a result, tissues would necessarily differ among small fragments in vitro and larger 510

intact colonies in situ. Changes in biomass composition and energy (Fig. 6, Fig. S2) independent 511

of bleaching history may also relate to shared physiological challenges confronting both 512

bleaching susceptible and resistant corals (i.e., gene regulation, stress protein synthesis) (Kenkel 513

et al. 2013) and complex seasonal (Fitt et al. 2000) and site-specific environmental contexts (i.e., 514

light availability) (Patton et al. 1977; Anthony 2006) juxtaposed atop bleaching stress. Indeed, 515

while tissue composition (i.e., % proteins, lipids, carbohydrates) did not differ among bleached 516

and non-bleached corals at either time point, total biomass (mg cm-2) was reduced in bleached 517

phenotypes of both species in October 2014 (Fig. 5). While P. compressa and M. capitata can 518

rely on lipid catabolism to recover from bleaching (Grottoli et al. 2004; Rodrigues and Grottoli 519

2007), our results highlight the established role of total biomass as a metric for coral 520

performance and show biomass quantity may change substantially during bleaching and recovery 521

without appreciable change in its biochemical composition (g gdw-1) or energetic value (kJ gdw-522

1). 523


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524

Nutritional plasticity and tissue isotopic composition 525

The isotopic values of an organisms is linked to the constitutive biochemical composition of 526

tissues and the substrates acquired through diet and broken down in metabolism. In corals and 527

Symbiodinium the δ13C and δ15N values reflect the acquisition of nutrients through carbon 528

fixation, internal nutrient cycling, heterotrophic feeding, and the isotopic values of the inorganic 529

carbon and external nutrient pool (Swart et al. 2005b). Short-term increases in heterotrophic 530

nutrition, however, can be difficult to verify due to uncertainty in rates of tissue turnover and 531

changes in tissue composition, especially following physiological stress (Rodrigues and Grottoli 532

2006; Logan et al. 2008). Isotopic inference on nutritional plasticity are also made complicated 533

in corals by the translocation/recycling of metabolites between symbiotic partners (Reynaud et al. 534

2002; Einbinder et al. 2009), kinetic isotope fractionation in biological reactions (i.e., metabolic 535

isotope effects) (Land et al. 1975), and the isotopic composition of internal resource pools (Swart 536

et al. 2005b). For instance, the recovery of tissue biomass reserves in bleached corals is 537

compound specific (Rodrigues and Grottoli 2007; Schoepf et al. 2015) and the nutritional inputs 538

(i.e., autotrophy vs. heterotrophy) responsible for biomass growth differ among species and 539

according to time post-bleaching (Baumann et al. 2014). Changes in growth rates also influence 540

isotope values. In Symbiodinium and other microalgae δ13C values are influenced by rates of 541

photosynthesis and cell growth, where elevated rates of photosynthesis and growth produce 542

carbon limitations (Laws et al. 1995; Swart et al. 2005a) that reduce isotopic discrimination and 543

increase δ13C values. Conversely, light attenuation (Muscatine et al. 1989; Heikoop et al. 1998) 544

and decreased photosynthesis can increase 13C-discrimination and reduce δ13C values (but see 545

also, Rost et al. 2002). 546


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547

Isotopic discrimination and the preferential loss of light nitrogen (i.e., 14N) as metabolic waste 548

lead to consumer δ15N values being ~ 3.5 ‰ enriched relative to food sources (Minagawa and 549

Wada 1984). In corals, Symbiodinium assimilate 15N-depleted ammonium excreted by the host, 550

which in turn translocate 15N-depleted photosynthates to the coral host (Wang and Douglas 551

1998), thus producing an attenuated trophic enrichment factor of ca. 1 – 2 ‰ (Reynaud et al. 552

2009). Variance in δ15N values in Symbiodinium can reflect the assimilation of distinct nitrogen 553

species, (i.e., ammonium, nitrate), the rates of nitrogen flux in and out of cells, and enzymatic 554

reactions, particularly the reduction of nitrate (Granger et al. 2004). For instance, δ15N values of 555

Symbiodinium are predicted to increase when growth rates are elevated and nitrogen availability 556

is limited (Rodrigues and Grottoli 2006), although this depends on whether photosynthesis is 557

resource limited and growth is balanced (Granger et al. 2004). δ15N values of nitrogen sources at 558

the base of the food web can also influence Symbiodinium δ15N values (Heikoop et al. 2000) and 559

influence the isotopic composition of internal nutrient pools through contributions of isotopically 560

distinct metabolic end members (i.e., CO2, NH4+). δ15N values of DIN in coastal waters integrate 561

natural (δ15N 0 to 4 ‰) and anthropogenic nitrogen sources, including those from wastewater 562

sewage (7 to 38 ‰) and/or agriculture (-4 to 5 ‰) (see Dailer et al. 2010) delivered through 563

runoff and SGD (Richardson et al. 2017). Microbially mediated processes such as the 564

assimilation (i.e., phytoplankton) or removal (i.e., denitrification) of 14N can increase DIN δ15N 565

values, whereas newly fixed nitrogen inputs (δ15N of -1 to 0 ‰) (Sigman and Casciotti 2001) can 566

reduce DIN δ15N values. In Kāne‘ohe Bay, northern reefs experience greater oceanic and SGD 567

influence along with shorter seawater residence (Lowe et al. 2009; Dulai et al. 2016), whereas 568

southern reefs are exposed to high stream input (30 % of bay total) and legacy effects of sewage 569


https://doi.org/10.1101/414086

dumping (1951 – 1978) (Smith et al. 1981). Therefore, site-specific patterns in δ15N values in 570

both coral species observed here indicate the influence of seawater hydrodynamics and nutrient 571

sources on baseline stable isotope values across Kāne‘ohe Bay. 572

573

M. capitata host and symbiont δ13C values showed different responses. δ13CH values were higher 574

in bleached corals, while δ13CS values were low during bleaching in October 2014 and increased 575

during recovery in January 2015 (Fig. 7a-b). M. capitata δ13CH-S values were also higher in 576

bleached corals throughout the study, but were higher in. Conversely, effects on P. compressa 577

host and symbiont δ13C were limited to January alone, where δ13CS values were higher in 578

bleached versus non-bleached colonies at all sites but only at HIMB for δ13CH. Lower δ13C 579

values can result from greater feeding on particles (i.e., plankton, organic particles) with low-580

δ13C values (Levas et al. 2013; Grottoli et al. 2017) and the preferential utilization of 581

heterotrophic nutrition in lipid biosynthesis (Alamaru et al. 2009; Baumann et al. 2014), or 582

reduced photosynthesis rates and greater 13C-discrimination (Muscatine et al. 1989; Laws et al. 583

1995; Swart et al. 2005b). In this case, M. capitata δ13CH-S values do not support a greater 584

reliance heterotrophic feeding in bleached corals, but instead suggest differences in host tissue 585

δ13C during both bleaching and recovery along with seasonal effects on δ13CS independent of 586

bleaching history. For P. compressa, however, δ13C values were dependent on colony bleaching 587

history as well as site-specific effects on the host, especially at HIMB where PAR is greatest, 588

seawater residence times are prolonged, and chlorophyll recovery was incomplete (Fig. 7g-h). In 589

both species, changes in the proportion of proteins, lipids, carbohydrates, and their isotopic 590

composition may be particularly salient in explaining δ13C variance. 591

592


https://doi.org/10.1101/414086

Organism bulk δ13C values are affected by their biochemical compositions (Logan et al. 2008; 593

Alamaru et al. 2009). Isotope mass balance calculations (Fig. 8) show that the majority of 594

variance in M. capitata and P. compressa δ13CHolobiont values (88% and 55%, respectively, Fig 8) 595

can be explained by changes in the relative proportions of compounds (i.e., proteins, lipids, 596

carbohydrates) independent of bleaching history. However, it should be acknowledged that 597

changes in tissue growth/metabolism and the sources for biosynthesis also alter compound-δ13C 598

values (Baumann et al. 2014). Corals are lipid rich, and lipids are depleted in 13C relative to bulk 599

tissues (Hayes 2001). The breakdown of lipids during bleaching, therefore, is expected to lead to 600

small increases in δ13C values of remaining lipid fraction and organism δ13C (DeNiro and 601

Epstein 1977). However, bleached corals can catabolize isotopically light lipids, leaving residual 602

tissues relatively enriched in 13C (Grottoli and Rodrigues 2011). Should tissue lipids depart from 603

predicted relationships (Hayes 2001) during bleaching and recovery – bring either 3 ‰ lower or 604

higher than non-bleached corals – the predictive power of our modeled relationship in observed- 605

and expected-δ13C values is lessened [48 and 67 % (M. capitata) and 27 and 36 % (P. 606

compressa) variance explained, respectively]. Therefore, changes in the relative proportions of 607

proteins, lipids, and carbohydrates and not their isotopic composition may best explain changes 608

in the bulk δ13C values of corals in this study. Changes in compound-δ13C values can occur 609

under physiological stress (Grottoli and Rodrigues 2011) and in response to changing resources 610

availability (Alamaru et al. 2009), however, few examples of compound-specific isotope values 611

for coral tissues exist [lipids (Alamaru et al. 2009; Grottoli and Rodrigues 2011), coral skeletal 612

organic matrix (Muscatine 2005)]. Nevertheless, changes in biomass composition effectively 613

explain patterns in δ13C values of both species used in this study, albeit an understanding of 614


https://doi.org/10.1101/414086

baseline isotopic values for coral tissue compounds is needed to better discern effects of habitat, 615

environment, and nutrition in reef corals. 616

617

Unlike most predator-prey relationships, greater heterotrophic nutrition in corals does not lead to 618

appreciable higher δ15N in coral relative to Symbiodinium (Reynaud et al. 2009). M. capitata 619

and P. compressa δ15N values were highest at HIMB relative to other sites, but were within the 620

range of δ15N values of nitrate in Kāne‘ohe Bay (4 – 5 ‰) (Table S2), and δ15N values here 621

support spatial variability in the sources and isotopic values of DIN δ15N (Heikoop et al 2000; 622

Nahon et al. 2013). Similar patterns of higher δ15N values in southern Kāne‘ohe Bay were also 623

seen in juvenile brown stingray (Dasyatis lata) known to have a fairly constant diet (Dale et al. 624

2011), indicating conservation in δ15N spatial patterns among trophic levels. Higher δ15NH 625

values in all P. compressa in January – driven largely by corals at HIMB – may also be 626

influenced by nitrogen acquisition deficits or changes in amino-acid synthesis/deamination, 627

reductions in prey capture (Reynaud et al. 2009), and changes in nitrogen concentration of 628

heterotrophic prey (Haubert et al. 2005) and autotrophic products (Tanaka et al. 2006). 629

630

P. compressa δ15NS values differed from the host, being higher in October relative to January, 631

and in particular, 2 ‰ higher in non-bleached Reef 25 P. compressa relative to bleached colonies. 632

At the same time, the predicted +1.5 ‰ enrichment (i.e., δ15NH-S) reversed and was negative for 633

bleached P. compressa at Reef 25 and HIMB colonies, suggesting disruption of nitrogen 634

recycling (Wang and Douglas 1998) in bleached corals and/or contributions of nitrogen not 635

originating from animal metabolism. These low δ15NS values may indicate a greater utilization 636

of a 15N-depleted DIN source, possibly from N2-fixation by coral-associated diazotrophs 637


https://doi.org/10.1101/414086

(Bednarz et al. 2017) or decreased rates of growth and nitrogen demand in non-bleached coral 638

symbionts (Heikoop et al. 1998; Baker et al. 2013). Increased δ15NS values in bleached P. 639

compressa agrees with other studies (Rodrigues and Grottoli 2006; Bessell-Browne et al. 2014; 640

Schoepf et al. 2015) that suggest increased rates of mitotic cell division and photopigment 641

synthesis post-bleaching increases Symbiodinium nitrogen demand resulting in reduced nitrogen 642

isotope fractionation (Heikoop et al. 1998). An increase in δ15NS values at the time of bleaching 643

is intriguing, as this suggests symbiont repopulation proceeds rapidly following peak thermal 644

stress. The capacity for rapid nitrogen assimilation post-bleaching may be an important factor in 645

physiological resilience of corals, and may be shaped by Symbiodinium functional diversity 646

(Baker et al. 2013), properties of the coral host (Loya et al. 2001), and the extent of physiological 647

stress. 648

649

Conclusion 650

The biochemical and isotopic composition of coral host and Symbiodinium biomass differ among 651

coral species in response to changes in physiological condition and site-specific environmental 652

contexts experienced by the holobiont. Our analyses of bleached and non-bleached corals during 653

and after a regional bleaching event at three reef sites revealed tissue biomass and chlorophyll to 654

be most affected by bleaching. Photopigment and total biomass recovery was rapid in M. 655

capitata but lagged in P. compressa, suggesting longer post-bleaching recovery times for this 656

species. Surprisingly, bleaching history did not significantly affect energy reserves in either 657

species. Instead, protein (M. capitata) and lipids (P. compressa) declined over time, and showed 658

significant differences among sites (P. compressa). Significant spatiotemporal effects on δ13C 659

values in both species were largely explained by changes in the relative proportions of proteins, 660


https://doi.org/10.1101/414086

lipids, and carbohydrates, and neither species appeared to increase heterotrophic nutrition. δ15N 661

values indicated baseline differences in the isotopic composition of nitrogen sources. However, 662

bleaching conditions influenced P. compressa δ15NS but did not affect M. capitata. Taken 663

together, these results shed light on the dynamic effects of bleaching and post-bleaching recovery 664

and emphasize the biology of bleached and non-bleached corals (during and following thermal 665

stress) are determined by environmental contexts that may vary over small spatial scales or 666

seasonal periods. Finally, our results identify the need to further quantity effects of changing 667

tissue composition on isotopic values in corals, as this may reveal insights into the metabolism, 668

nutrition, and performance of reefs corals across space and time. 669

670

Acknowledgments 671

The author’s thank A. Grottoli, L. Rodrigues, and J. Sparks for discussions on stable isotope, N. 672

Wallsgrove, C. Lyons, and W. Ko for stable isotope analyses, W. Ellis and J. Davidson for 673

laboratory support, C. Hunter and NOAA Marine Education and Training Grant 674

(NA17NMF4520161) for assistance in seawater nutrient analysis, and A. Amend, M. Donahue, 675

A. Moran, and E.A. Lenz for constructive comments. CBW was supported by an Environmental 676

Protection Agency (EPA) STAR Fellowship Assistance Agreement (FP-91779401-1). The 677

views expressed in this publication have not been reviewed or endorsed by the EPA and are 678

solely those of the authors. 679

680

References 681

Alamaru, A., Y. Loya, E. Brokovich, R. Yam, and A. Shemesh. 2009. Carbon and nitrogen 682utilization in two species of Red Sea corals along a depth gradient: Insights from stable 683


https://doi.org/10.1101/414086

isotope analysis of total organic material and lipids. Geochim. Cosmochim. Acta 73: 6845333–5342. 685

Anthony, K. R. N. 2006. Enhanced energy status of corals on coastal, high-turbidity reefs. Mar. 686Ecol. Prog. Ser. 319: 111–116. 687

Anthony, K. R. N., M. O. Hoogenboom, J. A. Maynard, A. G. Grottoli, and R. Middlebrook. 6882009. Energetics approach to predicting mortality risk from environmental stress: a case 689study of coral bleaching. Funct. Ecol. 23: 539–550. 690

Anthony, K. R. N., S. R. Connolly, and B. L. Willis. 2002. Comparative analysis of energy 691allocation to tissue and skeletal growth in corals. Limnol. Oceanogr. 47: 1417–1429. 692

Anthony, K. R. N., S. R. Connolly, and O. Hoegh-Guldberg. 2007. Bleaching, energetics, and 693coral mortality risk: effects of temperature, light, and sediment regime. Limnol. Oceanogr. 69452: 716–726. 695

Anthony, K. R., and K. E. Fabricius. 2000. Shifting roles of heterotrophy and autotrophy in coral 696energetics under varying turbidity. J. Exp. Mar. Bio. Ecol. 252: 221–253. 697

Bahr, K. D., K. S. Rodgers, and P. L. Jokiel. 2017. Impact of Three Bleaching Events on the 698Reef Resiliency of Kāne‘ohe Bay, Hawai‘i. Front. Mar. Sci. 4: 435. 699

Bahr, K. D., P. L. Jokiel, and K. S. Rodgers. 2015. The 2014 coral bleaching and freshwater 700flood events in Kāne’ohe Bay, Hawai'i. PeerJ 3: e1136. 701

Baker, D. M., J. P. Andras, A. G. Jordán-Garza, and M. L. Fogel. 2013. Nitrate competition in a 702coral symbiosis varies with temperature among Symbiodinium clades. ISME J. 7: 1248–7031251. 704

Bates, D., M. Maechler, B. Bolker, and S. Walker (2015). Fitting Linear Mixed-Effects Models 705Using lme4. J. Stat. Softw. 67:1–48. 706

Baumann, J., A. G. Grottoli, A. D. Hughes, and Y. Matsui. 2014. Photoautotrophic and 707heterotrophic carbon in bleached and non-bleached coral lipid acquisition and storage. J. 708Exp. Mar. Bio. Ecol. 461: 469–478. 709

Bednarz, V. N., R. Grover, J.-F. Maguer, M. Fine, and C. Ferrier-Pagès. 2017. The assimilation 710of diazotroph-derived nitrogen by scleractinian corals depends on their metabolic status. 711MBio 8: e02058-16. 712

Bessell-Browne, P., M. Stat, D. Thomson, and P. L. Clode. 2014. Coscinaraea marshae corals 713that have survived prolonged bleaching exhibit signs of increased heterotrophic feeding. 714Coral Reefs 33: 795–804. 715

Bodin, N., F. Le Loc’h, and C. Hily. 2007. Effect of lipid removal on carbon and nitrogen stable 716isotope ratios in crustacean tissues. J. Exp. Mar. Bio. Ecol. 341: 168–175. 717


https://doi.org/10.1101/414086

Claar, D. C., L. Szostek, J. M. McDevitt-Irwin, J. J. Schanze, and J. K. Baum. 2018. Global 718patterns and impacts of El Niño events on coral reefs: A meta-analysis. PLoS One 13: 719e0190957. 720

Coles, S. L., and P. L. Jokiel. 1977. Effects of temperature on photosynthesis and respiration in 721hermatypic corals. Mar. Biol. 43: 209–216. 722

Connolly, S. R., M. A. Lopez-Yglesias, and K. R. N. Anthony. 2012. Food availability promotes 723rapid recovery from thermal stress in a scleractinian coral. Coral Reefs 31: 951–960. 724

Couch, C. S., J. H. R. Burns, G. Liu, K. Steward, T. N. Gutlay, J. Kenyon, C. M. Eakin, and R. K. 725Kosaki. 2017. Mass coral bleaching due to unprecedented marine heatwave in 726Papahānaumokuākea Marine National Monument (Northwestern Hawaiian Islands). 727PLoS One 12: e0185121. 728

Cunning, R., R. Ritson-Williams, and R. D. Gates. 2016. Patterns of bleaching and recovery of 729Montipora capitata in Kāne‘ohe Bay, Hawai‘i, USA. Mar. Ecol. Prog. Ser. 551: 131–139. 730

Dailer, M. L., R. S. Knox, J. E. Smith, M. Napier, and C. M. Smith. 2010. Using δ15N values in 731algal tissue to map locations and potential sources of anthropogenic nutrient inputs on the 732island of Maui, Hawai’i, USA. Mar. Pollut. Bull. 60: 655–671. 733

Dale, J. J., N. J. Wallsgrove, B. N. Popp, and K. N. Holland. 2011. Nursery habitat use and 734foraging ecology of the brown stingray Dasyatis lata determined from stomach contents, 735bulk and amino acid stable isotopes. Mar. Ecol. Prog. Ser. 433: 221–236. 736

DeNiro, M. J., and S. Epstein. 1977. Mechanism of Carbon Isotope Fractionation Associated 737with Lipid Synthesis. Science 197: 261–263. 738

Drupp, P., E. H. De Carlo, F. T. Mackenzie, P. Bienfang, and C. L. Sabine. 2011. Nutrient inputs, 739phytoplankton response, and CO2 variations in a semi-enclosed subtropical embayment, 740Kaneohe Bay, Hawaii. Aquat. Geochem. 17: 473–498. 741

DuBois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric 742Method for Determination of Sugars and Related Substances. Anal. Chem. 28: 350–356. 743

Dulai, H., A. Kleven, K. Ruttenberg, R. Briggs, and F. Thomas. 2016. Evaluation of submarine 744groundwater discharge as a coastal nutrient source and its role in coastal groundwater 745quality and quantity, p. 187–221. In A. Fares [ed.], Emerging Issues in Groundwater 746Resources. Springer International Publishing. 747

Edmunds, P. J., R. D. Gates, and D. F. Gleason. 2003. The tissue composition of Montastraea 748franksi during a natural bleaching event in the Florida Keys. Coral Reefs 22: 54–62. 749

Einbinder, S., T. Mass, E. Brokovich, Z. Dubinsky, J. Erez, and D. Tchernov. 2009. Changes in 750morphology and diet of the coral Stylophora pistillata along a depth gradient. Mar. Ecol. 751Prog. Ser. 381: 167–174. 752


https://doi.org/10.1101/414086

Ferrier-Pagès, C., C. Rottier, E. Beraud, and O. Levy. 2010. Experimental assessment of the 753feeding effort of three scleractinian coral species during a thermal stress: Effect on the 754rates of photosynthesis. J. Exp. Mar. Bio. Ecol. 390: 118–124. 755

Fitt, W. K., F. K. McFarland, M. E. Warner, and G. C. Chilcoat. 2000. Seasonal patterns of 756tissue biomass and densities of symbiotic dinoflagellates in reef corals and relation to 757coral bleaching. Limnol. Oceanogr. 45: 677–685. 758

Fitt, W. K., H. J. Spero, J. Halas, M. W. White, and J. W. Porter l. 1993. Recovery of the coral 759Montastrea annularis in the Florida Keys after the Caribbean “bleaching event.” Coral 760Reefs 12: 57–64. 761

Garrison, G. H., C. R. Glenn, and G. M. McMurtry. 2003. Measurement of submarine 762groundwater discharge in Kahana Bay, O’ahu, Hawai'i. Limnol. Oceanogr. 48: 920–928. 763

Gates, R. D., G. Baghdasarian, and L. Muscatine. 1992. Temperature Stress Causes Host Cell 764Detachment in Symbiotic Cnidarians: Implications for Coral Bleaching. Biol. Bull. 182: 765324–332. 766

Gnaiger, E., and G. Bitterlich. 1984. Proximate biochemical composition and caloric content 767calculated from elemental CHN analysis: a stoichiometric concept. Oecologia 62: 289–768298. 769

Granger, J., D. M. Sigman, J. A. Needoba, and P. J. Harrison. 2004. Coupled nitrogen and 770oxygen isotope fractionation of nitrate during assimilation by cultures of marine 771phytoplankton. Limnol. Oceanogr. 49: 1763–1773. 772

Grottoli, A. G., and L. J. Rodrigues. 2011. Bleached Porites compressa and Montipora capitata 773corals catabolize δ13C-enriched lipids. Coral Reefs 30: 687. 774

Grottoli, A. G., D. Tchernov, and G. Winters. 2017. Physiological and biogeochemical responses 775of super-corals to thermal stress from the northern Gulf of Aqaba, Red Sea. Front. Mar. 776Sci. 4: 215. 777

Grottoli, A. G., L. J. Rodrigues, and J. E. Palardy. 2006. Heterotrophic plasticity and resilience in 778bleached corals. Nature 440: 1186–1189. 779

Grottoli, A. G., M. E. Warner, S. J. Levas, M. D. Aschaffenburg, V. Schoepf, M. McGinley, J. 780Baumann, and Y. Matsui. 2014. The cumulative impact of annual coral bleaching can 781turn some coral species winners into losers. Glob. Chang. Biol. 20: 3823–3833. 782

Haubert, D., R. Langel, S. Scheu, and L. Ruess. 2005. Effects of food quality, starvation and life 783stage on stable isotope fractionation in Collembola. Pedobiologia 49: 229–237. 784

Hayes, J. M. 2001. Fractionation of carbon and hydrogen isotopes in biosynthetic processes. Rev. 785Mineral. Geochem. 43: 225–277. 786

Heikoop, J. M., J. J. Dunn, M. J. Risk, I. M. Sandeman, H. P. Schwartz, and N. Waltho. 1998. 787Relationship between light and the δ15N of coral tissue: Examples from Jamaica and 788Zanzibar. Limnol. Oceanogr. 43: 909–920. 789


https://doi.org/10.1101/414086

Heikoop, J. M., J. J. Dunn, M. J. Risk, T. Tomascik, H. P. Schwarcz, I. M. Sandeman, and P. W. 790Sammarco. 2000. δ15N and δ13C of coral tissue show significant inter-reef variation. 791Coral Reefs 19: 189–193. 792

Heron, S. F., J. A. Maynard, R. van Hooidonk, and C. Mark Eakin. 2016. Warming Trends and 793Bleaching Stress of the World’s Coral Reefs 1985–2012. Sci. Rep. 6: srep38402. 794

Hoogenboom, M. O., S. R. Connolly, and K. R. N. Anthony. 2011. Biotic and abiotic correlates 795of tissue quality for common scleractinian corals. Mar. Ecol. Prog. Ser. 438: 119–128. 796

Houlbrèque, F., and C. Ferrier-Pagès. 2009. Heterotrophy in Tropical Scleractinian Corals. Biol. 797Rev. Camb. Philos. Soc. 84: 1–17. 798

Hughes, A. D., and A. G. Grottoli. 2013. Heterotrophic compensation: a possible mechanism for 799resilience of coral reefs to global warming or a sign of prolonged stress? PLoS One 8: 800e81172. 801

Hughes, T. P., J. T. Kerry, M. Álvarez-Noriega, and others. 2017. Global warming and recurrent 802mass bleaching of corals. Nature 543: 373–377. 803

Innis, T., R. Cunning, R. Ritson-Williams, C. B. Wall, and R. D. Gates. 2018. Coral color and 804depth drive symbiosis ecology of Montipora capitata in Kāne‘ohe Bay, O‘ahu, Hawai‘i. 805Coral Reefs 37: 423–430. 806

Oksanen J., and others (2017). vegan: Community Ecology Package. R package version 2.4-2. 807

Jeffrey, S. W., and G. F. Humphrey. 1975. New spectrophotometric equations for determining 808chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. 809Physiol. Pflanz. 167: 191–194. 810

Kenkel, C. D., and M. V. Matz. 2016. Gene expression plasticity as a mechanism of coral 811adaptation to a variable environment. Nat Ecol Evol 1: 14. 812

Kenkel, C. D., E. Meyer, and M. V. Matz. 2013. Gene expression under chronic heat stress in 813populations of the mustard hill coral (Porites astreoides) from different thermal 814environments. Mol. Ecol. 22: 4322–4334. 815

Kuznetsova, A., P. B. Brockhoff, and R. H. B. Christensen. 2017. lmerTest package: tests in 816linear mixed effects models. J. Stat. Softw. 82: 1–26. 817

LaJeunesse, T. C., D. J. Thornhill, E. F. Cox, F. G. Stanton, W. K. Fitt, and G. W. Schmidt. 2004. 818High diversity and host specificity observed among symbiotic dinoflagellates in reef 819coral communities from Hawaii. Coral Reefs 23: 596–603. 820

Land, L. S., J. C. Lang, and B. N. Smith. 1975. Preliminary observations on the carbon isotopic 821composition of some reef coral tissues and symbiotic zooxanthellae1. Limnol. Oceanogr. 82220: 283–287. 823


https://doi.org/10.1101/414086

Laws, E. A., B. N. Popp, R. R. Bidigare, M. C. Kennicutt, and S. A. Macko. 1995. Dependence 824of phytoplankton carbon isotopic composition on growth rate and [CO2]aq: Theoretical 825considerations and experimental results. Geochim. Cosmochim. Acta 59: 1131–1138. 826

Lenth R. V. 2016. Least-Squares Means: The R Package lsmeans. J. Stat. Softw. 69: 1–33. 827

Levas, S. J., A. G. Grottoli, A. Hughes, C. L. Osburn, and Y. Matsui. 2013. Physiological and 828biogeochemical traits of bleaching and recovery in the mounding species of coral Porites 829lobata: implications for resilience in mounding corals. PLoS One 8: e63267. 830

Levas, S., A. G. Grottoli, V. Schoepf, M. Aschaffenburg, J. Baumann, J. E. Bauer, and M. E. 831Warner. 2016. Can heterotrophic uptake of dissolved organic carbon and zooplankton 832mitigate carbon budget deficits in annually bleached corals? Coral Reefs 35: 495–506. 833

Levitan, D. R., W. Boudreau, J. Jara, and N. Knowlton. 2014. Long-term reduced spawning in 834Orbicella coral species due to temperature stress. Mar. Ecol. Prog. Ser. 515: 1–10. 835

Logan, J. M., T. D. Jardine, T. J. Miller, S. E. Bunn, R. A. Cunjak, and M. E. Lutcavage. 2008. 836Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical 837extraction and modelling methods. J. Anim. Ecol. 77: 838–846. 838

Lowe, R. J., J. L. Falter, S. G. Monismith, and M. J. Atkinson. 2009. A numerical study of 839circulation in a coastal reef-lagoon system. J. Geophys. Res. 114: C06022. 840

Loya, Y., K. Sakai, K. Yamazato, Y. Nakano, H. Sambali, and R. van Woesik. 2001. Coral 841bleaching: the winners and the losers. Ecol. Lett. 4: 122–131. 842

Maier, C., M. G. Weinbauer, and J. Pätzold. 2010. Stable isotopes reveal limitations in C and N 843assimilation in the Caribbean reef corals Madracis auretenra, M. carmabi and M. 844formosa. Mar. Ecol. Prog. Ser. 412: 103–112. 845

Marubini, F., and P. S. Davies. 1996. Nitrate increases zooxanthellae population density and 846reduces skeletogenesis in corals. Mar. Biol. 127: 319–328. 847

Manzello, D. P., M. Brandt, T. B. Smith, D. Lirman, J. C. Hendee, and R. S. Nemeth. 2007. 848Hurricanes benefit bleached corals. Proc. Natl. Acad. Sci. USA 104: 12035–12039. 849

Minagawa, M., and E. Wada. 1984. Stepwise enrichment of 15N along food chains: Further 850evidence and the relation between δ15N and animal age. Geochim. Cosmochim. Acta 48: 8511135–1140. 852

Muscatine, L., C. Goiran, L. Land, J. Jaubert, J.-P. Cuif, and D. Allemand. 2005. Stable isotopes 853(δ13C and δ15N) of organic matrix from coral skeleton. Proc. Natl. Acad. Sci. USA 102: 8541525–1530. 855

Muscatine, L., J. W. Porter, and I. R. Kaplan. 1989. Resource partitioning by reef corals as 856determined from stable isotope composition. I. δ13C of zooxanthellae and animal tissue vs 857depth. Mar. Biol. 100: 185–193. 858


https://doi.org/10.1101/414086

Nakamura, T., and R. van Woesik. 2001. Water-flow rates and passive diffusion partially explain 859differential survival of corals during the 1998 bleaching event. Mar. Ecol. Prog. Ser. 212: 860301–304. 861

Nahon, S., N. B. Richoux, J. Kolasinski, M. Desmalades, C. Ferrier Pages, G. Lecellier, S. 862Planes, and V. Berteaux Lecellier. 2013. Spatial and temporal variations in stable carbon 863(δ13C) and nitrogen (δ15N) isotopic composition of symbiotic scleractinian corals. PLoS 864One 8: e81247. 865

NOAA. 2017. Tides and Currents. Mokuoloe, Hawaii, Station ID: 1612480. National Oceanic 866and Atmospheric Administration, USA. [accessed March 01 2017] 867https://tidesandcurrents.noaa.gov/stationhome.html?id=16124 80. 868

NOAA. 2018. Central Pacific Hurricane Center, National Atmospheric and Oceanic 869Administration. [accessed: May 21 2018] 870http://www.prh.noaa.gov/cphc/tcpages/archive.php?stormid=CP022014. 871

NOAA Coral Reef Watch. 2018. Main Hawaiian Islands 5-km Bleaching Heat Stress Gauges 872(Version 3). Jan 2014 – Jan 2015. College Park, Maryland, USA: NOAA Coral Reef 873Watch. [accessed: May 21 2018] https://coralreefwatch.noaa.gov/vs/gauges/hawaii.php 874

Oku, H., O. K. U. Hirosuke, H. Yamashiro, K. Onaga, H. Iwasaki, and K. Takara. 2002. Lipid 875distribution in branching coral Montipora digitata. Fish. Sci. 68: 517–522. 876

Palardy, J. E., L. J. Rodrigues, and A. G. Grottoli. 2008. The importance of zooplankton to the 877daily metabolic carbon requirements of healthy and bleached corals at two depths. J. Exp. 878Mar. Bio. Ecol. 367: 180–188. 879

Patton, J. S., S. Abraham, and A. A. Benson. 1977. Lipogenesis in the intact coral Pocillopora 880capitata and its isolated zooxanthellae: Evidence for a light-driven carbon cycle between 881symbiont and host. Mar. Biol. 44: 235–247. 882

Pinzón, J. H., B. Kamel, C. A. Burge, C. D. Harvell, M. Medina, E. Weil, and L. D. Mydlarz. 8832015. Whole transcriptome analysis reveals changes in expression of immune-related 884genes during and after bleaching in a reef-building coral. R. Soc. Open. Sci. 2: 140214. 885

Porter, J. W. 1976. Autotrophy, Heterotrophy, and Resource Partitioning in Caribbean Reef-886Building Corals. Am. Nat. 110: 731–742. 887

Porter, J. W., W. K. Fitt, H. J. Spero, C. S. Rogers, and M. W. White. 1989. Bleaching in reef 888corals: Physiological and stable isotopic responses. Proc. Natl. Acad. Sci. USA 86: 9342–8899346. 890

Reynaud, S., C. Ferrier-Pagès, R. Sambrotto, A. Juillet-Leclerc, J. Jaubert, and J. P. Gattuso. 8912002. Effect of feeding on the carbon and oxygen isotopic composition in the tissues and 892skeleton of the zooxanthellate coral Stylophora pistillata. Mar. Ecol. Prog. Ser. 238: 81–89389. 894


https://doi.org/10.1101/414086

Reynaud, S., P. Martinez, F. Houlbrèque, I. Billy, D. Allemand, and C. Ferrier-Pagès. 2009. 895Effect of light and feeding on the nitrogen isotopic composition of a zooxanthellate coral: 896role of nitrogen recycling. Mar. Ecol. Prog. Ser. 392: 103–110. 897

Richardson, C. M., H. Dulai, and R. B. Whittier. 2017. Sources and spatial variability of 898groundwater-delivered nutrients in Maunalua Bay, Oʻahu, Hawai‘i. J. Hydrol. Reg. Stud. 89911: 178–193. 900

Ritson-Williams, R., and R. D. Gates. 2016a. Kaneohe Bay Light Data 2014 and 2015. 901doi:10.5281/zenodo.160214. 902

Ritson-Williams, R., and R. D. Gates. 2016b. Kaneohe Bay Seawater Temperature Data 2014 903and 2015 - superseded.doi:10.5281/zenodo.46366. 904

Rodrigues, L. J., and A. G. Grottoli. 2006. Calcification rate and the stable carbon, oxygen, and 905nitrogen isotopes in the skeleton, host tissue, and zooxanthellae of bleached and 906recovering Hawaiian corals. Geochim. Cosmochim. Acta 70: 2781–2789. 907

Rodrigues, L. J., and A. G. Grottoli. 2007. Energy reserves and metabolism as indicators of coral 908recovery from bleaching. Limnol. Oceanogr. 52: 1874–1882. 909

Rodrigues, L. J., A. G. Grottoli, and T. K. Pease. 2008. Lipid class composition of bleached and 910recovering Porites compressa Dana, 1846 and Montipora capitata Dana, 1846 corals 911from Hawaii. J. Exp. Mar. Bio. Ecol. 358: 136–143. 912

Rosset, S., J. Wiedenmann, A. J. Reed, and C. D’Angelo. 2017. Phosphate deficiency promotes 913coral bleaching and is reflected by the ultrastructure of symbiotic dinoflagellates. Mar. 914Pollut. Bull. 118: 180–187. 915

Rost, B., I. Zondervan, and U. Riebesell. 2002. Light-dependent carbon isotope fractionation in 916the coccolithophorid Emiliania huxleyi. Limnol. Oceanogr. 47: 120–128. 917

Sampayo, E. M., T. Ridgway, P. Bongaerts, and O. Hoegh-Guldberg. 2008. Bleaching 918susceptibility and mortality of corals are determined by fine-scale differences in symbiont 919type. Proc. Natl. Acad. Sci. U. S. A. 105: 10444–10449. 920

Sawall, Y., A. Al-Sofyani, E. Banguera-Hinestroza, and C. R. Voolstra. 2014. Spatio-temporal 921analyses of Symbiodinium physiology of the coral Pocillopora verrucosa along large-922scale nutrient and temperature gradients in the Red Sea. PLoS One 9: e103179. 923

Scheufen, T., W. E. Krämer, R. Iglesias-Prieto, and S. Enríquez. 2017. Seasonal variation 924modulates coral sensibility to heat-stress and explains annual changes in coral 925productivity. Sci. Rep. 7: 4937. 926

Schoepf, V., A. G. Grottoli, S. J. Levas, M. D. Aschaffenburg, J. H. Baumann, Y. Matsui, and M. 927E. Warner. 2015. Annual coral bleaching and the long-term recovery capacity of coral. 928Proc. Biol. Sci. 282. doi:10.1098/rspb.2015.1887 929

Selph, K. E., E. Goetze, M. J. Jungbluth, P. H. Lenz, and G. Kolker. 2018. Microbial food web 930connections and rates in a subtropical embayment. Mar. Ecol. Prog. Ser. 590: 19–34. 931


https://doi.org/10.1101/414086

Shick, J. M., M. P. Lesser, and P. L. Jokiel. 1996. Effects of ultraviolet radiation on corals and 932other coral reef organisms. Glob. Chang. Biol. 2: 527–545. 933

Sigman, D. M., and K. L. Casciotti. 2001. Nitrogen isotopes in the ocean, p. 1884–1894. In J.H. 934Steele [ed.], Encyclopedia of Ocean Sciences. Academic Press. 935

Silbiger, N. J., C. E. Nelson, K. Remple, J. K. Sevilla, Z. A. Quinlan, H. M. Putnam, M. D. Fox, 936and M. J. Donahue. 2018. Nutrient pollution disrupts key ecosystem functions on coral 937reefs. Proc. Biol. Sci. 285. 938

Smith, P. K., R. I. Krohn, G. T. Hermanson, and others. 1985. Measurement of protein using 939bicinchoninic acid. Anal. Biochem. 150: 76–85. 940

Smith, S. V., W. J. Kimmerer, E. A. Laws, R. E. Brock, and T. W. Walsh. 1981. Kaneohe Bay 941sewage diversion experiment: perspectives on ecosystem responses to nutritional 942perturbation. Pac. Sci. 35: 279–395. 943

Stimson, J., and R. A. Kinzie. 1991. The temporal pattern and rate of release of zooxanthellae 944from the reef coral Pocillopora damicornis (Linnaeus) under nitrogen-enrichment and 945control conditions. J. Exp. Mar. Bio. Ecol. 153: 63–74. 946

Swart, P. K., A. Saied, and K. Lamb. 2005a. Temporal and spatial variation in the δ15N and δ13C 947of coral tissue and zooxanthellae in Montastraea faveolata collected from the Florida reef 948tract. Limnol. Oceanogr. 50: 1049–1058. 949

Swart, P. K., A. Szmant, J. W. Porter, R. E. Dodge, J. I. Tougas, and J. R. Southam. 2005b. The 950isotopic composition of respired carbon dioxide in scleractinian corals: implications for 951cycling of organic carbon in corals. Geochim. Cosmochim. Acta 69: 1495–1509. 952

Tanaka, Y., T. Miyajima, I. Koike, T. Hayashibara, and H. Ogawa. 2006. Translocation and 953conservation of organic nitrogen within the coral-zooxanthella symbiotic system of 954Acropora pulchra, as demonstrated by dual isotope-labeling techniques. J. Exp. Mar. Bio. 955Ecol. 336: 110–119. 956

Thomas, L., and S. R. Palumbi. 2017. The genomics of recovery from coral bleaching. Proc. Biol. 957Sci. 284. 20171790 doi:10.1098/rspb.2017.1790 958

Thornhill, D. J., R. D. Rotjan, B. D. Todd, and others. 2011. A connection between colony 959biomass and death in Caribbean reef-building corals. PLoS One 6: e29535. 960

Vega Thurber, R. L., D. E. Burkepile, C. Fuchs, A. A. Shantz, R. McMinds, and J. R. Zaneveld. 9612014. Chronic nutrient enrichment increases prevalence and severity of coral disease and 962bleaching. Glob. Chang. Biol. 20: 544–554. 963

Wall, C. B., R. A. B. Mason, W. R. Ellis, R. Cunning, and R. D. Gates. 2017. Elevated pCO2 964affects tissue biomass composition, but not calcification, in a reef coral under two light 965regimes. R. Soc. Open Sci. 4: 170683. 966

Wang, J., and A. E. Douglas. 1998. Nitrogen recycling or nitrogen conservation in an alga-967invertebrate symbiosis? J. Exp. Biol. 201: 2445–2453. 968


https://doi.org/10.1101/414086

Weis, V. M. 2008. Cellular mechanisms of Cnidarian bleaching: stress causes the collapse of 969symbiosis. J. Exp. Biol. 211: 3059–3066. 970

Wiedenmann, J., C. D’Angelo, E. G. Smith, A. N. Hunt, F.-E. Legiret, A. D. Postle, and E. P. 971Achterberg. 2012. Nutrient enrichment can increase the susceptibility of reef corals to 972bleaching. Nat. Clim. Chang. 3: 160–164. 973


https://doi.org/10.1101/414086

Wall, C.B., et al (in review) Spatial variation in the biochemical and isotopic composition of

corals during bleaching and recovery.

Tables

Table 1. Statistical analysis of bleached and non-bleached Montipora capitata and Porites

compressa at three Kāne‘ohe Bay patch reefs during bleaching and recovery*.

Response variable Species Montipora capitata Porites compressa

Oct ’14: Bleaching Jan ’15: Recovery Oct ’14: Bleaching Jan ‘15: Recovery

chlorophylls B < NB — B < NB HIMB: B < NB biomass B < NB — B < NB proteins 2014 > 2015 — — lipids — — HIMB > R44 = R25 carbohydrates — — energy content — 2014 > 2015 δ13CH B > NB — HIMB: B > NB δ13CS 2014 < 2015 — B > NB

δ13CH-S 2014 > 2015 B > NB —

δ15NH HIMB > R25 2014 < 2015 HIMB > R44 = R25

δ15NS — B > NB — HIMB > R44

δ15NH-S — B < NB — C:NH — B > NB B < NB B > NB C:NS — — *Periods are October 2014 bleaching and January 2015 recovery. Sites (north to south) are Reef 44 (R44), Reef 25 (R25) and the Hawai’i Institute of Marine Biology (HIMB). Corals are described according to their physiological condition in October 2014, being bleached (B) or non-bleached (NB); condition designators from October (i.e., B/NB) were retained in January after corals regained pigmentation. Subscripts indicate either host (H) or symbiont (S) tissues, or their relative difference (H-S). Dashed lines indicate no significant effects (p > 0.05).


https://doi.org/10.1101/414086

Figures

Fig. 1. (a) Map of Kāne‘ohe Bay on the windward side of O‘ahu, Hawai‘i, USA, showing study sites Reef 44, Reef 25, and HIMB (Hawai’i Institute of Marine Biology). Bleached and non-bleached (b) Montipora capitata and (c) Porites compressa during a regional thermal stress event in October 2014. Photo credit (b-c): CB Wall


https://doi.org/10.1101/414086

Fig. 2. Dissolved inorganic nutrient concentrations (November 2014 – February 2015) and sedimentation rates (January 2015 – January 2016) at Reef 44, Reef 25, and HIMB in Kāne‘ohe Bay. (a) Phosphate (PO4

3-), (b), ammonium (NH4+), (c) nitrate + nitrite (NO3

- + NO2-), and (d)

silicate (Si(OH)4) concentrations in seawater, and the (e) short-term and (f) annual sedimentation rates at the three reef sites. Symbols (*) indicate significant site effects (p ≤ 0.05).

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https://doi.org/10.1101/414086

Fig. 3. Multivariate non-metric multidimensional scaling (NMDS) plots for bleached (B) and non-bleached (NB) Montipora capitata at three reefs [Reef 44 (R44), Reef 25 (R25), HIMB] during bleaching (left panel) and recovery (right panel) a regional bleaching event. Polygons are standard error of point means (x symbols). (a, c) NMDS with site × condition effect. (b, d) NMDS with condition effect alone, with vectors showing significant responses (p ≤ 0.05) among bleached and non-bleached corals.

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https://doi.org/10.1101/414086

Fig. 4. Multivariate non-metric multidimensional scaling (NMDS) plots for bleached (B) and non-bleached (NB) Porites compressa at three reefs [Reef 44 (R44), Reef 25 (R25), HIMB] during bleaching (left panel) and recovery (right panel) a regional bleaching event. Polygons are standard error of point means (x symbols). (a, c) NMDS with site × condition effect. (b, d) NMDS with condition effect alone, with vectors showing significant responses (p ≤ 0.05) among bleached and non-bleached corals.

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https://doi.org/10.1101/414086

Fig. 5. Chlorophyll and total biomass in bleached (gray) and non-bleached (black) Montipora capitata (left panel) and Porites compressa (right panel) at three reefs [Reef 44 (R44), Reef 25 (R25), HIMB] during bleaching and recovery. Area-normalized (a, c) chlorophyll (a + c2) and (b, d) ash-free dry weight of tissue biomass. Values are mean ± SE (n = 5). Symbols indicate significant differences (p ≤ 0.05) between periods (‡) and bleached and non-bleached corals within a period (*') and within a site (*).

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https://doi.org/10.1101/414086

Fig. 6. Biomass composition and energy content in bleached (gray) and non-bleached (black) Montipora capitata (left panel) and Porites compressa (right panel) at three reefs [Reef 44 (R44), Reef 25 (R25), HIMB] during bleaching and recovery. (a, e) Proteins, (b, f) lipids, (c, g) carbohydrates, (d, h) energy content (kJ) normalized to grams of ash-free dry weight (gdw-1). Values are mean ± SE (n = 4 – 5). Symbols indicate significant (p ≤ 0.05) period effects (‡); letters indicate differences between sites within periods of bleaching (lowercase) or recovery (uppercase).

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https://doi.org/10.1101/414086

Fig. 7. Isotopic analysis of bleached (gray) and non-bleached (black) Montipora capitata (left) and Porites compressa (right) host and symbiont tissues at three at three reefs [Reef 44 (R44), Reef 25 (R25), HIMB] during bleaching and recovery. Carbon (δ13C) and nitrogen (δ15N) isotopic values for (a, g, d, j) coral host (δ13CH, δ15NH) (b, h, e, k) symbiont algae (δ13CS, δ15NS) and (c, i, f, l) their relative difference (δ13CH-S, δ15NH-S). Values are permil (‰) relative to standards for carbon (Vienna Pee Dee Belemnite: v-PDB) and nitrogen (air). Values are mean ± SE (n = 5); small SE may be masked by points. Symbols indicate significant (p ≤ 0.05) period (‡) and site effects (*S), and differences among bleached and non-bleached corals within a period (*') or a site (*).

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https://doi.org/10.1101/414086

Fig. 8 Relationship between observed and expected δ13CHolobiont for Montipora capitata (black circles) and Porites compressa (gray triangles) during post-bleaching recovery. Lines represent linear regression for M. capitata (solid line) and P. compressa (dotted line).

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R2 = 0.88, p <0.001R2 = 0.56, p <0.001


https://doi.org/10.1101/414086

1*, Raphael Ritson-Williams1,2, Brian N Popp3, Ruth D Gates1 · 4 Christopher B Wall1*, Raphael Ritson-Williams1,2, Brian N Popp3, Ruth D Gates1 5 6 *corresponding author: [email protected]

Documents

1, Raphael Ritson-Williams1,2, Brian N Popp3, Ruth D Gates1 · 4 Christopher B Wall1, Raphael Ritson-Williams1,2, Brian N Popp3, Ruth D Gates1 5 6 *corresponding author: [email protected]