Variation of the Symbiodinium Community Composition in Scleractinian Corals Along a Cross-Shelf and Depth Gradient

Variation of the Symbiodinium Community Composition in Scleractinian Corals along a Cross-shelf and Depth Gradient

Thesis by

Alejandro Mejia Restrepo

In Partial Fulfillment of the Requirements

For the Degree of

Master of Science

King Abdullah University of Science and Technology

Thuwal, Kingdom of Saudi Arabia

November, 2017

EXAMINATION COMMITTEE PAGE

The thesis of Alejandro Mejia Restrepo is approved by the examination committee.

Committee Chairperson: Michael Berumen Committee Members: Christian Voolstra, Timothy Ravasi

Alejandro Mejia Restrepo

ABSTRACT

Variation in the Symbiodinium Community Composition of Scleractinian Corals along a Cross-shelf and Depth Gradient

Alejandro Mejia Restrepo

Corals form a symbiotic relationship with photosynthetic zooxanthellae from the genus

Symbiodinium; the breakdown of this symbiosis results in the phenomenon known as coral bleaching. This relationship is especially vulnerable to high temperature stress, although corals may survive if they have resistant types of symbionts, or switch their community composition towards them. To assess the variation of the symbiont community in different environmental conditions, I recorded the temperature and collected samples from six scleractinian coral species and one calcifying hydrozoan, in two inshore, two mid-shelf, and two offshore reefs at 1, 15, and 30m depth, analyzing

Symbiodinium diversity using Next Generation Sequencing with the SymPortal profile typing approach. The temperature was very similar for all points in winter, when coral samples were collected, but variation between points increased until a maximum at summer, with the shallower parts of the inshore reefs showing higher temperatures and the points at 30m depth showing the lowest. The Symbiodinium composition was more similar between samples of the same host species than among samples of the same reefs or depths. Coral species from the Pocilloporidae family and Millepora dichotoma showed specific association with different profile types, specifically, intragenomic variants of

Symbiodinium type A1, which appears to be dominant in the Red Sea although it has not 5 been reported for these species in other regions. The other species showed specific associations with types previously reported in other regions, mostly from clade C and D, although also having different types and intragenomic variants. For most cases, certain profile types, which can reflect different species or populations, appeared to be dominant in particular environmental conditions, following a distribution related with depth, reef type, or both. In conclusion, this study showed that the Symbiodinium composition depends more on the host species than on the environmental conditions, and within each species the adaptation to environmental gradients can rely on tolerant symbiont species or populations characteristic of the Red Sea, or association with different types and clades that are common also in other regions.

ACKNOWLEDGEMENTS

I would like to thank Michael Berumen for the guidance and support throughout this research, and Christian Voolstra and Timothy Ravasi for being part of the thesis committee. I would also like to thank all the members of the Reef Ecology Lab, especially Roberto Arrigoni and Darren Coker for their advice and collaboration in the planning and development of the research and Tullia Terraneo, Rodrigo Villalobos,

Michael Wooster, Sara Wilson, and Emily Troyer for their help in the lab and in the field.

Benjamin Hume assisted with the data processing. Alan Barozzi, Ruben Diaz, and the staff of the BCL core lab helped with the sequencing process. The staff of CMRCL assisted in the fieldwork. I thank everyone that has helped me in one way or another.

TABLE OF CONTENTS

Page

EXAMINATION COMMITTEE PAGE 2

ABSTRACT 4

ACKNOWLEDGEMENTS 6

TABLE OF CONTENTS 7

LIST OF ABBREVIATIONS 9

LIST OF ILLUSTRATIONS 10

LIST OF TABLES 11

1. Introduction…..…………………………………...………..……………….………………………………...... 12

1.1 Objectives…………………………………………………………………………………………….……….…….18

2. Materials and Methods.……………………………………………………………….………………...... 19

2.1 Study area...... 19

2.2 Sample collection ...... …….……………… 20

2.3 Temperature measurement………………………………………………………….……………………..21

2.4 DNA extraction and PCR……………………………………………………………………….……………..21

2.5 Library preparation and Miseq ITS2 sequencing...... …….………….…………….. 22

2.6 Sequencing data processing...... …….………………….….….. 23

2.7 Data Analysis ...... …….…………………….….... 23

3. Results………………………………………………………………………….………………………………………25

3.1 Temperature ...... 25 8

3.2 Symbiodinium composition...... 27

4. Discussion……………….………………………………………………………………..…………………..…... 30

5. Conclusions ………………………………………………………………..………………………….………….. 37

BIBLIOGRAPHY……………………………………………………………...... …………………………………...39

APPENDICES…………………………………………………………….……………………………………………...45

LIST OF ABBREVIATIONS

DIV Defining intragenomic variants DGGE Denaturing Gradient Gel Electrophoresis ITS Internal Transcribed Spacer NGS Next generation sequencing OTU Operational taxonomic unit PCR Polymerase chain reaction PERMANOVA Permutational Analysis of Variance

LIST OF ILLUSTRATIONS

Figure 1. Map showing the location of the reefs sampled near Thuwal in the Saudi Arabian central Red Sea; two inshore reefs (Fsar and Tahla), two mid-shelf reefs (Al Fahal and Qita al Kirsh), and two offshore reefs (Shib Nazar and Abu

Madafi)………………………………………………...... …20

Figure 2. Monthly temperature averages in Celsius degrees measured at 1, 15 and 30m for one inshore reef (Tahla), two mid-shelf reefs (Al Fahal and Qita al Kirsh), and two offshore reefs (Abu Madafi and Shib Nazar) in the central Saudi Arabian Red

Sea……………………………………...... 26

Figure 3. Number of total Symbiodinuim type profiles per host species (Set Size), and shared or unique profile types between host species (Intersection) obtained using the

UpSetR online application...... 28

LIST OF TABLES

Table 1. Number of reads, Defining Intragenomic Variants (DIVs), and Type profiles for the different coral species obtained using SymPortal to analyze the MiSeq data of the Symbiodinium ITS2 region...... …………27

Table 2. PERMANOVA for type profiles with three factors, Species, Depth, and Reef Type...... 29

1. Introduction

Coral reefs are among the most productive and biologically diverse ecosystems, but they are also among the more vulnerable due to overexploitation and their susceptibility to perturbations like climate change (Wilkinson, 2008). This has led to a noticeable decline worldwide in the last decades, caused by increasing bleaching events (Silverstein et al.,

2015). Most corals depend on symbioses with photosynthetic dinoflagellates of the genus

Symbiodinium for supplying their nutritional needs, but this relationship can break down as a response to stress, generally from increasing temperature, leading to what is known as coral bleaching (Silverstein et al., 2015). The differences of susceptibility to bleaching can be related with the coral host phylogeny and colony size, the environmental conditions, (Glynn, 1990; Marshall and Baird, 2000; McClanahan, 2004), and also to the association with different types of Symbiodinium (Baker, 2003). In this last case, it is thought that corals may increase heat stress tolerance by changing their symbiont community due to the genetic, physiological and ecological diversity of this group (Baker et al., 2004; Thornhill et al., 2007). For this reason, the study of this community and its changes in different environmental conditions is necessary for understanding the fate of corals in a global warming scenario.

The taxonomy of Symbiodinium has been mostly defined using molecular tools in a physiological, rather than biological, concept of species since morphological differentiation is complicated and there is not much information about their life cycle

(Baker, 2003). The first attempts to use molecular tools for Symbiodinium classification 13 were done by Rowan and Powers (1991) using the small ribosomal subunit (SSU rDNA).

This led to the discovery of divergent lineages within the genus, known also as clades. So far, nine of these clades have been identified: A-I (Pochon et al., 2014), of which A-D are the most commonly found in scleractinian corals. Each of these clades includes several types, which are likely to be physiological different species ( LaJeunesse, 2001; Howells et al., 2009). For this finer-level distinction, researchers began to look at more variable regions of the genome, like the nuclear large subunit (Loh et al., 2001), the internal transcriber spacer regions ( LaJeunesse, 2001; Arif et al., 2014; Ziegler et al., 2017), the chloroplast large subunit (Pochon et al., 2006; Stat et al., 2011), the cytochrome oxidase b (Sampayo et al., 2009; Finney et al., 2010), and the cholorplast psbA noncoding region

(LaJeunesse and Thornhill, 2011). These techniques have a better resolution, but they all have some limitations that complicate agreement of which one is the most appropriate for species distinction.

Currently the ITS1 and ITS2 region of the rDNA is the most common genetic marker used to asses Symbiodinium diversity (e.g., Ziegler et al., 2017) due to its usefulness to distinguish types that differ in their ecological or physiological characteristics (LaJeunesse,

2001). This marker can provide enough resolution to find ecologically different

Symbiodinium species when the dominant intragenomic variants diagnostic of certain types are analyzed using the denaturing gradient gel electrophoresis (DGGE) technique to visualize the polymerase chain reaction (PCR) amplifications (LaJeunesse, 2002; Sampayo et al., 2009; Arif et al., 2014). Bacterial cloning and sequencing have also been used, but 14 this approach can overestimate diversity due to the tendency of recovering low abundant intragenomic variants and the introduction of artefacts generated during the process

(Thornhill et al., 2007). Some researchers have started to assess Symbiodinium diversity using next generation sequencing (NGS) by pyrosequencing (e.g., Arif et al., 2014) and

Ilumina MiSeq (e.g., Ziegler et al., 2017). These studies analyze the ITS2 data with an

Operational Taxonomic Unit (OTU) approach, but still using the DDGE method which has limited power to detect sequences below 10% of abundance, and can have problems for distinguishing sequences with the same melting profile (Neilson et al., 2013). This can be a problem when assessing community changes with environmental conditions, since this low abundance types can have a big role once conditions change.

SymPortal is a tool that can overcome this limitation, and can identify sequences several orders of magnitude less using the digital nature of NGS data. The SymPortal is like a digital version of DGGE based on an algorithmic approach to identify re-occurring sets of

ITS2 sequences that can include several intragenomic variants, named as ITS2 type profiles. (Hume et al. in prep.). These type profiles can be associated with different species or populations, since common intragenomic variants persist in the genomes among the individuals of a genetically recombining population over long evolutionary timescales through the process of concerted evolution (e.g., Arif et al., 2014). Since rare types can play an important role increasing their abundance under stress conditions and bleaching events (Boulotte et al., 2016), having the power to detect them, and taking 15 them in account to define the symbiotic community diversity, can be an advantage for understand community changes with environmental variation.

Some Symbiodinium types form specific symbiotic relations with certain coral host species, but in many cases the relationships are more generalized, with a host species harboring different types of symbionts and with the same Symbiodinium type present in several coral species. Host specificity is more common in corals with a brooder reproductive strategy, while broadcaster corals tend to be more generalist in the symbiotic associations (LaJeunesse et al., 2004: Bongaerts et al., 2013). This intraspecific symbiont diversity within a single species of host can provide great potential for phenotypic variability (Baker, 2003), which can help them thrive across different environmental gradients, and to adapt to changing conditions.

The type of symbiont present in a specific coral species depends greatly in the environmental conditions, varying between geographic regions (LaJeunesse et al., 2004;

Finney et al., 2010; Kennedy et al., 2016) and latitudinal gradients (Oliver and Palumbi,

2009; Huang et al., 2011; Sawall et al., 2014) to smaller scales such as cross-shelf (Howells et al., 2009; Tonk et al., 2014) and depth gradients (Bongaerts et al., 2013, 2015; Ziegler et al., 2015). There is also a seasonal variation in some cases (Hsu et al., 2012; Sawall et al., 2014), and the symbiont community can change in response to environmental stresses like increased temperature (Hsu et al., 2012; Keshavmurthy et al., 2014). This last factor that has been found to affect strongly this symbiosis, and it is usually related to coral 16 bleaching (Cooper et al., 2011; Silverstein et al., 2015). Since temperature generally varies with the environmental gradients studied, its relation with the Symbiodinium community changes along these gradients is key for understanding the distribution of this group.

The coral reefs of the Red Sea are known for developing under conditions that would be too stressful for corals in other parts of the world, with temperature values above global optimal value for coral reef development (Kleypas et al., 1999; Roik et al., 2016). In the

Red Sea region, previous studies have documented the Symbiodinium diversity in some coral species, looking at variation in the community through different gradients. Sawall et al. (2014) studied the latitudinal and seasonal variation of Symbiodinium in the coral

Pocillopora verrucosa, finding very similar composition along the latitudinal gradient for both winter and summer, with type A1 being dominant. One exception was the point farthest north in the Gulf of Aqaba that was dominated by types of clade C. This same pattern of a different Symbiodinium composition in the northern Red Sea compared to the central Red Sea was also found by Reimer et al. (2017) for the zoantharian Palythoa tuberculosa. Ziegler et al. (2015) looked at the Symbiodinium community variation for four different coral genera in a 60m depth gradient at the central Red Sea, finding that corals from the genus Porites, which were associated with Symbiodinium type C15, had a wider depth distribution than other genera like Podabacia and Pachyseris, which were mostly found associated with type C1. More recently, Ziegler et al. (2017) compared the

Symbiodinium diversity in different regions of the Arabian Peninsula, finding a different 17 composition for the same coral species between the Red Sea, the Sea of Oman, and the

Persian Gulf. These studies look at wide scale patterns within the region that have a relation with temperature, and have found some Symbiodinium types that appear to have a specific relation with certain host species, allowing them a wide distribution along the

Red Sea. However, there is lack of information about smaller-scale gradients, such as cross-shelf, and studying more coral species would lead to a better understanding of how the symbiont community relates to environmental conditions.

Major bleaching events occurred in the central Red Sea in 2010 and 2015, when the region experienced an unusual rise in water temperature (Furby et al., 2013; Monroe et al., in review). The patterns of this events were studied near Thuwal, Saudi Arabia, where Furby et al. (2013) found that the bleaching was more severe in the inshore reefs and in shallower depths than in the offshore reefs and deeper zones. The cross-shelf and depth gradient pattern was thought to be related with the water temperature, but this was not previously assessed. This study described the coral species that were more affected by the bleaching, but with no information about the Symbiodinium community before, during, or after the bleaching event. Although this cannot be assessed without having samples from the bleaching period, the relation of the temperature and the symbiont composition with the environmental gradients can be studied to understand better these patterns, and as a point of reference for future studies. Since some types of symbionts show a greater temperature tolerance, there is the possibility that changes in the symbiont community lead to the coral’s thermal acclimatization (Baker et al., 2004;

Thornhill et al., 2007). The temperature gradient may lead to the shallow parts and 18 inshore reef having types of symbionts with a greater temperature tolerance range, or a more diverse community in which certain types will predominate according to the conditions.

1.1 Objectives

The aim of this study was to assess the temperature variation in a cross-shelf and depth gradient of the central Red Sea, and to examine variation in the Symbiodinium composition of different coral species along this gradient. For this purpose, two inshore, two mid-shelf, and two offshore reefs were selected, measuring temperature for six months and taking coral samples of six different genera (Pocillopora, Stylophora,

Seriatopora, Galaxea, Gardineroseris, and Porites, with Millepora as an outgroup) at 1m,

15m, and 30m depth for each reef. Symbiodinium diversity was assessed using Next

Generation Sequencing to analyze the ITS2 rDNA region with the SymPortal analysis. I expected to find different Symbiodinium communities correlating with different environmental conditions. Particularly, I was expecting to find more stressful conditions related with higher temperatures in the inshore reefs and shallow depths, leading to a community reflecting greater thermal stress resistance in these sites.

2. Materials and methods

2.1 Study area

Six reefs near Thuwal, Saudi Arabia, in the central Red Sea were sampled to assess the temperature and Symbiodinium community variation along a depth and cross-shelf gradient. Two offshore reefs, Abu Madafi (22° 05.209’ N 38° 46.700’ E) and Shi’b Nazar

(22° 19.352’ N 38°51.257’ E), were located on the edge of the continental shelf, approximately 15-20km from shore. Two mid-shelf reefs, Qita al-Kirsh (22° 25.843’ N 38°

59.564’ E) and Al Fahal (22° 18.374’ N 38° 57.632’ E), were located approximately 8-9km from shore. Two inshore reefs, Tahla (22° 17.420’ N 39°03.271’ E) and Fsar (22° 13.957 N

39° 01.816’ E) were 2-4km away from shore (Figure 1). In the offshore reefs and Qita al

Kirsh, loggers were deployed and coral samples were taken at three different depths, 1-

5m, 10-15m and 25-30m, while in the other reefs only the first two depths were possible due to the reef topography. All the sampling and logger deployment was done in the exposed side of the reefs.

Figure 1. Map showing the location of the reefs sampled near Thuwal in the Saudi

Arabian central Red Sea; two inshore reefs (Fsar and Tahla), two mid-shelf reefs (Al

Fahal and Qita al Kirsh), and two offshore reefs (Shib Nazar and Abu Madafi)

2.2 Sample collection

Samples for six scleractinian coral species, Pocillopora verrucosa, Stylophora pistillata,

Seriatopora hystrix, Gardineroseris planulata, Galaxea fascicularis, and Porites spp., and one hydrozoan with calcareous skeleton, Millepora dichotoma, were collected during the first week of March 2017, at the reefs and the depths mentioned above. The coral species identification was done visually in the field. For each reef, depth, and coral species, five colonies at least 5m apart from each other were selected, and one fragment was collected for each using a hammer and chisel. Samples were transported in buckets filled with 21 seawater to the laboratory, where they were fixed in ethanol 96% the same day and stored until the DNA extraction.

2.3 Temperature measurement

For each depth and reef, one temperature logger was deployed the first week of March

2017 to register the temperature every hour. For 1 and 15m depths, HOBO pendant

Temp/Alarm, 64K loggers were deployed, while for 30m, HOBO Water Temp Pro v2 loggers (Onset Computer Corporation, Bourne, MA) were used, since the HOBO pendant loggers were not suitable for this depth. The loggers were collected the last week of July

2017, after almost six months of recording temperature

2.4 DNA extraction and PCR

The Symbiodinium DNA was extracted from the coral fragments using the Qiagen DNeasy

Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol with minor modifications. The coral fragment was crushed using mortar and pestle, and transferred to 1.5mL Eppendorf tubes containing 200 μl of sterile glass beads (BioSpec,

Bartlesville, OK) and 400 μl of Buffer AP1. Then 4 μl of RNase A was added to each sample and they were vortexed for one minute. The DNA extractions were continued according to manufacturer’s instructions, and the final product was analyzed using the Nanodrop 22

2000 Spectrophotometer (ThermoFisher Scientific, Wilmington, DE) to check the DNA concentrations and quality.

The PCR amplification of the ITS2 gene marker was performed using the primers

ITSintfor2 (LaJeunesse and Trench, 2000), and ITS2-reverse (Coleman et al., 1994). For each sample, three PCRs were performed using 11μl of Qiagen Multiplex PCR Master Mix

2X (Qiagen, Hilden, Germany), 9μl of RNase-free water, 2 μl of each primer at 10mM concentration, and 1μl of DNA extract. The PCR conditions were 15 min at 95 °C, followed by 27 cycles of 94 °C for 30 s, 51 °C for 30 s, 72 °C for 30s, and a final extension step of 10 min at 72 °C. PCR products were checked in the QIAxcel, with the DNA Screening Kit (2400) using a 3kb marker, and the AM320 method (Qiagen, Hilden, Germany) to visualize successful amplification. For each sample, 20μl of each PCR product was pooled together for a final volume of 60μl used for the library preparation.

2.5 Library preparation and MiSeq ITS2 sequencing

Pooled samples were cleaned with Agencourt AMPure XP magnetic bead system

(Beckman Coulter, Brea, CA). Nextera XT indexing and sequencing adapters were added via PCR (8 cycles, total PCR cycles for all samples = 35) following the manufacturer’s instructions. Samples were normalized using the SequalPrep Normalization Plate (96) Kit

(Invitrogen, Frederick, Maryland, USA), and then all samples for each library were pooled together and the library was concentrated to a final volume of 100 μl using CentriVap 23

Complete vacuum concentrator (Labconco, Kansas City, MO). Libraries were quantified on the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and qPCR

(ThermoFisher Scientific, Wilmington, DE) to check the concentration and average fragment size. The libraries were diluted to 7pM with 15% phiX for sequencing on the

Illumina MiSeq 2 x 300 bp paired-end version 3 according to the manufacturer’s specifications.

2.6 Sequencing data processing

The sequences from the Illumina Miseq were analyzed with the SymPortal engine.

SymPortal resolves Symbiodinium taxa through identification of unique combinations of the ITS2 gene copies using an algorithmic approach to identify re-occurring patterns of

ITS2 sequences. It searches through all of the samples it has data for and looks for re- occurring sets of ITS2 sequences known as ITS2 type profiles, which will only be considered as valid if they are found in four or more samples. The output obtained with

SymPortal was a two dimensional table representing which ITS2 type profiles and which

Defining Intragenomic Variants (DIVs) are found in which samples, with the number of reads and the relative abundance for each (Hume et al. in prep).

2.7 Data analysis

Temperature maximum and minimum values, as well as monthly and general averages with standard deviation were obtained to see differences between sites and throughout 24 time. To find if there was relation of the environmental gradients with the temperature, a Friedman test, as a non-parametric alternative to the repeated measures one-way

ANOVA, was done comparing each depth at each reef. Subsequently a Wilcoxon pairwise test was done to look at the differences between each pair. These analysis were done using PAST statistical program (Hammer et al., 2001)

To visualize the Symbiodinuim community composition, bar charts were plotted comparing the relative abundance of profile types at each depth and reef type for each host species. Significant differences of the symbiont composition between host species and environmental factors were analyzed through a permutational analysis of variance

(PERMANOVA), with Species as factor with seven levels (Pocillopora, Stylophora,

Seriatopora, Galaxea, Gardineroseris, Porites, and Millepora), Depth as a three level factor

(1m, 15m, and 30m), and Reef Type as a three level factor (Inshore, Mid-shelf, and

Offshore). The analysis was done following the Bray-Curtis method after applying the fourth root to the number of reads, using 999 permutations with ADONIS function in R

Vegan package (Oksanen et al., 2017). To visualize the intersection of the profile type between the different coral species, a plot was made using the UpSetR web application

(Lex et al., 2014).

3. Results

3.1 Temperature

The temperatures values registered were between 24.64 °C in Shib Nazar at 1m in March and 33.33 C in Tahla at 1m in July. The temperatures increased for all points from March to July, with the lowest monthly average and standard deviation between points in March

(25.48 ± 0.26 °C), and the highest values and differences in July (30.77 ± 1.16 °C) (Figure

1). There was a significant difference in the temperature between sites in the sampling period (Friedman test: chi2: 25157, p=0) with the exception of the two offshore reefs, and one mid-shelf reef, Qita al Kirsh, at 15m depth (Wilcoxon pairwise p>0.5).

The temperature values in winter were very similar between all reefs and depths, with differences less than 1°C for the monthly average of March. Nonetheless, the shallow points showed higher temperature, and this pattern increased as the months passed, getting to a difference of 3.5°C between the monthly average of an offshore reef at 30m depth and an inshore one at 1m. Since all the sites had very similar temperatures in winter, the variation for each site across time was also greater for the shallow points, reaching more than 6°C in Tahla reef at 1m, while it was less than 4°C at 30m.

In a cross-shelf gradient, the only inshore reef for which data was collected showed higher values for both 1 and 15m depth compared to the other reefs in the corresponding depths, and before July the temperature at 15m was even higher that at 1m for the other reefs. These differences were much smaller in the 15 and 30m averages of the mid-shelf and offshore reefs (Figure 2).

3.2 Symbiodinium composition

A total of 312 coral samples were collected and analyzed with NGS, of which 64 were from

Galaxea fascicularis, 63 from Pocillopora verrucosa, 56 from Porites spp., 41 from

Stylophora pistillata, 38 from Millepora dichotoma, 37 from Gardineroseris planulata and

13 from Seriatopora hystrix. In some cases, the species were not present, or there were not five colonies found in each sampling site. Also, some samples did not amplify properly, so by the end there were not the same number of samples for each species in each depth and reef (Appendix A).

A total of 22,093,272 reads were obtained from the NGS sequencing, with 85 defining intragenomic variants (DIVs) of Symbiodinium found in this study, 18 belonging to clade

A, 48 to clade C, and 19 to clade D. These were represented in 72 type profiles, 15 belonging to clade A, 33 to clade C, and 24 to clade D (Table 1). Pocillopora verrucosa had more type profiles and more unique type profiles than other species (Figure 3).

Table 1. Number of reads, Defining Intragenomic Variants (DIVs), and Type profiles for the different coral species obtained using SymPortal to analyze the MiSeq data of the Symbiodinium ITS2 region.

Species DIVs Type profiles Number of reads Gardineroseris planulata 82 35 2,237,573 Galaxea fascicularis 77 25 3,901,352 Millepora dichotoma 84 33 2,934,236 Porites spp. 83 39 3,059,323 Pocillopora verrucosa 85 44 5,517,337 Seriatopora hystix 81 19 1,361,387 Stylophora pistillata 85 40 3,082,064 Total 85 72 22,093,272

Figure 3. Number of total Symbiodinuim type profiles per host species (Set Size), and shared or unique profile types between host species (Intersection) obtained using the

UpSetR online application. The species Seriatopora hystrix, Galaxea fascicularis, Millepora dichotoma, Gardineroseris planulata, Porites spp., Stylophora pistillata, and Pocillopora verrucosa are represented by the genera name in the figure.

The distribution of these type profiles along the samples showed significant differences with the host species, the reef type, the depth, and the interaction among them. The greatest variation was found between species, followed by the interaction between species and the other factors (Table 2).

Table 2. PERMANOVA for type profiles with three factors, Species, Depth, and Reef Type.

The calculations were done following the Bray-Curtis method, after normalization with the fourth root of the counts, using 999 permutations with ADONIS function in R Vegan package (Oksanen et al., 2017).

Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)

Species 6 34.539 5.7566 20.953 0.24172 0.001

Depth 2 2.694 1.3469 4.9024 0.01885 0.001

Type 2 3.018 1.5088 5.4919 0.02112 0.001

Species:Depth 11 11.317 1.0288 3.7447 0.0792 0.001

Species:Type 11 8.943 0.813 2.9592 0.06259 0.001

Depth:Type 3 2.251 0.7502 2.7305 0.01575 0.001

Species:Depth:Type 11 7.322 0.6657 2.423 0.05125 0.001

Residuals 265 72.805 0.2747 0.50952

Total 311 142.889 1

4. Discussion

The temperature measurements showed evidence of a cross-shelf and temperature gradient, with the highest values in the shallow parts of inshore reefs and the lowest at

30m depth. The values are in the same range that the ones found between 7.5 and 9m in three of these same reefs by Roik et al. (2016) in 2012-2013, who also found higher temperatures in the inshore reef and similar values for a mid-shelf and offshore reef. This is in agreement with the bleaching pattern observed in 2010 at the central Red Sea, where the shallow parts and the inshore reefs had a higher bleaching impact than the offshore reefs and deeper parts (Furby et al., 2013). This pattern was thought to be related with temperature, and with these observations we can confirm that temperature is correlated to a depth and cross-shelf gradient, and that these differences become greater as temperature increases.

There was more variation in the Symbiodinium community explained by the host species than by the environmental variables. Between species, the ones more closely related shared more common types and had a more similar composition, especially at the clade level, although the type profiles differed in most cases. The three species belonging to the family Pocilloporidae (Pocillopora verrucosa, Stylophora pistillata, and Seriatopora hystrix) had mostly symbionts from clade A, with type A1 the most abundant in all cases.

This was also the case for Millepora dichotoma which, although it belongs to a different class, also exhibits a branching growth form. The A1 type, related with the species

Symbiodinium microadriaticum, has been found to be more tolerant to temperature 31 stress compared to types C1 and B1 (Hawkins et al., 2012). The A1 type has also been found to be dominant along the latitudinal gradient of the Red Sea in both summer and winter in Pocillopora verrucosa, presumably as the result of long-term acclimatization or adaptation of this symbiont to the conditions of the Red Sea (Sawall et al., 2014). With the dominance of this Symbiodinium type in other pocilloporid species and in Millepora dichotoma, this adaptation to the regional conditions of the Red Sea and a specific relation with certain hosts in this region becomes more evident.

Different defining intragenomic variants of A1 were found in each of the previously mentioned coral species, leading to differences in the type profiles, with some variants characteristic of samples in certain parts of the environmental gradients or particular reefs. Generally there is a high number of ITS2 variants, with one or two types dominant and several rare ones with low frequencies. Variants derived from the dominant ones are constantly generated, and rare variants are removed or replaced through concerted evolution, leading to evolutionary stability of the dominant variant among individuals of a genetically recombining population (Arif et al., 2014). The difference of this variants and the associated type profiles in the different species can reflect different Symbiodinium populations that can show specificity with particular host species. This can be limited to a small spatial scale like a reef for some cases, or have a wider distribution that can be limited by environmental factors such as depth or cross-shelf gradients.

In the case of Millepora there was a relation of the intragenomic variants with depth. At

1 and 15m for the inshore and offshore reefs this host species had mostly symbionts from 32 type A1k, while at 30m and at 15m for the offshore reefs, type A1 was dominant. This first variant and the associated profile type A1k/A1-A1b were only found in this species in 1 and 15m, which is a sign of specificity and might indicate that this type is related to a population with higher thermal tolerance, since it is absent in the deeper parts where the type A1-A1b takes over. Other species from this genus have been found associated with other types of clade A in the Indo-Pacific (Lajeunesse et al., 2003) and in the Caribbean, where they also harbor types from clade B (Finney et al., 2010), showing that the specificity of the types found is also related to the biogeographic region.

For P. verrucosa, there was not much difference between depths, sharing the same types in similar proportions for all cases. Some exceptions were the presence of types from clade C in all samples of Abu Madafi at 30m, which in this case could be more related to the reef than to the depth, and the presence of type A1j, and the related profile type

A1j/A1, only in the shallowest depth of the inshore reefs, indicating a possible relation of this type with tolerance to thermal stress. The amount of intragenomic variation can differ between species (Thornhill et al., 2007), and it appears to be greater in this species, which had more profile types than the others of the study. The most common one, which was present in most samples but absent in the rest of the species, had five different defining variants, A1-A1c-A1h-A1q-A1i. This can reflect a Symbiodinium population that has a wider distribution and tolerance to a wider range of environmental conditions, although physiological studies should be done to test this possibility. 33

Samples from S. hystrix and S. pistillata also showed characteristic intragenomic variants of A1, with A1r and A1u the most abundant for the former, and A1g, A1l, and A1s the most abundant for the latter. These species, which both have a brooding reproductive strategy, also had a noticeable representation from other clades, with types from clade C being dominant at 30m but almost absent at shallower depths, where type A1 was dominant. The association with clade C is the most common for these species in other parts of the Indo-Pacific (Loh et al., 2001; Lajeunesse et al., 2003), and maybe a more beneficial relation for the coral in lower temperature conditions, since this clade has been shown to be associated with higher growth rates and photosynthetic production compared to more thermally-resistant clade D (Cantin et al., 2009; Jones and Berkelmans,

2010). Moreover, in Taiwan, Keshavmurthy et al. (2014) found that S. hystrix and S. pistillata were the only coral species that did not show associations with clade D and were absent in a thermally stressful locality, which had temperature ranges similar to the ones found in the shallow depths of this study. This further indicates the importance of the role of A1 in the Red Sea, where it can allow these pocilloporid species to colonize environments with high temperature conditions, which might not be suitable for clade C symbionts.

For Porites, there was a symbiont community change with depth and a cross-shelf gradient, with around half of the samples in the inshore reefs having dominant types of clade D, and this clade being present in just one sample of a mid-shelf and one of the offshore reefs, while the deepest reefs contained only profile types with C15 and several 34 of its variants. Previous studies have found Porites to be associated with C15 throughout the Indo-Pacific (LaJeunesse, 2005; Keshavmurthy et al., 2014) and in the central Red Sea

(Ziegler et al., 2015). In this study, type C15 was also the most abundant and was present in most samples but it was almost absent in other species, while type D1 was only present in some samples from 1m and 15m. This confirms the host specificity of C15 found in previous studies, in which codistribution with Porites in all depths can be related with physiological traits of this symbiont that allows it to occur and perform photosynthesis even in shallow waters exposed to high light intensity, although having lower maximum photosynthetic yield than other types like C1 (Ziegler et al., 2015). Nonetheless, the association with type D1, especially in the inshore reefs, shows that the acclimation of this species does not rely only on the physiology of a specific symbiont type, but can be facilitated by harboring different symbiont types in more stressful conditions.

Galaxea and Gardineroseris show a very similar composition upon a first consideration of the defining intragenomic variants. However, a closer examination of the type profiles reveals that the two species have a different symbiont community. This was very specific for G. fascicularis, with the profile type C1/C39-C1b-C41-C1af-C39a being the most common in all reefs and depths, while it was only present in one sample of Gardineroseris.

Samples with types from clade D, characterized by a combination of variants of D1 and

D4 were found only in the inshore reefs and in Al-Fahal (a mid-shelf reef), while just one sample contained both clades. Galaxea fascicularis has been found associated with types

C1, C21, D1, and D1a throughout the Indo-Pacific, with type D1 limited to lower latitudes 35 and generally associated with higher water temperature locations (Huang et al., 2011;

Keshavmurthy et al., 2014). Although D1a had been found associated with deep parts of reefs (LaJeunesse et al., 2004), in this study it was only found at 1 and 15m, and in this case it appears to be more related with its tolerance to higher temperatures.

Nonetheless, the presence of type C1/C39-C1b-C41-C1af-C39a in all depths and reefs, whose defining variants had not been previously found on other regions, can indicate that populations with variants of C1, might allow the host to live in high temperature conditions with symbionts from this clade.

There was not a clear depth or cross-shelf gradient for the symbiont community of

Gardineroseris planulata, with types from clade C and D present in all depths at the mid- shelf and offshore reefs, and only one sample collected in the inshore reefs. The lack of samples in the inshore reefs, which could explain the absence of relation with the gradients, might be due to the high susceptibility to bleaching of this coral. This family was amongst the most affected in 2010, showing more than 75% bleaching in the inshore reefs (Furby et al., 2013). In other parts of the Indo-Pacific this species had been found associated with types from clade C, including C1 and other types not found in this study, but with no reports for clade D (Baker and Rowan, 1997; Fabina et al., 2013). This might be an indication that the presence of clade D types might be important for this species in the Red Sea, which according to the bleaching patterns might have lower thermal tolerance than the others. However, the presence of samples containing just types from clade C, especially combinations of C1, C39, and C116, and samples with combination of 36 both clades C and D, might indicate some adaptation of these specific types to the warmer conditions of the Red Sea, but that might not be enough to resist higher temperatures like the ones leading to bleaching events.

Since the samples for this study were collected during winter, when the water temperature is lower and shows less difference between depths and reefs, the symbiont community might be mostly represented by symbiont types that are not the most resistant to high temperatures. This could explain the low abundance of Symbiodinium types that are commonly known to be thermally resistant such as members of clade D, like D1, which might take over only in stressful conditions, and remain in low abundance the rest of the time (Tonk et al., 2014). Nonetheless, the very low abundances in species where clade A was predominant can indicate that the association with types from this clade, which is almost absent in other locations such as Caribbean (Finney et al., 2010;

Bongaerts et al., 2015), the Great Barrier Reef (Lajeunesse et al., 2003; Tonk et al., 2017), and the rest of the Indo-Pacific (Baker et al., 2004; Keshavmurthy et al., 2014), confers the host corals resistance to high temperature water conditions of the Red Sea.

Nonetheless, to clarify if the types found are adapted to the thermal variation along the year or if there is a switch of community when water temperature increases, the results from this study should be compared with a sampling done in summer for the same coral species in the same site.

5. Conclusions

In conclusion, this study showed that the host species determines the Symbiodinium community more than the environmental conditions. Although there are some generalist

Symbiodinium types and all coral species hosted more than one type of symbiont, host specificity of certain defining intragenomic variants and profile types was found for all species in this study, with some types or variants associated with certain depths, reefs, or reef types. The environmental gradients assessed were related to temperature as expected, with the shallower and inshore sampling points showing more variation along the year and higher values than the deeper and offshore ones.

There is a relation between the profile types, the host species and environmental gradients, which could reflect adaptation of certain symbiont populations or species to different conditions. For Millepora dichotoma and corals from the family Pocilloporidae, the association with Symbiodinium type A1, and the type profiles characterized by different defining intragenomic variants for each species, appears to play an important role in their survival at the higher temperature conditions of the reefs in the central Red

Sea, while for the rest of the species this is achieved by the association with different types from clades C and D.

This study indicates the importance of considering the intragenomic variation of the ITS2 region for defining the Symbiodinium diversity at a finer scale. The use of NGS sequencing and approaches like SymPortal to analyze this data can show ecological differences within 38 the established clades and, with further physiological studies, can lead to a better understanding of this group and its symbiotic relationship with corals.

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APPENDICES

Appendix A. Number of samples collected for each reef, depth and coral species.

Reef/Depth/Species # Samples Reef/Depth/Species # Samples Qita al Kirsh 75 Abu Madafi 66 15m 23 15m 25 Porites spp. 4 Porites spp. 5 Gardineroseris planulata 5 Gardineroseris planulata 3 Galaxea fascicularis 5 Galaxea fascicularis 5 Pocillopora verrucosa 5 Pocillopora verrucosa 5 Millepora dichotoma 4 Millepora dichotoma 5 1m 30 Stylophora pistillata 2 Porites spp. 5 1m 21 Gardineroseris planulata 5 Porites spp. 1 Galaxea fascicularis 5 Gardineroseris planulata 5 Pocillopora verrucosa 5 Galaxea fascicularis 5 Millepora dichotoma 5 Pocillopora verrucosa 5 Stylophora pistillata 5 Stylophora pistillata 5 30m 22 30m 20 Porites spp. 4 Porites spp. 5 Galaxea fascicularis 5 Galaxea fascicularis 5 Pocillopora verrucosa 5 Pocillopora verrucosa 4 Millepora dichotoma 4 Stylophora pistillata 5 Seriatopora hystrix 4 Seriatopora hystrix 1 Al-Fahal 27 Fsar 50 15m 17 15m 27 Porites spp. 4 Porites spp. 5 Gardineroseris planulata 3 Galaxea fascicularis 5 Galaxea fascicularis 5 Pocillopora verrucosa 4 Pocillopora verrucosa 5 Millepora dichotoma 4 1m 10 Stylophora pistillata 4 Gardineroseris planulata 1 Seriatopora hystrix 5 Galaxea fascicularis 2 1m 23 Pocillopora verrucosa 4 Porites spp. 5 Millepora dichotoma 3 Gardineroseris planulata 1 Shib Nazar 60 Galaxea fascicularis 5 15m 24 Pocillopora verrucosa 4 Porites spp. 5 Millepora dichotoma 4 Gardineroseris planulata 5 Stylophora pistillata 4 46

Galaxea fascicularis 5 Tahla 34 Pocillopora verrucosa 4 15m 17 Millepora dichotoma 1 Porites spp. 2 Stylophora pistillata 4 Galaxea fascicularis 5 1m 19 Pocillopora verrucosa 4 Porites spp. 3 Millepora dichotoma 3 Gardineroseris planulata 5 Seriatopora hystrix 3 Galaxea fascicularis 1 1m 17 Pocillopora verrucosa 2 Porites spp. 4 Millepora dichotoma 3 Galaxea fascicularis 2 Stylophora pistillata 5 Pocillopora verrucosa 5 30m 17 Millepora dichotoma 1 Porites spp. 4 Stylophora pistillata 5 Gardineroseris planulata 4 Galaxea fascicularis 4 Pocillopora verrucosa 2 Millepora dichotoma 1 Stylophora pistillata 2 Total 312

Appendix B. 100% Stacked bar charts comparing the Symbiodinium diversity, represented by profile types composed by characteristic defining intragenomic variants

(DIVs), between depths, 1, 15, and 30m, and reef type (inshore, mid-shelf, and offshore) for each host species. Coral species are mentioned in the left side of each graph and the profile types with their respective color codes are in the right side. 47

Seriatopora hystrix Seriatopora

Gardineroseris planulata Gardineroseris

Stylophora pistillata Stylophora

Porites spp. Porites

Galaxea fascicularis Galaxea

Pocillopora verrucosa Pocillopora

Millepora dichotoma Millepora