THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE

DEPARTMENT OF BIOLOGY

DIVERSITY OF SYMBIOTIC DINOFLAGELLATES IN NORTHERN LATITUDE CORALS

MADISON TAYLOR SPRING 2018

A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biology with honors in Biology

Reviewed and approved* by the following:

Todd LaJeunesse Associate Professor of Biology Thesis Supervisor

James Marden Professor of Biology and Associate Director, Huck Institutes of the Life Sciences Honors Adviser

* Signatures are on file in the Schreyer Honors College. i

Abstract

Reef building corals are widespread throughout the tropics and subtropics; however,

some species exist at high temperate latitudes. Temperate conditions such as high seasonality in temperature and turbidity impact coral physiology and likely affect the endosymbiotic dinoflagellates (Symbiodinium) that live in their host tissues. The identities of endosymbionts from corals at extreme northern latitudes are poorly characterized, therefore we used molecular

genetic approaches to examine Symbiodinium collected across a broad host taxonomic range

around Honshu Island, Japan. We observed patterns of high specificity between coral and

symbiont as well as a few generalist symbionts. In addition, our data provide evidence that many

high latitude Symbiodinium species are new species well adapted to the seasonal fluctuations of

temperate environments like Japan. These findings suggest a long-term co-evolution of

symbionts that are highly adapted to the intracellular environment of particular coral species at

high latitudes. This research improves our understanding of Symbiodinium diversity in high

latitude coral communities and begins to elucidate to what extent this diversity is unique relative

to the symbionts that thrive in coral populations at lower latitudes. Moreover, characterizing

these symbioses is important for the future of coral reef conservation by establishing a baseline

of biodiversity at northern latitudes, as corals may find protection from the effects of climate

change by colonizing at northern latitudes like Japan.

ii

Table of Contents

List of Figures ...... iii

List of Tables ...... iv

Acknowledgements ...... v

Introduction ...... 1

The significance of coral-algal symbioses ...... 1 Defining Species of Symbiodinium ...... 3 Patterns of Symbiodinium evolution ...... 6 Symbiodinium distribution and ecology ...... 7 High vs. low latitude: the importance of studying symbioses at extremes ...... 8 Investigating the diversity of Symbiodinium in northern latitude corals ...... 11

Materials and Methods ...... 13

Sample collection and preservation ...... 13 DNA extraction and Polymerase Chain Reaction (PCR) ...... 14 Denaturing Gradient Gel Electrophoresis (DGGE) and fingerprinting ...... 15 Sanger sequencing and phylogenetic analysis ...... 16 Coral ...... 17

Results...... 19

Symbiont diversity quantified with genetic evidence ...... 19 DGGE analysis of Symbiodinium common to Acropora ...... 19 Ecological distribution of symbiont diversity...... 21 Host diversity and phylogeny ...... 24

Discussion ...... 26

High latitude lineages of Symbiodinium ...... 26 Ecological niche and host specificity ...... 28 Generalist species ...... 29 Effect of climate change on high latitude coral symbioses ...... 30 Limitations and further research ...... 31

Supplemental Table ...... 33

Supplemental Figure ...... 36

Bibliography ...... 37 iii

List of Figures

Figure 1. The host-symbiont relationship between coral and Symbiodinium ...... 3

Figure 2. Collection sites on Honshu Island, Japan ...... 14

Figure 3. Phylogeny of Symbiodinium collected from Japan based on the ribosomal marker LSU20

Figure 4. Phylogeny of Symbiodinium collected from Japan based on the mitochondrial marker cob ...... 21

Figure 5. Phylogeny of Symbiodinium collected from Japan based on the chloroplast marker cp23s ...... 22

Figure 6. Phylogeny of coral host families and their associated symbiont types ...... 25

Figure 7. DGGE ITS2 results containing coral hosts Acropora and Montipora ...... 36

iv

List of Tables

Table 1. List of sampled corals from Japan, their , species, and the number of specimens collected ...... 33

v

Acknowledgements

“We just don’t get to be competent human beings without a lot of different investments from

others” –Mister Rogers

I have had so many incredible people supporting me through the thesis process.

First, I’d like to express my gratitude to the entire LaJeunesse Lab Team for three years

of incredible opportunities to learn what hands-on science looks and feels like. Todd,

thank you for guiding me from the basics to where I am now, with a lot of memorable

conversations in between. To Daniel, for being a smart friend and fellow marine scientist.

Thanks to Kira, Hannah, and Allison for being boss women in science. I couldn’t have

had better role models to experiment and do science with. Kira, thank you for being an

inspiration both in the lab and out on the trails. Hannah, thanks for sharing office space

with me all of the Fall semester and for always pulling my samples from the PCR

machine. Last but not least, I’d like to express my endless gratitude to Allison for her

patience, guidance, and mentorship over the years. Thank you for fielding my many

questions, the FaceTime calls, shared laughs and honest talks about where we’re both

going next.

Thank you also to Dr. Marden for being a fantastic resource and honors advisor.

Our trip to Costa Rica will forever be a huge influence on my future career; thank you for

showing me how challenging yet rewarding fieldwork can be.

Lastly, thank you to the Schreyer Honors College for academic support, thesis

boot camps, and the opportunity to culminate my research in a body of work that I am

proud of. 1

Introduction

The significance of coral-algal symbioses

Coral ecosystems are home to a wide array of marine organisms, earning their name of the “rainforests of the sea”. They provide a habitat and nutrition for fish, invertebrates, marine mammals, and plants, among other organisms. Coral reefs are also critical to aspects of human society: tourism, coastal and island community cultures, food security, and the conservation of biodiversity. Coral colonies are the foundation of reef ecosystems and if harmed, numerous other

species and aspects of human society suffer as well.

Currently, coral populations are threatened by environmental degradation. Climate

change, ocean warming and acidification, pollution, and unsustainable resource management are

putting stress on these and their symbiotic partners. Some of these threats cause massive

bleaching events and the eventual death of reefs. Therefore, there is a pressing need to study

corals, in order to inform future conservation practices.

Coral reef ecosystems are widespread across the planet. They are the dominate ecosystem

in shallow tropical marine environments, especially in western parts of the major oceans.

Environments in these regions are often characterized by warm and clear waters with few

nutrients. However, coral communities can also live in temperate, high latitude ecosystems

characterized by high seasonality in temperature, nutrients, and water turbidity.

Reef-building corals owe much of their existence to their mutualistic symbioses with

photosynthetic dinoflagellates belonging to the genus Symbiodinium (Coffroth & Santos, 2005). 2 Symbiodinium are unicellular micro-algae that commonly dominate the tissues of corals and other cnidarians including sea anemones, jellyfish, and sponges (Rowan, 1998). The mutualistic association between a heterotrophic invertebrate (coral ) and autotroph (single-celled algal symbiont) underlies coral productivity and drives the formation of coral reefs. The foundation of this obligate mutualism is nutrition. Symbiodinium provide photosynthetic products, mainly glycerol and glucose, to aid the host in growth, reproduction, and metabolic processes (Buck,

Rosenthal, & Saint-Paul, 2002). It is estimated that 65-90% of a coral’s energy comes from their symbionts which provide energy for coral reproduction and growth and calcification

(Houlbrèque & Ferrier-Pagès, 2009). In return, the symbionts receive key limited nutrients such as phosphorous, carbon, and nitrogen from their host corals as well as a protected intracellular habitat (Davy, Allemand, & Weis, 2012). Symbiodinium grow in extreme densities ranging from hundreds of thousands to millions per square centimeter (Stimson, Sakai, & Sembali, 2002).

Most hosts are located in shallow water, as the photosynthetic efficiency of many symbionts increase with greater access to the sun (Holland, Dawson, Crow, & Hofmann, n.d.).

3

Figure 1. The host-symbiont relationship between coral and Symbiodinium

a) The whole coral organism b) An individual coral polyp in dense association with Symbiodinium (Marine Genomics Unit of OIST) c) Light image of Symbiodinium cells in hospite (“Creative Commons light micrograph Image” by T. C. LaJeunesse licensed under CC BY-SA 4.0)

Defining Species of Symbiodinium

Within the tree of life, eukaryotes are unicellular or multicellular organisms with a

nucleus and other organelles enclosed inside a membrane. Symbiodinium, commonly referred to

as zooxanthellae, are classified as dinoflagellates, one of the largest groups of marine micro-

eukaryotes. Many are photosynthetic and most are free-living (Stoecker, 1999). Because they lack sufficient morphological traits, understanding the diversity, ecology, and evolution of these symbionts was difficult prior to the application of genetic methods.

There are several favored concepts in biology used to define species. Among these are

the Biological Species Concept and Morphological Species Concept. The Biological Species 4 Concept states that species are identified as a group of interbreeding natural populations that is

reproductively isolated from other groups. (Baker & Bradley, n.d.). The Biological Species

Concept cannot be assessed directly because there are no techniques available to manipulate

Symbiodinium into undergoing sexual recombination under controlled experimental conditions.

In terms of the Morphological Species Concept, the inherently small size and a lack of discernible features in the non-motile coccoid (spherical) life history of Symbiodinium development makes it difficult to distinguish between species. Therefore, morphological features of Symbiodinium are rarely useful in definitively identifying species types.

Symbiodinium research must turn to molecular genetics to investigate the existence of distinct species, and to identify the ecological, physiological, and biochemical differences between them (Todd C. LaJeunesse, 2001). At this time, a Phylogenetic or Molecular Species

Concept is most appropriate for making taxonomic distinctions. Fixed differences in DNA sequences of various genes in Symbiodinium indicate that phylogenetically distinct lineages are reproductively isolated, and therefore justify their classification as different species.

Symbiodinium encompasses numerous species lineages, many of which form symbiotic relationships with marine invertebrates (Blank & Trench, 1986). To date, nine arbitrarily defined

Clades, or groups, of Symbiodinium have been assigned, each with varying levels of species diversity (Coffroth & Santos, 2005). The earliest genetic data on the genus Symbiodnium were reported by Rowan & Powers (Rowan & Powers, 1992) , who, using nuclear small subunit ribosomal nr18s sequences, broadly defined three phylogenetic groupings A-C. Molecular techniques have since progressed to make use of the more variable large subunit ribosomal nr28s

“benchmark” sequences to lead to more specific classifications in nine Clades, A-I (Pochon,

Putnam, & Gates, 2014). Past studies have shown that corals and cnidarians in general associate 5 primarily with Symbiodinium from Clades A-D , and with members from F and G in extremely

rare cases (Todd C. LaJeunesse, 2001).

Sequence comparisons of nuclear ribosomal and chloroplast DNA identify hundreds of

different Symbiodinium lineages or types. Multiple genetic markers are currently used to delimit

evolutionarily distinct lineages (i.e. species) that exhibit differences in ecology and biogeography

(Finney et al., 2010; T. C. Lajeunesse, Parkinson, & Reimer, 2012). The assessment of symbiont diversity across a community of corals is often first investigated by analzying rDNA internal

transcribed spacer region 2 (ITS2), which is located between small and large subunit of rRNA.

The ITS2 region is widely used for categorizing diversity because it is easy to amplify and

usually has a high degree of variation between Symbiodinium types, or species (Todd C.

LaJeunesse, 2001). However, there are some instances in the literature where ITS2 sequences are

the same for two or more species of Symbiodinium (LaJeunesse, personal communication). In

these cases, additional markers are used to provide higher resolution of species boundaries.

Several genetic markers are currently used to study the diversity and ecology of coral

symbionts. These include large subunit nuclear rDNA (LSU), which enables the determination of

phylogenetic relatedness among closely and distantly related symbionts (T. LaJeunesse & Loh,

2003). LSU is described as a useful marker for species identification, especially when combined

with the resolving power of mitochondrial markers such as COX1 (Sonnenberg, Nolte, & Tautz,

2007).

Cp23s-rDNA is a “benchmark” chloroplast gene from the large subunit ribosomal DNA

(Pochon, Putnam, Burki, & Gates, 2012). It is regarded as a faster evolving target gene that

complements LSU sequence data and can address questions of small-scale, within-clade patterns

of specificity (Pochon et al., 2012). 6

Cytochrome oxidase B (cob) is a mitochondrial marker and protein-encoding gene. Cob

is a widely used genetic marker for dinoflagellates and can further inform phylogenetic analyses,

especially when combined with cp23s sequence data (Sampayo, Dove, & LaJeunesse, 2009).

PsbA minicircle (psbAncr) is a plastid marker that describes the non-coding region of

chloroplast genes. PsbAncr provides the highest level of phylogenetic differentiation. It distinguishes the most closely related Symbiodinium species and is able to resolve distinct genotypes within a species (Moore, Ferguson, Loh, Hoegh-Guldberg, & Carter, 2003).

The use of multiple markers in combination more accurately distinguishes between

species and provides insight into the ecological distributions and evolutionary history of

symbiotic relationships.

Patterns of Symbiodinium evolution

The evolutionary history of Symbiodinium is marked by recurrent episodes of adaptive

radiations, where a common ancestor undergoes diversification creating numerous species over

several million years or less. These adaptive radiations appear to coincide with major changes in

environment, which opens niche space to species adapted to the new environmental conditions

(Dolph Schluter, 2000) . Classic systems like Darwin’s finches and Anolis lizards have evolved

via adaptive radiation (Grant & Grant, 2008; Losos, 2009 as cited by Thornhill et al., 2014).

For dinoflagellate symbionts, different coral hosts present unique environments in which

to inhabit and proliferate, representing a strong selective filter leading to the evolution of new

species. Most species of Symbiodinium are not randomly distributed among coral hosts, and 7 previous studies have shown that members of the same host species typically harbor the same

Symbiodinium species (Coffroth & Santos, 2005; Thornhill et al., 2014). This suggests a dynamic interaction between host and symbiont, as well as local environments leading to the evolution of partner specificity. Different habitats promote different pairings of partners based on environmental factors like depth, irradiance, temperature gradients, and latitude.

Speciation via ecological selection is a major theme of Symbiodinium evolution. A study by Thornhill et al. (2014) examined the evolutionary relationships among ecologically dominant

Clade C Symbiodinium and their affinity to particular host taxa. Patterns of host association corresponded with certain types of Clade C Symbiodinium indicating that symbiont speciation is driven first by host specialization and then by regional isolation. Host habitat is considered the first axis of lineage diversification, as seen by gene combinations specific to intracellular environments and host characteristics (Thornhill et al., 2014). Beyond host associations, external habitat is a secondary driving force in Symbiodinium diversification. Factors such as depth, temperature, and geographic location drive the occurrence of symbiont types.

Symbiodinium distribution and ecology

The stability and specificity of the mutualism between Symbiodinium and host corals depend on the identity of both organisms and the environmental setting (Stat, Morris, & Gates,

2008). While most hosts exhibit extreme specificity for particular Symbiodinium species, some corals have the ability to form symbioses with more than one species or type of symbiont (T.

LaJeunesse & Loh, 2003). On the symbiont side, specificity can be a 1:1 species-species interaction in nature, meaning that one symbiont type associates with only one coral host. 8 Alternatively, specificity can also mean that one symbiont type associates with a monophyletic

(closely related) group. This is likely due to ecological specialization to a single host lineage

comprising of diverse but closely-related species (Thornhill et al., 2014).

Generalist symbiont species also exist. These symbionts have a broader ecological niche.

They are capable of establishing a symbiosis with phylogenetically diverse coral taxa, meaning

the coral hosts they associate with are not monophyletic.

One example of a previously identified generalist is S. goreaui, as described in Thornhill

et al. (2014). As a true host-generalist, S. goreaui associates with a number of host genera over a

broad geographic range. The ITS2 sequence characteristic of this species, “C1,” has been

identified across the western Atlantic Ocean and the Indo-Pacific region (Thornhill et al., 2014).

Another example of a host generalist Symbiodinium thermophilum, an ITS2 type from the C3 adaptive radiation. S. thermophilum is thermally tolerant, helping various species of coral in the

Persian/Arabian gulf survive some of the warmest sea surface temperatures on earth (Hume et al., 2015).

High vs. low latitude: the importance of studying symbioses at extremes

The distribution of a particular symbiont is dictated in part by environmental conditions such as temperature and light. Most research on the algal symbionts of corals has focused on low latitude regions where coral diversity is high, sea surface temperature is warm, and light availability is seasonably stable. However, given the increasing potential for the pole-ward migration of coral populations because of global warming, the study of high latitude coral populations is important now more than ever. Characterizing high latitude symbioses establishes 9 a baseline of biodiversity in these regions. Indeed, high latitude ocean habitats are experiencing

greater temperature disturbances and thus communities adapted to temperate climates may

exhibit greater vulnerability to continued climate change (Beger, Sommer, Harrison, Smith, &

Pandolfi, 2014). Furthermore, knowledge gained by the study of coral communities in high- latitude environments has the potential to make inferences about how coral populations respond to environmental change. There are a few examples in the literature that have investigated the ecology of high-latitude coral reef ecosystems. This project aims to provide a more complete

ecological and evolutionary picture of symbioses in high latitude coral communities.

LaJeunesse et al. (2008) reported on high latitude coral-algal symbioses from the Sea of

Cortez in the eastern Pacific Ocean. Many of the coral taxa living in the region harbored Clade C

Symbiodinium types consistent with findings from most other regions of the Pacific Ocean (T. C.

LaJeunesse et al., 2008). The corals in this area also showed high levels of host-symbiont specificity. This could be because coral diversity is less in the Sea of Cortez and environmental conditions are more variable, resulting in the evolution of specific and stable symbiont-host combinations.

Other studies on the diversity and ecology of Symbiodinium have been conducted around

Hawaii, another northern latitude site. Hawaii is geographically remote, making it a unique place to study host-symbiont pairings. Researchers found high specificity in this region with the majority of the hosts examined harboring Clade C symbionts (Todd C. LaJeunesse et al., 2004).

There was no clear generalist symbiont among these Hawaiian corals.

Researchers in South Korea conducted a study on Symbiodinium diversity on Jeju Island, close in proximity to Honshu Island, Japan (De Palmas et al., 2015). A high level of specificity was observed between host genera and Symbiodinium despite existing in a temperate 10 environment with large variability in seasonal temperature and light. Only a few symbiont

species variations were identified despite broad sampling efforts, suggesting high specificity at

these latitudes. According to DePalmas et al., marginal areas such as high-latitude ocean habitats

in Japan often represent the outer edges of a species’ range, a place where adaptive genetic and

phenotypic characteristics can potentially occur, thus explaining the apparent lack of diversity.

However, only one marker ITS2, was used, resulting in the potential to overlook genetic diversity

that becomes apparent when additional markers are applied.

Perhaps most informative is a study by Lien et al. and their investigation into

zooxanthellae and corals in the temperate region of Japan. The authors observed that most of the

corals sampled harbored Clade C Symbiodinium, and rarely did they observe symbioses

involving Clades D and F (Lien, Fukami, & Yamashita, 2013). Tropical corals also commonly

contain Clade C Symbiodinium, which is interesting because the environmental characteristics

between the two regions are vastly different. However, only one genetic marker, ITS2, was used,

and the sequence variation measured was not considered to be biologically important. The

hypothesis presented here is that the diversity of Clade C reported from temperate Japan is

genetically different, and most likely physiologically different, than the Clade C Symbiodinium

found in tropical and sub-tropical latitudes (Lien et al., 2013).

The repeating characteristics of high northern latitude sites – high specificity and Clade C

dominance – have been identified in a number of studies and potentially describe high latitude coral assemblages of Japan, though more investigation via genetics is needed. 11 Investigating the diversity of Symbiodinium in northern latitude corals

The core motivation for this project lies in the fact that the biological diversity of endosymbionts from corals at extreme northern latitudes is poorly characterized beyond one genetic marker. Corals species exhibit broad geographic distributions, existing at both high and low latitude reefs, yet little is known about whether these corals associate with identical endosymbionts across their distribution. Endosymbiont species are commonly zoned by depth, adapted to particular light levels, and influenced by geography. We investigate the nature of coral dinoflagellate symbioses at northern latitudes, specifically the temperate waters surrounding Japan. We seek to categorize the diversity of northern latitude symbionts and compare them to symbionts found at tropical and subtropical latitudes.

Marginal environments like Japan are predicted to experience greater disturbances with climate change than lower latitude reefs; therefore it is important to categorize symbiont diversity in these regions to inform the best methods of coral reef conservation (Beger et al.,

2014). Ultimately, northern latitude coral reef communities are poised for intense modification if the current environmental trends continue (Beger et al., 2014). Characterizing the nature of host- symbiont partnerships of corals in areas like Japan offers an important step toward understanding the capacity and limitations in the response of these animals to climate change.

In this study, we use molecular genetic approaches to examine Symbiodinium collected across a broad host taxonomic range living around Honshu Island, Japan. We hypothesize that while Clade C Symbiodinium are the dominant group of symbionts, thorough genetic analyses will show that the Clade is represented by numerous regionally endemic species lineages and most are likely to exhibit host-specific pairings. We also hypothesize that the high latitude environment has selected for symbionts species that do not occur at lower latitudes. 12 To investigate these hypotheses, we employ six different genetic markers in tandem, five used to describe Symbiodinium diversity and one used to describe host diversity, to distinguish phylogenetic relationships.

13 Materials and Methods

Sample collection and preservation

Symbiotic corals were collected from Northwest Pacific reefs from Honshu Island, Japan.

Collections were made at two locations along the South-Eastern coast of Japan including,

Kushimoto Marine Aquarium (33˚ 28’ 47.9” N 135˚ 44’ 44.6” E) and Kushimoto Turbid Bay

(33˚ 28' 31.77" N 135˚ 46' 16.79" E) (Fig. 2). Small skeletal fragments (2-3 cm) were sampled by

SCUBA using hammer and chisel at a depth of approximately 4-6m and then later preserved in

20% dimethylsulfoxide (DMSO) buffer, 0.25 mol L-1 EDTA, in sodium chloride-saturated water solution (Seutin, White, & Boag, n.d.). In an effort to survey a high diversity of coral and symbionts, we collected a total of a total of 86 samples, representing 36 families comprised of 54 species of (Supplemental Table 1).

14

Figure 2. Collection sites on Honshu Island, Japan Coral samples were acquired from reefs surrounding the Kushimoto Marine Park and Turbid Bay on Honshu Island, Japan. Latitude and longitude coordinates are shown to give context to northern latitude ocean habitats.

DNA extraction and Polymerase Chain Reaction (PCR)

Genomic DNA was extracted from preserved fragments using a modified Promega

Wizard DNA extraction protocol (T. LaJeunesse & Loh, 2003). Symbiont diversity was investigated by first targeting the multi-copy rDNA internal transcribed spacer region 2 (ITS2), which is located between small and large subunit of rRNA. Fixed sequence variation in the ITS2 region is often diagnostic for delimiting species of Symbiodinium and is therefore a useful first step in characterizing the diversity of Symbiodinium in a newly studied region. In addition, the 15 ITS2 rDNA has been used widely for categorizing Symbiodinium and thus allows for direct comparison of diversity over large geographic areas (Todd C. LaJeunesse, 2001).

Polymerase Chain Reaction (PCR) was used to amplify regions of interest in

Symbiodinium and coral host. PCR involves thermal cycling, repeated cycles of heating and cooling to anneal and replicate the DNA. The result is an exponential number of copies of DNA that can be used for further analysis. DNA polymerase and free nucleotides are also needed to catalyze the highly specific, enzyme-driven temperature dependent PCR reactions. Specific primer sets, or complementary DNA sequences to the target region of amplification, are used for each genetic marker.

The internal transcribed spacer region 2 (ITS2) was amplified using the primer set “ITS2 clamp” and “ITSintfor2ITS2” (T. Lajeunesse & Trench, 2000) with a touch-down thermal cycle described in LaJeunesse & Loh (2003). The purpose of touch-down PCR is to gradually lower the annealing temperature to promote specificity. A clamp refers to the long GC-rich chain of nucleotides attached to the end of either the reverse or forward primer but always the 5’ end. To assess success of the PCR reaction, gel electrophoresis was run on a 1% agarose gel and visualized under UV light to confirm bands of amplified DNA.

Denaturing Gradient Gel Electrophoresis (DGGE) and fingerprinting

Successfully amplified ITS2 sequences were prepared for Denaturing Gradient Gel

Electrophoresis (DGGE) analysis by combining 5 µl of DGGE dye with 20 µl of the PCR product. Samples were electrophoresed on denaturing gradient gels (45-80%) using a

CBScientific system (Del Mar, CA). The denaturing gradient of the gel allows small base pair 16 fragments of DNA to separate by sequence (T. Lajeunesse & Trench, 2000). A GCGC clamp is

also needed for DGGE to aid separation in the gel, as the long tail of high GC content makes it

very difficult for the amplified PCR product to dissociate. DGGE is useful because it allows for comparison of generated fingerprinting patterns to a known ladder, the extraction of dominant

ITS2 variant types for sequencing, and identifying instances of corals hosting more than one species of Symbiodinium simultaneously. Symbiodinium types included in the ladder are C21 and

C40 from Clade C and D1a from Clade D. Ultimately, DGGE provides insight into genotype and which samples contain identified and unidentified symbionts.

The prominent bands of each unique PCR-DGGE generated fingerprints were excised, re- amplified, and sequenced following the protocol described by LaJeunesse et al. (2003). The brightest and lowest bands were cut and sequenced in order to target the highest-copy number and most informative ITS2 sequence variant from each unique observed DGGE fingerprint.

Multiple bands were excised from one sample when there appeared to be codominant sequences present within a genome at the same relative intensity across samples (T. LaJeunesse & Loh,

2003). The identity of symbionts in each host coral were beginning to be deduced, however, further analysis through sequencing is necessary to determine true identity and evolutionary relationship to other Symbiodinium.

Sanger sequencing and phylogenetic analysis

Additional phylogenetic markers were applied to Symbiodinium to increase the resolution among putative species lineages identified using the ITS2 marker. Nuclear rDNA LSU (600-700

bp) and ITS2 (360 bp), chloroplast genes cp23s (670 bp) and psbAncr (536-601 bp), and 17 mitochondrial cob were used as symbiont markers. Previous studies have shown that the use of multiple genetic markers more completely reveals the identity of a symbiont species, for more accurate inferences about their ecology and evolution. A lineage-based approach, combining sequences of mitochondrial, chloroplast, plastid, ribosomal genes, and spacer regions can classify Symbiodinium into fundamental biological units (T. C. Lajeunesse et al., 2012; T. C.

Lajeunesse & Thornhill, 2011).

Samples were prepared for sequencing following the protocol outlined in LaJeunesse (T.

C. LaJeunesse, 2002) and the LSU region was amplified from each sample according to the primers and settings described in LaJeunesse & Loh (2003). Products were directly sequenced at the Pennsylvania University Genomics Core Facility on an Applied Biosciences sequencer

(Foster City, CA).

The analysis software Geneious version 6.1 (Kearse et al., 2012) was used to edit the symbiont DNA sequences and form alignments. Chromatograms were visually inspected for accuracy across sequences. The aligned data set was analyzed with PAUP to produce both coral host and symbiont phylogenetic trees. Methods of bootstrapping, maximum parsimony, and anchoring trees with outgroups were used to assess evolutionary relationships and patterns of specificity from the samples.

Coral taxonomy

In addition to the five symbiont markers, the mitochondrial genetic marker COX1 (607 bp) was targeted to confirm host coral identity. Cytochrome c oxidase I is part of the main subunit of the cytochrome c oxidase complex. In the literature, COXI is commonly used to 18 identify animals because it is more variable than nuclear genes and contains conserved regions for primer design (Lin et al., 2009)

19 Results

Symbiont diversity quantified with genetic evidence

Gene sequence analyses of LSU rDNA, cob, and cp23s identified 13 genetically distinct

lineages of Symbiodinium from 20 host genera (Figures 3, 4 and 5). Most lineages belong to

Clade C, with one belonging Clade D and another Clade G (Figure 3). The most comprehensive

sequenced marker LSU-rDNA resolved most but not all lineages (Figure 3). The additional

analysis of slower evolving mitochondrial and chloroplast genes substantiated the findings based

on LSU and provided further genetic resolution in some cases (Figures 4 and 5).

Mitochondrial marker cob differentiated about half the number of symbionts resolved by

LSU-rDNA (Figure 4). Like cob, cp23s clearly resolved some but not all LSU lineages (Figure

5). However, cp23s resolved one lineage identified by a single fixed difference for hosts

Cephastrea, Xenia, and Porites not observed by LSU analyses (Figure 5).

DGGE analysis of Symbiodinium common to Acropora

A single Clade C lineage termed Generalist 1 based on LSU, cob and cp23s data was

found in all nine species of Acropora sampled (Figures 3, 4 and 5). PCR-DGGE fingerprinting

analysis of ITS2 rDNA confirmed that Generalist 1 is just one species lineage characterized by

possessing two co-dominant ITS2 sequences in its genome. Generalist 1 produced a consistent

and unique banding pattern (Supplemental Figure 7).

20

Figure 3. Phylogeny of Symbiodinium collected from Japan based on the ribosomal marker LSU

Branch length numbers indicate the number of base pair changes between samples. Fixed differences are based on the LSU DNA phylogeny and identify host-specific and host generalist lineages. Host specific symbionts are marked with labels and generalist symbionts are marked with gray-scale boxes. (#) after host genus indicates the number of samples of that coral genus. Maximum parsimony analysis was used to construct the phylogeny.

21

Figure 4. Phylogeny of Symbiodinium collected from Japan based on the mitochondrial marker cob

Branch length numbers indicate the number of base pair changes between samples. Host specific symbionts are marked with labels and generalist symbionts are marked with gray-scale boxes. (#) after host genus indicates the number of samples of that coral genus. Maximum parsimony analysis was used to construct the phylogeny.

22

Figure 5. Phylogeny of Symbiodinium collected from Japan based on the chloroplast marker cp23s Branch length numbers indicate the number of base pair changes between samples. Host specific symbionts are marked with labels and generalist symbionts are marked with gray-scale boxes. (#) after host genus indicates the number of samples of that coral genus. Maximum parsimony analysis was used to construct the phylogeny. 23 Ecological distribution of symbiont diversity

Two of the 11 Clade C lineages characterized exhibited associations with multiple host

genera and therefore were the most prevalent ecologically. The most common of these,

Generalist 2 “ITS2 type C1”, associated with coral colonies representative of the scleractinian

genera Coscinaraea, Cyphastrea, Leptastrea, Pavona, Psammocora, Stylocoeniella and

Turbinaria as well as the soft coral genera Cladiella and Sinularia (Figure 3). Coscinaraea and

Psammocora belong to Siderastreidae (Budd, Fukami, Smith, & Knowlton, 2012), while

Leptastrea and Cyphastrea are in the Faviidae family (Lien et al., 2013). Pavona and Turbinaria

and are classified in two different families.

The second C lineage called Generalist 1 exhibited a narrower host distribution and

consistently associated with corals in the genus Acropora and Acanthastrea as well as the

zoantharian genus Palythoa (Figure 3). Acropora and Acanthastrea each belong to different

coral families. Acropora belongs to Acroporidae and Acanthastrea belongs to Mussidae (Budd et

al., 2012). Palythoa is in the order Zoantharia belonging to the family Spehnopidae.

Specialist C2 appeared to be broadly distributed to multiple coral hosts including Astrea,

Coelastrea, Dipsastrea, Favites, Oulophyllia, Physophyllia, and Platygrya (Figure 3). All of these corals belong to a single family, the (Budd et al., 2012). Specialist C3

associated with colonies of Hydnophora and Paragoniastrea, also members of the family

Merulinidae (Figure 3).

Most genetically distinct lineages of symbiont were narrowly distributed to a single host

genus or species. Pocillopora only associates with Specialist C5, while Stylophora associates

with Specialist C7 (Figure 3). Colonies of Oulastrea associated with Symbiodinium boreum from

Clade D (Figure 3). 24 Many non-scleractinian symbiotic cnidarians surveyed in this study also associated with host-specific symbionts. The hosts of the genus Nausithoe classified under the jellyfish family

Nausithoeidae possessed Specialist C6. Moreover, the giant sea anemone Heteractis also harbored a unique symbiont type, Specialist C4. Finally, Capnella, a soft coral in the family

Nephtheidae, associates with a Symbiodinium in Clade G.

Host diversity and phylogeny

A wide diversity of Cnidarians, including hard and soft corals, jellies, and sea anemones were obtained for this study. The mitochondrial COX1 gene was used as a host marker in order to identify evolutionary relationships between Cnidarian hosts from this region. Using this host marker, we confirmed that we collected 36 species representing 28 genera and 15 families of

Cnidaria (Figure 6).

25

Figure 6. Phylogeny of coral host families and their associated symbiont types

This coral phylogeny is based on the mitochondrial marker COX1. Maximum parsimony analysis was used to construct the phylogeny. Branch length numbers indicate the number of base pair changes between samples. Host specific symbionts are marked with text and generalist symbionts are marked with gray-scale boxes. (#) after host genus indicates the number of samples of that coral genus. The subfamilies for each coral species is marked by the colored bars. Asterisks indicate unknown symbiont type. 26

Discussion

High latitude lineages of Symbiodinium

Evidence from multiple genetic markers resolves numerous phylogenetically distinct

lineages of Symbiodinium, many of which likely represent biological species. The consistent phylogenetic relationships between symbionts across independent nuclear ribosomal, mitochondrial, and chloroplast genes indicate that each is reproductively isolated and evolutionarily divergent. Many of these “species” appear endemic to this high latitude

Northwestern Pacific region and few exist in lower latitude reef ecosystems. Ecologically, most

exhibited high host specificity, suggesting a long-term co-evolution of symbionts that are highly adapted to the intracellular environment of particular coral species at high latitudes.

Coral symbionts from Symbiodinium Clade C are found in the majority of hosts living in tropical and sub-tropical locations around the world, especially in the Indo-Pacific, which encompasses Japan. This largely explains the high diversity and prevalence of Clade C symbionts in the corals from Honshu Island, Japan. The host taxa Oulastrea (stony coral) and

Capnella (soft coral) were the only taxa that possessed symbionts outside of Clade C. Oulastrea was found to associate with Symbiodinium boreum from Clade D. This particular Clade D species occurs in this host at subtropical and high latitude environments (Todd C. LaJeunesse et al., 2014). The only colony of Capnella surveyed during this study harbored a rare kind of symbiont from Clade G. 27 These results echo the findings of other researchers in Japan and the Gulf of California

who also observed Clade C symbiont dominance in high latitude corals (T. C. LaJeunesse et al.,

2008; Lien et al., 2013). However, earlier studies of this kind only used a single genetic marker,

potentially limiting the recognition of a rich species diversity found among high latitude hosts.

Lien et al. also observed three Clades (C, D, and G) including novel lineages of Clade C, further

supporting our evidence for potential new high latitude species of symbionts. While Clade C

dominance seems to be a common theme throughout high latitude symbioses, researchers in

South Korea observed a rare Clade F symbiont (De Palmas et al., 2015). Because sequence

diversity within a Symbiodinium Clade was not always viewed as biologically important in the

past, researchers missed the ecological and evolutionary significance of their findings.

The northward-flowing Kuroshio Current connects many of the ocean ecosystems of interest in this study. It begins in the Philippines, eventually reaching the Japanese coast before

turning east into the North Pacific Ocean (Minchin, Peter R. and Vernon, 1992). This strong flow

of warm water provides a mechanism for coral migration to temperate latitudes. There are three

important aspects of the Kuroshio Current that may influence coral and symbiont distribution in

Japan. First, the current is unidirectional, flowing adjacent to the collection sites in this study.

Second, its dampens seasonal temperature variation and maintains relatively hospitable

temperatures at high latitudes like Japan (Minchin, Peter R. and Vernon, 1992). While the

Kuroshio Current connects low and high latitude regions, we find that the symbiont community

in Japan is distinct from many of the symbionts common to the corals from tropical and

subtropical environments (LaJeunesse, unpublished data). While it is possible that connectivity

occurs between sub-tropical and temperate coral ecosystems, there still seems to be a long-

standing selection pressure from different latitudinal ecosystems on the symbiont community. 28 Ecological niche and host specificity

The strong correspondence between host identity and symbiont species distribution is

typical for most coral communities especially for animal-algal symbioses at high latitudes (De

Palmas et al., 2015; T. C. LaJeunesse et al., 2008; Todd C. LaJeunesse et al., 2004). In many

cases, we found high host-symbiont specificity, where a single host is found to associate with

only one symbiont species (for example, Heteractis and Specialist C4). In other cases, several

closely related host taxa from the same family or subfamily were found to associate with a single symbiont species. These examples of host specificity are likely a result of long-term (several millions of years) niche diversification and the evolution of ecologically specialized species highly adapted to the intracellular habitats of a single host species or monophyletic group living under a particular environmental setting.

Closely related specialists 5 and 7 associate with host corals Pocillopora and Stylophora respectively, which belong to the same coral family Pocilloporidae. This raises an interesting question of why two closely related corals have different symbionts. A potential explanation for the Pocilloporidae family is that Pocillopora corals transmit their symbionts vertically while

Stylophora has been shown to transmit symbionts both vertically and horizontally (Byler, Carmi-

Veal, Fine, & Goulet, 2013). Vertical transmission is when corals acquire their symbionts

through direct inheritance whereas horizontal transmission occurs when corals acquire their

symbionts from the surrounding environment. The two modes of transmission observed in

Stylophora may be indicative of an adaptive mechanism that allows these corals to acquire novel

symbionts. If advantageous, natural selection can act upon this symbiont variability and pass the

novel symbionts along to progeny via vertical transmission (Byler et al., 2013). 29 Generalist species

In this study we documented two dominant host generalists found to associate with many taxonomically diverse coral taxa. Generalist 1 appears to be regionally endemic to the North

Western Pacific while the other, Generalist 2 “ITS2 type C1” is known to occur in tropical locals

(LaJeunesse, personal communication). The high-latitude Generalist 1 associates with three distinct genera, two from the order and one from the coral subfamily Sphenopidae, order Zoantharia. There is no obvious explanation for why it associates with only these hosts and not any others. While Generalist 2 “ITS2 type C1” occurred in many host taxa, corals hosting highly specific symbiont “species” maintained their unique associations. Interestingly, there was no evidence of mixed communities involving more than one symbiont in the samples we analyzed further indicating a high degree of specificity, which may be influenced by competitive displacement and partner incompatibility (Brown & Wilson, 1956).

Our finding of two host generalists contrasts the work of researchers in Hawaii who documented only high specificity between corals and symbionts (Todd C. LaJeunesse et al.,

2004). While we did observe high specificity in Japan’s coral symbiont relationships, we distinguished clear generalists as well. Although Hawaii is technically a high latitude site, it is not geographically close to Japan and the host diversity is considerably lower and dominated by coral taxa that rely on the vertical transmission of symbionts, possibly explaining the contradictory finding. 30 Effect of climate change on high latitude coral symbioses

Seawater temperature has a strong effect on the survival of reef-building corals and their symbionts, making it a major determinant of latitudinal distributions. The observations presented here are potentially important for understanding the effect of climate change and the importance of high latitude habitats in acting as coral refuges, as tropical regions warm to temperatures that are above the threshold of these symbioses. Corals and their symbionts may be able to cope with the negative impacts of climate change and increasing sea surface temperatures by further colonizing at high latitude regions with suitable habitats like Japan (Grupstra et al., 2017).

Additionally, the ability of many coral species to associate with more than one kind of

Symbiodinium over their geographic ranges may facilitate acclimatization and the persistence of various coral populations.

Coral ecosystems are vulnerable in the face of climate change. Largely understudied and with little conservation priority, high-latitude coral reef communities serve as a buffer from the effects of climate change because of their unique characteristics and composition of coral species

(Beger et al., 2014). High-latitude coral communities are compositionally very different from their tropical counterparts, because they are characterized by overlap of taxa at the margins of their range, some endemic species, and seasonality in species composition. These marginal environments are predicted to undergo greater changes with climate than lower latitude reefs, resulting in community re-assembly through shifts in range, survival, and habitat loss.

Ultimately, northern latitude coral reef communities are poised for intense modification if the current environmental trends continue (Beger et al., 2014). When thinking about the best way to anticipate these changes, conservation strategies must switch their focus to fostering ecosystem resilience. Understanding the nature of host-symbiont partnerships of corals in areas like Japan 31 offers an important step toward understanding the capacity and limitations of the response of these animals to climate change.

Limitations and further research

In any ecological study, sampling an entire ecosystem is not feasible. Despite this, we

believe we have provided a comprehensive picture of Symbiodinium species from Japan. Even

though we may have missed a host species during sampling efforts, there will always be cryptic

hosts to discover. In addition, characterizing Symbiodinium to the species level requires

molecular evidence based on multiple markers, including conserved and rapidly evolving genes,

analyzed from a wide variety of samples (T. C. Lajeunesse et al., 2012). Therefore, further

research requires a full investigation of every lineage identified here and formal descriptions to

be considered true species. Lastly, genetic markers may not truly resolve organisms to the

species level. For example, Generalists 1 and 2 may contain some cryptic host-specific

symbionts that require higher resolution markers like psbAncr to resolve.

Future research should involve detailed comparisons of the symbiont diversity in coral

assemblages at locations further south along the Kuroshio Current, including both high latitude and sub-tropical regions. In order to classify the symbionts identified here as high latitude types, it is necessary to study other high latitude ecosystems to see if these patterns are consistent across latitude. For example, comparison to another high latitude location such as Kenting

National Park in Taiwan, would strengthen our findings. Additionally, this work can be compared to the symbiont diversity from low latitude coral reefs such as Palau, a tropical location, near the start of the Kuroshio Current. Such latitudinal comparisons will provide further 32 insight into how coral-algal combinations may change with increasing latitude and changes in environment.

In summary, this survey of biodiversity of symbiotic algae in corals from Japan yielded previously unidentified species not found in low latitude regions. In addition, we found host- symbiont pairings that are highly host specific and other symbionts that are seemingly host generalists. Lastly, symbionts belonging to Symbiodinium Clade C were found to be the most common symbiont group for Scleractinia from Japan. Our findings inform coral reef conservation by starting to establish a baseline of biodiversity at northern latitudes, as corals may find protection from the effects of climate change by colonizing at northern latitudes like Japan.

33 Supplemental Table

Table 1. List of sampled corals from Japan, their genus, species, and the number of specimens collected 34

35

36

Supplemental Figure

Figure 7. DGGE ITS2 results containing coral hosts Acropora and Montipora

The ladder contains known Symbiodinium species S. trenchii (D1a) from Clade D and C21, C40, and C15 from Clade C. ‘C?’ stands for a currently unidentified symbiont in Acropora belonging to the ITS2 C1 lineage.

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ACADEMIC VITA Madison Taylor | [email protected] EDUCATION

Penn State University, Schreyer Honors College B.S. Honors in Biology 2018 Area of Concentration: Ecology Minor: Global Health Honors Thesis: Diversity of Symbiotic Dinoflagellates in Northern Latitude Corals INTERNATIONAL EDUCATION

Muhimbili University of Health and Allied Sciences, Tanzania Global Health Fieldwork Summer 2017 Six weeks of global health fieldwork, shadowing different medical and public health specialties in hospitals, clinics, and collaborating in a mental health community assessment in rural villages. Penn State University, San José, Costa Rica Tropical Field Biology Course January 2017 Three-week tropical field biology course, contributing to long-term existing and independent research projects. Penn State University, Lima, Peru Service-Learning Course March 2015 Two-week service learning course in the Amazon rainforest

RESEARCH EXPERIENCE

Dar es Salaam and Dodoma, Tanzania Global Health Fieldwork Summer 2017 Collaborated on the Muhimbili University of Health and Allied Science’s community mental health assessment in the Mkonze community outside of Dodoma, Tanzania. Penn State University, State College, PA Undergraduate Research Assistant, LaJeunesse Lab 2015-2018 Member of the Symbiosis Ecology and Evolution Laboratory team, assisting in research on Symbiodinium, a common symbiont of corals. Trained in DNA extraction, DNA sequencing, phylogenetics, basic microscopy, and culture maintenance.

TEACHING EXPERIENCE

Penn State University, State College, PA Teaching Assistant, Leadership JumpStart 2015 – 2018 Three-year Teaching Assistant for a freshman honors course that teaches leadership through service and experiential learning. Penn State University, State College, PA Teaching Assistant, Society and Global Disease Management 2017 – 2018 First Teaching Assistant for a new course at Penn State, helped design, implement, and teach a new cross-disciplinary course between biology and management. Overland Summers, Williamstown, Massachusetts Outdoor Educator Summer 2016 Leader for Overland’s Mountains & Sea Adventure, six weeks of trips for middle school students. Provided outdoor pursuits, teaching wilderness survival skills, and opportunities for experiential learning. LEADERSHIP EXPERIENCE

Penn State University, State College, PA Founder and President of Schreyer for Women 2017 – 2018 The first president of the Honors College’s first female-focused professional organization. Penn State University, State College, PA Presidential Leadership Academy 2015 – 2018 Member of an exclusive three-year academy at Penn State for developing a working philosophy of leadership and critical thinking skills through classes with University leadership and extracurricular activities. COMMUNITY SERVICE INVOLVEMENT

Hekima Place Home for Girls, Kiserian, Kenya Volunteer 2013 – Present Serving as a tutor and mentor, organizing creative activities for eighty girls ages Pre-K through University. Point Prevention Pittsburgh, Pittsburgh, PA Volunteer 2015 – Present Assisting in a needle exchange clinic in the city of Pittsburgh with the aim of providing harm reduction services to injection drug users.

MEMBERSHIPS

Penn State Triathlon Club The Co.Space, Home for Change-Makers

AWARDS

Fulbright English Teaching Assistantship 2018 – 2019 The Headings Scholarship for Excellence in the Eberly College of Science 2017 – 2018 Student Engagement Network Grant 2016 – 2017 Eberly College of Science Global Experiences Scholarship 2016 – 2017 Presidential Leadership Academy International Travel Grant 2015 – 2017 Schreyer Honors College Travel Ambassador Grant 2015 – 2017 Eberly College of Science Undergraduate Research Grant Recipient 2015 – 2016 Dean’s List 2014 – 2018 Parkin Fellowship 2013 – 2014