Fig. 2 in Elisesione imajimai Jimi & Eibye-Jacobsen & Salazar-Vallejo 2018, sp. nov.

© 2018 Academia Sinica, Taiwan Open Access Repeated and Prolonged Temperature Anomalies Negate Symbiodiniaceae Genera Shuffling in the Coral Platygyra verweyi (Scleractinia; Merulinidae) Kuo-Wei Kao1,2, Shashank Keshavmurthy1,*, Cing-Hsin Tsao1,2, Jih-Terng Wang3, and Chaolun Allen Chen1,2,4,* 1Academia Sinica, Biodiversity Research Center, Nankang, Taipei 11529, Taiwan. E-mail: [email protected] (Kao); [email protected] (Keshavmurthy); [email protected] (Tsao); [email protected] (Wang) 2National Taiwan University, Institute of Oceanography, Taipei 106, Taiwan 3Tajen University of Science and Technology, Institute of Biotechnology, Pingtung 90741, Taiwan 4Academia Sinica, Taiwan International Graduate Program (TIGP) - Biodiversity, Taipei 11529, Taiwan (Received 16 May 2018; Accepted 15 October 2018; Published 20 November 2018; Communicated by Benny K.K. Chan) Citation: Kao KW, Keshavmurthy S, Tsao CH, Wang JT, Chen CA. 2018. Repeated and prolonged temperature anomalies negate Symbiodiniaceae genera shuffling in the coral Platygyra verweyi (Scleractinia; Merulinidae). Zool Stud 57:55. doi:10.6620/ZS.2018.5755. Kuo-Wei Kao, Shashank Keshavmurthy, Cing-Hsin Tsao, Jih-Terng Wang, and Chaolun Allen Chen (2018) With climate change, global average sea surface temperatures are expected to increase by 1.0-3.7°C by the end of this century. Even a 1.0°C increase in seawater temperature from local long-term summer maxima lasting for weeks to months results in bleaching and/or mortality in reef-building corals. Studies on coral resistance mechanisms have proposed a correlation between shuffling of different Symbiodiniaceae genera (changing the dominant Symbiodiniaceae genera) and putative thermal tolerance in corals. Although it was suggested that some corals can increase their tolerance by 1.0-1.5°C through shuffling to thermally tolerant Durusdinium trenchii (formerly D1a), the effects of accumulated thermal stress due to prolonged high temperatures on the survival of corals that have shuffled have not been investigated. We show herein that prolonged exposure to high temperature (> 10.43-degree heating weeks) can drastically reduce coral survival rate even after it has shuffled to stress-tolerant Symbiodiniaceae genera. Our study suggests that there is a limit to the capacity of for shuffling, and hence is likely to lose its efficacy in the future as repeated and prolonged thermal stress events become more frequent and pronounced. Key words: Climate change, Seawater temperature fluctuations, Degree of heating weeks, Reciprocal transplantation, Kenting-Taiwan. *Correspondence: E-mail: [email protected]; [email protected] BACKGROUND In response to rising CO2 emissions, up to 90% of global coral reefs may suffer from annual bleaching by 2055 (IPCC 2013; van Hooidonk et al. 2014; Hoegh-Guldberg 1999; Frieler et al. 2012). In recent years, the frequent bleaching of corals worldwide has indicated that adaptation is not keeping up with environmental changes (HoeghGuldberg et al. 2007; Camp et al. 2016). In order to monitor and predict global coral bleaching events, data on degree heating weeks (DHW; °C-weeks) are often based on regional satellite temperature records against long-term summer maxima (Liu et al. 2003; Howells et al. 2013; Ridgway et al. 2016). Typically, a reef site with a DHW value of 4°C-weeks experiences significant coral bleaching, whereas a value of 8°C-weeks may cause widespread coral Zoological Studies 57: 55 (2018) doi:10.6620/ZS.2018.57-55 1 © 2018 Academia Sinica, Taiwan bleaching accompanied by mortality (Liu et al. 2003). Recently, DHW has been used to reflect the accumulated thermal stress on corals under experimentally manipulated conditions and assess thermal tolerance (Schoepf et al. 2015). Thermal tolerance in scleractinian corals, depending on the environmental conditions, is the result of a combination of the coral host and Symbiodiniaceae genera resistance mechanisms. There is evidence indicating an advantage for corals in overcoming stress when they associate with Durusdinium species. For example, corals are more thermally tolerant when associated with symbiont D. trenchii compared to conspecifics associated with Cladocopium C3 (Silverstein et al. 2015; Keshavmurthy et al. 2012). Generally, species in the genus Durusdinium are considered to be heat tolerant and species in the genus Cladocopium as stress sensitive, with the exception of some Cladocopium species (e.g. inhospite Cladocopium C15), which are relatively stress tolerant (Fisher et al. 2012). The proposed shuffling mechanism involves changes in the relative abundances of different symbiont types within the coral host depending on the temperature (Cunning et al. 2015b); this allows “background” genera of Symbiodiniaceae to become dominant. A transition from thermally sensitive to thermally tolerant dominant symbionts offers a greater likelihood of corals surviving thermally induced bleaching (Bay et al. 2016). This acclimatization mechanism, although with its limits, is one of the strategies that may help corals survive the effects of global warming in the near future (Berkelmans and van Oppen 2006; Palumbi et al. 2014). In an earlier study, we gave evidence that coral species compositions change with long-term exposure to high temperatures and concurrent associations with tolerant Durusdinium spp. (Keshavmurthy et al. 2014); however, this does not mean that Symbiodiniaceae genera are driving thermal tolerance. Many studies have proposed that the potential for symbiont shuffling during coral acclimatization helps corals survive the effects of climate change (Silverstein et al. 2015; Bay et al. 2016; Berkelmans and van Oppen 2006; Palumbi et al. 2014). However, studies have also shown that, after shuffling their Symbiodiniaceae genera, corals revert back to a relationship with their pre-stress symbionts if conditions change (see LaJeunesse et al. 2010). In this study, we show that shuffling between Symbiodiniacceae genera may not always benefit corals. We used a field-based study to demonstrate the influence of different temperature patterns on the behavior of a coral species, Platygyra verweyi, associated with different Symbiodiniceae genera and conclude that shuffling between sensitive and tolerant Symbiodiniaceae genera is insufficient for corals to survive repeated and prolonged thermal stress. We conducted two sets of in situ reciprocal transplant experiments (RTEs) - one in 2014 (2014RTE) and the other in 2015 (2015RTE) using cores of the coral P. verweyi collected from sites proximal to a nuclear power plant outlet and a nuclear power plant inlet, and Wanlitung in Kenting National Park, Taiwan (Fig. 1a). We calculated DHW values for each experimental group from in situ temperature records at each site and assessed Symbiodiniaceae genera compositions to test our hypothesis that prolonged thermal stress will influence the survival of a coral host even after shuffling to a stress-tolerant Symbiodiniaceae genus. In Kenting National Park, P. verweyi was associated with Durusdinium glynii (ITS type D1), D. trenchii (ITS type D1a), Cladocopium C3 (ITS2 type C3) and Cladocopium C3cc (ITS2 type C3cc), either specifically or in combination depending on the location (Keshavmurthy et al. 2012). MATERIALS AND METHODS Experimental design The coral species used in this study, Platygyra verweyi, is a massive coral species inhabiting shallow waters (2-4 m) at Kenting National Park. Individual colonies can host each symbiont alone or in combination with another type. In this study, all Cladocopium sp.-dominated colonies associated with both Cladocopium C3 and Cladocopium Ccc and all Durusdinium sp.- dominated colonies associated with both D. glynii and D. trenchii. Three sites were included in this study, Nuclear Power Plant Outlet (OL), Nuclear Power Plant inlet (IL), and Wanlitung (WLT), in Nanwan, south Taiwan (Fig. 1a). Among the three sites, OL (21°55'54.4"N, 120°44'42.7"E) and IL (21°57'20.3"N, 120°45'14.2"E) are located within Nanwan in Kenting National Park, Taiwan. A recent long-term (2007 to 2010, and 2013) seawater temperature data set obtained from the deposited underwater data loggers shows that the average summer (June to August) daily seawater temperature at OL (29.31 ± 1.36°C) is approximately 1°C higher than at IL (28.14 ± page 2 of 14Zoological Studies 57: 55 (2018) © 2018 Academia Sinica, Taiwan 1.18°C). Due to the tidally induced upwelling in Nanwan (Lee et al. 1997), the maximum daily seawater temperature fluctuation at OL and IL can be more than 8°C in summer (Fig. 1b). The third site, WLT (21°59'41.0"N, 120°42'19.6"E), is located on the west coast of Kenting National Park with average summer (June to August) seawater temperature (28.99 ± 0.74°C) similar to OL while the intervals of extreme temperature events (≥ 30°C) and the daily seawater temperature fluctuations are shorter and less extreme than OL (Fig. 1b, c). In the first reciprocal transplant experiment conducted in 2014 (2014RTE, Fig. 1a), two sets of reciprocal transplant experiments (RTE) were carried out - one between OL and Wanlitung WLT, and the other between OL and IL - in Kenting National Park, Taiwan in 2014. On March 2014, 25 mm-dimeter cores from 5 colonies of P. verweyi from each study site (OL: 21 cores/ colony, WLT: 16 cores/colony, IL: 10 cores/colony) at a depth of 1 to 2 m were sampled underwater using a pneumatic drill and placed in Ziploc bags underwater before being transferred to a wet lab at the National Museum of Marine Biology & Aquarium (NMMBA). The difference in sample size between OL and the other two sites (WLT and IL) was due to mortality of tagged mother colonies in WLT and small population size of P. verweyi at IL. Coral cores were maintained in indoor seawater tanks with constantly filtered seawater input. For RTEs set between the OL and WLT, coral cores from each site were randomly assigned to racks (a native group and a transplant group). Each rack contained 40 cores (5 colonies × 8 replicates). For another set between OL and IL, each rack contained 25 cores (5 colonies × 5 replicates). In the case of the native group of OL, the same rack was used for both sets of experiments. Fewer replicates were used in the RTE set between OL and IL due to the small size of P. verweyi colonies at IL. Coral cores were attached to PVC adapters with underwater epoxy to be fixed onto the acrylic rack and were transferred back to their original, respective sampling sites at similar depths (1-2 m) as sampled colonies for 1.5-month recovery to ensure coral health before being transplanted. On April 2014, all the racks were retrieved and transported to NMMBA and stained with Alizarin Red S (Sigma-Aldrich, USA) 20 mg/L, 24 hrs (LeGore et al. 1989) for the analysis of skeleton growth. Subsequently, all the racks were put back Fig. 1. Reciprocal transplant experiment (RTE) designs and temperature regimes at each study site. (a) Study sites in Kenting National Park. Blue arrows represent the 2014RTE and 2015RTE. (b) Weekly average temperatures recorded through time. (c) Daily seawater temperature fluctuations (°C) at each site. NA = no data. Different lowercase letters indicate significant differences in daily temperature ranges among sites (Kruskal-Wallis test, Dunn’s post-hoc test, Bonferroni adjusted p values at α = 0.05). Bars within each box represent the median with boundaries representing the 25 to 75th percentiles. Whisker boundaries represent the 1.5x interquartile range and black dots represent outliers with values beyond that range. OL, nuclear power plant outlet; IL, nuclear power plant inlet; WLT, Wanlitung. N (a) (b) (c) page 3 of 14Zoological Studies 57: 55 (2018) © 2018 Academia Sinica, Taiwan to the study sites with treatment groups being reciprocally transplanted. Sampling was conducted every 4 months, with 5 coral cores (1 cores from each colony) from each rack being retrieved each time. The sampled cores were cut into three parts. Tissue from the first part was removed by scraping the surface and stored in 95% Ethanol for DNA extraction. The second part was wrapped in aluminium foil and stored at -20°C for chlorophyll concentration measurement and total symbiont density count, and the last part was snap-frozen in liquid nitrogen and stored for protein analysis. Monthly maintenance of the racks underwater was performed by cleaning the macroalgae attached to coral cores and the racks components to minimize any competition effect. The second reciprocal transplant experiment of Platygyra verweyi was conducted in 2015 (2015RTE, Fig. 1a) between OL and WLT with a sample size (n = 30 colonies from each site). IL was not included as one of the study sites in 2015RTE because not enough P. verweyi colonies were found to make a balanced design. Following identical procedures as 2014RTE, thirty colonies were sampled from OL and WLT in March 2015 and were reciprocally transplanted in April 2015 after onemonth of recovery. Coral cores of each group were retrieved (April, September, and January), subsampled, and preserved for DNA extraction (in 95% Ethanol), qPCR, and physiological parameters analysis. Seawater temperature The seawater temperature was recorded in situ at 30-minute intervals using data loggers (HOBO; PendantTM, USA) deployed underwater near the transplant racks (1-2 m) at each study site. The raw temperature data were transformed into Degree Heating Week (DHW) (IPCC 2013; Liu et al. 2003) to assess both the intensity and duration of the thermal stress for each experimental group. Although this indicator is typically used to reflect large-scale bleaching monitoring, its application to experimental manipulation has also been used to assess the cumulative thermal stress on heat-treated corals over daily scales (Schoepf et al. 2015). To calculate DHW, first, the maximum monthly mean temperature (MMM) was obtained from historical long-term data (recorded from 2007-2010 and 2013). Second, the weekly mean temperature during the RTEs for each study site was calculated. Finally, the weekly mean temperature was subtracted from MMM to get the temperature anomalies and those anomalies that were at least 1.0°C above MMM were summed, within the past 12-week windows, to obtain DHWs using following equation: DHWWLT-OL transfer = ∑ [(Ttransfer - MMMnative) ≥ 1°C] In the equation, Ttransfer is the weekly mean temperature at OL and MMMnative is the MMM of WLT, to calculate DHW for WLT-OL transfer. The projections of DHW in Nanwan, which is included in one of the 1° × 1° resolution grid reef cells locating at southern Taiwan, were obtained from van Hooidonk et al. (2014). Symbiont community dynamics DNA extraction DNA extraction was carried out using a modified salting-out method (Ferrara et al. 2006). Coral tissue (30 mg) was cut and incubated overnight at 56°C with 200 µL lysis buffer (1M Tris 25 mL, 0.5M EDTA pH8 10 mL, 20% SDS 10 mL, 5M NaCl 2 mL, ddH2O 53 mL) and 10 μL proteinase E (10 mg/mL). 7M NaCl (210 μL) was added to the tissue, centrifuged (6000 g, 30 sec), and transferred into B/T Genomic DNA Mini Column (Viogene, Taiwan). After a series washing with cold (-20°C) 70% ETOH and centrifugation, the column was dried at 37°C for 15 mins and finally, the DNA was eluted with 50 μL of preheated (65°C) 1X TE buffer and was isolated from the column after centrifugation (15000 g, 3 min). The concentrations of genomic DNA were determined using NanoDrop 2000 (Thermal Scientific, USA). Denaturing gradient gel electrophoresis The initial and final subclades (types) of the symbionts in the native and transplant groups in 2014RTE and 2015RTE were randomly selected and identified by amplifying the internal transcribed spacer 2 (ITS2) region of DNA samples with primer sets ITSintfor2 5'GAATTGCAGA ACTCCGTG-3' and ITS2clamp 5'CGCCCGCCGC GCCCCGCGC CCGTCCCGCCG CCCCCGCCC GGGATCCATA TGCTTAAGTT CAGCGGGT-3'. A touch-down PCR (LaJeunesse 2002) program was used: 92°C for 3 min followed by 20 cycles of 30 s at 92°C was carried out. Annealing temperature was set at 62°C and then decreased by 0.5°C in each cycle to a final temperature of 52°C, 30 secs at 72°C to ensure specificity. Denaturing gradient gels page 4 of 14Zoological Studies 57: 55 (2018) © 2018 Academia Sinica, Taiwan from 45% to 80% were used for electrophoresis under 115v for 15 hours using a CBS Scientific system (Del Mar, CA, USA). The gel was stained with SYBR Gold (Invitrogen, USA). Prominent bands were excised and amplified for sequencing. All sequences were aligned and compared with GioSymbio database (Franklin et al. 2012). Real-time quantitative PCR The copy numbers of symbiont Cladocopium sp. and Durusdinium sp. in P. verweyi samples were detected under LightCycler® 480 Instrument II (Roche, Switzerland) with the protocol modified from Mieog et al. (2007). Each 10 μL qPCR reaction consisted of 5 μL of 1x SYBR Fast Master Mix, 0.5 μL of UF primer (2 nM/μL), 0.5 μL of CR or DR primer (2 nM/μL), 7.5 μL of ddH2O, and 2.5 μL of DNA templates (equal to 1 ng of genomic DNA) with primer sets, ITS1 Cladocopium sp.-specific reverse primer (CR) 5-AAGCATCCCTCACAGCCAAA-3, Durusdinium sp.-specific reverse primer (DR) 5-CACCGTAGTGGTTCACGTGTAATAG-3, and universal forward primer (UF) 5-AAGGAGAAGTCGTAACAAGGTTTCC-3 (Ulstrup and van Oppen 2003). In each run, each sample was run in triplicate (technical replicates), and no-template control (NTC) was also run in triplicate with ddH2O to inspect any contamination in the reagent. Coral growth Tissue coverage growth Top-view photos of all the coral cores on the rack were photographed in situ with a scale, and the surface area covered by the coral tissue of each core was estimated using ImageJ software (1.48v, USA). Data were transformed into the relative percentage change of tissue coverage from the initial sampling (April 2014). Skeleton growth At the end of the experiment, all the retrieved coral cores were sliced and airbrushed to remove the remaining tissue, and the length of the newly accreted skeletons perpendicularly above the Alizarin Red S stain mark from 6-10 septa were measured under a microscope (SZ61 Olympus, Japan); the results were averaged to determine the skeleton growth for each nubbin. Skeleton growth was not measured in 2015RTE because no cores were retrieved for this group. The protocol for the analysis of physiological parameters is given in the supplementary file. Statistical analysis All the statistical analyses in this study were performed in R version 3.1.1. (R Core Team 2014). Differences in daily mean seawater temperatures and daily seawater temperature fluctuations between sites were tested using Kruskal-Wallis test followed by Dunn’s post hoc test with Bonferroni adjusted p-value. Data were presented as the mean ± standard deviation (S.D.). Relative symbiont abundances of each sample were assigned into categories, Cladocopium sp. or Durusdinium sp. dominant (i.e. either Cladocopium or Dururdinium ≥ 90% relative abundance) and Cladocopium sp. + Dururdinium sp. (10% < both Cladocopium and Durusdinium < 90% relative abundance) and differences in symbiont community distributions were tested using Fisher’s exact test. For photochemical efficiency and each physiological parameter, differences between each transfer group and its native group were tested using Student t-test. Data were box-cox transformed (Box and Cox 1964) if they failed to meet normality and/or homogeneity of variance assumptions. Wilcoxon rank sum test was performed on raw data if statistical assumptions were violated. All data were presented as the mean ± standard deviation (S.D.). For tissue coverage growth and skeleton growth in 2014RTE, differences between groups for each transplantation sets (WLT↔OL or OL↔IL) were tested using one-way ANOVA followed by Tukey’s post hoc test with Bonferroni adjusted p-value. For 2015RTE tissue coverage growth, differences between groups were tested using one-way ANOVA followed by Tukey’s post hoc test with Bonferroni adjusted p-value while the origin versus location effect was tested using two-way ANOVA. RESULTS Seawater temperature The maximum monthly mean for 3 sites were: MMMOL = 29.63°C; MMMWLT = 29.28°C; MMMIL = 28.37°C. In the 2014RTE, the average daily summer (June to August) seawater temperature at the OL (30.11 ± 1.07°C) was higher than that page 5 of 14Zoological Studies 57: 55 (2018) © 2018 Academia Sinica, Taiwan at WLT (29.59 ± 0.67°C; Dunn’s post-hoc test, p < 0.001) and the IL (28.61 ± 0.96°C; p < 0.001), and the weekly average temperature at the OL repeatedly exceeded P. verweyi’s bleaching threshold at WLT (30.28°C) and the IL (29.37°C; Fig. 1a). The daily seawater temperature fluctuation at the OL (2.23 ± 1.00°C; Fig. 1b) also differed from that at WLT (1.53 ± 0.58°C; p < 0.001) and the IL (1.80 ± 1.30°C; p < 0.001) during the summer, reaching a maximum of 9.12°C. In the 2015RTE, the average summer daily seawater temperature at the OL (29.77 ± 1.12°C) differed from that at WLT (29.52 ± 0.52°C; Wilcoxon ranksum test, W = 5097, p < 0.05; Fig. 1b) and the daily seawater temperature fluctuation at the OL (2.38 ± 1.04°C) differed from that at WLT (1.57 ± 0.70°C; Wilcoxon rank-sum test, W = 655557, p < 0.001; Fig. 1c). Degree heating weeks The prolonged thermal stress patterns observed during this study allowed us to predict P. verweyi survival in future warming scenarios. From DHW projections of the simulation model, the 1° × 1° grid reef cell containing Nanwan Bay was predicted to exceed 6°C-weeks at least twice in 2020-2030 under the RCP8.5 scenario (van Hooidonk et al. 2014), and to exceed 8°C-weeks at least twice in the same time interval (2020-2030; Table 1a). In the 2015RTE, DHW = 5.68°C-weeks (for coral cores transplanted from WLT to the OL), represented a “shuffle and survive” scenario similar to what can be expected at Nanwan Bay before 2020 (Table 1a, b), while in the 2014RTE, DHW = 10.43°C-weeks (for coral cores transplanted from WLT to the OL) represented a “shuffle but likely not to survive” scenario, which may become a common and frequent occurrence after 2030 (Table 1a, b). Data fitting considered the potential DHW threshold, which determines the survival of corals, i.e., DHW = 10.43°C-weeks could cause severe mortality, and in the present study, transplanted coral cores had already experienced a DHW of > 8.0°C-weeks. Table 1. Predicted degree heating weeks (DHW in °C-weeks) by year. (a) Future predictions for DHWs at Nanwan Bay under emission scenarios based on IPCC AR5. 2x-DHW conditions that may occur at least twice within a given time interval. 10x-DHW conditions that may occur every year within a given time interval. Bold texts represent worst-case scenarios. (b) Maximum DHW during the experiment and the percent of Symbiodinium shuffled in Platygyra verweyi transplanted to the nuclear power plant outlet (OL), and the subsequent coral core mortality rate. For the 2014 reciprocal transplant experiment (2014RTE), we only report the DHW for coral cores transplanted from Wanlitung (WLT) to the OL (WLT-OL transfer), since all coral cores transplanted from the nuclear power plant inlet (IL) to the OL (IL-OL transfer) died (a) RCP CO2 emissions DHW > 6 DHW > 8 2.6 2x Low 2010-20 2020-30 4.5 2x Median 2020-30 2030-40 6 2x Median 2020-30 2040-50 8.5 2x High 2020-30 2020-30 2.6 10x Low 2030-40 2040-50 4.5 10x Median 2050-60 2050-60 6 10x Median 2060-70 2070-80 8.5 10x High 2040-50 2050-60 Representative Concentration Pathway based on IPCC AR5 climate models. (b) 2015RTE 2014RTE Maximum DHW 5.68 10.43 Shuffling 73% 40% Mortality 0% 75% page 6 of 14Zoological Studies 57: 55 (2018) © 2018 Academia Sinica, Taiwan Reciprocal transplantation experiment - 2014 All coral cores transplanted from WLT to the OL (WLT-OL transfer) and those transplanted from the IL to OL (IL-OL transfer; n = 5) in the 2014RTE were initially associated with the dominant symbiont Cladocopium sp. and background Durusdinium spp. (Fig. 2a, b; Table S1). In contrast, OL corals were dominated by Durusdinium spp., and maintained the same dominant symbiont species even after being moved to the more moderate WLT and IL site. Thermal stress was first observed in both groups as a reduction in the photochemical efficiency (Fig. 3; Tables S2, S3) in early July 2014 when repeated seawater temperature anomalies occurred at the OL (Fig. 1b) causing the DHW to reach 4.41°C-weeks for WLT-OL transfer and 8.00°C-weeks for IL-OL transfer (Fig. 2a, b). The DHW continued to rise to maximum values of 10.43°C-weeks for WLT-OL transfer and 21.30°C-weeks for IL-OL transfer by early September 2014 (Fig. 2a, b), when both groups exhibited significant bleaching resulting in decreased symbiont cell densities and changes in other physiological parameters (Fig. 3; Tables S2, S3). Concurrently, Durusdinium spp. became dominant in 40% (n = 2) of WLT-OL transfer coral cores, Cladocopium + Durusdinium became codominant in 80% (n = 4) of IL-OL transfer colonies, and most colonies showed increased symbiont Durusdinium cell densities (104-105 cells cm-2; Fig. 2a, b; Table S4), despite no or few Durusdinium cells being detected initially. For both WLTOL transfer and IL-OL transfer, distributions of symbiont communities differed from their initial compositions (Fisher’s exact test, p < 0.01). However, only one colony of WLT-OL transfer survived after the thermal stress dissipated. In total, WLT-OL transfer and IL-OL transfer coral cores experienced 90% mortality (Fig. 2a, b; Table 2). Cladocopium remained dominant in all colonies Fig. 2. Durusdinium sp. dominance (%; symbols) and degree heating week (DHW in °C-weeks; shaded lines) of each group through time. (a) Transplanted group from Wanlitung (WLT) to the nuclear power plant outlet (OL) (WLT-OL transfer); (b) transplanted group from the nuclear power plant inlet (IL) to OL (IL-OL transfer); and (c) native group from the OL to OL (native OL), native group from the IL to IL (native IL), and native group from WLT to WLT (native WLT) for the 2014 reciprocal transplant experiment (2014RTE). (d) WLTOL transfer and (e) native OL and native WLT for the 2015RTE. Each symbol in the data represents a single Platygyra verweyi colony. For 2015RTE data, colonies with similar dominance were merged into larger groups with sample numbers shown above the circle. Circles represent symbiont type D dominance of each colony at its last sampling time. (f) Survival rates at WLT-OL transfer and IL-OL transfer for different final Durusdinium sp. dominance values under each maximum DHW (based on 5 colonies in 2014RTE). Horizontal lines are 4 and 8 DHW. April September January 14 15 8 4 10 15 20 5 0 75 10 25 15 20 5 0 Native OL Native IL Native WLT April September January 14 15 April September January 14 15 Native OL Native WLT Live Dead Live Dead 29 30 29 25 23 22 22 18 26 Degree Heating W Week) Degree Heating W Week) Durusdinium sp. relative dominance (%) April September January 15 16 April September January 15 16 10 25 15 20 5 0 8 4 10 15 20 5 0 0 20 40 60 80 100 0 20 40 60 80 100 (a) (b) 0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 100 MDH= 5.6 8 MDH=10.4 3 MDH=21.3 0 Final Durusdinium sp. relative dominance Survival rate (%) (c) (d) (e) (f) WLT-OL transfer IL-OL transfer WLT-OL transfer page 7 of 14Zoological Studies 57: 55 (2018) © 2018 Academia Sinica, Taiwan Fig. 3. Physiological parameters of experiment groups located at NPP-OL at each sampling time. (a) Total symbiont cell densities. (b) Chlorophyll a concentrations per cm2. (c) Total soluble protein concentrations. (d) Dark-adapted photochemical efficiency measured in 2014RTE. (n = 5 colonies for each group per sampling time unless stated otherwise). (e) Photochemical efficiency measured in 2015RTE. (n = 30 colonies for each group per sampling time unless stated otherwise). All data are presented as mean ± SD. Asterisks represent a significant difference of total symbiont density between each transplant group and the native group on each month. †All the samples were dead. 0 0.5 1.0 1.5 Apr-14 Sep Jan-15 0 1 20 0.3 0.6 A M J S O J 0 0.3 0.6 0 4 8 12 * * * * ** * * * * * ** ****** ** *** n=28 n=28 n=29 n=1 n=1 n=4 n=1 n=3 n=1 n=3 n=1 n=3 Total soluble protein (mg cm-2) Photochemical efficiency (Fv/Fm) Chlorophyll a (μg cm -2) Total symbiont density (106 cell cm -2) Photochemical efficiency (Fv/Fm) Apr-14 Sep Jan-15 Apr-14 Sep Jan-15Apr-14 Sep Jan-15 Apr-14 Sep Jan-15 Apr-14 Sep Jan-15 Apr-14 Sep Jan-15Apr-14 Sep Jan-15 Apr-14 Sep Jan-15 Apr-14 Sep Jan-15 Apr-14 Sep Jan-15Apr-14 Sep Jan-15 14 15 15 native WLT WLT-OL transfer native OL OL-WLT transfer OL-IL trasfer native IL IL-OL transfer native OL (a) (b) (c) (d) (e) † † † †† * n=17 n=23 * A M J S O J 14 15 A M J S O J 14 15 A M J S O J 14 15 A M J S N 15 A M J S N n=28 * n=25 page 8 of 14Zoological Studies 57: 55 (2018) © 2018 Academia Sinica, Taiwan of the native group at WLT (native WLT) and the IL (native IL) throughout the 2014RTE (Fig. 2c; Table S1), and Durusdinium remained dominant in all colonies of the native group at the OL (native OL) (Fig. 2c; Table S1). Reciprocal transplantation experiment - 2015 WLT-OL transfer coral cores in the 2015RTE were initially dominated by Cladocopium sp. (n = 26; Fig. 2d). Signs of bleaching were observed in coral cores of WLT-OL transfer with a decreasing photochemical efficiency (Fig. 3; Tables S6, S7) in early July, when seawater temperature anomalies occurring at the OL caused the DHW to reach 5.68°C-weeks for WLT-OL transfer (Figs. 1b; 2d). The prevalence of Durusdinium sp. increased in September as a result of 73% (n = 22) of WLTOL transfer colonies shuffling from Cladocopium sp. to Durusdinium spp. dominance (Fig. 2c). However, as the seawater temperature at the OL subsequently decreased due to several typhoon events (Fig. 1b), the DHWs of WLT-OL transfer did not further increase. The mortality of WLT-OL transfer colonies was only 23% after the thermal stress had dissipated (Fig. 2d; Table 2). In the end, the Symbiodiniaceae genera composition of WLT-OL transfer colonies differed from their initial composition (Fisher’s exact test; p < 0.001). All native WLT colonies retained their Cladocopium sp. dominance throughout the 2015RTE (Fig. 2e; Table S1), and all native OL colonies retained their Durusdinium spp. dominance (Fig. 2e; Table S1). Even though coral cores transplanted to the OL in both the 2014RTE and 2015RTE showed increasing relative abundances of Durusdinium spp., distinct mortality rates appeared (90% for the 2014RTE vs. 23% for the 2015RTE, Table x) after Table 2. Information on the coral cores used in the transplant experiment. Live cores versus total cores and final survival rate at each sampling time in; (a) 2014 reciprocal transplantation and (b) 2015 reciprocal transplantation experiment, respectively (a) Live cores/Total cores (Survival rate) Group 2014Apr 2014Sep 2015Jan Cores retrieved per sampling time native OL 40/40(100%) 34/34(100%)a29/29(100%) 5 OL-WLT transfer 40/40(100%) 35/35(100%) 30/30(100%) 5 OL-IL transfer 25/25(100%) 20/20(100%) 15/15(100%) 5 native WLT 36/36(100%) 31/31(100%) 19/25(76%)*c5 WLT-OL transfer 36/36(100%) 28/31(90.3%) 4/26(15.4%) 5 native IL 25/25(100%) 20/20(100%) 15/15(100%) 5 IL-OL transfer 25/25(100%) 16/18(88.9%)b0/13(0%) 5 a1 core was missing. b2 cores were missing. c1 core was missing. (b) Live cores/Total cores (Survival rate) Group 2015Apr 2015Sep 2016Jan native OL 30/30(100%) 30/30(100%) 30/30(100%) OL-WLT transfer 30/30(100%) 30/30(100%) 30/30(100%) native WLT 30/30(100%) 30/30(100%) 28/30(93.3%) WLT-OL transfer 30/30(100%) 30/30(100%) 23/30(76.7%) page 9 of 14Zoological Studies 57: 55 (2018)