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Study site and ecology of Orbicella annularis Colonies of Orbicella annularis were first sampled August 12th and 16th 1994 for DNA fingerprinting.\u00a0 In 1994, biopsies of tissue and skeleton were collected in two photoquadrats using a 13-mm diameter steel punch.\u00a0 To prevent cross-contamination, a sterile punch was used for each colony and each biopsy bagged individually underwater.\u00a0 Care was taken to sample biopsies from the horizontal upper surfaces of colonies, and the sampled holes were filled with non-toxic modeling clay.\u00a0 Photoquadrats were then photographed to allow sampled colonies to be located again and to evaluate the effects of sampling on these colonies.\u00a0 Biopsies were transported to the lab on ice (~30 min), and crushed in 2 ml of ice-cold guanadinium hydrochloride (GHCl) buffer (8 M GHCl, 0.1M sodium acetate (pH 5.2), 5 mM dithiothreitol, 0.5% N-lauryl sarcosine.\u00a0 After grinding, the slurry was stored at 4\u00b0C and transported at room temperature to California State University, Northridge (CSUN).\u00a0 Some samples were processed, while others were stored at 4\u00b0C until 2010.<\/p>\n Between August 5th and 8th 2010 (i.e., virtually the same time of year as in 1994) the same colonies of Orbicella annularis biopsied in 1994 were sampled with the objectives of genotyping the coral host and their Symbiodinium.\u00a0 To ensure identical colonies were sampled, the annual photoquadrats were used to track over time the shape, color, and orientation of the colonies first sampled in 1994.\u00a0 Based on the capacity to relocate colonies for which archived DNA from 1994 was available, and the constraints of permits issued by the Virgin Island National Park, 12 colonies were tracked from 1994 to 2009.\u00a0 In 2010, laminated prints of the photoquadrats were used underwater to locate the same colonies for a second sampling, and to ensure that the sampling orientations were the same as employed 16 years earlier.\u00a0 Sampling in the same position on each colony was important to reduce the likelihood that variation in Symbiodinium genotypes was caused by sampling different microhabitats across the colony surface.\u00a0 Our analysis therefore assumes that differences in Symbiodinium genotypes between the two samplings reflected the passage of time and not differences in sampling position within each colony.\u00a0<\/p>\n In 2010, the 12 colonies of O. annularis were sampled using 6-mm diameter punches, and the holes filled with non-toxic modeling clay.\u00a0 A sterile punch was used for each colony to prevent cross-contamination, and biopsies were bagged individually underwater, stored on ice, and returned to the lab for processing.\u00a0 Biopsies were stored in DNA extraction buffer (50% (w\/v) guanidinium isothiocyanate, 50 mM Tris pH 7.6, 10 \uf06dM ethylenediaminetetraacetic acid (EDTA), 4.2% (w\/v) sarkosyl, and 2.1 % (v\/v) \uf062-mercaptoethanol) and shipped at room temperature for processing at the University of Hawaii (Symbiodinium) or Florida State University (O. annularis host).<\/p>\n To evaluate growth, each of the 12 colonies was measured in the annual photoquadrats.\u00a0 Most colonies were found every year, although occasionally it was not possible to measure their size if they were obscured by the camera framer or gorgonians.\u00a0 Colonies were measured using ImageJ 1.42q software to outline coral tissue and calculate planar area (cm2).\u00a0 Each colony was tracked over 16 y and the trajectory of changing size used as a measure of success.\u00a0 Success of the 12 colonies was placed in a broader context by evaluating overall coral cover using the 30 photoquadrats sampled annually.\u00a0 Coral cover was measured using CPCe software and a grid of 200 randomly located points on each image.<\/p>\n DNA extraction Symbiodinium analysis We PCR amplified the nuclear Internal Transcribed Spacer 2 (nrITS2) region and the chloroplastic 23S domain V (cp23S) region of the ribosomal arrays using the nrITS2 primers \u2018its-dino\u2019 and \u2018its2rev2\u2019 and the cp23S primers \u201823S4F\u2019 and \u201823S7R\u2019 (after Stat et al. 2009, Pochon & Gates 2010).\u00a0 Each 50 microliters PCR reaction contained 1 microliters of DNA template, 5 microliters of 10x ImmoBuffer (Bioline, MA), 0.2 microliters IMMOLASE\u2122 Hot-Start DNA Polymerase (Bioline, MA), 2 microliters of 50 mM MgCl2, 1 microliters of 10 mM total dNTPs (10 mM each), 1 microliters of each primer (10 pmol each), and 38.8 microliters of deionized sterile water.\u00a0 A touchdown PCR protocol was performed on a BioRad iCycler\u2122 using the following conditions: 7 min at 95 degrees C, 20 cycles of 94 degrees C for 30 s, 62 degrees C for 30 s (decreased by 0.5 degrees C at each cycle), 72 degrees C for 1 min, and followed by 12 additional cycles with an annealing temperature set at 52\uf0b0C, and a final extension of 72\uf0b0C for 7 min.\u00a0 PCR products were puri\ufb01ed using the QIAquick\u2122 PCR Puri\ufb01cation Kit (Qiagen), and ligated into the pGEM-T Easy vector\u2122 (Promega).\u00a0 A minimum of 10 positive inserts per clone library were ampli\ufb01ed using plasmid-speci\ufb01c (M13) primers, and sequenced in both directions using the ABI Prism Big Dye\u2122 Terminator Cycle Sequencing Ready Reaction Kit and an ABI 3100 Genetic Analyzer (Perkin-Elmer Applied Biosystems).\u00a0 Bi-directional sequences were inspected and assembled using Sequencher v4.7 (Gene Codes Corporation, Ann Arbor, MI, USA), aligned using BioEdit v7.0.5.3, and Symbiodinium were identified to clade and subclade level using the Basic Local Alignment SearchTool (BLAST) in nrITS2 and cp23S databases generated from sequences archived in GenBank.\u00a0 This database is Supplementary Material as a fasta file.<\/p>\n The patterns of dominant-cloned Symbiodinium sequence types (i.e., the most abundant sequences in each sample) obtained using nrITS2 and cp23S were similar, but there were a few differences between the abundances in the cloned sequence numbers.\u00a0 This suggested that too few clones had been sequenced to fully capture the diversity of rare Symbiodinium sequence types.\u00a0 To address this issue, all DNAs were re-analyzed using multiplexed tag pyrosequencing of the nrITS2 amplicons (described as pyrosequencing hereafter).\u00a0 This method, pioneered in the field of microbial ecology, is replacing cloning and DGGE as the technique of choice for addressing diversity (Huse et al. 2008), and has been successfully employed for characterizing cryptic Symbiodinium diversity within O. faveolata and O. franksi in the Gulf of Mexico.\u00a0 We used pyrosequencing to increase the mean number of Symbiodinium sequences acquired per coral sample from ~10 to >1300.\u00a0 For pyrosequencing, 24 nrITS2 Symbiodinium amplicon libraries were submitted to Research and Testing Laboratory, LLC (Texas, USA) for analysis.\u00a0 Pyrosequencing involved ligating sample-specific tags for each of the 24 samples during the ITS2 amplification (cycling conditions described above, amplified separately from clone libraries), followed by amplicon purification, emulsion PCR, and high-throughput sequencing on a Roche GS FLX pyrosequencing system.\u00a0<\/p>\n As described above, we acknowledge that ITS2 does not provide the correct characteristics for use as an alpha diversity marker.\u00a0 Prior work has recognized the inability to link an individual biological entity to an individual sequences due to duplication in the ribosomal array resulting in the production of paralogs that preclude species assignment.\u00a0 ITS2 can however, be utilized in a comparative approach to determine patterns in the assemblage of sequences within a given sample.\u00a0 Our approach is primarily focused on identifying genetic variation over time and not to assign taxonomy to the sequences identified.\u00a0 Reconciling both ecological patterns and taxonomic identify for Symbiodinium must be a high priority if the full biological significance of Symbiodinium diversity is to appreciated for coral holobionts.<\/p>\n Bioinformatic analysis of pyrosequencing results Reads that were successfully assigned to a clade were subsequently compared using the Basic Local Alignment SearchTool (BLAST) in an nrITS2 database generated from sequences archived in GenBank and assigned the subtype of the reference with which they were most similar (97% similarity over 97% of their length).\u00a0 Similar to the clade assignment stage, the non-ambiguity requirement was enforced by requiring that the first hit have a higher raw bit score than that of the second hit.\u00a0 The sequencing reads lacking pairwise similarity with database entries were classified as putatively new and were manually investigated for known systematic biases.\u00a0 Short sequences were dropped from the analysis, while ambiguous sequences aligning with similar quality to two or more subtypes were assigned to the lowest common ancestor node in the phylogenetic tree of its clade and reported to clade level only.<\/p>\n
\nThe time-series analysis on the Tektite reef is based on three parallel 10-m transects along which 10 contiguous photoquadrats (1 x 1 m) are recorded annually (n = 30 photoquadrats y-1).\u00a0 Transects cross the flat upper surface of a single coral buttress and do not depart appreciably from 14-m depth across their length.\u00a0 Images were acquired on 35 mm film (Kodachrome 64) from 1987-2000, and digitally from 2001-2004 (3.34 megapixel), and 2005-2010 (6.1 megapixels). Images are archived (http:\/\/mcr.lternet.edu\/vinp\/overview\/<\/a>), and have been analyzed for percentage cover of the benthic community using CPCe software.\u00a0 In addition to photoquadrats, seawater temperature has been recorded in Great Lameshur Bay since 1989, initially at 9-m depth at Yawzi Point (~1 km away from the Tektite reef) from 1989 to 2011, and at the 14 m Tektite site since 2004.\u00a0 The temperature records used here include values from Yawzi Point from 1994-2003, and from Tektite from 2004-2009.\u00a0 Temperatures were recorded using a Ryan Industries Tempmentor (\u00b1 0.3\u00b0C [Ryan Industries, Redmond, WA]) from January 1992 to April 1997 and November 1997 to August 1999, an Optic Stowaway logger (\u00b1 0.2\u00b0C accuracy [Onset Industries, Bourne, MA]) at 9-m depth from May 1997 to October 1997, and from August 1999 to August 2001, and an Aquapro Logger (\u00b1 0.2\u00b0C accuracy [Onset Industries, Bourne, MA]) at 9-m depth from August 2001 to August 2010.\u00a0 Loggers recorded temperature every 15-30 mins and these data were collapsed by day, and described using the mean and interquartile ranges by year.<\/p>\n
\nGenomic DNAs (coral host and Symbiodinium combined) were extracted following Pochon et al. (2001).\u00a0 The 1994 samples were vortexed and 200 microliters of supernatant placed in tubes with 400 microliters of guanidinium buffer.\u00a0 These tubes, as well as the crushed coral biopsies from 2010, were incubated at 72 degrees C for 20 min, centrifuged at 16,000 g for 5 min, and 300 microliters of the supernatant mixed with an equal volume of isopropanol and incubated at -20 degrees C overnight.\u00a0 DNA was precipitated by centrifugation at 16,000 g for 15 min, and the DNA pellet washed in 70% ethanol, resuspended, and stored in Tris Buffer (0.1 M pH 8).<\/p>\n
\nTwo genes from different cellular compartments (nuclear and chloroplastic), and two analytical approaches (cloning and pyrosequencing) were used to genotype the Symbiodinium in the 24 coral biopsies (12 each from 1994 and 2010).\u00a0 These approaches added the support of multiple markers and methodologies to tests of our hypotheses.\u00a0 Moreover, pyrosequencing provided more resolution to the analysis of temporal variation in Symbiodinium assemblages and its effects on holobiont performance in comparison to studies employing fingerprinting gel-based analysis.\u00a0 Previous studies have focused on banding patterns generated by using gel electrophoresis to separate DNA from Symbiodinium based on denaturing gradients (i.e., DGGE fingerprints), and while such approaches have good resolution for identifying dominant patterns, they have limited capacity to more finely resolve genetic variation or to detect rare genotypes.\u00a0 While interpreting the biological significance of rare Symbiodinium genotypes remains controversial in light of the presence of intragenomic variation associated with ITS2, the increasing number of examples of Symbiodinium-host symbioses in which rare Symbiodinium genotypes have been found argue strongly for using techniques with the resolution to detect such forms.\u00a0 This argument was compelling to us in the decision to expand our analytical approach to tag pyrosequencing of nrITS2 amplicons, as was the success of this approach in advancing other fields requiring fine-grained genetic resolution of single-celled organisms (Sogin et al. 2006).\u00a0 An important limitation of this early-adoption of new techniques is we assume that the debate over the meaning of rare Symbiodinium genotypes will resolve in favor of their biological significance.<\/p>\n
\nThe 454 sequencing reads were first quality trimmed to a T threshold of 25, clustered using USEARCH (Edgar 2010), chimera-checked using UCHIIME, and de-noised based on a Quality score of 30 using a standard data analysis pipeline at the Research and Testing Laboratory, LLC.\u00a0 The full fasta file was demultiplexed and adapter-trimmed and subsequently filtered to remove any terminal regions with a PHRED quality score below 20.\u00a0 Sequences were filtered for length > 180 bp and maximum number of errors of 1 in forward primer, and trimmed of barcode, adapters, and forward and reverse primers with Integroomer (http:\/\/courge.ics.hawaii.edu\/inte\/groomer\/<\/a>).\u00a0 A Symbiodinium-specific bioinformatic pipeline (symTyper; M Belcaid unpublished method, https:\/\/github.com\/bingo11\/symTyper<\/a>) was then employed to assign each Symbiodinium sequence an identity. Briefly, using HMMER v3 (http:\/\/hmmer.org\/<\/a>), the resulting sequences were compared to a database of Symbiodinium clade HMM profiles, which were generated from the ITS2 database.\u00a0 The sequencing reads were subsequently compared to each clade-specific profile and a read was assigned to clade based on two rules: 1) the alignment was significant (e-value \u2264 1e-20 and sequence alignment over 95% of the read) and 2) the sequences was unlikely to have originated in another clade (e-value for the first hit was at least 5 orders of magnitude smaller than that of the second hit).\u00a0 Sequences failing the first rule were classified as unknown, whereas the sequences failing the second rule were considered ambiguous.\u00a0<\/p>\n