Sex and Symbionts New Discoveries in Local and Regional Patterns of Coral Reproduction and Ecology

Home , Capnella, Dendronephthya

Sex and symbionts New discoveries in local and regional patterns of coral reproduction and ecology

Micaela Hellström

Doctoral thesis in Marine Ecotoxicology Micaela Hellström, [email protected]

Department of Systems Ecology Stockholm University SE-10691 Stockholm, Sweden

©Micaela Hellström, Stockholm 2011

ISBN 978-91-7447-310-0

Printed in Sweden by US-AB, Stockholm 2011 Distributor: Department of Systems Ecology, Stockholm University

“The sea -once it casts its spell - holds you in its net of wonder forever.”

Jacques Yves Costeau (1910-1997)

List of papers The papers in this thesis are referred to in the text by their roman numerals: The use of paper I and II in this thesis by permission of Springer Link.

I: Hellström M, Benzie JAH (in press). Robustness of size measurements in soft corals. Coral Reefs. DOI: 10.1007/s00338-011-0760-4 II: Hellström M, Kavanagh KD, Benzie JAH (2010) Multiple spawning events and sexual reproduction in the octocoral Sarcophyton elegans (Cnidaria: Alcyonacea) on Lizard Island, Great Barrier Reef. Mar Biol 157:383–392 III: Hellström M, Kavanagh KD, Mallick S, Benzie JAH. Mode of vegetative reproduction and genetic identification of spawning groups, in the soft coral Sarcophyton elegans, Lizard Island, Northern Great Barrier Reef. (Manuscript) IV: Hellström M, Gross S, Hedberg N, Faxneld S, Cuong CT, Nguyen Ngai D, Huong Le Lan, Grahn M, Benzie JAH, Tedengren M. Symbiodinium spp. diversity in a Single Host Species, Galaxea fascicularis, Vietnam; Impact of Environmental Factors, Host Traits, and Diversity Hot Spots. (Manuscript) Author contribution to papers I-IV CONTRIBUTION Paper I Paper II Paper III Paper IV Wrote the paper: MH, JB MH MH, KK, SM, MH JB Co-authored after first KK, JB SG draft Field design MH MH MH MH, JB (MT) Choice of markers: - - JB MH Laboratory design: MH MH JB MH, JB, MG. Field collections: MH MH MH MH, NH, SG, CTC,NDN, LLH, SF, MT Performed the experi- MH MH MH MH, LLH, NH, ments: SG, SF Data preparation: MH MH MH, MH, NH, MG, SG, SF Data analysis: MH, JB MH MH, KK, SM, MH JB Maps and figures: MH MH, KK MH,KK NH, SG, MH Reagents/ materials/ MH MH MH, JB MT, MG lab

Abbreviations: CTC, Chu The Cuong; JB (note JAHB in the papers), John Benzie; LLH, Le Lan Huong; MG, Mats Grahn; MH, Micaela Hellström ; NDN, Ngai D Nguyen; NH, Nils Hedberg; SF, Suzanne Faxneld; SG, Susanna Gross; KK, Kathryn Kavanagh; SM, Swapan Mallick; MT, Michael Tedengren

Additional publications by the author of this thesis: Bugiotti L, Hellström AM, Primmer C (2006) Characterization of the first growth hor- mone gene sequence for a passerine bird - the pied flycatcher (Ficedula hypoleuca). DNA Seq. 17 (6), 401-406 Lindström K, Hellström AM (1993) Male size and parental care in the sand goby, Po- matoschistus minutus (Pallas). Ethology Ecology and Evolution 5: 97-106

Abstract

Coral reefs belong to the most diverse and the most threatened ecosystems on earth. Anthropogenic stressors and climate change have led to mortalities at levels unprecedented in modern times. The aims of this thesis are to investigate aspects of the corals’ ability to reproduce, disperse, adapt and survive in order to maintain themselves. Papers II-III study reproduction in a common soft coral species, Sarcophyton elegans, with previously unknown reproductive modes. Paper IV investigates genetic distribution of coral-symbiont associations in Galaxea fascicularis focusing on adaptation to the environment along the coastline of Vietnam.

S. elegans is a gonochoric broadcast spawner with a 1:1 sex ratio. Reproduction is strictly size dependent (Paper II- III, method; Paper I). Oogenesis takes 19-24 months, with a new cycle commencing every year. Spermatogenesis takes 10-12 months. The majority of gametes were released during the annual austral mass spawning event after full moon in November, but spawning also occur between August and February. The polyps at the outer edge of the colonies released their gametes first, followed by polyps situated closer to the center during subsequent months. Colonies upstream in the prevailing current spawn earlier than those downstream (Paper II). The colonies were arranged in clusters of alternating males and females, which spawned simultaneously and were of the same genotype (PaperIII). Fission and buddying is a common mode to expand locally. Additionally, females undergoing fission divided into the most fecund size classes (Paper III).

The G. fascicularis and their associated symbionts were not genetically coupled to each other but to environmental factors (Paper IV). The host displayed an inshore- offshore zonation, with higher diversity offshore. The D1a symbiont exhibited an inshore- offshore zonation. In contrast; the 5 different C symbiont types showed a latitudinal distribution gradient, which shifted in dominance north to south. The study highlights the importance of protecting resilient coral and algal genotypes in stressed areas (Paper IV) and the need to understand reproductive modes (Papers II-III) for coral conservation.

Keywords: Indo-Pacific, S. elegans, G. fascicularis, Symbiodinium, size, reproduction, allozymes, ITS2, mtDNA, geography, environment

Svensk sammanfattning

Korallreven som även kallas ’havens regnskogar’ hör till de mest artrika och hotade ekosystemen på jorden. Antropogena stressfaktorer och klimatförändringar har orsa- kat massdöd av koraller som saknar motstycke i modern tid. Målet med denna av- handling är att undersöka aspekter i korallernas förmåga att överleva genom förök- ning, spridning, samt anpassning till snabba förändringar. Artikel I-III undersöker sexuell och könlös förökning hos en vanlig mjukkorall art, Sarcophyton elegans, i Australien med tidigare okänd förökningsstrategi. Artikel IV undersöker hur den revbyggande korallen Galaxea fascicularis samt algen den lever i symbios med anpassar sig genetiskt till en miljö i förändring längs Vietnams kustlinje.

S. elegans är tvåkönad och befruktningen sker i vattnet utanför föräldrakolonierna. Könsmognaden samt den könlösa förökningen styrs av korallstorlek istället för ål- der. Äggcellerna utvecklas över två år, men en ny cellcykel börjar varje år. Spermie utvecklingen tar ett år i anspråk. Könscellerna frigörs i vattnet under årliga spektaku- lära mass-förökningar som styrs av fullmånen i november/december. Könscellerna som bildas i polyperna längst ut i kolonierna (perifera polyper) avger sina könsceller flera månader tidigare än de som befinner sig längre in i korallkolonin. Detta feno- men var tidigare okänt och har konsekvenser för korallforskningen eftersom antalet könsceller som produceras kan användas för att räkna ut korallens potential till över- levnad. Tidigare har man antagit att de perifera polyperna inte förökar sig. Kolonier uppströms avger sina könsceller tidigare än de nedströms, vilket möjligen styrs av kemiska signaler. S. elegans förökar sig könslöst genom att dela sig i hälften samt genom att bilda utväxter som faller av föräldrakolonin.

Korallen G. fascicularis upptar algsymbionter helt oberoende av genetisk uppsätt- ning. Däremot styrs förekomsten av genotyper hos både korallvärd och alg av miljö- faktorer. Reven nära kusten som utsätts för mycket föroreningar, visar genetiska uppsättningar av alger som är stresstålig (typ D) och genotyper av koraller som skiljer sig mellan förorenade och rena miljöer. Algtypen C visade sig bestå av många genetiska varianter som visade en ändring av utbredning i nord-sydlig rikt- ning. Mångfalden av alger i G. fascicularis kan bero på att artens larver upptar algerna från vattnet (horisontell upptagning) och inte från föräldrakolonin (vertikal upptagning). Detta är en direkt konsekvens av yttre befruktning. Denna studie visar vikten av att förstå korallers reproduktionsstrategier, samt behovet att skydda korall- arter i stressade områden eftersom de uppvisar tåliga genotyper hos både alg och korallvärd.

Contents

Introduction 13 Background 13 Thesis objectives 15 Study species 17  Position in the tree of life: Scleractinia, Alcyonacea and Dinoflagellata 17  Sarcophyton elegans 18  Galaxea fascicularis 18  Symbiodinum sp. 18 Reproduction in Soft Corals 19  Sexual reproduction 19  Asexual reproduction 20 Materials and Methods 21 Study area 21  Australia 21  Vietnam 21  Oceanographic background data 22 Paper I: Colony measurements 24 Paper II:Sexual reproduction in S. elegans 25  Colony sexual identity, sex ratio 25  Gametogenic cycle 25  Temporal spawning dynamics 26 Paper III: Asexual reproduction in S. elegans 26  Modes of asexual reproduction 26  Genetic analysis 26 Paper IV: Genetic distribution of Symbiodinium sp. in G.fascisularis 27  Sample collection and DNA extractions 27  Genotyping 27 Statistical Analysis 28 Major Findings – Summary of papers 31 To wrap it up 42 Literature cited 44 Acknowledgements 52

Introduction

Background Coral reefs belong to the most diverse and the most threatened (Bellwood et al. 2004) ecosystems on earth, often referred to as rainforests of the sea (Connell 1978), not just because of the estimated 800 to 1000 species of reef building corals themselves (Veron 1993, 1995, 2000, Knowlton 2008) and the thousands of closely related taxa of soft corals (Benayahu and Loya 1977, 1981 Dinesen 1985, Fabricius and Alderslade 2001), but because of the 1-9 million other species living mainly or entirely in relation to them (Readka-Kulda 1997, Knowlton 2008). The reef community includes 32 of the 33 phyla inhabiting marine ecosystems, compared to 17 phyla in total hosted by terrestrial ecosystems (Norris 1993). The coral reefs cover only 0.1-0.5% of the ocean floor, still, an estimated 30% of marine fish live on coral reefs (Moberg and Folke 1999). The reef building corals and a majority of the tropical soft corals exist in a close nutritional partnership with unicel- lular algae (Protista) of the genus Symbiodinium called zooxanthellae. The symbiosis is a key factor behind the high levels of productivity on the reef (Muscatine 1990, Yellowlees et al. 2008). The acquisition strategies of the symbionts are either vertical (transfer to the offspring from the parent colony) or horizontal (acquisition from the surrounding environment) and depend on the reproductive modes of the corals (brooders or spawners) which determine the portfolio of symbiont types within the host (LaJeunesse 2005, Stat et al. 2008, Stat et al. 2011b). Regional differentiation patterns of these taxa may be affected by the type of acquisition strategy (LaJeunesse et al. 2010a).

Coral reefs are of crucial importance for the daily livelihood of the human populations living around them, including some of the poorest populations in the world (Fisher et al. 2011). The reefs provide essential ecological goods such as; food production for both local consumption and export, protection of the shoreline from wave erosion and income from recreational activities (Mo- berg and Folke 1999, United Nations Environment Programme 2008). Ten per cent of the fish consumed by humans originate from the reefs (Smith 1978). In 2003 the economic net gain from the coral reefs was estimated to exceed USD 30 billion (Cesar et al. 2003). Additionally soft corals on the reefs produce highly complex compounds with medicinal potential, the scope of which is still to be explored (Carte 1996, Sella and Benayahu 2010).

In recent decades the reefs have changed profoundly due to anthropogenic stressors such as; increased sedimentation, nutrient loading, pollution and destructive fishing (dynamite fishing, cyanide fishing and over-fishing) (Hughes et al. 2003, Pandolfi et al. 2003, Nyström et al. 2008, Burke et al. 2011). Since the mid 1970‘s and 1980‘s climate change and elevated water temperatures have led to local and global coral bleaching followed by mass mortality of reef building corals (Wilkinson 2000, 2004). Negative human impact in combination with increasing and decreasing SSTs (sea surface temperatures) (Hoegh-Guldberg 1999, LaJeunesse 2010a), and increased ocean acidity (Hoegh-Guldberg et al. 2007, Knowlton and Jackson 2008) can make the conditions for the symbionts unsuitable, and they leave their host (mass bleaching; Glynn 1993, Glynn et al. 1996). A consequence of this is high levels of coral mortality with devastating consequences for the organisms living on the reefs (loss of habitats) and for the human populations who depend on them (Moberg and Folke 1999, Nyström et al. 2000). In the late 1990‘s and early 2000‘s following a period of unusually high SSTs, levels of coral bleaching unprecedented in modern times, caused the demise of 30%- 50% of the world‘s coral reefs and alterations to coral communities species compositions (Wilkinson 2000, 2004, Hoegh-Guldberg et al. 2007). During the past 30 years a reduction of live coral cover by 50-95% on a global scale has been estimated (Jackson 2008, Jackson 2010, Burke et al. 2011). These events have also locally reduced soft coral cover by 50% (Bastidas et al. 2004) to 95% (Benayahu 2007) compared to the initial area covered before bleaching events.

Increased environmental stressors make the corals more susceptible to patho- gens (Palmer et al. 2010, Haapakylä et al. 2011), bleaching, and reduce their reproductive output (Michalek-Wagner 2001, Baird and Marshall 2002, Ward et al. 2002). These events have a long lasting effect on the coral reef ecosystems and raise the questions about ecology, biogeography and evolution of the algal-coral symbiotic partnerships.

If bleaching continues to intensify, entire soft coral species may go extinct before they even are discovered (Benayahu 2007) and their pivotal biological, chemical and physical roles in the ecosystems on the coral reefs may not be fully realized before it is too late. The soft coral taxonomy is even more complex and unknown than that of scleractinian corals, and therefore soft coral research has been overshadowed by scleractinian research. In recent years attempts to farm soft corals in order to extract their secondary compounds for the industry (Sella and Benayahu 2010) have underlined the importance of understanding their life history characteristics.

Surveys on coral reef management and population genetics to date have con- centrated on regions in the Caribbean, Hawaii, the Red Sea and the Great Barrier Reef whilst high diversity reefs surrounding poor nations in the cen-

tral Indo-Pacific and Southeast Asia are heavily underrepresented (reviewed by Fisher et al. 2011). The latter areas are heavily populated and face environmental challenges as rapidly growing human populations (depending on the goods from the reefs) and emerging economies put these ecosystems under pressure. Today more than 60% of the coral reefs worldwide are threatened and 50% of these classified as highly threatened, the corresponding rates in South East Asia are 95% and 50% (Burke et al. 2011).

The close association of corals, bacteria, symbiont algae and fungi together form the ‗holobiont‘ (Rohwer et al. 2002, Stat and Gates 2011) which is highly interconnected to processes such as; reproduction of the host (uptake of symbionts determined by brooders or spawners) (LaJeunesse 2005, La- Jeunesse et al. 2010b), predation of corallivorous fish (disperse the symbionts trough excrements) (Porto et al. 2008) ocean currents for dispersal, and environmental factors such as SST‘s and pollution. The importance of understanding the different ecological processes that maintain the coral reef ecosystems is pertinent for efficient conservation efforts of these threatened ecosystems.

Thesis objectives The objective of this thesis is to gain further insight into two important inter- linked aspects of coral reef research crucial for assessing the maintenance and survival of coral populations. The first half of the thesis focuses on sexual and asexual reproduction in a common species of soft coral Sarcophyton elegans on the Great Barrier Reef (Papers II and III) with previously unknown reproductive modes. Reproduction is a key for survival trough recombination, ability to disperse and is also reported to determine the uptake of life supporting symbionts The data collected are based on observations before global coral reef mass mortality events and is therefore a potential baseline study for post bleaching research. The study also suggests a reliable metric of soft corals for repeated measure studies (Paper I). The second part of the thesis focuses on genetic diversity and distribution in a scleractinian coral species, Galaxea fascicularis, and its associated symbionts in relation to environmental factors along a vast latitudinal and environmental range and is the first study of its kind in the South China Sea (Paper IV). The reefs in Vietnam are disintegrating at an alarming rate mainly due to anthropogenic stressors and a rapidly growing population. This thesis highlights the importance of understanding different biological processes such as genetic adaptation to environmental factors and the consequences of modes of sexual and asexual reproduction for dispersal and survival.

Paper I was set out to determine a valid measurement for different growth forms of soft corals, which might be applicable to other erect fleshy soft coral species and other soft bodied invertebrates. Our hypothesis was that the aggregated cemented spicules at the colony base provide more structure to

the colony as the base is closely attached to the substrate and therefore less likely to be affected by water intake and expulsion than other parts of the colonies. The study aimed to find a reliable measure related to biomass and reproductive potential without showing too much temporal fluctuations due to the variations of water intake of the hydroskeleton by:  Measuring variation of different dimensions in soft corals of contrasting morphology to find a stable measure over 24 hours.  Establishing the rate of variation/stability in measures used in earlier studies.  Establishing ecological relevance of measure by correlating the metric to body volume (a proxy for potential fecundity) after water displacement.

The objectives of Papers II-III were to document in detail the sex differentiation, mode and timing of reproduction at different scales in an ecological- ly important soft coral species Sarcophyton elegans by:  Determining the modes sexual reproduction (Paper II) e.g. broadcast spawning or brooding, and asexual reproduction (Paper III) asexual planulae, fission, buddying, stolon formation and other forms of vegetative growth.  Estimating the life history characteristics such as sex ratio, gametogenesis (time and mode of oogenesis and spermatogenesis) and size (measured according to Paper I) at first reproduction (Paper II).  Distinguishing if the prevailing paradigm of one single mass- spawning event characterized the timing of reproduction or not, in monthly surveys over 2.5 years (Paper II).  Comparing the timing of spawning at different spatial scales, from within a single colony to between colonies and between local sizes and identifying possible correlations (Paper II).  Establishing possible sex, size and habitat dependent modes of asexual reproduction (Paper III).  Establishing contribution of asexual and sexual reproduction to one of the study populations by examining genetic differences using allozyme gel electrophoresis (Paper III)

Paper IV tested hypotheses regarding the relationship between several environmental factors and diversity and genetic differentiation among latitudinal- ly separated populations of corals and symbiotic zooxanthellae. The study determined distribution of zooxanthellate ITS2 types within one broadcast spawning coral species, Galaxea fascicularis (Harrison 1988) with horizontal symbiont uptake (Baker et al. 2008) in relation to:  Inshore (anthropogenic stressors) and offshore (less stressed) reef habitats.  Latitude over a 3200 km range of the coast of Vietnam, covering 11 degrees of latitude. 16

 Host characteristics (mtDNA genotype).  Environmental factors (sea surface temperatures and chlorophyll a derived from satellite data, depth and visibility).

Study Species Position in the tree of life: Scleractinia, Alcyonacea and Dinoflagellata The common characteristics for corals, jellyfish, sea anemones and hydro- zoans belonging to the phylum Cnidaria are radial symmetry, possessing nematocysts which are stinging capsules (cnidae) for capturing prey, true tissue (dipoblastic) and lack of a body cavity. The class Anthozoa includes hard and soft corals, sea pens, sea anemones and zoanthids and possesses tentacles in the adult form. Hard or scleractinian corals belong to the subclass hexacorallia (they possess a multiple of 6 tentacles at the oral end of the polyps) and represent approximately 800-1000 species (Veron 2000), and soft corals belong to the subclass octocorallia (they possess 8 tentacles at the oral end of the polyps) with more than 3000 species divided between three main orders; Pennatulacea (sea pens), Helioporacea (blue corals) and Alcyonacea (Daly et al. 2007).

Many of the tropical hard and soft corals live in symbiosis with dinoflagellates (genus Symbiodinium) called zooxanthellae. The zooxanthellae types within the gastrodermal cells of the animal host are photosynthetic. The vast majority (approx.95%) of the assimilated organic carbon (‗photosynthate‘) is typically translocated to the coral, contributing substantially to its carbon and energy needs (Trench 1993, Yellowlees et al.2008). In return the symbionts receive protection from external predators and receive host derived sub- + strates, principally carbon dioxide (CO2(aq)) and ammonium (NH4 ) (Trench 1993, Yellowlees et al.2008). The organic carbon from the algae enables the host to form large CaCO3, deposits. This relationship is the basis for the coral reefs which are the largest structures on earth constructed by a living organism.

Soft corals do not form large CaCO3 structures like scleractinian corals but form species specific spicules instead. This is due to differences in the three dimensional structure of the CaCO3 molecules in the two subclasses.

Dinoflagellates belong to the Kingdom Protozoa, parvkingdom Alveolata and phylum Dinoflagellata (Cavalier-Smith 1993). About half of all aquatic prot- ists in the phylum Dinoflagellata are photosynthetic, making up the largest group of eukaryotic algae aside from the diatoms (Lin 2011). The species of the Symbiodinium genera are both free-living and symbiotic, and cornerstones of the coral-algae-bacteria holobiont complex underlying the possibilities for reef building corals to exist (Patten et al. 2008, Stat and Gates 2011).

Sarcophyton elegans The fleshy soft corals of the genus Sarcophyton belong to the order Alcyo- nacea and are widespread on tropical reefs worldwide. Sarcophyton elegans is common in shallow lagoons and reef flats throughout the GBR, often ap- pearing in large monospecific aggregations (Papers I-III). Adults and juve- niles of S. elegans are relatively easy to distinguish from other Sarcophyton species by morphology and by examining the microscopic sclerites, which are species specific (Fabricius and Alderslade 2001). The colonies are mushroom shaped, and the disc-like polyp-bearing region (the polypary) has two different kinds of polyps; large autozooids that bear tentacles and smaller, more numerous siphonozooids lacking obvious tentacles (Fabricius and Alderslade 2001). Prior to this study the reproductive mode of this zooxanthellate soft coral was unknown.

Galaxea fascicularis The colonial scleractinian coral G. fascicularis is common on varying reef habitats and shows a wide distribution range. The species does not exist in the Caribbean but has been encountered on reefs everywhere else in the world (Veron 2000). The species has a reproductive mode called pseudo- gynodioecy: an unusual breeding system where the coral is gonochoric (i.e. separate male and female colonies) and hermaphroditic simultaneously. The female colonies produce viable egg cells in a yearly cycle; however, the males produce normal sperm cells and sterile pseudo-eggs within the same colony (Harrison 1988). The species is a broadcast spawner and during gamete release the pseudo-eggs in the hermaphroditic colonies function as floaters to push the sperm to the surface for fertilization with the viable egg cells from female colonies (Harrison 1988). The species is hermatypic and is reported to be resilient to bleaching (Yamazato 1999, Marshall and Baird, 2000, Huoang et al. 2011) and sedimentation (Philipp and Fabricius 2003). Due to its wide latitudinal distribution and presence in both disturbed and pristine habitats the species is ideal for wider population genetic studies.

Symbiodinium sp. When first discovered in the 1970‘s symbiotic zooxanthellae were thought to be only one, pandemic species named Symbiodinium microadriaticium (pre- sently classified as A1). Today, because of the advancements in molecular biology, 9 groups (A-I) of Symbiodinium have been identified as distinct lineages earlier called clades (based on Rowan and Powers 1991a, 1991b, LaJeunesse et al. 2003, 2004, Sampayo et al. 2007, Stat et al. 2009, Pochon and Gates 2010). Of these; A-D, F and G are known to inhabit corals (Baker 2003). The different endemic Symbiodinium lineages can be delineated to types (subclades, genetically designated with ITS1 and ITS2 rDNA, of which close to 100 have been discovered) and are in many cases distinct species; possibly even distinct on a higher taxonomic level (LaJeunesse 2001, 2005, Coffroth and Santos 2005).

It is generally perceived that certain Symbiodinium types (based on the ITS2 level) are ‗host generalists‘ associating with a wide range of hosts, whereas others are ‗host specialists‘ associating with only certain host species or genera (Sampayo et al. 2007, LaJeunesse et al. 2010a,Wicks et al. 2011). Some types are even shown to associate to different genotypes (based on the puta- tive control region) within one host (Bongaerts et al. 2010).The tolerance of corals to environmental stressors are partly attributed to the different symbiont groups with group D being more thermally tolerant than group C (e.g. Rowan and Knowlton 1995, Baker 2001, Berkelmans and vanOppen 2006). However, an increasing number of studies based on ITS2 types suggests varying function of types within the A- F or G group, and provide the possi- bility to further investigate coral symbiont interactions (i.e. LaJeunesse 2003, Sampayo et al. 2007, 2008, Frade et al. 2008, La Jeunesse et al. 2010b, LaJeunesse 2011).

Given the importance of the algal symbiosis to coral survival the number of symbiont types and the patterns of their occurrence among marine hosts are highly relevant to understanding the ability of the holobiont (coral-host- bacteria-fungi complex) to adapt to environmental change (Buddemeier and Fautin 1993). To date the majority of surveys determining the distribution of Symbiodinium in coral hosts have been based on an inclusion of very few colonies per species (1-5) in large multispecies surveys over a variety of geographic scales (e.g. LaJeunesse et al. 2010a, Wicks et al. 2010), or on one species of host within a limited geographic region (Bongaerts et al. 2010, Stat et al. 2011). Only a few studies have looked at within-species diversity of symbionts across broad geographic regions (Howells et al. 2011 (soft coral), Pinzon and LaJeunesse 2011).

Reproduction in Soft Corals Sexual reproduction: The mode and timing of reproduction are key life history characteristics influencing the population dynamics, ecology and evolution of organisms (Stearns 1992), but major characteristics of reproduction remain unknown for many marine species. For example, whilst most hard corals are hermaphroditic(male and females in the same colony) (Carlon 1999), it has only been established relatively recently that soft corals are primarily gonochoric (having separate males and females), as reviewed in Benayahu (1997) and in Hwang and Song (2007). Timing of reproduction is also variable among corals, but one general observation from the limited data available is that soft corals in temperate waters tend to show continuous gametogenesis, internal fertilization and brooding of developing embryos (Gohar 1940, Hartnoll 1975, Farrant 1985, Cordes et al. 2001, McFadden et al. 2001), however, recent studies in temperate environments have shown that reproduction in deep sea temperate corals is more complex (Mercier and Hamel 2011). In

contrast, tropical soft corals generally exhibit short seasonal synchronous spawning periods with external fertilization (Benayahu and Loya 1984a, Alino and Coll 1989, Benayahu et al. 1990, Benayahu 1997, Slattery et al. 1999).

Soft corals (Cnidaria: Alcyonacea) have this far shown three different modes of sexual reproduction including internal brooding (Achituv and Benayahu 1990), external surface brooding of planulae larvae (Dinesen 1985, Benaya- hu et al. 1990) and broadcast spawning of gametes with fertilization taking place in the water column (e.g. Alino and Coll 1989, Babcock 1990, Be- nayahu 1997). The mode of reproduction and length of breeding season va- ries both within genus (Hartnoll 1975, Dahan and Benayahu 1997, Hwang and Song 2007) and species (Benayahu and Loya 1986, Schleyer et al. 2004) depending on the geographical location of the corals.

Asexual reproduction Soft corals are reported to possess a large range of mechanisms of clonal propagation such as budding of daughter colonies in Sarcophyton gemmatum (Verseveldt and Benayahu 1978); fragmentation in Junicella fragilis (Walk- er and Bull 1983); colony fission in Capnella gaboenis (Farrant 1987), Xenia macrospiculata (Benayahu and Loya 1985), Alcyonim digitatum (McFadden 1986), and Sinularia flexibilis (Bastidas et al. 2004); formation of stolons in Efflatuonaria sp. (Dinesen 1985, Karlson et al. 1996), rapid autotomy of small fragments with root-like processes in Dendronephthya hemprichi (Da- han and Benayahu 1997) and production of asexual planulae (Fautin 2002 review). The ecological and biological circumstances in which a particular mode of asexual reproduction is favored are not well understood for any coral.

Asexual propagation is considered to be the most dominant mode of reproduction in soft corals (Verseveldt and Benayahu 1978, Fabricius 1997, Fa- bricius and Alderslade 2001). However, three recent studies showed that sexual reproduction may play a significant role in the population dynamics in soft coral species including A. digitatum (McFadden 1997), Sinularia flexibilis (Bastidas et al. 2001) and Clavularia koellikeri (Bastidas et al. 2002). Even so, the two latter studies concluded that asexual recruitment played a more important role for the population than sexual recruitment.

The variety of modes of reproduction revealed by a relatively small set of studies, usually limited in spatial and temporal extent, suggests that more detailed research is required to better document the reproductive strategies of soft coral species. However, comprehensive long term datasets on timing and mode of coral reproduction are extremely rare (Bastidas et al. 2002, Fuchs et al. 2006).

Materials and methods

Study Areas Australia The studies for this thesis were conducted in different parts of the Central Indo-Pacific. The soft coral studies (papers I-III) took place in the vicinity of Lizard island in the Northern Great Barrier Reef (14°40‘S, 145°28‘E): Site A was on shallow Loomis Reef at the western entrance of the Lizard Island lagoon. Site B was on a small wave-exposed patch reef between South Island and Palfrey Island of the Lizard Island group (Fig. 1b). The water depth at both sites ranged from 3 m at high tide to total exposure during maximum low tide in the austral spring. All field observations and collections were performed by snorkeling or SCUBA. Additionally for measurement studies in Paper I, specimens of the a zooxanthellate soft coral Dendronephthya sp. were collected from the deep wave exposed northern side of the island.

Vietnam Corals were collected in March 2009, August 2009 and in April 2010 from 11 sites using SCUBA and snorkel from 4 regions (North, North Central, Central and Southern) in Vietnam (Fig. 1a, Table1). Sample locations ranged over several degrees of latitude (09°55N-20°45N) and a maximum distance of 3200 km, and encompassed major gradients in seasonal temperatures and coral species number (Table 1). The distances from the sites to the mainland were recorded (km), and included sites with reduced visibility, lower light levels, and higher sediment load (usually nearer shore) and those with greater visibility, higher light levels and reduced sediment load (usually further offshore). The inshore sites were situated at 50 to 800 meters from the mainland whilst the offshore sites were situated 8-14 km from the shoreline.

Figure1. Map of study sites in the Indo-Pacific. The sites in (a) Vietnam (inserted enlarge- ment) are denoted 1-11 and described in detail in Table 1. The sites on (b) Lizard Island, Great Barrier Reef, Australia are denoted A (Lagoon site); and B (Exposed site, close to South Island). (Main map: Google Earth™ mapping service, insert of Lizard Island taken by Barry Goldman Australian Museum)

Oceanographic data Data for monthly Chl a (chlorophyll a, mg m-3) (a proxy for turbidity) and SSTs (sea surface temperatures ºC) measured between July 2002 and De- cember 2010 were retrieved from the Giovanni online data system, which is maintained by the NASA Goddard Earth Sciences Data and Information Services Center. SeaWiFS, NOAA/AVHRR measurements were averaged over 9 km2 from areas close to the sampling sites (Fig. 2). Local SSTs and additionally water temperatures at 5 and 15 m, and visibility data (kindly provided by local dive operators in Nha Trang, Phu Quoq and Hoi An and by IO and IMER divers on the other sites) were used to confirm the satellite- derived SSTs and Chl a data respectively. This local verification was useful because Chl a satellite data taken from near shore with pixels falling within a land-water interface are more unreliable than data taken offshore (Dien and Hai 2006, Maynard et al. 2008).

Figure 2. Average monthly values (±S.D.) between 2002 and 2010 of SSTs (a) and chlorophyll a concentrations (proxy for turbidity) for the locations based on satellite imaging. In- shore and offshore locations in Cat Ba and Nha Trang showed almost identical SST curves and therefore only one curve each present the SST‘s in Cat Ba and Nha Trang. I and O in panel b) denote Inshore and Offshore respectively.

Table 1. Background information on study sites including, region (North-South), number of scleractinian corals per region (Veron et al. 2009), GPS coordinates, distance to mainland (ML) in kilometers (km), inshore-offshore (IS, OS), maximum site depth, average (avg.) sea surface temperature (SST) (average from July 2002 to Dec 2010), average yearly SST range (monthly average SST‘s averaged over 2002 – 2010), avg Chlorophyll a (Chl a)(average from July 2002 to Dec 2010), average yearly Chl a range (monthly average Chl a averaged over 2002 – 2010) and avg. visibility range (based on daily data by dive operators averaged over 2005-2009) Chla # ML Depth SST Chla Vis SST range Region Coral Site Coordinates dist. max range (mg/ range (°C) (mg/ sp. (km) (m) (°C) m-3) (m) m-3) 1 .Cat 20°45'N, 0.84- North 182 0.8 2.8 25.5 17-32 2.4 0.5-4 Ba IS I 107°04'E 5.94 2. Cat 20°47'N, 0.84- Ba IS 0.1 2.2 25.5 17-32 2.4 0.5-4 107°06'E 5.94 II 3. Cat 20°37'N, 0.45- 1.5- Ba OS 14.2 6.7 25.5 17-32 1.6 107°09'E 3.00 10 I 4. Cat 20°37'N, 0.45- 1.5- Ba 13 2.8 25.7 17-32 1.6 107°08'E 3.00 10 OSII North 16°14'N, 0.65- 1.5- 334 5. Hue 0.3 6 27.2 18-32 2.4 Central 108°12'E 7.28 10 6.Hoi 15°56'N, 0.21- 1.0- 12 12 27.1 20-32 1.2 An 108°29'E 6.23 30 7. Nha 12°12'N, 0.05- Central 397 Trang 0.04 4.2 27.8 24-30 0.5 0.5-4 109°13'E 1.96 IS I 8. Nha 12°10'N, 0.05- 1.5- Trang 0.01 4 27.8 24-30 0.5 109°12'E 1.96 15 IS II 9. Nha 12°05'N, 0.16- 4.0- Trang 8.3 8.6 27.6 24-31 0.5 109°18'E 2.47 30 OSI 10.Nha 12°04'N, 0.16- 4.0- Trang 8.3 10 27.6 24-31 0.5 109°18'E 2.47 30 OS II 11.Phu 09°55'N, 0.42- 2.0- South 404 11 12 29.4 27-32 1.5 Quoc 103°59'E 3.30 15

Methods Paper I: Colony Measurements To test the hypotheses that the basal circumference is a more precise measure of colony size we used three different morphologies of soft corals represented by; Sinularia flexibilis (branching fleshy growth form with a very calcified base), Sarcophyton elegans (erect mushroom shaped growth form, with a relatively calcified base) and Dendronephthya sp. (erect growth form, with a mainly water filled interior). Ten individual colonies of each species at depths greater than 5 m were measured eight times over 24 hr providing coverage of two tidal cycles, and one diurnal period.

Colony height, oral disc diameter and basal stalk circumference (Fig. 2) were measured to the nearest cm with a measurement tape after the colonies were touched firmly to cause colony contraction (as described in Fabricius 1995).

In December 1993, a second set of observations aimed to determine which field measure of colony size correlated best with colony volume (after water 24

displacement in formalin). The best predictor of colony volume was examined using linear regression.

Figure 3. Illustration of the measurements of the colony size made for (a) Sarcophyton elegans, (b) Sinularia flexibilis and (c).Dendronephthya sp.

Methods Paper II: Sexual Reproduction in S. elegans Colony sexual identity, sex ratio, and size at onset of sexual reproduction At each study site (Fig. 1b), three 1 x 10 m belt-transects were laid down end to end on the reef. In December 1991 a detailed map was drawn and included sizes of all the colonies within the transects (Paper I) to determine colony size distribution.

In October 1991, several weeks before the predicted coral annual mass spawning (e.g., Harrison and Wallace 1990), the sex of the colonies were determined by making a small incision in the polypary which exposed the mesenteries containing the visible gonads (Alino and Coll 1989) The basal circumferences were measured according to Paper I. The collection was biased towards smaller colonies in order to detect the smallest size at maturity. Histological samples in the smaller colonies were processed as described below in order to confirm the absence of gametes in the colonies that ap- peared to be immature.

Gametogenic cycle To determine the length of the gametogenic cycle, 10 large male colonies and 10 large female colonies (> 20 cm in stalk circumference) outside the transects at sites A and B (Figure 1) were numbered with plastic markers .The marked colonies were sampled approximately monthly between Octo- ber 1991 and January 1994, by taking a small biopsy as outlined by Benaya- hu and Loya (1986). All samples were preserved in 10% formalin in seawater (v/v) for three days and then transferred to 70% ethanol for further analysis. The samples were stained with eosin and haematoxylin according to Winsor (1984). Diameters of the oocytes and spermaries were measured with a micrometer under a compound microscope. Fresh samples were examined under a stereomicroscope to establish the color difference between mature 25

and immature gonads. The gametogenic cycle was determined according to descriptions by Glynn et al. (1991) and Schleyer et al. (2004).

Temporal spawning dynamics The positions of the colonies along the belt transects were recorded and their reproductive state was noted monthly from October 1991 to January 1994. The colonies were monitored every two weeks for spawning activity after both the full moon and new moon, between August and March 1992 and 1993 and in January 1994 as higher reproductive activity occurred in those months. To obtain information on the spatial distribution of immature, mature and spawned mesenteries in individual polyparies, incisions were made at several points along the radius of the polypary to expose the mesenteries. A colony was recorded to have released its gametes when ripe gametes were recorded as present during a census and absent at the next sampling event. Colonies were observed releasing eggs and sperm on three separate dates, and the detailed times of gamete release within the transects were recorded at site A only, due to logistic reasons.

Methods Paper III: Asexual Reproduction in S. elegans Modes of asexual reproduction The study took place at the same sites as Paper II (Fig. 1b). All field observations and collections were performed by snorkeling or SCUBA. Prior to the annual mass spawning event in November 1991, the sex and size of the Sarcophyton elegans colonies were determined as outlined in Papers I and II. Detailed maps of the study sites with the size, sex and position of each colony were drawn for each transect, and the mode of asexual reproduction in each colony was recorded during each observation period. The transects were monitored on a quarterly basis between August 1991 and February 1994.

Genetic analyses Potential ramets (physically separated colonies of the same genotype) and genets (genetically different colonies) were identified in the field and the spawning groups (Paper II) by using used polymorphic allozyme markers.

The samples from 322 colonies were collected in the field and immediately transferred to liquid nitrogen. The samples were processed according to standard protocols. An initial screening of 32 enzymes using a variety of electrophoretic conditions determined that there were two reliably-scorable polymorphic loci; LGG and GPI.

Methods Paper IV: Genetic distribution of G. fascicularis and its associated symbionts Sample collection and DNA extractions Corals were collected in March 2009, August 2009 and in April 2010 from 11 sites using SCUBA and snorkel from 4 regions (North, North Central, Central and Southern) in Vietnam (Fig. 1a, Table1, Fig 2). One individual polyp was collected from each colony and stored in 95% ethanol until further DNA analysis.

DNAs for both coral and plant tissue were extracted simultaneously from the same tissue sample using the DNeasy Blood & Tissue Kit (Qiagen, Santa Clarita, Calif).

Genotyping The coral host was genotyped using a fragment of a non-coding intergenic region between cyt b and ND2 in the mitochondrial DNA. The Polymerase Chain Reaction (PCR) was conducted using the primers 188-2-F and 188-1- R as outlined in Watanabe et al. (2003) with an annealing temperature of 54°C.

The symbionts were genotyped by amplifying the Symbiodinium ITS2 region using the primers ITSintfor2 and ITS2no-clamp (LaJeunesse & Trench2000). PCR was amplified using a TD (Touch Down) protocol modi- fied after LaJeunesse & Trench (2000) and Porto (2008).

The PCR products were sequenced by direct sequencing in both directions using the reverse sequence as a reference for G. fascicularis and the forward sequence for the Symbiodinium to avoid unambiguities.

Statistical analysis

All the analyses were performed in R (version 2.10.1, R Foundation for Sta- tistical Computing) if not stated otherwise.

Paper I: Variation of measures over 24 hours were analyzed using the coefficient of variation. Paired student‘s t-test was used to compare the CV between the measures. Linear regression analysis was used to analyze correlation between colony volume and the different measures.

Paper II: Sex ratio was tested for any deviation from 1:1 using the Chi-square test.

Paper III: To determine the effects of reef exposure, mode of asexual reproduction, and sex of the colonies on size at time of budding or fission, we used a General Liner Model (GLM) with colony size versus sex (male or female), site (lagoon or exposed), replicate (transect 1, 2 or 3 within each site), signs of colony fission (present or absent) and production of buds (present or absent) as variables.

Allele frequencies and expected genotype frequencies were calculated in BIOSYS1. The probability of getting the observed genotype frequencies under the null assumption of Hardy-Weinberg equilibrium was calculated using a chi-squared statistic (Maddox et al. 1989).

Paper IV: Genotyping The chromatograms were aligned by hand using MEGA5 (Tamura et al 2011) The sequences were identified using BLAST search and aligned to the one existing published reference (AB109376) on GenBank, the other reference genotypes are described in Watanabe et al. (2003). The procedure for the Symbiodinium genotypes was as outlined above for the host; however the alignments were verified by sequences based on LaJeunesse et al. (2003, 2009). The different haplotypes were designated by DAMBE, thereafter for the host; analyses of diversity were performed using Maximum Parsimony (MP) analysis in MEGA5.0 (Tamura et al. 2011) under the delayed transition

Coupling between host and symbíont genotype Association between the Symbiodinium ITS2 and G. fascicularis mtDNA genotypes was tested by Pearson's Chi-square. Additionally we used SPSS Answer tree's Exhaustive CHAID (CHi-squared Automatic Interaction De- tector) and C&RT tree modules to detect potential couplings between Sym- biodinium and host genotypes.

Diversity indices ‗True diversity‘ (D) denotes the effective number (Jost 2007) of elements which is the effective number of ITS2 sequences. It is calculated by using the Shannon and Weaver diversity index, H‘ as follows: D = exp(H‘) s H‘ = - Σ i = 1 pi lnpi Where p is either the proportion of ITS2 sequences i out of s sequences in the sample for Symbiodinium, or the proportion of mtDNA sequences i out of s sequences in the sample for the host. Comparisons of D among the 11 different sites were analysed using analysis of variance (ANOVA) with Fisher‘s LSD post hoc testing of population pairwise differences.

Environmental parametes The environmental variables at each site analysed in the study were Chl a (mean, min, max and range), SST (mean, min, max and range), visibility (mean, min, max and range from local dive measurements), site depth (mean, min, max and range), number of coral species in the region (based on Veron et al. 2009) and latitude (Y). The variables were checked for normal- ity by Kolmogorov-Smirnov test, and were log-transformed when needed to obtain a normal distribution. Relationships between the variables were assessed using a principal component analysis (PCA) in R (version 2.10.1, R Foundation for Statistical Computing). PCA results were summarized in a bi-plot containing the distribution of environmental parameters in two- dimensional space, and their correlations with the PCA axes. For each variable the measure with the highest eigenvalue (i.e. Chl a choosing Chl a max etc.) was chosen for further analysis.

The effect of the environmental variables on the presence/absence of different ITS2 types (with n > 30) was also tested separately by using a general- ized linear model (GLM). As the ITS2 type data were binary (presence/absence), a binomial regression was applied to the presence/absence data in the construction dataset. All linear and quadratic terms were included as potential predictors in the building of the model. Co-variance between each variable was assessed using pair plots and only variables with co- variance > 0.8 were considered for the GLM. In order to select the model that explained the most variation using the fewest number of variables, a ‗backwards stepwise‘ procedure was used (R software version 2.10.1, R Foundation for Statistical Computing). The statistic used to select the final

linear model was the Akaike‘s Information Criterion (AIC—Chambers and Hastie, 1997).

Major findings – summary of papers

The most reliable measure of S. elegans and the two other soft coral species described in Paper I is basal stalk circumference. The measure correlated strongly with volume after water displacement and additionally showed the smallest variation of change over 24 hours (Figs. 4 and 5).

Figure 4. Histograms showing mean coefficient of variation (CV) and minimum and maximum CV (bars) over 24 hours in Sarcophyton elegans (a), Sinularia flexibilis (b) and Den- dronephthya sp. (c). White bars denote basal circumference, dark grey bars colony height and light grey bars oral disc diameter.

Colony size is rather than age (Hughes and Jackson 1985) a key metric in coral life history studies and an important predictor of growth and reproductive potential. The size structure of a population can be used to estimate rates of recruitment and size specific mortality (e.g. Harvell and Grosberg 1988; Babcock 1990; McFadden 1997; Gutierrez-Rodrıguez and Lasker 2004). Previous work has demonstrated that soft coral size estimates vary over a period of days (Cordes et al. 2001) or weeks (Fabricius 1995), whilst Paper I demonstrates that soft coral size estimates vary considerably within a 24 h period. The difficulty in obtaining a reliable measure is acknowledged among soft coral scientist (Fabricius and Alderslade 2001, Cordes et al. 2001). The consequences of an imprecise metric can give inaccuracies in growth, shrinkage and size frequency distribution data. The results from Paper I suggests it may be useful to establish a metric for colony size prior to long term studies on life histories and dynamics in soft corals.

Figure 5. Linear regressions showing the relationship between the linear size measurements (on the x-axis) of basal stalk circumference (filled diamond, solid line), colony height (open diamond, dashed line), and oral disc diameter (open circle, dotted line), with colony volume after water displacement by formalin (y-axis). Regression equations and significance of the relationships are illustrated for (a) Sarcophyton elegans (R2 = 0.75, p < 0.0001; R2 = 0.74, p< 0.0001 and R2 = 0.48. p = 0.01) for basal stalk circumference, disc diameter and colony height respectively), (b) Sinularia flexibilis (R2 = 0.51, p =0.01 and R2 = 0.42, p = 0.02 for basal stalk circumference and colony height respectively) and (c) Dendronephthya sp. (R2 = 0.87, p=0.0002 and R2 = 0.34, p = 0.66 for basal stalk circumference and colony height).

Paper II revealed that S. elegans is a typical tropical alcyonarian coral as it is a gonochoric broadcast spawner with a sex ratio of 1:1. Sexual reproduction was strictly controlled by colony size with first reproduction at 130 mm basal stalk circumference for females and 120 mm for males. Oogenesis takes 19-24 months, with a new cycle commencing every year (Figs 6a, 7a). Figure 6a shows the different simultaneous development stages during oogenesis. Spermatogenesis takes 10-12 months (Fig 6b, 7b).

The majority of gametes are released during the annual austral mass spawning event after full moon in November, but gametes are also released each month after the full moon between August and January. This unusual pattern of gamete release was achieved by a novel mechanism of gamete production, reported here for the first time. All autozooid polyps participate in reproduction, but those at the outer edge of the colonies release the gametes first and during subsequent months the polyps situated closer to the center of the colonies release their gametes. This strategy represents a new manner in which to accommodate the demands of feeding and reproduction, in contrast to separation of roles for individual polyps observed for other corals.

Figure 6. Monthly measurements of diameters (µm) of, upper panel, oocytes (n = 520) and; lower panel, spermaries (n = 460) of Sarcophyton elegans colonies between October 1991 and February 1994. Months start at October 1991 through January 1994. No data were collected in May 1992 and July 1993 due to bad weather.

Colonies upstream in the prevailing current initiated and ceased spawning, up to one month earlier than those further downstream. S. elegans appears to have a strategy that allows for the protection of releasing gametes during mass spawning of a number of species, but to hedge bets by spawning over an extended period to avoid loss of investment in gametes in case of cata- strophic events (storms etc.) The results also gave some new insights to the spectacular annual multi species mass spawning event taking place on the Great Barrier Reef in Australia, and indicated that the fecundity concept for future studies needs to be revisited.

Figure 7. a) Oogenic development of Sarcophyton elegans. c coenenchyme, f follicular layer (under the coenenchyme), oI stage I primordial oocyte, oII stage II oocyte, oIII stage III oocyte, oIV stage IV mature oocyte, p pedicle, pc polyp cavity. Stages as in Glynn et al. (1991) and Schleyer et al. (2004). Scale bars = 500 µm. b) Spermatogenic development A: Sperma- togonia and spermatocysts B: Spermatocyst attached with pedicle C: Spermaries with spermatids. p pedicle, sI stage I spermatogonia, sII stage II spermaries, sIII stage III spermaries with spermatids. Scale bars = 250 µm

During the study we also observed that some scelractinian hard corals of the genus Acropora released their gametes from the edge of the polyps during the full moon in the months leading up to the annual mass spawning event. That means that previous calculations of fecundity in corals may be under- estimated because most fecundity studies have taken place just before the annual mass spawning event and concluded that some coral species exhibit polyps in sterile zones, while others do not. Our results therefore may indicate that coral species including Acropora species may not exhibit a sterile zone at the edge of the colonies (Hall and Hughes 1996).

A more detailed study into the mechanisms underlying ‗split-spawning‘ would be of interest as hormonal cues (Atkinson and Atkinson 1992, Slattery 1995, Tarrant 2005) may regulate the timing of spawning and therefore control the repeated spawning patterns. In a larger context it is known that the cues to spawning are regulated by the full moon, water temperature, solar irradiation and recently Levy et al. (2007) found that the expression of the Cry-2 gene is activated by the faint blue light of the full moon. Even this study was conducted close to the main annual mass spawning event, but the gene may as well be activated during other months close to the full moon.

Paper III showed that the mode of asexual reproduction in S. elegans, were budding and fission (Fig 8). Budding was fairly rare, with only 8% of colo- 34

nies producing buds over the study period. Fission was commonly used in these populations, with 38% of colonies observed splitting and was strictly size dependent with larger colonies dividing into smaller more fecund size classes. Fission took more than 30 months to complete and the complete process was beyond the time scale of this study, buddying took 3-6 months to complete.

Figure 8. Sarcophyton elegans. Modes of asexual reproduction observed in colonies. (a) Binary fission starting from the polypary, eventually producing two moderately sized colonies. (b) Binary fission starting from the base of the stalk. (c) Buddying sequence.

The genetic study revealed thirteen (possibly up to 22 if no sex change among colonies is assumed) genotypes in the 30 m2 area studied (Fig 10), which complemented the pattern of clonal expansion seen within individual sex-specific spawning groups having genetically identical colonies.

Papers I-III are based on data collected prior to the escalating global bleaching events that altered many coral reefs on a global scale (Wilkinson 2000, 2004), and therefore the two papers in this study may function as a reference study for future post-bleaching studies. The discoveries in the thesis showed that soft corals exhibit an even larger spectrum of reproductive traits than described before in the order (Paper II).

Figure 9. Genotypes (a) and sex (b) of single-sex colony-groups of Sarcophyton elegans on site A. Each circle corresponds to a spawning group (described in Paper II)

In Paper IV the study sites were visited both in 2009 and 2010 to obtain efficient sample sizes. The differences between sites 1 and 2 in 2009 and 36

2010 were substantial. Site 1 was subjected to dynamite fishing in 2010 and no live corals remained. In 2009 Site 2 was dominated by G. fascicularis (40% of coral cover) and approximately 40 other scleractinian coral species (Ngai et al unpublished data). In 2010 the site was altered to large sand banks covered with plastic baskets used for clam culture, with hardly any corals remaining.

The mtDNA sequences in G. fascicularis revealed six new haplotypes which were closely related to those obtained from Japan (Fig 10). The majority of haplotypes represented the S01 type which was even more dominant on inshore than on offshore reefs (Fig 11). The samples from the inshore reefs from site 1 and 2 fell apart and their skeletons dissolved quickly in alcohol, whereas the samples from other sites remained intact. This may be an effect of heavy pollutants - such as arsenic, dioxins, increasing sediment loads and reportedly untreated acidic waste waters in the nearby Sông Hồng river (Red River) (Duong et al. 2008, 2010, Faxneld 2011, Winkel et al. 2011) - on coral calcification.

LA ORIGINAL

30 LD cytb+ND2 LE cytb+ND2 23 L01 25 L02 58 LC cytb+ND2

32 LB cytb+ND2 SB cytb+ND2(7) 23 SC cytb+ND2(8) SA ORIGIN cytb+ND2(6) S01 S02

72 S03 87 S04

0.001 Figure 10. Molecular phylogenetic analysis of host mtDNA haplotypes using the Neighbor- Joining method (MEGA 5.0) Percentages of replicate trees where associated taxa clustered together in the bootstrap test are shown (500 replicates). Evolutionary distances were com- puted by Jukes-Cantor method using number of substitutions per site as units. The analysis involved 6 nucleotide sequences from Vietnam (S01-S04, L01 and L02) and 8 from Japan (SA, SB, LA-LE from Watanabe et al. 2005). All ambiguous positions were removed for each sequence pair. There were a total of 696 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.0. (Tamura et al. 2011).

Figure 11. Observed geographic distribution patterns of Galaxea fascicularis mtDNA intergenic genotypes across regions and sites along the coast of Vietnam. Pie charts are scaled by area to match sample numbers (1) Cat Ba Inshore I, Ba Trai Dao (n = 5); (2) Cat Ba Inshore II, Van Boi (n= 12); (3) Cat Ba Offshore I,Vung Cao Bai (n =16); (4) Cat Ba OffshoreII,Vung Tau (n = 12); (5) Hue (n =24); (6) Hoi An (n =12); (7) Nha Trang IS I, Nha Trang Port (n = 20); (8) Nha Trang ISII, Diamond Bay, (n = 13); (9) Nha Trang OSI, Mooray-Rainbow (n = 20); (10) Nha Trang OSII, Lan Beach-Mama Hahn (n = 17); (11) Phu Quoc (n = 22).

Rare alleles were restricted to one or two populations, but individual haplotypes did not appear to have any geographical pattern at regional scales (Figure 11) and no significant genetic structure was found on a regional scale (FST=-0.044, p=0.91, AMOVA). On the other hand, there was a significant genetic structure differentiating inshore and offshore sites (FST=0.093, p=0.021, AMOVA).

The diversity indices of the hosts showed a strong inshore offshore distribution (Fig 12). The diversity indices of the symbionts showed a strong latitudinal distribution with a tendency of higher diversity further South (Fig 14). There was no coupling between host and symbiont genotypes (Pearson's chi- square test; χ2 = 1.72, p = 0.19) (No associations detected with Answer tree) and no association between host diversity and latitude (2-way ANOVA; p >0.40). 38

Figure 12. Genetic diversity of G. fascicularis based on mtDNA (non-coding intergenic region between cyt b and ND2) at inshore and offshore sites along the coastline of Vietnam. Genetic diversity measured by Shannon Weaver‘s true diversity index (‗D‘) averaged (±S.D.) across sites.

The ITS2 genotypes of the Symbiodinium sequences (Paper IV) belonged to group D (n = 142) and group C (n = 132) which has been reported from China and Spratley Islands (Dong et al. 2009, Huang 2011). The previous studies in China are based on group level only and do not cover the area in Vietnam. There were strong differences in the latitudinal distribution of the Symbiodinium ITS2 types (Fig. 13). Group D was present in all regions dis- playing only one ITS2 type; D1a and was found consistently on inshore sites. Group C was divided into five ITS2 genotypes; C1 (S. goreaui), C3, C3u, C21, and C27 (La Jeunesse et al. 2003, 2009). Group C was represented by one ITS2 types in the north region (C1), two types in the North Central regions (type C3u in Hue and type C1 in Hoi An), all six types in the Central Region (Nha Trang) and three types in the South region (types C1, C3, C3u).

The types changed dominance from C1 in the north to C3u in the south. C1 one is reportedly tolerant to temperature fluctuations and regarded as a resilient Symbiont type. The C1 presence may be an effect of a more fluctuating temperature regimes and higher turbidity and sedimentation in the north compared to the South Central and South Regions. However the division may also be a result of the different monsoons in northern and southern Vietnam with alternating currents resulting in a cut off at the Hue-Hoi An region, causing a dispersal barrier between symbionts. Further research using microsatellite markers may be needed to evaluate the barrier hypothesis.

Figure 13. Observed geographic distribution patterns of Symbiodinium types (ITS-2) across regions and sites along the coast of Vietnam. (details in fig 11 and table 1). Pie charts are scaled by area to match sample numbers (1) n = 23, (2) n = 20, (3) n = 27, (4) n = 21, (5) n = 21, (6) n = 23, (7) n = 27, (8) n = 23, (9) n = 21 (10) n = 13, (11) Phu Quoc n = 21.

6,00 5,00 4,00 3,00 2,00

Diveristy index D index Diveristy 1,00 0,00 North North North North Hue Hoi Nha Nha Nha Nha Phu IS1 IS2 OS1 OS2 An Trang Trang Trang Trang Quoc IS 1 IS2 OS2 OS1

Sites

Figure 14. Genetic diversity of Symbiodinium (ITS-2) types hosted by G. fascicularis in the different sites ranging from north to south along the coastline of Vietnam. Genetic diversity measured by Shannon Weaver‘s true diversity index (‗D‘) across sites in the regions. Site information is provided in full in Table 1.

Reefs closer to the shore are likely to undergo more stress caused by human activities as pollution, eutrophication and siltation as well as natural stress such as freshwater runoff (Latypov 2005). This, in turn, can cause differences in the zooxanthellae composition in the host. The findings in the present study confirm the role of D1a and C1, where D1a took over dominance in extremely stressed environments seemingly unsuitable for other symbionts. Corals with horizontal uptake are thought to be more flexible in their ex- change of symbionts than brooding corals with vertical uptake (Baker et al. 2008), therefore horizontal uptake in areas with higher diversity of corals and free living symbionts should result in higher diversity of symbionts within the species. This is provided the conditions are not stressed and of no an advantage to the opportunistic D1a type. The results in our study confirm this theory and suggest that dominance of D1a is an indicator of a stressed reef. Interestingly the sampling design in the present study showed that approximately 50% of G. fascicularis colonies in the southern South China Sea harboured clade D due to inshore-offshore patterns possibly created by anthropogenic factors, thus questioning previous conclusions stating that group C is more prevalent in area (Baker 2004, Huang et al 2009, Huang et al. 2011).

Barriers, such as strong currents between inshore and offshore areas, different depths and difference in climate between the sites are also plausible to influence the difference in symbiont types (LaJeunesse et al. 2010a). The shifts in dominance of Symbiodinium types over latitudinal and on an inshore offshore gradient is consistent with these general findings, and that some Symbiodinium types appear to be more resilient than others. The different host haplotypes in our study also showed differences in frequency related to anthropogenic stressors. In some instances sampling only a few sites might show an apparent relationship between coral genotype and Symbiodinium type. However, in our study we were able to demonstrate, that while there were regional differences in symbiont type, these were related to environmental differences and not to genetic characteristics in the coral G. fascicularis.

To wrap it up:

In order to understand how reefs adapt and persist or go extinct in the ‗Anth- ropocene‘ (Jackson 2008) it is important to understand the wider aspects of holobiont maintenance and the interactions between reproduction of the host, environment, and presence of symbionts in the water. These interactions need to be determined in detail among both brooders and spawners on large scales of both time and space to enable predictions of the future states of reefs.

Species diversity and abundance are indicators of the present state as well as the future existence of the reef. However, a species may be abundant but exhibit low genetic variation. Low genetic variation is a potential vulnerability to organisms as fewer genotypes have a smaller chance to recombine and adapt to environmental changes, and may be wiped out in the face of environmental change. Preliminary studies elsewhere indicate that the genetic diversity in corals is lower in disturbed areas than in pristine areas (Souter 2007). Therefore, it is relevant to determine genetic variation in soft and reef building corals over larger geographical scales in relation to both geographic distance and varying human disturbance. These types of studies in combination with detailed life history data, and species diversity data, are needed to convey a wider and more accurate picture of the present state of the reefs.

The marine ecosystems are facing rapid changes and enormous threats due to destructive anthropogenic impact. The world population increased from 2 billion people in the 1950‘s to 6.8 billion today (Global Footprint Network 2010, Jackson 2010). Renewable resources are already overexploited and a changing climate is a threat for the future of marine ecosystems, of which some have collapsed locally. Toxic pollution, sediment run-off to the oceans, dynamite fishing, cyanide fishing (mainly for export of ornamental fish), overfishing and unsustainable aquaculture take a toll on the reef environment (Worm et al. 2009, Jackson 2010, Burke et al. 2011). These are some factors behind the fast demise of the reefs today, where 60% are threatened globally and 95% in South East Asia (Burke et al. 2011). As discovered at repeated visits among our study sites (Paper IV), human-induced destruction of reef habitats is ongoing in North to South Vietnam. Baseline data before human impact is necessary for defining the more natural states of reefs as well as facilitating detection of ongoing reef destruction. Data collected at the Great Barrier Reef in Australia for Papers I, II and III are also potential baseline studies for future post bleaching research.

Furthermore, this thesis provides a reliable method for estimating central life history traits of soft corals (size in Paper I). Thesis explores fundamental processes (such as reproduction Paper II & III, genetic adaptation and distribution Paper IV) and environmental factors Paper IV (such as potential stressors) that may determine the survival of coral populations. Further knowledge of such fundamentals will be necessary for building effective conservation strategies. However, these efforts are only sufficient if placed in a larger context where this research, politics and economy can connect on local as well as international levels. It is possible to reduce pollution (e.g. eutrophication) in harmony with economic development using modern prac- tices such as waste water treatment, usage of less polluting fish feed in fish farms, shoreline protective zones in farmland and urban areas, and generally less environmentally destructive technologies. While non-sustainable illegal fishing methods with e.g. dynamite and cyanide calls for crucial changes in attitude so that these illegal enterprises are made societally unacceptable. I believe that education and "awareness" campaigns involving local communities, schools, entrepreneurs (local scientists, industry, fishermen, dive operators, and tourism entrepreneurs) may be a first step in the right direction. But can enough joint efforts be mobilized in time to conserve and secure future sustainable use of these reefs?

Clavularia koellikeri on the Great Barrier Literature cited Reef. Coral Reefs 21:233–241.

Bastidas C, Benzie JAH, Uthicke S, Fabricius KE (2001) Genetic differentia- Achituv Y, BenayahuY (1990) Polyp tion among populations of a broadcast dimorphism and functional sequential spawning soft coral, Sinularia flexibilis, hermaphrodism in the soft coral Heterox- on the Great Barrier Reef. Mar Biol enia fuscescens (Octocorallia). Mar Ecol 138:517–525. Prog Ser 64: 263-269. Bastidas C, Willis BL, Fabricius KE Alino PM, Coll JC (1989) Observations (2004) Demographic processes in the soft on the synchronized mass spawning and coral Sinularia flexibilis leading to local postsettlement activity of octocorals on dominance on coral reefs. Hydrobiologia the Great Barrier Reef, Australia: Biolog- 530: 433-441. ical aspects. Bull Mar Sci 45: 697-707. Bellwood DR, Hughes TP, Folke C, Babcock RC (1990) Reproduction and Nyström M (2004) Confronting the coral development of the blue coral Heliopora reef crisis. Nature 429: 827-33. coreulea (Alcyonaria: Coenothecalia) Benayahu Y (1997) Developmental epi- Mar Biol 104: 475-481. sodes in reef soft corals: Ecological and th Baird AH, Marshall PA (2002) Mortality, cellular determinants. Proc 8 Int Coral growth and reproduction in scleractinian Reef Sym 2: 1213 – 1218. corals following bleaching on the Great Benayahu Y, Loya Y (1977) Space parti- Barrier Reef. Mar Ecol Prog Ser 237: tioning by stony corals, soft corals and 133-141. benthic algae on the coral reefs of the Baker AC (2001) Reef corals bleach to northern Gulf of Eilat, Red Sea. Bull Mar survive change. Nature 411: 765-766. Sci 31. 514-522. Baker AC (2003) Flexibility and specific- Benayahu Y, Loya Y (1981) Competition ity in Coral-Algal Symbiosis: Diversity, for Space Among Coral-Reef Sessile Ecology, and Biogeography of Symbiodi- Organisms at Eilat, Red Sea. Bull Mar Sci nium. Annu Rev Ecol Evol Syst 34: 661- 31: 514-522. 689. Benayahu Y, Loya Y (1984) Life history Baker AC (2004) Diversity, distribution studies on the Red Sea soft coral Xenia and ecology of Symbiodinium on coral macrospiculata (Gohar 1940). I. Annual reefs and its relationship to bleaching dynamics on gonadal development. Biol resistance and resilience. In: Coral Health Bull 166: 32-43. and Disease (eds Rosenberg E, Loya Y), Benayahu Y, Loya Y (1985) Settlement pp. 177–194. Springer-Verlag, New and recruitment of a soft coral: Why is York, Berlin. Xenia macrospiculata a successful colo- nizer? Bull Mar Sci 36: 177-188. Baker AC, Smith LW, Wirshing HH (2008) Coral symbiont diversity along a Benayahu Y, Loya Y (1986) Sexual seawater temperature gradient. reproduction of a soft coral: synchronous and brief annual spawning of Sarcophy- Babcock RC (1991) Comparative Demo- ton glacum (Quoy and Gaimard, 1883). graphy of 3 Species of Scleractinian Biol Bull 70: 32-42. Corals Using Age-Dependent and Size- Dependent Classifications. Ecol Monogr Benayahu Y, Weil D, Kleinmann M 61: 225-244. (1990) Radiation of broadcasting and brooding patterns in coral reef alcyona- Bastidas C, Benzie JAH, Fabricius KE ceans. Adv Invertebrate Reprod 5: 323- (2002) Genetic differentiation among 328. populations of the brooding soft coral 44

Ben-David-Zaslow R, Benayahu Y Connell JH (1978) Diversity in tropical (1998) Competence and longevity in rain forest and coral reefs. Science planulae of several species of soft corals. 199:1302-1310. Mar Ecol Prog Ser 163:235–243. Cordes EE, Nybakken JW, VanDykhui- Ben-David-Zaslow R, Henning G, Hof- zen G (2001) Reproduction and growth of mann DK, Benayahu Y (1999) Reproduc- Anthomastus ritteri (Octocorallia: Alcyo- tion in the Red Sea soft coral Heteroxenia nacea) from Monterey Bay, California, fuscescens: seasonality and long-term USA. Mar Biol 138:491–501. record (1991 to 1997). Mar Biol 133:553– 559. Dahan M, Benayahu Y (1997) Clonal propagation by the azoohanthellate soft coral Dendronephthya hemprichi. Coral Bongaerts P, Riginos C, Ridgway T, Reefs 16: 5-12. Sampayo EM, van Oppen MJH, et al. (2010b) Genetic Divergence across Habi- Daly M, Brugler MR, Cartwright P, tats in the Widespread Coral Seriatopora Collins AG, Dawson MN, et al. (2007) hystrix and Its Associated Symbiodinium. The phylum Cnidaria: A review of phylo- PLoS One 5: e10871. genetic patterns and diversity 300 years after Linnaeus. Zootaxa: 127-182. Buddemeier RW, Fautin DG (1993) Coral bleaching as an adaptive mechanism: a Dien TV, Hai HP (2006) Using SeaWiFS testable hypothesis. Bioscience 43:320– satellite data for monitoring algal bloom 326. in Vietnam waters, the South China Sea. Coast Mar Sci 30: 44-48. Burke L, Reytar K, Spalding M, Perry A, Cooper E, et al. (2011) Reefs at Risk Dinesen ZD (1985) Aspects of the life Revisited. Washington D.C. World Re- history of a stolon bearing species of sources Institute. 130 p. Efflatonuaria. Proc. 5th Int Coral Reef Conf, Tahiti. 6: 89-94. Carlon DB (1999). The evolution of mating systems in tropical reef corals. Dong ZJ, Huang H, Huang LM, Li YC Trends Ecol Evol 14: 491–495. (2009) Diversity of symbiotic algae of the genus Symbiodinium in scleractinian Carte BK (1996) Biomedical potential of corals of the Xisha Islands in the South marine natural products. Bioscience 46: China Sea. J Syst Evol 47: 321-326. 271-286. Duong HA, Berg M, Hoang MH, Pham Cavalier-Smith T (1993) Kingdom proto- HV, Gallard H et al. (2010) Triha- zoa and its 18 phyla. Microbiol Mol Biol lomethane Formation by Chlorination of Rev. 57: 953-994. Ammonium- and Bromide-containing Cesar H, Burke ,L Pet-Soede L (2003) Groundwater in Water Supplies of Hanoi, The economics of worldwide coral reef Vietnam. Water Res 37: 3242–3252. degradation. Arnhem, The Netherlands: Duong HA, Pham NH, Nguyen HT, Cesar Environmental Economics Consult- Hoang TN, Hoang TT et al. (2008) Oc- ing. currence, Fate and Antibiotic Resistance Chambers JM, Hastie TJ (1997) Statistic- of Fluoroquinolone Antibacterials in al Models in Science. Chapman and Hall, Hospital Wastewaters in Hanoi, Vietnam. New York. Chemosphere 72: 968–973.

Coffroth MA, Santos SR (2005) Genetic Excoffier L, Laval G, Schneider S (2005) diversity of symbiotic dinoflagellates in Arlequin ver. 3.0: an integrated software the genus Symbiodinium. Protist 156: 19– package for population genetics data 34. analysis. Evol Bioinform Online 1:47–50 Fabricius KE (1995) Slow population turnover in the soft coral genera Sinularia 45

and Sarcophyton in mid- and outer-shelf Gohar HAF 1940. Studies on the Xenii- reefs of the Great Barrier Reef. Mar Ecol dae of the Red Sea. Publ Mar Biol Stn Prog Series 126: 145-152. Ghardaqa 2: 27-118. Fabricius KE (1997) Soft coral abundance Hall VR, Hughes TP (1996) Reproductive in the central Great Barrier Reef: effects strategies of modular organisms: compar- of Acanthaster planci, space availability ative studies of reef-building corals. and aspects of the physical environment. Ecology 77. 950-963. Coral Reefs 16: 159-167. Hanh DC, Dong NT (2008) The Current Fabricius KE and Alderslade P (2001) State of River Basins in Vietnam - Pollu- Soft Corals and Sea Fans: A comprehen- tion and Solution. 3rd WEPA Forum, sive guide to the tropical shallow water Putrajaya, Malaysia Oct 23-24 2008. genera of the central-west Pacific, the Indian Ocean and the Red Sea. Australian Haapkylä J, Unsworth RKF, Flavell M, Institute of Marine Science. 264 p Bourne DG, Schaffelke B, et al. (2011) Seasonal Rainfall and Runoff Promote Farrant P (1985) Reproduction in the Coral Disease on an Inshore Reef. PLoS temperate soft coral Capnella gaboensis. ONE 6(2): e16893. Proc. 5th Int Coral Reef Cong, Tahiti 4: 319-324. Harrison PL (1988) Pseudo-gynodioecy: an unusual breeding system in the scle- Farrant P (1987) Population dynamics of ractinian coral Galaxea fascicularis. Proc. the temperate Australian soft coral Cap- 6th. Int. Coral Reef Symp. 2:699–705. nella gaboensis. Mar Biol 96: 401-407. Harrison PL, Wallace CC (1990) Repro- Faxneld S, Lund Jörgensen T, Nguyen duction, dispersal and recruitment of ND, Nyström N, Tedengren M (2011) scleractinian corals. In: Dubinsky Z (ed) Differences in physiological response to Ecosystems of the world, 25th edn. El- increased seawater temperature in near- sevier, Amsterdam, the Netherlands, shore and offshore corals in northern pp133–207. Vietnam. Mar Env Res 71: 225- 233. Hartnoll RG (1975) The annual cycle of Fisher R, Radford BT, Knowlton N, Alcyonium digitatum. Eustarine Coastal Brainard RE, Michaelis FB, et al. (2011) Mar Sci 3: 71-75. Global mismatch between research effort and conservation needs of tropical coral Harvell CD, Grosberg CD (1988) The reefs. Conserv Lett 4: 64-72. timing of sexual maturity in clonal ani- mals. Ecology 69:1855–1864. Frade PR, De Jongh F, Vermeulen F, van Bleijswijk J, Bak RPM (2008) Variation Hoai PM, Ngoc NT, Minh NH, Viet PH, in symbiont distribution between closely Berg M et al. (2010) Recent levels of related coral species over large depth organochlorine pesticides and polychlori- ranges. Mol Ecol 17: 691-703. nated biphenyls in sediments of the sewer system in Hanoi, Vietnam. Environ Pollut Fuchs Y, Douek J, Rinkevich B, Ben- 158: 913-920 Shlomo R (2006) Gene Diversity and Mode of Reproduction in the Brooded Hoegh-Guldberg O, Carter DA (2008) Larvae of the Coral Heteroxenia fusces- Symbiont acquisition strategy drives host– cens. J Heredity 2006 97(5):493-498. symbiont associations in the southern Great Barrier Reef. Coral Reefs 27: 763- Glynn PW (1993) Coral bleaching: eco- 772. logical perspectives. Coral Reefs 12: 1– 17 Hoegh-Guldberg O, Mumby PJ, Hooten AJ, Steneck RS, Greenfield P, Gomez E, Glynn PW (1996) Coral reef bleaching: Harvell, CD, Sale PF, Edwards AJ, Cal- facts, hypotheses and implications. Glob- deira K, Knowlton N, Eakin CM, Igle- al Change Biol 2: 495–509. sias-Prieto R, Muthiga N, Bradbury RH, 46

Dubi A, Hatziolos ME (2007 ) Coral reefs Knowlton N, Jackson JBC (2008) Shift- under rapid climate change and ocean ing baselines, local impacts, and global acidification. Science 318: 1737-1742. change on coral reefs. PLoS Biology 6: 215-220. Hull VR, Hughes TP, Reproductive Strat- egies of Modular Organisms: Compara- Lajeunesse TC (2001) Investigating the tive Studies of Reef- Building Corals. biodiversity, ecology, and phylogeny of Ecology 77: 950-963. endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS re- Hoegh-Guldberg, O. (1999) "Coral gion: in search of a "species" level mark- bleaching, Climate Change and the future er. J Phycol 37:866–880. of the world's Coral Reefs." Review, Mar Freshw Res 50:839-866. LaJeunesse TC (2005) ―Species‖ radia- tions of symbiotic dinoflagellates in the Huang H, Donga Z, Huanga L, Yanga J, Atlantic and Indo-Pacific since the Mi- Dib B, Lic Y, Zhoua G, Zhanga C (2011) ocene-Pliocene transition. Mol Biol Evol Latitudinal variation in algal symbionts 22:570–581. within the scleractinian coral Galaxea fascicularis in the South China. Mar Biol LaJeunesse TC, Bhagooli R, Hidaka M, Res 7: 208 - 211. deVantier L, Done T, et al. (2004) Close- ly related Symbiodinium spp. differ in Hughes TP, Baird AH, Bellwood DR, relative dominance in coral reef host Card M, Connolly SR, et al. (2003) Cli- communities across environmental, lati- mate change, human impacts, and the tudinal and biogeographic gradients. Mar resilience of coral reefs. Science 301: Ecol Prog Ser 284: 147-161. 929-933. LaJeunesse TC, Loh WKW, van Woesik Hughes TP, Jackson JBC (1985) Popula- R, Hoegh-Guldberg O, Schmidt GW, et tion-Dynamics and Life Histories of al. (2003) Low symbiont diversity in Foliaceous Corals. Ecol Monogr 55: 141- southern Great Barrier Reef corals, rela- 166. tive to those of the Caribbean. Limnol Hwang SJ, Song JI (2007) Reproductive Oceanogr 48: 2046-2054. biology and larval development of the LaJeunesse TC, Loh WKW, van Woesik temperate soft coral Dendronephthya R, Hoegh-Guldberg O, Schmidt GW et al. gigantea (Alcyonacea: Nephtheidae). Mar (2009) Low symbiont diversity in south- Biol 152:. 273-284. ern Great Barrier Reef corals, relative to Jackson JBC (2008) Ecological extinction those of the Caribbean. Limnol Oceanogr and evolution in the brave new ocean. 48: 2046-2054. PNAS 105: 11458-11465. LaJeunesse TC, Pettay DT, Sampayo EM, Jackson JBC (2010) The future of the Phongsuwan N, Brown B, et al. (2010a) oceans past. Phil Trans R Soc B 365: Long-standing environmental conditions, 3765-3778. geographic isolation and host-symbiont specificity influence the relative ecologi- Karlson RH, Hughes TP, Karlson SR cal dominance and genetic diversification (1996) Density-dependent dynamics of of coral endosymbionts in the genus soft coral aggregations: the significance Symbiodinium. J Biogeogr 37: 785-800. of growth and form. Ecology 77:1592- 1599. LaJeunesse TC, Smith R, Walther M, Pinzon J, Pettay DT, et al. (2010b) Host- Knowlton N (2001). The future of coral symbiont recombination versus natural reefs. Proceedings of Natl. Acad Sci 98 selection in the response of coral- (10): 5419-5425. dinoflagellate symbioses to environmental disturbance. Proc R Soc B-Biol Sci Knowlton N (2008) Coral reefs. Curr Biol 277: 2925-2934. 18: 18-21.

LaJeunesse TC, Trench RK (2000) Bio- tion in the soft coral genus Alcyonium. geography of two species of Symbiodi- Evolution 55(1):54-67. nium (Freudenthal) inhabiting the inter- tidal sea anemone Anthopleura elegantis- Mercier A, Hamel JF (2011) Contrasting sima (Brandt). Biol Bull 199: 126-134. reproductive strategies in three deep-sea octocorals from eastern Canada: Primnoa Latypov YY (2005) Reef-building corals resedaeformis, Keratoisis ornata, and of Vietnam as a part of the Indo-Pacific Anthomastus grandiflorus Coral Reefs reef ecosystem. Biol Morya 31: 75-81 DOI 10.1007/s00338-011-0724-8 Michalek-Wagner K, Willis BL (2001) Latypov YY (2006) Changes in the Com- Impacts of bleaching on the soft coral position and Structure of Coral Com- Lobophytum compactum I. Fecundity, munities of Mju and Moon Islands, Nha fertilization and offspring viability. Coral Trang Bay, South China Sea. Russian J Reefs 19: 231 - 239. Mar Biol 32: 269–275. Moberg F, Folke C (1999) Ecological Levy O, Appelbaum L, , Leggat W, Goth- Goods and Services of Coral Reef Eco- lif Y, Hayward DC, MillerDJ, Hoegh- systems. Ecol Econ 29: 215-233. Guldberg O (2007) Light-Responsive Cryptochromes from a Simple Multicellu- Muscatine L (1990) The role of symbiotic lar Animal;the Coral Acropora millepora. algae in carbon and energy flux in reef Science 318: 467-470 corals. Coral Reefs 25: 1-29. Lin S (2011) Genomic understanding of Norris EA (1993) Global marine biologi- dinoflagellates. Res Microbiol (In Press) cal diversity: a strategy for building conservation into decision making. Island Maddox GD, Cook RE, Wimberger PH, Press, Washington Gardeschu S (1989) Clone structure in four Solidago altissina (Asteraceae) Nyström M, Folke C, Moberg F (2000) populations; rhizome connections within Coral reef disturbance and resilience in a genotypes. Am J Bot 76: 318-326. human-dominated environment. Trends Ecol Evol 15: 413-417. Marshall PA, Baird AH (2000) Bleaching of corals on the Great Barrier Reef: diffe- Nystrom M, Graham NAJ, Lokrantz J, rential susceptibilities among taxa. Coral Norstrom AV. 2008. Capturing the cor- Reefs 19: 155-163. nerstones of coral reef resilience: linking theory to practice. Coral Reefs 27: 795- Maynard JA, Turner PJ, Anthony KRN, 809. Baird AH, Berkelmans R, et al. (2008) ReefTemp : An interactive monitoring Nyström M, Graham NAJ, Lokrantz J, system for coral bleaching using high- Norstrom AV. 2008. Capturing the cor- resolution SST and improved stress pre- nerstones of coral reef resilience: linking dictors. Geophys Res Lett 35: 1-5. theory to practice. Coral Reefs 27: 795- 809 McFadden CS (1986) Colony fission increases particle capture rates of a soft Oliver TA, Palumbi SR (2011) Many coral; advantage of being a small colony. corals host thermally resistant symbionts J Exp Mar Biol Ecol 103: 1-20. in high temperature habitat. Coral Reefs 30: 241-250. McFadden CS (1997) Contributions of sexual and asexual reproduction to popu- Palmer CV, Bythell JC, Willis BL (2010) lation structure in the clonal soft coral, Levels of immunity parameters underpin Alcyonium rudyi Evolution, 51: 112-126. bleaching and disease susceptibility of reef corals. FASEB 24 : 1935-1946. McFadden CS, Donahue R, Hadland BK, Weston R. (2001) A molecular phyloge- Pandolfi JM, Bradbury RH, Sala E, netic analysis of reproductive trait evolu- Hughes TP, Bjorndal KA, et al. (2003) Global trajectories of the long-term decline 48

of coral reef ecosystems. Science 301: Rowan R, Powers DA (1991b) A molecu- 955-958. lar genetic classification of zooxanthellae and the evolution of animal-alga sym- Patten NL, Harrison PL, Mitchell JG ( bioses. Science 251: 1348–1351. 2008) Prevalence of virus-like particles within a staghorn coral (Acropora muri- Sampayo EM, Franceschinis L, Hoegh- cata) from the Great barrier Reef. Coral Guldberg O, Dove S (2007) Niche parti- Reefs 27: 569-580. tioning of closely related symbiotic dinoflagellates. Mol Ecol 16: 3721-3733. Pinzon JH, LaJjeunesse TC (2011) Spe- cies delimitation of common reef corals Sampayo EM, Ridgway T, Bongaerts P, in the genus Pocillopora using nucleotide Hoegh-Guldberg O (2008) Bleaching sequence phylogenies, population genet- susceptibility and mortality of corals are ics and symbiosis ecology. Mol Ecol 20: determined by fine-scale differences in 311-325. symbiont type. PNAS 105: 10444-10449. Pochon X, Gates RD (2010) A new Sym- Schleyer MH, Kruger A, Benayahu Y biodinium clade (Dinophyceae) from (2004) Reproduction and the unusual soritid foraminifera in Hawaiʼi. Mol condition of hermaphroditism in Sarco- Phylogen Evol 56: 492-497. phyton glaucum (Octocorallia, Alcyonii- dae) in KwaZulu-Natal, South Africa. Porto I, Granados C, Restrepo JC, San- Hydrobiologia 530/531: 399-409. chez JA (2008) Macroalgal-Associated Dinoflagellates Belonging to the Genus Sella I, Benayahu Y (2010) Rearing Symbiodinium in Caribbean Reefs. Plos cuttings of the soft coral Sarcophyton One 3. glaucum (Octocorallia, Alcyonacea): towards mass production in a closed Porto I, Granados C, Restrepo JC, San- seawater system. Aquac Res 41:1748– chez JA (2008) Macroalgal-Associated 1758. Dinoflagellates Belonging to the Genus Symbiodinium in Caribbean Reefs. Plos Slattery M, Hines GA, Starmer J, Paul VJ One 3. (1999) Chemical signals in gametogenesis, spawning, and larval settlement and Porto I, Granados C, Restrepo JC, Sán- defense of the soft coral Sinularia poly- chez JA (2008) Macroalgal-associated dactyla. Coral Reefs 18: 75-84. dinoflagellates belonging to the genus Symbiodinium in Caribbean reefs. PloS Smith SV (1978) Coral-Reef Area and ONE 3: e2160. Contributions of Reefs to Processes and Resources of Worlds Oceans. Nature 273: Reaka-Kudla ML (1997) In Biodiversity 225-226Rohwer F, Seguritan V, Azam F, II: Understanding and protecting our Knowlton N (2002) Diversity and distri- biological resources, eds Reaka-Kudla bution of coral-associated bacteria. Mar M.L, Wilson D.E., Wilson E.O. (Joseph Ecol-Prog Ser 243: 1-10. Henry Press, Washington DC) pp 83-108. Souter P (2007) Causes and consequences Rohwer F, Seguritan V, Azam F, Knowl- of spatial genetic variation in two species ton N (2002) Diversity and distribution of of scleractinian coral in East Africa. PhD coral-associated bacteria. Mar Ecol-Prog thesis, University of StockholmStat M, Ser 243: 1-10. Loh WKW, Rowan R, Knowlton N (1995) Intraspe- Stat M, Bird CE, Pochon X, Chasqui L, cific diversity and ecological zonation in Chauka LJ, et al. (2011) Variation in coral-algal symbiosis. PNAS 92: 2850- Symbiodinium ITS2 Sequence Assem- 2853. blages among Coral Colonies. Plos One Rowan R, Powers DA (1991a) Molecular 6: Article No.: e15854 genetic identification of symbiotic dinof- Stat M, Bird CE, Pochon X, Chasqui L, lagellates (zooxanthellae). Mar Ecol Prog Chauka LJ, et al. (2011) Variation in Ser 71: 65–73. 49

Symbiodinium ITS2 Sequence Assem- London, England, UK. ISBN: 0-8014- blages among Coral Colonies. Plos One 8263-1 xiii+321p. 6: Article No.: e15854. Veron JEN (2000) Corals of the world. Stat M, Bird CE, Pochon X, Chasqui L, Townsville: Australian Institute of Ma- Chauka LJ, et al. (2011a) Variation in rine Science. 1382 p. Symbiodinium ITS2 Sequence Assemblag- es among Coral Colonies. PLoS ONE 6: Veron JEN, Devantier LM, Turak E, e15854. Green AL (2009) Delineating the Coral Triangle. Galaxea JCRS 11: 91-100. Stat M, Gates R (2011) Clade D Symbiodi- nium in Scleractinian Corals: A ―nugget‖ Verseveldt J, Benayahu Y (1978) De- of hope, a selfish opportunist, an omnious scription of one old and five new species sign, or all of the above. J Mar Biol 1-9. of Alcyonacea (Coelenterata: Octocoral- lia) from the Red Sea. Zool Meded, Lei- Stat M, Gates RD (2011b) Clade D Symbi- den 53: 57-74. odinium in Scleractinian Corals: A ―Nug- get‖ of Hope, a Selfish Opportunist, an Walker TA, Bull GD (1983) A newly Ominous Sign, or All of the Above? J Mar discovered method of reproduction in Biol 2011: 1-9. gorgonian coral. Mar Ecol Prog Ser 12 (1983), pp. 137–143. Stat M, Loh WKW, LaJeunesse TC, Hoegh-Guldberg O, Carter DA (2009) Watanabe T, Nishida M, Watanabe K, Stability of coral-endosymbiont associa- Wewengkang DS, Hidaka M (2005) tions during and after a thermal stress Polymorphism in the nucleotide sequence event in the southern Great Barrier Reef. of a mitochondrial intergenic region in Coral Reefs 28: 709-713. the scleractinian coral Galaxea fascicularis. Mar Biotechnol 7: 33–39. Stearns SC (1992) The evolution of life histories. Oxford University Press, Ox- Wicks LC, Hill R, Davy SK (2010a) The ford England influence of irradiance on tolerance to high and low temperature stress exhibited Tamura K, Peterson D, Peterson N, by Symbiodinium in the coral, Pocillopo- Stecher G, Nei M, and Kumar S (2011) ra damicornis, from the high-latitude reef MEGA5: Molecular Evolutionary Genet- of Lord Howe Island. Limnol Oceanogr ics Analysis using Maximum Likelihood, 55: 2476-2486. Evolutionary Distance, and Maximum Parsimony Methods. Mol Bio Evol (sub- Wicks LC, Sampayo E, Gardner JPA, mitted). Davy SK (2010b) Local endemicity and high diversity characterise high-latitude The R Development Core Team (2010) coral–Symbiodinium partnerships. Coral R: A Language and Environment for Reefs 29: 989-1003. Statistical Computing. Version 2.12.0 (2010-10-15). Wilkinson C., editor (2000) Status of Coral Reefs of the World: 2000. Australian Trench RK (1993) Microalgal- Institute of Marine Science, Townsville, invertebrate symbioses: a review. En- Queensland. docyt Cell Res Vol. 9, pp. 135– 175.United Nations Environment Pro- Wilkinson C., editor (2004) Status of Coral gramme 2007 Annual Report. 2008. 121 Reefs of the World: 2004, vol 1. Australian pages. ISBN No: 978-92-807-2907-8. Institute of Marine Science, Townsville, Queensland. Veron JEN (1993) Corals of Australia and the Indo-Pacific. University of Ha- Winkel LHE, Trang PTK, Mai Lan VM, waii Press Honolulu 644 p Stengela C, Aminia M et al. (2010) Ar- senic pollution of groundwater in Viet- Veron JEN (1995) Corals in space and nam exacerbated by deep aquifer exploi- time: The biogeography and evolution of tation for more than a century. PNAS the Scleractinia. Cornell University Press, 108: 1246–1251. 50

Winsor L (1984) (Compilation) Manual of basic zoological microtechniquesn for light microscopy. School Biol Sci, James Cook University of North Queensland, Towns- ville, Australia. Worm B, Hilborn R, Baum JK, Branch TA, Collie JS, et al. (2009) Rebuilding Global Fisheries. Science 325: 578-585. Worm B, Hilborn R, Baum JK, Branch TA, Collie JS, et al. (2009) Rebuilding Global Fisheries. Science 325:578-585. Yellowlees D, Rees TA, Leggat W (2008) Metabolic interactions between algal symbionts and invertebrate hosts. Plant Cell Environ 31: 679–94.

Acknowledgements

The path to my thesis went under the heading: ―We live in interesting times,‖ and was a fun journey of science, people, cultures, late nights, coffee, new insights and far too little time with 2.5 years. A great number of people and organizations have made this possible. I especially want to thank: The generous sponsors of this research; Academy of Finland, Australian European Awards Program, Oskar Öflund Foundation, E & G Ehrnrooth Foundation, Wallen- berg Foundation, AEW Smitts Foundation (SU), Swedish International Develop- ment Agency (SIDA, awarded to M Tedengren). Nils ‗Nisse‘ Kautsky at the Department of Systems Ecology for giving that amazing lecture on tropical ecosystems and encouraging me to come to Stockholm, for be- lieving in me and for always giving time to answer questions during your busy days. I learned a lot from you. The ‗Three Musketeers‘ who were my academic supervisors during the course of the thesis and who all three trusted me to design projects, choose tools and learn how to use them on my own accord; Michael ‗Micke‘ Tedengren (SU) for making it possible to go to Vietnam, for find- ing funding, being supportive and making me laugh. You believe that amazing things can be done – and therefore they happen. John Benzie (AIMS and Cork Uni- versity), you were there all the way from Australia to Stockholm. You believed in my ideas and encouraged me when the results were different from anything we had seen before - that made me brave. Despite your busy schedule you never declined to give sound advice, either in person or over the phone from remote airports whilst in transit. Mats Grahn (Södertörn University College) who alongside with John is a wizard of genetics; for providing me with molecular genetic facilities. For sharing your knowledge, brilliance and enthusiasm. The staff at Lizard Island Research Station for making my stay there a pleasure and for collecting samples when I was not on the island. For sunset drinks on the beach. Leigh Winsor at James Cook University for invaluable histological advice. Phil. Alderslade (Northern Territory Museum, Darwin) for confirming identification of the soft coral species. JCU crowd for taking me out to witness the coral spawning for the very first time. Kathryn Kavanagh (JCU/Harvard University/Dartmouth College); for being a friend colleague, brilliant co-author and dive buddy in Australia and Vietnam. Steve Palumbi (Harvard University) for teaching me the secrets of PCR in his lab and for encouraging me to go back to Europe to get this done. Maria Milicich for your friendship, solving the world‘s problems over a glass of wine and making Townsville such a fun place. The field assistants at Lizard Island for spending long hours in the water. Many of you became friends for life after spending weeks on a remote island. Marie Egerrup, Helmut Grossman, Keith Martin-Smith, Rob Rowan, Chris Ryan, Evizel Seymour, Guy Smith, Jean-Luc Solandt, Ken Okaji and Sven Uticke - you really made life fun on the island. To my ―fellow Scandinavian Down Under‖ -Marek Cuhra grat dive buddy and underwater photographer. 52

The diving teams and support groups in Vietnam for making the trips so fantastic, interesting and fun; Director Dr Long and Dr Le Lan Huong at the Institute of Ocea- nography (IO) in Nha Trang, and Dr Lan at the Institute of Marine and Environmen- tal Research (IMER) in Haiphong for logistic support and help with research permits in the field. Cuong, Ngai and the scientific divers at IMER for sample collections in the North, Hoi An and in Hue and for introducing me to Vietnamese cuisine and way of life, for friendships and company. Huong, Lan Vo Tran Tuan Linh, Thi Minh Hue and Â Phan Kim Hoang for assistance in the field in South Central Vietnam and extracting DNA on site. Huong (again) for setting up a genetics lab with me in Nha Trang, and for guiding me through the cultural codes in Nam and becoming a dear friend in the process. Nisse, Kathy, Sussie , Suzanne and Alex for long days in the field. Extra thanks to Suzanne for introducing me to Vietnam and making it all so easy and fun during the first fieldtrip. Steve Reid at Blue Coral Divers in Hoi An and Jeremy Stone at Rainbow Divers in Nha Trang, with dive teams, for generously supporting this project and providing invaluable background data series, and for taking such a great interest in this work. My co-authors (in alphabetical order); Cuong, Huong, Kathy, John, Mats, Micke, Ngai, Nisse H, Shop, Sussie and Suzanne. The crowd at AIMS. The crowd at SU; My inspiring fellow PhD students who are too many to mention individually. Johan N (for Vietnamese coffee and for providing a haven in your office with the talking fridge). Jenny H, Ben, Ola and Angelina -my office mates for sharing space and life wisdom. Siw, Barbara & Co at the admin for everything connected to official paperwork. The crowd at Södertörn; Jussan - lab wizard, statistical heroine and friend, Patrick stat wiz, Emma - fun, Oskar - happy, Petter - hilarious comments, Stefan - Spielberg in a fishy world, Niklas - with all your grand projects in life. Future professors Morten Limborg and Nina Biermann - Abba rules (not)! Nisse H and Sussie for being the best master students one can ever wish for. Joel Jonasson at Eurofins for helping with primer orders and fine tuning of primer design. Johan Spens for hours of editorials and stats advice, and for everything else. Mamma and pappa for being the wise and loving people you are. Thanks for filling my life with laughter and for being so incredibly adventurous. To my super heroes - ‗The Fantastic Four‘ e.g. my siblings with families; for being my best friends always, for discussing corals and diving, sharing fabulous dinners. The two most significant persons in my life; my fantastic children Alex and Hannah, it is an honor to be your mum. Universe in the eyes of a growing teenager and a lively pre-schooler gives life a new dimension. I love you! You - for reading this. Now you can flick through the rest of this book 