Shifting baselines: Physiological legacies contribute to the response of reef corals to frequent heatwaves

Abstract

Global climate change is altering coral reef ecosystems. Notably, marine heatwaves are producing widespread coral bleaching events that are increasing in frequency, with projections for annual bleaching events on reefs worldwide by mid‐century.
Responses of corals to elevated seawater temperatures are modulated by abiotic factors (e.g. environmental regimes) and dominant Symbiodiniaceae endosymbionts that can shift coral traits and contribute to physiological legacy effects on future response trajectories. It is critical, therefore, to characterize shifting physiological and cellular states driven by these factors and evaluate their influence on in situ bleaching (and recovery) events. We use back‐to‐back bleaching events (2014, 2015) in Hawai'i to characterize the cellular and organismal phenotypes of Montipora capitata corals dominated by heat‐sensitive Cladocopium or heat‐tolerant Durusdinium Symbiodiniaceae at two reef sites.
Despite fewer degree heating weeks in the first‐bleaching event relative to the second (7 vs. 10), M. capitata bleaching severity was greater [bleached cover: ~70% (2014) vs. 50% (2015)] and environmental history (site effects) on coral phenotypes were more pronounced. Symbiodiniaceae affected bleaching responses, but immunity and antioxidant activity was similar in all corals, despite differences in bleaching phenotypes.
We demonstrate that repeat bleaching triggers cellular responses that shift holobiont multivariate phenotypes. These perturbed multivariate phenotypes constitute physiological legacies, which set corals on trajectories (positive and/or negative) that influence future coral performance. Collectively, our data support the need for greater tracking of stress response in a multivariate context to better understand the biology and ecology of corals in the Anthropocene.

A free Plain Language Summary can be found within the Supporting Information of this article.

Full text

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wileyonlinelibrary.com/journal/fec Functional Ecology. 2021;35:1366–1378.© 2021 British Ecological Society
Received: 30 July 2020
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Accepted: 24 March 2021
DOI: 10.1111/1365-2435.13795
RESEARCH ARTICLE
Shifting baselines: Physiological legacies contribute to the
response of reef corals to frequent heatwaves
Christopher B. Wall1,2 | Contessa A. Ricci3 | Alexandra D. Wen1,4 |
Bren E. Ledbetter3 | Delania E. Klinger3 | Laura D. Mydlarz3 | Ruth D. Gates1 |
Hollie M. Putnam5
1Hawai'i Institute of Marine Biology,
University of Hawai'i at Mā noa, Kāne'ohe,
HI, USA
2Pacific Biosciences Research Center,
University of Hawai'i at Mā noa, Honolulu,
HI, USA
3Depar tment of Biolog y, University of Texas
at Arlington, A rling ton, T X, USA
4Rosenstiel School of Mar ine and
Atmospheric Science, University of Miami,
Miami, FL , USA
5Depar tment of Biological Sciences,
University of Rhode Island, Kingston, RI,
USA
Correspondence
Christopher B. Wall
Email: chris.wall@hawaii.edu
Funding information
U.S. Environmental Protection Agency,
Grant/Award Number: FP- 91779401- 1;
Nationa l Science Foundation (USA ), Grant/
Award Number: NSF 1756623
Handling Editor: Adriana Verge s
Abstract
1. Global climate change is altering coral reef ecosystems. Notably, marine heat-
waves are producing widespread coral bleaching events that are increasing in
frequency, with projections for annual bleaching events on reefs worldwide by
mid- century.
2. Responses of corals to elevated seawater temperatures are modulated by abi-
otic factors (e.g. environmental regimes) and dominant Symbiodiniaceae en-
dosymbionts that can shift coral traits and contribute to physiological legacy
effects on future response trajectories. It is critical, therefore, to characterize
shifting physiological and cellular states driven by these factors and evalu-
ate their influence on in situ bleaching (and recovery) events. We use back-
to- back bleaching events (2014, 2015) in Hawai'i to characterize the cellular
and organismal phenotypes of Montipora capitata corals dominated by heat-
sensitive Cladocopium or heat- tolerant Durusdinium Symbiodiniaceae at two
reef sites.
3. Despite fewer degree heating weeks in the first- bleaching event relative to the
second (7 vs. 10), M. capitata bleaching severity was greater [bleached cover:
~70% (2014) vs. 50% (2015)] and environmental history (site effects) on coral phe-
notypes were more pronounced. Symbiodiniaceae affected bleaching responses,
but immunity and antioxidant activity was similar in all corals, despite differences
in bleaching phenotypes.
4. We demonstrate that repeat bleaching triggers cellular responses that shift holo-
biont multivariate phenotypes. These perturbed multivariate phenotypes con-
stitute physiological legacies, which set corals on trajectories (positive and/or
negative) that influence future coral performance. Collectively, our data support
the need for greater tracking of stress response in a multivariate context to better
understand the biology and ecology of corals in the Anthropocene.
KEYWORDS
bleaching, El Niño, immunity, physiology, Symbiodiniaceae

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1 | INTRODUCTION
Long- term trends in ocean warming and marine heatwaves are
destabilizing the symbiosis between corals and their algae en-
dosymbionts (Symbiodiniaceae) and increasing coral bleaching
events (Hughes, Anderson, et al., 2018). Cumulative impacts of
episodic heatwaves not only shift ecological assemblage and eco-
system function baselines (Hughes, Kerry, Baird, Connolly, Chase,
et al., 2019; McWilliam et al., 2020), but may also produce effects
on coral biology that modulate responses to subsequent distur-
bances, such as bleaching events (Guest et al., 2012; Thompson
& van Woesik, 2009). The influence of chronic environmental
change or more acute perturbations may therefore drive positive
and/or negative organism acclimatization, which we refer to as
‘physiological legacies’.
It is growing increasingly clear that coral bleaching suscepti-
bility is based on interactions between environmental histories
(Brown et al., 200 0; Safaie et al., 2018) and holobiont traits, includ-
ing endosymbiont communities, coral host genetics and physiol-
ogy (Barshis et al., 2013; Palmer et al., 2010; Palumbi et al., 2014;
Sampayo et al., 2008). Thermotolerant Symbiodiniaceae can confer
bleaching resistance in some corals (Sampayo et al., 2008); however,
host properties, including constitutive immunity (Palmer, 2018a)
and antioxidant capacity (Barshis et al., 2013), are also central to
maintaining cellular homeostasis and preventing bleaching mor-
tality (Palmer et al., 2010). As such, immunological processes are
implicated as targets for natural selection and are integral to the
future of reef- building corals in the face of climate change (Mydlarz
et al., 2010; Palmer, 2018a; Pinzón et al., 2014). The nexus of these
influential properties of coral stress responses – symbiont commu-
nity and host immunity – and their role in repetitive natural bleaching
and recovery events remains to be fully understood, especially with
respect to the role of environmental history (Palumbi et al., 2014;
Safaie et al., 2018).
Shifts in the biology and function of corals can be exam-
ined through various pertinent response variables as a ‘multi-
variate phenotype’ (i.e. multivariate trait space; e.g. McWilliam
et al., 2020; Van Straalen, 2003), a term which we use throughout
this manuscript. Ecological trait space approaches have been ap-
plied to track changes in reef coral communities and representa-
tive trait diversity following bleaching (Hughes, Kerry, et al., 2018;
McWilliam et al., 2020), and to distinguish cellular c ascades during
bleaching in the laboratory (Gardner et al., 2017). However, these
trait space approaches have yet to be broadly applied in under-
st andin g ble achin g respo nses (b ut se e, Ma yfi eld et al. , 2017 ), espe -
cially in a time- series context. The characterization of multivariate
phenotypes in time- series can complement ecological time- series
and allow for integration across cellular, ecological and evolution-
ary scales to improve the mechanistic understanding of linkages
pertinent to coral resilience.
Assessment of multivariate phenotypes through time addition-
ally provides both the opportunity to understand phenotype shifts
(e.g. Figure 1a), as well as to test mechanistic hypotheses underpin-
ning resistance or susceptibility to repeated stress (Figure 1b). For
FIGURE 1 (a) Multivariate analyses identif y changes in organism multivariate phenotype trait space through time by accounting for
the variation in multiple traits (Van Straalen, 2003). Transitions of organisms can occur from initial (α0) to stressed states (β1), and recover
to initial (α0; i.e. physiological resilience) or altered states (α1). Alternatively the absence of trait space shifts may be obser ved in stress
resistant individuals (i.e. maintained at α0). (b) Conceptual model of melanin and antioxidant activity in coral and Symbiodiniaceae under
heat stress (indicated by lightning bolts). Reactive oxygen species (ROS), oxygen singlets (1O2) and superoxide (
O−
2
), generated by symbiont
photochemical dysfunction and host mitochondria membrane damage, are neutralized by a combination of antioxidant scavenger enzymes
[e.g. superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX)]. Host immunity via the melanin- synthesis pathway can also scavenge
ROS during intermediate steps that lead to the synthesis of melanin (MEL). Oxidative bursts during melanin- synthesis can also create ROS
that act as antimicrobials, which may lead to antioxidant enzymes upregulation (shown in bold letters and dark lines)
Mutlivariate axis1
Mutlivariate axis 2
α
0
α
1
β
1
(a) (b)
Coral
O
2
–
1
O
2
Antioxidant scavengers
Immunity
MEL pathway
O
2
–O
2
–
H
2
O
2
SOD
H
2
OO
2
CAT
POX
SOD
CAT
POX
H
2
O
PPO
PO
MEL
ROS
SOD, CAT, POX
H
2
OO
2
heat
O
2
ROS
ROS
Damage
PSI / PSII
Calvin
Cycle
O2ATP
NADPH

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WALL et AL.
instance, higher constitutive immunity (i.e. immune activity neces-
sary for cellular homeostasis) reduces coral thermal sensitivity and
disease susceptibility (Palmer et al., 2010). Thermal stress can also
trigger immune responses modulated by energetic requirements
(Fuess et al., 2018; Palmer, 2018b; Pinzón et al., 2015), stress fre-
quency (i.e. acute, chronic, repeated stress; Ainsworth et al., 2016;
Schoepf et al., 2015), or as a function of a history of biotic and
abiotic challenges that modulate constitutive immunity (Mydlarz
et al., 2009; Palmer, 2018b; Wall et al., 2018). These cellular mech-
anisms (i.e. antioxidant and immunity strategies) can also be based
on environmental histories, such as the lower melanin synthesis and
higher antioxidant activity observed in heat- stressed corals from a
reef with high pCO2- variability relative to a low pCO2- variability
reef (Wall et al., 2018). Thus, pairing bleaching metrics, like symbi-
ont cell densities and chlorophyll concentrations, with immune ac-
tivity and antioxidant s provides powerful and tractable mechanistic
assessments of coral performance within an integrative, multivari-
ate framework.
The Main and Northwestern Hawaiian Islands experienced severe
bleaching in 2014 and 2015 (Bahr et al., 2017; Couch et al., 2017).
An ecologically abundant reef coral, Montipora capitata (Dana 1846),
showed significant bleaching in both events (Figure 2a). Within
Kāne'ohe Bay, O'ahu, Hawai'i, differences in bleaching responses of
colonies of M. capitata were observed across sites and events (Bahr
et al., 2017), linked to thermally sensitive (Cladocopium sp.) or re-
sistant (Durusdinium glynnii) Symbiodiniaceae (Cunning et al., 2016).
Using the opportunity presented by these natural bleaching events
(Figure 2d), we tracked coral bleaching and recovery phenotypes
using a multivariate trait space approach, selecting response vari-
ables indicative of cellular bleaching and symbiosis integrity (sym-
biont density, areal- and cell- specific chlorophyll a, holobiont total
protein and total biomass), host immunity (prophenoloxidase, mel-
anin) and host antioxidant activity (peroxidase, catalase, superox-
ide dismutase). We focused on the melanin- synthesis pathway, as
it is an important component of host immunity for wound healing
and pathogen invasion that may also serve as a photoprotectant
FIGURE 2 Montipora capitata cover
and bleaching patterns at two reefs in
relation to periods of thermal stress
and recover y. (a) A bleached M. capitata
colony. (b) Benthic surveys of total coral
cover (left), M. capitata cover (middle) and
the proportion of bleached M. capitata
colonies (right) at Lilipuna and Reef 14
sites during bleaching (B1, B2) and during
post- bleaching recovery (R1, R2) in
2014– 2015 and 2015– 2016. Values are
mean ± SD, n = duplicate transects, and
symbols indicate differences among sites
within a period (*) and among sites (†). (c)
Overlay of mean daily temperatures at
Lilipuna and Reef 14 and the NOAA- HIMB
Moku o Lo'e buoy from January 2014
to January 2016, and (d) a comparison
of temperature ramping and cooling
(top lines, left axis) and degree heating
weeks [(DHW ) bottom lines, right axis]
between the first and second bleaching
events. Vertical dashed lines indicate coral
collections during bleaching and recovery
periods in the first (black, 2014– 2015) and
second (gray, 2015– 2016) events
Period and time
(a) (b)
(c)
(d)

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mitigating bleaching stress (Mydlarz & Palmer, 2011; Palmer
et al., 2010; Figure 1b). Our sampling design was tailored to identify
changes in multivariate phenotypes of a common reef coral (M. cap-
itata) at the population level, and therefore encompasses changes in
the pool of coral genotypes persisting in the face of environmental
selection.
We quantified the response of M. capitata in Kāne'ohe Bay to
the back- to- back bleaching events of 2014 and 2015 (i.e. bleaching
in October 2014, recovery in February 2015, bleaching in October
2015, recover y in February 2016) at two Kāne'ohe Bay reefs with
contrasting environmental histories relating to seawater residence
time (10– 20 days vs. >30 days), pCO2 variab ility (c. 30 0 vs. 60 0 μatm
pCO2 diel flux), and proximity to shore (0.15 vs. 1.50 km; Drupp
et al., 2011, 2013; Lowe et al., 2009). Reefs were selected because
previous work demonstrated lower melanin synthesis and higher
antioxidant activity in thermally stressed corals from the high pCO2-
variability site (Wall et al., 2018). We posited that: (H1) immunity
and antioxidant contributions to bleaching responses would be in-
fluenced by site environmental histories and symbiont communities,
such that corals from the high pCO2- variability site and those as soci-
ated with thermally sensitive Symbiodiniaceae (i.e. Cladocopium sp.)
would exhibit greater bleaching and attenuated immune responses.
We also expected (H2) immunit y and antioxidant activity/concen-
tration would increase in corals in subsequent bleaching events
due to legacy effects on acclimatory and/or stress response path-
ways over time. Considering the wide- ranging functions of melanin
in host immunity (Mydlarz et al., 2008; Palmer et al., 2010; Wall
et al., 2018), we expec ted melanin synthesis to act as an acute and
broad- spectrum defence in physiologically stressed corals, with an-
tioxidant s having a more specialized response after chronic or repeat
bleaching stress. Finally, while the thermal tolerance of symbiont
communities is vital to coral bleaching sensitivity, evidence for dif-
ferential immunity or antioxidant capacities in coral holobionts asso-
ciated with thermally tolerant Symbiodiniaceae is lacking. Given that
M. capitata associates with two genetically and physiologically con-
trasting Symbiodiniaceae (Cunning et al., 2016; Wall et al., 2020), we
predicted (H3) that holobionts harbouring heat- tolerant Durusdinium
sp. would show lower immune and antioxidant responses (less con-
centrations/activity) due to their resistance to thermal stress. This
multivariate and cross- scale approach allows for a holistic quantifi-
cation of coral phenotypes and can reveal population- level pheno-
typic patterns through bleaching and recovery periods.
2 | MATERIALS AND METHODS
2.1 | Site description
Coral bleaching and recover y were monitored at two reef systems:
a fringing reef [hereafter, Lilipuna (21°25′36.8′′N, 157°47′24.0′′W)]
in southern Kāne'ohe Bay adjacent to the Hawai'i Institute of
Marine Biology (HIMB) on Moku o Lo'e, and an inshore patch reef
[hereafter, Reef 14 (21°27′08.6′′N, 157°48′04.7′′W)] in central
Kāne'ohe Bay. These reef sites were chosen due to their unique en-
vironmental history of seawater pCO2 and hydrodynamics (Drupp
et al., 2013). Seawater pCO2 adjacent to both locations is compa-
rable (c. 450 μatm); however, diel pCO2 flux is significantly higher
( 1 9 6 – 9 7 6 μatm pCO2) in central Kāne'ohe Bay near Reef 14 relative
to the Bay's southern basin proximate to Lilipuna (225– 671 μatm;
Drupp et al., 2011, 2013). Thus, we refer to these sites as low pCO2-
variability (Lilipuna) and high pCO2- variability (Reef 14) locations
(sensu, Wall et al., 2018).
2.2 | Environmental monitoring
Temperature and photosynthetic active radiation (PAR) loggers were
placed at Lilipuna and Reef 14 at a depth of 1- m. Temperature and
PAR were recorded continuously at 15- min intervals from October
2014 to February 2016. Gaps in logger temperature data were sup-
plemented with NOAA temperature data from the Moku o Lo'e sta-
tion at HIMB (NOAA , 2019). Light loggers were calibrated against
a LI- 1400 quantum meter attached to a cosine LI- 192 underwater
quantum sensor. Instantaneous light data (μmol photons m−2 s−1)
were used to calculate daily integrated light integrals (DLI) for each
site by calculating the mean hourly PAR and converting to a 24- hr
day (mol photons m−2 d−1; Figure S1). Temperature loggers were cali-
brated with a certified digital thermometer (5- 077- 8, ±0.05°C ac-
curacy) and cross- calibrated against each other for standardization.
Degree heating weeks (DHW) for the southern portion of
Kāne'ohe Bay where our corals were collected were calculated using
in situ Moku o Lo'e temperature data (NOAA, 2019), with the dif-
ference between mean half- week temperatures (i.e. mean hourly
temp over 3.5 days) and the maximum monthly mean temperature of
27.7°C, (Jokiel & Brown, 2004) to determine ‘hotspots’. DHW were
determined as the number of hotspots >1 across a rolling 12- week
window (i.e. 24 half- weeks; NOAA, 2020). DHW for windward O'ahu
and the Hawaiian Islands during the 2014 and 2015 bleaching events
have been previously repor ted (Bahr et al., 2017; Sale et al., 2019).
The purpose of our calculations were to quantify the incurred heat
stress using calculation of DHW from temperature data collected
proximate to our two reef locations.
2.3 | Benthic surveys and coral collections
Four sampling times were identified as corresponding to a ‘bleaching
period’ following the point of maximum thermal stress (10 October
2014 and 12 October 2015) and a post- bleaching ‘recovery period’
approximately 4 months after peak seawater warming (11 February
2015 and 26 February 2016). In each time period, benthic surveys
at 1- m depth were conducted at each reef site using two 20- m tran-
sects and a line- point- intersect at 1- m intervals (see Supporting
Information). Coral bleaching states were either non- bleached (i.e.
appearing fully pigmented), or bleached (i.e. exhibiting degrees of
tissue paling/pigment variegation or being wholly white). Total coral

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cover (% benthic cover) and M. capitata benthic cover were calcu-
lated, with M. capitata bleaching extent calculated as the proportion
of total M. capitata colonies bleached.
In each sampling period, coral branches were collected from 40
M. capitata coral colonies (n = 1 fragment/colony) selected at ran-
dom along the reef crest at each site at a depth of 1- m. Our sam-
pling methodology was designed to track the population of corals
surviving at each of the two study sites. Immediately post collection,
corals were snap- frozen in liquid nitrogen, returned to HIMB and
stored at −80°C. While frozen, each colony was split in half along
its longitudinal axis. One- half of each coral fragment remained at
HIMB (−80°C) until processing for physiological assays and qPCR.
The corresponding fragment halves were shipped to the University
of Texas at Arlington using a dry- shipper charged with liquid nitro-
gen for immunology and antioxidant assays.
2.4 | DNA extraction and symbiont community
analysis
Symbiodiniaceae DNA was extracted by adding an isolate of coral
tissue (500 μl) to 500 μl DNA buffer with 2% (w/v) sodium dodecyl
sulphate, following a modified CTAB- chloroform protocol (Cunning
et al., 2016; https://doi.org/10.17504/ proto cols.io.dyq7vv). The
dominant Symbiodiniaceae genera Cladocopium and Durusdinium
(na mely, IT S2 type s C31, wit h C17 and C21 and D1- 4- 6 [Durusdinium
glynnii, Wham et al., 2017]) known to be numerically dominant in
Kāne'ohe Bay M. capitata were assessed by quantitative PCR
(qPCR; Cunning et al., 2016). Coral colonies were determined to be
Cladocopium- or Durusdinium- dominated based on numerical abun-
dance (>0.5 proportion) of each genus from qPCR analysis (Wall
et al., 2020; see Supporting Information).
2.5 | Physiological metrics
Coral tissue was removed from the skeleton using an airbrush filled
wit h 0.2- μm filtered seawater, yielding ~10– 30 ml of tissue slurr y.
Extracted tissues were briefly homogenized and subsampled for the
following physiological metrics: symbiont cell densities, chlorophyll
a concentrations, protein biomass and the total organic biomass de-
termined from the ash- free dry weight of holobiont (coral + algae)
tissues. All physiological metrics were normalized to surface area
(cm2) of coral skeleton using the paraffin wax- dipping technique
(Stimson & Kinzie, 1991).
Symbiont cell counts (cells/cm2) were measur ed by rep lic at e cell
counts (n = 6– 10) on a haemocytometer. Chlorophyll a was quan-
tified by extracting pigments in 100% acetone for 36 hr in dark-
ness at −20°C. Spectrometric absorbance were measured (630 and
663 nm) using a 96- well quartz microtiter plate with two technical
replicates; chlorophyll concentrations (μg chlorophyll a cm−2 and pg
chlorophyll a per sym bio nt ce l l) we r e qua ntifie d usi ng the eq uat ion s
for dinoflagellates in 100% acetone (Jeffrey & Humphrey, 1975)
and normalized for path length of the well plate (0.6 cm). Total ho-
lobiont protein concentration (mg protein cm−2; soluble + insoluble)
was quantified using a BCA Protein Assay Kit and measured spec-
trophotometrically (562 nm) in a 96- well plate with three technical
replicates against a bovine serum albumin standard. Total biomass
(mg/c m2) wa s mea su red by drying a subs amp le of cor al tiss ue sl ur r y
at 60°C (48 hr) followed by burning to ash at 450°C . The difference
between the dried and burned masses is the ash- free dry weight
(AFDW).
2.6 | Immunity and oxidative stress metrics
Immunology and oxidative stress metrics were determined using
previously published protocols for coral host tissues (Mydlarz
et al., 2009; Mydlarz & Palmer, 2011; Palmer, McGint y, et al., 2011;
Wall et al., 2018). Additional information can be found in the
Supporting Information.
A 3– 4 ml aliquot of coral tissue slurry was obtained by airbrush-
ing with an extraction buffer. Tissue was homogenized on ice (1 min),
and an aliquot (1 ml) was freeze- dried for 24 h r for melanin concen-
tration estimates. The remaining slurry was centrifuged to remove
cellular debris and Symbiodiniaceae cells to achieve a host- enriched
cell- free extract. All colorimetric measurements were calculated
spectrophotometrically using a microplate reader. Total protein con-
centration of each coral cell- free extract was determined using the
RED660 protein assay with a bovine serum albumin standard curve.
Melanin was extracted from freeze- dried tissue and the concen-
tration of melanin (490 nm) was determined using a standard curve
of commercial melanin and presented as mg melanin mg of tissue−1
(Fuess et al., 2018). Prophenoloxidase activity was determined by
adding trypsin to activate prophenoloxidase, and the reaction was
initiated by L- 1,3- dihydroxyphenylalanine. Change in absorbance
(490 nm) over time was measured and normalized to mg protein
and time for each sample (ΔAbs490 nm mg protein−1 min−1; Fuess
et al., 2018; Mydlarz & Palmer, 2011).
Coral host oxidative stress was determined by measuring the
scavenging activity of the coral cell- free extracts to different sub-
strates specific to the antioxidants: peroxidase, catalase and super-
oxide dismutase. Peroxidase activity (EC 1.11.1.7) was determined
by adding guaiacol and initiating the reaction initiated with 30%
H2O2. Change in absorbance (470 nm) over time was calculated
and normalized to mg protein in each sample and represented as
ΔAb s 47 0 nm mg pro tei n−1 min−1 (My dla rz & Ha r vell, 20 07) . Catal a se
activity (EC 1.11.1.6) was determined by using coral cell- free ex-
tract s and adding H2O2 to activate the reaction. Change in absor-
bance (240 nm) over time was calculated and converted to μmol
H2O2 and presented as μmol H2O2 scavenged mg protein– 1 min– 1
(Palmer, McGinty, et al., 2011). Superoxide dismutase (EC 1.15.1.1)
was determined using the SOD Assay Determination Kit- WST
(450 nm). One SOD unit of activity (U) equates to 50% inhibition
of superoxide anion, and data are presented as SOD U mg/protein
(Krueger, Fisher, et al., 2015).

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2.7 | Statistical analysis
Permutational multivariate analysis of variance (PERMANOVA)
and non- metric multidimensional scaling (NMDS) were performed
using a balanced matrix of all physiology and immunity/antioxidant
responses (n = 10 responses) with the package vegan (Oksanen
et al., 2019). PE RM ANOVA ana ly si s wa s co nd uc ted on Euclidean ca l-
culations of pairwise distances using adonis2 based on a scaled and
centred matrix. Prior to NMDS ordination, the data were double-
standardized using a Wisconsin and square root transformation with
Euclidean distances using metaMDS. Response variables showing
significant correlations (p < 0.05) with NMDS ordination were plot-
ted as vectors using envfit command in vegan. Coral multivariate
phenotypes were defined by convex hulls, with borders defined by
the range of points in the multivariate trait space (i.e. NMDS1 and
NMDS2). Phenotype trait spaces were grouped categorically using
either sampling periods (in trajector y plots), or the interactions of
Site, Symbiont and Period; centroids were used in trajector y plots
and calculated as the arithmetic mean of NMDS1 and NMD2 for
each category.
Ecological benthic data (total coral cover, M. capitata cover,
and bleached M. capitata cover) were tested using nonparametric
Kruskal- Wallis tests to evaluate Period (two bleaching and two re-
covery events) and Site (Lilipuna, Reef 14) effects. Physiology, im-
munity, and antioxidant response variables were analysed using a
linear model with Periods, Sites and Symbiont community composi-
tion (Cladocopium- or Durusdinium- dominated) as fixed effects (see
Suppor ting Information). All analyses were performed in R version
3.6.1 (R Core Team, 2019).
3 | RESULTS
In 2014, DHW began accumulating on 30 August, with maximum
sustained DHW of 7.1 (peaked 04 October – 19 November). In 2015,
DHW began accumulating 01 July, with a maximum of 10.2 DHW
sustained (peaked 12– 19 November; Figure 2c,d). Coral cover at the
two sites ranged from (mean ± SD) 63 ± 11% to 93 ± 11% across
2014– 2016, and mean coral cover decreased over time ( p = 0.029;
Figure 2b). Mean M. capitata cover declined over time (p < 0.001) as
well, but was more stable at Lilipuna (~40%) compared to Reef 14,
which declined in the first bleaching event and over time (91 ± 4%
in October 2014 to 51 ± 7% in February 2016). Greater propor tions
of corals and M. capitata colonies (p = 0.001) bleached in the first
bleaching event (October 2014: 62– 75 ± 9%) relative to the second
(October 2015: 43– 55 ± 16%). Additionally, proportions of bleached
M. capitata corals were greater at Reef 14 compared to Lilipuna
during thermal stress (p < 0.001) and Reef 14 colonies retained
higher bleaching/paling scores during recovery periods (p = 0.013;
Figure 2b).
PERMANOVA testing of the effects of Period (bleaching event),
Site (Lilipuna vs. Reef 14) and dominant Symbiodiniaceae community
(Cladocopium sp. or Durusdinium sp.) revealed significant dif ferences
of the interaction of Period- by- Site ( p < 0.001) and Period- by-
Symbiont community (p < 0.0 01; Figures 3 and 4; Table 1) and all
main effects (p < 0.001). In each of the four periods, coral multivar-
iate phenotypes occupied unique positions in multidimensional trait
space (Figure 3), with location and trajectory of trait space convex
hulls in each period principall y being a func tion of symbiont commu-
nity, and to a lesser extent, environmental histories associated with
sites of collection (Figures 3 and 4).
Multivariate phenotypes of bleached corals relative to re-
covered corals showed stronger separation in 2014– 2015, which
was apparent at both reefs (Figure 4a,b) and paralleled patterns
of greater bleaching prevalence in the first- bleaching event
(Figure 2b). Similarly, site effects on corals were most pronounced
in the first bleaching event (Figure 4; Table 1). Despite greater
bleaching at Reef 14 in 2014 (Figure 2b), shifts in bleaching and
recovery trait space were most distinct at Lilipuna (Figure 4a,c).
At the physiological level (Figure 5a– e), separation of multivari-
ate phenotypes during and after thermal stress was driven by
bleaching sensitivity and symbiont cells/chlorophyll concentra-
tions (p < 0.001, Figures 4 and 5a– c; Table S1). ‘Bleached’ corals
observed during temperature stress events (October 2014 and
2015) generally were dominated by Cladocopium sp., although
in the first event coral phenotypes at Reef 14 did not separate
by symbiont communities (Figure 4b). Nevertheless, across all
sampling periods M. capitata colonies dominated by Durusdinium
symbionts had higher symbiont cell densities, less variable areal
FIGURE 3 Non- metric multidimensional scaling (NMDS)
analyses of coral trait space during bleaching stress and post-
bleaching recovery. Montipora capitata corals dominated by
Cladocopium sp. (red) or Durusdinium sp. (blue) symbionts from
two sites (solid lines Lilipuna, dashed lines Reef 14). Convex hulls
represent multivariate phenotypes (i.e. NMDS point clusters) of
all corals in each time period, with points indicating the mean
centroid for all samples in each group. Lines show trajectories of
mean centroids for each group across times from the first bleaching
period (white- filled circles, Bleaching 2014) to the trajector y termini
at the final recovery period (triangle arrowheads, Recovery 2016)
Bleaching
2014
Bleaching
2015
Recovery
2015
Recovery
2016
2D stress = 0.20
C-Lilipuna
C-Reef 14
D-Lilipuna
D-Reef 14
−0.1 0.0 0.1
NMDS2
−0.1 0.00 0.1
NMDS1

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WALL et AL.
chlorophyll concentrations, and lower chlorophyll per symbiont
cell compared to colonies dominated by Cladocopium symbionts
(p < 0.001, Figure 5a– c). Coral protein and total biomass were
variable across the study, but each showed positive correlations
with Durusdinium- dominated corals from Lilipuna in bleaching and
recovery periods (Figures 4a,c and 5d,e). Overall, protein was 9%
higher in Durusdinum- dominated colonies (p = 0.031) and 32%
higher in Lilipuna corals during first bleaching (October 2014), but
equivalent at all sampling points thereafter (p = 0.002, Figure 5d).
Total biomass was lower during recovery periods (p < 0.001) in
addition to being 20%– 40% higher at Lilipuna compared to Reef
14 in all periods (except in February 2015 recover y; p < 0.001)
and ~30% higher in Durusdinium- dominated colonies during the
second bleaching, but equivalent across all colonies in other peri-
ods (p = 0.012, Figure 5e).
Immunity and antioxidant metrics differed through time in re-
sponse to repeat bleaching and recovery (Figures 4a,b and 5f– j;
p < 0.001), while the environmental influence from the Site and
Symbiont communities were less pronounced ( Table S2). In the first
event, corals had high levels of melanin (with corresponding low
prophenoloxidase precursors) and catalase. As corals recovered,
prophenoloxidase and superoxide dismutase increased and mela-
nin and catalase declined (Figures 4a,b and 5f– j; Table S2; Figure 6;
post- hoc test, bleaching 2014 vs. recovery 2015 = p < 0.001). The
melanin pathway increased in all corals in the 2014 bleaching
event regardless of bleaching sensitivity (i.e. loss or retention of
FIGURE 4 Non- metric multidimensional scaling (NMDS) analyses of coral multivariate phenotypes separated by dominant symbiont
type (colours), bleaching- recovery periods (lighter and darker shades), sites (columns) and events (rows). Montipora capitata corals dominated
by Cladocopium sp. or Durusdinium sp. symbionts from Lilipuna (left panel: a, c) and Reef 14 (right panel: b, d) during bleaching (Bleach) and
recovery (Recov) periods. Biplot vectors (black arrows) represent significant physiology and immunity responses (p < 0.05) according to
squared correlation coefficients (r2). AFDW, ash- free dry weight biomass (mg/gdw); CAT, catalase; MEL , melanin; POX, peroxidase; PPO,
prophenoloxidase; SOD, superoxide dismutase; chla, chlorophyll a µg/c m2; chla cell, chlorophyll a per symbiont cell; prot, protein mg/cm2;
symb cells, symbionts/cm2

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WALL et AL.
symbionts/chlorophyll), or symbiont community, and was a signifi-
cant cellular response shaping multivariate phenotypes in the first
bleaching event (Figure 5f). Corals in the second bleaching (October
2015) experienced a sixfold increase in melanin and corresponding
declines in prophenoloxidase, relative to concentrations measured
in February 2015 (Figure 5f,g). However, melanin activity was sig-
nificantly lower compared to the high melanin activit y obser ved in
the first bleaching period (0.015 vs. 0.0 03 mg melanin mg tissue−1
in first- vs. second- bleaching period). There was a significant peak
in catalase activity in the second bleaching (Figures 5i and 6), reach-
ing the highest level observed across study periods (post- hoc test,
Periods = p < 0.001). The 2015 bleaching peak in catalase corre-
sponded with the lowest obser ved peroxidase activity (Figure 5h),
particularly for Cladocopium- dominated colonies (post- hoc test:
Symbiont community effect, p = 0.007). Subsequently in the 2016
recovery period, catalase declined by ~70% and peroxidase activ-
ity doubled, reaching peak activity similar to those observed in time
periods (Figure 5i). Superoxide dismutase increased progressively
through time, peaking in the 2016 recovery (p < 0.0 01, Figure 5j).
4 | DISCUSSION
Gaining a more complete understanding of environmental history
and legacy effects in ameliorating or exacerbating coral bleaching
is vital as marine heatwaves intensif y (Oliver et al., 2018, 2021).
At our study sites, the fringing reef habitat of Lilipuna, with close
proximity to shore and silt- dominated backreef benthos, contrasts
the patch reef pinnacle of Reef 14 in the middle of the Kāne'ohe
TABLE 1 Result s of PERMANOVA testing the effects of
repeated bleaching and recovery periods on Montipora capitata
corals hosting two distinct symbiont communities at two reef
locations
Factor df SS R2F p
Period 3855.740 0.297 48.491 <0.0 01
Site 160.996 0.021 10.369 <0.001
Symbiont 1202.349 0.070 34.399 <0.001
Period × Site 369. 0 03 0.024 3.910 <0.001
Period ×
Symbiont
360.101 0.021 3.406 <0.001
Site ×
Symbiont
16. 861 0.002 1.166 0.293
Period × Site
× Symbiont
319.048 0.0 07 1.079 0.360
Residual 273 1605.902 0.558
Tot al 288 2,880.000 1.000
Bold p values represent significant effects (p < 0.05).
Abbreviations: Period, sequential bleaching and recover y events from
October 2014 to February 2016; Site, Lilipuna or Reef 14; Symbiont,
Cladocopium sp. or Durusdinium sp. dominated symbiont communit y; SS,
sum of squares; df, degrees of freedom.
FIGURE 5 Physiology, immunity and antioxidant metrics for Montipora capitata corals dominated by Cladocopium sp. or Durusdinium sp.
symbionts (C or D- dominated) from t wo reefs in Kāne'ohe Bay (Lilipuna, Reef 14) during repeat bleaching (B- 1, B- 2) and recovery periods
(R- 1, R- 2). Area- normalized (a) symbiont cell densities and (b) chlorophyll a concentrations, (c) chlorophyll a per symbiont cell, (d) area-
normalized protein concentrations and (e) ash- free dry weight biomass, (f) melanin (MEL), (g) prophenoloxidase (PPO), (h) peroxidase (POX),
(i) catalase (CAT) and (j) superoxide dismutase (SOD). Values are mean ± SE (n = 11– 28). Symbols represent post- hoc tests for highest order
interactions: Period × Symbiont (*) and Period × Site (†), Period × Site × Symbiont (*†)
Period and time
Chlorophyll a (µ g/cm2)
Chlorophyll a (pg/cell)
Protein
( mg/cm2)
Total biomass (mg/cm2)
( mg melanin/mg tissue)
MEL
(U SOD/mg prot)
SOD activity
(a)
(f) (g)(h) (i)
(b) (c) (d)(e)

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Bay lagoon. Environmental conditions linked to bleaching severity
(seawater temperature, DHW or light availability) did not differ be-
tween Lilipuna and Reef 14 within our period of study. While these
similarities may be driven by the shallow depths where corals were
sampled, these reef sites do experience significant differences in
hydrodynamics and seawater residence that produce disparate pat-
terns in pCO2 variability, with greater daily pCO2 variabilit y at Reef
14 compared to Lilipuna (daily range c. 800 vs. 450 μatm pCO2;
Drupp et al., 2011, 2013). Together, long- term hydrodynamic and
biogeochemistry conditions between these reefs represent distinct
environmental histories, which we show are influencing multivariate
phenotypes in resident M. capitata and their response to, and recov-
ery from, thermal stress. We observed a negative relationship be-
tween DHW and percent bleached coral cover, suggesting a greater
role for legacy effects (through selection, acclimatization, adapta-
tion or a combination therein) over differences in the extent of ther-
mal stress between years. Specifically, our calculations and others
(Bahr et al., 2017), support that DHW in Kāne'ohe Bay were lower
in the first bleaching event relative to the second, while bleached
coral cover was higher in 2014 than 2015 at our study sites, and
in Kāne'ohe Bay as a whole [45%– 77% (2014) vs. 30%– 55% (2015),
Bahr et al., 2017; Ritson- Williams & Gates, 2020].
Legacy effects on coral physiological responses parallel concepts
of ‘ecological memory ’ (Hughes, Kerry, Baird, Connolly, Dietzel,
et al., 2019), where responses to heatwaves are dependent on previ-
ous events, and ‘environmental memory’ (Brown et al., 2015), where
coral physiology is modulated by prior environmental exposure.
Here, physiological legacies apparent between Lilipuna and Reef 14
corals demonstrate the combination of environmental histor y (years
to decades) and recent heatwave effects at the population level.
Wh ile our st udy ca nno t dif ferentiate betwe en se lec tion (i .e. stoc has-
tic, differential bleaching sensitivity) and acclimatization responses
in corals surviving back- to- back bleaching and recovery events, we
show corals occupying distinct trait spaces in each sampling period
and a clear trajectory migration through time. Multivariate shifts
were influenced by environmental forces at each site and patterns
FIGURE 6 Schematic of observed immunity and antioxidant responses of Montipora capitata corals to repeat bleaching and recover y.
In the first event (top row) thermal stress led to a substantial increase in melanin (MEL) along with modest spikes in catalase (CAT) and
superoxide dismutase (SOD). Corals in the first recovery showed increases in prophenoloxidase (PPO; as the precursor to the melanin-
pathway) as well as SOD and peroxidase (POX), while CAT activity declined. In the second event (bottom row), CAT and SOD spike with
modest contributions of MEL and a general decline in POX. Corals in the second recovery had the highest levels of SOD across all time
points, an increase in POX and a sharp decline in CAT Measurements were made on host cell- free extract and are therefore shown in the
‘coral’ compartment; the influence of Site and Symbiont community are omitted from the schematic
Damage
PSI / PSII
MEL pathway
O2–
O2–
H2O2
SOD
O2
O2
PPO
PO
MEL
ROS
heat
H2O
Bleaching Recovery
Coral Coral
H2O
POX
CAT
Calvin
Cycle
2014 – 2015
2015 – 2016
O2–
O2–
CAT> SOD > POX
PSI / PSII
O2
ATP
NADPH
Calvin
Cycle
ROS
MEL pathway
PPO
PO
MEL
ROS
SOD > POX > CAT
ROS
H2O2
O2 H2O
POX
O2–
O2–
SOD
CAT
O2
ATP
NADPH
ROS
Damage
PSI / PSII
MEL pathway
O2–
O2–
H2O2
SOD
O2
O2
ROSH2O
Coral Coral
H2O
POX
CAT
Calvin
Cycle
O2–
O2–
PSI / PSII
Calvin
Cycle
MEL pathway
PPO
PO
MEL
ROS
H2O2
O2 H2O
POX
O2–
O2–
SOD
CAT
O2
ATP
NADPH
ROS
SOD >POX > CAT
ROS
PPO
PO
MEL
O2
ATP
NADPH
O2
H2O
O2
H2O
CAT, SOD > POX
ROS
ROS
Antioxidants
Antioxidants
Antioxidants
Antioxidants
O2–
O2–
1O2
1O2
O2–
H2O2
O2–
H2O2
heat
ROS
C
C
C
C

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WALL et AL.
of symbiont association and loss/repopulation, though the signifi-
cance of these predictors were not uniform across time. Therefore,
symbiont communities are integral to bleaching responses and mul-
tivariate phenotypes (Suggett et al., 2017), but their relative impor-
tance are tempered by environmental conditions and site- specific
histories.
Changes in the symbiont community— via differential perfor-
mance of genera and types (Sampayo et al., 2008), and the poten-
tial for switching/shuffling (Baker, 2003) to more thermally tolerant
species— provides one clear example of how environmental his-
tory can shift holobiont performance. Here, the role of dominant
Symbiodiniaceae on the holobiont was present, but effects were
often period- or site- dependent. Additionally, while high symbi-
ont and chlorophyll a densities in M. capitata colonies dominated
by Durusdinium sp. across time aligns with the paradigm of high
thermotolerance within this genus (Cunning et al., 2016; Lesser
et al., 2013; Silverstein et al., 2017; Wham et al., 2017), antioxidant
and immunity metrics were equivalent between functional distinct
symbiont communities (Cladocopium- vs. Durusdinium- dominated)
and countered expectations that these traits would be modulated
by symbiont- derived bleaching resistance. In fact, similarities in an-
tioxidant s and immunity were present despite greater loss of symbi-
ont cells and photopigmentation in Cladocopium- dominated colonies
relative to Durusdinium- dominated colonies. Host mechanisms reg-
ulating redox status are critical to coral thermal stress responses
[e.g. reactive oxygen species (ROS) release via symbiont PSII pho-
todamage (Weis, 20 08)], though host antioxidant responses can be
decoupled from symbiont photophysiological function (Krueger,
Hawkins, et al., 2015). Thus, our work supports an integral role of
host mechanisms in the biology of corals during thermal bleaching
and post- bleaching recovery, of which there is a growing apprecia-
tion (Gardner et al., 2017; Mydlarz et al., 2010).
Observed differences in antioxidant and immune activity in
corals during repeat bleaching and recover y reveal shifts in cellu-
lar priorities and mechanisms for coping with bleaching stress. For
instance, the melanin pathway (collectively here as the prophe-
noloxidase reservoir and the melanin product) was the primary
cellular response in both populations in the first- bleaching event
regardless of symbiont community (Figure 6). Engagement of the
melanin pathway is an im port ant generalized response to periodic
stress (Mydlarz et al., 2008; Palmer et al., 2010; Palmer, Traylor-
Knowles, et al., 2011; Wall et al., 2018), and though its decline
may indicate the melanin cascade becoming exhausted during
the first bleaching event, it more likely represents a shift away
from melanin synthesis in favour of antioxidant activity, as sup-
ported by the recover y of the prophenoloxidase reservoir during
the first recovery period. Thus, we interpret these results as in-
dicating a specialized nature for antioxidants in mitigating cellular
damage and maintaining coral holobiont homeostasis (Murphy
et al., 2019).
Increases in catalase and superoxide dismutase after the first
bl each ing even t are pre sen t amon g both po p ula tio ns, wi t h a grea ter
catalase response from Lilipuna corals. Previously, we showed that
M. capitata exposed to thermal stress showed higher antioxidant
activity, but lower melanin when primed by a histor y of high pCO2
variability (Wall et al., 2018). Though the influence of pCO2 on coral
thermal responses is uncertain, with studies showing both negative
and null effects (Anthony et al., 20 08; Noonan & Fabricius, 2016;
Wall et al., 2014), this contrasting pattern further highlights the
need to ac co un t fo r legac y eff ect s when ex amining cor al response s
and planning management effort s. Likewise, both total biomass
and superoxide dismutase activity increased through time in both
populations, suggesting progressive increases in constitutive anti-
oxidant activity (i.e. superoxide dismutase) that may contribute to
biomass maintenance and enhanced potential for overall survival
(Thornhill et al., 2011). Superoxide dismutase contributions may
work through combating ROS originating from damage to photoma-
chinery and host mitochondria, which together can trigger apopto-
sis (Weis, 2008). Thus, we see both an immediate and acclimatory
mechanism (i.e. catalase), as well as ‘cellular memory’ to thermal
stress (i.e. superoxide dismutase) that may carry over to buffer fu-
ture oxidative stress (Barshis et al., 2013; Brown et al., 2015). This
provides evidence in support of constitutive frontloading (Barshis
et al., 2013) as an important strategy for maintaining cellular ho-
meostasis during repetitive thermal stress; however, it is unclear if
persistence of a high antioxidant state in these populations is sus-
tainable long- term. Building on these findings, future works should
continue to test for coral physiotype changes at the individual and
population levels in response to repeated thermal stress an d deter-
mine whether high antioxidant states are indicative of either stress
acclimation and physiological resilience or chronic stress and holo-
biont dysbiosis.
Cor al st ress response s are based on a net work of dynamic in terac-
tions at biological and environmental levels (Suggett & Smith, 2020)
that can influence responses to physiological challenges posed by
a warming planet. In an era of increasing frequency and magnitude
of thermal stress event s, the ongoing examination of legacy effects
on corals is of great importance. Our study highlights how cumula-
tive impacts of stress, history and subsequent responses can result
in fundamentally different molecular and physiological states, even
within a short period of time. Thus, we provide further evidence that
environmental memory shapes the homeostatic strategies of cor-
als, ultimately dictating a coral's abilit y to respond to future stress
events.
ACKNOWLEDGEMENTS
The authors acknowledge funding support from an Environmental
Protection A gency STAR Fellowship Assistance Agreement (FP-
91779401- 1) to C.B.W. and NSF 1756623 (Biological Oceanography,
Integrative and Ecological Physiology and EPSCoR) to H.M.P. The
views expressed in this publication have not been reviewed or en-
dorsed by the EPA and are solely those of the authors. We also thank
P.J. Edmunds and reviewers for insightful comments on manuscript
drafts and W. Ellis and R.A.B. Mason for laborator y and field assis-
tance. This is SOEST contribution number 11302 and HIMB contri-
bution number 1849.

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CONFLICT OF INTEREST
The authors declare they have no competing interests.
AUTHORS’ CONTRIBUTIONS
C.B.W., C. A.R., L.D.M., R.D.G. and H.M.P. designed the projec t;
C.B.W., C.A.R., L.D.M. and H.M.P. wrote the manuscript and C.B.W.
statistically analysed the data; Coral collections were performed by
C.B.W. and A.D.W. Laborator y analyses were per formed by C.B.W.,
C.A .R., A.D.W., B.E.L. and D.E.K.
DATA AVA ILAB ILITY STATE MEN T
All data and code to generate figures and perform analyses are archived
and openly available at Github (https://github.com/cbwal l/Gates - Mydla
rz- bleac hing- recovery) and Zenodo (Wall & Putnam, 2021).
ORCID
Christopher B. Wall https://orcid.org/0000-0002-7164-3201
Contessa A. Ricci https://orcid.org/0000-0002-2202-1449
Hollie M. Putnam https://orcid.org/0000-0003-2322-3269
REFERENCES
Ainswor th, T. D., Heron, S. F., Ortiz, J. C., Mumby, P. J., Grech, A., Ogawa,
D., Eakin, C. M., & Leggat, W. (2016). Climate change disables coral
bleaching protec tion on the Great Barrier Reef. Science, 352 (6283),
338– 342. https://doi.org/10.1126/scien ce.aac7125
Anthony, K. R. N., Kline, D. I., Diaz- Pulido, G., Dove, S., & Hoegh- Guldberg,
O. (2008). Ocean acidification causes bleaching and productivity loss
in coral re ef builders. Proceedings of the National Academy of Sciences
of the United States of America, 105(45 ), 17442– 17446 . ht tps ://doi .
org /10.1073/pnas.08044 78105
Bahr, K. D., Rodgers, K. S., & Jokiel, P. L. (2017). Impact of three bleach-
ing events on the reef resiliency of Kāne'ohe Bay. Hawai'i. Frontiers
in Marine Science, 4, 435. https://doi.org/10.3389/fmars.2017.00398
Baker, A. C. (2003). Flexibility and specificity in coral- algal symbiosis:
Diversity, ecology, and biogeography of Symbiodinium. Annual Review
of Ecology, Evolution, and Sys tematics, 34, 661– 689.Retrieved from
http://www.jstor.org/stabl e/30033790
Barshis, D. J., Ladner, J. T., Oliver, T. A ., Seneca, F. O., Traylor- Knowles,
N., & Palumbi, S. R . (2013). Genomic basis for coral resilience to cli-
mate change. Proceedings of the National Academy of Sciences of the
United States of A merica, 110(4), 1387– 1392. https://doi.org/10.1073/
pnas.12102 24110
Brown, B. E., Dunne, R. P., Edwards, A. J., Sweet , M. J., & Phongsuwan, N.
(2015). Decadal environmental ‘memory’ in a reef coral? Marine Biology,
162( 2 ) , 4 7 9 – 4 8 3 . h t t p s : / / d o i . o r g / 1 0 . 1 0 0 7 / s 0 0 2 2 7- 0 1 4 - 2 5 9 6 - 2
Brown, B. E., Dunne, R. P., Goodson, M. S., & Douglas, A. E. (2000).
Bleaching patterns in reef corals. Nature, 404(6774), 142– 143. https://
doi.org/ 10.1038/350 04 657
Couch, C. S., Burns, J. H. R., Liu, G., Steward, K., Gutlay, T. N., Kenyon,
J., Eakin, C. M., & Kosaki, R . K . (2017). Mass coral bleaching due to
unprecedented marine heatwave in Papahānaumokuākea Marine
National Monument (Northwestern Hawaiian Islands). PLoS ONE,
12(9), e0185121. https://doi.org/10.1371/journ al.pone.0185121
Cunning, R., Ritson- Williams, R., & Gates, R. D. (2016). Patterns of
bleaching and recovery of Montipora capitata in K āne‘ohe Bay,
Hawai‘i, USA. Marine Ecology Progress Series, 551 , 131– 139. https://
doi.org/10.3354/meps1 1733
Drupp, P., De Carlo, E. H., Mackenzie, F. T., Bienfang, P., & Sabine, C. L.
(2011). Nutrient inputs, phytoplankton response, and CO2 variations
in a semi- enclosed subtropical embayment, Kaneohe Bay, Hawaii.
Aquatic Geochemistry, 17(4), 473– 498. htt ps: //doi.or g/10.1007/s1049
8 - 0 1 0 - 9 1 1 5 - y
Drupp, P. S., De C arlo, E. H., Mackenzie, F. T., Sabine, C. L., Feely, R. A., &
Shamberger, K. E. (2013). Comparison of CO2 dynamics and air– sea gas
exchange in differing tropical reef environments. Aquatic Geochemistry,
19( 5 ) , 3 7 1 – 3 9 7. h t t p s : / /d o i . o r g / 1 0 . 1 0 0 7/ s 1 0 4 9 8 - 0 1 3 - 9 2 1 4 - 7
Fues s, L. E., Ma nn, W. T., Jink s, L. R. , Bri nkhui s, V., & Mydl arz , L. D. (20 18 ).
Transcriptional analyses provide new insight into the late- stage im-
mune response of a diseased Caribbean coral. Royal Society Open
Science, 5(5), 172062. https://doi.org/10.1098/rsos.172062
Gardner, S. G., Raina, J.- B., Nitschke, M. R., Nielsen, D. A., Stat, M., Motti,
C. A., Ralph, P. J., & Petrou, K. (2017). A multi- trait sys tems approach
reveals a response cascade to bleaching in corals. BMC Biology, 15(1),
1 1 7 . h t t p s : / / d o i . o r g / 1 0 . 1 1 8 6 / s 1 2 9 1 5 - 0 1 7 - 0 4 5 9 - 2
Guest , J. R., Baird, A. H., Maynard, J. A., Muttaqin, E., Edwards, A. J.,
Campbell, S. J., Yewdall, K., Affendi, Y. A., & Chou, L. M. (2012).
Contrasting patterns of coral bleaching susceptibility in 2010 sug-
gest an adaptive response to thermal stress. PLoS ONE, 7(3), e33353.
https://doi.org/10.1371/journ al.pone.0033353
Hughes, T. P., Anderson, K. D., Connolly, S. R., Heron, S. F., Kerry, J. T., Lough,
J. M., Baird, A. H., Baum, J. K., Berumen, M . L., Bridge, T. C., Claar, D. C.,
Eakin, C. M., Gilmour, J. P., Graham, N. A. J., Harrison, H., Hobbs, J.- P. A.,
Hoey, A. S., Hoogenboom, M., Lowe, R. J., … Wilson, S. K. (2018). Spatial
and temporal patterns of mass bleaching of corals in the Anthropocene.
Science, 359(6371), 80– 83. https://doi.org/10.1126/scien ce.aan8048
Hughes, T. P., Kerry, J. T., Baird, A. H., Connolly, S. R., Chase, T. J., Dietzel,
A., Hill, T., Hoey, A. S., Hoogenboom, M. O., Jacobson, M., Kerswell,
A., Madin, J. S., Mieog, A., Paley, A. S., Pratchett, M. S., Torda, G.,
& Woods, R. M. (2019). Global warming impairs stock– recruitment
dynamics of corals. Nature, 568(7752), 387– 390. https://doi.
o r g / 1 0 . 1 0 3 8 / s 4 1 5 8 6 - 0 1 9 - 1 0 8 1 - y
Hughes, T. P., Kerry, J. T., Baird, A. H., Connolly, S. R., Dietzel, A.,
Eakin, C. M., Heron, S. F., Hoey, A. S., Hoogenboom, M. O., Liu, G.,
McWilliam, M . J., Pears, R. J., Pratchett , M. S., Skirving, W. J., Stella,
J. S., & Torda, G. (2018). Global warming transforms coral reef as-
semblages. Nature, 556 (7702), 492– 496. https://doi.org/10.1038/
s 4 1 5 8 6 - 0 1 8 - 0 0 4 1 - 2
Hughes, T. P., Kerr y, J. T., Connolly, S. R., Baird, A. H., Eakin, C. M.,
Heron, S. F., Hoey, A. S., Hoogenboom, M. O., Jacobson, M., Liu, G .,
Pratchett, M. S ., Skirving, W., & Torda, G. (2019). Ecological mem-
ory modifies the cumulative impact of recurrent climate extremes.
Nature Climate Change, 9(1), 40– 43. https://doi.org/10.1038/s4155
8- 018- 0351- 2
Jeffrey, S. W., & Humphrey, G. F. (1975). New spectrophotometric equa-
tions for determining chlorophylls a, b, c1 and c2 in higher plants, algae
and natural phytoplankton. Biochemie Un d Physiologie Der Pf lanzen: BPP,
167( 2 ) , 1 9 1– 1 9 4 . h t t p s : / / d o i . o r g / 1 0 . 1 0 1 6 / S 0 0 1 5 - 3 7 9 6 ( 17 ) 3 0 7 7 8 - 3
Jokiel, P. L., & Brown, E. K. (200 4). Global warming, regional trends
and inshore environmental conditions influence coral bleaching in
Hawaii. Global Change Biology, 10(10), 1627– 1641. https://doi.org/
10.1111/j.1365- 2486.2004.00836.x
Krueger, T., Fisher, P. L., Becker, S., Pont asch, S., Dove, S., Hoegh-
Guldberg, O., Leggat, W., & Davy, S. K. (2015). Transcriptomic char-
acterization of the enz ymatic antioxidants FeSOD, MnSOD, APX and
KatG in the dinoflagellate genus Symbiodinium. BMC Evolutiona ry
Biology, 15, 4 8 . h t t p s : / / d o i . o r g / 1 0 . 1 1 8 6 / s 1 2 8 6 2 - 0 1 5 - 0 3 2 6 - 0
Krueger, T., Hawkins, T. D., Becker, S., Pontasch, S., Dove, S., Hoegh-
Guldberg, O., Leggat, W., Fisher, P. L., & Davy, S. K. (2015). Differential
coral bleaching – Contrasting the activity and response of enzymatic
antioxidants in symbiotic par tners under thermal stress. Comparative
Biochemistry and Physiology, Part A, 19 0(C), 15– 25. https://doi.org/
10.1016/j.cbpa.2015.08.012
Lesser, M. P., Stat, M., & Gates, R. D. (2013). The endosymbiotic dinofla-
gellates (Symbiodinium sp.) of corals are parasites and mutualist s. Coral
Reefs, 32( 3 ) , 6 0 3 – 6 1 1 . h t t p s : // d o i . o r g / 1 0 . 1 0 0 7 / s 0 0 3 3 8 - 0 1 3 - 1 0 5 1 - z

|
1377
Functional Ecolog
y
WALL et AL.
Lowe, R. J., Falter, J. L., Monismith, S. G., & Atkinson, M. J. (2009). A nu-
merical study of circulation in a coastal reef- lagoon system. Jour nal of
Geophysical Research, 114(C6), 997. https://doi.org/10.1029/2008J
C005081
May fi eld, A . B. , Chen, C.- S., & Demps ey, A . C. (2017). The molecu lar eco -
physiology of closely related pocilloporid corals of New Caledonia.
Platax, 14, 1– 45.
McWilliam, M., Pratchett, M. S., Hoogenboom, M. O., & Hughes, T. P.
(2020). Deficits in functional trait diversity following recover y on
coral reefs. Proceedings of the Royal Society B: Biological Science s,
287(1918), 20192628. https://doi.org/10.1098/rspb.2019.2628
Mur phy, J. W. A. , Colli er, A . C., & Ri chmond, R. H. (2019). Antioxi dant en-
zyme cycling over reproductive lunar cycles in Pocillopora damicornis.
Pee rJ, 7, e7020. https://doi.org/10.7717/peerj.7020
Mydlarz, L . D., Couch, C. S., Weil, E., Smith, G., & Har vell, C. D. (2009).
Immune defenses of healthy, bleached and diseased Montastraea
faveolata during a natural bleaching event. Diseases of Aquatic
Organisms, 87(1– 2), 67– 78. https://doi.org /10.3354/dao02088
Mydlarz, L. D., & Harvell, C . D. (20 07). Peroxidase activity and inducibil-
ity in the sea fan coral exposed to a fungal pathogen. Comparative
Biochemistry and Physiology. Part A , Molecular & Integrative Physiology,
146 (1), 54– 62. https://doi.org/10.1016/j.cbpa.2006.09.005
Mydlarz, L. D., Holthouse, S. F., Peters, E. C., & Harvell, C. D. (2008).
Cellular responses in sea fan corals: Granular amoebocytes react to
pathogen and climate stressors. PLoS ONE, 3(3), e1811. https://doi.
org/10.1371/journ al.pone.0001811
Mydl arz, L. D., McGi nty, E. S. , & Harve ll, C. D. (2010). What are the phys-
iological and immunological responses of coral to climate warming
and disease? The Journal of Experimental Biology, 213(6), 934– 945.
https://doi.org/10.1242/jeb.037580
Myd la r z, L. D., & Palme r, C. V. (20 11). Th e pr es ence of multipl e ph enolox-
idases in Caribbean reef- building corals. Comparative Biochemistry
and Physiology. Part A , Molecular & Integrative Physiology, 159(4), 372–
378. https://doi.org/10.1016/j.cbpa.2011.03.029
NOAA . (2019, August 14). National Data Buoy Center, Station MOKH1 -
1612480 - Mokuoloe, HI. Retrieved from https://www.ndbc.noaa.gov/
stati on_page.php?stati on=mo kh1
NOAA . (2020, April 20). Coral Reef Watch, NOAA Satellite and
Information Services. Retrieved from https://coral reefw atch.noaa.
gov/satel lite/metho dolog y/metho dology.php
Noonan, S. H. C., & Fabricius, K. E. (2016). Ocean acidification affects
productivity but not the severity of thermal bleaching in some tropi-
cal corals. ICES Journal of Marine Science, 73 (3), 715– 726. https://doi.
org /10.1093/icesj ms/fsv127
Oksanen, J., Guillaume, F. B., Friendly, M., Kindt, R., Legendre, P., McGlinn,
D., … Wagner, H . (2019). vegan: Com munity ecology package (pp. 631–
637). Retrieved from https://cran.r- proje ct.org/web/packa ges/ vegan/
index.html
Oliver, E. C. J., Benthuysen, J. A ., Darmaraki, S., Donat, M. G., Hobday,
A. J., Holbrook, N. J., Schlegel, R. W., & Sen Gupta, A. (2021). Marine
heatwaves. Annual Review of Marine Science, 13, 313– 342. https://
d o i . o r g / 1 0 . 1 1 4 6 / a n n u r e v - m a r i n e - 0 3 2 7 2 0 - 0 9 5 1 4 4
Oliver, E. C. J., Donat, M. G., Burrows, M. T., Moore, P. J., Smale, D. A.,
Alexander, L. V., Benthuysen, J. A., Feng, M., Sen Gupta, A., Hobday,
A. J., Holbrook, N. J., Perkins- Kirkpatrick, S. E., Scannell, H. A .,
Straub, S. C., & Wernberg, T. (2018). Longer and more frequent ma-
rine heat waves over the past century. Nature Communications, 9(1),
1 3 2 4 . h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 1 8 - 0 3 7 3 2 - 9
Palmer, C. V. (2018a). Immunity and the coral crisis. Communications
Biology, 1( 9 1 ) . h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 4 2 0 0 3 - 0 1 8 - 0 0 9 7 - 4
Palmer, C. V. (2018b). Warmer water affects immunity of a tolerant reef
coral. Frontiers in Marine Science, 5(266). https://doi.org/10.3389/
fmars.2018.00253
Palmer, C. V., Bythell, J. C., & Willis, B. L. (2010). Levels of immunity
parameters underpin bleaching and disease susceptibility of reef
corals. FASEB Journal, 24(6), 1935– 1946. https://doi.org/10.1096/
f j . 0 9 - 1 5 2 4 4 7
Palmer, C. V., McGinty, E. S., Cummings, D. J., Smith, S. M., Bartels, E.,
& Mydlarz, L. D. (2011). Patterns of coral ecological immunology:
Variation in the responses of C aribbean corals to elevated tem-
perature and a pathogen elicitor. The Journal of Experimental Biology,
214( 24), 4240– 4249. https://doi.org/10.1242/jeb.061267
Palmer, C. V., Traylor- Knowles, N. G., Willis, B. L., & Bythell, J. C. (2011).
Corals use similar immune cells and wound- healing processes as
those of higher organisms. PLoS ONE, 6(8), e23992. https://doi.org/
10.1371/journ al.pone.0023992
Palumbi, S. R., Barshis, D. J., Traylor- Knowles, N., & Bay, R. A. (2014).
Mechanisms of reef coral resistance to future climate change. Science,
344(6186), 895– 898. https://doi.org/10.1126/scien ce.1251336
Pinzón C., J. H., Beach- Letendre, J., Weil, E., & Mydlarz, L. D. (2014).
Relationship between phylogeny and immunity sug gests older
Caribbean coral lineages are more resistant to disease. PLoS ONE,
9(8), e104787. https://doi.org/10.1371/journ al.pone.0104787
Pinzón, J. H., Kamel, B., Burge, C. A., Harvell, C. D., Medina, M., Weil,
E., & Mydlarz, L. D. (2015). Whole transcriptome analysis reveals
changes in expression of immune- related genes during and after
bleaching in a reef- building coral. Royal Society O pen Scie nce, 2(4),
140214. https://doi.o rg /10.1098/rsos .140214
R Core Team. (2019). R: A language and environment for statis tical comput-
ing. R Foundation for Statistical Computing. Retrieved from https://
w w w . R - p r o j e c t . o r g
Ritson- Williams, R., & Gates, R. D. (2020). Coral community resilience to
successive years of bleaching in Kāne'ohe Bay, Hawai'i. Coral Reefs,
39( 3 ) , 7 5 7 – 7 6 9 . h t t p s : / / d o i . o r g / 1 0 . 1 0 0 7 / s 0 0 3 3 8 - 0 2 0 - 0 1 9 4 4 - 4
Safaie, A., Silbiger, N. J., McClanahan, T. R., Pawlak, G., Barshis, D. J.,
Hench, J. L., Rogers, J. S., Williams, G. J., & Davis, K. A. (2018). High
frequency temperature variability reduces the risk of coral bleaching.
Nature Communications, 9(1), 1671. https://doi.org/10.1038/s4146 7-
0 1 8 - 0 4 0 7 4 - 2
Sale, T. L., Marko, P. B., Oliver, T. A., & Hunter, C. L. (2019). Assessment
of acclimatization and subsequent survival of corals during repeated
natural thermal stress events in Hawai'i. Marine Ecology Progress
Series, 624, 65– 76. https://doi.org/10.3354/meps1 3031
Sampayo, E. M., Ridgway, T., Bongaert s, P., & Hoegh- Guldberg, O. (2008).
Bleaching susceptibility and mortality of corals are determined by
fine- scale differences in symbiont type. Proceedings of the National
Academy of Sciences of the United States of America, 105 (30), 10444–
104 49. htt ps://doi.or g/10.1073/pna s.0708 0 49105
Schoepf, V., Grottoli, A. G., Levas, S. J., Aschaffenburg, M. D., Baumann, J.
H., Matsui, Y., & Warner, M. E. (2015). Annual coral bleaching and the
long- term recover y capacity of coral. Proceedings of the Royal Society
B: Biological Sciences, 282(1819), 20151887. https://doi.org/10.1098/
rspb.2015.1887
Silvers tein, R. N., Cunning, R., & Baker, A. C . (2017). Tenacious D:
Symbiodinium in clade D remain in reef corals at both high and
low temperature extremes despite impairment. The Journal of
Experimental Biology, 220(7), 1192– 1196. https://doi.org/10.1242/
jeb.148239
Stimson, J., & Kinzie, R. A. (1991). The tempor al pattern and rate of
release of zooxanthellae from the reef coral Pocillopora damicornis
(Linnaeus) under nitrogen- enrichment and control conditions. Journal
of Exper imental Marine Biology an d Ecology, 153(1), 63– 74. ht tps://doi .
o r g / 1 0 . 1 0 1 6 / S 0 0 2 2 - 0 9 8 1 ( 0 5 ) 8 0 0 0 6 - 1
Sugget t, D. J., & Sm ith , D. J. (2020 ). Co ral bleaching patterns are the out-
come of complex biological and environmental networking. Global
Change Biology, 26(1), 68– 79. https://doi.org/10.1111/gcb.14871
Sugget t, D. J., Warner, M. E., & Leggat, W. (2017). Symbiotic dinofla-
gellate functional diversity mediates coral survival under ecologi-
cal crisis. Trends in Ecology & Evolution, 32 (10), 735– 745. https://doi.
org/10.1016/j.tree.2017.07.013

1378
|
Functional Ecology
WALL et AL.
Thompson, D. M., & van Woesik, R. (20 09). Corals escape bleaching in
regions that recently and historically experienced frequent ther-
mal stress. Proceedings of the Royal Society B: Biologic al Sciences,
276 (1669), 2893– 2901. https://doi.org/10.1098/rspb.2009.0591
Thornhill, D. J., Rotjan, R . D., Todd, B. D., Chilcoat, G. C., Iglesias- Prieto,
R., Kemp, D. W., LaJeunesse, T. C., Reynolds, J. M. C., Schmidt, G.
W., Shannon, T., Warner, M. E., & Fit t, W. K. (2011). A connection
between colony biomass and death in Caribbean reef- building
corals. PLoS ONE, 6(12), e29535. https://doi.org/10.1371/journ al.
pone.0029535
Van Straalen, N. M. (2003). Ecotoxicology becomes stress ecology.
Environmental Science & Echnology, 37(17 ), 324A– 33 0A . https://doi .
org /10.1021 /es032 5720
Wall, C. B., Fan, T.- Y., & Edmunds, P. J. (2014). Ocean acidification has no
effect on thermal bleaching in the coral Seriatopora caliendrum. Coral
Reefs, 33( 1 ) , 1 1 9 – 1 3 0 . h t t p s : / / d o i . o r g / 1 0 . 1 0 0 7 / s 0 0 3 3 8 - 0 1 3 - 1 0 8 5 - 2
Wall, C. B., Kaluhiokalani, M., Popp, B. N., Donahue, M. J., & Gates, R.
D. (2020). Divergent symbiont communities determine the physiol-
ogy and nutrition of a reef coral across a light- availabilit y gradient.
The ISME Journal, 14(4), 945– 958. https://doi.org/10.1038/s4139
6 - 0 1 9 - 0 5 7 0 - 1
Wall, C., & Putnam, H. (2021). cbwall/Gates- Mydlarz- bleaching- recovery:
Repeat bleaching coral physiotypes (Version vpub). Zenodo, h t tps://
doi.org/10.5281/zenodo.4647959
Wall, C. B., Ricci, C. A., Foulds, G. E., Mydlarz, L. D., Gates, R. D., &
Put na m, H. M. (2018). Th e ef fect s of environmental histor y and ther-
mal stress on coral physiology and immunity. Marine Biology, 165 (3),
5 6 . h t t p s : / / d o i . o r g / 1 0 . 1 0 0 7 / s 0 0 2 2 7 - 0 1 8 - 3 3 1 7 - z
Weis, V. M. (2008). Cellular mechanisms of Cnidarian bleaching: Stress
causes the collapse of symbiosis. The Journal of Experimental Biology,
211(19), 3059– 3066. https://doi.org/10.1242/jeb.009597
Wham, D. C., Ning, G., & LaJeunesse, T. C. (2017). Symbiodinium glynnii
sp. nov., a species of stress- tolerant symbiotic dinoflagellates from
pocilloporid and montiporid corals in the Pacific Ocean. Phycologia,
56, 396– 4 09. https://doi.org/10.2216/16- 86.1
SUPPORTING INFORMATION
Additional suppor ting information may be found online in the
Supporting Information section.
How to cite this article: Wall CB, Ricci CA , Wen AD, et al.
Shifting baselines: Physiological legacies contribute to the
response of reef corals to frequent heatwaves. Funct Ecol.
2021;35:1366– 1378. ht tps://doi.org/10 .1111/1365- 2435.13795