You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction
============

Reef-building corals (Scleractinia) show diverse coloration ([@evy122-B17]). It has been proposed that fluorescence is a major determinant of color in corals ([@evy122-B7]; [@evy122-B12]; [@evy122-B21]). The sources of fluorescence in corals are fluorescent proteins (FPs), which emit visible light, and chlorophyll-a from symbiotic dinoflagellate algae, which emit near infrared light ([@evy122-B20]; [@evy122-B18]). FPs are excited by environmental light and emit a longer wavelength than the excitation spectrum ([@evy122-B11]). The color of an FP is determined by its amino acid sequence ([@evy122-B8]). The center of the region determining light emission in FPs comprises the tripeptide "--X--Y--G--" (termed a chromophore), where the first peptide X varies among FPs ([@evy122-B10]). Based on the emission spectra, FPs are classified into four groups: cyan (CFP), green (GFP), yellow (YFP), and red (RFP) ([@evy122-B15]; [@evy122-B1]). Nonfluorescent chromoprotein (CP) is also classified as an *FP* gene family member based on its amino acid sequence similarity ([@evy122-B15]).

*FP* genes show signatures of diversifying selection, suggesting that FPs with different spectral ranges may have distinct roles in corals ([@evy122-B8]). Although many biological roles of FPs have been predicted, the biological roles of FPs with different spectral properties remain unclear. Originally, a photo-protective role of FPs was proposed ([@evy122-B27]), and roles in photo-protection in algal symbionts have subsequently been proposed for RFP ([@evy122-B9]) and CP ([@evy122-B31]). An antioxidant role has been suggested for CFP, GFP, RFP, and CP; however, FP mutants lacking a chromophore show greater antioxidant activity than that of wild-type FP ([@evy122-B22]).

*Acropora* is one of the most abundant coral genera in coral reefs of the Indo-Pacific region ([@evy122-B34]) and the whole genome of *Acropora digitifera* has been decoded ([@evy122-B28]). The natural variation of FPs provides an opportunity to analyze the biological role of FPs. FP-mediated color polymorphisms within *Acropora* species have been reported. For example, the photo-inhibitory effect is thought to be substantially weaker in *Acropora palifera* individuals with the fluorescent phenotype than in those with the nonfluorescent phenotype ([@evy122-B27]). *A. aspera* with the blue phenotype is less sensitive to changes in light than *A. aspera* with the light blue or cream phenotype at normal temperatures, but *A. aspera* with the blue phenotype is more sensitive to increases in temperature ([@evy122-B6]). In *A. nobilis*, individuals with the blue phenotype show greater zooxanthellae and photosynthetic pigment concentrations than those of individuals with the brown phenotype ([@evy122-B31]). In *A. valida*, individuals with the purple phenotype show a weaker photo-inhibitory effect than individuals with the brown phenotype ([@evy122-B31]). In vitro analyses have demonstrated that CP affects the algal chlorophyll excitation spectrum, supporting a role for CP in the photo-protection of zooxanthellae ([@evy122-B31]). However, CP expression is not vital for shallow-water corals, as evidenced by the adjacent growth of individuals with different color phenotypes in a shallow-water reef ([@evy122-B31]). In *A. millepora*, a fluorescence polymorphism has been observed in both larval and adult stages ([@evy122-B13]; [@evy122-B9]; [@evy122-B32]). Redder *A. millepora* larvae exhibit diapause-like physiological characteristics and are less responsive to settlement cues than greener larvae, suggesting that red fluorescence might be a marker of long-range larval dispersal ([@evy122-B13]; [@evy122-B32]). Adult *A. millepora* exhibit high, medium, and low redness levels under the same light levels in shallow water, and the expression levels of RFP are correlated with reduced photo-damage of zooxanthellae under light stress ([@evy122-B9]). These expression levels are correlated with the number of particular *RFP* sequences with a particular promoter type ([@evy122-B9]). Although color polymorphisms mediated by different FPs have been reported in many *Acropora* species, the genetic differences have only been analyzed for *RFP* sequences in *A. millepora*.

Several *FP* sequences have been reported for a single species in the genus *Acropora* ([@evy122-B1]; [@evy122-B2]; [@evy122-B9]). In *A. digitifera*, the *FP* multigene family has been comprehensively described. More than 30 *FP* gene copies per individual have been reported in *A. digitifera*, and these copies can be separated into short-/middle-wavelength emission (S/MWE including CFP and GFP), middle-/long-wavelength emission (M/LWE including GFP and RFP), and chromoprotein (CP) clades ([@evy122-B33]). All known FPs from *Acropora* species belong to one of these three clades in the *FP* multigene family ([@evy122-B33]). Although *FP* gene copy numbers and several *FP* sequences have been reported in *A. digitifera*, the sequences in this family have not been comprehensively identified. It is necessary to determine all *FP* gene sequences in each individual to understand the genetic basis of fluorescence polymorphisms and the biological roles of FPs.

In this study, we focused on exon 3 of the *FP* gene, because it is the longest exon and contains nucleotides coding for a chromophore. We examined all exon 3 sequences from *FP* genes and their expression patterns in adult and larval *A. digitifera.* The presence and absence of highly expressed *FP* sequences contributed to the total expression differences among individuals.

Materials and Methods
=====================

Specimen Collection and Species Identification
----------------------------------------------

A branch fragment was collected from each of 11 *A. digitifera* colonies in Sesoko, Okinawa, Japan. In total, 11 coral fragments (sample ID: S1601--08 and S1610--12) were preserved in RNAlater (Thermo Scientific, Waltham, MA). In addition, five different colonies of *A. digitifera* (sample ID: S1401--05) were collected from the field and subsequently maintained at the Sesoko Station aquarium (Tropical Biosphere Research Center, University of the Ryukyus). Bundles of gametes from each of five colonies of *A. digitifera* were mixed to allow fertilization. Larvae were reared by daily transfer to fresh seawater and maintained at ∼26°C. A single larva was preserved in RNAlater (Thermo Scientific). Species were identified based on morphology. This study was approved by the Aquaculture Agency of Okinawa Prefecture (permit numbers 26-9 and 28-31).

DNA Extraction and Preparation of DNA Libraries for Hybridization
-----------------------------------------------------------------

Genomic DNAs were extracted from 12 coral fragments of *A. digitifera* (sample ID: S1403, S1601--08, and S1610--12) using DNeasy Blood & Tissue Kits (QIAGEN, Hilden, Germany). Genomic DNAs were fragmented to 500 bp using the Covaris M220 (Covaris, Inc., Woburn, MA). Following the manufacturer's instructions, DNA libraries of 11 *A. digitifera* samples (sample ID: S1601--08 and S1610--12, [supplementary table S1](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online) were constructed using the TruSeq Nano DNA Library Preparation Kit (Illumina, Inc., San Diego, CA).

Preparation of Hybridization Probes for *FP* Gene Capture
---------------------------------------------------------

The *FP* gene family in *A. digitifera* can be divided into three clades (S/MWE, M/LWE, and CP) ([@evy122-B33]). *FP* gene sequences were amplified using TaKaRa Ex Taq (Takara, Shiga, Japan) and the following primer sets: MiA_MWEe2_F1 and AdiFP2XbaI_R\_L for the *FP* gene in the S/MWE clade, MiA_MLWEe2_F3 and MiA_MLWe5_R2 for the *FP* gene in the M/LWE clade, and AdiFP8L_KpnIF_L and AdiFP8XbaI_R2_L for the *FP* gene in the CP clade. All primer sequences are shown in [supplementary table S2](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online. Genomic DNAs of *A. digitifera* (sample ID: S1403) and *Acropora* sp. were used as templates. PCR was performed using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). PCR conditions for the amplification of *FP* gene sequences were as follows: denaturation for 3 min at 94°C, followed by 35 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 50°C, and extension for 3 min at 72°C.

Following the manufacturer's instructions, *FP* gene sequences in all three clades were labeled with biotin using the *Label* IT Biotin Labeling Kit (Takara). Biotin-labeled DNA fragments were used as hybridization probes.

*FP* Gene Capture
-----------------

*FP* gene capture was performed using the SeqCap EZ Hybridization and Wash Kit (Roche, Basel, Switzerland), SeqCap EZ Accessory Kit v2 (Roche), SeqCap HE-Oligo Kit (Roche), and SeqCap EZ Pure Capture Bead Kit (Roche) following the manufacturer's instructions for SeqCap EZ Library SR (Roche), with minor modifications. Briefly, Biotin-labeled *FP*-gene fragments were used as hybridization probes, instead of the SeqCap EZ library (Roche). Equal amounts of all libraries (in total, 1 µg) were mixed with 135 ng of Biotin-labeled *FP*-gene fragments and were hybridized at 46°C for 72 h. Other procedures were performed in accordance with the manufacturer's instructions.

Sequencing of Captured DNA Libraries and Extraction of Exon 3 Sequences
-----------------------------------------------------------------------

Short DNA sequences (paired-end, 250 bp) were determined from the captured libraries using the Illumina Hiseq2500 platform. The nucleotide sequences were deposited in the DDBJ Sequenced Read Archive under accession number DRR120550--DRR120560. Since the average insert size of the libraries was 500 bp, the overlapping paired reads were merged into one sequence read using CLC Genomics Workbench (<https://www.qiagenbioinformatics.com/>; last accessed June 30, 2018), after the removal of the adaptor sequences and low-quality reads (quality score \<20). When the paired reads did not overlap, both reads were used, without merging. Merged and unmerged reads were used for sequence identification.

The longest exon containing nucleotides encoding a chromophore, that is, exon 3 (e3), was used for subsequent analyses. To identify e3 sequences in the *FP* gene, the ancestral sequences of *FP* genes (explained below) were used as a reference for mapping. To construct the ancestral sequences, full-length coding regions of *FP* genes of *Acropora* species were collected from the NCBI database. The accession numbers of the collected sequences are shown in [supplementary table S3](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online. Using these *FP* sequences, the ancestral sequences of two subclades in the S/MWE, M/LWE, and CP clades ([@evy122-B33]) were estimated by the maximum likelihood method with a preset tree topology using MEGA ver. 7 ([@evy122-B14]). The ancestral sequences are listed in the [supplementary material](#sup1){ref-type="supplementary-material"} online.

Short DNA reads from each sample were mapped to the reference sequences composed of the e3 ancestral sequences of two subclades in the S/MWE, M/LWE, and CP clades using CLC Genomics Workbench. Reads showing similarity (\>80%) to the e3 sequence (S/MWE and M/LWE clades: length, 225 bp \[accessions: BR000963 and XM_015914911\]; CP clade: length, 219 bp; [supplementary fig. S1](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online) were mapped to reference sequences. Reads mapped to the ancestral sequences of each of the three clades were separately extracted and assembled using ATGC (GENETYX CORPORATION, Tokyo, Japan) (matching percentage = 100%), and unassembled reads were removed. Assembled contigs for each of the three clades were aligned, and contigs covering the full length of e3 were selected. Sequences in intronic regions were removed from contigs. For each sample, DNA reads (merged reads and other reads) were mapped (complete match) to these assembled e3 contigs using CLC Genomics Workbench. The e3 contigs in which at least two reads covered the full length were identified as e3 sequences, those containing insertions or deletions that cause frameshifts and/or premature stop codons were regarded as pseudogene (pseudo-e3) sequences. Among all e3 sequences isolated from 11 individuals, identical sequences were removed. The phylogenetic tree of e3 sequences was constructed using the maximum likelihood method based on the Kimura 2-parameter mode with gamma distributed rates and invariant sites using MEGA ver. 7 ([@evy122-B14]). Bootstrap support for each clade was obtained based on 1,000 replicates. E3 sequences were deposited in GenBank under accession numbers LC349488--LC349725.

Validation of the Absence of Highly Expressed Exon 3 Sequences
--------------------------------------------------------------

Following the manufacturer's instructions, DNA libraries of three *A. digitifera* samples (sample ID: S1601, S1603, and S1606) were constructed using the TruSeq DNA PCR-Free Library Preparation Kit (Illumina, Inc.). Short DNA sequences (paired-end, 125 bp) were determined from these three and from the DNA libraries of eight *A. digitifera* samples (sample ID: S1602, S1604--5, S1607--8, and S1610--S1612) described under Materials and Methods (DNA extraction and preparation of DNA libraries for hybridization) by the Illumina HiSeq2500 platform. The nucleotide sequences were deposited in the DDBJ Sequenced Read Archive under accession numbers DRR108003--DRR108012 and DRR108024. To verify for the absence of the highly expressed sequences in the genomes of individuals in which these sequences were not isolated, short reads from the genomes of each individual were mapped to all isolated e3 sequences. Short DNA reads showing similarity (100%) with 80% read lengths were mapped to e3 sequences using CLC Genomics Workbench. When the full length of e3 was covered, with at least one coverage without any mismatches, the e3 sequence was regarded as positive in the genome of the individual. When any reads mapped to the e3 without any mismatches, the e3 sequence was regarded as negative in the genome of the individual. If a few or several reads were mapped to e3, but the full length of e3 was not covered, the e3 sequences were classified by the second mapping condition. The second mapping was performed under the same criteria as the moderate mapping condition (similarity 100% with 40% of read length) using the same short DNA reads and e3 sequences. When the full length of e3 was covered without any mismatches under the moderate mapping condition, the e3 sequence was regarded as positive. The remaining e3 sequences were categorized as unclassified.

Estimation of *A. digitifera FP* Gene Copy Numbers
--------------------------------------------------

*FP* gene copy numbers in the *A. digitifera* genome were estimated by quantitative PCR (qPCR) using 11 *A. digitifera* specimens. *FP* gene copy number estimation was performed using previously described methods ([@evy122-B33]) with minor modifications. Briefly, two new reverse primers (MLWE_qPCR_R1-3 for M/LWE and MiA_CP_e3_R1-3 for CP) were used for qPCR. Primer sequences are shown in [supplementary table S2](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online. qPCR was performed using the Thermal Cycler Dice TP800 (Takara). Genomic DNAs of 11 samples (S1601--8, S1610--12) were used as templates.

RNA Extraction and Sequencing
-----------------------------

Total RNAs were extracted from 11 adult coral fragments of *A. digitifera*, the same individuals used for DNA extraction, and a single *A. digitifera* larva using TRIzol reagent (Thermo Fisher Scientific). cDNAs were synthesized from total RNA using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). RNAs from 11 adults were used to construct libraries for high-throughput RNA sequencing using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Bio Labs, Ipswich, MA). Short DNA sequences (paired-end, 125 bp) were determined from the libraries using the Illumina HiSeq2500 platform. The nucleotide sequences were deposited in the DDBJ Sequenced Read Archive under accession numbers DRR108013-DRR108023.

Expression Levels of Exon 3 Sequences of *FP* Genes
---------------------------------------------------

After the removal of the adaptor sequences and low-quality reads (quality score \<20), RNA-seq reads from 11 individuals were mapped to *A. digitifera* genome assembly ver.1.1. In addition to the 11 adult individuals, RNA-seq reads from *A. digitifera* larvae (12.1 Gbp, accession: SRX1534820) were downloaded from a public database (DDBJ) and mapped to *A. digitifera* genome assembly ver.1.1. Reads showing high sequence similarity (\>90%, \>112 bp) were mapped to query sequences and expression values were calculated using CLC genomic workbench. RPKM (Reads Per Kilobase of exon model per Million mapped reads) were used to normalize expression values to identify candidate housekeeping genes. First, house-keeping genes that showed stable expression among individuals and developmental stages were selected as internal controls for normalization of *FP* gene expression levels. In particular, the variance indexes for RPKM values (SD/average RPKM) were calculated for all genes among 11 adults and larvae. Thirty-four genes with average RPKM values among 11 adults and larvae of ≥50 and with a variance index of RPKM among 11 adults and larvae of \<1.0 were selected as house-keeping genes ([supplementary table S4](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online).

To calculate expression values for e3 sequences, RNA-seq reads from 11 adults and larvae (accession: SRX1534820 and DRR054773) were mapped to all isolated e3 sequences using CLC Genomics Workbench. Reads showing high similarity to corresponding e3 sequences (100%, \>119 bp) were mapped to those sequences. To normalize mapped reads, the total numbers of reads mapped to the 34 house-keeping genes were used as internal controls. The normalized expression values for *FP* sequences were calculated as follows: (Mapping coverage of the *FP* e3 sequence/Mapped reads on the internal control) × 100,000, where the mapping coverage of the *FP* e3 sequence was estimated as the reads mapped to the e3 sequence divided by the e3 length (bp) and mapped reads on the internal control indicates reads mapped to the 34 house-keeping genes. The e3 sequences of FPs in the S/MWE, M/LWE, and CP clades were 225,225, and 219 bp in length, respectively.

Identification of *Symbiodinium* Clades in Each Sample
------------------------------------------------------

To identify the clades of *Symbiodinium* in each sample, RNA-seq reads of 11 individuals were mapped to the nucleotide sequences of nuclear 28S (nr28S) and chloroplast 23S (cp23S) ribosomal DNA from eight clades of *Symbiodinium* ([@evy122-B23]) using CLC Genomics Workbench. The accession numbers for each sequence are shown in [supplementary table S5](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online.

Expression Analyses of *Symbiodinium* Genes
-------------------------------------------

To analyze gene expression in symbiotic algae living in all coral individuals, RNA-seq reads of 11 coral individuals showing high similarity (\>80%) and lengths of \>100 bp were mapped to the transcriptome of *Symbiodinium goreaui* (clade C) ([@evy122-B4]) using CLC Genomics Workbench. The following comparisons were evaluated according to gene expression and gene presence--absence patterns: 1) high expression of *S/Me3_a\_hi1* and the absence of this sequence in the genome; 2) presence and absence of *S/Me3_a\_hi1* in the genome; 3) expression of *M/L-De3_a* and the absence of this sequence in the genome; 4) expression and no expression of M/LWE clade TYG-type sequences; 5) high (expression values ≥10) and low expression (expression values ≤5) of total CP sequences. The contigs with significant expression differences (p \< 0.05 with FDR correction) between two groups were selected.

Identification and Cloning of *FP* cDNA Sequences, Purification, and Spectroscopic Analyses of Recombinant FP Proteins
----------------------------------------------------------------------------------------------------------------------

Full-length cDNAs of *FP* genes were amplified by PCR. Using the cDNA of an *A. digitifera* larva as a template, S/MWE-*FP* full-length cDNAs were amplified using PrimeSTAR GXL DNA Polymerase (Takara). M/LWE-*FP* full-length cDNAs were amplified using PrimeSTAR GXL DNA Polymerase (Takara) from the cDNAs of an *A. digitifera* larva and an adult (sample ID: S1603). CP full-length cDNAs were amplified using TaKaRa Ex Taq from the cDNAs of an *A. digitifera* larva and an adult (sample ID: S1606). PCR conditions for the amplification of full-length cDNAs were as follows: denaturation for 3 min at 94°C, followed by 30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 1 min at 72°C. All primer sets and primer sequences are shown in [supplementary tables S2 and S6](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online. PCR products were cloned into the T-Vector pMD20 vector (Takara), and the sequences were verified using the Applied Biosystems Automated 3130xl Sequencer (Foster City, CA).

To construct recombinant FP proteins, vectors with cloned *FP* sequences were used as templates for subcloning into expression vectors. Full-length *FP* cDNAs were subcloned into the pCold I expression vector (Takara) and then used to transform BL21 *Escherichia coli* cells (Takara). Each clone was grown in 20 mL of LB medium supplemented with ampicillin and IPTG overnight, and the recombinant proteins were extracted by sonication and purified using TALON beads with poly-histidine tags (Takara).

Emission and excitation spectra of purified recombinant FP proteins in 50 mmol/L phosphate buffer solution with 500 mmol/L imidazole, pH 7.0, were measured using the RF-6000 spectro-fluorophotometer (SHIMAZU, Kyoto, Japan). Absorption spectra of purified recombinant CP proteins were measured using a UV-1800 spectrophotometer (SHIMAZU). Measurements were obtained at least three times for each FP.

Identification of *FP* Genes in the Three *A. digitifera* Subpopulations
------------------------------------------------------------------------

The publicly available genomic DNA reads of *A. digitifera* collected from the southern Ryukyu Archipelago located in southwestern Japan were used ([supplementary fig. S2](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). These individuals have been separated into four subpopulations; Okinawa, Kerama, Yaeyama-South, and Yaeyama-North ([@evy122-B29]). Individuals with mapping read coverage ≥6 were selected for our analysis. The mapping read coverages were based on those in a previous report ([@evy122-B29]). A total of 91 sets of genomic DNA reads from 40 individuals belonging to Okinawa, 39 individuals belonging to Kerama, and 12 individuals belonging to Yaeyama-North were downloaded. A subpopulation (Yaeyama-South) was excluded from our analysis, because there were only two individuals with mapping read coverage ≥6 in this subpopulation. The accession numbers of downloaded genomic DNA reads are shown in [supplementary table S7](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online. Validation of the existence of highly expressed e3 sequences in each individual from the three subpopulations was performed using the method explained under Materials and Methods (Validation of the absence of highly expressed exon 3 sequences).

Live Coral Fluorescence Measurements
------------------------------------

Light emission, including reflectance and fluorescence spectra, was measured from each of 11 *A. digitifera* colonies that were used for DNA and RNA extraction. Visual observation did not reveal clear color differences among 11 *A. digitifera* colonies. An LED source with a peak wavelength of 448 nm was used as the excitation light source. The distances from the excitation light and measurement probe to objects were 6 cm, and spectra were recorded using the Jaz Spectrometer (Ocean Optics, Dunedin, FL) in the dark. For all measurements, light emitted at longer than 660 nm was not attributed to coral fluorescence because chlorophyll *a* from the symbiotic dinoflagellate algae living within the coral tissues emit light with a primary peak wavelength ∼685 nm and a secondary peak at 730 nm ([@evy122-B20]; [@evy122-B18]). Fluorescence was estimated by excitation (LED)--emission spectral subtraction.

Results
=======

Copy Number Variation and Sequence Diversity in the *FP* Gene Family
--------------------------------------------------------------------

We quantified *FP* gene copy numbers in 11 *A. digitifera* genomes by qPCR. We detected 14--24 *FP* gene copies in the S/MWE clade, 5--13 in the M/LWE clade, and 9--15 in the CP clade ([fig. 1](#evy122-F1){ref-type="fig"}), indicating copy number variation among individuals.

![---*FP* gene copy numbers and total numbers of sequences obtained in each clade. *FP* gene copy numbers and exon 3 sequence numbers in the MWE (*A*), M/LWE (*B*), and CP (*C*) clades from 11 adult colonies of *Acropora digitifera* are given. Copy numbers are shown in light gray with SDs obtained from three replicates. Numbers of intact exon 3 sequences are shown in dark gray and pseudo-exon 3 sequences are shown in white. S1601--S1612 indicate sample IDs.](evy122f1){#evy122-F1}

Despite the high estimated copy numbers, only ten *FP* genes (including nine partial genes) were identified in the assembled genome sequence of *A. digitifera* ([@evy122-B30]). This inconsistency may reflect the difficulty of multigene family assembly ([@evy122-B16]). To identify sequence diversity in *FP* genes, we determined the exon 3 (e3) sequences of *FP* genes without de novo assembly. We chose e3 because it has the longest exon among *FP* genes (those of the S/MWE, M/LWE, and CP clades were 225, 225, and 219 bp in length, respectively) and contains the region that encodes a chromophore. DNA fragments containing *FP* genes in libraries were condensed by *FP* gene capture, and short DNA sequences (paired-end, 250 bp) were determined (2,141,708--2,850,976 reads) from 11 samples ([supplementary table S1](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). These reads from each library were mapped to e3 of S/MWE *FP* (1,069--1,532 reads), M/LWE *FP* (215--655 reads), and CP (628--1,698 reads) genes. Using mapped reads, e3 sequences of *FP* genes were determined from 11 *A. digitifera* genomes ([fig. 1](#evy122-F1){ref-type="fig"}). We detected different e3 nucleotide sequences from 11 individuals, including 111 intact and 19 pseudosequences in the S/MWE clade, 29 intact and one pseudosequence in the M/LWE clade, and 49 intact and 29 pseudosequences in the CP clade ([table 1](#evy122-T1){ref-type="table"}). The intact e3 sequences were translated into amino acid sequences. The number of amino acid sequences were 67, 17, and 38 in the S/MWE, M/LWE, and CP clades, respectively. Based on the first peptide of the chromophore, we divided the e3 sequences in the M/LWE clade into two types, TYG-type and DYG-type. The phylogenetic trees based on the nucleotide sequences of e3 are shown in [supplementary figure S3](#sup1){ref-type="supplementary-material"}*A--C*, [Supplementary Material](#sup1){ref-type="supplementary-material"} online. Among the three clades, we detected the most e3 sequences at both the nucleotide and amino acid sequence levels in the S/MWE clade. Table 1Number of Exon 3 Sequences Isolated in Three CladesCladeIntact exon 3Pseudo-exon 3NucleotideAmino AcidNucleotideS/MWE1116719M/LWE29171CP493829

Expression Differences for Exon 3 Sequences of *FP* Genes among Adult Individuals
---------------------------------------------------------------------------------

We examined the expression levels of each e3 sequence based on RNA-seq reads from 11 adult individuals (41,007,102--109,525,174 reads). In RNA-seq read mapping, 36--50% of RNA-seq reads were mapped to the *A. digitifera* genome ver. 1.1 ([supplementary table S9](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). Expression levels of e3 sequences were normalized by the expression levels of 34 house-keeping genes and categorized into six levels ([fig. 2](#evy122-F2){ref-type="fig"}). The expressed e3 sequences in larval (see below) and adult stages are summarized in [table 2](#evy122-T2){ref-type="table"}. Table 2Number of Expressed Sequences in the Larval and Adult StagesCladeAdults and LarvaeOnly AdultsOnly LarvaeS/MWE1343M/LWE_TYG217M/LWE_DYG018CP4168

![---Expression of each *FP* sequence in adults and larvae. Rows indicate the exon 3 sequences belonging to S/MWE clade (*A*), M/LWE clade (*B*), and CP clade (*C*). Columns indicate the normalized expression values for *FP* sequences calculated by the expression of the internal control genes (see Materials and Methods) in 11 adults and larvae. Sample IDs S1601--S1612 are indicated as 01--12. L indicates larvae. A gray box indicates the absence of the sequence in a genome of a sample and a slash mark indicates that the sequence data were not available. Exon 3 sequences were aligned based on the phylogenetic trees in [supplementary figure S2](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online (UPGMA based on amino acid differences) to show amino acid differences. Black arrow indicates a branch supported by the ML tree in [supplementary figure S3](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online. The first peptide of the chromophore for each exon 3 sequences is shown in a rectangle. An asterisk indicates the exon 3 sequence used for the recombinant protein analysis.](evy122f2){#evy122-F2}

We detected variation in total expression levels in each clade or type of M/LWE among 11 individuals ([table 3](#evy122-T3){ref-type="table"}). In the S/MWE clade, among the individuals with high overall *FP* gene expression ([table 3](#evy122-T3){ref-type="table"}, total expression of the S/MWE clade sequences in S1603, S1605, and S1611), one e3 sequence (SMe3_010) was highly expressed ([fig. 2*A*](#evy122-F2){ref-type="fig"}). We refer to this e3 sequence as "*S/Me3_a\_hi1*"; it was also detected in four other individuals ([fig. 2*A*:](#evy122-F2){ref-type="fig"} S1602, S1606--7, and S1610), but at lower levels than those in the three high-expression individuals ([table 3](#evy122-T3){ref-type="table"}). In the other four individuals ([fig. 2*A*:](#evy122-F2){ref-type="fig"} S1601, S1604, S1608, and S1612), we did not detect *S/Me3_a\_hi1* in genomic DNA (250 bp reads) or RNAseq reads. In addition to *S/Me3_a\_hi1*, the other e3 sequence (e3 ID: SMe3_079) in a single individual ([fig. 2*A*:](#evy122-F2){ref-type="fig"} S1603) showed high sequence similarity with *S/Me3_a\_hi1* and high expression. We refer to this e3 sequence as "*S/Me3_a\_hi2.*" Among three individuals with high overall *FP* gene expression in the S/MWE clade, the expression of *S/Me3_a\_hi1* and *S/Me3_a\_hi2* sequences accounted for \>90% of the total expression ([fig. 3*A*](#evy122-F3){ref-type="fig"}). The absence of *S/Me3_a\_hi1* and *S/Me3_a\_hi2* in the genomes of individuals shown by the 250 bp reads from genomic DNA was verified using short reads (paired-end, 125 bp). These results indicate that each of the *S/Me3_a\_hi1* and *S/Me3_a\_hi2* sequences were present in one individual and absent from another individual, thus, there were presence--absence polymorphisms in these 11 individuals. Table 3Expression Values for Specific Exon 3 SequencesSampleS/MWEM/LWE (DYG)M/LWE (TYG)CP*S/Me3_a\_hi1-2*Other Than *S/Me3_a\_hi1-2*Total*M/L-De3_a*Other Than *M/L-De3_a*TotalTotalTotalS16010120120000113S1602567713300008S16031,002861,0884804806S16040898900003S1605304530900014S1606118818941041143S16071621800005S16080242400015S16104619123783083010S1611514551900018S16120212100027Larvae023923901,2361,2361,186830

![---Proportions of highly expressed exon 3 sequences in the S/MWE and M/LWE clades. The normalized expression values for *FP* sequences calculated by the expression of the internal control genes (see Materials and Methods) in the (*A*) S/MWE clade (*S/Me3_a\_hi1* and *S/Me3_a\_hi2*) and (*B*) M/LWE clade (*M/L-De3_a*) and the expression of all *FP* sequences in each clade, except highly expressed sequences shown in black and gray, respectively. The *y* axis indicates the normalized expression value. S1601--S1612 indicate sample IDs.](evy122f3){#evy122-F3}

In the M/LWE clade, we observed that 4 of 29 e3 nucleotide sequences were expressed in at least one adult. In four adult individuals ([fig. 2*B*:](#evy122-F2){ref-type="fig"} S1602, S1604, S1605, and S1607), we did not detect the expression of any e3 sequence. In adults, only one DYG-type sequence (e3 ID: MLe3_009) was expressed in three samples ([fig. 2*B*:](#evy122-F2){ref-type="fig"} S1603, S1606, and S1610), and little expression of TYG-type sequences was observed. We refer to this expressed DYG-type e3 sequence as "*M/L-De3_a*"; we identified this sequence in four individuals and verified these findings using short reads (paired-end, 125 bp) of genomic DNA from these individuals ([supplementary table S7](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). Four individuals had *M/L-De3_a*, among which one individual ([fig. 3*B*;](#evy122-F3){ref-type="fig"} ID: S1602) did not express this sequence. The other six individuals did not possess this sequence in their genome ([supplementary table S7](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online), indicating a presence--absence polymorphism of *M/L-De3_a*.

In the CP clade, we detected the expression of 20 out of 49 e3 nucleotide sequences in at least one adult. In one sample (ID: S1606), total expression in the CP clade was relatively high ([table 3](#evy122-T3){ref-type="table"}), and this expression was attributed to five e3 sequences ([fig. 2*C*](#evy122-F2){ref-type="fig"}). We did not identify an e3 sequence with high expression in the CP clade ([fig. 2*C*](#evy122-F2){ref-type="fig"}).

Expression Differences in Exon 3 Sequences of *FP* Genes between Adults and Larvae
----------------------------------------------------------------------------------

We compared the expression of each e3 sequence between planula larvae and adults using publicly available RNA-seq reads of larvae (accession: SRX1534820) and RNA-seq reads of 11 adults, respectively. In the S/MWE clade, we observed larval expression of four closely related e3 sequences, and we did not detect the expression of these four e3 sequences in adults, except for low expression in one individual ([fig. 2*A*](#evy122-F2){ref-type="fig"}), indicating differences in the regulation of *FP* gene expression between adults and larvae. The total expression of sequences in this clade was lower in larvae than in the adults with the three highest expression levels ([table 3](#evy122-T3){ref-type="table"}).

In the M/LWE clade, we observed higher total expression of both TYG-type and DYG-type sequences in larvae than in adults ([table 3](#evy122-T3){ref-type="table"}). Although we observed lower expression of TYG-type than DYG-type sequences in 11 adults, we detected similar total expression values for TYG-type and DYG-type sequences in larvae. We observed high expression of two ([fig. 2*B*:](#evy122-F2){ref-type="fig"} MLe3_004, and MLe3_008) and one ([fig. 2*B*:](#evy122-F2){ref-type="fig"} MLe3_012) TYG-type and DYG-type e3 sequences, respectively. Hereafter, we refer to these e3 sequences as "*M/L-Te3_l\_hi1*," "*M/L-Te3_l\_hi2*," and "*M/L-De3_l\_hi.*" Among 11 adult individuals, we isolated *M/L-Te3_l\_hi1, M/L-Te3_l\_hi2*, and *M/L-De3_l\_hi* from six, four, and two individuals, respectively ([fig. 2*B*](#evy122-F2){ref-type="fig"}), and the presence and absence of these sequences in the genome were verified by short reads (paired-end, 125 bp) of genomic DNA ([supplementary table S7](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). These results indicate that highly expressed sequences in larvae showed presence--absence polymorphisms in adult individuals.

In the CP clade, we observed higher total expression of CP clade sequences in larvae than in adults ([table 3](#evy122-T3){ref-type="table"}). We detected the expression of 12 e3 sequences in larvae, and no or low expression of these e3 sequences in 11 adults ([fig. 2*C*](#evy122-F2){ref-type="fig"}). These results indicate differences in *CP* sequences expression between adults and larvae.

The genetic differences in *FP* genes (copy number variation and presence--absence polymorphisms) in individuals used as "adult samples" and "larval samples" might affect the expression patterns of *FP* genes at different life stages. To examine this possibility, we compared two independent RNA-seq data sets of *A. digitifera* larvae (accessions: SRX1534820 and DRR054773). The results showed that two RNA-seq data sets from larvae had similar expression patterns, and that the patterns were different from those of adults ([supplementary fig. S5](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). This expression pattern difference was most likely the result of a difference in expression between adults and larvae rather than the result of a difference in genetic background.

In all three clades, some e3 sequences exhibited no expression in both adults and larvae ([fig. 2*A*--*C*](#evy122-F2){ref-type="fig"}). It is possible that these unexpressed sequences are parts of pseudogenes and/or are expressed at different life stages (e.g., developmental stages) or in specific conditions.

Functional Differences among FPs
--------------------------------

To evaluate functional differences among *FP* e3 sequences, we cloned full-length *FP* coding regions, constructed recombinant FPs, and measured emission and excitation or absorption spectra. In the S/MWE clade, we cloned four different full-length cDNAs of *FP* genes, that is, *S/Mcd_l* expressed in larvae, *S/Mcd_a* expressed in adults, and *S/Mcd_a\_hi1* and *S/Mcd_a\_hi2*, which are highly expressed in adults. The e3 sequences of full-length cDNAs of *FP* genes were identical to the e3 sequences we determined, and the correspondence between full-length cDNA names and e3 sequence IDs is shown in [supplementary table S6](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online. The emission peak of recombinant *S/Mcd_l* expressed in larvae was 519 ± 0 nm ([fig. 4*A*](#evy122-F4){ref-type="fig"}), whereas the emission peaks of two recombinant proteins expressed in adults (*S/Mcd_a* and *S/Mcd_a\_hi1*) were 488 ± 0 nm and 483 ± 0 nm, respectively ([fig. 4*B*](#evy122-F4){ref-type="fig"}). Thus, recombinant FPs encoded by *FP* sequences highly expressed in larvae and adults exhibited different emission peaks.

![---Emission and absorption spectra of FP recombinant proteins. Absorption (dotted line) and emission (solid line) spectra of recombinant proteins belonging to the S/MWE clade (*A*, *S/Mcd_l*; *B*, *S/Mcd_a* and *S/Mcd_a\_hi1*; and *C*, *S/Mcd_a\_hi2*), M/LWE clade (*D*, *M/L-Tcd_i\_hi2*; *E*, *M/L-Dcd_l2*; and *F*, *M/L-Dcd_a*), and CP clade (*G*, *CPcd_a*; *H*, *CPcd_l1*; *I*, *CPcd_l2*) are shown. Horizontal and vertical axes indicate wavelengths (nanometers) and normalized light intensities, respectively.](evy122f4){#evy122-F4}

Furthermore, we detected similar emission peaks for recombinant *S/Mcd_a\_hi1* (highly expressed) and *S/Mcd_a* ([fig. 4*B*](#evy122-F4){ref-type="fig"}). Recombinant *S/Mcd_a\_hi1* was excited by light under 380 nm, whereas *S/Mcd_a* was not highly excited by light in this range. The excitation and emission spectra of recombinant *S/Mcd_a\_hi1* (shown in black in [fig. 4*B*](#evy122-F4){ref-type="fig"}) and *S/Mcd_a\_hi2* ([fig. 4*C*](#evy122-F4){ref-type="fig"}) were identical, indicating that these two highly expressed e3 sequences in adults share the same function in the wavelength of excitation and emission spectra of FP.

In the M/LWE clade, we cloned three different full-length cDNAs of *FP* genes, that is, *M/L-Tcd_l\_hi2* (TYG-type expressed in larvae), *M/L-Dcd_l2* (DYG-type with identical amino acid sequences expressed in larvae), and *M/L-Dcd_a* (DYG-type expressed in adults). Emission peaks for the recombinant *M/L-Tcd_l\_hi2, M/L-Dcd_l2*, and *M/L-Dcd_a* were 519 ± 0 nm, 613 ± 2 nm, and 606 ± 1 nm, respectively ([fig. 4*D--F*](#evy122-F4){ref-type="fig"}).

In the CP clade, we cloned three different full-length cDNAs of *FP* genes, that is, *CPcd_l1* expressed in larvae, *CPcd_l2* expressed in larvae, and *CPcd_a* expressed in adults. Absorption peak values for recombinant *CPcd_l1, CPcd_l2*, and *CPcd_a* were 585 ± 0 nm, 603 ± 0 nm, and 579 ± 0 nm, respectively ([fig. 4*G--I*](#evy122-F4){ref-type="fig"}). These CPs absorbed long wavelength light.

Identification of Highly Expressed *FP* Sequences in Three *A. digitifera* Subpopulations
-----------------------------------------------------------------------------------------

In S/MWE, we detected high *S/Me3_a\_hi1* and *S/Me3_a\_hi2* expression in three adult individuals with high total *FP* gene expression. Additionally, in the M/LWE clade DYG-type, expression differences among 11 adult individuals were caused by differences in *M/L-De3_a* expression. In larvae, *M/L-Te3_l\_hi1, M/L-Te3_l\_hi2*, and *M/L-De3_l\_hi* in the M/LWE clade were highly expressed.

To confirm whether these sequences existed in *A. digitifera* subpopulations in the southern Ryukyu Archipelago, we examined 102 individuals from three subpopulations (Okinawa, Kerama, and Yaeyama-North) ([@evy122-B29]). We detected the presence of each of *S/Me3_a\_hi1, M/L-De3_a*, *M/L-Te3_l\_hi1, M/L-Te3_l\_hi2*, and *M/L-De3_l\_hi* in the genomes of individuals from the three subpopulations ([table 4](#evy122-T4){ref-type="table"} and [supplementary table S7](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). We detected the presence and absence of each of *S/Me3_a\_hi1, M/L-Te3_l\_hi2*, and *M/L-De3_l\_hi* in the genomes of individuals from the three subpopulations ([table 4](#evy122-T4){ref-type="table"} and [supplementary table S7](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online), indicating that the presence--absence polymorphic state of these sequences was shared by the three subpopulations. We found *S/Me3_a\_hi2* in two subpopulations, but not in the Yaeyama-North subpopulation containing six individuals without this sequence or with partially covered (categorized as unclassified, see Materials and Methods). In addition, we found only individuals with *M/L-Te3_l\_hi1* or unclassified individuals in Kerama and Yaeyama-North. In these cases, we were unable to determine whether or not *S/Me3_a\_hi2* and *M/L-Te3_l\_hi1* were presence--absence polymorphic in these subpopulations. We found individuals with *M/L-De3_a*, but no individual without *M/L-De3_a* or unclassified individuals in Yaeyama-North, suggesting that this sequence is fixed or exists at high frequencies in this subpopulation. However, since the number of individuals in Yaeyama-North used in this analysis was smaller than in the other two subpopulations, it could have occurred by chance. Table 4Number of Individuals That Possess or Did Not Possess Specific Exon 3 Sequences in Their GenomeFP e3 SequenceSubpopulationNumber of IndividualsPolymorphic StatePositiveNegativeUnclassified*S/Me3_a\_hi1*Okinawa (n=51)28716PolymorphicKerama (n=39)2748PolymorphicYaeyama_North (n=12)354Polymorphic*S/Me3_a\_hi2*Okinawa (n=51)11139PolymorphicKerama (n=39)31026PolymorphicYaeyama_North (n=12)066Unknown*M/L-De3_a*Okinawa (n=51)132711PolymorphicKerama (n=39)9237PolymorphicYaeyama_North (n=12)1200(Presence-fixed)*M/L-Te3_l\_hi1*Okinawa (n=51)31515PolymorphicKerama (n=39)3207UnknownYaeyama_North (n=12)408Unknown*M/L-Te3_l\_hi12*Okinawa (n=51)13335PolymorphicKerama (n=39)11226PolymorphicYaeyama_North (n=12)237Polymorphic*M/L-De3_l\_hi*Okinawa (n=51)131424PolymorphicKerama (n=39)19614PolymorphicYaeyama_North (n=12)444Polymorphic

Differences of *FP* Gene Expression Were Consistent with Differences in Fluorescence Emitted from Coral Individuals
-------------------------------------------------------------------------------------------------------------------

The emission spectra (including reflectance and fluorescence) from 11 *A. digitifera* colonies excited by LED (448 nm spectrum peak) were measured in the sea. In individuals with high expression of *S/Me3_a\_hi1*, fluorescence spanned ∼475--570 nm ([supplementary fig. S4](#sup1){ref-type="supplementary-material"}*A--C*, [Supplementary Material](#sup1){ref-type="supplementary-material"} online).

We did not observe fluorescence from four individuals without *S/Me3_a\_hi1* in their genomes ([supplementary fig. S4](#sup1){ref-type="supplementary-material"}*D--G*, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). Two of the four individuals with low expression of *S/Me3_a\_hi1* (ID: S1607, 1610) did not show fluorescence ([supplementary fig. S4](#sup1){ref-type="supplementary-material"}*J* and *K*, [Supplementary Material](#sup1){ref-type="supplementary-material"} online*).* The remaining two individuals (ID: S1602, 1606) with low expression of *S/Me3_a\_hi1* showed fluorescence with a peak at 516 nm ([supplementary fig. S4](#sup1){ref-type="supplementary-material"}*H* and *I*, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). The fluorescence spanning ∼500--590 nm in one individual (ID: 1606) ([supplementary fig. S4](#sup1){ref-type="supplementary-material"}*I*, [Supplementary Material](#sup1){ref-type="supplementary-material"} online) may have been emitted by different FPs, because the emission peak of the FP encoded by *S/Me3_a\_hi1* was 483 nm ([fig. 4*B*](#evy122-F4){ref-type="fig"}). The fluorescence from one individual (ID: 1602) ([supplementary fig. S4](#sup1){ref-type="supplementary-material"}*H*, [Supplementary Material](#sup1){ref-type="supplementary-material"} online) may have been emitted by a mixture of FPs encoded by *S/Me3_a\_hi1* and other sequences. These results show that fluorescence polymorphisms existed among 11 *A. digitifera*; individuals with fluorescence spanning ∼475--570 nm, one individual with fluorescence spanning ∼500--590 nm, and individuals with no fluorescence. Coral fluorescence spanning ∼475--570 nm was consistent with the high expression of *S/Me3_a\_hi1* and absence of *S/Me3_a\_hi1* in the genome.

*FP* Expression Does Not Affect Gene Expression in Symbiotic Algae
------------------------------------------------------------------

Photo-protective effects of FPs on photosynthetic algal symbionts have been proposed ([@evy122-B27]; [@evy122-B31]; [@evy122-B9]). However, we observed differences in *FP* expression among individuals collected from the same shallow water light environment. To compare the conditions of symbiotic algae living in each coral individual, we evaluated the levels of all expressed genes in symbiotic algae by RNA-seq.

First, we identified the clade of symbiotic algae in the 11 individuals that we collected using the mapping results for RNA-seq reads to the nucleotide sequence list of nr28S and cp23S ribosomal DNA from eight clades of algae. In all individuals, RNA-seq reads were mapped to nr28S and cp23S of the clade C, and few reads were mapped to sequences of other clades ([supplementary table S5](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). This result indicates that most symbiotic algae living in the 11 individuals of *A. digitifera* belonged to clade C.

To compare gene expression, RNA-seq reads from 11 adult individuals were mapped to the transcriptome of *S. goreaui* (clade C) ([@evy122-B4]). Approximately 20--34% of RNA-seq reads were mapped to the contigs ([supplementary table S9](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). For all comparisons, the expression levels of algal genes were strongly correlated between groups (*r *=* *0.98--1.00, [supplementary table S8](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). In the comparisons based on presence and absence of *S/Me3_a\_hi1* (comparisons \#1 and 2) and the expression level of *M/L-De3_a* (comparison \#3), no gene showed significant differences in expression. In the comparisons based on the expression level of M/LWE TYG-type sequences (comparison \#4), we detected significant differences (p \< 0.05 with FDR correction) in the expression levels of one contig annotated as cytochrome b6-f complex subunit four between groups ([supplementary table S10](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). In the comparisons based on the expression level of CP sequences (comparison \#5), we detected significant differences (p \< 0.05 with FDR correction) in the expression levels of one unannotated contig between groups ([supplementary table S10](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online).

Discussion
==========

Genetic Mechanism Underlying the Fluorescence Polymorphism among Individuals of *A. digitifera*
-----------------------------------------------------------------------------------------------

*Acropora* FPs are thought to be involved in the protection of photosynthetic algal symbionts against sunlight ([@evy122-B27]; [@evy122-B31]; [@evy122-B9]). However, intraspecific FP-mediated color polymorphisms have been found within the same light environment ([@evy122-B31]; [@evy122-B9]). This natural variation raises questions about the importance of FPs for photo-protection. To address this question, it is necessary to determine the mechanism that generates the intraspecific fluorescence polymorphism.

The expression of *FP* genes in adult *Acropora* individuals is influenced by external stimuli, such as light, heat, and injury. CFP, GFP, RFP, and CP levels increase according to the light intensity ([@evy122-B2]; [@evy122-B26]), and CFP is down-regulated in dark stress ([@evy122-B5]). Heat stress down-regulates GFP ([@evy122-B24]). The expression of CFP increases in response to injury ([@evy122-B3]). However, these factors cannot explain variation in fluorescence in corals living in the same environment, without injury.

Previously, we reported variation in the sequences and total copy number of particular *FP* genes in *A. digitifera* ([@evy122-B33]); therefore, we analyzed copy number variation in 11 adult individuals of *A. digitifera*, living in the same light environment in shallow water. We detected copy number variation for *FP* genes from each of three clades (S/MWE, M/LWE, and CP). The numbers of e3 sequences and the e3 sequence repertories in each of three clades also differed among individuals. The numbers of e3 sequences were generally higher than the estimated gene copy numbers in the S/MWE and CP clades, suggesting that *FP* genes in these clades exhibit high allelic diversity. Hence, there were large genetic differences in *FP* genes in each of two clades among individuals of *A. digitifera*.

In each of three clades, we detected variation in the total expression levels of *FP* genes among 11 adult individuals. In the S/MWE clade, one sequence (*S/Me3_a\_hi1*) showed a presence--absence polymorphism, and high expression of this sequence contributed to the total expression levels in the individuals with the top three overall expression levels of *FP* sequences in the S/MWE clade. In this clade, four individuals possessed *S/Me3_a\_hi1*, but the expression levels were lower than those of the other three individuals with high expression. This lower expression may be due to differences in gene regulation or promotor sequence differences. In the M/LWE clade DYG-type, expression differences among 11 adult individuals were explained by the expression of one sequence (*M/L-De3_a*) in three individuals that possessed this sequence. These results suggest that particular *FP* sequences contribute to differences in total *FP* gene expression among individuals. In the CP clade, we did not observe a highly expressed sequence in adult samples. A comparative analysis of the emission spectra and expression of *FP* sequences indicated that fluorescence emission and nonemission of corals were consistent with high expression of *S/Me3_a\_hi1* and the absence of *S/Me3_a\_hi1* in the genome, respectively. According to these results, we conclude that the presence--absence differences of particular *FP* sequences with high expression contribute to the fluorescence differences among individuals in *A. digitifera*.

*FP* Gene Expression Differences between Adults and Larvae
----------------------------------------------------------

The overall expression of *FP* genes in each clade differed substantially between adults and larvae, especially for both DYD- and TYG-type in the M/LWE clade and in the CP clade. High expression of the M/LWE clade in larvae was consistent with the high proportion of RFP in the larval transcriptome of *A. millepora* ([@evy122-B19]). These results suggest that FPs in the M/LWE and CP clades have important roles in larvae. However, several adult individuals did not possess the *FP* sequences with high expression in larvae in the M/LWE and CP clades. This result suggests that the individuals without highly expressed sequences in larvae did not express these sequences when they were larvae. In other words, the intensity of fluorescence and the expression of *FP* genes by highly expressed sequences might be different among larvae. Indeed, a difference in fluorescence has been reported among larvae of *A. millepora* ([@evy122-B13]; [@evy122-B32]).

In the S/MWE clade, the expressed *FP* sequences were different between adults and larvae. In the S/MWE clade, the FP sequence with high expression in larval stage was absent in several adult individuals, suggesting that the expression of this *FP* gene might differ among larval individuals. Hence, the expression of FPs was different between adults and larvae.

Hypothetical Biological Roles of FPs in *A. digitifera*
-------------------------------------------------------

The biological roles of FPs have been proposed based on natural variation in fluorescence in corals ([@evy122-B27]; [@evy122-B31]; [@evy122-B9]). However, observed fluorescence variation can be explained by genetic differences or by differences in the regulation of gene expression among individuals. In this study, we demonstrated *FP* genetic variation among individuals of *A. digitifera*. Based on genetic differences, it was possible to separate individuals into different groups. Therefore, we examined two proposed biological roles of FPs using genetic differences and RNA-seq data containing reads from algal symbionts.

FPs in adult *Acropora* species have a proposed role in the photo-protection of algal symbionts ([@evy122-B27]; [@evy122-B31]; [@evy122-B9]). A supportive role in photosynthesis by algal symbionts has also been proposed ([@evy122-B27]). These two biological roles predict that algal photosynthesis for individuals with high *FP* gene expression is more active than that for individuals with very low expression. In cultured symbiotic algae (genus *Symbiodinium*), up-regulation of a transcript encoding intrinsic membrane-bound light harvesting proteins is caused by the high intensity of light illumination, although most of transcripts encoding genes related to photosynthesis did not show light intensity-regulated expression ([@evy122-B35]).

We compared the expression of symbiotic algae within a coral host (*in hospite)* with respect to the presence and absence of highly expressed genes in the S/MWE and M/LWE (TYG- and DYG-type) clades in their genomes. In the case of CP, the groups were categorized by their expression values. In comparisons of S/MWE, M/LWE DYG-type, and CP clades, no gene related to algal photosynthesis showed differential expression, suggesting that *FP* gene expression in the S/MWE, M/LWE DYG-type, and CP clades observed in this study did not have strong effects on transcriptional regulatory processes of genes related to algal photosynthesis. A contig annotated as cytochrome b6-f complex subunit 4 was significantly more highly expressed (p \< 0.05 with FDR correction) in individuals without M/LWE TYG-type expression than in individuals with M/LWE TYG-type expression. However, genes encoding the other subunits of cytochrome b6-f complex did not show differential expression ([supplementary table S11](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). Therefore, we could not conclude the effects of M/LWE TYG-type on algal photosynthesis based on the differential expression of only one subunit of the cytochrome b6-f complex. In this study, we only studied 11 individuals; thus, it is possible that RNA-seq analysis with larger data sets might detect some more subtle differences.

One finding in our study was that there was a difference in FP function between adults and larvae. Adult individuals with fluorescence emission mainly expressed *FP* sequences of the S/MWE clade excited by short wavelength light, including UV light, and emitted fluorescence with a peak under 490 nm. Larvae mainly expressed *FP* genes in the S/MWE clade excited by short wavelength light (visible light, no UV) and emitted fluorescence with a peak at a longer wavelength (519 nm). Among the FP recombinants that we analyzed, both the excitation and emission spectra of larval FPs had longer wavelengths than those of adult FPs. The same shift in fluorescence has been observed in *Seriatopora hystrix* ([@evy122-B25]), suggesting that FPs may have similar roles in adults and larvae of distantly related coral species. In addition to the S/MWE clade, larvae mainly expressed *FP* genes in the M/LWE and CP clades. The excitation and emission wavelengths were middle (TYG-type) and long (DYG-type) in the M/LWE clade. CPs absorbed long wavelength light. In summary, it is possible to hypothesized that adults utilized UV to short wavelength light and larvae utilized middle to long wavelength light via FPs, based on the functions of the highly expressed sequences in adults and larvae. These different light usages may provide insight into the biological roles of FPs.

The other key feature of *FP* genes in *A. digitifera* was the pattern of polymorphisms. The highly expressed sequences (*S/Me3_a\_hi*1 and *M/L-De3_a)* in adults from the S/MWE and M/LWE clades were presence--absence polymorphisms in three *A. digitifera* subpopulations in the southern Ryukyu Archipelago with one exception, *M/L-De3_a*, in Yeyama-North. In addition, two highly expressed sequences in larvae from the M/LWE clade were presence--absence polymorphisms in all subpopulations, and polymorphisms in fluorescence in larvae were predicted. These polymorphisms suggest that the presence--absence status of highly expressed *FP* sequences has been maintained in *A. digitifera* subpopulations, at least in the southern Ryukyu Archipelago. In other words, the presence and absence of fluorescence emission in adults (short wavelengths) and in larvae (middle to long wavelengths) has persisted throughout the evolution of *A. digitifera* subpopulations. This polymorphic status may be beneficial for the survival of immobile adult corals in various environments, for example, different light conditions, depths, currents, and attached substrates. In larvae, the polymorphic status may be beneficial for different dispersal distances, as suggested in *A. millepora* ([@evy122-B13]; [@evy122-B32]). In summary, the presence--absence polymorphisms of highly expressed *FP* sequences were a notable feature and will be key to unraveling the biological roles of FPs in corals.

In this study, we demonstrate the genetic basis of differences in fluorescence among individuals in *A. digitifera.* Comparisons between *FP* genotypes and the habitats or external environments of corals will provide a basis for understanding the biological roles of FPs in corals in the near future.

Supplementary Material
======================

[Supplementary data](#sup1){ref-type="supplementary-material"} are available at *Genome Biology and Evolution* online.

Supplementary Material
======================

###### 

Click here for additional data file.

This work was supported by an internal SOKENDAI grant to Y.T., a Center for the Promotion of Integrated Sciences (CPIS) of SOKENDAI grant to Y.T., and a Grant-in-Aid for JSPS Fellows to S.T.-K. We thank Drs Masayuki Hatta (Ochanomizu University) for help with sampling and Mutsumi Nishida (University of the Ryukyus) for arranging the sampling.

[^1]: **Data deposition:** The nucleotide sequences were deposited in GenBank under accession numbers LC349480--LC349725 and in the DDBJ Sequenced Read Archive under accession numbers DRR120550--DRR120560, DRR108013--DRR108023, DRR108003--DRR108012, and DRR108024. Four ancestral sequences estimated in this study are provided in the supporting text file.