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Transgenerational inheritance of thermal tolerance in two coral species in the Florida Keys
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Transgenerational inheritance of thermal tolerance in two coral species in the Florida Keys
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Content
Transgenerational Inheritance of Thermal Tolerance in Two Coral Species in the
Florida Keys
By
Yingqi Zhang
A Dissertation Presented to the
FACULTY OF THE USC GRADUA TE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MARINE BIOLOGY AND BIOLOGICAL OCEANOGRAPHY)
August 2023
Copyright 2023 Yingqi Zhang
ii
DEDICATION
To all my family members who have always been my biggest fans and sources of support
since the very beginning. I am especially grateful for my parents, Chongli Zhang and Haili Xue,
who are undoubtedly the absolute GOAT. Thank you for supporting my decision to come to the
United States in 2014 and growing with me throughout my academic and life journey despite
being thousands of miles apart. I would never become who I am today without your
unconditional love.
To my husband, my partner in crime, the love of my life, Nick Tunnicliffe. You have
brought so much joy and light into my life. I cannot wait to see what the future holds for us as we
embark on this crazy parenthood journey.
To my newborn son, Isomer Tunnicliffe. I love you so much and can tell that you are
going to be a riot. May you find your own sparks in life someday, whether they are related to
natural sciences or not (no pressure). I will be incredibly honored to guide and support you along
the way.
iii
ACKNOWLEDGEMENTS
I attribute much of my success and accomplishment in my graduate career to my beloved
advisor, Dr. Carly Kenkel. From publishing my first peer-reviewed paper to leading my first
coral spawning trip in the Florida Keys, you not only provided me with the critical research
support and guidance, but also instilled in me the confidence and grit to become a better scientist
and human being. I have so much respect and appreciation for you. I will be forever grateful for
your mentorship and friendship from the past, present, and future. Pursuing a Ph.D. is by no
means an easy path, but you certainly made it a lot more enjoyable for me and so many others.
I would like to thank my committee members for your support, insight, and expertise that helped
improve this thesis and expanded my scientific horizon.
I would like to thank the staff at Mote Marine Laboratory for your assistance in the field and lab,
especially Erich Bartels. I would also like to thank our collaborators at NOAA and University of
Alabama at Birmingham. It was my pleasure working on cool science projects with you and
sharing fun field and lab memories.
I would also like to thank the past and present CEE lab members, especially Dr. Maria Ruggeri,
Dr. Holland Elder, Dr. Wyatt Million, Dr. Xuelin Zhao, Emily Aguirre and Jenna Dilworth, for
your endless support and inspiration in my academic journey and beyond. You all are incredible
scholars and friends. I am so grateful that our paths crossed.
iv
TABLE OF CONTENTS
DEDICATION ................................................................................................................................ ii
ACKNOWLEDGEMENTS ........................................................................................................... iii
LIST OF FIGURES ....................................................................................................................... vi
ABSTRACT ................................................................................................................................. viii
CHAPTER 1. INTRODUCTION ................................................................................................... 1
1.1 Coral Bleaching in the Context of Global Climate Change ................................................................ 1
1.2 Factors Determining Thermal Tolerance in Corals ............................................................................. 1
1.3 Life History Cycle and Reproductive Strategies in Corals ................................................................. 3
1.4 Focal Species and Study System ......................................................................................................... 5
1.5 Thesis Outline ..................................................................................................................................... 7
CHAPTER 2. FAMILY MATTERS: V ARIATION IN THE PHYSIOLOGY OF BROODED
PORITES ASTREOIDES LARV AE IS DRIVEN BY PARENT COLONY EFFECTS ................ 8
2.1 Abstract ............................................................................................................................................... 8
2.2 Introduction ......................................................................................................................................... 9
2.3 Methods............................................................................................................................................. 14
2.3.1 Coral Collections and Spawning Conditions ............................................................................................. 14
2.3.2 Daily Release Data .................................................................................................................................... 15
2.3.3 Thermal Tolerance Experiments ................................................................................................................ 15
2.3.4 Larval V olume ............................................................................................................................................ 16
2.3.5 Symbiont Density, Chlorophyll, and Protein Content ............................................................................... 17
2.3.6 Statistical Analyses .................................................................................................................................... 18
2.4 Results ............................................................................................................................................... 19
2.4.1 Daily Variation in Larval Physiology by Family ....................................................................................... 19
2.4.2 Physiological Response to Moderate Thermal Stress ................................................................................ 21
2.4.3 Survival under Acute Thermal Stress ........................................................................................................ 22
2.5 Discussion ......................................................................................................................................... 24
2.6 Supplementary Materials .................................................................................................................. 30
CHAPTER 3. CROSS-GENERATION HERITABILITY ANALYSIS OF PHYSIOLOGICAL
TRAITS IN PORITES ASTREOIDES ACROSS AN INSHORE-OFFSHORE GRADIENT IN
THE LOWER FLORIDA KEYS .................................................................................................. 35
3.1 Abstract ............................................................................................................................................. 35
3.2 Introduction ....................................................................................................................................... 36
3.3 Methods............................................................................................................................................. 39
3.3.1 Spawning Collection and Larval Thermal Stress Experiment ................................................................... 39
3.3.2 Adult and Recruit Thermal Stress Experiments ......................................................................................... 42
3.3.3 Physiological Assays ................................................................................................................................. 43
3.3.4 Statistical Analyses .................................................................................................................................... 45
3.4 Results ............................................................................................................................................... 47
3.5 Discussion ......................................................................................................................................... 49
v
3.6 Supplementary Materials .................................................................................................................. 56
CHAPTER 4. PERFORMANCE OF ORBICELLA FA VEOLATA LARV AL COHORTS DOES
NOT ALIGN WITH PREVIOUSLY OBSERVED THERMAL TOLERANCE OF ADULT
SOURCE POPULATIONS ........................................................................................................... 62
4.1 Abstract ............................................................................................................................................. 62
4.2 Introduction ....................................................................................................................................... 63
4.3 Methods............................................................................................................................................. 66
4.3.1 Sites and Temperature Data ....................................................................................................................... 66
4.3.2 Spawning, Cross Design and Larval rearing ............................................................................................. 67
4.3.3 Thermal Stress Challenges ........................................................................................................................ 68
4.3.4 Physiological Assays ................................................................................................................................. 70
4.3.5 Statistical Analysis of Physiological Trait Data and Survival under Acute Stress .................................... 71
4.3.6 RNA Extraction, Library Preparation, and Sequencing ............................................................................. 72
4.3.7 Bioinformatics Pipelines ............................................................................................................................ 73
4.3.8 Statistical Analysis of Gene Expression .................................................................................................... 73
4.4 Results ............................................................................................................................................... 76
4.4.1 Temperatures and bleaching history at study sites ..................................................................................... 76
4.4.2 Larval survival under acute heat stress ...................................................................................................... 76
4.4.3 Gamete (2019 and 2021) and larval ecophysiology (2021 only) .............................................................. 77
4.4.4 Major drivers of transcriptional variation .................................................................................................. 78
4.4.5 Conservation of expression networks and their functional significance ................................................... 79
4.4.6 Origin-specific responses to thermal stress ............................................................................................... 81
4.5 Discussion ......................................................................................................................................... 83
4.5.1 Impaired larval performance may result from reduced reproductive investment ...................................... 84
4.5.2 Stress tolerance rather than a front-loaded stress response is associated with enhanced larval survival .. 87
4.5.3 Conserved transcriptomic signatures of population origin and response to treatment .............................. 88
4.5.4 Implications for adaptive management ...................................................................................................... 90
4.6 Supplementary Materials .................................................................................................................. 90
CHAPTER 5. CONCLUSION ..................................................................................................... 99
REFERENCES ........................................................................................................................... 101
APPENDICES ............................................................................................................................ 121
APPENDIX A: 2bRAD PARENTAGE ANALYSIS OF PORITES ASTREOIDES LARV AE ........... 121
vi
LIST OF FIGURES
Figure 1. 1 Two P. astreoides adult collection sites in the lower FL Keys and two O. faveolata
gamete collection sites in the upper FL Keys. Inshore and offshore sites are marked in red and
blue respectively. ............................................................................................................................. 7
Figure 2. 1 Larvae volume for each family during 5 days of release (mean ± SEM). Families 1-
15 are inshore-origin parents and families 26-41 are offshore-origin parents. The y-axis indicates
sequential days in which planulation was observed, with day 1 indicating the first day of larval
collection and continuing for a total of five days of observation. ................................................ 20
Figure 2. 2 Concentration of chlorophyll a during the moderate temperature stress experiment by
family and treatment (mean ± SEM). Families 2, 4 and 15 are inshore-origin parents and families
26-29 are offshore-origin parents. ................................................................................................. 22
Figure 2. 3 Mortality curves of larvae shown as mean survival (± SEM) over time for the acute
thermal stress experiments consisting of (A) twelve families and (B) six families. Families 1-15
are inshore-origin parents and families 26-41 are offshore-origin parents. .................................. 23
Figure 3. 1 Experimental design and timeline including ten Porites astreoides families. ........... 41
Figure 3. 2 Standardized physiological parameters in response to experimental conditions shared
across all life stages separated by reef origin. Boxplots represent the 50th (median), 25th, and
75th percentiles (± 1.5 × IQR). Each column represents a distinct life stage (left to right: adult,
larvae, and recruit) and each row represents a distinct trait (top to bottom: symbiont density,
chlorophyll a concentration, total soluble protein content). Natural logarithmic transformation
was performed on values that were not normally distributed. ...................................................... 47
Figure 3. 3 Broad-sense heritability estimate (mean ± 95% C.I.) for three shared physiological
traits at different life stages. .......................................................................................................... 49
Figure 4. 1 Time series of running 30-day mean sea surface temperature (SST) for Cheeca Rocks
(red line) and Horseshoe Reef (blue line) from 2017 to 2021. Bleaching threshold for Cheeca
Rocks (monthly mean SST ≥ 31.3°C) shown as dashed black line. ........................................... 76
Figure 4. 2 Number of surviving larvae (mean ± standard error of the mean [SEM]) across time
in the acute temperature stress experiment for a) 2019 and b) 2021 cohorts. Survivorship was
grouped by larval origin and treatment condition. ........................................................................ 77
Figure 4. 3 Concentration (mean ± SEM) of (a) different lipid classes (PL: phospholipid, TAG:
triacylglycerol, WAX: wax ester) and (b) total lipids standardized by individual gamete bundle
collected from Cheeca Rocks (n = 5) and Horseshoe Reef (n = 5) in 2019 and 2021. No
replicates were available for CR gametes in 2021. ....................................................................... 78
vii
Figure 4. 4 Principal component analysis (PCA) on rlog-transformed read counts in a) 2019 and
b) 2021 larval datasets. Points are colored by origin and shaped by treatment. The percentage
variance explained by each PC is reflected on the axis label. ...................................................... 79
Figure 4. 5 Weighted gene co-expression network analysis (WGCNA) module-trait relationships
identified in a) 2019 and b) 2021 larval cohort. Correlation values range from 1 (red) to -1 (blue)
and the associated p values were included in the parenthesis below for modules showing a
significant trait association, with color of each block determined by strength and direction of the
correlation between given module and trait. ................................................................................. 80
Figure 4. 6 Hierarchical clustering of ontology terms enriched by genes up-regulated (red) or
down-regulated (blue) in 2019 heat-treated (a) CR x CR larvae and (b) HR x HR larvae
compared to their respective untreated control, summarized by biological process (BP). Font size
indicates level of statistical significance (FDR-corrected). Term names are preceded by fractions
indicating the number of individual genes within each term differentially regulated with respect
to treatment (unadjusted p < 0.05). (c) Density plots showing distribution of global expression
across samples from the two origins along the temperature responsive axis (linear discriminant 2
[LD2], Fig. S7) based on discriminant analysis of principal components (DAPC) performed on
variance stabilized data (VSD) grouped by treatment and origin. ................................................ 83
viii
ABSTRACT
Driven by increasing sea surface temperature, coral bleaching events have become more
severe and frequent across the globe since the early 1980s. Yet not all coral population responses
are equal. Certain populations can outperform their conspecifics under similar heat stress by
adapting and/or acclimatizing to local thermal conditions. In the Florida Keys, populations closer
to shore are generally more resilient to heat stress than neighboring offshore populations
potentially due to naturally higher and more variable temperature regimes that are characteristic
of the inshore reef environment. Most current studies on local adaptation and/or acclimatization
focus on adults, but the implications of parental adaptation and/or acclimatization on offspring
life stages and even generations remain relatively unexplored. To address this important
knowledge gap, this dissertation aims to investigate the heritability of population-specific
thermal tolerance traits across generations in Porites astreoides (Chapters 2 and 3) and Orbicella
faveolata (Chapter 4), two ecologically significant coral species in Florida and the wider
Caribbean. Ultimately, understanding the genetic and physiological contribution to fitness-
related phenotypic differences among spatially distinct populations will help us predict how
corals will respond to selection under future global warming scenarios and determine the best
restoration as well as management strategies.
The first study was designed to understand family effects on larval physiology in Porites
astreoides. We found that the majority of variation in baseline physiological and thermal
tolerance traits was explained by larval family, as opposed to previously implicated factors such
as day of release and reef origin. This study inspired the next chapter which further investigated
the extent of familial effects in multiple life stages by performing similar assays on adults,
larvae, and juvenile recruits. High broad sense heritability was identified for multiple
ix
physiological traits among the different life stages, highlighting potentially significant genetic
underpinnings and the adaptive potential of local populations to environmental change. The final
chapter investigated whether larvae produced by a more thermally tolerant inshore adult
Orbicella faveolata population outperformed those originating from less thermally tolerant
offshore-origin parents. Contrary to our expectations, inshore O. faveolata larvae were less heat
tolerant than the offshore larvae on both physiological and molecular levels, which may be
driven by reproductive trade-offs caused by the prior history of thermal stress that the inshore
adults experienced. This finding sheds light on the negative consequences of bleaching events on
coral reproductive output and presents valuable perspectives on implementing selective breeding
methods to reseed declining reefs
1
CHAPTER 1. INTRODUCTION
1.1 Coral Bleaching in the Context of Global Climate Change
Scleractinian corals, the foundation of coral reef ecosystems, have undergone
unprecedented global declines in recent decades (Hughes et al., 2018). Elevated seawater
temperature due to increased atmospheric CO2 is the major threat to reef health and persistence
(Hoegh-Guldberg et al., 2007). The most common stress response in corals is bleaching, which
refers to the whitening of coral tissues resulting from the loss of their endosymbiotic
dinoflagellates in the family Symbiodiniaceae and/or reduction in algal photosynthetic pigments
(Lesser, 2011). Numerous field observations and laboratory studies have demonstrated that
thermal stress is the primary cause for extensive coral bleaching (Heron et al., 2016; Jokiel &
Coles, 1990; Tolleter et al., 2013). Mechanistically, the breakdown in the symbiotic relationship
between coral host and algal symbiont may result from the accumulation of reactive oxygen and
nitrogen species during thermal stress (Weis, 2008). If symbionts are unable to re-populate, the
host will eventually starve to death as up to 90% of its nutritional requirements are met by the
symbionts (Muscatine & Porter, 1977). The subsequent loss of coral cover and structural
complexity can lead to the collapse of the entire reef tract, jeopardizing the associated
biodiversity and ecosystem services (Moberg & Folke, 1999).
1.2 Factors Determining Thermal Tolerance in Corals
However, different coral taxa have different susceptibilities to thermal stress (P. A.
Marshall & Baird, 2000). Different populations within the same species are capable of adapting
and/or acclimatizing to their local thermal environment (Thomas et al., 2018; Torda et al., 2017).
Adaptation results in the increase of thermally resistant genotypes by changing allele frequencies
in a given population. This process requires existing genetic variation in thermal tolerance for
2
natural selection to act upon. A distinct suite of alleles at multiple loci are associated with
increased survival and bleaching resistance in corals exposed to thermal stress (Bay & Palumbi,
2014; Fuller et al., 2020; Smith-Keune & van Oppen, 2006). However, the accumulation of
adaptive genotypes occurs slowly in nature unless selection is strong and recombination rate is
high (Barrick & Lenski, 2013). Acclimatization, on the other hand, is achieved through changing
phenotypic traits (including behavior, morphology, and physiology) during an individual’s
lifespan in response to specific environmental stimuli (Gates & Edmunds, 1999). Since
acclimatization typically operates on a shorter time scale, it can buffer populations from rapid
environmental changes while buying time for adaptation to occur over the longer term (Munday
et al., 2013).
In addition to the coral host, symbiotic algae and other microbial partners also play a
crucial role in determining the thermal tolerance of the organism as a whole. Certain symbiont
types are more resistant to heat stress than others and the relative abundance of each symbiont
can change (or “shuffle”) following a bleaching event, which may increase the colony’s
resistance to future warming episodes (Baker, 2003). Symbiodiniaceae in the genus Durusdinium
(formerly clade D), specifically D. trenchii, are generally considered extremophiles (LaJeunesse
et al., 2018), enabling their associated hosts to experience a higher bleaching threshold during
heat exposure (Berkelmans & van Oppen, 2006; Oliver & Palumbi, 2011). Nonetheless,
associating with D. trenchii has been shown to come at a physiological and energetic cost to its
host, notably reduced carbon acquisition and growth (A. Jones & Berkelmans, 2010). However,
several coral species are able to shuffle to Durusdinium- dominated community structure after
major thermal disturbances (Baker et al., 2004; Cunning et al., 2015) and recent evidence
3
suggests that the shuffled community can be inherited by the next generation in corals which
transmit their symbionts vertically (Quigley et al., 2019).
1.3 Life History Cycle and Reproductive Strategies in Corals
Organisms that experience multiple life history stages typically encounter different
selective pressures as a result of the interactions with different environments, which could yield
profound ecological and evolutionary consequences that affect more than one generation (D. J.
Marshall & Morgan, 2011). Corals exemplify the typical life history of many marine organisms
where sessile adults produce planktonic larvae that eventually recruit back to the benthic
substrate (Richmond & Hunter, 1990a). Since larvae represent the only stage where large-scale
dispersal is possible, larval settlement and subsequent survival are key processes that determine
local demographic patterns and population genetic structures (Palumbi, 2003). In general, the
larval stage in most marine invertebrates is considered the most vulnerable stage due to higher
mortality rate from developmental failure and predation, as well as susceptibility to
environmental stressors including temperature, salinity, pH, and pollution (Pineda et al., 2012;
Putnam & Gates, 2015).
Given the aforementioned genetic basis for heat tolerance in corals, it is not surprising
that thermally resistant adults are likely to produce resistant offspring. Indeed, the performance
of larvae and recruits is strongly influenced by the environmental history of the adult population.
Lipid metabolism differed between Pocillopora damicornis larvae from Taiwan and Moorea
during a heat exposure, suggesting variation in thermal tolerance (Rivest et al., 2017). Larvae of
Acropora millepora exhibited a 10-fold increase in survival odds under heat stress when their
parents came from a warmer reef (G. B. Dixon et al., 2015). To date, the majority of studies on
local adaptation and acclimatization have focused on a single life stage, with fewer studies that
4
incorporated multiple stages (G. B. Dixon et al., 2015; Kenkel, Setta, et al., 2015; Putnam &
Gates, 2015; Quigley & van Oppen, 2022; Wong et al., 2021). More research is needed to track
phenotypes across multiple life stages to better understand the ecological relevance of adaptive
traits related to heat tolerance and predict population persistence under global change.
Within a local population, unique genetic identities of parents and the level of maternal
provisioning can contribute to variation in offspring fitness and their adaptive potential. Traits
that have been shown to be influenced by parental genotypes include fertilization rate, larval
swimming speed, affinity to settlement cues, gene expression patterns, and symbiont community
composition (Baums et al., 2013; Meyer et al., 2009; Quigley et al., 2019). Compared to paternal
effects, maternal effects (which, for the purpose of this thesis, are broadly defined as the causal
influence of the maternal genotype or phenotype on the phenotype of the offspring, see (Wolf &
Wade, 2009)) might be more dominant in corals as has been observed in other marine organisms
(D. J. Marshall, 2008). Maternal investment likely also varies between species that have different
modes of sexual reproduction (Baird et al., 2009). Approximately 85% of scleractinian coral
species reproduce through broadcast spawning where sperm fertilizes eggs in the water column,
with ~80% of these species acquiring their symbionts horizontally from the environment (Baird
et al., 2009). Alternatively, fertilization occurs internally for brooding corals and mature larvae
are subsequently released over several days during monthly or seasonally reproductive cycles
(Fadlallah, 1983), usually with symbionts inherited from the maternal colony. Vertically-
transmitted symbiont communities are speculated to have lower diversity but higher fidelity
(Baker, 2003; Fabina et al., 2012).
5
1.4 Focal Species and Study System
The major goals of this thesis are to investigate the inheritance of thermal tolerance in
two focal coral species exhibiting different life history strategies, Porites astreoides (de
Lamarck, 1838) and Orbicella faveolata (Ellis & Solander, 1786), in the Florida Keys, USA, and
to explore the underlying genetic and physiological mechanisms contributing to variation in
performance (Fig. 1.1). Transgenerational studies on marine organisms have examined the
heritability of morphological and life history traits of economically valuable aquaculture species,
such as Atlantic salmon (Powell et al., 2008) and Pacific Oyster (Evans et al., 2009). Human-
induced environmental stressors that coral reefs face can be regarded as new agents of selection.
Understanding the heritability of bleaching resistance will help us evaluate the potential of local
populations to cope with ongoing ocean warming and subsequently guide conservation and
management decisions.
I chose P . astreoides and O. faveolata primarily because they are among the dominant
reef-builders in the wider Caribbean region and support important ecological functions (Green et
al., 2008; Rippe et al., 2017). Populations of P . astreoides have seen a positive growth over the
past few decades (Green et al., 2008), despite the overall coral cover losses that plagued the
Florida Keys in the same time frame (Precht & Miller, 2007). Such success was attributed to its
weedy lifestyle and the ability to tolerate suboptimal habitats including shallow and turbid
waters. O. faveolata, on the other hand, is characterized by slower growth and longer lifespan
(Gladfelter et al., 1978). Since the 1980s, O. faveolata populations have suffered from a sharp
decline due to slow recovery from repeated bleaching and disease-driven mortality events,
further exacerbated by persistent recruitment failure (van Woesik et al., 2014). The entire genus
Orbicella was listed as threatened under the Endangered Species Act in 2014. Another
6
consideration is that these two species engage in different modes of sexual reproduction; P .
astreoides broods (McGuire, 1998) and O. faveolata broadcast spawns (Szmant, 1991).
Therefore, studying both species in similar experimental and analytical frameworks will allow
me to compare the relative contribution of different mechanisms to thermal resistance in distinct
reproductive types and potentially apply my findings to a wider range of coral species.
The Florida Keys provides an ideal ecosystem to study the adaptation and/or
acclimatization of corals to local environmental regimes. Inshore patch reefs experience high
temperature variation, turbidity, nutrient input, and sedimentation (Kenkel, Almanza, et al., 2015;
Lirman & Fong, 2007). In comparison, the offshore reef tract is buffered by the Florida Current
and is more distant from land-based pollution, and thus is characterized by less varied
temperatures and low turbidity (Kenkel, Almanza, et al., 2015). Inshore coral populations
generally exhibit higher thermal tolerance than offshore populations despite inhabiting a more
stressful physical environment. P . astreoides colonies from inshore sites bleached less severely
than their offshore counterparts in response to a common heat stress (Kenkel et al., 2013).
Inshore O. faveolata also suffered less from bleaching and recovered faster after recent mass
bleaching events than offshore colonies (Manzello et al., 2015a, 2019). However, strong reef
origin effects have only been documented in the adult populations and it is largely unknown
whether such effects can be passed on to the later life stages. To my best knowledge, only three
studies to date have found origin-dependent variability in thermal tolerance in P . astreoides
larvae and recruits (Kenkel, Setta, et al., 2015; Zhang et al., 2019, 2022), and heritability of
thermal tolerance in O. faveolata offspring has yet to be examined.
7
1.5 Thesis Outline
This thesis was driven by the following research questions: 1. Are the origin-dependent
thermal tolerance patterns observed in adult populations evident in the offspring generation? 2.
What is the relative contribution of maternal vs. paternal components to trait heritability? 3.
What are the underlying physiological and/or molecular mechanisms as well as their ecological
and evolutionary implications? In Chapter 2, I quantified the scope of family-level physiological
variation in P . astreoides larvae that derived from the two reef sites in the lower FL Keys (Fig.
1.1). I then followed up on the results from Chapter 2 by including additional adult and recruit
life stages in Chapter 3, and estimated broad-sense heritability (H
2
) values specific to each life
stage for the various physiological traits examined. In Chapter 4, pure-bred and hybrid O.
faveolata larvae were created by collecting gametes from adult colonies at the two sites in the
Upper FL Keys (Fig. 1.1) to evaluate their thermal tolerance in lab-based heat stress experiments
using physiological metrics and gene expression profiling. In the appendix, a reciprocal
transplant field experiment was conducted to gain a better understanding of the reproductive
biology of P . astreoides using the 2bRAD genotyping method. Preliminary data suggested that
larvae can be a product of both sexual and asexual reproduction, although further method
development is needed to derive reliable inferences.
Figure 1. 1 Two P. astreoides adult collection sites in the lower FL Keys and two O. faveolata gamete collection sites in the upper
FL Keys. Inshore and offshore sites are marked in red and blue respectively.
8
CHAPTER 2. FAMILY MATTERS: VARIATION IN THE PHYSIOLOGY OF
BROODED PORITES ASTREOIDES LARVAE IS DRIVEN BY PARENT
COLONY EFFECTS
Yingqi Zhang, Wyatt C. Million, Maria Ruggeri, Carly D. Kenkel
2.1 Abstract
The planktonic larval phase of scleractinian coral life-history represents a crucial stage
when dispersal takes place and genetic diversity among populations is maintained.
Understanding the dynamics influencing larval survival is especially relevant in the context of
climate change, as larvae may be more vulnerable to environmental disturbances than adults.
Several physiological parameters of coral larvae have been shown to vary by release time and
past environmental history. However, the contribution of parental or genetic effects is largely
unknown. To investigate these potential familial effects, we collected adult Porites astreoides
colonies in April 2018 from two reef zones in the lower Florida Keys and quantified
physiological traits and thermal tolerance of the newly released larvae. Family accounted for
more variation than day of release and reef origin, with > 60% of the variation in chlorophyll a
and protein content explained by family. The survivorship of larvae under 36 °C acute
temperature stress was also tightly linked to what parent colony they were released from. During
a 32 °C moderate temperature stress experiment, inshore larvae tended to bleach less than
offshore larvae, mirroring the enhanced bleaching resistance previously observed in inshore adult
coral populations. The significant familial effects identified in the present study suggest that
researchers should be cautious when interpreting results of studies which pool larvae among
families, and that future studies should take care to account for this variation.
9
2.2 Introduction
Modern oceans are currently undergoing a period of rapid environmental change
(Burrows et al., 2011; Zalasiewicz et al., 2011). Marine organisms have the potential to cope
with these challenges through some combination of genetic adaptation, physiological
acclimatization, and/or migration to a more suitable environment or microclimate (Foo & Byrne,
2016; Gienapp et al., 2008; Palumbi et al., 2014). The general consensus is that the accumulation
of beneficial mutations occurs slowly in nature unless selection is strong and recombination rate
is high (Barrick & Lenski, 2013; Kimura, 1964). An increasing body of literature has identified
phenotypic plasticity as an important acclimatization mechanism that enables populations to
persist in new regimes, while buying time for adaptation to occur over multiple generations
(Hendry, 2016; Munday et al., 2013). Migration or dispersal to more suitable habitats will
facilitate the persistence of a species, but may result in localized extinction of populations
(Gienapp et al., 2008; Pellerin et al., 2019). The extent to which one or a combination of these
strategies is employed depends in large part on life history (Meyers & Bull, 2002; Moreno &
Møller, 2011).
Marine organisms exhibit an incredible diversity of life histories, many of which include
a planktonic larval stage or stages (D. J. Marshall & Morgan, 2011; R. R. Strathmann, 1985). For
species in which adults are sessile, planktonic larvae represent the only life stage in which large-
scale dispersal is possible (Levin, 2006; R. Strathmann, 1974). When considering the impacts of
environmental stress on marine organisms, larval stages are generally considered to be most
sensitive (Pineda et al., 2012; Putnam et al., 2010). However, variation in larval phenotypes can
facilitate survival and reproductive success in spatially and temporally variable environments
(Beaumont et al., 2009; Clobert et al., 2009). When recruitment occurs, higher quality larvae
10
produce higher quality juveniles (Emlet & Sadro, 2006; Giménez, 2010; Jarrett, n.d.; D. J.
Marshall et al., 2003). Consequently, variation in larval phenotypes resulting from either
adaptation or acclimatization can significantly impact subsequent adult population dynamics
(Burgess & Marshall, 2011; Davis & Marshall, 2014). As these mechanisms may operate at
fundamentally different rates, disentangling the adaptive or acclimatory mechanisms driving the
sensitivity or resilience of marine larvae to environmental change will be critical for
understanding and predicting population and species-level responses to climate change.
Reef-building corals are a valuable study system in which to explore these mechanisms
because they exemplify the classic marine life-history dichotomy of sessile adult populations
linked by a dispersive planktonic larval stage (Richmond & Hunter, 1990a). While coral are
unquestionably susceptible to climate change stressors on a broad scale (Hughes et al., 2018), an
increasing number of studies has demonstrated their capacity for adaptation and acclimatization
to local environmental variation (Barshis et al., 2013; G. B. Dixon et al., 2015; Kenkel & Matz,
2016; Palumbi et al., 2014). The capacity for dispersal is largely dependent on the reproductive
type of the focal coral species (Ayre & Hughes, 2004). The majority of corals reproduce by
broadcast spawning eggs and sperm during an annual reproductive event, which fertilize
externally and must develop in the water column for some days before reaching recruitment
competency (Kerr et al., 2011; Richmond & Hunter, 1990a). Brooding corals, on the other hand,
release sperm into the water column, but fertilization and early larval development are internal,
with maternal colonies releasing competent larvae over many days per reproductive cycle, and
typically exhibiting multiple reproductive cycles per year (McGuire, 1998; Richmond & Hunter,
1990a).
11
In brooding species, the timing of planulation has significant implications for post-release
performance. Several qualities of planula larvae, including size, energetic status, symbiont
density, and photosynthetic potential, are known to vary by day of release (Cumbo et al., 2012;
de Putron et al., 2017; P. Edmunds et al., 2001; Putnam et al., 2010; Rivest & Hofmann, 2015).
Larvae released early within a spawning cycle were shown to be smaller and less likely to settle
compared to peak-release larvae and such within-brood divergence may persist into later
development (Cumbo et al., 2012). On a finer time scale, larvae of Favia fragum released near
dawn exhibited higher rates of substrate testing behaviors and settlement success than those
released near sunset (Goodbody-Gringley, 2010). Temporal variation in larval performance has
also been reported for four broadcasting species when comparing batches of larvae generated
through bulk fertilizations over multiple nights of spawning (Nozawa & Okubo, 2011).
Multiple abiotic factors affecting the physiology and behavior of brooded larvae have
been identified (reviewed in (Gleason & Hofmann, 2011)), but climate change related stressors
have received the most attention to date. Larvae of Porites astreoides were found to exhibit non-
linear variation in larval size and symbiont density when subjected to a 24 h exposure to a linear
temperature gradient (P. J. Edmunds et al., 2005). Warmer temperatures have been consistently
shown to reduce the proportion of swimming larvae but enhance their settlement (Putnam et al.,
2008; Randall & Szmant, 2009; Serrano et al., 2018). Simulated ocean acidification significantly
reduced metabolic rate in both P . astreoides and P . damicornis and settlement in P . astreoides
larvae (Albright & Langdon, 2011; Rivest & Hofmann, 2014). In addition, susceptibility of
brooded larvae to simulated climate change conditions (temperature and pH) has also been
shown to vary by day of release (Cumbo et al., 2013).
12
For both brooding and broadcast spawning species, larval performance is also influenced
by parental reef origins that are characterized by distinct physical environments, suggesting an
important role for adaptation and/or acclimatization. Larvae of the broadcast spawning Acropora
millepora exhibited a 10-fold increase in survival odds under heat stress when their parents came
from a warmer reef (G. B. Dixon et al., 2015). Similarly, brooded P . astreoides from upper-
mesophotic reef sites in Bermuda were found to produce more competent larvae with higher
settlement and survival rates compared to shallower sites (Goodbody-Gringley et al., 2018).
Brooded larvae of Agaricia agaricites responded to ultraviolet radiation differently depending on
what depths their parents originated from (Gleason & Wellington, 1995). Pocillopora damicornis
larvae from Taiwan and Moorea exhibited differences in lipid metabolism early in their dispersal
period, which may contribute to differences in temperature tolerance (Rivest et al., 2017). Larval
performance can also be modified by short-term adult pre-conditioning. Pre-conditioning adult P .
damicornis to high temperature and pCO2 increased larval performance in those same conditions
compared to larvae released from naive parent colonies (Putnam & Gates, 2015).
Finally, variation among families within reef environments has also been shown to affect
larval performance in broadcast spawning corals. Genetically unique families of A. millepora
larvae exhibited substantial variation in metabolism and gene expression which were attributable
to both fixed genetic differences among parent colonies and maternal effects (Meyer et al.,
2009). Similarly, significant family and maternal effects were reported for larval survival under
an acute thermal stress (G. B. Dixon et al., 2015). However, the contribution of family and/or
maternal effects have rarely been quantified in any brooding coral species. Specific brooding
behaviors necessary to facilitate the survival of embryos internally, for example, those involved
in oxygen transport, can increase offspring fitness at the expense of maternal fecundity
13
(Fernández et al., 2008; D. J. Marshall et al., 2008). Consequently, maternal investment may be
greater in brooding coral species as larval development occurs within the maternal colony. This
suggests that on top of potential variation due to genetic differences, variation in maternal
condition may substantially alter larval performance. Maternal effects may also contribute to the
differential survival and growth of offspring during the first few weeks following release,
although earlier work on growth rate in P . astreoides recruits shows that effects diminish over
time (Kenkel, Setta, et al., 2015), potentially reflecting the depletion of maternal nutritional
provisioning. A handful of studies have reported normalizing the contribution of different
families to their experimental larval pool, but none examined differences among family groups
(Cumbo et al., 2012; Hartmann et al., 2018; Rivest et al., 2017). Whereas (Chamberland et al.,
2017) found substantial variation in larval size, symbiont number, and symbiont density among
spawning colonies, suggesting that this family-level variation may be an important driver of
variation in larval performance.
This study aimed to quantify the importance of family-level variation (which we define
for the purposes of this study as both maternal and additive genetic effects) on larval
performance in a brooding species, Porites astreoides, using a system in the Florida Keys in
which strong effects of reef origin have previously been established. Inshore environments in the
Florida Keys are marked by higher temperature variation, turbidity, and nutrient input compared
to offshore environments (Kenkel, Almanza, et al., 2015; Lirman & Fong, 2007)Boyer and
Briceno, 2011; (Kenkel, Almanza, et al., 2015; Lirman & Fong, 2007). Orbicella faveolata
populations from inshore sites in the upper Keys exhibit higher calcification rates, greater
resistance to and faster recovery from thermal stress events than offshore corals (Manzello et al.,
2015a, 2019). Similarly, adult colonies of P . astreoides from inshore reefs in the lower Florida
14
Keys have repeatedly been shown to exhibit higher thermal tolerance than their offshore
counterparts (Kenkel et al., 2013; Kenkel, Setta, et al., 2015). We obtained parental colonies
from both an inshore and an offshore reef in the lower Florida Keys and collected planula larvae
released during the April 2018 spawning event. We characterized changes in physiological traits
by day of larval release and examined larval thermal tolerance. For all performance metrics
examined, variation attributable to larval family exceeded variation attributable to day of release
or parental environment of origin, highlighting the dominant role of family in shaping
physiology during the larval life history stage.
2.3 Methods
2.3.1 Coral Collections and Spawning Conditions
Porites astreoides are hermaphroditic brooding corals which fertilize eggs internally and
release competent planula larvae in monthly reproductive cycles (McGuire, 1998; Richmond &
Hunter, 1990a). In the Florida Keys, peak larval release months are April and May, with
planulation occuring around the new moon of each month.
On 12 April 2018, three days before the new moon, 31 adult P . astreoides coral colonies
were collected from a depth of 2–3 m in the lower Florida Keys. Sixteen colonies were collected
from an offshore site (Big Pine Ledges: 24° 33.174′ N, 81° 22.809′ W) and fifteen from an
inshore site (Summerland Shoals Patch: 24° 36.346′ N, 81° 25.742′ W), under permit #FKNMS-
2018-033. Colonies were transported to Mote Marine Laboratory's Elizabeth Moore International
Center for Coral Reef Research and Restoration and placed in a shaded (70% photosynthetically
active radiation reducing) flow-through seawater system (i.e. raceway) where water temperatures
averaged 26.3 °C, consistent with seasonal temperatures naturally experienced at inshore and off-
shore reefs (Kenkel, Almanza, et al., 2015). To track colony identity, plastic numerical labels (1–
15
15 for inshore, 26–41 for offshore) were affixed to the bottom of each coral using marine epoxy
putty (All-Fix). Each night before sunset for 5 nights following collection, corals were placed
into flow through larval collection chambers following (Kuffner et al., 2006). The following
morning, the identity of spawning colonies was recorded, and all larvae were collected in 3-L
bowls identified by parent colony number.
2.3.2 Daily Release Data
For five mornings following colony collection, ten larvae from each colony with
sufficient larval release were aliquoted into individual wells in 6-well plates, fixed in 5%
formalin and immediately photographed using a stereomicroscope. Digital photographs were
retained for subsequent quantification of larval volume. An additional three replicates of 10
larvae per family were aliquoted into 1.5 mL tubes, excess seawater was removed and tubes were
frozen at −80 °C for subsequent quantification of soluble protein content and Symbiodininaceae
cell density. Remaining larvae were retained for subsequent thermal tolerance experiments in
individual 3-L bowls, pooled by family, with gentle aeration and water changes occurred on
alternate days.
2.3.3 Thermal Tolerance Experiments
Thermal tolerance among larval families was quantified in two ways, through an acute
challenge which quantified differential mortality rates in response to 36 °C over time and a 4-day
exposure to moderate thermal stress (32 °C) to quantify changes in larval physiology among
families. The acute thermal challenge was conducted twice, with the first experiment beginning
on 14 April and the second on 17 April. For each experiment, two Nally bins were filled with 30
L seawater and outfitted with SL381 submersible pumps. In each bin, 3 × 10 larvae per family
were aliquoted into replicate 70 uM cell strainers (Grenier BioOne, 542,070) which were used as
floating netwells (sensu (G. B. Dixon et al., 2015), n = 30 larvae total per family per bin). One
16
bin was left at room temperature (mean = 24.3 °C, range = 23.4–24.8 °C, hereafter referred to as
the control). This temperature is lower than the average temperature larvae would experience on
reefs, but not outside of the range of temperature variation previously observed on reefs at this
time of year (http://serc.fiu.edu/wqmnetwork/FKNMS-CD/). For the heat treatment, a 100-W
aquarium heater was used to ramp the temperature to 36°C at a rate of ~1°C per hour (Fig. S2.1).
Mortality rate was quantified by counting the number of larvae remaining in each netwell at
multiple time-points over a 48 h cumulative exposure. In the first acute challenge experiment,
three inshore (Families 2, 3, and 4) and three offshore (26, 27, 28) families were represented,
while in the second four inshore (3, 4, 14, and 15) and eight offshore (26, 27, 28, 29, 31, 35, 39,
41) had released sufficient larvae to also be included.
In the moderate thermal challenge, three replicate 30 L Nally bins were set up for each
temperature treatment as in the acute challenge, and 5 × 10 larvae per family were aliquoted into
replicate netwells by bin (n = 50 larvae total per family per bin). Temperatures in the 32 °C
treatment bins were ramped over the course of 24 h and maintained for four days (Fig. S2.2). At
the end of the exposure period, swimming larvae were removed from netwells and placed into a
1.5 mL tube, seawater was removed, and larvae were frozen for subsequent physiological
analyses.
2.3.4 Larval Volume
The length and width of each larvae was measured in ImageJ (Schneider et al., 2012).
Assuming an elliptical shape, volume was determined by V =
!
"
𝜋𝑎𝑏
#
, where a is
$
#
length and b is
$
#
width (Isomura & Nishihira, 2001).
17
2.3.5 Symbiont Density, Chlorophyll, and Protein Content
For both the daily release and thermal stress experiment samples, three batches of 10
larvae per family were frozen in −20°C until analysis. After the samples were thawed, 100μL
extraction buffer (50 mM phosphate buffer, pH 7.8, with 0.05 mM dithiothreitol) was added to
each replicate group, which was then homogenized by back pipetting to release symbionts from
host animal cells. The total volume of the slurry was measured to account for any residual
seawater, and 20 μL was removed and immediately mixed with 20 μL 20% formalin solution to
perform Symbiodiniaceae cell counts. Symbiont density was determined using triplicate
hemocytometer counts per batch. The remaining slurry was centrifuged for 3 min at 1500 xg to
pellet symbiont cells for chlorophyll quantification. The supernatant was transferred to a new 1.5
mL tube for protein quantification. The three biological replicates per family were separately
tested for chlorophyll and protein across different spectrophotometer runs. Symbiont cell pellets
were resuspended in 90% acetone, shaken with metal beads in a TissueLyser II (Qiagen) for 90 s
to further break down zooxanthellae cells, and incubated at −20 °C overnight. The sample was
centrifuged for 5 min at 10,000 x g at 4 °C and 50 μL of the resultant supernatant was measured
in triplicate for absorbance at 630, 647, and 664 nm using Synergy H1 microplate reader
(Biotek). Chlorophyll a concentration was de- termined by applying the following equation, Chla
(μg/ ml) = −0.3002*A630 + −1.7538*A647 + 11.9092*A664 (Ritchie, 2008). Soluble host
protein was quantified with BCA Protein Assay Kit II following the manufacturer's instructions
(BioVision). Protein concentrations were calculated by comparing sample absorbance to a
standard curve of serial dilutions of bovine serum albumin. Symbiont density, chlorophyll a, and
protein content were multiplied by the initial slurry volume to yield total content, which was then
normalized per larvae.
18
2.3.6 Statistical Analyses
Daily variation in physiological traits were analyzed using ANOV As with day of release
(levels: 1, 2, 3, 4, 5; specified as sequential days in which planulation was observed, with day 1
indicating the first day of larval collection and continuing for a total of 5 days of observation),
reef origin (levels: inshore, offshore), and larval family as fixed effects in R 3.5.2 (R Core Team,
2017). Models were evaluated for normality and homoscedasticity using diagnostic plots, and
symbiont cell density was log-transformed to meet the assumption of normality. Families 15 and
29 only produced enough larvae for one biological replicate for the symbiont density, chlorophyll
a and protein content measures on Day 3 and these data were excluded prior to statistical
analysis. Tukey HSD post hoc tests were used to identify significant differences among factor
levels when p < .05. Survival analysis was used to model time of death in the acute thermal stress
experiments as a function of reef origin, including a random effect of larval family and
individual netwell using the coxme package (Therneau, 2018). Mortality was coded as a binary
trait (dead = 1, alive = 0) and time of death as an integer (the re-survey time point in hours).
Variation in physiological traits in the moderate stress experiment were modeled as a function of
origin (levels: inshore, offshore) and treatment (levels: control, heat), including a random effect
of larval family using the lme4 package (Nakagawa & Schielzeth, 2013). Schielzeth, 2013).
Models were again evaluated for normality and homoscedasticity, and protein concentration was
log-transformed to meet the assumption of normality. A series of linear regressions was used to
evaluate relationships between symbiont density and chlorophyll content, as well as between
physiological trait values and hazard ratios from the survival analysis. In addition, we explored
relationships between fecundity (the number of larvae released per parent daily and cumulative
release over the entire observation period), physiological trait measures and familial hazard
ratios.
19
2.4 Results
2.4.1 Daily Variation in Larval Physiology by Family
Of the 31 colonies collected, 8 inshore and 13 offshore released larvae during our
window of observation (Fig. S2.3). Fecundity varied among planulating colonies, both in terms
of the number of larvae released and days over which planulation was observed. Although both
inshore and offshore colonies planulated for a similar number of days on average (inshore: 2.75
days, offshore: 2.54 days), there was significant variation among individual colonies, with some
producing larvae daily for the entire observation window and others planulating only once (Fig.
S2.3). The mean number of larvae released per colony was also similar between reef zones
(inshore: 2.75 days, offshore: 2.54 days; t-test: p > .05), however, summing across all families
and days of release, offshore origin corals released 40% more larvae than inshore origin corals,
attributable to the greater number of planulating colonies overall. No relationships were evident
between the number of larvae released per day and the resulting daily larval trait measures
(volume, symbiont density, chlorophyll a, or protein content).
Larval volume differed significantly by day of release, reef origin, and family, with each
fixed effect explaining 6%, 4%, and 17% of the variance in larval volume, respectively (day: F =
11.263, df = 4, p < .001; origin: F = 31.135, df = 1, p < .001; family: F = 7.174, df = 18, p
< .001). Larvae became progressively smaller in later release days, except for one family (Fam
30) that displayed the opposite trend (Fig. 2.1). Inshore larvae were on average larger than
offshore larvae by 22%. Families 1 and 12 produced larger larvae than families 28 and 40 (p
< .01, Tukey HSD).
20
Figure 2. 1 Larvae volume for each family during 5 days of release (mean ± SEM). Families 1-15 are inshore-origin parents and
families 26-41 are offshore-origin parents. The y-axis indicates sequential days in which planulation was observed, with day 1
indicating the first day of larval collection and continuing for a total of five days of observation.
Day of release, reef origin, and family also had significant effects on symbiont density,
explaining 19%, 8%, and 48% of the variance, respectively (day: F = 12.337, df = 4, p < .001;
origin: F = 23.385, df = 1, p < .001; family: F = 28.339, df = 14, p < .001). Symbiont densities
were increased by 39% in larvae released from the inshore families in comparison to those from
offshore families. Symbiont density was generally higher later in the planulation window (Fig.
S2.4). However, larvae from several families experienced a slight decrease in symbiont density
during Day 3. Larvae from families 3 and 29 contained more symbionts on average than those
from families 27 and 41 (p < .001, Tukey HSD).
A similar trend was observed in chlorophyll a content where again, all three effects were
significant (day: F = 7.728, df = 4, p < .001; origin: F = 34.25, df = 1, p < .001; family: F =
19.221, df = 14, p < .001). Origin accounted for 8% of the variance in chlorophyll a content.
Inshore larvae contained on average 36% more chlorophyll a than offshore larvae. Day of release
21
accounted for 7% of the variance in chlorophyll a content, with higher concentrations associated
with later releases (Fig. S2.5). Family accounted for 63% of the variance in chlorophyll a
content. Larvae from families 3 and 29 contained more chlorophyll a than those from families 27
and 39 (p < .001, Tukey HSD). Symbiont density explained 61% of the variation in chlorophyll a
content across days (Fig. S2.6).
Only reef origin and family had significant effects on host protein content (family: F =
12.814, df = 14, p < .001). Origin explained 5% of the total variance in protein content. Inshore
larvae had higher protein content on average than their offshore counterparts, by 0.51 μg/ larvae
(F = 13.496, df = 1, p < .001; Fig. S2.7). Family explained 61% of the variance in host protein
content, with larvae from families 1, 3, and 29 containing more protein on average than those
from families 27 and 28 (p < .01, Tukey HSD).
2.4.2 Physiological Response to Moderate Thermal Stress
Mean survival among replicates in the moderate thermal stress experiment was 97% or
greater, save for Family 29 in the control treatment, where average larval survival was 87%, and
Family 27 in the heat treatment, where survival declined to 79% (Fig. S2.8). No changes in
symbiont density (Fig. S2.9) or protein content (Fig. S2.10) were detected in response to either
treatment or origin; however, temperature treatment had a significant impact on chlorophyll a
content. On average, larvae exhibited a 19% reduction after 4 days at 32 °C in comparison to
paired controls (F = 12.544, df = 1, p = .001; Fig. 2.2). A marginally significant interaction term
was also detected, due to offshore larvae tending to lose more chlorophyll a post heat stress than
inshore larvae (p = .075). Similar to the daily release data, the majority of the variance in
chlorophyll a content was explained when a random effect of family was included in the model
(R
2
= 0.809 vs R
2
= 0.169 for the fixed effects only model).
22
Figure 2. 2 Concentration of chlorophyll a during the moderate temperature stress experiment by family and treatment (mean ±
SEM). Families 2, 4 and 15 are inshore-origin parents and families 26-29 are offshore-origin parents.
Symbiont density explained 43% of the variation in chlorophyll a at the end of the
experiment (Fig. S2.6). No relationships were detected between cumulative fecundity (the total
number of larvae released prior to beginning the experiment) and the change in trait values in
response to temperature treatment.
2.4.3 Survival under Acute Thermal Stress
Temperature treatment significantly impacted mortality, with > 99.7% of larvae surviving
in controls, whereas only 25% of larvae survived 46 h of exposure to 36 °C in the experiment
encompassing 12 larval families. Reef origin (inshore or offshore) did not significantly impact
mortality risk. However, a substantial familial effect was evident, with outcomes ranging from
less than half (hazard ratio [HR] = 0.45, Family 31) to over 3.4 times (HR = 3.45, Family 27) the
average familial risk for the experiment (Fig. 2.3A).
While absolute mortality rates differed between the two replicate acute stress experiments
(Fig. 2.3, e.g. fraction surviving at 22 vs 24 h), the same general patterns were detected in the 6
family experiment. Again, 99.4% survival was observed in control treatments, whereas survival
was reduced to 34% under elevated temperature. Similarly, no effect of reef origin was evident,
23
but familial effects were large, with a range of less than a third (HR = 0.27, Family 26) to nearly
10 times (HR = 9.87, Family 27) the average mortality risk (Fig. 2.3B). For families represented
in both experiments, the rank order of hazard ratios was highly consistent (R
2
= 0.85, P < .05;
Thermal tolerance ranking: 26~28>3>4>27,Fig. 2.3).Families 26 and 28 were generally tolerant
of elevated temperature, whereas family 27 always exhibited 100% mortality in the 36 °C
treatment.
Figure 2. 3 Mortality curves of larvae shown as mean survival (± SEM) over time for the acute thermal stress experiments
consisting of (A) twelve families and (B) six families. Families 1-15 are inshore-origin parents and families 26-41 are offshore-
origin parents.
No significant relationships were detected in regressions of familial hazard ratios on daily
release phenotypes averaged by family (volume, host protein content, symbiont density or
chlorophyll a) or bleaching in the moderate thermal stress experiment, calculated as the
difference in chlorophyll a content between control and heat-treated larvae by family. Nor was
there a relationship between familial hazard ratios and cumulative fecundity, calculated as the
total number of larvae released prior to beginning the experiment.
24
2.5 Discussion
Variation in larval quality can facilitate survival and reproductive success in spatially and
temporally variable environments, contributing to adult population dynamics in a changing
climate (Beaumont et al., 2009; Burgess & Marshall, 2011). We found that several larval traits,
including volume, symbiont density, chlorophyll a, and protein content varied by day of release,
reef zone of origin, and individual P . astreoides family. In particular, family was the dominant
factor influencing differences in larval physiology. Of the four traits that were quantified, >60%
of the variation in chlorophyll and protein content was explained by family. In comparison, day
of release and origin explained <20% of the total variation. This result has substantial
implications for the interpretation of prior studies which focused on variation in the physiology
of brooded coral larvae. To our knowledge, the majority of work which examined larval
physiology by day of release, reef origin, or other experimental treatment (e.g. (de Putron et al.,
2017; P. Edmunds et al., 2001; P. J. Edmunds et al., 2011; Goodbody-Gringley et al., 2018;
Putnam et al., 2010; Putnam & Gates, 2015) relied on pooling larvae from different maternal
colonies and performing analyses on the composite. Only a handful of studies clarified that an
equal number of larvae were sampled from different families to control for this source of
variation (Cumbo et al., 2012; Hartmann et al., 2018; Rivest et al., 2017). Given the variation in
planulation among individual parent colonies over a single reproductive cycle and the large
contribution of individual family in explaining the physiological variances examined here, it is
possible that a large proportion of the variation previously attributed to day of release or origin
may in fact have been the result of familial effects, if care was not taken to normalize the
representation of different families in the larval pools.
25
Family also played a significant role in determining the probability of larval survival
under 36 °C acute temperature stress. The rank order of thermal resilience among families was
preserved in the two technical replicates of this experiment, despite incorporating larvae from
different days of release (Fig. 2.3). Larvae released from families 26 and 28 were generally more
tolerant than those from families 27 and 4. A prior study which used this same acute stress assay
to quantify thermal tolerance of A. millepora larvae also found a significant family effect, but a
significant portion of the variation in larval performance was also attributable to the parent's reef
of origin: parents from a warmer location conferred significantly higher thermo-tolerance to their
offspring in comparison to parents from the cooler location (G. B. Dixon et al., 2015). Adult P .
astreoides in the lower Florida Keys exhibit differences in thermal tolerance depending on their
reef of origin, with inshore-origin corals exhibiting greater bleaching resistance than offshore-
origin corals (Kenkel et al., 2013; Kenkel, Setta, et al., 2015; Kenkel & Matz, 2016). Although
survival was clearly linked to family, we did not find any association between survival and
parental reef origin. The most (26 and 28) and least tolerant (27) families all originated from the
offshore reef site.
One explanation for this finding is that larval thermal tolerance differs fundamentally
from adult thermal tolerance, possibly as a consequence of significant maternal effects
(Lockwood et al., 2017). Another possibility is that our assay examining survival under acute
thermal stress is quantifying a different aspect of thermal tolerance than the traditional bleaching
response metrics, such as the reduction in symbiont or chlorophyll concentrations. The moderate
thermal stress experiment which included these most and least tolerant offshore families (26, 27
and 28) in addition to a set of inshore families, showed that while family 27 again exhibited the
greatest mortality under heat stress, larval bleaching responses (quantified as the change in
26
chlorophyll a content) were similar to previously reported adult bleaching phenotypes (Kenkel et
al., 2013; Kenkel, Setta, et al., 2015); offshore larvae tended to lose more chlorophyll than
inshore larvae (Fig. 2.2). Moreover, survivorship under acute stress was unrelated to changes in
chlorophyll a content under this milder temperature stress, nor was it explained by mean daily
release phenotypes. The ecological significance of larval survival under acute temperature stress
remains unclear. However, it may be an important phenotype to study with ongoing global
warming when extreme temperature regimes such as heat waves occur more frequently (Jentsch
et al., 2007).
In Pocillopora damicornis, elevated temperature reduced maximum quantum yield of
photosystem II in larvae but not adult colonies, suggesting that early life stages might be more
vulnerable to thermal stress (Putnam et al., 2010). While we did observe a loss of chlorophyll a
following a 4-day exposure to 32 °C, the density of Symbiodiniaceae cells and soluble host
protein content were unaffected. The weaker correlation between chlorophyll a and symbiont cell
density following the moderate thermal stress exposure (R
2
= 0.43) in comparison to the daily
release measures (R
2
= 0.61), suggests a decoupling of bleaching mechanisms. Prior work has
shown that loss of symbiont cells can occur without pigment reduction (R. J. Jones, 1997) and
decreases in chlorophyll a content can occur without losing symbionts (Venn et al., 2006). Our
results suggest the occurrence of the latter, where chlorophyll a is eliminated through photo-
oxidative reactions that yield colorless compounds during pigment loss, or photobleaching,
which may not necessarily lead to symbiont expulsion (Venn et al., 2006). The propensity for
photobleaching in Symbiodiniaceae can also be altered by thermal acclimation (Takahashi et al.,
2013), suggesting that variable thermal tolerance may influence the extent of bleaching.
Similarly, although larval families exhibited significant variation in mortality during our acute,
27
36°C exposure, significant differences in mortality were not observed until larvae had
experienced these conditions for > 24 h (Fig. 2.3). Similar patterns were observed in
aposymbiotic A. millepora larvae, where 36 °C exposure times in excess of 24 h were necessary
to elicit significant mortality (G. B. Dixon et al., 2015). Combined, this suggests that larvae of
certain coral species can withstand these excessive temperatures, several degrees above typical
summer maxima (Kenkel, Almanza, et al., 2015; Manzello et al., 2019), for prolonged periods
and calls for additional investigation into the thermal tolerance limits of larval life stages and
how this impacts subsequent life stages. For example, negative latent effects were reported in
brooded A. humilis larvae, where minimal mortality was observed in larvae exposed to high
temperature but subsequent reductions in settlement rate and post-settlement survival were
evident (Hartmann et al., 2013).
Not all families released larvae every day during the 5-day observation period and the
number of larvae released varied greatly (Fig. S2.3), suggesting variation in the reproductive
capacity of individual parent colonies. However, we did not detect any predictive relationships
among the number of larvae released per parent colony and variation in daily release phenotypes,
the change in physiological trait values in response to moderate thermal stress, or familial hazard
ratios under acute thermal stress. Although our observations occurred during the peak
planulation window for P. astreoides, this species is capable of releasing larvae through
September (McGuire, 1998). In a multi-site survey along the Great Barrier Reef, fecundity varied
significantly among colonies of A. millepora within a site, yet colony was a good predictor of
fecundity (Tan et al., 2016). Conversely, Agaracia humilis was reported to exhibit interannual
variation in fecundity in Curaçao, although individual colonies were not tracked (Hartmann et
al., 2018). If individual P. astreoides colonies vary in their reproductive investment over time it
28
may be that our fecundity measures do not accurately reflect individual reproductive output, and
future studies should investigate this possibility.
Quantification of additional energetic traits may yield more in formative relationships
among daily release phenotypes or thermal tolerance responses. For example, lipids are
important energy reserves and contribute to buoyancy of planktonic coral larvae (Harii et al.,
2007; Richmond, 1987). Correlations between larval size and lipid content have been previously
reported (de Putron et al., 2017; Hartmann et al., 2013). A positive relationship between
triacylglycerol content and temperature treatment was observed in P. damicornis larvae, in
addition to increased metabolism of both protein and lipid (Rivest & Hofmann, 2015).
Population-level variation in lipid catabolism has also been observed (Rivest et al., 2017). Lipid
reserves are predicted to influence mortality and recovery in adult corals in response to bleaching
(Anthony et al., 2009). Consequently, evaluating the relationship between lipid content and
family level variation in larval thermal tolerance represents an important avenue of future
research.
To our knowledge, this study provides the first evidence of size differences in P.
astreoides larvae from different reef zones in the Florida Keys. Earlier work showed that
offshore-origin recruits were significantly smaller than inshore-origin recruits (Kenkel, Setta, et
al., 2015), which aligns with the pattern we observed in larvae. In contrast to a study on P.
astreoides in Bermuda that documented smaller individuals released earlier in the lunar cycle (de
Putron et al., 2017), most families released larger larvae early in the April spawning event except
for one offshore family (Fig. 2.1), although more exceptions might have been captured if more
families were included. Size variation may determine settlement success in later developmental
stages (Hartmann et al., 2013). Larger larvae are also more likely to contain higher endogenous
29
energetic reserves and thus disperse farther in the field (D. J. Marshall & Keough, 2007). Larvae
of three Pocilloporid coral species exhibited longer lifetimes, defined as time from free-
swimming to death, in larger individuals than smaller ones (Isomura & Nishihira, 2001).
Interestingly, the trend in symbiont density we observed in the current study (Fig. S2.3) agrees
with a previous finding that symbiont abundance was higher in the last 3 days than the first 3
days of collection (de Putron et al., 2017; P. J. Edmunds et al., 2011; Putnam et al., 2010). If
symbiont density and chlorophyll a content are reliable predictors of energy reserves, our results
showed small but energy-rich offspring are likely to be released later in the spawning window.
Compared to the observation of larger and more energy-rich larvae that are late releases in P.
damicornis (Putnam et al., 2010), such deviation may indicate the unique physiology of P.
astreoides larvae from the lower Florida Keys.
In conclusion, we find that the majority of variation in the physiology of brooded P.
astreoides larvae is explained by larval family. These effects may be the result of either fixed
genetic differences, or maternal effects, and future work should aim to conduct additional genetic
analyses to help distinguish among these mechanisms. Prior work established that symbiont
community composition in P. astreoides is largely uniform in the Florida Keys, regardless of
parent population origin (Kenkel et al., 2013; Thornhill et al., 2006), but finer-scale genotyping
of Symbiodiniaceae is also needed to evaluate the contribution of maternally inherited symbiont
types. Furthermore, additional data on other energetic and life-history traits may help shed light
on variation among families. Regardless of the ultimate mechanism, it will be important for
future studies to control for this variation by either tracking the familial origin of larvae or taking
care to pool larvae originating from different families in equal proportions. Additional studies
examining interactive effects between family and other previously identified sources of variation
30
(e.g. day of release) will provide additional insights. Future work will also benefit from including
juveniles and recruits in the phenotyping assay to allow for the dissipation of maternal effects
and gain a better understanding of how parental effects influence later developmental stages.
2.6 Supplementary Materials
Figure S2. 1 Temperature profile for the acute thermal stress experiment, recorded using HOBO Pendant Temperature Loggers
(Onset, Bourne, MA) set to record every 10 min.
Figure S2. 2 Temperature profile for the sub-lethal thermal stress experiment, recorded using HOBO Pendant Temperature
Loggers (Onset, Bourne, MA) set to record every 10 min.
31
Figure S2. 3 Fecundity for each family during 5 days of release. Families 1-15 are inshore-origin parents and families 26-41 are
offshore-origin parents.
Figure S2. 4 Symbiont density for each family during 5 days of release (mean ± SEM). Families 1-15 are inshore-origin parents
and families 26-41 are offshore-origin parents. The y-axis indicates sequential days in which planulation was observed, with day
1 indicating the first day of larval collection and continuing for a total of 5 days of observation. Families 15 and 29 only produced
enough larvae for one biological replicate on Day 3 and thus lack error bars.
32
Figure S2 .5 Chlorophyll a concentration for each family during 5 days of release (mean ± SEM). Families 1-15 are inshore-
origin parents and families 26-41 are offshore-origin parents. The y-axis indicates sequential days in which planulation was
observed, with day 1 indicating the first day of larval collection and continuing for a total of 5 days of observation. Families 15
and 29 only produced enough larvae for one biological replicate on Day 3 and thus lack error bars.
Figure S2. 6 Chlorophyll a content per larvae as a function of symbiont density (cells/larvae) across days of larval release and
following the moderate thermal stress experiment.
33
Figure S2. 7 Soluble host protein content for each family during 5 days of release (mean ± SEM). Families 1-15 are inshore-
origin parents and families 26-41 are offshore-origin parents. The y-axis indicates sequential days in which planulation was
observed, with day 1 indicating the first day of larval collection and continuing for a total of 5 days of observation. Families 15
and 29 only produced enough larvae for one biological replicate on Day 3 and thus lacked error bars.
Figure S2. 8 Mean number of larvae by family and treatment at the end of the moderate thermal stress experiment. Note that the
number of surviving larvae exceeds the original 10 for some family by treatment averages, these were instances where 11 larvae
were recovered at the end of the experiment, presumably due to an error in initial counts.
34
Figure S2. 9 Symbiont density during the moderate temperature stress experiment by family and treatment (mean ± SEM).
Families 2, 4 and 15 are inshore-origin parents and families 26-29 are offshore-origin parents.
Fig. S2.10 Soluble host protein concentration during the moderate temperature stress experiment by family and treatment (mean
± SEM). Families 2, 4 and 15 are inshore-origin parents and families 26-29 are offshore-origin parents.
35
CHAPTER 3. CROSS-GENERATION HERITABILITY ANALYSIS OF
PHYSIOLOGICAL TRAITS IN PORITES ASTREOIDES ACROSS AN
INSHORE-OFFSHORE GRADIENT IN THE LOWER FLORIDA KEYS
Yingqi Zhang, Shelby J. Barnes, Carly D. Kenkel
3.1 Abstract
Estimating the heritable genetic variation in fitness-related traits is key to projecting the
adaptive evolution of organisms in response to a changing environment. While heritability
studies on reef-building corals to date support adaptive capacity, little is known about the
dynamics of trait heritability across life stages in which distinct selective pressures can have
long-lasting effects both within and across generations. In this study, we obtained heritability
estimates for energetic and thermal stress response traits in adult, larval, and recruit Porites
astreoides from two populations in the Lower Florida Keys. To induce bleaching phenotypes
among individual families, larvae were exposed to a 4-day thermal stress at 32 ˚C, whereas
adults and recruits received the same treatment for 22 days. Origin-dependent tolerance was
observed in two life stages where offshore recruits lost more symbiont cells under heat than
inshore recruits compared to their respective controls and heat-treated offshore adults suffered a
greater loss in total protein content. Surprisingly, larvae appeared to be largely insensitive to heat
regardless of origin. Broad sense heritability (H
2
) estimates varied greatly among traits and life
stages, which may reflect changes in the relative importance of genetic and environmental
variation throughout development. Over 50% of the variation in all larval traits, adult symbiont
density and chlorophyll a concentration, and recruit protein content can be attributable to genetic
factors. The overall moderate to high H
2
estimates measured here suggest family-level variation
36
can persist across different life stages and these corals may be equipped with considerable
potential to adapt to environmental change.
3.2 Introduction
Understanding the adaptive potential of marine organisms is essential to predict
population dynamics and persistence in light of rapidly changing ocean temperature and
chemistry (Munday et al., 2013). For quantitative traits, heritability, which is defined as the
proportion of total phenotypic variation attributed to genetic variation, is often used as a metric
to estimate the evolvability of those traits under selection (Falconer and Mackay 1996).
Specifically, broad sense heritability (H
2
) includes all genetic factors, including both additive
genetic effects and non-additive genetic effects such as dominance and epistasis. In contrast,
narrow sense heritability (h
2
) is a fraction of H
2
and only accounts for additive genetic factors,
which are guaranteed to be inherited by the next generation via sexual reproduction. Therefore,
h
2
describes the true adaptive potential of a given trait but requires knowledge of relatedness or
pedigree.
Scleractinian corals are foundation species in reef ecosystems but have undergone severe
population declines in recent decades due to increased anthropogenic activities (Hoegh-Guldberg
et al., 2007; Hughes et al., 2017). The most common stress response in corals is bleaching, which
refers to the whitening of tissues resulting from the dissociation of endosymbiotic algae from the
coral host (Lesser, 2011). Elevated temperature has been repeatedly shown to be a major cause of
mass bleaching events worldwide (Hughes et al., 2018; Jokiel & Coles, 1990). Factors that
influence the thermal tolerance of corals include host genetics (Bay & Palumbi, 2014), diversity
and flexibility of endosymbiont (family Symbiodiniaceae) (Berkelmans & van Oppen, 2006),
other members of the microbial community (V oolstra et al., 2021), and prior acclimatization to
thermal stress (Ainsworth et al., 2016), which may be mediated by epigenetic changes (G. Dixon
37
et al., 2018). Each component of the coral holobiont is characterized by vastly different
generation time (e.g. 3-74 days for symbiont in hospite vs. 4-20 years for coral host) (Babcock,
1991; Muscatine et al., 1984), thus the rate of evolutionary change in each symbiont partner may
not align, further complicating the projection of future reefs as sea surface temperature continues
to increase.
Similar to other marine invertebrates, corals exhibit complex life cycles that oscillate
between the planktonic larval stage and the benthic juvenile/adult stages (Richmond & Hunter,
1990a). Different life stages encounter distinct selective pressures but phenotypic changes in a
given life stage resulting from interactions with its environment can have long-lasting effects
both within and across generations (D. J. Marshall & Morgan, 2011; Putnam, 2021). The
majority of work to date has focused on the effects of abiotic stressors on the physiology and
critical developmental processes of one distinct life stage and only a handful of studies have
compared phenotypic response to similar stress among multiple life stages (Kenkel, Setta, et al.,
2015; Putnam et al., 2010; Putnam & Gates, 2015; Wong et al., 2021). Even less is known about
how variable the heritability of a given trait may be across a coral’s life span (Bairos-Novak et
al., 2021). Additive genetic variance (and hence h
2
) of fitness-related traits has been shown to
increase with age in birds (mute swan and collared flycatcher) and terrestrial mammals (red deer
and soay sheep), likely explained by the accumulation of deleterious mutations, or positive
selection on genes that increase fitness at earlier life stages but have the opposite effects later in
life (A. J. Wilson et al., 2008). Life history traits highly related to fitness can also undergo
stabilizing selection in a specific life stage and become less heritable due to reduced additive
genetic variance (Gustafsson, 1986). Carlon et al. (2011) documented lower h
2
estimates of coral
skeletal traits in juvenile Favia fragum than in adult colonies and attributed the differences to
38
environmental effects. More work is needed to shed light on the dynamics of heritability over
distinct coral life stages to obtain a holistic understanding of the adaptive potential of critical
phenotypes, as only focusing on a single life stage can be misleading (Albecker et al., 2021).
Heritability estimates across life stages may also be impacted by other aspects of coral
life-history or environmental variation, including when and how symbionts are acquired.
Lingering maternal effects may inflate heritability estimates in larvae, particularly for coral
species in which fertilization and larval development are completed within the maternal colony,
termed ‘brooding’ species (Richmond & Hunter, 1990b). Given the role that algal endosymbionts
play in holobiont thermal tolerance traits (Fuller et al., 2020), heritability of such traits will likely
also be driven by the extent of genomic fidelity between partners. For species in which offspring
directly inherit algal endosymbionts from maternal colonies, known as ‘vertical transmitters’,
there may be greater correlation in thermal tolerance between generations and thus higher
heritability (Baker 2003). However, more recent work has suggested that symbiont communities
in vertical transmitters may be more flexible (Quigley et al., 2018) and moreover, that symbiont
acquisition in species which must acquire algal endosymbionts anew each generation likely has a
genetic and thus heritable component to symbiont specificity (Quigley et al., 2017). Finally,
heritability is dependent on population-specific parameters and it is not unusual for heritability
estimates to vary substantially among populations (Visscher et al., 2008). Given increasing
examples of environmental variation influencing coral phenotypes among populations, even
across small spatial scales (Kenkel, Almanza, et al., 2015; Thomas et al., 2018), it will be
imperative to consider how heritability estimates may be affected by these different drivers.
In this study, we aimed to quantify thermal performance of Porites astreoides at adult,
larval, and recruit life stages as well as estimate broad sense heritability of various physiological
39
traits shared across all stages. We utilized two distinct populations originating from an inshore
and an offshore reef environment in the Lower Florida Keys. Compared to offshore reefs at
similar latitudes, inshore reefs experienced greater thermal variation and more extreme summer
temperatures (Kenkel, Almanza, et al., 2015; Lirman & Fong, 2007). Nonetheless, inshore coral
populations were characterized by greater thermal tolerance, higher cover, higher growth and
calcification rate (Kenkel et al., 2013; Manzello et al., 2015a, 2019). Prior studies on inshore and
offshore P . astreoides populations revealed distinct host genetic structures and transcriptomic
profiles but shared symbiont type (ITS2 type A4/A4a), indicating that the animal host might play
a bigger role in conferring thermal tolerance (Kenkel et al., 2013; Kenkel & Matz, 2016).
Although it is important to note that physiological variation among symbiont strains can exist
despite a lack of variation in ITS2-type profiles (Hoadley et al., 2021). We aimed to take
advantage of standing genetic variation and origin-specific thermal traits between the two
populations to answer the following two questions: 1. Are inshore larvae and recruits also more
heat resistant than the offshore offspring? 2. Are traits associated with thermal tolerance more or
less heritable depending on life stage?
3.3 Methods
3.3.1 Spawning Collection and Larval Thermal Stress Experiment
A total of 30 adult Porites astreoides colonies were collected from an inshore reef site
(Summerland Shoals Patch: 24° 36.346′ N, 81° 25.742′ W, n=15) and an offshore reef site (Big
Pine Ledges: 24° 33.174′ N, 81° 22.809′ W, n=15) five days before the new moon on April 29,
2019 under permit #FKNMS-2018-033. Colonies were transported to Mote Marine Laboratory's
Elizabeth Moore International Center for Coral Reef Research and Restoration and kept in a
shaded (70% PAR reducing) raceway to monitor for larval release. Plastic numerical tags were
40
attached to the bottom of colonies using marine epoxy putty (All-Fix) to track individual
identities. Colonies were then placed into flow-through larval collection chambers before sunset
according to (Kuffner et al., 2006) and exposed to ambient moonlight to mimic the natural
planulation processes. Beaker traps were checked for larvae the following morning (April 30,
2019). Most inshore colonies produced enough larvae to be included in the subsequent
experiment while only one offshore colony (Family 34) had sufficient planulation. As a result,
collection chambers were deployed for a second night to capture more spawning colonies that
originated from the offshore site.
A total of ten colonies (five from inshore and five from offshore) were used in the
following cross-generational thermal stress experiments. Ten larvae from each colony were
immediately fixed in 5% formalin post release and photographed under a stereomicroscope for
size estimate. Three groups of ten larvae (n=3x10 per family) from each colony were then
subjected to a moderate heat stress challenge beginning on May 1 following the same methods
described in (Zhang et al., 2019) (Fig. 3.1). Briefly, three replicate plastic bins were set up for
both control (26 ˚C) and treatment (32 ˚C) groups under a 12hr:12hr light:dark cycle (Figure
S3.1). Treatment was set to 32 ˚C because inshore reefs commonly experience this temperature
during late summer months (Aug-Sept) when bleaching is likely to occur (Kenkel, Almanza, et
al., 2015). Additionally, 32 ˚C induces a level of stress that can produce distinct bleaching
responses among individuals (Zhang et al., 2019). Each bin was filled with 30 L seawater and
equipped with a SL381 submersible water pump to maintain circulation, a 100 W aquarium
heater, and a HOBO temperature logger (Onset). One group of 10 larvae per spawning colony
was added to a 70 uM cell strainer (Grenier Bio-One) which served as a floating netwell in each
replicate bin (n=3x10 larvae per family per temperature). Target temperature for the heat
41
treatment bins was achieved by increasing the temperature by 0.5 ˚C per hour over a 12-hour
window and maintained for 4 days. No water change was performed due to the short
experimental duration and high water volume to biomass ratio, but bins were periodically topped
off with distilled water to compensate for evaporation. At the end of the exposure period,
swimming larvae in each netwell were sampled on the same day (with seawater removed) and
frozen at -20 ˚C for subsequent physiological assays.
Remaining larvae were settled onto aragonite plugs preconditioned with crustose
coralline algae (CCA) in ambient temperature by family, with the goal of recruiting between 3
and 4 individuals on the top side of each plug to avoid fusion of recruits. For each larval family,
19 plugs were secured by two staggered egg crates placed in a 1.2 L plastic food storage
container. After adding seawater to the containers, larvae were gently pipetted onto the plugs and
into the water column. Containers were monitored daily and water changes were performed
every two days, after which new larvae were introduced to facilitate further recruitment. To
securely transport recruits to the University of Southern California (USC), plugs with ideal
settlement densities were superglued to the caps of 50 mL falcon tubes, which were then filled up
with seawater to displace any air bubbles. Adult colonies that produced larvae were wrapped in
wet bubble wrap and transported to USC along with the recruits.
Figure 3. 1 Experimental design and timeline including ten Porites astreoides families.
42
3.3.2 Adult and Recruit Thermal Stress Experiments
Adult coral fragments and their recruits were allowed a 37-day recovery and acclimation
period in a 500 L holding tank at the Cnidarian Evolutionary Ecology Lab aquarium room at
USC (26 ˚C, 80 μmol photons m
−2
s
−1
, 5 cm s
−1
). After a month of recovery (June 5), six ca. 15
cm2 fragments were obtained from each adult colony and mounted on clean aragonite plugs
using superglue. Adult fragments were then returned to the acclimation tank. To quantify the
thermal tolerance of adult and recruit life stages, three replicate 50 L tanks were set up for both
control (26 ˚C) and treatment (32 ˚C) groups. On June 13, 2019, one adult fragment per colony
and an average of 8.5 recruits per family were randomly assigned to each tank. Recruit numbers
as well as settlement patterns varied across families (Table S3.1), thus each tank received
between 1 and 6 plug(s) to achieve an even distribution of recruits across tanks. The
experimental tanks were individually outfitted with submersible water pumps (Sicce Syncra
Silent 0.5, 185 GPH), 150W HMA-S thermal-regulated heaters (Finnex), and AI Prime HD lights
(Aqua Illumination) programmed to imitate the light cycle in FL, with an average daytime PAR
of 85-115 μmol photons m
−2
s
−1
reaching the tank bottom. Adult and recruit plugs were
acclimated to their individual tank conditions with recirculating flow for 4 days. Adult fragments
and recruit plugs were cleaned using a small brush every two days for the duration of the
experiment to remove any filamentous algal growth. The thermal stress challenge started on June
17, when recirculating flow was turned off for each tank to prevent pseudo replication.
Temperature in the individual heat treatment tanks was slowly ramped to 32 ˚C over 6 days (1 ˚C
increase per day) and maintained for 22 more days until July 15 (Figure S3.2). HOBO
temperature loggers were deployed to track the temperature profile throughout the exposure. A
10% water change was performed every week and the concentrations of potential nitrogen
metabolic wastes (including NH₄⁺, NO3
-
, and NO2
-
) were measured daily using commercial
43
aquarium test kits. Levels of NH₄⁺, NO3
-
, and NO2
-
were consistently under 0.25, 1, 0.05 ppm
respectively. Salinity was maintained at 35 ppt by topping off with distilled water (300-500 ml
for control tanks and 500-1300 ml for heat tanks per day).
To track bleaching status and recruit growth over the course of the thermal stress
exposure, photographs were taken from a top-down view under identical illumination using an
Olympus Tough camera TG-4/TG-5 (Olympus America Inc.) at the initial timepoint (T0), final
timepoint (T28), and 6 additional time points in between (T7, T11, T14, T18, T21, T25). Starting with
the control group, PVC racks were removed from each tank and immediately placed into a Nally
bin filled with seawater. To minimize the warping effect along the edges of the photos, racks
were divided into 4 quadrants and each quadrant was photographed individually to make sure the
corals were centered (Figure 3.1). A subset of the Coral Health Chart (Siebeck et al., 2006) E1-
E6 and B1-B6, was zip tied to an empty rack that was placed against the coral racks to serve as a
color and size reference. Each quadrant was photographed three times with the camera lens just
submerged. After all four quadrants were covered, the coral racks were immediately transferred
back to their respective treatment tank. The Nally bin was then heated to 32 ˚C before corals
from the heat treatment group were transferred and photographed.
After 22 days of exposure, recruits were scraped off the plugs using a razor blade and
pooled into 1.5 ml Eppendorf tubes by family per replicate tank. Adult fragments were removed
from their plugs and wrapped in pre-labeled tin foil. All samples were frozen immediately at -80
˚C for subsequent physiological assays.
3.3.3 Physiological Assays
Similar to (Zhang et al., 2019), larvae (3 replicates of 10 larvae per family) and recruit (3
replicates of between 2 and 15 recruits per family, see Table S3.1) samples were thawed on ice
44
and mixed with 100 µl extraction buffer (50 mM phosphate buffer, pH 7.8, with 0.05 mM
dithiothreitol). The mixture was further homogenized by back pipetting to free symbiont cells
from host tissues and the final volume of the homogenate was recorded to account for residual
seawater. For recruit samples, skeletal debris was allowed to settle for a minute and the aqueous
tissue layer was further transferred to a new set of tubes. To quantify symbiont cell density, 20 µl
of the homogenate was fixed in 20 µl of a 20% formalin solution (10% final concentration) and
triplicate 10 µl aliquots were assessed under a compound microscope at 100x magnification
using a hemocytometer. The remaining slurry was centrifuged for 3 min at 1500 x g at 4 ˚C to
pellet symbiont cells for chlorophyll quantification and isolate host tissue supernatant for protein
analysis. Symbiont cell pellets were resuspended in 90% acetone and further broken down by
shaking with metal beads in a TissueLyser II (Qiagen) for 90 s. After an overnight incubation at
−20 °C, the solution was centrifuged for 5 min at 10,000 x g at 4 °C. Triplicate 50 µl aliquots of
the resultant supernatant were measured for absorbance at 630, 647, and 664 nm using Synergy
H1 microplate reader (Biotek). Chlorophyll a concentration was determined using the equation
specified in (Ritchie, 2008). Soluble host protein was measured colorimetrically using triplicate
10 ul aliquots of the host supernatant with the RED 660
TM
protein assay kit (G-Biosciences)
following the manufacturer’s instructions. Measured concentrations were multiplied by initial
homogenate volume to account for differential dilution. Larval traits were normalized by total
volume of larvae as the average larval size across different colonies varied (i.e. family #34
produced substantially bigger larvae than the rest, Figure S3.3). Recruit traits were normalized
by total recruit surface area (see details below) as individual recruit size was also quite variable.
The same workflow also applied to adult sample processing with a few modifications:
each thawed adult fragment was mixed with 10 ml extraction buffer in a plastic zipper bag
45
(Plymor) over ice and airbrushed to remove the tissue, which was then homogenized by a tissue
homogenizer (VWR
®
Model 200) for 1 min following (Palmer et al., 2010) and the final volume
was recorded. Symbiont cells were fixed by mixing 250 µl of homogenate with 125 µl 20%
formalin solution (6.7% final concentration). One ml of homogenate was aliquoted to conduct
chlorophyll analysis. Host tissue was isolated by centrifuging the remaining homogenate (ca.
8.75 ml) at 1500 xg. Skeletons were cleaned using 10% bleach and air dried. Surface area was
assessed using the single wax dipping method (Veal et al., 2010) and was used to standardize
adult traits.
Adult and recruit photos were analyzed in ImageJ (Schneider et al., 2012). To assess
visual bleaching, color scores were assigned to individuals at each time point following (Siebeck
et al., 2006). Briefly, the Coral Health Chart color reference was used to generate a standard
curve of mean grayness values (or an average of R, G, B values) from the standardized color
scores (D1-D6) within each photo (Grottoli et al., 2021). The entire surface area of recruits was
traced and three unshaded subsets of adult fragments were selected to measure mean grayness
value, which was then converted to a color score using the standard curve. A recruit was
considered dead (recorded as NA) if no visible tissue remained. Recruit size was determined by
tracing the outline of each individual and recording the pixel area. A line of known distance on
the Coral Health Chart was used as a reference to convert pixel area to metric area. Growth of
individual recruits was calculated as the difference in size between initial and final timepoints.
3.3.4 Statistical Analyses
All statistical analyses were conducted in R 4.0.3 (R Core Team, 2020). Linear mixed
effects models (lme4 package, (Bates et al., 2014)) were used to analyze all physiological traits,
including symbiont density, chlorophyll a concentration, total protein content, change in color
46
score, and recruit growth. Treatment (levels: control and heat) and origin (levels: inshore and
offshore) were included as fixed effects. The interaction of these two effects was also included in
the model. Family and tank (for adults and recruits only) were included as random effects. Larval
volume was modeled as a function of origin, with family included as random effect. Models were
evaluated for normality and homoscedasticity using diagnostic plots. Natural logarithmic
transformation was performed on trait data that did not satisfy the model assumptions of
normality and absence of heteroscedasticity of residuals. The null hypothesis was rejected at an
alpha of 0.05. Survival analysis was used to model time of death in the recruit heat stress
experiments as a function of treatment and origin, including a random effect of family and tank
using the coxme package (Therneau, 2018). Specifically, mortality was coded as a binary trait
(dead = 1, alive = 0) and time of death as an integer (timepoints at which photos were taken in
days). Correlation coefficients were calculated for traits that were shared between life stages to
investigate the strength of familial effect across stages (Hmisc package, (Harrell & Harrell,
2019)). Trait data were grouped by family and treatment and thus each comparison consisted of
20 data points.
Broad sense heritability (H
2
) of measured traits was estimated using MCMC models
(MCMCglmm package, (Hadfield, 2010)) for each life stage, including treatment and origin as
fixed effects and family as a random effect. Traits were log-transformed as necessary to meet the
normality requirement. All models were run for 100,000 iterations, with the first 10,000
discarded to ensure convergence and every 20 subsequent parameter values sampled to minimize
autocorrelation. Model convergence and autocorrelation were evaluated by plotting trace of
mean and variances. The resulting effective sample size was 4,500. H
2
values were calculated as
the ratio of variance attributable to the random familial effect over total variance.
47
3.4 Results
There was a significant interactive effect of treatment x origin on recruit symbiont
density, where recruits originating from inshore families lost fewer symbionts than offshore
recruits, by 5% on average (t = -2.86, df = 48, p < 0.01, Figures 3.2c). Adults, larvae, and recruits
all experienced a significant decline in symbiont density in response to heat stress (p < 0.05,
Figure 3.2a-c). Specifically, heat-exposed adult corals lost 62% of their symbionts compared to
the control group, followed by 9% in both larvae and recruits. Mean adult symbiont density
correlated positively with mean recruit symbiont density across families (r = 0.66, p < 0.01) but
no correlations with larvae were apparent.
Figure 3. 2 Standardized physiological parameters in response to experimental conditions shared across all life stages separated
by reef origin. Boxplots represent the 50th (median), 25th, and 75th percentiles (± 1.5 × IQR). Each column represents a distinct
life stage (left to right: adult, larvae, and recruit) and each row represents a distinct trait (top to bottom: symbiont density,
chlorophyll a concentration, total soluble protein content). Natural logarithmic transformation was performed on values that were
not normally distributed.
48
Chlorophyll a concentration declined by 85% (t = -3.86, df = 48, p < 0.001, Figure 3.2d)
in heat-treated adults and tended to decrease (t = -1.97, df = 45, p = 0.055, Figure 3.2f) in heat-
treated recruits compared to their respective controls. Larval chlorophyll a was not affected by
treatment, origin, or the interaction between the two factors. No significant correlations were
found between any two life stages in terms of chlorophyll a concentration.
Significant interaction was detected in adults where offshore coral lost 23% more protein
after the heat exposure period compared to inshore coral (t = -2.18, df = 48, p < 0.05, Figure
3.2g). However, no fixed effect of origin or treatment was observed for total soluble host protein
content in any life stage, save for a marginal treatment effect in recruits where protein content
tended to be reduced in response to heat stress (p = 0.059, Figure 3.2i). No significant
correlations were found between any two life stages in terms of protein content.
The overall survival rate of recruits after the 28-day heat stress was 89%. No significant
difference in mortality risk was found between treatment or origin. The standardized color score
of adult and recruit corals decreased over time (Figure S3.4). Heat-treated individuals
experienced a greater degree of paling, by more than 2-fold in both life stages at the end of the
experiment compared to the beginning (p < 0.05, Figure S3.5). The correlation between change
in color score over time in adults and recruits was not significant. Individual recruit size also
decreased after the exposure period, but recruits from the heat group suffered less of a decrease
than those from the control group (t = 3.05, df = 48, p < 0.01, Figure S3.6).
Significant heritability estimates were detected for multiple physiological traits but varied
greatly across life stages. The average broad sense heritability estimate for symbiont density was
highest in the larval stage (H
2
= 0.75, 95% confidence interval (CI): 0.54, 0.93), followed by
adult (H
2
= 0.65, 95% CI: 0.27, 0.96) and recruit stages (H
2
= 0.32, 95% CI: 0.0009, 0.61) (Figure
49
3.3). The highest H
2
value was detected for chlorophyll a concentration in larvae (H
2
= 0.67, 95%
CI: 0.43, 0.90), indicating 67% of the variance in chlorophyll a was explained by genetic factors,
which include additive genetic effects, epistasis, and maternal effects. In comparison, the H
2
estimate for chlorophyll a yielded a lower value of 0.53 (95% CI: 0.27, 0.84) in adults and a
much lower value of 0.07 (95% CI: 3x10
-5
, 0.22) in recruits. Heritability estimates for protein
content exhibited the opposite trend where recruits had the highest value of 0.67 (95% CI: 0.42,
0.92), followed by larvae (H
2
= 0.61, 95% CI: 0.35, 0.88) and adults (H
2
= 0.26, 95% CI: 3x10
-7
,
0.58). The H
2
of change in color score between final and initial time points was 0.35 (95% CI:
0.03, 0.66) for adults and 0.11 (95% CI: 0.01, 0.25) for recruits.
Figure 3. 3 Broad-sense heritability estimate (mean ± 95% C.I.) for three shared physiological traits at different life stages.
3.5 Discussion
Heat induced a significant bleaching response in all three life stages, as indicated by the
reduction in symbiont density and/or chlorophyll a concentration after treatment (Figure 3.2a-f).
Adult corals and their recruits in the 32 ˚C group also lost more pigmentation as indicated by
larger decreases in color score (Figures S3.4 and S3.5). Interestingly, larvae appeared to be more
heat resistant compared to adults and recruits. In particular, symbiont density was unaffected by
50
treatment in several larval families (5, 17, 38, 39) and even slightly elevated in two offshore
families 32 and 34 (Figure S3.7b). No treatment effect was detected at all for larval chlorophyll
concentration (Figure 3.2e). One possible explanation for the muted response to heat in larvae is
a shorter exposure period (i.e., 4 days as opposed to 22 days for adults and recruits). From a
developmental standpoint, it would be difficult to extend the exposure window as P . astreoides
larvae become competent quickly after release (Ritson-Williams et al., 2016) and high
temperature tends to expedite settlement (Nozawa & Harrison, 2005). Another possibility may be
that larvae are indeed more tolerant of thermal stress than later life stages, although prior
evidence has been mixed and studies emphasizing both the vulnerability and robustness of coral
larvae can be found. Earlier studies showed that larval survivorship and settlement in broadcast
spawning coral species Diploria strigosa and Acropora palmata were significantly reduced when
subjected to sublethal thermal stress (30-32 ˚C) (Bassim & Sammarco, 2003). Symbiotic larvae
of Pocillopora damicornis, a brooding coral like P . astreoides, suffered from impaired
metabolism under a combination of high temperature and CO2 stresses (Rivest & Hofmann,
2014). Another study on the same Pocillopora species reported an over 50% reduction in dark-
adapted maximum quantum yield of photosystem II of heat-exposed larvae compared to adults
under the same treatment (Putnam et al., 2010). Conversely, our previous study highlighted
incredible thermal tolerance of P . astreoides larvae where significant mortality under 36 ˚C was
only observed after >24 hours of exposure (Zhang et al., 2019) mirroring the survival data for
aposymbiotic Acropora millepora larvae subjected to a similar level of stress (G. B. Dixon et al.,
2015). A. millepora larvae also survived well and underwent normal development after a 5-day
exposure to 32 ˚C (Meyer et al., 2009). Larval thermal tolerance is therefore likely species- and
phenotype- specific. More cross-generational or even multi-generational studies are needed to
51
clarify these conflicting results. Ultimately, the performance of larvae also needs to be
contextualized in a wider developmental framework, as short-term sublethal thermal stress
experienced by larvae can negatively affect recruitment and post-recruitment survival (P.
Edmunds et al., 2001). In turn, the environment that the parents experience during the brooding
period can also have carry-over effects on newly-released larvae (Wong et al., 2021).
Population-specific responses to heat stress were observed in both adults and recruits.
Notably, inshore adult corals lost less protein content and inshore recruits lost fewer symbiont
cells respectively in comparison to their offshore counterparts (Figure 3.2g and 3.2c). Adult P .
astreoides colonies from the inshore environment in the Lower Florida Keys have been
repeatedly shown to exhibit higher bleaching tolerance based on multiple phenotypes, including
bleaching color score, brightness in the red channel, and photochemical yield of the algal
endosymbiont (Kenkel, Almanza, et al., 2015; Kenkel et al., 2013). In this study, surprisingly,
inshore adults failed to outperform the offshore adults in response to heat based on the two
measured bleaching phenotypes. Altered environmental pressures may have induced different
physiological responses among populations, for example, repeated thermal stress events could
reduce the resilience of inshore populations or increase the tolerance of offshore populations
through acclimation (Ainsworth et al., 2016). The current study did occur after the back-to-back
mass bleaching events of 2014 and 2015(Manzello et al., 2019). Paling and partial bleaching was
repeatedly observed in late August to early September between 2016 and 2019 although both
inshore and offshore reefs appeared to be equally impacted (The Florida Keys BleachWatch
Program). This result could also be explained by the comparatively smaller sample size and
noticeable variation among individual colonies; families 11 and 17 were more similar to offshore
families whereas family 39 was more inshore-like (Figure S3.7a). Although among-family
52
variation in thermal tolerance of P . astreoides adults originating from the same location has not
been investigated before, our prior study documented significant family effects on larval survival
and physiology, where a greater percentage of phenotypic variation was due to parental colony
identity than other factors such as day of release and reef origin (Zhang et al., 2019). In addition,
the significant positive correlation in symbiont density between individual adult and recruit
families suggest strong family effects can persist into the following life stage. Most importantly,
the maintenance of symbiont densities under elevated temperature presents the first evidence of
enhanced bleaching tolerance in inshore offspring. This may indicate that difference in tolerance
between inshore and offshore recruits has a heritable basis given that these recruits lacked prior
exposure to different thermal regimes, which is further supported by the analysis of heritability.
Reduction in recruit size was likely due to lack of feeding throughout the experimental
period (Figure S3.6). Feeding Pocillopora acuta with brine shrimp (Artemia spp.) three times a
week doubled colony growth and increased symbiont density as well as their maximum quantum
yield (Huang et al., 2020). We chose not to feed the corals mainly due to the concern of increased
fouling in a small, closed system. More importantly, neither treatment group was provisioned
with external food sources, so it is still valid to make conclusions on population level differences
in heat tolerance by comparing control and heat-treated corals, although results may differ under
replete conditions. Corals derive the majority of their daily energetic requirements from
symbiotic Symbiodiniaceae (Muscatine & Porter, 1977). However, certain species rely more
heavily on heterotrophic feeding during bleaching events when their autotrophic source is limited
(Grottoli et al., 2006). It is unclear whether our focal species P . astreoides has flexible
heterotrophic capacities and therefore hard to predict the actual effects of feeding. Based on the
53
limited trophic plasticity in its Pacific congeners P . compressa and P . lobata (Grottoli et al.,
2006), feeding might have a minor role in mitigating the effect of bleaching in P . astreoides.
In general, we found moderate (H
2
= 0.25~0.50) to high (H
2
> 0.50) broad sense
heritability estimates across all traits examined (Figure 3.3). This finding reinforces the main
conclusion of a recent meta-analysis by (Bairos-Novak et al., 2021) which found that the
majority of coral physiological traits (with few exceptions such as gene expression and
photochemistry) exhibited relatively high heritability. For instance, our H
2
estimate of adult
symbiont density (0.65) is on par with an estimate of 0.71 for the same trait in adult Orbicella
faveolata during a natural bleaching event (Manzello et al., 2019). These findings may indicate
adaptive capacity, which will project a positive outlook for the persistence of coral communities
under changing environmental conditions. One important caveat, however, is that the heritability
values derived from this study likely overestimate the true adaptive potential of our focal traits
because h
2
is only a subset of H
2
(Falconer & Mackay, n.d.). Nonetheless, h
2
for highly heritable
traits in certain life stages, for instance, symbiont density and chlorophyll a in larvae and protein
in larvae and recruits, is expected to be significant as well, as h
2
is typically only 1.4-2.5 fold
lower than H
2
(Bairos-Novak et al., 2021). High fidelity inheritance of symbiont communities
and host-symbiont co-evolution will be important for rapid adaptation, although additional high-
resolution genotyping and phenotyping of symbionts is needed to verify both heritability and the
extent to which symbionts contribute to holobiont thermal tolerance in this system(Hoadley et
al., 2021; Quigley et al., 2018). Another factor that may bias estimates is the assumption that
each spawning colony possesses a unique genetic identity. Given the rapid propensity for
settlement in P . astreoides larvae, they are expected to have short dispersal potential and
therefore high local retention (Jones et al., 2009). It is likely that clones or sibling clusters group
54
over a small spatial scale as larvae of other brooding species have been documented to settle
within meters from their parents (Carlon & Olson, 1993). If phenotypically similar colonies were
clones and their larval/recruit families were siblings, true heritability values could possibly be
higher than our estimates. Conversely, true heritability for a given trait may be lower than
estimated if phenotypically dissimilar individuals shared the same genetic identity. In the
absence of genotyping data we are unable to rule out the possibility of clones, however, it is
unlikely that we collected clonal or sibling adult colonies because we sampled individuals that
were at least 10 m apart in the field, a greater distance compared to 1 m used in other studies
(Riquet et al., 2022; Serrano et al., 2018).
Despite overall moderate to high heritability levels, estimates differed among life stages
and across different physiological traits within a given stage. Although the confidence interval
associated with most heritability values is not trivial, it is comparable to similar error estimates in
previous analyses (Jury et al., 2019; Kenkel, Setta, et al., 2015) and in future work can be
potentially reduced by larger sample sizes and increased replication. Symbiont density tended to
be more heritable in the adult and larval stages in comparison to the recruit stage (Figure 3.3).
Similarly, recruits had the lowest heritability estimate for chlorophyll a concentration (H
2
=0.07),
whereas over half of the variation in adults and larvae was attributed to genetics. In general,
older age in various vertebrate species is associated with high genetic variance in fitness-related
traits due to the accumulation of somatic mutation over time (Wilson et al., 2008). A recent study
estimated that the table coral Acropora hyacinthus accumulates mutations at a similar rate (2.6
mutations per gigabase per year in its coding region) as human somatic cell lines (López &
Palumbi, 2020). Given this parallel, it is reasonable to expect increased mutations and genetic
variation in older corals with bigger colony sizes. Moreover, those potentially deleterious
55
mutations are suspected to be purged before gamete production (Orive, 2001), which likely
contributes to the relatively lower genetic variation in the offspring. A potential explanation for
high heritability estimate of larval symbiont and chlorophyll traits is that maternal effects may be
dominant during early life stages as energetic reserves are maternally provisioned (Richmond &
Hunter, 1990a), thus inflating the overall estimate of genetic effects. Moreover, brooding species
like P. astreoides likely experience larger maternal effects since brooded larvae are subject to
maternal environment during early development and inherit symbionts directly from the mother
(Richmond & Hunter, 1990a). However, the rank order of H
2
estimates is completely reversed
for soluble protein content, where higher values were observed in larvae and recruits rather than
adults (Figure 3.3). Based on an interspecific comparison, the genetic component of protein
content was about four times higher in P. astreoides larvae (H
2
= 0.60) than in A. millepora adults
(H
2
= 0.15) (Bairos-Novak et al., 2021), which could be determined by the lack of maternal
effects in A. millepora as it reproduces by broadcasting gametes, leading to external fertilization
and embryonic development. Interestingly, despite the general expectation that maternal effects
attenuate over time (Dufty et al., 2002), the estimate of heritability of protein content was similar
between recruits and larvae. Studies on small mammals and birds have found that phenotypic
variance components constantly fluctuate across developmental stages, resulting in divergent
heritability estimates depending on sampling time (Atchley, 1984; Bourret et al., 2017). Additive
genetic variance (VA), in particular, can be modified by relative expression at specific loci and/or
the phenotypic effects of those loci especially during early ontology (Atchley, 1984). Indeed, loci
involved in metabolism and proteolysis were differentially expressed in A. millepora planula
larvae and newly-settled polyps (Hayward et al., 2011). It is also possible that different life
stages in P . astreoides use different sets of genes to respond to environmental stimuli (Ruggeri et
56
al., 2022), which may further contribute to the difference in VA observed between larvae and
recruits.
In summary, we observed population-level differences in the response to heat stress in
both adult and offspring generations of P . astreoides derived from two distinct reef zones in the
Lower Florida Keys, highlighting the potential contribution of genetic adaptation and
physiological acclimatization to thermal tolerance. Larvae tended to be more bleaching tolerant
than adults and recruits from the same lineages, although more studies involving multiple life
stages need to be conducted to validate this hypothesis. It is also important to identify an
appropriate level and duration of heat stress for each life stage so that responses are comparable.
Moreover, we also found that broad sense heritability of a given trait is widely divergent among
different life stages. Two bleaching phenotypes, symbiont density and chlorophyll a
concentration, were highly heritable in adults and larvae, whereas protein content was highly
heritable in larvae and recruits, which likely reflects the fluctuating dynamics between genetic
variation and environmental variation as organisms undergo different developmental phases. The
overall significant heritability levels of bleaching- and nutrient- associated traits suggest strong
familial influence on those traits that is evident across generations. If these traits are truly
heritable (determined by additive genetic effects), it bodes well for the local P . astreoides
populations, as they may be capable of keeping up with the rapidly changing ocean temperatures
through adaptation.
3.6 Supplementary Materials
Table S3.1 Number of recruit replicates by experimental factors.
57
Table S3.2 Summary table of linear mixed effects models. Significant p-values (<0.05) are marked with an asterisk.
58
Figure S3. 1 Temperature profile of control and heat treatment during larval thermal stress experiment.
Figure S3. 2 Temperature profile of control and heat treatment during adult and recruit thermal stress experiment.
59
Figure S3. 3 Size of individual larvae (mean ± SEM) post release across different families. No significant origin effect was
observed.
Figure S3. 4 Change in color score (relative to T0) (mean ± SEM) across 7 timepoints grouped by treatment and origin for adult
and recruit corals. Positive values indicate increase in pigmentation and negative values indicate decrease in pigmentation (or
bleaching).
60
Figure S3. 5 Change in color score between final and initial timepoint (T28 - T0) for adult and recruit corals in response to
experimental conditions separated by reef origin. Boxplots represent the 50th (median), 25th, and 75th percentiles (± 1.5 × IQR).
Positive values indicate increase in pigmentation and negative values indicate decrease in pigmentation (or bleaching).
Figure S3. 6 Change in size between final and initial timepoint (T28 - T0) for recruit corals (mean ± SEM) in response to
experimental conditions separated by reef origin.
61
Figure S3. 7 Standardized symbiont density (mean ± SEM) of individual Porites astreoides adult, larval, and recruit families in
response to experimental conditions separated by reef origin. Values were log-transformed if not normally distributed.
62
CHAPTER 4. PERFORMANCE OF ORBICELLA FAVEOLATA LARVAL
COHORTS DOES NOT ALIGN WITH PREVIOUSLY OBSERVED THERMAL
TOLERANCE OF ADULT SOURCE POPULATIONS
Yingqi Zhang, Shelby E. Gantt, Elise F. Keister, Holland Elder, Graham Kolodziej, Michael S.
Studivan, Dana E. Williams, Dustin W. Kemp, Derek P. Manzello, Ian C. Enochs, Carly D.
Kenkel
4.1 Abstract
Orbicella faveolata, commonly known as the mountainous star coral, is a dominant reef-
building species in the Caribbean, but populations have suffered sharp declines since the 1980s
due to repeated bleaching and disease-driven mortality. Prior research has shown that inshore
adult O. faveolata populations in the Florida Keys are able to maintain high coral cover and
recover from bleaching faster than their offshore counterparts. However, whether this origin-
specific variation in thermal resistance is heritable remains unclear. To address this knowledge
gap, we produced purebred and hybrid larval crosses from O. faveolata gametes collected at two
distinct reefs in the Upper Florida Keys, a nearshore site (Cheeca Rocks, CR) and an offshore
site (Horseshoe Reef, HR), in two different years (2019, 2021). We then subjected these
aposymbiotic larvae to severe (36 °C) and moderate (32 °C) heat challenges to quantify their
thermal tolerance. Contrary to our expectation based on patterns of adult thermal tolerance, HR
purebred larvae survived better and exhibited gene expression profiles that were less driven by
stress response under elevated temperature compared to purebred CR and hybrid larvae. One
potential explanation could be compromised reproductive output of CR adult colonies due to
repeated summer bleaching events in 2018 and 2019, as gametes originating from CR in 2019
contained less storage lipids than those from HR. These findings provide an important counter-
63
example to the current selective breeding paradigm, that more tolerant parents will yield more
tolerant offspring, and highlight the importance of adopting a holistic approach when evaluating
larval quality for conservation and restoration purposes.
4.2 Introduction
Global ecosystems are undergoing unprecedented structural and functional changes as
atmospheric CO2 level and temperature continue to rise in the Anthropocene (Steffen et al.,
2007). One ecosystem that is particularly vulnerable to these changes is coral reefs, because most
reef-building corals are found in the tropics (Spalding & Brown, 2015) and already live close to
their upper thermal limits (Baker et al., 2008). A small temperature increase, as little as 1 °C
above the maximum monthly mean temperature for a period of four weeks, or four degree
heating weeks (Liu et al., 2005), can lead to the breakdown of the symbiotic relationship between
the cnidarian animal host and their intracellular photosynthetic dinoflagellate algae. This
phenomenon is commonly known as coral bleaching (Hoegh-Guldberg et al., 2007; Lesser,
2011). Worldwide, coral cover is estimated to have declined by 20% over the past 30 years and
reefs will continue to be threatened by large-scale bleaching events even with climate
intervention strategies (Hoegh-Guldberg et al., 2019). Similar to the pattern observed in the
wider Caribbean region (Gardner et al., 2003), coral reefs in the Florida Keys have experienced
drastic population declines since the early 1980s mostly due to bleaching and disease (Dustan &
Halas, 1987; Precht & Miller, 2007). The two most recent large-scale bleaching events to affect
this region occurred in 2014 and 2015 when maximum temperatures exceeded local bleaching
thresholds for over 4-8 weeks (Eakin et al., 2019; Smith et al., 2019).
However, not all corals are equally susceptible to bleaching. Coral populations inhabiting
thermally-challenging environments, characterized by elevated temperatures and/or greater
64
temperature variabilities, have been repeatedly shown to exhibit higher tolerance to heat stress
(Howells et al., 2016; Thomas et al., 2018). Mechanistically, increased temperature tolerance can
be the result of adaptation and/or acclimatization on the part of coral hosts, their dinoflagellate
endosymbionts, or other members of the microbiome (Ainsworth et al., 2016; Berkelmans & van
Oppen, 2006; Palumbi et al., 2014; Santoro et al., 2021). Along the Florida Keys reef tract,
inshore patch reefs experience higher annual temperature fluctuations and elevated mean
temperature during bleaching-prone summer months in comparison to offshore reefs at similar
latitudes (Kenkel et al., 2015; Manzello et al., 2015a, 2015b). This spatially-defined thermal
heterogeneity has been theorized to support elevated heat tolerance of inshore corals, which
aligns with lab-based experiments and field-based observations of reduced bleaching severity of
inshore coral populations (Gintert et al., 2018; Kenkel et al., 2013). During the back-to-back
bleaching events in 2014 and 2015, inshore Orbicella faveolata colonies in the Upper and Lower
Florida Keys demonstrated lower bleaching prevalence and higher recovery rate than colonies at
paired offshore sites (Manzello et al., 2019). Due to the lack of distinct genetic structure among
inshore and offshore host populations, the increased heat tolerance of the inshore corals was
attributed to the significantly greater prevalence of heat tolerant symbionts (Durusdinium
trenchii) in these corals versus those at offshore sites (Manzello et al., 2019). The host role in
shaping holobiont thermotolerance in this system remains unclear.
Establishing the degree to which the coral host contributes to thermal tolerance is
essential for modeling adaptive potential and implementing intervention strategies. Assisted
evolution was proposed as a suite of intervention approaches to mitigate the decline and
degradation of reef systems, given that adaptive changes that occur naturally might not be able to
keep pace with the rapidly-changing climate (Van Oppen & Oliver, 2015). Studies in multiple
65
Indo-Pacific coral species provide compelling evidence for host genomic heritability of traits to
enhance the tolerance to heat, ocean acidification, and disease (Dixon et al., 2015; Drury et al.,
2022; Howells et al., 2021; Quigley et al., 2020). Additional data is needed to better understand
the tradeoffs of selecting for a single trait in corals, given their exposure to multiple
environmental challenges (Ladd et al., 2017). This approach may also lead to outbreeding
depression and genetic swamping, which can threaten the survival and fitness of their offspring
(Aitken & Whitlock, 2013). Significant differences in the decline of coral species have been
observed in the Pacific and Caribbean regions (Tebbett et al., 2023). It has been suggested that
impaired colony physiology in corals could contribute to suboptimal larval performance that
causes recruitment failure (Hughes & Tanner, 2000; Williams et al., 2008).
We investigate how the source population affects the performance of O. faveolata
offspring. To do this, we created larval cohorts sourced from parent colonies living in thermally
distinct reef sites in the Upper Florida Keys. We then exposed these aposymbiotic larvae to
severe and moderate temperature stress. O. faveolata is one of the major reef-building corals in
the Florida Keys and its populations are highly connected throughout the wider Caribbean
seascape (Rippe et al., 2017). However, O. faveolata populations have suffered sharp declines in
the past few decades (Edmunds, 2015). These declines are largely due to bleaching and disease,
making recovery challenging (Gladfelter et al., 1978). Orbicella faveolata is a hermaphroditic
broadcast-spawning coral species that sexually reproduces during late summer months when
water temperatures are maximal (Szmant, 1991). Symbiotic dinoflagellates are acquired from the
environment during metamorphosis (Coffroth et al., 2001). By working with aposymbiotic
larvae, we can study the physiological and transcriptomic basis for heat tolerance in the animal
host without the confounding effects of symbiosis. Understanding the physiological and genetic
66
factors underlying origin-dependent bleaching resistance in O. faveolata (and congeners) is
crucial because they were listed as threatened under the Endangered Species Act in 2014. By
studying this, we can assess the impact on future generations and estimate adaptive capacity,
enabling informed conservation efforts.
4.3 Methods
4.3.1 Sites and Temperature Data
Two well-monitored sites in the Upper Florida Keys were chosen for subsequent
spawning collections in 2019 and 2021, including Cheeca Rocks (CR, 24.8977°N, 80.6182°W)
and Horseshoe Reef (HR, 25.1388°N, 80.3133°W). Temperature was measured every 3 hrs at the
Cheeca Rocks Moored-Autonomous pCO2 buoy (MApCO2, depth = 1 m) using a conductivity–
temperature sensor (Model SBE-16 plus v. 2.2, Seabird Electronics). Data were collected every
30 min from HR (depth = 3.4 m) using the following loggers: Pendant (from 1/1/2017 to
8/6/2019 09:00 h) and Tidbit MX2204 (8/6/19 09:30 h to 12/31/21). From these data, daily
average temperature and the running 30-day mean temperatures were calculated for each site
from January 1, 2017 to December 31, 2021.
The bleaching thresholds for CR were determined as previously described (Gintert et al.,
2018). Bleaching thresholds can be estimated for reef sites in the Florida Keys by taking the
average of the maximum monthly mean sea surface temperature (SST) during a non-bleaching
year and the minimum monthly mean SST during a bleaching year (Manzello et al., 2007). Every
Florida Keys-wide mass bleaching event since 2005 has been predicted in near-real-time by
calculating the running 30-day mean SST from the Molasses Reef Coastal Marine Automated
Network (C-MAN) station and using that site as a proxy for the rest of the Florida Keys offshore
67
reef sites (Manzello, 2015). This technique has also been used to predict every bleaching event
that has occurred at CR since 2012.
The bleaching threshold for CR is a monthly mean SST ≥ 31.3°C, such that once the
running 30-day mean SST reach this value, bleaching has been observed in 2014, 2015, 2018,
and 2019 (Gintert et al., 2018; Manzello et al., 2019) (Fig. 4.1). The monthly mean bleaching
threshold for offshore reefs in the Florida Keys is ≥ 30.4°C, nearly 1°C lower than CR. We lack
sufficient coverage of both bleaching and non-bleaching year temperature data for HR to
determine a local bleaching threshold for this site. However, we can deduce that temperatures
experienced at HR are warmer than the values experienced at the far offshore reefs used for
calculation of the Florida Keys-wide threshold, but cooler than CR (Fig. 4.1).
4.3.2 Spawning, Cross Design and Larval rearing
Spawning collections were conducted under permit FKNMS-2018-163-A1. The first
spawn occurred on August 22, 2019, seven days after the full moon. Gamete bundles were
collected from five adult colonies within each site using spawning tents with 50 mL collection
tubes following standard protocols (Marhaver et al. 2017). Collection tubes were removed from
tents after ~5 mL of gametes had been collected, immediately capped, and transported back to
the boat by divers. Gametes were then diluted to reach a sperm concentration of ~ 10
6
cells/mL in
individual 5 gallon buckets filled with 0.2 μm filtered seawater (FSW). Three replicate bulk
crosses were created on each boat by mixing equivalent aliquots of diluted gametes from each
colony (n = 5) at each site (n = 2). Approximately 1.5 hrs post spawning, diluted gametes
released from two CR colonies and three HR colonies were mixed in the laboratory to create
three replicate hybrid bulk crosses. Note that given the optimum fertilization window for
gametes (~2 hrs), there was not sufficient time to separate eggs from sperm to attempt a diallel-
68
type crossing design. Therefore, the bulk hybrid crosses could include true CR x HR and HR x
CR reciprocal larvae as well as CR x CR and HR x HR fertilizations.
A second round of O. faveolata gamete collection from each site occurred six (28 August,
HR only) and seven (29 August, CR and HR) days after the full moon in August 2021. Gametes
were again obtained from five colonies at HR on day 6, and handled as in 2019, resulting in three
bulk crosses of HR x HR cultures. No spawning was observed at CR on day 6. On day 7, only a
single colony was observed spawning at each site, which restricted our fertilization design to the
creation of hybrid crosses only. Gamete bundles from each site were returned to the Key Largo
Marine Research Laboratory and separated into eggs and sperm by filtration through an 80 μm
nitex mesh, followed by rinsing with FSW. Individual eggs and sperm were crossed to create
three culture replicates of each of two hybrid crosses (CR sperm x HR egg, HR sperm x CR egg).
For all crosses in each year, successful fertilization was confirmed through observation of
initial cell division under 100x magnification following ~2 hrs of incubation. Developing
embryos were gently rinsed 3x in FSW to remove excess sperm and transferred to 6 L culture
bins at a density of ~1 embryo per mL in FSW. Healthy developing larvae were rinsed and
transferred to fresh FSW twice daily until reaching the planula stage, after which water changes
were performed every other day. Larval cultures were maintained at 29°C by placing filled bins
in shallow 32 L polycarbonate (Rubbermaid) water baths equipped with 100 W aquarium heaters
and SL381 submersible pumps (Domica) to maintain ambient temperature consistent with field
temperature profiles.
4.3.3 Thermal Stress Challenges
Two thermal stress experiments were conducted at the Key Largo Marine Research
Laboratory in 2019 after mature swimming larvae were observed in all cultures (CR x CR, HR x
69
HR, putative hybrid cross), which occurred on day 3 post fertilization: an acute stress at 36 °C
and a moderate stress at 32 °C following (Zhang et al., 2022). For each experiment, six replicate
6 L polycarbonate larval bins (Vigors) were filled with 0.2 μm FSW and placed into a set of two
shallow 32 L polycarbonate (Rubbermaid) water baths for temperature control (n = 3 larval bins
per bath). Each water bath was filled with ~15 L water and equipped with a SL381 submersible
water pump to maintain circulation and a 100 W aquarium heater. Each larval rearing bin was
fully filled and two of three bins were equipped with HOBO temperature loggers (Onset). For the
acute stress experiment, each larval rearing bin received two groups of ten larvae per bulk cross
(n = 6 per cross type per treatment) that were aliquoted into floating netwells (70 μm cell
strainers, Grenier Bio-One). The control bath remained at 29 °C and the treatment bath was
heated up to 36 °C over 24 hrs (Fig. S4.1a). Mortality was assessed every 24 hrs by counting the
number of surviving larvae in the netwells. The experiment was terminated once mortality
reached more than 50% for the majority of the netwells. For the moderate stress experiment, a
total of 20 larvae from each bulk cross replicate were aliquoted to each netwell. The control bin
remained at 29 °C and the treatment bin was heated up to 32 °C over 24 hrs (Fig. S4.1a). After 4
days of exposure, swimming larvae were removed from each netwell, flash frozen in liquid
nitrogen, and stored at -80 °C for RNA extraction.
Similar acute and moderate stress experiments were conducted with larvae reared in 2021
(HR x HR, CR x HR, HR x CR). All cultures were transported to the Experimental Reef Lab
(ERL) at The University of Miami’s Cooperative Institute for Marine and Atmospheric Studies
(CIMAS) on day 4 post fertilization. Larvae were packed into 50 mL centrifuge tubes with no air
bubbles and stored in coolers at ambient temperature during transit. Upon arrival at ERL, larvae
were re-distributed into 6 L culture bins (n = 3 per cross) filled with 0.2 μm FSW. Temperature
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control was accomplished using the ERL aquaria with individual treatment tanks serving as
water baths (Enochs et al., 2018). For the stress experiments, netwells were floated directly in the
temperature-controlled flow-through aquaria. For each bulk cross, two groups of 10 larvae were
allocated to each treatment tank in the acute stress treatment (n = 6 per cross type per treatment)
and three groups of 20 larvae were allocated to each treatment tank in the moderate stress (n = 9
per cross type per treatment). The control temperature for both experiments was set to 27 °C.
Heat ramps started 5 days post fertilization (note this represented different calendar days for HR
x HR vs. CR x HR and HR x CR to account for differences in developmental age) and target
temperatures were reached over 48 hrs (Fig. S4.1b). Lights were maintained at 180 µmol s
-1
·m
-2
for a 12:12 hr light dark cycle. The acute stress assay was monitored every 12 hrs for survival
and the experiment was terminated once mortality reached more than 50% for the majority of the
netwells. For the moderate duration experiment, larvae were retrieved from 1 netwell per cross
per replicate tank following 4 days of exposure using a pipette, counted, and transferred to a
cryovial for RNA extraction. Excess seawater was removed and larvae were snap frozen in liquid
nitrogen and stored at -80 °C until processing. Additional replicate samples were taken from the
remaining 2 netwells for protein and lipid analyses.
4.3.4 Physiological Assays
Gametes from both years, as well as 2021 fertilized larvae, were collected for lipid
analyses. Gametes from each parent colony were sampled in duplicate. Gametes and larvae were
counted under a dissection microscope before being transferred to combusted glass tubes and
frozen at -20 °C until lipid processing. Total lipids were extracted and determined
gravimetrically using a modified Folch method (Folch et al., 1957), as described in (Keister et
al., 2023). Subsequently, 100% chloroform was added to total lipids to achieve a 10 mg mL-1
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concentration, to standardize samples. Lipid classes were quantified by spotting, in duplicate, 1
μL of extracted lipids on silica Chromarods
®
before being developed using thin-layer
chromatography via a two-step solvent system (Conlan et al., 2014, 2017; Nichols et al., 2001),
as described in (Keister et al., 2023). Developed rods were then dried at 100 °C for 10 min in an
IsoTemp Oven (Fisher Scientific) before being run on an Iatroscan MK 6S thin-layer flame
ionization detector (TLC-FID). Known concentrations of lipid compounds, ranging from 0.1–
10.0 mg mL
-1
, were used to calibrate the Iatroscan for the following lipid classes:
phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylinositol (PS-PI),
phosphatidylcholine (PC), lysophosphatidylcholine (LPC), wax esters (WAX), triacylglycerols
(TAG), sterols (ST), and diacylglycerols (DAG). All phospholipid lipid classes (PE, PS-PI, PC,
LPC) were grouped and analyzed as one unit. All total lipid and lipid class values were presented
as μg per gamete or μg per larva, respectively.
Protein samples were thawed on ice and homogenized by back pipetting in FSW for at least 60 s
until no visible cellular debris was present. Total homogenate volume was recorded. Soluble host
protein was quantified in triplicate with a BCA Protein Assay Kit II (BioVision) following the
manufacturer's protocol. Final protein concentration was multiplied by the initial homogenate
volume and then standardized by the number of total sampled larvae.
4.3.5 Statistical Analysis of Physiological Trait Data and Survival under Acute Stress
All statistical analyses were conducted in R 4.2.1 (R Core Team, 2022). Traits were
evaluated for normality using the Shapiro-Wilks test and log-transformed if not normally
distributed. A two-way ANOV A was performed to analyze the effects of treatment (levels:
control and heat) and origin (levels: CR x CR, HR x HR) on 2019 gamete lipid content. No
statistical analysis of 2021 gametes was possible given that only one CR colony spawned. Larval
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protein and lipid content for the 2021 cohorts were analyzed using a two-way anova to analyze
the effect of origin (levels: HR x HR, CR x HR, HR x CR) and treatment (levels: control and
heat). A mixed effects Cox model (Therneau, 2018) was used to model time of death for the
acute stress experiment from both years as a function of treatment and origin, including a random
effect of replicate bulk cross. Due to low mortality rate, HR x HR and control were chosen as the
reference level for each fixed effect to calculate hazard ratio for other levels. A hazard ratio
above 1 means a higher mortality risk and below 1 means a lower mortality risk compared to the
reference levels. The null hypothesis was rejected at an alpha of 0.05.
4.3.6 RNA Extraction, Library Preparation, and Sequencing
Total RNA was extracted from frozen samples using the Aurum Total RNA Mini Kit
(Bio-Rad). Samples were back pipetted to ensure complete homogenization after Lysis Solution
was added. Genomic DNA was removed by adding DNAse I on-column according to the
manufacturer's instructions. RNA concentration was quantified using a Take2 plate on a Synergy
H1 microplate reader (Biotek) and only samples with > 10 ng/µl concentration were used to
generate tag-based RNA-seq libraries, following protocols modified for sequencing on the
Illumina platform https://github.com/ckenkel/tag-based_RNAseq.
Libraries from 2019 (n = 45 samples) were sequenced on the NextSeq 550 in 2020 by the
USC Genome Core using a 1x75bp HO kit. Libraries from 2021 (n = 30 samples) were
sequenced on the NextSeq 2000 in 2022 in two replicate separate runs by the USC Norris
Comprehensive Cancer Center Molecular Genomics core using a NextSeq 2000 P2 Reagents v3
kit, after which reads were concatenated to reach a comparable sequencing depth. The average
sequencing depth per sample for the two libraries was 5.9 M reads (± 0.2 M) and 5.4 M reads (±
0.3 M) for the 2019 and 2021 datasets, respectively.
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4.3.7 Bioinformatics Pipelines
Downstream bioinformatic processing was performed on USC’s Center for Advanced Research
Computing (CARC) following protocols described in https://github.com/ckenkel/tag-
based_RNAseq. Briefly, a custom perl script was used to discard PCR duplicates and reads
missing adapter sequences. Poly-A tails and adapter sequences were trimmed and only high
quality reads (PHRED score ≥ 20 over 90% of the read) were retained. A total of 2.7 M (± 0.09
M) and 2.4 M (± 0.1 M) reads per sample remained for the 2019 and 2021 datasets respectively
after quality filtering. Filtered reads were mapped to an adult Orbicella faveolata transcriptome
(Supplemental Materials) using the gmapper command from SHRiMP2 (Rumble et al., 2009).
Read counts were summed by isogroup using a custom perl script, resulting in a per sample
mapped read average of 1.5 M (± 0.05 M) and 1.3 M (± 0.07 M), for the 2019 and 2021 datasets
respectively.
4.3.8 Statistical Analysis of Gene Expression
Larval samples from the two years were first compiled, where isogroups shared between
the two year cohorts that had fewer than 2 counts across 90% of the samples were discarded,
resulting in a total of 28,675 high quality isogroups remaining. Counts were then transformed
using the rlog function in DESeq2 (Love et al., 2014) and outlier samples were identified
through a sample network with a standardized connectivity score of less than -2.5, based on
which one sample from the 2019 dataset and one from the 2021 data set were filtered. The
filtered dataset was then separated by year to form two subsets, on which principal component
analysis (PCA) was applied to visualize global gene expression pattern for each larval cohort
using the R package FactoMineR (Lê et al., 2008).
74
Gene co-expression analysis was conducted using the WGCNA package in R (Langfelder
& Horvath, 2008) following the standard tutorial (Langfelder & Horvath 2016) and a pipeline for
performing meta-analysis of two microarray datasets (Miller 2011). Briefly, a signed gene co-
expression network was constructed with a soft power of 4. Module assignment was generated
based on the 2019 dataset and imposed onto the 2021 dataset to assess how well those modules
were preserved across datasets. The eigengenes of these shared modules were correlated with
origin (levels for 2019: CR x CR, HR x HR, Hybrid; levels for 2021: HR x HR, CR x HR, HR x
CR) and treatment (levels: control, heat).
DESeq2 (Love et al., 2014) was used to examine genes that were differentially expressed
in larvae by origin and treatment, with each year’s cohort being analyzed separately. Isogroups
that had fewer than 2 counts across 90% of the samples were discarded, resulting in a total of
27,894 high quality reads in the 2019 dataset and 28,960 reads in the 2021 dataset. A two-factor
grouping was created by combining origin and treatment and fed into the DESeq2 model as the
design, from which results were extracted with specific contrasts to obtain differentially
expressed genes (DEGs) in response to treatment in a particular larval origin (e.g. CR x CR heat
vs. CR x CR control). Significance testing was determined using a Wald test after independent
filtering using a false discovery rate-corrected (FDR) threshold of 0.1. Multiple test correction
was applied to raw p-values following Benjamini & Hochberg (Benjamini & Hochberg, 1995)
and adjusted p-values less than 0.1 were considered significant. Signed log-p values were
generated based on the adjusted p-values to serve as input for gene ontology (GO) enrichment
analysis described below.
To explore the possibility that heat-responsive genes were front- or back-loaded in the
CR x CR or HR x HR populations which would preclude their identification as significantly
75
differentially expressed genes (Barshis et al., 2013), we identified the overlap between
significantly up-regulated/down-regulated genes in response to heat in one population and the
significantly up-regulated/down-regulated genes between groups in control conditions (i.e.
constitutively differentially expressed) as candidate front-/back-loaded genes in the other
population. For example, genes identified as significantly up-regulated in CR x CR larvae but not
HR x HR larvae under heat that were also significantly up-regulated under control in HR x HR
vs CR x CR larvae were identified as potentially front-loaded genes in HR x HR larvae. The
same approach was repeated for determining back-loaded genes as well as front-loaded/back-
loaded genes in CR x CR larvae.
To understand the functional implications of conserved gene modules identified in
WGCNA, a categorical gene ontology (GO) enrichment analysis was performed using binary
values (1 or 0) to indicate module membership in the WGCNA set followed by a Fisher’s exact
test and false discovery rate correction. For heat-responsive DEGs by larval origin, signed log p-
values using adaptive clustering of GO categories and a two-sided Mann-Whitney U-test was
applied, followed by a false discovery rate correction. GO scripts can be found at
https://github.com/z0on/GO_MWU. Heatmaps of hierarchically clustered GO terms were
generated using the pheatmap package (Kolde, 2012) in R.
Discriminant analysis of principal components (DAPC) was performed to explore the
relative changes in global expression between treatment in different populations from 2019 and
2021 using the R package adegenet (Jombart, 2008). Variance stabilized data (VSD) were used to
create the model and the number of PCs was chosen to capture at least 80% of transcriptional
variance. Distribution of samples grouped by origin and treatment was visualized in density
plots.
76
4.4 Results
4.4.1 Temperatures and bleaching history at study sites
SST was always cooler at HR than CR (Fig. 3.1) during the data collection period,
although bleaching occurred at both sites in 2014 and 2015 (Gintert et al., 2018; Manzello et al.,
2019). Bleaching was observed at CR in 2018 and during gamete collection in 2019 at CR, but
not at HR. CR gametes were collected from colonies that didn’t show visual bleaching. In 2021,
bleaching was not observed at either site, in line with the cooler temperature patterns (Fig. 3.1).
In total, from the start of the most recent mass global bleaching event in 2014 to the 2021 spawn,
CR experienced four bleaching events, whereas HR only experienced two events. Notably, HR
did not experience bleaching between 2015 and 2021.
Figure 4. 1 Time series of running 30-day mean sea surface temperature (SST) for Cheeca Rocks (red line) and Horseshoe Reef
(blue line) from 2017 to 2021. Bleaching threshold for Cheeca Rocks (monthly mean SST ≥ 31.3°C) shown as dashed black line.
4.4.2 Larval survival under acute heat stress
To achieve a reasonable separation of survivorship among the different groups, the 36 °C
acute temperature stress lasted for 96 hrs in 2019 and 141 hrs in 2021. The average survival rate
at the end of each year’s experiment for the control vs. heat group was 77% vs. 47% and 86% vs.
33% respectively (Fig. 4.2). Exposure to 36 °C significantly increased larval mortality in both
years, resulting in hazard ratios of 3.1 (z = 4.16, p < 0.001) and 28.7 (z = 4.11, p < 0.001) for
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heat-treated larvae from 2019 and 2021, respectively. Larval origin also had a significant effect
on survival in both years. In 2019, the CR x CR cross experienced almost double the mortality
risk (HR = 1.9, z = 2.18, p < 0.05) in comparison to the HR x HR cross, and in 2021 the CR x
HR cross experienced more than 10 times the risk (HR = 11.8, z = 2.18, p < 0.001) of the HR x
HR cross.
Figure 4. 2 Number of surviving larvae (mean ± standard error of the mean [SEM]) across time in the acute temperature stress
experiment for a) 2019 and b) 2021 cohorts. Survivorship was grouped by larval origin and treatment condition.
4.4.3 Gamete (2019 and 2021) and larval ecophysiology (2021 only)
Total lipid content of gametes collected from CR and HR during 2019 spawning season
did not differ between the two sites (Fig. 4.3b). However, HR gametes contained 2.2 times more
wax esters (F = 6.27, df = 1, p < 0.05) and 1.5 times more phospholipids (F = 6.07, df = 1, p <
0.05) than CR gametes (Fig. 4.3a). No differences in triacylglycerol content by origin were
apparent.
Qualitatively, lipid content tended to be higher in the CR gametes in 2021 (Fig. 4.3b), but
as only one colony spawned, formal significance was not evaluated. No differences were
detected in total lipid content, lipid classes, or total soluble protein content of 2021 larvae
78
between origin, treatment conditions or their interaction (Fig. S4.2, S4.3; Table S4.2). Protein
and lipid data were unavailable for 2019 larvae exposed to the 32 °C moderate stress.
Figure 4. 3 Concentration (mean ± SEM) of (a) different lipid classes (PL: phospholipid, TAG: triacylglycerol, W AX: wax ester)
and (b) total lipids standardized by individual gamete bundle collected from Cheeca Rocks (n = 5) and Horseshoe Reef (n = 5) in
2019 and 2021. No replicates were available for CR gametes in 2021.
4.4.4 Major drivers of transcriptional variation
Principal component analysis (PCA) on all high expression genes (count > 2 in 90%
samples) within each dataset showed that larval origin was an important driver of transcriptional
variance in addition to temperature treatment (Fig. 4.4). Samples clustered by larval origin along
PC2 in the 2019 dataset, which accounted for 5.6% of the overall variance in expression (Fig.
4.4a). Putative hybrid samples generally clustered mid-way between HR x HR and CR x CR
origin larvae. In the 2021 dataset, larval origin was the primary driver of expression variation, as
cross types were clustered along the first PC which accounted for 10.4% of the overall variance
(Fig. 4.4b). No significant differences in clustering were apparent between the reciprocal hybrids
(CR x HR vs. HR x CR) but both were distinct from the HR x HR origin larvae. Clustering by
temperature treatment was apparent along PC2 in the 2021 dataset, which explained 9.6% of the
variance in overall expression (Fig. 4.4b), and along PC3 in the 2019 dataset, which explained
5.1% of transcriptional variance (Fig. S4.4).
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Figure 4. 4 Principal component analysis (PCA) on rlog-transformed read counts in a) 2019 and b) 2021 larval datasets. Points
are colored by origin and shaped by treatment. The percentage variance explained by each PC is reflected on the axis label.
4.4.5 Conservation of expression networks and their functional significance
WGCNA was used to investigate whether and to what degree expression patterns were
conserved between the two datasets and to further explore their relationships with experimental
factors. Four modules, pink (n = 400 genes), purple (n = 268), magenta (n = 283), and red (n =
514), were highly correlated with origin in both years, although the directions of the correlations
were not always conserved (Fig. 4.5). Specifically, genes within the pink and purple modules
were strongly negatively correlated, or down-regulated, in 2019 CR x CR larvae (pink: Pearson’s
r = -0.87, pcor = 5e
-15
; purple: Pearson’s r = -0.74, pcor = 8e
-9
) and up-regulated in 2019 HR x HR
larvae (pink: Pearson’s r = 0.81, pcor = 1e
-11
; purple: Pearson’s r = 0.69, pcor = 1e
-7
), while the
opposite relationship was observed in the magenta (CR x CR: Pearson’s r = 0.71, pcor = 6e
-8
; HR
x HR: Pearson’s r = -0.86, pcor = 7e
-14
) and red modules (CR x CR: Pearson’s r = 0.88, pcor = 2e
-
15
; HR x HR: Pearson’s r = -0.76, pcor = 1e
-9
). In comparison, the magnitude and direction of
module-trait correlations remained similar for the pink and red modules in 2021, with pink
module genes again showing strong up-regulation in HR x HR larvae and red module genes
showing strong down-regulation (pink: Pearson’s r = 0.98, pcor = 3e
-21
; red: Pearson’s r = -0.98,
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pcor = 3e
-21
). While there were still highly significant correlations observed for the purple and
magenta modules with respect to origin, the direction of the association was completely reversed,
with strong down-regulation of genes in the purple module (Pearson’s r = -0.98, pcor = 9e
-20
) and
up-regulation of genes in the magenta module (Pearson’s r = 0.96, pcor = 5e
-17
). Modules
significantly associated with treatment exhibited more moderate correlation coefficients
(Pearson’s r range: ± 0.4 to ± 0.6), but their expression patterns were more strongly conserved
across years. Genes in the black (n = 408 genes), greenyellow (n = 268), salmon (n = 160), and
yellow (n = 574) modules were consistently up-regulated in heat-treated larvae whereas genes in
the cyan (n = 152) module were consistently down-regulated in heat-treated larvae across years.
Figure 4. 5 Weighted gene co-expression network analysis (WGCNA) module-trait relationships identified in a) 2019 and b) 2021
larval cohort. Correlation values range from 1 (red) to -1 (blue) and the associated p values were included in the parenthesis
below for modules showing a significant trait association, with color of each block determined by strength and direction of the
correlation between given module and trait.
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Categorical functional enrichment analysis of genes assigned to the significant origin
modules (pink, purple, red, magenta) revealed few significant GO terms, likely due to small
module size. Two terms in the molecular function category (GO:0031683, G-protein
beta/gamma-subunit complex binding; GO:0050780, dopamine receptor binding) and one term
in the cellular components category (GO:0005834; GO:1905360, GTPase complex) were
significantly enriched (p ≤ 0.05) among genes in the pink module (Table S4.3). GO analysis of
treatment modules identified enrichment of genes associated with heat response pathways, such
as immune response, defense response, and response to external stimulus in the black module
which was up-regulated in response to heat treatment (p < 0.01, Fig. S4.5, Table S4.3). Whereas
the cyan module, which was down-regulated in response to heat, showed enrichment of genes
associated with amide and peptide metabolic and biosynthetic processes (p < 0.05, Fig. S4.6,
Table S4.3). These processes were also enriched among genes in the salmon module, which was
up-regulated under heat (Table S4.3). One term in the molecular function category
(GO:0031210; GO:0050997, phosphatidylcholine binding) was significantly enriched (p = 0.05)
among genes in the yellow module, and was also up-regulated under heat treatment (Table S4.3).
No significant functional enrichments were detected for the greenyellow module.
4.4.6 Origin-specific responses to thermal stress
DESeq2 analysis of the 2019 dataset showed that a total of 561 genes were up-regulated
and 436 were down-regulated in heat-treated larvae relative to controls (Table S4.4). When
further partitioned by origin, 133 heat-responsive genes were up-regulated and 144 were down-
regulated in 2019 CR x CR larvae, whereas 376 were up-regulated and 158 were down-regulated
in 2019 HR x HR larvae. The putative hybrid larvae up-regulated 12 genes and down-regulated 9
genes under heat and thus were excluded from the downstream functional enrichment analysis
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due to a limited number of differentially expressed genes (DEGs). Among these origin-specific
heat-responsive genes, 28 (11 annotated) were front-loaded and 47 (31) were back-loaded in
2019 HR x HR larvae, while 82 (48) were front-loaded and 23 (14) were back-loaded in 2019
CR x CR larvae (Table S4.5). In the 2021 dataset, 706 heat-responsive genes were up-regulated
and 953 were down-regulated in HR x HR larvae, 228 were up-regulated and 54 were down-
regulated in CR x HR larvae, and 124 were up-regulated and 101 were down-regulated in HR x
CR larvae.
Subsequent ontology analysis of these DEGs showed that biological processes including
immune response, peptide hormone processing, defense response, and response to stimulus were
enriched among heat-responsive up-regulated genes in 2019 CR x CR larvae (FDR < 0.01),
while RNA metabolic process and macromolecule biosynthetic process were enriched among
down-regulated genes (FDR < 0.01, Fig. 4.6a, Table S4.6). In the 2019 HR x HR larvae,
phospholipid catabolic process was enriched among genes up-regulated in heat (FDR < 0.05),
while microtubule-based movement/process and protein-DNA complex subunit organization
were enriched among downregulated genes (FDR < 0.01, Fig. 4.6b, Table S4.7). Discriminant
analysis of principal components (DAPC) for heat responsive genes revealed a greater
transcriptional response in 2019 HR x HR larvae compared to the CR x CR larvae from the same
year (Fig. 4.6c).
In the 2021 cohort, genes up-regulated in response to heat in HR x HR larvae were
enriched for biological processes including DNA metabolic process, organelle localization, and
cellular response to DNA damage stimulus (FDR < 0.01, Fig. S4.8, Table S4.8). Processes
enriched among down-regulated genes included a suite of metabolic processes of small and large
molecules, such as organic acids, fatty acids, and lipids (FDR < 0.01). For the two hybrid
83
crosses, upregulated genes were enriched for NF-κB signaling regulation, immune and defense
response, as well as regulation of cell death (FDR < 0.05, Fig. S4.9, S4.10, Table S4.9, S4.10).
Downregulated genes were enriched for lipids and protein metabolic/catabolic processes (FDR <
0.05). DAPC for heat responsive genes identified a greater response in HR x CR larvae
compared to the HR x HR and CR x HR larvae from the same year (Fig. S4.11).
Figure 4. 6 Hierarchical clustering of ontology terms enriched by genes up-regulated (red) or down-regulated (blue) in 2019 heat-
treated (a) CR x CR larvae and (b) HR x HR larvae compared to their respective untreated control, summarized by biological
process (BP). Font size indicates level of statistical significance (FDR-corrected). Term names are preceded by fractions
indicating the number of individual genes within each term differentially regulated with respect to treatment (unadjusted p <
0.05). (c) Density plots showing distribution of global expression across samples from the two origins along the temperature
responsive axis (linear discriminant 2 [LD2], Fig. S7) based on discriminant analysis of principal components (DAPC) performed
on variance stabilized data (VSD) grouped by treatment and origin.
4.5 Discussion
Adult O. faveolata from Cheeca Rocks exhibit elevated thermal tolerance in response to
natural bleaching events (Gintert et al., 2018; Manzello et al., 2019) suggesting that they have
acclimatized or adapted to naturally higher and more variable temperatures characteristic of
inshore reef sites in the Florida Keys (Kenkel et al., 2015; Kenkel & Matz, 2016). Contrary to
84
the current paradigm of inherited and/or enhanced thermal tolerance in adults experiencing more
extreme thermal regimes (Dixon et al., 2015; Putnam & Gates, 2015; Strader & Quigley, 2022),
we found that offspring of these more tolerant inshore colonies were more susceptible to thermal
stress, exhibiting reduced survival and stronger expression signatures of a stress response in
comparison to larvae from offshore colonies (Fig. 4.2, 4.6). The observed total lipid data suggest
robust bleaching resistance in adults may come at the cost of reproductive investment (Fig. 4.3),
although patterns are inconsistent across years. These findings represent an important counter-
example to the rationale underpinning selective breeding approaches (Drury et al., 2022): that
tolerant parents can be counted on to produce tolerant offspring.
4.5.1 Impaired larval performance may result from reduced reproductive investment
Reef origin (or cross type) and temperature treatment played important roles in driving
physiological traits in O. faveolata larvae. As expected, larvae were more likely to die under heat
treatment than in the ambient control (Fig. 4.2), but the origin response was unexpected.
Horseshoe Reef (HR) purebred larvae appeared to be the best performers in both years, while
Cheeca Rocks (CR) purebred larvae in 2019 and 2021 CR x HR hybrids experienced
significantly higher mortality in comparison (Fig. 4.2). Previous studies conducted on Acropora
millepora and Acropora tenuis from the Great Barrier Reef showed that larval survival under a
similar level of acute thermal stress (35.5 °C) was enhanced in offspring of parents from warmer
source reefs (Dixon et al., 2015; Strader & Quigley, 2022). Yet, in our study system, the HR x
HR cross, produced by the less tolerant parents sourced from a cooler reef environment,
repeatedly outperformed the CR cross produced by more tolerant parents from a warmer reef
environment (Gintert et al., 2018; Manzello et al., 2019). This suggests that the host genetic
85
contribution to thermal tolerance may be minimal or overpowered by other factors such as recent
and concurrent heat stress, or maternal provisioning.
Prior thermal stress and bleaching has been linked to negative reproductive outcomes,
including both fecundity and gamete quality, in multiple coral species (Jones & Berkelmans,
2011; Szmant & Gassman, 1990; Ward et al., 2002). Although the severity of reproductive
impacts is thought to be related to the severity of bleaching and rate of recovery,
resistant/resilient colonies do not necessarily exhibit latent effects (Leinbach et al., 2021;
Szmant, 1991). Notably, the HR source population suffered from both a reduced number of
spawning colonies and total gametes released as a result of the back-to-back bleaching events in
2014 and 2015 (Fisch et al., 2019). Previous research consistently demonstrates that adult
colonies from nearshore reef environments display higher resistance and resilience to thermal
stress compared to those from offshore reefs (Gintert et al., 2018; Manzello et al., 2019). Based
on these findings, we anticipated that the offspring of these resilient adults would also exhibit
enhanced thermal tolerance, in line with previous studies. At the time of spawning collections in
2019, temperature at CR had surpassed the local bleaching threshold and a majority of the
colonies showed some degree of paling (i.e., onset of bleaching), while HR experienced cooler
temperatures and all the colonies appeared fully pigmented (Fig. 4.1). Recently, (Leinbach et al.,
2021) showed that Acropora hyacinthus which resisted bleaching maintained higher reproductive
capacity than recovered coral. Although we only collected gametes from CR colonies without
apparent signs of physiological stress in 2019 (i.e. thermally resistant adults), it is likely that the
energetic state of individual colonies was already compromised due to the heat stress, which may
have impacted maternal provisioning.
86
Scleractinian coral gametes are largely composed of lipids with wax esters, phospholipids
and triacylglycerols being the most abundant classes (Figueiredo et al., 2012). Mean total lipids
were higher for 2019 HR gametes compared to CR gametes (Fig. 4.3b). Lipid class analyses, of
the 2019 gametes, showed CR gametes contained less wax esters and phospholipids than HR
gametes (Fig. 4.3a). Wax esters take longer to metabolize, supporting their role in long-term
energy storage, and are important for larval development (Lee et al., 2006; Richmond, 1987;
Rivest et al., 2017), indicating 2019 HR gametes had greater energy reserves available for
dispersal and settlement. Additionally, greater wax ester stores lower gamete/larval density,
possibly contributing to extended dispersal (Richmond, 1987). Phospholipids were significantly
different across sites in 2019, but this lipid class encompasses many structural lipid compounds,
thus muddling the implications. While triacylglycerols can be rapidly hydrolyzed and likely
support immediate energetic needs (Figueiredo et al., 2012; Sewell, 2005), no significant
differences were detected across sites in either 2019 or 2021. Similar lipid class patterns were not
observed in the 2021 cohort, possibly due to lack of replication in CR gametes (Fig. 4.3a). The
general deficiency of 2019 CR gametes in all these classes suggests that bleaching tolerance and
resilience of CR adults may come at the cost of reproductive investment, potentially contributing
to reduced performance of CR larvae.
Further supporting a compromised reproductive output of nearshore coral populations
was the absence of mass spawning on CR in the summer of 2021, despite no observed bleaching
(Fig. 4.1), which limited our ability to re-assess performance of CR x CR larvae and additional
hybrid crosses. This implies that reproductive capacity of CR adults (likely among other
marginal nearshore populations) could be jeopardized by the persistent latent effects of
accumulated stress. As thermal anomalies increase in magnitude and frequency in the Florida
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Reef Tract (Manzello, 2015), it is important to consider any latent effects beyond visible stress
responses that could affect the next generation and ultimately the persistence of reef
communities.
4.5.2 Stress tolerance rather than a front-loaded stress response is associated with enhanced larval
survival
In addition to origin-specific physiological differences, larvae originating from different
reef sites mounted different transcriptional responses after four days of 32 °C heat challenge. In
the 2019 CR purebred larvae, we observed functional enrichment in a wide range of stress
responses (e.g., immunity, defense, inflammation) among the significantly upregulated genes,
whereas metabolic and biosynthetic processes (e.g., various RNA molecules, peptide, cellular
nitrogen compounds) were enriched among downregulated genes (Fig. 4.6a). Similarly,
underperforming CR x HR larvae in 2021 showed pronounced upregulation of defense and
immune response pathways and downregulation of metabolic processes (Fig. S4.9). Upregulation
of stress response genes and concomitant downregulation of growth-related processes, such as
rRNA metabolism, is a hallmark of the environmental stress response (López-Maury et al.,
2008). In contrast, fewer enriched GO categories were identified among the heat responsive
DEGs in the 2019 and 2021 HR purebred crosses (Fig. 4.6b, S4.7), and more interestingly, the
enrichments did not highlight stress response pathways like their CR x CR (2019) or hybrid
(2021) counterparts, but cellular homeostatic processes instead (Fig. 4.6b).
The lack of an apparent stress response in HR purebreds does not appear to be due to an
inability to detect differential expression as a result of transcriptional front-loading, or higher
baselines expression of stress response genes (Barshis et al., 2013) We tested for front-loading
and found that comparatively fewer genes were front-loaded in HR x HR larvae and none of
those were annotated as stress response genes (Table S4.5). Among the annotated genes that were
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front-loaded in CR x CR larvae, two were related to protein ubiquitination (Ube2g1 and
ZNF598, Table S4), which may indicate constitutive expression of stress response pathways.
Additionally, HR purebreds had a more robust expression response to thermal stress, exhibiting
lower baseline expression, but achieving the same magnitude of overall transcriptional plasticity
in response to thermal stress as 2019 CR x CR larvae (Fig. 4.6c). In 2021 HR x HR and CR x HR
larvae showed similarly elevated levels of baseline expression in comparison to HR x CR hybrid
(Fig. S4.11). Taken together, this suggests the 2019 HR x HR larvae may be more resistant to
thermal stress not because they were pre-conditioned for stressful conditions, but because they
were able to strongly and rapidly acclimate their physiology, possibly as a result of having more
energy reserves to devote towards a stress response (Fig. 4.3). Such a robust response may also
be followed by a rapid return to baseline expression, or transcriptomic resilience (Rivera et al.,
2021), when stress abates, although additional time-course data are needed to test this
hypothesis.
4.5.3 Conserved transcriptomic signatures of population origin and response to treatment
In addition to survival and gamete lipid content, global gene expression profiles of
aposymbiotic O. faveolata larvae also revealed a strong signature of reef origin. The 2019
samples were organized into 3 distinct clusters based on origin along PC2, while origin was the
predominant driver of clustering in the 2021 samples (Fig. 4.4). Treatment appeared to be a
weaker driver in both datasets, clustering samples along PC3 in 2019 and PC2 in 2021 (Fig.
S4.4, 4.4b). Despite a difference in developmental age of the two larval cohorts at the time of
sampling (7 vs. 9 days post fertilization for 2019 vs. 2021), a WGCNA meta-analysis identified
highly conserved gene modules significantly correlated with larval origin although the
magnitude and direction of expression patterns was not always conserved (Fig. 4.5). Corals
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exhibit waves of transcription during early development consistent with zygotic genome
activation and degradation of maternal transcripts (Chille et al., 2021; Hayward et al., 2011).
Differences in the magnitude and direction of select modules between the 2019 and 2021
datasets (purple and magenta, Fig. 4.5) may reflect these temporal transcriptional waves,
although a more thorough time course is needed to test this hypothesis. Nevertheless, evidence of
such strong module preservation implies the existence of a core group of origin-specific genes
that have a lasting effect throughout the organisms’ development and these modules may be
linked to baseline differences in larval physiology. However, little information on the functional
implications of these gene sets was retrieved from the GO enrichment analysis, which may be
attributable to small module size and/or an insufficient number of annotated genes. This could be
a worthwhile investigation for future studies as annotations improve and better enrichment
methods become available.
WGCNA meta-analysis also identified highly conserved gene modules that were
significantly associated with temperature treatment. Similar to the origin-specific modules, the
majority of these treatment modules lacked significant functional enrichment. For the two
modules that did have sufficient enrichment, biological processes including immune response,
defense response, and response to external stimulus were enriched in genes associated with the
black module, which were up-regulated in heat (Fig. S4.5), while amide and peptide metabolic
and biosynthetic processes were enriched in genes associated with the cyan module that were
downregulated in heat (Fig. S4.6). Therefore, upregulation of genes involved in stress response
pathways and downregulation of metabolic processes related to growth and development in heat-
treated larvae aligns with the cellular stress and cellular homeostasis response profiles identified
90
in corals (and other marine and terrestrial organisms) during short- to medium-term
physiological stress (Kenkel et al., 2014; Kültz, 2005).
4.5.4 Implications for adaptive management
The documented resilience of adult corals to recurrent heat stress and bleaching in
environments with high and variable temperatures may come with highly consequential trade-
offs. In this study, we show that larvae from a site that routinely experiences and recovers from
heat-induced bleaching, significantly underperformed relative to larvae from a cooler, less
variable reef site. The unexpected outcome that more thermally tolerant O. faveolata produced
poorer quality offspring challenges the prevailing paradigm that breeding vulnerable populations
with thermally tolerant individuals can contribute to genetic rescue (Bay et al., 2017), which also
serves as the theoretical justification for selective breeding approaches (Drury et al., 2022).
Moreover, natural adaptive capacity is likely already impaired as CR x CR larvae and CR x HR
hybrids exhibited greater mortality risk even under ambient conditions (Fig. 4.2). These findings
also align with the long-term pattern of recruitment failure in Caribbean coral (Hughes & Tanner,
2000; Williams et al., 2008)
4.6 Supplementary Materials
Orbicella faveolata reference transcriptome assembly and annotation
Replicate fragments from seven coral colonies representing three inshore and four
offshore host genotypes (Manzello et al. 2019) were collected in July 2017 and subjected to a
short and moderate-duration thermal stress experiment (Table S1). Briefly, samples were
acclimated for seven days to control conditions (30°C) in Experimental Reef Lab (ERL) aquaria
followed by a 7-day temperature ramp for fragments assigned to thermal stress treatments to
reach the moderate (32°C) and short (33°C) targets. Fragments in the short-duration treatment
91
were sacrificed by snap-freezing in liquid nitrogen after 5 days at the target temperature and after
31 days in the moderate-duration treatment. Total RNA was extracted using the RNAqueous kit
(AM1912, Life Technologies) and sent on dry ice to the Duke Center for Genomic and
Computational Biology for library preparation and sequencing. Libraries were sequenced in a 75
bp PE run on the NextSeq500 High Output flow cell returning a total of 574 M raw reads.
Adapters and low-quality reads were removed in Trimmomatic v0.36 (phred33, quality score >
20, 4 bp sliding window; Bolger, Lohse, and Usadel 2014) yielding a total of 498 M high quality
reads used for a de novo assembly in Trinity v2.5.1 (Grabherr et al. 2011), yielding a total of
491,454 contigs (N50 = 1,192) in the metatranscriptome. The metatranscriptome was filtered
using BLASTx (e-value < 1e−5) searches against two coral host proteomes, and BLASTn (e-
value < 1e−5) searches against four Symbiodiniaceae genomes and transcriptomes (corals:
Acropora digitifera, (Shinzato et al. 2011); Orbicella faveolata, (Prada et al. 2016);
Symbiodiniaceae: Symbiodinium microadriaticum, (Aranda et al. 2016; Bayer et al. 2012),
Breviolum minutum (Shoguchi et al. 2013; Bayer et al. 2012), Cladocopium goreaui, (Liu et al.
2018), Durusdinium trenchii, (Dougan et al. 2022)). Transcripts were sorted based on their
lowest e-value and highest bit score, retaining only transcripts with best hits to host references.
The results from a GenBank nr (non-redundant) database (Sayers et al. 2019) search (e-value <
1e
−4
) were used to identify contigs matching metazoan proteomes to corroborate candidate host
transcripts. Transcripts with no hits to the nr database were also retained if they matched the
coral databases. A total of 87,440 contigs (N50 = 2,228, mean GC content: 42%) were assigned
to Orbicella faveolata. Host transcripts were annotated using BLASTx and BLASTp searches
against the uniprot database (e-value cut-off = 1e
−4
, Bateman et al. 2017) using the Trinotate
v3.1.1 pipeline (Bryant et al. 2017). Comparison against the Benchmarking Universal Single-
92
Copy Ortholog set (BUSCO) revealed that 91% of the core metazoan orthologs were represented
(Simão et al. 2015; Nishimura, Hara, and Kuraku 2017).
Figure S4. 1 Thermal profile of the 36 °C acute stress (AS) and 32 °C moderate stress (MS) experiments conducted in a) 2019
and b) 2021. Note that heat ramp for 2021 CR x HR and HR x CR larvae (MSHeat1) started on 9/3/2021 15:00 due to the 24-hr
developmental delay.
Figure S4. 2 Concentration (mean ± standard error of the mean [SEM]) of (a) different lipid classes (PL: phospholipid, TAG:
triacylglycerol, WAX: wax ester) and (b) total lipids standardized by individual larvae in 2021. Points are colored by origin and
shaped by treatment.
93
Figure S4. 3 Concentration (mean ± SEM) of total host soluble protein standardized by individual larvae in 2021. Points are
colored by origin and shaped by treatment.
Figure S4. 4 Principal component analysis (PCA) on rlog-transformed read counts in 2019 larval dataset displaying PC2 and
PC3. Points are colored by origin and shaped by treatment. The percentage variance explained by each PC is reflected on the axis
label.
94
Figure S4. 5 Hierarchical clustering of ontology terms enriched by genes included in the back module determined by WGCNA,
summarized by biological process (BP).
Figure S4. 6 Hierarchical clustering of ontology terms enriched by genes included in the cyan module determined by WGCNA,
summarized by biological process (BP).
Figure S4. 7 Discriminant analysis of principal components (DAPC) on variance stabilized data (VSD) in a) 2019 and b) 2021
larval datasets. Points are colored by origin and shaped by treatment. The percentage variance explained by each linear
discriminant (LD) is reflected on the axis label.
95
Figure S4. 8 Hierarchical clustering of ontology terms enriched by genes up-regulated (red) or down-regulated (blue) in heat-
treated 2021 HR x HR larvae compared to their untreated control, summarized by biological process (BP). Font size indicates
level of statistical significance (FDR-corrected). Term names are preceded by fractions indicating the number of individual genes
within each term differentially regulated with respect to treatment (unadjusted p < 0.05).
96
Figure S4. 9 Hierarchical clustering of ontology terms enriched by genes up-regulated (red) or down-regulated (blue) in heat-
treated 2021 CR x HR larvae compared to their untreated control, summarized by biological process (BP). Font size indicates
level of statistical significance (FDR-corrected). Term names are preceded by fractions indicating the number of individual genes
within each term differentially regulated with respect to treatment (unadjusted p < 0.05).
97
Figure S4. 10 Hierarchical clustering of ontology terms enriched by genes up-regulated (red) or down-regulated (blue) in heat-
treated 2021 HR x CR larvae compared to their untreated control, summarized by biological process (BP). Font size indicates
level of statistical significance (FDR-corrected). Term names are preceded by fractions indicating the number of individual genes
within each term differentially regulated with respect to treatment (unadjusted p < 0.05).
98
Figure S4. 11 Density plots showing distribution of global expression across samples from the three origins along the temperature
responsive axis (LD2, Figure S4. 7) based on discriminant analysis of principal components (DAPC) performed on variance
stabilized data (VSD) grouped by treatment and origin.
99
CHAPTER 5. CONCLUSION
As our climate continues to change, it is becoming increasingly imperative to understand
the evolutionary and ecological factors that drive variation in thermal tolerance among local
coral populations. Additionally, predicting the response of their offspring to future bleaching
events is equally, if not more crucial as the persistence of coral reef ecosystems is heavily
dependent on the resistance and resilience of many generations to come. My thesis as a whole
provides a nuanced perspective on the heritability of fitness-related traits across generations in
locally adapted and/or acclimated populations in two coral species. My work also uncovers
limitations on the easily simplified belief that tolerant parents will produce tolerant offspring.
With the frequency of marine heatwaves projected to increase in the upcoming decades (IPCC
2021, (Jentsch et al., 2007), it is possible that once tolerant populations (such as the FL Keys
inshore populations examined in this thesis) might lose their competitive edge when
environmental conditions become too extreme to stay below their thermal maximum, or suffer
from severe growth or reproductive trade-offs to the point where they are no longer viable. My
research highlights the need to frequently re-evaluate the dynamics of our current study systems
and hopefully provides future scholars a solid framework with which to achieve this goal.
One promising avenue of future research stemming from this dissertation would be to
investigate the role of algal symbionts in determining holobiont thermal performance. O.
faveolata is an ideal system to experiment with because it naturally associates with a diverse
Symbiodiniaceae community including the four most common symbiotic genera: Symbiodinium,
Breviolum, Cladocopium, and Durusdinium (Manzello et al., 2019). In addition, symbionts are
acquired horizontally from the environment, providing opportunities for introducing different
combinations of symbiont taxa in unique proportions. Given the pronounced host effects
100
observed in Chapter 4, examining how larval physiological and performance under heat stress is
further modulated by symbionts in later life stages is the next critical step toward a more accurate
understanding of their true thermal tolerance and their adaptive and/or acclimation potential
under changing environmental conditions. I experimented with infecting both aposymbiotic
larvae and recruits with Breviolum minutum, Durusdinium trenchii, and a combination of the two
species following the 2021 heat stress experiments (Chapter 4.3.2). Both species were able to
establish symbiosis with O. faveolata larvae, but the establishment was unstable and disappeared
after ~24 hrs. My recommendation is to infect aposymbiotic recruits and test their performance
under heat stress after symbiosis is established.
101
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APPENDICES
APPENDIX A: 2bRAD P ARENTAGE ANALYSIS OF PORITES ASTREOIDES LARVAE
A1. Background
Maternal effects have long been recognized as an important determinant of offspring
phenotype in terrestrial ecosystems and parallel investigation in marine systems burgeoned in the
past decade (D. J. Marshall et al., 2008). While both sexes contribute genetic material, mothers
typically invest more in the offspring by producing bigger gametes, which usually correlates
positively with nutritional storage and the level of epigenetic control via small non-coding RNAs
(Harii et al., 2007; Torda et al., 2017). In brooding organisms, offspring might also be affected by
the maternal environment via hormone exposure (McCormick, 1999) and oxygen provisioning
(Naylor et al., 1999). Interestingly, despite the theorized greater maternal provisioning in
brooding species, maternal effects have only been directly examined in broadcast spawning
corals, likely because fertilization can be easily manipulated. For instance, variation in survival
rates of heat stressed Acropora millepora larvae was largely explained by dam (or maternal)
identity (G. B. Dixon et al., 2015). Strong maternal effects also underpinned larval settlement
success and juvenile survival in Acropora tenuis (Quigley et al., 2016).
The first goal of this study was to further investigate the significant parental effects
observed in Chapters 2 and 3 by focusing on the contribution of maternal genetics and
environment on larval physiology in Porites astreoides. Although P. astreoides are
hermaphroditic (with exceptions, see (Chornesky & Peters, 1987)), colonies that brood larvae act
as mothers regardless of whether they contribute to the paternity of other nearby broods. To
separate maternal from paternal components, I aimed to have the same maternal colony fertilized
by different paternal colonies by fragmenting colonies and reciprocally transplanting replicate
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halves across two reef environments. This creative approach allowed me to quantify maternal
effects in isolation for the first time in a Caribbean species, and even more broadly among
Caribbean brooding corals.
Another goal was to gain a better understanding of the reproductive biology in Porites
astreoides. Our current knowledge suggests that P . astreoides is capable of self-fertilization and
the average inbreeding rate for wild P . astreoides populations calculated using random amplified
polymorphic DNA (RAPD) was reported to be 34% (Gleason et al., 2001). Interestingly, this
appears to be a fairly binary trait, as evidenced by high selfing rates (>80%) in some colonies
and extremely low rates (~0%) in others (Brazeau et al., 1998). Nevertheless, the RAPD assay
used in both prior studies is limited in that it relies on fingerprint-like banding patterns to
distinguish individuals rather than true allele calls. The development of modern next generation
sequencing technology calls for a new perspective on this major knowledge gap. A more recent
microsatellite-based parentage analysis conducted on another brooding coral species,
Seriatopora hystrix, found that the same maternal colony could be fertilized by multiple paternal
sources, resulting in varied offspring genotypes within the same brood (Warner et al., 2016). To
determine the level of selfing and identify the potential for multiple paternity, I extracted DNA
from single P . astreoides larva from 9 x 2 maternal colony pairs and used the 2bRAD method
(Wang et al., 2012) to generate high quality SNPs to determine larval parentage and genealogy
with greater accuracy.
A2. Methods
A2.1 Field collections, reciprocal transplant, and spawning
A total of 50 adult Porites astreoides colonies with surface area >250 cm
2
were collected
from an inshore site (n = 25, Summerland Shoals Patch: 24° 36.346′ N, 81° 25.742′ W) and an
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offshore site (n = 25, Cudjoe Ledges: 24° 32.304′ N, 81° 27.203′ W) from depth 2-4m on Sept
14, 2021 under Permit #FKNMS-2020-189. Colonies were wrapped in wet bubble wraps and
transported in coolers to Mote Marine Laboratory’s Elizabeth Moore international center for
coral reef research and restoration. All colonies were split in half using a tile saw, resulting in a
total of 100 coral fragments. To track the identity of each fragment, inshore fragments were
labeled 1-50 and offshore fragments were labeled 51-100 using cow tags epoxied to the bottom,
with two fragments from the same family labeled consecutively. All fragments were returned to
their native reef sites on Sept 16, 2021 to allow for recovery. Specifically, fragments were affixed
to the substrate with marine epoxy (All-Fix, USA) in a 15 m x 15 m radius and their associated
tags were attached to the substrate adjacent to the fragments using 3’’ square masonry nails. Two
HOBO temperature loggers were deployed at each site to track temperature profiles.
P . astreoides release male gametes around the full moon which are taken up by
neighboring female/hermaphrodite coral, and larvae are released around the new moon in each
lunar cycle (Chornesky & Peters, 1987; McGuire, 1998). In order to catch planulation in May
2022 (one of the peak release months), half of the fragments were reciprocally transplanted
between the two sites about two months in advance, on Mar 17, 2022, to capture two prospective
gametogenesis and fertilization cycles. Even numbers from 1-50 were transplanted to the
offshore site and from 51-100 were transplanted to the inshore site. To minimize the impacts on
the seafloor and ensure all fragments remained close to cross-fertilize, newly transplanted
fragments were affixed to the same spot as the original fragments. Three days before the new
moon, on May 27, 2022, all fragments (along with HOBO loggers) were retrieved from the two
sites and transported to Mote Marine Laboratory, where fragments were kept in 4 raceways
within the Climate and Acidification Ocean Simulator (CAOS) system at 27.7 ˚C, consistent with
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the ambient field conditions. Colonies were placed in flow-through larval collection devices
every night before sunset for 3 consecutive nights following (Kuffner et al., 2006). A total of 3
inshore pairs (# 3 + #4, #21 + #22, #49 + #50) and 5 offshore pairs (#53 + #54, #61 + #62, #71 +
#72, #79 +# 80, #91 + #92) had sufficient planulation to be included in the subsequent
physiological assays and genotyping. After the spawning period, all adult tissues from each
outplant site were sampled for genotyping.
A2.2 Physiological trait measures
For each parent fragment, ten larvae were aliquoted into a 6-well petri dish and
immediately photographed using a stereomicroscope. Assuming that coral larvae have an
elliptical shape, volume was calculated based on the length and width of the ellipse measured in
ImageJ (Schneider et al., 2012). Photographed larvae were then transferred to individual PCR
strip tubes for individual genotyping. Excess seawater was removed and tubes were stored at -80
˚C until further processed.
An additional three groups of ten larvae per parent fragment were sampled and stored at -
20 ˚C until further processing for symbiont density, chlorophyll a concentration, and protein
content. The same protocols were used as detailed in Chapter 3.3.3 Physiological Assays. Traits
were normalized by larval volume as the average larval size across different colonies varied (see
Fig. A1).
A2.3 DNA extraction, 2bRAD library preparation, and sequencing
Adult DNA was extracted using Wayne’s DNA extraction method (K. Wilson et al., 2002)
followed by a Zymo column-based clean-up procedure (Zymo Genomic DNA Clean &
Concentrator-10, Zymo Research, USA). Individual larval DNA was extracted using a
customized proteinase K digest and sodium acetate precipitation protocol without additional
column-based clean-up due to low concentrations. Reduced representation 2bRAD libraries were
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prepared separately for adult and larval samples using a hybrid protocol described in
https://github.com/z0on/2bRAD_denovo, targeting
$
%
of BcgI sites. Libraries were sequenced on
the NextSeq 2000 in 2022 by the USC Norris Comprehensive Cancer Center Molecular
Genomics core. The average sequencing depth per sample was 0.8 M reads.
A2.4 Bioinformatics pipelines
Downstream bioinformatic processing was performed on USC’s Center for Advanced
Research Computing (CARC) following protocols described in
https://github.com/z0on/2bRAD_denovo. Reads were first de-multiplexed based on internal
ligation adaptor barcodes to separate individual samples from sample pools, followed by adaptor
trimming and PCR duplicate removal using a custom perl script. Reads were quality filtered to
retain 99% accurate base calls over 100% of the read using the fastx-toolkit and those containing
adaptor sequences were removed. High quality reads were then competitively mapped to a
combined P . astreoides host genome (Wong & Putnam, 2022) and symbiont Symbiodinium
microadriaticum (ITS2 type A1) reference genome (Aranda et al., 2016) using Bowtie2
(Langmead & Salzberg, 2012). Reads mapped to the host genome were subset from reads
mapped to the symbiont genome for subsequent analysis.
ANGSD 0.933 (Korneliussen et al., 2014) was used to determine quality filtering
thresholds based on read depth, coverage, and SNP quality. A total of 261 adult and larval
samples were retained after removing 7 low coverage samples (defined as <20% of sites having
at least 5x coverage). Genotype likelihoods were estimated to retain high confidence SNPs (-
uniqueOnly 1 -removebads 1 -minMapQ 20 -minQ 25 -minInd 248 -snp_pval 1e
-5
) with at least
2x coverage in at least 95% of samples and a minimum minor allele frequency of 0.05.
Additional filters were incorporated to remove sites with either extremely high depth of coverage
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(10x number of samples) or strongly deviating from Hardy-Weinberg equilibrium (HWE) (p <
1e
-5
). An Identity by State (IBS) matrix and a covariance matrix were generated based on 88 high
quality SNPs across all samples. Dendrograms of the IBS matrix were generated for a larval
family with its putative parents based on transplant location, using the pvclust function (Suzuki
& Shimodaira, 2006) to perform hierarchical clustering with the average method and euclidean
distance. The approximately unbiased (AU) p-value threshold was determined between offspring
and the known maternal colony. Paternity can be identified at the first node up from any
offspring given that the node is above the AU p-value threshold and that only one adult is at that
node (Miller-Crews et al. 2021).
A2.5 Statistical analyses
All four physiological traits for newly released larvae were modeled as a function of
origin (levels: inshore, offshore) and transplant site (levels: inshore, offshore), including a
random effect of larval family (~maternal colony) using the lme4 package (Nakagawa &
Schielzeth, 2013). Models were evaluated for normality and homoscedasticity. Larval volume
and chlorophyll a concentration were log-transformed to meet the assumption of normality.
A3. Results & Discussion
Overall, larval traits varied depending on which family they are released from (Figs. A2-
5). No significant origin or transplant effects were found for the majority of the traits, except that
larvae from an offshore-origin maternal coral contained more protein (t = 3.15, df = 6, p < 0.05)
than those from an inshore-origin maternal coral (Fig. A5). This result echoes the main
observation from Chapter 2 where majority of the trait variation in daily released cohorts of P .
astreoides larvae was driven by family. The significant origin effect on larval volume, symbiont
density, and chlorophyll a identified in Chapter 2 was absent here, which could be explained by
the different standardization methods used in the two projects (per larva vs. per mm
3
larvae).
127
Given the substantial differences in larval volume (i.e. family 62 was significantly smaller and
family 72 was significantly larger than the rest of the families, Fig. A2), volume standardization
appears to be a more appropriate normalization for this dataset. There was also an absence of
transplant effects for all traits, possibly due to the relatively short time period spent in the
transplanted site (~2 months), although there appears to be a trend for inshore-transplanted
offshore colonies to produce larvae with higher symbiont cell densities (Fig. A3). This could be
due to the lack of a strong temperature difference between the two sites during the transplant
period (Fig. A1). One factor that could improve the statistical power of this analysis is by
increasing the number of larval family pairs. Even though we had a quite large collection of adult
colonies, only a small fraction of pairs ended up producing enough for physiological analyses
and genotyping. Fragmentation might have jeopardized the reproductive output, although recent
work suggests reproduction is not a function of size but rather of overall colony age (Rapuano et
al. 2023). Alternatively, reproduction occurs monthly in this species with some annual variation
(McGuire 1998) and May 2023 could have been a lean reproductive month.
Two technical replicates for adult fragment #87 paired at a secondary node with an AU
value of 100 (Fig. A6). Such high fidelity was replicated in other known biological pairs,
including #31 and #32, #17 and #18, #69 and #70, as well as #91 and #92. Empirically, an AU
value higher than 80 is considered an acceptable threshold for determining a successful node
(Miller-Crews et al., 2021). Applying that threshold, the majority of the good clusters were
composed of fragments from the same origin, indicating highly similar genetic profiles or
possibly clones. However, outliers did exist as evident by clusterings of inshore and offshore
individuals that should theoretically be separated due to the geographical barrier, including #78
and #11 (AU = 87), #95 and #44 & #43 (AU = 91).
128
Using the same method to determine the parentage of single larvae produced mixed
results. Instead of clustering closely with its maternal colony #3, larva 6 released from inshore
colony #3 shared the first closest node with AU above 80 with an offshore colony #88 (Fig. A7).
It is possible that #88 fertilized #3 after being transplanted to the inshore site, but the separation
between the larva and its maternal colony made it hard to interpret. In comparison, larva 1
released from colony #4 correctly clustered with its maternal colony and clustered closely with
two other inshore colonies #46 and #24 (Fig. A8). Based on the adult lineage assignments, #23,
#24, #45, and #46 are potential clones (Fig. A6). Therefore, larva 4-1 was likely a product of
high level of inbreeding or asexual reproduction.
In summary, using the IBS matrix to determine the parentage of individual larva might
not be the best approach as results are inconclusive and not always align with the biological
reality. One next step would be to build IBS matrices on only one larval family and potential
parents and enforcing more stringent SNPs filtering criteria could yield more accurate results.
Meanwhile, different parentage assignment methods need to be explored in case there is a better
method that fits our data and study design. In addition, symbiont community composition also
contributes to maternal effects as it is vertically transmitted. P . astreoides in the lower Florida
Keys were shown to exclusively associate with one Symbiodinium type (formerly clade A4/A4a)
but different haplotypes do exist (Kenkel et al., 2013).
129
Figure A1 Continuous temperature profile of inshore and offshore sites during recovery and transplant periods. The gray shaded
area highlights the transplant period.
Figure A2 Size of individual larvae (mean ± SEM) post release across different families (note that sequential numbers indicate
the same maternal colony transplanted to two different reef sites). Data points were colored by origin and shaped by transplant
site.
130
Figure A3 Symbiont cell density (mean ± SEM) of larvae across different families (note that sequential numbers indicate the
same maternal colony transplanted to two different reef sites). Data points were colored by origin and shaped by transplant site.
Figure A4 Chlorophyll a concentration (mean ± SEM) of larvae across different families (note that sequential numbers indicate
the same maternal colony transplanted to two different reef sites). Data points were colored by origin and shaped by transplant
site.
131
Figure A5 Protein content (mean ± SEM) of larvae across different families (note that sequential numbers indicate the same
maternal colony transplanted to two different reef sites). Data points were colored by origin and shaped by transplant site.
Figure A6 IBS matrix dendrogram of all adult fragments. Each node is labeled with AU (Approximately Unbiased, red) p-value
and BP (Bootstrap Probability, green) value.
132
Figure A7 IBS matrix dendrogram of larva #6 from family 3 (“3_6”) with its known mother (“3”) (both highlighted in blue) and
potential fathers that were transplanted to the inshore site. Each node is labeled with AU (Approximately Unbiased, red) p-value
and BP (Bootstrap Probability, green) value.
Figure A8 IBS matrix dendrogram of larva #1 from family 4 (“4_1”) with its known mother (“4”) (both highlighted in blue) and
potential fathers that were transplanted to the inshore site. Each node is labeled with AU (Approximately Unbiased, red) p-value
and BP (Bootstrap Probability, green) value.
Abstract (if available)
Abstract
Driven by increasing sea surface temperature, coral bleaching events have become more severe and frequent across the globe since the early 1980s. Yet not all coral population responses are equal. Certain populations can outperform their conspecifics under similar heat stress by adapting and/or acclimatizing to local thermal conditions. In the Florida Keys, populations closer to shore are generally more resilient to heat stress than neighboring offshore populations potentially due to naturally higher and more variable temperature regimes that are characteristic of the inshore reef environment. Most current studies on local adaptation and/or acclimatization focus on adults, but the implications of parental adaptation and/or acclimatization on offspring life stages and even generations remain relatively unexplored. To address this important knowledge gap, this dissertation aims to investigate the heritability of population-specific thermal tolerance traits across generations in Porites astreoides (Chapters 2 and 3) and Orbicella faveolata (Chapter 4), two ecologically significant coral species in Florida and the wider Caribbean. Ultimately, understanding the genetic and physiological contribution to fitness- related phenotypic differences among spatially distinct populations will help us predict how corals will respond to selection under future global warming scenarios and determine the best restoration as well as management strategies.
The first study was designed to understand family effects on larval physiology in Porites astreoides. We found that the majority of variation in baseline physiological and thermal tolerance traits was explained by larval family, as opposed to previously implicated factors such as day of release and reef origin. This study inspired the next chapter which further investigated the extent of familial effects in multiple life stages by performing similar assays on adults, larvae, and juvenile recruits. High broad sense heritability was identified for multiple physiological traits among the different life stages, highlighting potentially significant genetic underpinnings and the adaptive potential of local populations to environmental change. The final chapter investigated whether larvae produced by a more thermally tolerant inshore adult Orbicella faveolata population outperformed those originating from less thermally tolerant offshore-origin parents. Contrary to our expectations, inshore O. faveolata larvae were less heat tolerant than the offshore larvae on both physiological and molecular levels, which may be driven by reproductive trade-offs caused by the prior history of thermal stress that the inshore adults experienced. This finding sheds light on the negative consequences of bleaching events on coral reproductive output and presents valuable perspectives on implementing selective breeding methods to reseed declining reefs.
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Asset Metadata
Creator
Zhang, Yingqi
(author)
Core Title
Transgenerational inheritance of thermal tolerance in two coral species in the Florida Keys
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Biology (Marine Biology and Biological Oceanography)
Degree Conferral Date
2023-08
Publication Date
07/19/2023
Defense Date
05/24/2023
Publisher
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climate change,Corals,larvae,OAI-PMH Harvest,omics,Physiology,thermal tolerance
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Kenkel, Carly (
committee chair
), Bottjer, David (
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), Edmands, Suzanne (
committee member
), Gracey, Andrew (
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)
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yingqizh@usc.edu,yzhang2@colgate.edu
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