Color Morphs of the Coral, Acropora Tenuis, Show Different Responses to Environmental Stress and Different Expression Profiles of Fluorescent- Protein Genes

bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

[Genes/Genomics/Genetics: Investigations]

Color morphs of the coral, Acropora tenuis, show different responses to environmental stress and different expression profiles of fluorescent- protein genes

Noriyuki Satoh1,+,* Koji Kinjo2,+, Kohei Shintaku3,+, Daisuke Kezuka3,

Hiroo Ishimori3, Atsushi Yokokura4, Kazutaka Hagiwara5,

Kanako Hisata1, Mayumi Kawamitsu6, Koji Koizumi7

Chuya Shinzato8, Yuna Zayasu1

1 Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan.

2 Umino-Tane Co. LTD, Yomitan, Okinawa 905-0888, Japan.

3 IDEA Consultants, Inc., Okinawa Branch Office, Naha, Okinawa 900-0003, Japan

4 IDEA Consultants, Inc., Institute of Environmental Informatics, Yokohama, Kanagawa 224-0025, Japan

5 Okinawa Environmental Research Co., Ltd., Naha, Okinawa 900-0003, Japan

6 DNA Sequencing Section, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan.

7 Imaging Section, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan.

8 Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Chiba

277-8564, Japan. *e-mail: [email protected]: ORCID ID, 0000-0002-8520-8876.

+ Equal contribution.

Keywords Acropora tenuis・Okinawa coast・Color morphs・Thermal stress response・Symbiotic zoothanthella・Fluorescent protein gene expression

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ABSTRACT Corals of the family Acroporidae are key structural components of reefs that support the most diverse marine ecosystems. Due to increasing anthropogenic stresses, coral reefs are in decline. Along the coast of Okinawa, Japan, three different color morphs of Acropora tenuis have been recognized for decades. These include brown (N morph), yellow-green (G) and purple (P) forms. The tips of axial coral polyps exhibit specific fluorescence spectra. This attribute is inherited asexually, and color morphs do not change seasonally. In Okinawa Prefecture, during the summer of 2017, the N and P morphs experienced bleaching, in which some N morphs died while P morphs recovered. In contrast, G morphs successfully withstood the stress. Symbiotic dinoflagellates are essential symbiotic partners of scleractinian corals. Photosynthetic activity of symbionts was reduced in July in N and P morphs; however, the three color- morphs host similar sets of Clade-C zoothanthellae, suggesting that beaching of N and P morphs cannot be attributed to differences in symbiont clades. The decoded Acropora tenuis genome includes five genes for green fluorescent proteins (GFP), two for cyan fluorescent proteins (CFP), three for red fluorescent proteins (RFP), and seven genes for chromoprotein (ChrP). A summer survey of gene expression profiles demonstrated that (a) expression of CFP and REP was quite low in all three morphs, (b) P morphs expressed higher levels of ChrP, (c) both N and G morphs expressed GFP highly, and (d) GFP expression was reduced in N morphs, compared to G morphs, which maintained higher levels of GFP expression throughout the summer. Although further studies are required to understand the biological significance of these color morphs of Acropora tenuis, our results suggest that thermal stress resistance is modified by genetic mechanisms that coincidentally lead to diversification of color morphs.

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One of the most critical issues facing the human race is warming of our planet. Anthropogenic activities have harmed the environment in various ways, and coral reefs have been especially impacted (Hoegh-Guldberg et al. 2007; Hughes et al. 2017). In spite of the fact that coral reefs occupy only 0.2% of the ocean area, they are estimated to harbor about one-third of all described marine species (Knowlton et al. 2010; Fisher et al. 2015). Coral reefs are the most diverse marine ecosystems on Earth (Wilkinson 2008). Scleractinian corals, a keystone component of calcium-carbonate based reefs, form obligate endosymbiosis with photosynthetic dinoflagellates or zoothanthellae, which supply the vast majority of their photosynthetic products to the host corals (Yellowlees et al. 2008). However, corals now face a variety of environmental stresses, including increasing surface seawater temperatures, decimation by outbreaks of crown- of-thorns starfish, and acidification and pollution of oceans (Hoegh-Guldberg et al. 2007; Uthicke et al. 2009; Burke et al. 2011; Uthicke et al. 2015; Hughes et al. 2017). Loss of coral reefs is potentially catastrophic.

Faced with such environmental changes, not only are corals diminished by bleaching and other causes, but to survive they must adapt somehow to increasingly stressful environments (Skelly et al. 2007). Acropora tenuis is one of the most important scleractinian corals along the coast of Okinawa, Japan (Omori et al. 2016). Most A. tenuis appear brownish (Fig. 1a, b), reflecting the color of the symbiotic algae that they host. Acropora tenuis exhibits faster growth than other Acropora species, forming colonies approximately 30 cm in diameter within 3~5 years (Fig. 1f) (Iwao et al. 2010). In 1998, along the Okinawa coast, various Acropora species suffered extensive bleaching and many died (Reef Conservation Committee of Japanese Coral Reef Society, 2014). After several years, they gradually recovered. However, while color morphs of Acropora tenuis have been described (Nishihira and Veron 1995), divers in Okinawa noticed the re-appearance of greenish-yellow and purple A. tenuis morphs (Fig. 1c, d) (Fig. 1e).

In corals, fluorescent protein-mediated color polymorphism is thought to contribute to their acclimatization potential (Dove et al. 2001; Dove 2004; Kelmanson and Matz

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2003; Salih et al. 2010; Smith et al. 2013; Paley 2014; Gittins et al. 2015; Jarret et al. 2017). Therefore, we thought that the color polymorphism of Acropora tenuis might be associated with its potential to resist to stressful environments. We describe here the characteristics of the three color-morphs and their capacity to accommodate rising summer surface seawater temperatures. Since the genome of Acropora tenuis has been decoded (Shinzato et al. 2020), we surveyed genes for fluorescent proteins to document the presence of genes for green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP) and chromoprotein (ChrP). We examined gene expression profiles in the three color morphs in response to rising summer surface seawater temperatures.

MATERIALS AND METHODS

Acropora tenuis colonies with different color morphs Various Acropora tenuis color morphs exist along the Okinawa coast. The “wild-type” color morph (N) looks brownish, presumably reflecting the color of its symbiotic algae. Depending upon the color of axial polyp tips, they are further subdivided into NO (the morph is brownish overall, but the tips of axial polyps are orange; Fig 1a) and NB (a brownish morph with bluish axial polyp tips; Figs. 1b and 2a). The color morph that looks greenish (G) is also subdivided into GO (axial polyp tips look orange; Fig. 1c) and GB (axial polyp tips look bluish; Figs. 1d and 2b). Yet another color morph is purple (Figs. 1e and 2c). The five morphs were collected in about 2005/2006 and have been maintained in the “Sea Seed Aquarium” (a non-governmental organization founded by K. Kinjo) at Yomitan, Okinawa, Japan.

Branches of wild specimens of the five different color morphs were broken off and raised for several years in the Sea Seed Aquarium. Nursery-raised corals more than 3 cm in diameter were then transplanted back into the ocean by attaching them to rocks along the shallow coast (2~4 m in depth) in the South China Sea. After several years, they became mature colonies more than 20 cm in diameter (Fig. 1f), which were used for field observations mentioned below.

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For molecular biological analyses, three to five colonies of each color morph were cultured in outdoor pools of the Sea Seed Aquarium under continuous natural seawater supply. Previously, we developed a microsatellite genotyping method that can be widely applied to Acropora species, including A. tenuis, to identify individual colony by assessing specific heterogeneity (Shinzato et al. 2014). This method was employed to determine genetic relationships among the five color-morphs used in this study. Each morph had an independent genotype, and genotypes of the color morphs reproduced asexually were identical (data not shown). That is, it is unlikely that color polymorphism arose from a single founder or a small group of founders. Rather, it appears that each morph was derived from widespread diversification of different ancestral morphs.

Two or three branches approximately 2 cm in length were cut from NB, GB, GO and P

color-morphs on June 9th, July 26th, August 15th, and September 14th, 2017. Branches were immediately fixed in RNAlater in 50-mL Falcon tubes and were maintained at - 80℃ until use. Temperature changes of the outdoor pools were recorded with a HOBO

Pendant Temperature logger (Onset) from July 27th to September 27th. Temperatures of the pools were kept slightly lower than those of the ocean itself.

Fluorescent Emissions Fluorescence spectra of axial polyp tips of A. tenuis color morphs were examined using a Carl Zeiss LSM780 inverted confocal microscope with a 5x Fluar lens. Live branches of NB, GB and P colonies were embedded in a 1% agarose gel in a glass-bottomed dish. Lasers of four wavelengths (405 nm, 488 nm, 561 nm and 633 nm) were employed. Z- stack images, including spectral information from 409 – 695 nm were acquired using 32-channel detectors. Spectra data were displayed with wavelength color codes. Three- dimensional images were constructed with ImageJ (http://imagej.nih.gov/ij/).

Field observations Field observations were conducted during the summers of 2016 and 2017. An ortho- image of the research field constructed with Agisoft Metashape is shown in Fig. S1.

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Surface seawater temperatures at approximately 2 m depth were automatically recorded with a Compact CT (Fig. S2a) (JFE Advantech Co., LTD, Nishinomiya, Japan). The photosynthetic photon flux density (PPFD) was also recoded automatically using a DEFI2-L (Fig. S2b) (JFE Advantech Co., LTD). In 2017, changes in temperature and insolation were observed for 171 days from June 11 to November 28.

Field survey sites employed 2m X 2m quadrats (Fig. S1). Bleaching status of 169 colonies (125 NO, 35 NB, 3 GO, 3 GB, and 3 P) was determined by direct observation by divers. The degree of bleaching was quantified as the bleached surface area relative to the total surface area of each morph. Bleaching outcome (death or survival) was also recorded.

Photosynthetic activity Photosynthetic activity of Acropora tenuis colonies of different color morphs was continuously monitored at each field site. Chlorophyll fluorescence measurements were performed with a pulse amplitude-modulated fluorometer, Diving-PAM (Heinz Walz Company, Germany). Photosynthetic activity was measured by maximum quantum yield of PSII, determined as Fv/Fm. Fv was obtained as Fm-Fo, where Fo is the minimum fluorescence obtained under the measuring beam of PAM (weak pulsed light

< 1μmol photons m-2 s-1) in dark-adapted condition, and Fm is the maximum fluorescence detected using a short, saturating pulse. Data were analyzed with ANOVA using JMP software (https://www.jmp.com). Statistical significance of differences was tested with the Steel-Dwass method.

Identification of symbiotic dinoflagellates Symbiotic dinoflagellate clades from each color morph were identified using RNA-seq data, following the method of Shinzato et al. (2018). Briefly, quality trimmed (QV>20) sequencing reads were aligned to the curated database of Symbiodiniaceae internal transcribed spacer 2 from NCBI using BLASTN (Camacho et al. 2009) with an e-value

cutoff of 1e-20, and the number of reads that ensured that the BLASTN bit score of the best alignment was greater than the second-best alignment was counted.

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The Acropora tenuis genome, isolation of genes for fluorescent proteins, and molecular phylogeny A draft genome of Acropora tenuis was sequenced using Illumina technology by Shinzato et al. (2020). The approximately 407-Mbp A. tenuis genome is estimated to contain 23,119 protein-coding genes with a BUSCO score of 97.4% (92.2% completeness). Acropora tenuis genes for green (GFP), cyan (CFP), and red (RFP) fluorescent proteins, and non-fluorescent blue/purple chromoprotein (ChrP) were identified by BLAST searching of the genome using A. digitifera genes as queries for GFP, CFP, RFP and ChrP, respectively (Shinzato et al. 2012). Orthologous relationships of genes of the two Acropora species were confirmed by molecular phylogenetic analysis, carried out using RAxML 8.2.11 (Stamatakis 2016)

Expression levels of genes for GFP, CRP, RFP and ChrP. Total RNA was extracted from all specimens using an RNA plant mini Kit (Qiagen). cDNA libraries were produced using a TruSeq Standard mRNA Library Prep Kit for NeoPrep (Illumina). Library sequencing was carried out on a Hi-seq4000 (Illumina) according to the method described by Pertea et al. (2015). Sequencing adapters and low-quality (

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RESULTS

Color morphs of Acropora tenuis at the Okinawa coast The Ryukyu Archipelago, and especially the Okinawa coast, experienced very severe coral bleaching in 1998. Afterward, local divers and researchers noticed that recovering A. tenuis exhibited different color morphs (Fig. 1). These included green (GO and GB) and purple morphs (P), as well as the predominant brown (NO and NB) color morphs (Fig. 1). These were collected and maintained at the Sea Seed Aquarium. Nubbins of each color morph were asexually reproduced by cutting branches from original colonies, nursing them for a few years at the Aquarium, and culturing them in the ocean (average depth, 2~4 m) adjacent to the Aquarium (Fig. 1f). Since then, the five color morphs have maintained their originals colors and features.

To characterize the color morphs, fluorescence spectral imaging was carried out on the tips of axial polyps of NB, GB, and P morphs. Lasers (405, 488, 561, and 633 nm) were employed, and three-dimensional images were constructed using ImageJ. Polyps of all color morphs fluoresced green inside the mouth when excited at 488 nm (Fig. 2g-i). On the other hand, the response to 405-nm excitation differed among color morphs. Body walls of NB and P morphs fluoresced blue (Fig. 2d, f). In contrast, body walls of GB morphs fluoresced green (Fig. 2e). Fewer differences in red and deep red fluorescent emissions were detected among the three color morphs when excited at 561 and 633 nm (Fig. 2j-o). Although detailed molecular and biochemical mechanisms remain to be explored, these differences in fluorescence emission may be associated with differences in the color of A. tenuis.

Responses of color morphs to summer environmental stress To examine whether the three color morphs differ in their potential to respond to environmental changes, especially the rise of surface seawater temperatures, we followed bleaching profiles of the five morphs in the summers of 2016 and 2017. A similar trend in the mode of bleaching was observed in both years. Since the seawater

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temperature rise was higher and coral bleaching was more severe in 2017 than 2016, we describe here results from 2017.

Changes in surface seawater temperatures (Fig. S2a) and photosynthetic photon flux density (PPFD) (Fig. S2b) were automatically recorded using a Compact CT and a DEFI2-L, respectively. The surface seawater temperatures rose to over 30℃ in early July, a temperature that continued until early September. Forty-six of 171 days exceeded 30℃, with a maximum of 34.2℃ on July 27 (Fig. S2a). The PPFD also increased toward mid-July, and a high level of PPFD continued until late September,

although daily variations occurred (Fig. S2b). The highest was 4,187  mol/m-2 sec-1 on August 25. Bleaching status was observed by diving on July 26, August 30, September 29, and November 29.

The degree of bleaching was highest in NO and NB, then P (Fig. 3). More than half of NO colonies commenced bleaching in July and August (Fig. 3a). Although 20~30% of bleached NOs recovered their color morphs in September and November, 10~15% of them died after bleaching (Fig. 3a). The percentage of bleached NBs in July and August was less than that of NOs, but many of the bleached NBs did not recover and died (Fig. 3b). Nearly 30% of NBs died by late November (Fig. 3b). Nearly 20% of the P morph surface area appeared dead (Fig. 3e). Bleaching occurred in 30% of P morphs in late August, but most of them recovered in late September (Fig. 3e). In contrast, G morphs, especially GB, did not bleach at all (Fig. 3d), although a few GO colonies died for unknown reasons (Fig. 3c). This suggests that G morphs have the greatest potential resistance to increasing summer surface seawater temperatures in Okinawa A. tenuis.

Coral bleaching is caused by the escape of symbiotic dinoflagellates from corals or by their death inside corals. Decreased photosynthetic activity of symbiotic dinoflagellates may degrade their physical condition, and in turn, that of coral hosts. Photosynthetic activity of symbionts can be measured by chlorophyll fluorescence (Fv/Fm). We examined whether the three color-morphs show different Fv/Fm profiles (Fig. 4). The observed Fv/Fm value was nearly the same in all five color morphs in June, August,

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September and November, but in July, GB and GO maintained their levels of Fv/Fm, whereas it was reduced in NO, and was lowest in P and NB (Fig. 4a). GB and GO maintained photosynthetic activity at the level of June (Fig. 4a). While activity was reduced in P and NB, both recovered their photosynthetic rates in August to the same level as in GB and GO (Fig. 4a). The decrease in Fv/Fm of NB and P in July was statistically significant, compared to those of GB and GO (p<0.05) (Fig. 4b). The positive correlation between the rate of bleaching and photosynthetic activity confirms that coral bleaching is caused at least to some extent by decreased photosynthetic activity of symbiotic dinoflagellates, as shown by previous studies (e.g., Smith et al. 2013; Gittins et al. 2015).

Clade distribution of symbiotic dinoflagellates among color morphs One possible explanation for the differing potentials of the various color morphs to resist thermal stress is that they host different clades of symbiotic dinoflagellates. The zoothanthella family Symbiodiniaceae has recently been reorganized into nine clades or genera, according to new analyses of combinatorial data (LaJeunesse et al. 2018). It has been suggested that corals that host some species of Clade D (Durusdinium) are more heat-resistant than those that host species of Clade C (Cladocopium). Therefore, we examined Symbiodiniaceae clades hosted by A. tenuis to see whether the clade differs among the five color morphs.

RNA-Seq analyses of the internal transcribed spacer 2 (ITS2) sequence showed that all color morphs hosted only Clade C (Cladocopium) zoothanthellae (Fig. 5). No other clades were detected. Among them, group C1 was most abundant, and C50 was next, with smaller numbers of various other Clade-C subgroups (Fig. 5). NB and GB morphs hosted more C3k, whereas P morph hosted more C1i compared with other color morphs, although these differences were not discrete. These symbiont profiles did not change during the study period from late June to early September 2017. Acropora tenuis appeared to incorporate higher numbers of C1 and C50 zoothanthella in August and September than in June and July, and this tendency was pronounced in GO color morphs. Nonetheless, these results indicate that shifts in symbiont clades are not the main cause of color differences and/or thermal-resistance of the color morphs, although

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their levels of photosynthetic activity are likely involved in bleaching of NG and P morphs (Fig. 4).

Genes for fluorescent proteins Vivid coloration of corals has been attributed to fluorescent proteins, including green (GFP), red (RFP) and cyan (CFP) proteins, and non-fluorescent blue/purple chromoprotein (ChrP) (e.g., Dove et al. 2001; Kelmanson et al. 2003; Salih et al. 2010; Smith et al. 2013). Since genome decoding of A. digitifera (Shinzato et al. 2011), it has been shown that A. digitifera contains five GFP genes, one RFP, one CFP, and three genes for ChrP (Shinzato et al. 2012). The approximately 407-Mbp genome of Acropora tenuis has also been decoded (Shinzato et al. 2020). BLAST searches employing A. digitifera fluorescent-protein gene sequences as queries demonstrated the presence in the A. tenuis genome of five genes for GFP (Gene ID: s0077.g62, s0297.g27, s0297.g28, s0297.g29, and s0366.g7) (shown by green in Fig. 6a), two genes for CFP (s0010.g7 and s0025.g63) (black in Fig. 6b), three genes for RFP (s0024.g131, s0024g134 and s0217.g35) (red in Fig. 6c), and seven genes for ChrP (s0096.g1, s0096.g3, s0096.g4, s0152.g9, s0152.g11, s0152.g15, and s0182.g24) (purple in Fig. 6d), respectively (https://marinegenomics.oist.jp/gallery/gallery/index.). Molecular phylogeny supports orthologous relationships of the Acropora tenuis genes with genes of fluorescent proteins and non-fluorescent chromoproteins of other corals (Fig. S3). Transcriptomes corresponding to these genes have been identified during genome decoding (Shinzato et al. 2020), and were used to determine their expression profiles (see below).

Expression profiles of fluorescent protein genes Ten colonies of four color-morphs (three NB, two GB, three GO and two P) were cultured in an outdoor pool at the Sea Seed Aquarium from late June to early September 2017. We sampled specimens at three time points to examine expression profiles of fluorescent protein genes in each of the ten colonies. Three color morphs (N, G and P) showed different expression profiles of fluorescent protein-related genes during the summer (Fig. 7). First, the two CFP genes and three RFP genes showed very low expression levels with almost no detectable changes during the summer (Fig. 7). This

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indicates that genes for the four fluorescence-related proteins do not necessarily show similar responses to environmental changes, including surface seawater temperature rise, and that genes for CFP and RFP are unlikely to be regulated by surface seawater temperatures. On the other hand, GFP and ChrP genes did show detectable changes in expression profiles.

Two GFP genes (s0297.g27 and s0297.g29) showed distinctive changes in expression profile, while expression levels of the other three genes (s0077.g62, s0297.g28 and s0366.g7) remained low, with little change (Fig. 7). This indicates that even among genes that encode the same fluorescent protein and that are located in close proximity on the same chromosome (Fig. 6), their responses to SST are not always the same, suggesting complicated individual gene regulation in response to environmental stresses. In addition, s0297.g27 and s0297.g29 exhibited an interesting shared expression profile during the study period. First, the expression level of s0297.g29 was distinctively higher in GB and GO than in NB and P (Fig. 7). In all three wild-type NB1, 2 and 3 genotypes, s0297.g29 showed moderate levels of expression in June, but diminished levels with rising temperatures in August and September. A similar expression profile of s0297.g29 was detected in GB1, GO1, GO3, and P1. However, gene expression was distinctively different between N and G. The expression level was much higher in GB and GO than in NB. In addition, the higher level of s0297.g29 expression in GB2 and GO2 was maintained throughout the summer (Fig. 7). Although less obvious, a similar expression profile was observed in s0297.g27 (Fig. 7).

The purple morphs exhibited another expression profile of these genes, especially in relation to genes for non-fluorescent blue/purple chromoprotein (ChrP). Of seven ChrP genes, s0096.g1, s0096.g3, s0096.g4 and s0182.g24 exhibited an interesting expression profile (Fig. 7). Although the level of the three other genes, s0152.g9, s0152.g11 and s0152.g15 (localized in the same scaffold) was comparatively low (Fig. 7), the former four ChrP genes were expressed at higher levels in P1 and P2 than in NB, GB, and GO (Fig. 7). Although expression level differed among the four genes, this pattern was shared by all four color morphs and relative levels of the four genes were maintained

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during the summer as surface seawater temperatures rose (Fig. S2a). s0096.g4 showed significantly higher expression in both P1 and P2 as September approached (Fig. 7). This tendency was also seen in s0182.g24. These results indicate that in contrast to the role of GFP in N and G colonies, ChrP is very likely involved in coloration of P colonies and has potential to resist surface seawater temperature rise.

DISCUSSION

There are at least three color morphs of Acropora tenuis along the Okinawa coast, brown, green and purple (Nishihira and Veron 1995) (Fig. 1). They exhibit different profiles of fluorescence in the axial polyps when excited at 405 nm (Fig. 2). We expect that differences in fluorescence are associated with coloration of this coral, although detailed molecular, biochemical, and biophysical mechanisms underlying these differences should be addressed in future studies. The color morphs are stable as far as we have observed for a decade, since color morphs show no seasonal alternation (e.g., Paley 2014), although the brightness of color changes. Color is asexually heritable. Although it remains to be determined whether it is also sexually heritable, color polymorphism is likely caused by genetic / genomic variation.

Field studies in 2017 showed that N morphs underwent extensive bleaching (Fig. 3a, b), and 10~15% of NB morphs eventually died (Fig. 3b). Bleaching occurred in P morphs in late August, but many of them recovered in subsequent months (Fig. 3e). In contrast, G morphs did not show bleaching (Fig. 3c, d). Therefore, it is evident that sensitivity to thermal stress differs among the three color morphs. N is the most sensitive, P is moderate, and G is resistant. Along the Okinawa coast, the N color morph is most abundant (as seen in Fig. 3 and Fig. S1), while G and P are not as common. This suggests that the N morph is probably ancestral, while the G and P color morphs are more recent. If this feature is heritable, it is tempting to speculate that in Okinawa, A. tenuis has acquired the capacity to resist thermal stresses by developing new color morphs. In other words, polyp color polymorphism may be a strategy to survive severe

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summer environmental stresses, provided that the current rate of temperature change does not overwhelm the coral’s capacity to adapt.

Bleaching responses are complicated, varying across colonies, taxa, and events (e.g. van Woesik et al. 2011). Scleractinian corals form obligate endosymbioses with photosynthetic dinoflagellates or zoothanthellae, and host-symbiont interactions contribute to differential bleaching susceptibility (Enrıquez et al. 2005; Hawkins et al. 2014; Wooldridge 2014). Since bleaching is usually caused by escape and/or death of symbionts, declining photosynthetic activity of zoothanthellae may portend bleaching. Indeed, decreased photosynthetic activity was detected in June in NB and P morphs (Fig. 4). In contrast, in July, GO and GB morphs maintained photosynthetic activity comparable to that in other months. This suggests that G color morphs provide a suitable physiological environment for symbionts to remain in the host despite the high thermal stress. Genetic and molecular mechanisms involved in this relationship are intriguing, and should be addressed in future studies.

The zoothanthella family Symbiodiniaceae has recently been reorganized into nine clades or genera (A-I), according to new analyses of combinatorial data (LaJeunesse et al. 2018). It has been suggested that in general, corals hosting Clade D (Durusdinium) are more heat-resistant than those hosting Clade C (Cladocopium) or that specific Symbiodinium phylotypes, such as D1, D1-4, C15 and A3 are exceptionally thermotolerant, while others (e.g. C3, C7, B17, A13) are thermosensitive (e.g. Silverstein et al. 2011; Tonk et al. 2014). Therefore, differing capacities of A. tenuis color morphs to resist higher surface seawater temperatures may be due to different zoothanthella hosted by the three color morphs. However, this is not the case in Okinawa A. tenuis, because all three host a very similar repertoires of Clade C Symbiodinium, especially C1 and C5 types (Fig. 5).

Acropora tenuis is useful as an experimental system because its ~400-Mb genome has been decoded, and is thought to contain 22,802 protein-coding genes (Shinzato et al. 2020). Moreover, approximately 95% of the gene models are confirmed with

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corresponding mRNAs. Therefore, we were able to investigate not only the genes for GFP, CFP and RFP and ChrP, but also their expression profiles in colonies with different color morphs during the summer of 2017. We found that all three morphs possess an identical array of fluorescence-related genes and that expression of these genes was confirmed in all morphs using RNA-seq. This indicates that color polymorphism is not caused by mutations in the genes themselves. Instead, molecular and genic mechanisms that control transcriptional activity or quantitative regulation over gene products may produce the three color morphs. Genetic and genic mechanisms underlying fluorescent protein-mediated color polymorphisms and adaptation potential to variable environmental conditions have been studied in Acropora millepora as an experimental system (D’Angelo et al. 2008; Smith et al. 2013; Gittins et al. 2015). In this species, an RFP gene, amiFP597, is essential for color polymorphism. The gene, amiFP597, is multicopy with a particular promoter type in the genome, and the number of gene copies is strongly correlated with the level of gene expression. Higher levels of gene expression are found in more intensely red morphs, which show higher resistance to strong insolation (Gittins et al. 2015 ). This suggests the presence of variable genetic mechanisms in color polymorphisms, depending on coral species.

Interestingly, the P morph exhibits a gene expression profile different from those of the N and G morphs. Specifically, the P morph shows higher ChrP gene expression, especially s0096.g4 (Fig. 7). In addition, the N and G morphs differ in expression levels of GFP genes, especially s0297.g29 and s02897.g27 (Fig. 7). Higher gene expression was evident in the G morph compared to the N morph (Fig. 7). Because the G morph is the most resistant to high SSTs, it is likely that stress resistance varies with different expression levels of GFP genes. We expect that different control mechanisms of GFP gene expression in N and G morphs explain their different thermal tolerance. We will address this question in future studies.

Coral bleaching is caused by multiple, complex environmental stresses. Except for outbreaks of crown-of-thorns starfish, typhoons, and diseases, the most deleterious influence is surface seawater temperature warming. Strong solar radiation also likely

15 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

contributes to environmental stress. Seawater in Okinawa Prefecture is very transparent; thus, UV irradiation acts in concert with higher sea temperatures to cause bleaching. Fluorescent proteins and chromoproteins absorb UV radiation, rendering corals and their symbionts more resistant to solar stress (Dove et al. 2001; Dove 2004; Banaszak and Lesser 2009; Salih et al. 2010). We need to better understand the relationship between solar stress and different expression profiles of genes for GFP and ChrP.

Divers and marine researchers have noticed that some corals, sometimes called “Super corals” (https://phys.org/news/2019-05-super-corals-glimmer-world-dying.html) and/or “super coral reefs” (Dance 2019), are able to survive severe environmental stresses. Nonetheless, almost nothing is known about how such super corals have appeared in the reefs. Our present results regarding different thermal tolerances of three Acropora tenuis color morphs may help to explain the resilience of these super corals.

ACKNOWLEDGEMENTS

This work was supported by Okinawa Prefecture Project of Coral Preservation to KS, DK, HI, AY, KH and NS. The Sumiko Imano Memorial Foundation by Sumio Imano, Haruo Imano and Hiromichi Imano to NS and OIST support to Marine Genomics Unit are much appreciated. YZ and CS are supported by JSPS grants (17K15179 and 19K15902 to YZ, and 17K07949, 17KT0027 and 20H03235 to CS). The DNA Sequencing and Imaging Sections of OIST are gratefully acknowledged. We thank Dr. Steven D. Aird for editing the manuscript.

16 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

LITERATURE CITED

Banaszak, A.T. and M.P. Lesser, 2009 Effects of solar ultraviolet radiation on coral reef organisms. Photochem. Photobiol. Sciences 8: 1276-1294. Bolger, A.M., M. Lohse and B. Usadel, 2014 Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114–2120. Burke, L., K. Reytar, M. Spalding and A. Perry, 2011 Reefs at risk revisited, World Resources Institute. Camacho, C., G. Coulouris, V. Avagyan, N. Ma, J. Papadopoulos, K. Bealer, T.L. Madden, 2009 BLAST+: architecture and applications. BMC Bioinformatics 10: 42. Dance, A., 2019 Hope for coral reefs. Nature 575: 580-582. D’Angelo C., A. Denzel, A. Vogt, et al., 2008 Blue light regulation of hot pigment in reef building corals. Marine Ecology Progress Series 364: 97-106. Dove, S.G., O. Hoegh-Guldberg and S. Ranganathan, 2001 Major colour patterns of reef-building corals are due to a family of GFP-like proteins. Coral Reefs 19:197-204. Dove, S.G., 2004 Scleractinian corals with photoprotective host pigments are hypersensitive to thermal bleaching. Marine Ecology Progress Series 272: 99- 116. Enriquez, S., E.R. Mendez and R. Iglesias-Prieto, 2005 Multiple scattering on coral skeletons enhances light absorption by symbiotic algae. Limnology and Oceanography 50: 1025-1032. Fisher, R., R.A. O’Leary, S. Low-Choy, K. Mengersen, N. Knowton, R.E. Brainard and M.J. Caley, 2015 Species Richness on Coral Reefs and the Pursuit of Convergent Global Estimates. Curr. Biol. 25: 500-505. Gittins, J.R., C. D’Angelo, F. Oswald, R.J. Edwards and J. Wiedenmann, 2015 Fluorescent protein-mediated color polymorphism in reef corals: multiple genes extend the adaptation/acclimination potential to variable light enviroment. Molecular Ecology 24:453-465. Hawkins, T.D., T. Krueger, S. Becker, P.L. Fisher and S.K. Davy, 2014 Differential nitric oxide synthesis and host apoptotic events correlate with bleaching susceptibility in reef corals. Coral Reefs 33: 141-153. Hoegh-Guldberg, O., P.J. Mumby, A.J. Hooten, R.S. Steneck, P. Greenfield et al., 2007 Coral reefs under rapid climate changes and ocean acidification. Science 318: 1737-1742.

17 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Hughes, T.P., J.T. Kerry, M. Alvarez-Noriega, J.G. Alvarez-Romero, K.D. Anderson et al., 2017 Global warming and recurrent mass bleaching of corals. Nature 543: 373-377. Iwao, K., M. Omori, H. Taniguchi and M. Tamura, 2010 Transplanted Acropora tenuis (Dana) spawned first in their life 4 years after culture from eggs. Galaxea, J Coral Reef Studies, 12, 47. LaJeunesse, T.C., J.E. Parkinson, P.W. Gabrielson, H.J. Jeong, J.D. Reimer, C.R. Voolstra and S.R. Santos, 2018 Systematic revision of Symbiodiniacea highlights the antiquity and diversity of coral endosymbionts. Curr. Biol. 28: 2570-2580. Jarett, J.K., M.D. MacManes, K.M. Morrow, M.S. Pankey and M.P. Lesser, 2017 Comparative genomics of color morphs in the coral Montestraea cavernosa. Scientific Reports 7:16039. Knowlton, N., R.E. Brainard, R. Fisher, M. Moews, L. Plaisance and M.J. Caley, 2010 Coral reef biodiversity, in Life in the World’s Oceans: Diversity, Distribution, and Abundance, ed McIntyre AD (Chichester: Wiley-Blackwell), pp. 65–79. Kelmanson, I.V. and M.V. Matz, 2003 Molecular basis and evolutionary origins of color diversity in great star coral Montastraea cavernosa (Scleractinia: Faviida). Mol. Biol. Evol. 20: 1125–1133. Liao, Y., G.K. Smyth and W. Shi, 2014 featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30: 923-30. Love, M.I., W. Huber and S. Anders, 2014 Moderated estimation of fold change and dispersion for RNA‐seq data with DESeq2. Genome Biology 15: 550. McCarthy, D.J., Y. Chen and G.K. Smyth, 2012 Differential expression analysis of multifactor RNA‐Seq experiments with respect to biological variation. Nucleic Acids Research 40: 4288–4297. Nishihira, M. and J.E.N. Veron, 1995 Hermatypic corals of Japan, Kaiyusya, Tokyo. Omori, M., Y. Higa, C. Shinzato, Y. Zayasu, T. Nagata et al., 2016 Development of active restoration methodologies for coral reefs using asexual reproduction in Okinawa, Japan. Proceedings of the 13th International Coral Reef Symposium, 369-387. Paley, A.S., 2014 Colour polymorphism and its role in stress tolerance in the coral Acropora millepora in the Great Barrier Reef. The degree of Doctor of Philosophy, James Cook University. Pertea, M., D. Kim, G. Pertea, J.T. Leek and S.L. Salzberg, 2014 Transcript-level

18 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nature Protocols 11: 1650-67. Pertea, M., G.M. Pertrea, C.M. Antonescu, T-C. Tsuagm et al., 2015 StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnol. 33: 290–295. Reef Conservation Committee of Japanese Coral Reef Society, 2014. Coral bleaching in the summer of 2013. Salih, A., A. Larkum, G. Cox, M. Kühl and O. Hoegh-Guldberg, 2010 Fluorescent pigments in corals are photoprotective. Nature 408: 850-853. Schmieder, R. and R. Edwards, 2011 Quality control and preprocessing of metagenomic datasets. Bioinformatics 27: 863-864. Shinzato, C., E. Shoguchi, T. Kawashima, M. Hamada, K. Hisata et al., 2011 Using the Acropora digitifera genome to understand coral responses to environmental change. Nature 476: 320-323. Shinzato, C., E. Shoguchi, M. Tanaka and N. Satoh, 2012 Fluorescent protein candidate genes in the coral Acropora digitifera genome. Zool. Sci. 29: 260-264. Shinzato, C., Y. Yasuoka, S. Mungpakdee, N. Arakaki, M. Fujie, Y. Nakajima and N. Satoh. 2014 Development of novel, cross-species microsatellite markers for Acropora corals using next-generation sequencing technology. Frontiers in Marine Science 1: 11 Shinzato, C., Y. Zayasu, K. Kanda, M. Kawamitsu, N. Satoh, H. Yamashita and G. Suzuki, 2018 Using seawater to document coral-zoothanthella diversity: a new approach to coral reef monitoring using environmental DNA. Frontiers Marine Sci 5: 28 Shinzato, C., K. Khalturin, J. Inoue, Y. Zayasu et al., 2020 Eighteen coral genomes reveal the evolutionary origin of Acropora strategies to accommodate environmental changes. Mol. Biol. Evol. in press Silverstein, R.N., A.M.S. Correa, T.C. Lajeunesse and A.C. Baker, 2011 Novel algal symbiont (Symbiodinium spp.) diversity in reef corals of Western Australia. Marine Ecology Progress Series 422: 63-75. Skelly, D.K., L.N. Joseph, H.P. Possingham, L.K. Freidenburg, T.J. Farrugia, M.T. Kinnison and A.P. Hendery, 2007 Evolutionary responses to climate change. Conservation Biology 21: 1353-1355. Smith, E.G., C. D’Angelo, A. Salih and J. Wiedenmann, 2013 Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae. Coral Reefs 32: 463-474

19 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Stamatakis A., 2016 The RAxML v8.2.X Manual. Tonk, L., E.M. Sampayo, T.C. Lajeunesse, V. Schrameyer and O. Hoegh-Guldberg, 2014 Symbiodinium (Dinophyceae) diversity in reef-invertebrates along an offshore to inshore reef gradient near Lizard Island, Great Barrier Reef. Journal of Phycology 50: 552-563. Uthicke, S., M. Logan, M. Liddy, D. Francis, N. Hardy and M. Larmare, 2015 Climate change as an unexpected co-factor promoting coral eating seastar (Acanthaster planci) outbreaks. Scientific Reports 5. Uthicke, S. B. Scharffelke and M. Byrne, 2009 A boom-bust phylum? Ecological and evolutionary consequences of density variations in echinoderms. Ecological Monographs 79: 3-24. van Woesik, R., K. Sakai, A. Ganase and Y. Loya, 2011 Revisiting the winners and the losers a decade after coral bleaching. Marine Ecology Progress Series, 434: 67- 76. Wilkinson, C., 2008 Status of Coral Reefs of the World: 2008, Global Coral Reef Monitoring Network and Reef and Rainforest Research Centre, Townsville, Australia. Wooldridge, S. A., 2014 Differential thermal bleaching susceptibilities amongst coral taxa: re-posing the role of the host. Coral Reefs 33: 15-27. Yellowlees, D., T.A. Rees and W. Leggat, 2008 Metabolic interactions between algal symbionts and invertebrate hosts. Plant Cell Environ 31:679-694.

20 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure Legends

Figure 1 Color morphs of Acropora tenuis along the Okinawa coast. (a, b) Two “wild-type” morphs, NO (a) and NB (b). Both appear generally brownish while the tips of axial polyps are orange in NO (a) and blue in NB (b). (c, d) Two greenish morphs, GO (c) and GB (d). Tips of axial polyps are orange in GO (c) and blue in GB (d). (e) A purple colony (P). (f) Branches originating from color morphs are growing in shallow water off the coast of Okinawa.

Figure 2 Spectral imaging of fluorescent emission at the tips of axial bodies of NB, GB, and P morphs in A. tenuis. Morphology of tip regions of the axial bodies of NB (a), GB (b) and P (c) color morphs. Lasers of four different wavelengths, 405 nm (d-f), 488 nm (g-i), 561 nm (j-l) and 633 nm (m-o), were used for excitation. Spectra data are displayed with wavelength color codes. Three-dimensional images were constructed with ImageJ.

Figure 3. Bleaching and subsequent death of Acropora tenuis color morphs in the summer of 2017 along the Yomitan, Okinawa coast. The surface area of color morphs that exhibit bleaching is shown in white, with that of healthy areas is green. Dead areas after bleaching are in red. Colonies that disappeared due to wave action or unknown causes are shown in grey. Because the numbers of each color morph examined differ,

areas are shown by different values on the y-axis (m2). It is evident that GO and GB were little affected by environmental stresses, compared to NO, NB and P.

Figure 4. Photosynthetic activity (Fv/Fm) of five color morphs of Acropora tenuis during the summer of 2017. (a) Activity profiles in June, July, August, September and November. Differences in activity were evident in July among the five color morphs. (b) Comparison of activities in July among the five color morphs. n, the number of colonies examined. Bars indicate one standard deviation. * and ** show statistically- relevant differences between GO/GB and NB/P, using the Steel-Dwass method.

Figure 5. Symbiotic zoothanthellae hosted by five color morphs of Acropora tenuis. Zoothanthella clades were identified based upon ITS-2 sequences, determined by RNA- seq analysis. Numbers of sequencing reads are shown by color density (right upper column) as log2 (number of valid alignments +1). All sub-populations hosted only

21 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Clade C zoothanthellae (Cladocopium), numbers of which did not change during the summer examined.

Figure 6. Genes for fluorescent proteins in the Acropora tenuis genome. The A. tenuis genome contains 13 fluorescent protein-related genes. (a) Three scaffolds, s0077, s0297 and s0366, contain one, three, and one gene for green fluorescent proteins (GFP; shown in green). Other genes (grey) in scaffolds are named if the gene was annotated. Arrowheads indicate the transcription direction. (b) Two scaffolds, s0010 and s0025, contain one gene each for cyan fluorescent proteins (CFP; shown in black). (c) Two scaffolds, s0024 and s0217, contain two and one gene, respectively, for red fluorescent proteins (RFP; shown in red). (d) Three scaffolds, s0096, s0152 and s0182, contain three, three, and one gene for chromoproteins (shown in purple). Molecular phylogeny of these genes is shown in Supplementary Fig. S3.

Figure 7. An expression profile of genes for fluorescent proteins in four Acropora tenuis color morphs during the summer of 2017. Expression levels of four GFP genes (green), two CFP genes (black), three RFP (red) and seven chromoprotein genes (purple) are shown with their densities (right upper column) as log2 (number of valid alignments +1). Gene IDs are identical to those in Fig. 6. NB1, 2, and 3 are three genotypes of the NB morph, GB1 and 2 are two genotypes of the GB morph, GO1, 2, and 3 are three genotypes of the GO morph, and P1 and 2 are two genotypes of the P morph.

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Supplementary Figure S1 An ortho-image of a sample site of Acropora tenuis to monitoring bleaching of the five color morphs in 2017. Two quadrats (2m X 2m), enclosed with yellow, contain seven (left) and five morphs with different colors, which were used for PAM measurement of photosynthetic activity. Bleaching was observed on all morphs in the quadrats.

Supplementary Figure S2 Changes in surface seawater temperature (SST) (a) and photosynthetic photon flux density (b) during the summer and autumn of 2017. Both were automatically measured. June 11, July 11, August 10, September 9, October 9 and November 8 indicate dates of coral bleaching observations. SSTs >30℃ continued from early July to early September. The dotted red line indicates 30℃.

Supplementary Figure S3 Molecular phylogeny of coral fluorescent proteins of Acropora tenuis. Phylogenetic relationships of four GFPs (green bold letters), two RFPs (red bold letters) and seven chromoproteins (purple bold letters) are shown. CFPs are not included due to their extreme divergence from the others. The tree was constructed using the NJ method and bootstrap values (% of 1,000 replicates) are shown for major branches. The scale bar indicates a 0.5% substitution rate.

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Figuer 1

a b c d e

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Figuer 2

Color morph NB GB P a b c

Daylight

Excitation d e f

405

g h i 488

j k l 561

m n o 633

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Figuer 3

a NO b NB )

8 ) 1.5

2 m

m disappeared

(

a r

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o C

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a e

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Figure 3. Bleaching and subsequent death of Acropora tenuis color morphs in the summer of 2017 along the Yomitan, Okinawa coast. The surface area of color morphs that exhibit bleaching is shown in white, with that of healthy areas is green. Dead areas after b leaching are in red. Colonies that disappeared due to wave action or unknown causes are shown in grey. Because the numbers of each color morph examined differ, areas are shown by different values on the y-axis (m2). It is evident that GO and GB were little affected by environmental stresses, compared to NO , NB and P.

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figuer 4

a b

0.8 0.8

0.7 0.7 *

0.6 0.6 m

m *

F /

/ 0.5 0.5

v F F ** 0.4 ● NO 0.4 ** ● NB 0.3 0.3 ● GO ● GB ● P 0.2 n=3 n=4 n=3 n=2 n=2 0.2 Jun Jul Aug Sep Nov NO NB GO GB P

Figure 4. Photosynthetic activity (Fv/Fm) of five color morphs of Acropora tenuis during the summer of 2017. (a) Activity profiles in June, July, August, September and November. Differences in activity were evident in July among the five color morphs. (b) Com parison of activities in July among the five color morphs. n, the number of colonies examined. Bars ind icate one standard deviation. * and ** show statisti - cally-relevant differences between GO/GB and NB/P, using the Steel-Dwass method.

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 5

Jul 7

NO Aug 6 Sep 5 Jun 4 NB Jul Aug 3 Sep 2 Jun 1 Jul GO 0 Aug Sep Jun GB Jul Aug Sep Jun P Jul Aug Sep

C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 3 4 5 5 5 6 6 6 6 7 8 9 9 9 9 s 0 0 0 1 2 2 2 5 _ c i m 0 1 3 4 5 .1 7 _ h k t 2 0 4 6 1 2 4 9 9 9 1 2 3 p 5 6 7 3 3 4 5 _ g ● ● .b 4 n a _ a a ● ● _ _ ● _ d ● a ● ● g ro C C 1 e _ g _ C C g g C g C ● C C ro u 4 w g ro g 7 6 ro ro 9 ro 1 C 1 1 u p 5 5 ro u ro 0 9 u u 2 u 0 1 1 2 p _ 2 u p u a p p a p 6 0 4 6 g p p a 7 ro b u p

Figure 5. Symbiotic zoothanthellae hosted by five color morphs of Acropora tenuis. Zoothanthella clades were identified based upon ITS-2 sequences, determined by RNA-seq analysis. Numbers of sequen cing reads are shown by color density (right upper column) as log2 (number of valid alignments +1). All sub-populations hosted only C lade C zoothanthellae (Cladocopium), numbers of which did not change during the summer examined.

28 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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Supplementary Figuer 1

GB1

E NB3 NO1 W GO2 GO3

NB2 P2

GB2 GO1 NB2 NO2

NB4

NO3

Supplementary Figure S1 An ortho-image of a sample site of Acropora tenuis to monitoring bleaching of the five color morphs in 2017. Two quadrats (2m X 2m), enclosed with yellow, contain seven (left) and five morphs with dif ferent colors, which were used for PAM measurement of photosynthetic activity. Bleaching was observed on all morphs in the quadrats.

31 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

32 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272823; this version posted August 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is

made available under aCC-BY-NC-ND 4.0 International license.

Supplementary Figuer 3

a 0.5% substitution rate.

the others.

letters), two RFPs (red bold letters) and seven chromoproteins (purple bold letters) are shown. CFPs are not included due to their extreme diver

Supplementary Figur

The tree was constructed using the NJ method

e S3 Molecular

phylogeny of coral fluor

AAK71342.1 cgigFP-g AAK71342.1 Condylactis gigantea

and bootstrap values (% of 1,000 replicates) are shown for major branches.

AAN05449.1 red fluorescent protein FP611 Entacmaea quadricolor Entacmaea FP611 protein fluorescent red AAN05449.1

escent pr

AAL27541.1 GFP-like chromoprotein Condylactis passiflora

oteins of

AAM10625.

AAU06854.1 chromoprotein Acropora millepora Acropora chromoprotein AAU06854.1

BAM10197.1 fluorescent protein 8 Acropora digitifera Acropora 8 protein fluorescent BAM10197.1

aten

FAA00744.1 TPA: fluorescent protein 7 partial Acropora digitifera Acropora partial 7 protein fluorescent TPA: FAA00744.1

aten

ABB17949.1 GFP-like chromoprotein Goniopora djiboutiensis

FAA00745.1 TPA: fluorescent protein 9 partial Acropora digitifera Acropora partial 9 protein fluorescent TPA: FAA00745.1

3 green fluorescent protein Dendronephthya sp. SSAL-2002 sp. Dendronephthya protein fluorescent green 3

aten

ABB17955.1 cyan fluorescent GFP-like protein Mycedium elephantotus

Acropora tenuis.

s0096.

s0182.

ACD13194.1 green fluorescent GFP-like protein Platygyra lamellina Platygyra protein GFP-like fluorescent green ACD13194.1

s0096.

AAG16224.1 red fluorescent protein Discosoma sp. SSAL-2000

aten

4.t

24.t

aten

AAF03370.1 fluorescent protein FP483 Discosoma striata Discosoma FP483 protein fluorescent AAF03370.1

s0152.

3.t

s0096.

11.t

AAS18271.1 green fluorescent protein 2 Astrangia lajollaensis Astrangia protein2 fluorescent green AAS18271.1

aten

1.t

Phylogenetic relationships of four GFPs (green bold

aten_s0152.g15.t1

s0152.

aten

AAU06852.1 red fluorescent protein Acropora millepora Acropora protein fluorescent red AAU06852.1

ACH89429.1 red fluorescent protein FP597 Acropora millepora Acropora FP597 protein fluorescent red ACH89429.1

AAT77753.1 colorless GFP-like protein Acropora millepora Acropora protein GFP-like colorless AAT77753.1

9.t

ACH53607.1 red fluorescent-like protein partial Acropora millepora Acropora partial protein fluorescent-like red ACH53607.1

aten

FAA00746.1 TPA: fluorescent protein 10 Acropora digitifera Acropora 10 protein fluorescent TPA: FAA00746.1

aten

s0297.

s0024.

s0297.

XP 001634522.1 predicted protein Nematostella vectensis Nematostella protein predicted 001634522.1 XP

s0217.

ACH89427.1 green fluorescent protein FP497 Acropora millepora Acropora FP497 protein fluorescent green ACH89427.1

s0025.

AAR85351.1 green fluorescent protein 2 Anthomedusae sp. SL-2003 sp. Anthomedusae 2 protein fluorescent green AAR85351.1

XP 001633713.1 predicted protein Nematostella vectensis Nematostella protein predicted 001633713.1 XP

FAA00740.1 TPA: fluorescent protein 3 Acropora digitifera Acropora 3 protein fluorescent TPA: FAA00740.1

ACH89426.1 cyan fluorescent protein FP484 Acropora millepora Acropora FP484 protein fluorescent cyan ACH89426.1

ACH89428.1 green fluorescent protein FP512 Acropora millepora Acropora FP512 protein fluorescent green ACH89428.1

s0024.

AAR85350.1 green fluorescent protein 1 Anthomedusae sp. SL-2003 sp. Anthomedusae 1 protein fluorescent green AAR85350.1

AAU06846.1 green fluorescent protein Acropora millepora Acropora protein fluorescent green AAU06846.1

AAU06849.1 cyan fluorescent protein Acropora millepora Acropora protein fluorescent cyan AAU06849.1

AAU06851.1 cyan fluorescent protein 2 Acropora nobilis Acropora 2 protein fluorescent cyan AAU06851.1

FAA00738.1 TPA: fluorescent protein 1 Acropora digitifera Acropora 1 protein fluorescent TPA: FAA00738.1

FAA00739.1 TPA: fluorescent protein 2 Acropora digitifera Acropora 2 protein fluorescent TPA: FAA00739.1

FAA00741.1 TPA: fluorescent protein 4 Acropora digitifera Acropora 4 protein fluorescent TPA: FAA00741.1

28.t

FAA00742.1 TPA: fluorescent protein 5 partial Acropora digitifera Acropora partial 5 protein fluorescent TPA: FAA00742.1

ABB17973.1 green fluorescent GFP-like protein Acropora millepora

aten

ACH53606.1 green fluorescent-like protein partial Acropora millepora Acropora partial protein fluorescent-like green ACH53606.1

134.t

27.t

35.t

63.t

FAA00743.1 TPA: fluorescent protein 6 partial Acropora digitifera Acropora partial 6 protein fluorescent TPA: FAA00743.1

131.t

s0297.

aten

29.t

s0366.

s0077.

AAQ01187.1 green fluorescentgreen AAQ01187.1 Pontellaprotein2 meadi

The scale bar indicates

AAQ01186.1 green fluorescentgreen AAQ01186.1 Pontellaprotein1 meadi

BAE78442.1 greenprotein fluorescent Chiridius poppei

aten

7.t

62.t

s0010.

gence from

7.t 1