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台灣產微孔珊瑚共生體之共生藻群聚多樣性與熱逆境生理

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(1)命 科 學 系. 國立臺灣師 範大學. 生. 國立臺灣師範大學生命科學系碩士論文. 碩 士 論 文. 台灣產微孔珊瑚共生體之共生藻群聚多樣性 與熱逆境生理. 台灣產微孔珊瑚共生體之 共生藻群 聚 多樣性與熱逆境生理. Symbiodinium diversity and physiological responses to thermal stress on Porites holobionts in Taiwan.. 研 究 生: 陳彥嘉 . 陳彥嘉 撰. Yen-Chia Chen 指導教授:陳昭倫 博士  Chaolun Allen Chen 林思民 博士 . 3 0 1. 學年度. Si-Min Lin. 中 華 民 國. 1 0 3. 年. 7. 月.

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(3) Table of contents Table of contents……………………………………………...……….… 謝辭….........……...……...……………………………………………...  Abstract…..……………...........................................................................III 中文摘要………………………………………………….……………..V. Introduction………………………………………………………………1 Material and method ……………………………………………………..4 Results………………………………………………………….………..10 Discussion…………………………………………………….…………15 Conclusion………………………………………………………………22 Reference………………………………………………………………..23 Table legends……………………………………………………………29 Figure legends……………………………………………………….…..38. I.

(4) 謝辭 首先,感謝我的父母在這兩年的支持,讓我沒有後顧之憂的念書 以及做任何想做的事。感謝陳昭倫老師的指導與支持,讓我在科學的 思維上有大大的進步,每每與您討論總是能被您對珊瑚的熱情感染, 十分感謝您給我機會能在貴實驗室學習。感謝國科會的經費支持,讓 我有機會能深入探討珊瑚與共生藻的奇妙世界。感謝實驗室嘉閔學長 極有耐心且深入的教我珊瑚養殖技巧、水缸實驗的架設與設計,讓我 一個養殖新手也能享受架設水缸的成就感,若沒有你的幫助與教導我 是 不 可 能 完 成 這 本 論 文 的 。 謝 謝 實 驗 是 佳 妍 學 姐 、 Shashank Keshavmurthy 在實驗設計上的建議,Vianney Denis、Stéphane de PALMAS、Lauriane deulofeu 在統計上的協助以及實驗設計的討論還 有生活上的調劑。謝謝高國偉、曹景昕、鄭念昀、林妤芬、鍾愛琪在 潛水採樣的協助,讓每次的出差雖然累但有你們一起苦中作樂也都讓 我甘之如飴,希望各位好戰友們往後的發展都能一帆風順、平步青雲! 謝謝雁文和文華在出差及行政上的諸多幫忙,謝謝南北蔡教練在出差 時提供舒適的住宿也給我們諸多協助和方便。謝謝口委王志騰老師、 林思民老師指出我論文的缺失並給予我許多建議,是我的論文更加完 整,真的非常感謝您們! 謝謝師大草魚實驗室的李閣桓、曾威、王浥 璋、林彥伯在課業上的協助與討論,謝謝林展蔚學長在統計分析的指 點。 最後,感謝這兩年來所遇到的人、事、物,謝謝你們參與我的生 命,讓能力如此有限的我能完成一本論文,謝謝你們讓我的生命更加 完整豐盛。謝謝自己面對這兩年的困難沒有放棄逃避的念頭。 II.

(5) Abstract Corals and their symbiotic algae (genus Symbiodinium), collectively known as coral holobionts, live close to their physiologically-limit of sea surface temperature (SST) between 28 0C to 30 0C in the tropical and subtropical water. Increasing of 0.5-1 0C above the sea surface temperature (SST) will cause physiological stress of coral holobionts and, as the consequence, breakdown of symbiotic relationship (also known as “bleaching”). Understanding how coral holobionts with different SST “background” respond to the thermal stress is the key to identify the strategies of future survival of coral holobionts and function of coral reef ecosystem under the impact of climate change. In this study first conducted Symbiodinium diversity surveys of Porites corals from the Bietou (BT) in the northern Taiwan and Nuclear power plant outlet (OL) of Kenting National Park, in southern Taiwan, where yearly mean SST 23.8 ± 3.8 0C and 27.5 ± 1.7 0C, respectively. 37 coral samples were examined, C15, C55, C55-1, and six C15-related new Symbiodinium types were identified in Porites. A significant difference in Symbiodinium type compositions was found in Porites between BT and NPP-OL with the six C15-related types dominant in BT. Analysis of the maximum quantum yield (Fv / Fm) in Porites exposed to different temperature treatments in the tanks showed that Porites of NPP-OL was more sensitive to the thermal treatments than those of the BT during thermal stress. In addition, NPP-OL Porites collected from 7 m displayed a significantly higher Fv / Fm than those from 3 m, which might due to difference of the host species. Analysis of Symbiodinium density and chlorophyll pigments concentration showed that BT population displayed higher concentration of chlorophyll pigments than. OL. population.. However, III. NPP-OL. population. increased.

(6) Symbiodinium density instead of chlorophyll pigments when facing thermal stress. Results from this thesis suggested that Porites living in different thermal background could associate with different Symbiodinium C15-related types. Meanwhile, different C15-related types might have variety of photosynthesis responses in assisting coral hosts to survive under the thermal stress caused by rising sea surface temperature.. Key words: Porites, thermal physiology, Symbiodinium composition, coral holobionts. IV.

(7) 中文摘要 熱帶與亞熱帶造礁珊瑚與其體內共生藻所組成的“共生體”,其生 理極限溫度約在攝氏 28~30 度左右,當海表面溫度升高攝氏 0.5-1 度 將對珊瑚共生體造成生理上的熱逆境,熱逆境持續的結果將造成珊瑚 產生“白化”現象。面對氣候變遷可能在本世紀末引以海水溫度升高攝 氏 1.8~4 度,探討生活不同“溫度背景”的珊瑚共生體在熱逆境下如何 調適或適應作出反應及其可能的機制,是珊瑚礁保育與永續重要的課 題。本論文以在不同海域溫度背景的團塊型微孔珊瑚共生體為對象探 討共生藻的多樣性,以及是否展現不同的熱逆境生理反應能力。將台 灣南部恆春核三廠出水口(年均溫攝氏 27.5 ± 1.7 度)與北部鼻頭角 (年均溫攝氏 23.8 ± 3.82 度)的團塊型微孔珊瑚共生體採回實驗室, 已變性梯度電泳與核苷酸序列定序分析微孔珊瑚體內共生藻型的多 樣性,並以水缸實驗探討原生溫度背景相異的微孔珊瑚共生體對熱逆 境的反應。共生藻多樣性的調查顯示,在 37 個微孔珊瑚樣本中可鑑 定出 C15 型、C55、C55-1 與六種未命名的共生藻單型,而微孔珊瑚 體內共生藻型在臺灣南北的地理分佈上有顯著差異,南部以 C15 原 型為主,而北部多為未知的新共生藻單型為主。以共生藻最大光合作 用效率量測微孔珊瑚共生體對熱逆境的反應結果顯示,鼻頭角的微孔 珊瑚對於熱逆境的耐受度顯著優於核三廠出水口的群體。分析共生藻 密度及葉綠素濃度隨時間的變動顯示,鼻頭角的微孔珊瑚共生體的葉 綠素濃度隨著時間而增加,但共生藻密度下降,相反的,核三廠出水 口的微孔珊瑚共生體體內共生藻密度隨時間而增加,葉綠素濃度並改 變。綜合以上的結果顯示,團塊微孔珊瑚(P. lobata)在台灣南北海域不 同的背景溫度下已發展出與不同型共生藻的共生能力,而且其對於熱 V.

(8) 逆境的反應策略也有所不同。此外,由於珊瑚宿主及環境不同,核三 廠出水口三米水深與七米水深的微孔珊瑚共生體在光合作用效率上 存在著本質上的差異,但其對於熱逆境的反應是相同的。總結本論文 從分子與生理證據皆證實,即使是高度共生藻藻型專一性的團塊微孔 珊瑚在小尺度的地理差異上也發現因適應不同的環境溫度導致共生 藻組成族群之差異,而不同共生藻型對於熱逆境的光合生理也發展出 不同的策略。 關鍵字: 熱逆境生理、最大光合作用效率、變性梯度電泳、微孔屬珊 瑚. VI.

(9) Introduction. The relationship between coral reefs and holobionts Coral reefs are one of the marine ecosystems hosting high biodiversity [1]. It is suggested that the mutualistic relationship between scleractinian corals and photosynthetic dinoflagellates (also known as zooxanthellae), belonging to genus Symbiodinium, play a major role in creating and maintaining the coral reef ecosystem. Coral harbor intercellular photosynthetic dinoflagellates since the Triassic is credited a long term successful strategy to survive at shallow, tropical, nutrient-poor environments [2, 3]. The mutualistic relationship established by reciprocal transportation of nutrient form coral host to Symbiodinium and photosynthetic products from Symbiodinium to hosts, collectively known as “holobionts” [4]. However, this successful symbiosis is sensitive to temperature that coral holobionts may lose its Symbiodinium causing ‘bleaching’ while 1 or 2 0C higher than summer maximum temperature. Mass coral bleaching has caused extremely high mortality of corals during 1998 in the world and 2002 in the Great Barrier Reef (GBR) due to a longlasting summer maximum [5]. According to the intergovernmental panel on climate change (IPCC) predicted till the end of twenty-first century, sea surface temperature (SST) will increase 1.8-4.0 0C depend on the emission of greenhouse gas [6]. Therefore, whether coral holobionts could adapt or acclimatize to warming ocean is a serious concern.. 1.

(10) The Symbiodinium diversity and thermal tolerance The thermal tolerance of coral holobionts depend on varied Symbiodinium clades. The taxonomy of Symbiodinium is established by the molecular genetic markers, such as large- and small-subunit of ribosomal DNA (rDNA), restrict fragment length polymorphisms (RFLPs) or DNA sequences since 90s in the last century [7] with nine highly divergent ‘clades’, A to I, currently identified [8]. Within each of the clades, numerous subclades or types of Symbiodinium were identified by fastevolving markers including denaturing gradient gel electrophoresis (DDGE), single-strand conformation polymorphism (SSCP), and DNA sequences of internal transcribed spacer 1 (ITS1) and 2 (ITS2) of rDNA [24], and plastid and chloroplast (cp23S) [25]. Different Symbiodinium clades already have been proved that varied thermal tolerance [9-11], There were lots of evidence that corals with clade D symbiont are more resistant to thermal stress [12, 13]. In scleractinian corals, there are many case showed multiple symbiosis. Jones, 2008 noted that Acropora millepora changed Symbiodinium into thermal tolerance clade D after bleaching [14]. Some reef-building corals would shuffle their symbiont compositions in response to the thermal stress [15, 16]. Therefore, the ability to associate with multiple symbiont clades is tolerant feature in scleractinian corals [9]. This result has been inferred as an evidence of corals to acclimatize to the future rising seawater temperature under the impact of climate change [17, 18]. Nevertheless this have be challenged in coral Porites genus. Lots of studies already showed that Symbiodinium composition of Porites genus around the Pacific Ocean almost exclusively associated with C15 and reveals the highly environmental specific (Tab. 1, Fig. 1). 2.

(11) The holobionts resistance capacity of thermal stress The resistance capacity of coral holobionts can be inferred separately into two parts, coral host and Symbiodinium. It is traditionally viewed that bleaching tolerance differs among species during mass bleaching event suggesting the coral host is the major contributor to thermal tolerance of holobionts [19]. On the contrary, Fisher et al. 2012 showed the photophysiology of Symbiodinium C3 type showed lower value than C15, provide evidence for varied temperature tolerance depend on types of Symbiodinium but the effect of host still need to confound [20, 21]. Most of the papers focused on different coral species, the thermal tolerance of population level was still uncertain [22]. In Figure 2, Yehliu and Kenting represented subtropics and tropic areas and showing 5-10 0C temperature difference in winter and obviously different of daily seawater temperature fluctuation between BT and OL (Kenting), those showing strong geographic effect between those two populations. Because association with C15 is a “common” characteristics of Porites, but the large environmental difference in small geographic scale, BT and OL Taiwan, still uncertain, therefore I wonder the Symbiodinium composition of those geographic populations whether difference due to distinct ambient environments. In addition, the third nuclear power plant (NPP) have recharge hot water to Outlet since 1985. The hot water affected shallow water coral populations almost 30 years due to temperature difference between depths [23]. Therefore, whether different populations with ambient temperature background were tested to know how it effect on the thermal tolerance.. 3.

(12) Materials and methods. Study sites and seawater temperature Coral bleaching occurred at OL and many other sites in southern Taiwan in 2007, while only few corals bleaching found in northern Taiwan. In order to compare the temperature profiles between northern and southern Taiwan, seawater temperatures data at depths of 2m and 7 m from sites Yehliu (YL: 25°12'10.8"N, 121°40'48.6"E) northern Taiwan and OL (OL: 21°55'53.3"N, 120°44'41.6"E) southern Taiwan collected in 2007 with 60 min-interval using temperature loggers (Hobo, Onset Corporation, precision ± 0.1. 0. C) were analyzed. At another site Beitou (BT:. 25°07'34.0"N 121°54'53.7"E) northern Taiwan was also monitored at depth 2m between October 2009 to February 2010. Sample collection For Symbiodinium diversity analysis, totally 36 colonies massive Porites spp. were collected by scuba diving from BT and Longdon (LD: 25°06'47.1"N, 121°55'11.5"E) which were about 25 km away from YL in July 2012. Coral colonies were photographed for morphological identification, while a small piece (<5 cm in diameter) of sample was chiseled off from each colony then preserved in 70% ethanol for molecular analysis. Hereafter, coral skeletons were bleached and dried for species identification by scanning electronic microscope. Genomic DNA extraction Genomic DNA was extracted using the high salt solution by the method in Ferrara et al 2006 [26]. Between 20-30 mg coral tissue was 4.

(13) placed into 2 ml microcentrifuge tubes with 200 µl lysis buffer [0.25 m Tris, 0.05 M EDTA at pH 8.0, 2% sodium dodecylsulfate (SDS) and 0.1 m NaCl and 5 µl proteinase E (10 mg ml-1) for overnight at 60 0C NaCl (210 µl at 7 M) was added to the lysed tissue in the tube, and the sample was mixed carefully by inverting the tube. The solution was then transferred to a 2-mL collection tube that contained a DNA spin column (Viogene, USA) and centrifuged at 8000 rpm for 1 min. The lysate was washed twice with 500 µl of ethanol (70%) by centrifuging at 8000 rpm for 1 min at each step, with an additional centrifugation step at 8000 rpm for 3 min to dry the spin column. The column was dried further at 37 0C for 15 min and then the DNA was eluted by adding 50 μl of distilled water, with a final centrifugation at 14 000 g for 3 min. The quality of genomic DNA was checked using a 1% agarose gel. Polymerase chain reaction (PCR) and Denaturing gradient gel electrophoresis (DGGE) Polymerase chain reaction (PCR) was performed in a 50 µl of final volume with a concentration of 1X Buffer (200nM Tris-HCl pH=8.4, 500mM KCl), 0.1mM of each dNTP, 0.1mM for each primer, 3 mM MgCl2, and 0.5U Taq DNA Polymerase (Invitrogen). The internal transcribed spacer 2 region (ITS-2) was then amplified for DGGE analysis. Primers for PCR-DGGE analyses were designed by LaJeunesse and Trench, 2000 [27] to amplify a 340-bp product including the ITS 2. The forward primer ITSintfor2 (5’-GAATTGCAGA ACTCCGTG-3’) anneals to a conserved region of the 5.8S rDNA and is paired with the ITS reverse primer [28]. The reverse primer was modified with 39 base pairs GC clamp, called the ‘‘ITS2CLAMP’’ (5’-CGCCCGCCGC GCCCCGCGCC CGTCCCGCCG CCCCCGCCC GGGATCCATA TGCTTAAGTT CAGCGGGT-3’). A 5.

(14) ‘‘touchdown’’ amplification protocol with annealing conditions 10 0C above the final annealing temperature of 52 0C was designed to ensure specificity [29]. The annealing temperature was decreased 1 0C every two cycles. After 20 cycles, the annealing temperature was maintained at 52 0C for another 15-18 cycles. All PCR-DGGE products were loaded with a 2% ficoll loading buffer (2g Ficoll-400, 10 mM Tris-HCL pH 7.8, 1 mM EDTA, 1% bromophenol blue) onto an 8% polyacrylamide denaturing gel containing a gradient of 3.15 M urea/18% deionized formamide to 5.6 M urea/37% deionized formamide. PCR products of the identified Symbiodinium types were mixed and loaded onto the gels as the molecular markers (Fig 1). The PCR products were separated by electrophoresis for 9.5 h at 150 V and a constant temperature of 60 0C. The gel was stained in the Syber Green® with 15 ml TAE buffer for 25 min then photographed under Gel Doc™ IM system (Bio Rad, USA). Isolation of PCR products and sequencing from the DGGE gel The PCR-DGGE gels were visualized under Gel Doc™ IM system (Bio Rad, USA). Selected bands were cut and eluted by 80 μl ddH2O and stored overnight at 4 0C. Re-amplification the eluted DNA by the ‘‘ITSintfor2’’ forward primer and the reverse primer with a GC clamp. A touchdown PCR protocol described above was modified with a change of 12–14 final cycles at 52 0C Sequence alignment, parsimony networks and phylogenetic analysis DNA sequences were assembled using the SeqMan (DNASTAR, USA). All sequence alignment and analyses were conducted using MEGA v5 (http://www.megasoftware.net/). The ITS sequences were trimmed to a 6.

(15) length of 267 bp. Molecular phylogenetic networks were constructed using the TCS v1.21 which implemented Statistical Parsimony method [30]. The ITS 2 sequence was aligned with all Indo-Pacific Symbiodinium species which were published by Thornhill et al 2005 [7]. The gap in phylogenetic network constructed as the fifth character. Heat stress experiments In April 2014, massive Porites spp. colonies commonly found at sites BT and OL were chosen for physiologically thermal stress experiment. Five colonies from BT at 3 m depth and 3 colonies from OL at each depth of both 3 m and 7 m were chosen. The BT colonies was contained by fore P. lobata and one P. lutea after skeleton identification. To avoid the effect of coral species, the P. lutea was exclued from photophysical analysis. The shallow OL colonies were P. lobata and deep colonies were P. lutea. Coral cores in 2.5 cm diameters were collected (35 cores per colony) by drilling and transported back to laboratory for acclimation. Unfortunately, one of shallow OL colonies was unhealthy after one month acclimation and consequent the sample size reducing when data analysis. All nubbins were distributed evenly in three experimental tanks submerged in water tables after one month acclimation. Heat stress experiments were performed in the 2-liter tanks at the aquarium system as open system in Academia Sinica (Fig 7). All nubbins were distributed evenly in three experimental tanks submerged in water tables after one mouth acclimation. The temperatures were set up into three conditions, including: 1) control temperature (26 0C); 2) middle temperature (29 0C); 3) high temperature (32 0C). According to seawater temperature data in OL, above temperature individually represent winter average, summer average 7.

(16) and yearly maximum, therefore the tank setting was based on the year seawater temperature fluctuation at the hottest sampling location. Light provided average underwater intensity 10-25 μmol photons m -2 s -1 for 12 hours per day. Expect 29 0C, the temperature of 26 0C and 32 0C treatments were gently increase two degrees after 96 hours. Over the next one week after acclimation, nubbins were assessed and collected at 0, 6, 12, 24, 48, 96 hours and 1week as end point for photo physiological parameters as describe below. Samples were freeze in liquid nitrogen immediately and preserved in -80 0C for future analyzed. Physiological parameters measurement Photochemical performance Nubbins were measured for chlorophyll fluorescence by Diving-PAM (Walz, Germany) to collect maximum and effective quantum yields of PSII (Fv / Fm). The Fv / Fm refers to the maximum photochemical efficiency of PSII, and requires complete relaxation of the photochemical energy conversion, usually measured after dark adaption. The Fv / Fm has been applied to analysis of short-term response to temperature, light and osmotic stress etc. the lower value represents the lower photochemical performance which was affected by environmental stresses. Measurements of two maximum yields of PSII (Fv /Fm) were taken at 09:00 and 22:00 each day. At each desired time period of incubation in experimental tank, nubbins were froze in liquid nitrogen immediately and stored at -80 0C for future analysis. Chlorophyll pigments measurement Whole tissue were removed from skeleton by water-pick with autoclaved sea water and divided into five for chlorophyll pigments, 8.

(17) Symbiodinium density measurements and back up. Chlorophyll pigments that were extracted with 90% acetone and centrifuged at 18,000g in 4 0C. The absorption of samples was measured at 630nm, 647nm, 664nm, and 750nm, with 90% acetone as blank. Chlorophyll a and chlorophyll c2 were calculated using the equations of Jeffery and Humphrey and normalized to surface area of nubbin which calculated with the nubbin radius [31]. Symbiodinium density After isolated Symbiodinium from coral, 10 microliter of vortex suspended zooxanthellae of each sample was used for calculating density. Number of zooxanthellae was counted in replicates (n=5) with a brightline hemocytometer (Improved Neubauer) and normalized to surface area of coral nubbins. Data statistics analysis The sea water temperature at the different sites were calculated from January 2007 to December 2007 and compared the mean between locations and depths by using Student’s t test by JMP v.11 software. The concentration of chlorophyll pigments concentration and the Fv / Fm was calculated from chlorophyll fluorescence data which refer to it as Fv / Fm were physiological performance of zooxanthellae. The Fv / Fm was determined by nonparametric Steel-Dwass pairs tests which calculated by JMP. v.11. software.. Chlorophyll. pigments. concentration. and. Symbiodinium density were determined by one-way ANOVA and Welch’s test depend on the equal variance. On the other hand, the sample size of OL 3 m population was limitation, therefore the chlorophyll pigments and density cannot proceed statistical analysis. So, the pigments concentration and density only BT has been done. 9.

(18) Results. Seawater temperatures variations Greater temperature variations are clearly found in northern Taiwan than southern Taiwan (Fig. 2). The maximum daily average SST at depth 2m of OL was 32.2  1.1 0C in 26 July 2007 and at depth 2m of YL was 30.2  1.1 0C in 2 August 2007. Monthly means temperature ranged between 25.2  1.3 to 30.6  1.5 0C at OL and 19.1  1.0 to 29.5  1.0 0C at YL (Fig. 2). Differences between the warmest and the coolest month in 2007 reached 10.4 0C at YL and 5.4 0C at OL. Overall, the yearly mean SST of OL was 3.8 ± 2.2 0C higher compared to YL in 2007 (2m: paired ttest, t= 3.14, p < 0.01; 7 m: t= 3.02, p < 0.01, Fig. 3). In addition, significant temperature variation between depths was also found between 2m and 7 m at OL (paired t-test, t=1.96, p < 0.001, Fig. 3). Daily variation (∆T= daily maximum temperature- daily minimum temperature) also plays an important role on affecting the physiology of coral holobionts, especially in the place with recurrent upwelling bringing cool water to shallow reefs. The daily variation between BT and OL at 2m between October 2009 to February 2010 was significant different (paired t-test, t=3.01, p < 0.01, Fig. 4).. 10.

(19) The Symbiodinium composition of massive Porites spp. from north and south Totally, 36 specimens were analyzed with 24 collected from BT and LD, while 12 ones were from OL. The minimum spanning network reconstruction analyzed ITS2 sequence and totally identified seven haplotypes from north and south. Those haplotypes not only developed from original C15 types following one base pair difference but also independent with known C15 related types (Fig. 6). The undescribed types are labeled with “H” and the number after alphabet shown the distance with original C15. Figure 6 presented that northern population associated with H2 to H8 while the south associated with original clade to H3. This result provided a view of the diversity of Symbiodinium composition associated with massive Porites spp. which inhabited distinct environment. Furthermore, not only the Symbiodinium composition between north and south population was significant different (chi-square test, p < 0.01) but also the band composition in DGGE profile was distinct, it clearly showed there were three varied bands to component the community of BT, on contrary, two close bands composited the OL. On the other hand, figure 5 displayed that the capacity of P. lobata to harbor distinct Symbiodinium compositions in different geographic location while same Symbiodinium composition was associated with P. lutea and P. lobata. Those results illustrated that geographic difference be showed on the Symbiodinium compositions (Fig. 5-6). Therefore, understanding the thermal physiology of holobionts whether different while associated with distinct Symbiodinium composition at those two geographic regions.. 11.

(20) 2.1 Maximum PSII quantum yields (Fv / Fm) 2.1.1 The thermal physiology of distinct geographic Symbiodinium populations The in situ photosynthetic performance of Symbiodinium was expressed by the maximum quantum yields (Fv / Fm) which measured at dark, represent as Fv / Fm. In 32 0C treatment, both OL and BT populations showed significant decrease at 12 hours (Steel-Dwass all pairs test, p < 0.05, Fig 11). The yields of BT population was 0.512  0.05 and OL was 0.455  0.05 at 24 hours. The recovery rate, a percentage recovery after 12 hours, the average recovery rate of BT population in 32 0C was 12.7  4.3 % while OL was -9.27  6.9 %. As shown in figure 12, the BT population recovered after 36 hours but the OL undulated at zero. Moreover, the average recovery rate of BT population in 29 0C was 12.5  7.2 % while OL was 0.33  5.6 %. It shown BT population quickly recovered from thermal stress compared to OL (Fig 12). 2.1.2 Fv / Fm from different depths of OL populations within treatments throughout time The Fv / Fm at 3 m population was significant lower than 7 m population under each treatments (Wilcoxon one-way test, p < 0.01, Tab. 6, Fig. 17). In field, the Fv / Fm of 3 m population was 0.607  0.02 and 7 m was 0.66  0.03, it highlights the difference of depths was origin from nature. Therefore I compared the change of difference between treatments throughout time and presented in Figure 18. The figure clearly shown that difference was gently reducing throughout time in 29 and 32 0C before 96 hours, and this pattern presented more obviously when the temperature 12.

(21) reached higher after 96 hours. Although associated with different coral hosts, the responses of Symbiodinium which from same geographic region to thermal stress were identical. Figure 11 the difference was -0.06, -0.02 and -0.05 individually represent 26 0C, 29 0C and 32 0C in 0 hour while shifted to 0.06, 0.07 and 0.09 in 12 hours, and it might displayed the environment of experiment tank was distinct with acclimation tank (Fig.18).. 2.2 Chlorophyll pigment concentration 2.2.1 Chlorophyll pigments concentration from different geographic regions Figure 13 presented significant decrease of BT population in 32 and 26 0C treatments during 48 hours (one-way ANOVA, p < 0.01, Fig. 13, Tab. 3), but OL population shown obviously increased in 32 0C (n= 2). Same pattern was display in chlorophyll pigment c in figure 14, but OL population did not present obviously difference in 32 and 29 0C under 48 hours treatment. Compared with the dynamic pattern during scale of 26 0C, 32 0C treatment presented opposite result. Due to the limit of sample size, it was hardly put scientific point. 2.2.2 Chlorophyll pigments concentration from different depths of OL population The result reflected in Figure 19 and 20 did not significantly difference between depths on thermal stress (Tukey-Kramer HSD pair test, p > 0.05), it suggested chlorophyll pigments concentration of distinct populations did not effect on thermal treatments. 13.

(22) 2.3 Symbiodinium density 2.3.1 Symbiodinium density from different geographic regions The results reflected in Figure 15 indicated that both populations density significant decrease under all treatments (One-way ANOVA test, p< 0.01, Tab.10), it suggested that the decline was affected by experimental tank rather than temperature treatments. Therefore, the density difference of time, calculated by 48 hours minus 0 hours, was compared and the result shown that generally decrease of Symbiodinium density in both populations under each treatments except the OL treated at 32 0C. Compared with BT, the decreasing margin was larger in OL population (Fig 16). 2.3.2 Symbiodinium density from different depths of OL population According to Figure 21, the density of 7 m did not displayed significant difference under 26 0C and 32 0C (Welch’s test, p> 0.05, Tab. 9), but shown significant difference under 29 0C during 48 hours (p< 0.01). The populations which inhabited different depths also did not show difference on thermal stress.. 14.

(23) Discussion. New haplotypes of Porites associated Symbiodinium Since LaJeunesse successfully used ITS2 region of the nuclear ribosomal DNA to identify major clade of Symbiodinium genus[9]. The ITS region has been used to identify the Symbiodinium clade because of its multicopy nature and can exhibit large levels of intragenomic variation [31, 32]. Researches over the past decade have revealed that different genotypic combinations of coral host and Symbiodinium display varied physiological characteristics such as thermal tolerance and bleaching resistance [4, 14, 16, 33, 34]. However, it was doubt that the constant Symbiodinium composition on Porites genus [7, 31, 35-37], which vertically transmit Symbiodinium to offspring [38]. Figure 1 presented the specific association of Symbiodinium C15 type with Porites at Pacific Ocean and this pattern was causing by the world distribution on Symbiodinium [39]. The C15 type was proved more thermal tolerant to heat compare to other C clade [22]. The Trophic of cancer and warm water supply make BT and NTT-OL to distinct environments, it provide me a unique opportunity to examine the question of the geographic effect on Symbiodinium composition of massive Porites spp. Surprisingly, not only new haplotypes be discovered but also the composition of Symbiodinium was significantly different between those geographic populations (Fig 5 and 6). Those do not support the previous point view on specific association of C15 type with Porites genus. The results discover six undescribed haplotypes which related with original C15 and differ from known C15 related haplotypes. Previous paper 15.

(24) indicated that the C15 and related types showing less than three nucleotide changes [40, 41]. In Figure 6 of this study showed there were four haplotypes presented more difference than this between described related types and original C15 type, it suggests that those new haplotypes showing high opportunity to be new Symbiodinium types associated with Porites by only small differentiation [35, 42]. In addition, the Symbiodinium associated with BT population displayed longer distance than the composition of OL population. In other words, the population which inhabits at warm environment associates more original haplotypes compare with the population from cool environment. This is the first study reporting the Symbiodinium community of massive Porites spp. diversified by environmental conditions. Those results displayed highly diverse of ITS region in clade C, however, the sequence analysis of a single gene barely resolves distinct lineages of C15 related clade [42]. In future study, more markers will be applied for identification and classification. Population differentiation of Symbiodinium cause different strategy to respond thermal stress According to the data, the opposite pattern was shown by Symbiodinium density and chlorophyll pigments concentration between BT population and OL population. (Fig. 13-16). It might suggest that the BT population increasing chlorophyll pigments to compensate for the decreasing of Symbiodinium density and photosynthetic efficiency. However, the OL increased the density to compensation, instead of chlorophyll pigments (Fig 11, 13-14). It suggests the different physiological strategies when facing thermal stress depend on distinct geographic. populations.. Furthermore,. the. phylogenetic. network. reconstruction displayed significant component difference of associated 16.

(25) Symbiodinium between those populations. Those results suggested that the associated Symbiodinium of P. lobata might show population genetic differentiation due to huge environmental difference in small geographic scale. Comparing to geographic effect on populations, the depths effect reveals relatively less significant although the host species is different between shallow and deep populations. It emphasizes that the inhabited environment effect on thermal tolerance might be stronger than host species effect. According to physiologic and molecular data on this study, these indicated that habitat environment might exhibit functional heterogeneity as a consequence of local adaption of symbiont associated with massive Porites spp. Most of the papers studying on thermal tolerance of Symbiodinium did comparisons between different clades [14, 33, 43] and concluded that coral holobionts show the capacity to acclimatize to future ocean warming[16] [44]. Yet functional variation among populations of the same type of Symbiodinium has been overlook [45]. In Howells et al, they presented the local temperature background making Symbiodinium populations showing better physiological response in its own ambient temperature and seem to adapt to local environment [45]. This founding provides viewpoint that the capacity to associate with divergent types of Symbiodinium be estimate at the corals which was considered absent in this trait [15], though lack of proof that showing the genetic difference between these Symbiodinium populations from distinct environments. It might present more advantage if contrary to the adaption of thermal tolerance clade, this adaption which fitted to local coral host population may not tradeoff between growth rate and thermal resistance[12].. 17.

(26) In aquatic asexual organisms (i.e. single-cell eukaryotic algae) already displayed the capacity to rapidly adapt to changes in environmental conditions caused by signal mutation [46],[47]. The finding of this study also presents similar phenomenon. According to this paper, selection of against genetic variants rising from somatic mutations in the order of 10−7– 10−5 resistant variants per cell division [47]. If Symbiodinium behaviors similar to single-cell eukaryotic algae, adaptive potential might exist because of wild populations, short generation time (days to weeks within coral hosts) [48] and large population in corals [49]. Not to mention the strong selection of coral bleaching, climate change and climate disturbance [50, 51]. Upwelling effect on the thermal tolerance of different geographic populations According to Fv / Fm data collected in experimental tanks, the coral holobionts that inhabit in OL displayed lower yields than those live in BT during thermal stress tank at 29 and 32 0C (Fig. 11). In addition, both of them shown significant drop at 12th hours but the recovery rate of OL population was the lowest. In other words, the results described above indicated that the population live in warm environment seems more sensitive to thermal stress than those inhabit in cool habitat although the photochemical efficiency measured directly after collected from the field did not showed physiological difference between geographic populations. Several papers, however, have pointed out that heat experience can help corals to survive or resist high temperature that would cause bleaching [5255]. The results of this study do not support this viewpoint. It should also be pointed out that there are some limitations in this study. These include 18.

(27) the small sample size of OL shallow populations (3m) which have only two colonies. Therefore, more sampling effort will be needed for further studies. While another possible explanation might be the daily SST fluctuation in Nanwan Bay, the southernmost embayment in Taiwan which includes OL, up to 8-9 0C during summer and spring tide upwelling, that is, the SST of OL be cool down each 10 days [56, 57]. The upwelling not only cool down SST at summer time but also supplies nutrients to surface layer which may be beneficial to coral reefs [58]. However, there is no upwelling effect in BT which do not display huge daily fluctuate compare to OL (Fig 4). As D’Croz (2004) estimated that the upwelling populations shown less thermal tolerance than non-upwelling populations in constant thermal stress and even at small geographic scale, this difference is concomitant with genetic differences of coral hosts [59]. The results in this study are identical to these finding and this may be the reason why OL population was more sensitive to constant thermal stress environment, though the population inhabit warm habitat and associated with original C15 type. Coral hosts species effect on thermal tolerance of different depth populations As the result displayed at figure 6, the difference of Fv / Fm between depths was found in field data and even after one month acclimation. According to skeleton and colony characters, the shallow water and deep water populations were P. lobata and P. lutea, respectively. Therefore, the differences of photo-physiological probably depend on host species and environmental difference. However, this difference has been reduced by thermal treatments (Fig. 17). The pattern was more obviously while two degrees increase and causing extreme high temperature at 32 0C treatment 19.

(28) after the 4th day. It seems that the response to thermal stress from different depths populations were similar, though the distinct host species and depths. Moreover, the density and chlorophyll pigments concentration between depths did not shown significant difference at 48 hours (Fig. 1921, Tab. 7-9). These results are the evidences for the same genetic background of Symbiodinium. Aquatic temperature and light effect on coral holobionts It should be mentioned that the temperature and light were not maintained very well in this experiment. The temperature of OL acclimation tank was three degrees higher than the BT three day before experiment due to out of order of heater, that is, the OL already pre-heat (Fig 8). In Mddlebrook (2008) demonstrated that pre-heat can positively modify the thermal threshold of corals [52]. However, the Fv / Fm of OL population in heat treatments showed opposite result, one of the reasons is that the photosynthetically active radiation (PAR) in OL sub-tank under 32 0. C treatment was slightly lower than BT sub-tank (Fig 11) and this light. difference of geographic populations might also cause the Fv / Fm difference. Papers already indicated that the Symbiodinium density and chlorophyll pigments were affected under weak light environment as increasing chlorophyll pigments concentration to compensate the decline of density [60, 61] However, the effect of this slightly difference was limited in the weak light environment which we provide since acclimation, hence the dramatic. changing. between. density. and. chlorophyll. pigments. concentration in this study might be due to the temperature treatments.. 20.

(29) Future work Many papers discovered that Symbiodinium clade D associated corals living at very shallow and high disturbance environment, clade D is though more stress tolerance than others [14, 39]. However, Abrego (2008) found that clade D is less thermal tolerance than type C1 when associated with Acropora tenuis juveniles. It suggests that species-specific interactions between symbiotic partners and a potential role for host factors in determining the physiological performance of reef corals [13]. It indicated the important and complex interaction between Symbiodinium and coral host of thermal tolerance. Future study will focus on transcription level, the early physiological responsese, compare the role between coral host and Symbiodinium by stress gene expression.. 21.

(30) Conclusion In this study, I present the results of different associated Symbiodinium compositions of P. lutea and P. lobata which inhabit different geographic environments. The varied dynamic between Symbiodinium density and chlorophyll pigments concentration depend on different geographic populations. Contrary to geographic effect, those dynamic was the same between different depths populations but the same location. It strongly proved that the difference of Symbiodinium compositions due to adaptation of huge environmental difference, such as temperature. Although the host species of depths populations is different, the response to thermal stress was identical. In conclusion, those results highlight that geographic effect might be stronger than host species effect for adaption.. 22.

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(36) 61.. 62.. from upwelling and non-upwelling environments in Panama. Coral Reefs, 2004. 23(4): p. 473-483. Steele, R.D., Light intensity as a factor in the regulation of the density of symbiotic zooxanthellae in Aiptasia tagetes (Coelenterata, Anthozoa). Journal of Zoology, 1976. 179(3): p. 387-405. Titlyanov, E., et al., Photo-acclimation dynamics of the coral Stylophora pistillata to low and extremely low light. Journal of experimental marine biology and ecology, 2001. 263(2): p. 211225.. 28.

(37) Table Table.1 Review of Symbiodinium types in genus of Porites. Abbreviation: Shallow water, WP: West Pacific; CP: Central Pacific; EP: East Pacific; WA: West Atlantic; GBR: Great Barrier Reef; Taxon Porites annae Porites cylindrica Porites rus Porites vaughani Porites lichen Porites nigrescens Porites massive Porites rus Porites cylindrica Porites lichen Porites massive Porites lutea Porites silimaniana Porites attenuata Porites australensis Porites cylindrical Porites lichen Porites monticulosa Porites rus Porites lobata Porites lutea Porites astereoides Porites astereoides Porites colonensis Porites divaricata Porites furcata Porites astereoides Porites astereoides Porites Porites divarieata Porites porites. Region WP. WP. WP. CP EP WA. WA WA. Reef GBR. Symbiodinium type C60, C15 C60, C15 C15 C60, C15 C15 C15 C15 Okinawa C15 C56a C56 C15 C15 C15 Palau C C C, D C D C Oahu, Hawaii C15, C55, C60 C15, C55, C60 Mexico A4a, A3, B1 Florida Keys A4a C1a C9 A4, B1, C4 Bahamas A4a A4a Caribbean C45 C1 C10. 29. Reference LaJeunesse et al, 2004. Fabricius et al., 2004. Apprill et al., 2007 LaJeunesse, 2002 Thornhill, 2006. LaJeunesse, 2002 Thornhill, 2006 LaJeunesse, 2006.

(38) Table 2. The Steel-Dwass all pairs test of maximum quantum yield (Fv / Fm) between temperature treatments in Bitou and Nuclear Power plant Outlet (NPP-OL). Significant level: *: p < 0.05 **: p < 0.01 ***: p < 0.001 N.A.: not applicable due to sample size was less than 3.. 12hr. Bietou. NPP-OL. Level 26 vs 29 29 vs 32 26 vs 32 26 vs 29 29 vs 32 26 vs 32. 60hr. Bietou. NPP-OL. Level 26 vs 29 29 vs 32 26 vs 32 26 vs 29 29 vs 32 26 vs 32. 24hr p 0.990 0.030* 0.020* N.A. N.A. N.A.. Level 26 vs 29 29 vs 32 26 vs 32 26 vs 29 29 vs 32 26 vs 32. 72hr p 0.99 0.56 0.40 0.94 0.55 0.73. Level 26 vs 29 29 vs 32 26 vs 32 26 vs 29 29 vs 32 26 vs 32. 36hr p 0.510 0.820 0.160 0.900 0.100 0.020*. Level 26 vs 29 29 vs 32 26 vs 32 26 vs 29 29 vs 32 26 vs 32. 96hr p 0.98 0.99 0.99 0.86 0.71 0.29. Level 26 vs 29 29 vs 32 26 vs 32 26 vs 29 29 vs 32 26 vs 32. 30. 48hr p 0.940 0.090 0.140 0.950 0.550 0.280. Level 26 vs 29 29 vs 32 26 vs 32 26 vs 29 29 vs 32 26 vs 32. 6Day p 1.00 0.99 0.96 0.95 0.74 0.44. Level 26 vs 29 29 vs 32 26 vs 32 26 vs 29 29 vs 32 26 vs 32. p 0.870 0.260 0.850 0.390 0.220 0.007** 8DAY. p 0.99 0.95 1.00 N.A. N.A. N.A.. Level 26 vs 29 29 vs 32 26 vs 32 26 vs 29 29 vs 32 26 vs 32. p 0.73 0.24 0.91 N.A. N.A. N.A..

(39) Table 3 one-way ANOVA of chlorophyll a concentration between treatments in Bietou. *: p < 0.05 **: p < 0.01 ***: p < 0.001 Treatment 26 29 32. Level 0 vs 48 0 vs 48 0 vs 48. Bietou F ratio p 0.006** 13.00 0.890 0.018 0.003** 35.31. 31. DF 1 1 1.

(40) Table 5 Welch’s test of chlorophyll c between treatments in Bietou population. *: p < 0.05 **: p < 0.01 ***: p < 0.001 Treatment 26 29 32. Level 0 vs 48 0 vs 48 0 vs 48. Bietou F ratio p 0.16 2.920 0.79 0.018 0.04* 7.820. 32. DF 1 1 1.

(41) Table 6. Wilcoxon / Kruskal-Wallis one-way test of the Fv / Fm of Porites from 3 m and 7 m in the NPP-OL. Significant level. *: p < 0.05 **: p < 0.01 ***: p < 0.001. N.A.: not applicable due to sample size were less than three.. Level 0hr 12hr 24hr 36hr 48hr 60hr 72hr 96hr 6D 8D. 3 m-7 m 3 m-7 m 3 m-7 m 3 m-7 m 3 m-7 m 3 m-7 m 3 m-7 m 3 m-7 m 3 m-7 m 3 m-7 m. 26 0C pvalue N.A. N.A. 0.01* 0.14 0.07 0.16 0.39 0.13 0.77 0.83. 29 0C. 32 0C. p-value. p-value. N.A. N.A. 0.004** 0.040* 0.010* 0.200 0.190 0.240 0.370 0.870. N.A. N.A. 0.001** 0.010* 0.008** 0.240 0.100 0.100 N.A. 0.870. 33.

(42) Table 7 one-way ANOVA test of chlorophyll a between treatments in NPP-OL deep population. Due to the limit of sample size, shallow population cannot be analysis. Treatment 26 29 32. Level 0 vs 48 0 vs 48 0 vs 48. NPP-OL (7 m) F ratio p 0.17 2.68 0.38 0.95 0.55 0.41. 34. DF 1 1 1.

(43) Table 8 one-way ANOVA test of chlorophyll c between treatments in NPP-OL deep population. Treatment 26 29 32. Level 0 vs 48 0 vs 48 0 vs 48. NPP-OL (7 m) F ratio p 0.81 0.06 0.02* 12.70 0.42 0.78. 35. DF 1 1 1.

(44) Table 9 Welch’s test density between treatments in NPP-OL deep population. Treatment 26 29 32. Level 0 vs 48 0 vs 48 0 vs 48. NPP-OL (7 m) F ratio p 0.720 0.15 0.002** 49.40 0.630 0.26. 36. DF 1 1 1.

(45) Table 10 One-way ANOVA test density between treatments in Bietou. Treatment 26 29 32. Level 0 vs 48 0 vs 48 0 vs 48. Bietou F ratio p 0.004** 14.79 0.900 0.01 0.030* 6.95. 37. DF 1 1 1.

(46) Figure. Figure 1. Review of Symbiodinium type diversity in Porites spp. reported from both the Atlantic and Pacific Ocean.. 38.

(47) Figure 2 Monthly averages (±SD) of seawater temperature measured in different depths from NPP-OL (south) and Yehliu (north) in 2007 which was the hottest year since the major bleaching event occurred during 1998-99... 39.

(48) Figure 3 box plot of seawater temperature measured in different depths from NPP-OL and Yehliu (north) in 2007. The circle represent outlier while the uper limit of box plot is 90 percentile and lower limit is 10 percentile. The bar inside box plot and dimaid individually repersent medium and average.. 40.

(49) Figure 4 the daily difference of Bietou and NPP-OL during Oct 2009 to Feb 2010. The arrow present typhoon cause huge decrease of temperature in BT. The ∆T was calculated by daily maximum minus daily minimum.. 41.

(50) Figure 5 PCR amplifications of the Symbiodinium ITS2 region. PCR products were electrophoresed on denaturing gradient gels to produce diagnostic fingerprints that exhibit its geographic distributions.. 42.

(51) Figure 6. Minimum spanning network of the Symbiodinium ribosomal intertranscribed spacer 2 (ITS2) types in Porites collected from NPP-OL (orange) and Bietou (blue). The published Symbiodinium C-related types from literatures are labeled. The undescribed types are labeled with “H” and the number after alphabet shown the distance with original C15. The size of circles represents the number of individual samples. The number of mutations (>2) is indicated by Roman numerals on the branch linking the types.. 43.

(52) (A). (B). Figure 7 Experimental tank setting and coral nubbins. The tanks were connected with chiller (Active Aqua, USA) and heater to control the temperature in water bath. Light was supplied by T-5 (Coral life, USA) with 12:12 hours day and night.(A) Each temperature set was divide into two sub-tank individually contain Bietou and NPP-OL populations. (B) The photo was taken at the 72 hours after experiment started.. 44.

(53) (A). (B). Figure 8 The temperature data of acclimation tanks. The black line represent as temperature and blue line is lntensity. (A) Bietou (B) NPPOL. 45.

(54) Temperature (0C). (A). Tank 32 29 26. 34 32 30 28 26 24 22 20. ‐7. ‐3. ‐1. 0. 1. 2. 3. 4. 5. 6. 7. 8. Day (B). Temperature (0C). 34. Tank 32 29 26. 32 30 28 26 24 22 20. ‐7. ‐3. ‐1. 0. 1. 2. 3. 4. 5. 6. 7. Day Figure 9 Average temperature data of experimental sub-tank. (A) NPPOL, (B) Bietou. Color presented different treatments. All treatments increase two degrees more after 96 hours. Temperature is expressed as mean. s.e. 46. 8.

(55) PAR (umol S‐1 m‐2). (A) 35 30 25 20 15 10 5. 32. 0. 24. 48. 72 Time. 29. 96. 26. 6D. 8D. PAR (umol S‐1 m‐2). (B) 35 30 25 20 15 10 5 0. 24. 48. 72 Time. 96. 6D. Figure 10 Daily average light intensity of different treated experimental sub-tank. (A) NPP-OL, (B) Bietou. 47. 8D.

(56) Fv / Fm. (A) 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0. 12. 24. 36. 0. 12. 24. 36. 48 60 Time. 72. 96. 6D. 8D. Fv / Fm. (B). 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 48 60 Time. 72. 96. 6D. Figure 11 The maxium quantum yield presented in treatments by two populaiton. (A) NPP-OL, (B) Bietou. The x-axis presenting time, D represents days and numbers represent hours. The triangle represent 32 0C treatment while square as 26 0C and circle is 29 0C.. 48. 8D.

(57) Recovery rate (%). 25 20 15 10 5 0 (5) (10) (15) (20) (25). N32 N29. 24. 36. 48. 60. 72. 96. 6D. 8D. S32 S29. Time. Figure 12 Recorvery rate between populaitons in thermal treatments throughout experiment time. The recorvery rate was cauculate how much it recorver after 12 hours, that is, the remaid time minus the 12th hour and devide by the 12th hour. The north: open circle, the south: open triangle, 32 0C: full line and 29 0C: dotted line. 49.

(58) (A). (B). (C). Figure 13. Chlorophyll a concentration of different geographic populations by treatments. (A) 32 0C (B) 29 0C (C) 26 0C. The sample size of south was too low to calculated stander deviation (n =2).. 50.

(59) (A). (B). (C). Figure 14 Chlorophyll c concentration between geographic populations by treatments. (A) 32 0C (B) 29 0C (C) 26 0C, the sample size of south was too low to calculated stander deviation (n =2). 51.

(60) (A). (B). (C). Figure 15 Symbiodinium density from different graphic regions under temperature treatments during 48hours. (A) 32 (B) 29 (C) 26 0C. 52.

(61) Time difference(48hr‐0hr). 0 ‐0.1. N. S. ‐0.2 ‐0.3 ‐0.4 ‐0.5 ‐0.6 26. 29. 32. Figure 16 Comparison of the Symbiodinium density difference of geographic populations between 0 and 48 hours by treatments. Patterns represented different experimental treatments. The difference was calculated by the 48 hours minus 0 hour and negative value means density decreasing while positive shows increasing during 48 hours treatment. Limitation of sample size, the southern population cannot present stander deviation (n =2).. 53.

(62) Figure 17 Fv / Fm from different depth under temperature treatments throughout time. (A) 26 0C, (B) 29 0C, (C) acclimation tank, (D) 32 0C. The diamond and cross repssent 7 m and 3 m of southern populaitons, indivireally. Star show significant level, *: p < 0.05 **: p < 0.01 ***: p < 0.001.. 54.

(63) Figure 18 Compared the depths difference of Fv / Fm from different treatments throuout time. Color represented treatments. The depths differnece of Fv / Fm couculated by 7 m populaiton minus 3 m populaiton. The black line means the temperature increased two degrees more after 96 hours.. 55.

(64) (A). (B). (C). Figure 19 Chlorophyll a concentration from different depths of OL population under temperature treatments during 48 hours. (A) 32 (B) 29 (C) 26 0C. 56.

(65) (A). (B). (C). Figure 20 Chlorophyll c concentration from different depths of OL population under temperature treatments during 48 hours. (A) 32 (B) 29 (C) 26 0C. 57.

(66) (A). (B). (C). Figure 21 Symbiodinium density from different depths of OL population under temperature treatments during 48 hours. (A) 32 (B) 29 (C) 26 0C. Limitation of sample size, the shallow species cannot present stander deviation (n =2).. 58.

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