Significance Reef corals in the Persian/Arabian Gulf (PAG) withstand exceptionally high salinity and regular summer temperatures of ∼35 °C that kill conspecifics elsewhere. These thermotolerant communities established themselves within only ∼6,000 y under the pressure of rapid climate change and can therefore inform how other coral reefs may respond to global warming. One key to the thermotolerance of PAG corals is their symbiosis with Symbiodinium thermophilum. Phylogeographic evidence indicates that this symbiont represents a stress-tolerant subpopulation of an ancestral taxonomic group with surprising genetic diversity that exists at barely detectable levels outside the PAG. Our results highlight the critical importance of present-day biodiversity for future adaptation to climate change for coral reefs and ecosystems in general.

Abstract Coral communities in the Persian/Arabian Gulf (PAG) withstand unusually high salinity levels and regular summer temperature maxima of up to ∼35 °C that kill conspecifics elsewhere. Due to the recent formation of the PAG and its subsequent shift to a hot climate, these corals have had only <6,000 y to adapt to these extreme conditions and can therefore inform on how coral reefs may respond to global warming. One key to coral survival in the world’s warmest reefs are symbioses with a newly discovered alga, Symbiodinium thermophilum. Currently, it is unknown whether this symbiont originated elsewhere or emerged from unexpectedly fast evolution catalyzed by the extreme environment. Analyzing genetic diversity of symbiotic algae across >5,000 km of the PAG, the Gulf of Oman, and the Red Sea coastline, we show that S. thermophilum is a member of a highly diverse, ancient group of symbionts cryptically distributed outside the PAG. We argue that the adjustment to temperature extremes by PAG corals was facilitated by the positive selection of preadapted symbionts. Our findings suggest that maintaining the largest possible pool of potentially stress-tolerant genotypes by protecting existing biodiversity is crucial to promote rapid adaptation to present-day climate change, not only for coral reefs, but for ecosystems in general.

Episodes of heat stress cause coral bleaching, the breakdown of the obligate symbiosis between the coral host and its algal partner that contributes to the global decline of coral reefs (1). Despite their capacity to acclimate to rising seawater temperatures (2), corals will suffer from high-frequency bleaching episodes by the end of this century, threatening their survival in the warmer oceans of the future (3). Changing to symbiotic associations with more thermally tolerant types like Symbiodinium trenchii (synonym type D1a/D1–4) may increase the heat stress tolerance of corals rapidly (4⇓–6). However, due to tradeoffs such as reduced calcification rates that can be associated with hosting alternative symbionts, reef ecosystems may ultimately fail to benefit from the increased thermal tolerance of their most important habitat-forming species (5, 6). Hence, it is unclear at present whether alternative symbiont associations will rescue reefs from their expected demise in response to global warming.

We are using the Persian/Arabian Gulf (PAG), the world’s hottest sea, as a natural laboratory where coral communities endure regular summer temperatures of up to ∼35 °C to address the question of how coral reefs relying on heat-tolerant Symbiodinium may respond to rapid climate change over a prolonged period. Due to rising sea levels associated with the last glacial retreat, the modern PAG started to form ∼12,500 y before present (BP) by ingression of the Indian Ocean into the previously dry basin, extending to present-day shorelines ∼6,000 y BP (7). By this time, the climate in the Middle East began to change from cooler and moister to warmer and more arid, reaching today’s conditions only ∼4,000 y ago (8⇓–10). Hence, the coral communities of the PAG, composed mostly of a subset of Indian Ocean species (11), have had to adjust rapidly to temperatures not expected to occur in other parts of the world’s oceans before the next century (12). We recently discovered that corals of the southern, hottest region of the PAG form a prevalent, year-round association with a thermally tolerant symbiont species, Symbiodinium thermophilum (13). Although the symbiosis with S. thermophilum might not be the sole cause for the heat tolerance, and others factors including the host physiology (14) and environmental conditions (such as the exceptionally high salinity in the relevant PAG regions) (15) may contribute to the resilience of the holobiont, the striking dominance of this symbiont in the southern PAG strongly suggests that it represents a key component to the success of corals in this extreme environment.

S. thermophilum can be identified by specific intragenomic variants of the nuclear ribosomal second internal transcribed spacer region (ITS2), which carry an 8-bp duplication indel named “S. thermo.-indel” (13). In close to pure S. thermophilum populations, these variants represent an average ∼16% of the total ITS2 sequences, however this proportion can vary considerably (Fig. S1). Additionally, this symbiont can be distinguished by its genetically disparate resolution from other closely related ITS2 C3 types using the noncoding region of the chloroplastic psbA gene (psbAncr) among other markers (13). As yet undetected in other parts of the world, our previous work suggested that symbionts characterized by the S. thermo.-indel and a disparate psbAncr resolution (hereafter S. thermophilum group) may not be endemic to the PAG (15). Because the understanding of heat tolerance in coral–symbiont associations is crucial in gauging the adaptation potential of coral reefs to global warming, we investigated the origin of S. thermophilum to assess whether this thermotolerant symbiont emerged as the result of an unexpectedly rapid evolution under the pressure of the extreme environmental conditions in the PAG or whether this species originated elsewhere.

Fig. S1. Proportion of S. thermo.-indel–containing variants in total ITS2 sequences from C3 predominated cnidarians (n = 116) from the PAG. Cnidarians included in the analysis contained >50% clade C Symbiodinium, of which >95% were C3 or closely related variants. Average % S. thermo.-indel–containing amplicons: 16.6%.

Methods Collection and DNA Extraction of Samples. Cnidarian samples from 46 genera were collected at 23 reefs in the PAG, the Gulf of Oman, and the Red Sea. Sampling locations were categorized as follows: water bodies (the PAG, the Gulf of Oman, and the Red Sea), sampling regions (made up of one or more reef), and reefs. A detailed list of the sampling locations and number of cnidarians collected at each site can be found in Table S1. All samples were collected by SCUBA (self-contained underwater breathing apparatus) diving with ∼1 cm2 of tissue sampled from the surface of each cnidarian. Samples collected at Eilat were placed into 5 mL of RNAlater, whereas all other samples were flash-frozen in liquid nitrogen before storage at –20 °C. Genomic DNA (gDNA) was extracted as described for environmental samples in Arif et al., 2014 (32), except for samples collected at Eilat. For Eilat samples, host and symbiont gDNA was extracted using a cetyl trimethylammonium bromide (CTAB) extraction. Before extraction, each of the samples was washed with 96% (vol/vol) ethanol to remove the majority of RNAlater storage buffer to minimize coprecipitation of salts during the DNA precipitation step of the extraction. Samples were then frozen in liquid nitrogen before being added to 1 mL of CTAB extraction buffer [2% (wt/vol) CTAB; 1.4 M NaCl; 0.5% 2-β-mercaptoethanol; 2% (wt/vol) polyvinylpyrrolidone (PVP); 20 mM EDTA; 100 mM Tris⋅HCl, pH 8.0] and beaten using a 5-mm stainless steel ball in a Tissue Lyser II (Qiagen) at maximum speed until the sample was completely homogenized. Samples were incubated at 60 °C for 30 min before three extractions in 1 mL of chloroform:isoamyl alcohol (IAA) (24:1), phenol:chloroform:IAA (25:24:1), and chloroform:IAA (24:1) with centrifugation after each extraction. The supernatant was added to an equal volume of isopropanol before incubation at –20 °C for 2 h. The DNA was pelleted through centrifugation before being washed in 750 µL of 96% ethanol and centrifuged. Supernatant was removed and pellets were dried before suspension in 50 µL of ddH 2 O. All centrifugation steps were carried out at 20,000 × g for 5 min at 4 °C. ITS2 Genotyping of Symbiodinium spp. Harbored by Coral Samples. The Symbiodinium nuclear ribosomal ITS2 region of all samples except those collected at Eilat was sequenced by 454 and MiSeq sequencing as detailed (32). The Symbiodinium ITS2 region of the Eilat-collected samples was sequenced by direct PCR sequencing (services provided by Eurofins MWG) using the internal primer SYM_VAR5.8SII (13) on the 18S-ITS1-5.8S-ITS2-28S amplicon amplified by SYM_VAR-FWD and SYM_VAR_REV as detailed (33), with the exception of an annealing temperature of 56 °C. Results of this sequencing revealed a mix of C15-cluster ITS2 sequences. Given that S. thermophilum group symbionts are characterized by an ITS2 type C3, these samples were excluded from further analysis. Screening of Corals for Associations with S. thermophilum Group. To identify the presence of S. thermophilum group symbionts, all corals were screened for the characteristic 8-bp indel sequence described in Hume et al., 2015 (13). To incorporate the possibility of PCR error and the existence of possible genetic variants, sequences 1 bp different (substitution only) from the originally described 8-bp sequences were also included in the results (hereafter referred to as the S. thermo.-indel) as indicative of the S. thermophilum group. Symbiont complements identified as having at least one ITS2 amplicon containing the S. thermo.-indel (hereafter referred to as S. thermo.-indel amplicons) were further categorized according to whether such amplicons made up more than or less than 1% of the total Symbiodinium amplicon sequences found in that organism (Fig. 1A). S. thermo.-indel–containing amplicons made up between 2% and 41% of the C3 type ITS2 amplicons in S. thermophilum samples (i.e., a coral sample hosting 100% S. thermophilum would likely have between 2% and 41% ITS2 amplicons containing the S. thermo.-indel; Fig. S1). As such, the 1% cutoff used in this study could potentially represent a 50% complement of S. thermophilum group symbionts, whereas symbionts containing >15% have a high likelihood of containing a close to pure complement of an S. thermophilum group symbiont. Verification of S. thermophilum Group by PCR Amplification, Sequencing, and Phylogenetic Analysis of the psbAncr. To validate the successful identification of the S. thermophilum group by the presence of the S. thermo.-indel, samples from the Gulf of Oman and Red Sea that contained more than 25% clade C ITS2 sequences and in which S. thermo.-indel amplicons made up more than 1% of the clade C sequences had the psbAncr analyzed by direct PCR sequencing as detailed in Hume et al., 2015 (13). Chromatograms were checked manually for miscalls. Chromatograms with multiple peaks were first assessed to determine whether the multiple peaks could be explained by a reading frame shift caused by indels by calling secondary peaks using the software Geneious 5.1.7 (www.geneious.com) before attempting to resolve indels using Indelligent 1.2 (dmitriev.speciesfile.org/indel.asp). If the multiple peaks could be resolved in this way, the majority sequence was associated with that sample. If multiple peaks were not explained by such “indel analysis,” chromatograms were characterized by no more than two peaks at each nucleotide location, and multiple peak locations clearly showed a predominant and lesser abundance of called nucleotide, then these predominant and lesser calls were used to identify a primary and secondary sequence, respectively. In this case, the primary sequence was associated with the sample. In cases where multiple-peaked chromatograms could not be explained by indel analysis or by identifying primary and secondary sequences, the sample genotype was not used in further analysis (1 out of 10 samples). Nine samples that successfully returned psbAncr sequences were aligned manually with additional sequences from the psbAncr alignment created by Hume et al., 2015 (13) that contained sequences from corals collected within the PAG and C3 radiation sequences (17) collected external to the PAG. Phylogenetic analysis was conducted by Bayesian inference using Mr. Bayes 3.2.2 (mrbayes.sourceforge.net/). Phylogenies were estimated using the Jukes–Cantor (JC) model with a gamma-shaped distribution (+G) with invariable sites (I) (according to Akaike Information Criterion using MEGA6; www.megasoftware.net/). Markov chain Monte Carlo (MCMC) analyses were run for 2.0 × 106 generations (SDs of split frequencies < 0.05), sampling every 1,000 generations. A relative burn-in of 0.25 was used in calculating a 50% majority rule consensus tree. To compare psbAncr genetic diversity between the S. thermophilum group and the two most divergent ITS2 types (C40 and C27) that resolve within the C3-radiation psbAncr phylogeny, as well as between S. thermophilum group sequences found within and external to the PAG, pairwise genetic distances and within-group genetic distance were calculated in MEGA6 using the JC model +G (Table S4). Variance was determined to be equal (F test) before conducting a two sample Student’s t test to compare the within-group genetic distances of the samples from sites internal and external to the PAG. Taxonomic Position of S. thermophilum Group Within Symbiodinium Clade C. To elucidate the taxonomic position of the S. thermophilum group within clade C, a selection of S. thermophilum group, ITS2 type C3, and C41 and C39 (two of the top four numerically common subclades) harboring corals (including some corals under culture at the Coral Reef Laboratory Experimental Mesocosm Facility) (34) had their symbiont complement genotyped with six additional genetic markers (35): the nuclear ribosomal large subunit (nr28S), the nuclear elongation factor 2 (elf2), the chloroplastic ribosomal large subunit (cp23S) domain V, the coding region of the plastid-encoded photosystem II protein D1 (psbAcds), the mitochondrial cytochrome oxidase I (coi), and the mitochondrial cytochrome b (cob). To assess fine-scale taxonomic resolution, the psbAncr was also amplified. Specifically, the following coral samples underwent this additional genotyping: all samples in the Gulf of Oman and the Red Sea characterized as S. thermophilum by psbAncr genotype and containing >99% clade C ITS2 sequences (eight samples); a selection of 23 samples collected in the PAG as part of studies by Hume et al., 2015 (13) and D’Angelo et al., 2015 (15), representing samples collected over >400 km within the PAG and as resolving in different positions within the psbAncr phylogeny of D’Angelo et al., 2015 (15); samples collected in the Red Sea and Gulf of Oman harboring ITS2 type C3 as their numerically dominant ITS2 amplicon and containing no S. thermo.-indel amplicons (three samples; OMD001, OMD002, and YBA008; Table S3); three ITS2-type C3-harboring Euphyllia spp. corals currently in culture at the Coral Reef Laboratory Experimental Mesocosm Facility (34) but originating from Indo-Pacific waters in proximity to Bali (EU2, EU3, and EU4; Table S3); and finally, four corals containing predominant ITS2 variants for C41 and C39 (representing the first and fourth most common ITS2 variants sampled, respectively) collected in the Red Sea and the Gulf of Oman (Table S3). All PCR conditions were as for the SYM_VAR_FWD/SYM_VAR_REV primer pair with cycles as detailed in Pochon et al., 2014 (35), except for the psbAncr region that was amplified according to Hume et al., 2015 (13). Sequences were attained through direct PCR sequencing, services provided by Eurofins, using forward (elf2, psbAcds, coi, and cob) and reverse (nr28S and cp23S) primers. Sequences from this study were added to a selection of reference sequences (Table S5) representing members from clades A–I, including subcladal C1, C15, C90, and C91, as well as sequence collections from Gymnodinium simplex and Polarella glacialis as outgroups. Sequences returned from the six additional genetic markers (i.e., not psbAncr) were aligned in MEGA 6 with the ClustalW algorithm and checked by eye. Hypervariable regions that prevented robust alignment were removed from the cp23S alignment sensu Pochon et al., 2006 (16). Phylogenies were estimated both from individual markers and from a concatenated supermatrix of all six markers. Phylogenies were estimated by Bayesian Inference in Mr. Bayes 3.2.2 using the following nucleotide substitution models: nr28S, Kimura 2-Parameter (K2) +G; Elf2, K2 +G +I; cp23S, Hasegawa Kishino Yano (HKY) +G; psbAcds, Generalized Time Reversible +G; coi, HYK +G; cob, HKY +G. The supermatrix analysis was partitioned with the separate nucleotide models being used for each marker’s region. MCMC analyses were run for 1.0 × 106 generations, sampling every 500 generations. A relative burn-in of 0.25 was used in calculating a 50% majority rule consensus tree. Alignments of the psbAncr sequences returned from the ITS2 type C3 (non-S. thermo.-indel–containing), C41, and C39 samples were not possible between ITS2 types from this study and with previous alignments by Hume et al., 2015 (13) (C3/S. thermophilum radiation) and Thornhill et al., 2014 (17) (C1 radiation) due to the dissimilarity of the sequences.

Acknowledgments We appreciate the help of Cornelia Roder, Sergey Dobretsov, Julia Schnetzer, Todd LaJeunesse, and Drew Wham with sample collection. A. Al-Hemeri (UAE Federal Environment Agency), A. Al-Cibahy (Environment Agency of Abu Dhabi), and the Oman Ministry of Environment & Climate Affairs kindly provided Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) export permits (no. 09FEA555) and collection permits. We acknowledge Tropical Marine Centre (London) and Tropic Marin (Wartenberg) for sponsoring the Coral Reef Laboratory at the University of Southampton. We thank the NYU Abu Dhabi Institute for supporting the 2012/2013 field workshops during which samples for this study were collected and the Interuniversity Institute for Marine Sciences in Eilat for field work support. The study was funded by Natural Environment Research Council Grant NE/K00641X/1 (to J.W.), the European Research Council under the European Union’s Seventh Framework Programme Grant FP7/2007-2013/ERC Grant Agreement 311179 (to J.W.), the King Abdullah University of Science and Technology (C.R.V.), and Israel Science Foundation Grant 341/12, United States Agency for International Development/Middle East Regional Cooperation (USAID/MERC) No. M32-037 (to Y.L.).

Footnotes Author contributions: B.C.C.H., C.R.V., and J.W. designed research; B.C.C.H., C.R.V., and C.A. performed research; B.C.C.H., C.R.V., J.A.B., G.E., Y.L., and J.W. performed field work; B.C.C.H., C.R.V., C.A., C.D., and J.W. analyzed data; and B.C.C.H., C.D., and J.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. KR996268–KR996464 and KT156647–KT156665).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1601910113/-/DCSupplemental.