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

and ecology

Micaela Hellström

Doctoral thesis in Marine Ecotoxicology

Micaela Hellström, micaela@ecology.su.se

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 repro-

duction 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,

JB

MH

Co-authored after first

draft

KK, JB SG

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-

ments:

MH MH MH MH, LLH, NH,

SG, SF

Data preparation: MH MH MH, MH, NH, MG,

SG, SF

Data analysis: MH, JB MH MH, KK, SM,

JB

MH

Maps and figures: MH MH, KK MH,KK NH, SG, MH

Reagents/ materials/

lab

MH MH MH, JB MT, MG

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 unprec-

edented 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 them-

selves. Papers II-III study reproduction in a common soft coral species, Sarcophyton

elegans, with previously unknown reproductive modes. Paper IV investigates genet-

ic 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 down-

stream (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, reproduc-

tion, 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

13

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 colo-

ny) or horizontal (acquisition from the surrounding environment) and depend

on the reproductive modes of the corals (brooders or spawners) which de-

termine 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).

14

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 organ-

isms 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 ecosys-

tems 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 com-

plex 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-

15

tral Indo-Pacific and Southeast Asia are heavily underrepresented (reviewed

by Fisher et al. 2011). The latter areas are heavily populated and face envi-

ronmental challenges as rapidly growing human populations (depending on

the goods from the reefs) and emerging economies put these ecosystems un-

der pressure. Today more than 60% of the coral reefs worldwide are threat-

ened 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 sym-

bionts 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 sex-

ual and asexual reproduction in a common species of soft coral Sarcophyton

elegans on the Great Barrier Reef (Papers II and III) with previously un-

known reproductive modes. Reproduction is a key for survival trough re-

combination, 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 anthropogen-

ic stressors and a rapidly growing population. This thesis highlights the im-

portance of understanding different biological processes such as genetic

adaptation to environmental factors and the consequences of modes of sex-

ual 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

16

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 con-

trasting 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 me-

tric to body volume (a proxy for potential fecundity) after water dis-

placement.

The objectives of Papers II-III were to document in detail the sex differen-

tiation, 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. broad-

cast 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, gameto-

genesis (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 asex-

ual reproduction (Paper III).

Establishing contribution of asexual and sexual reproduction to one

of the study populations by examining genetic differences using al-

lozyme gel electrophoresis (Paper III)

Paper IV tested hypotheses regarding the relationship between several envi-

ronmental 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 horizon-

tal 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.

17

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 sub-

class 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 dinoflagel-

lates (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).

18

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 mu-

shroom shaped, and the disc-like polyp-bearing region (the polypary) has

two different kinds of polyps; large autozooids that bear tentacles and small-

er, more numerous siphonozooids lacking obvious tentacles (Fabricius and

Alderslade 2001). Prior to this study the reproductive mode of this zooxan-

thellate 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 ga-

mete 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).

19

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 ge-

nera (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 sym-

biont 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 hermaph-

roditic(male and females in the same colony) (Carlon 1999), it has only been

established relatively recently that soft corals are primarily gonochoric (hav-

ing 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

20

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 repro-

duction 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).

21

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 great-

er visibility, higher light levels and reduced sediment load (usually further

offshore). The inshore sites were situated at 50 to 800 meters from the main-

land whilst the offshore sites were situated 8-14 km from the shoreline.

22

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).

23

Figure 2. Average monthly values (±S.D.) between 2002 and 2010 of SSTs (a) and chloro-

phyll 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.

24

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)

Region

#

Coral

sp.

Site Coordinates

ML

dist.

(km)

Depth

max

(m)

SST

(°C)

SST

range

(°C)

Chla

(mg/

m-3)

Chla

range

(mg/

m-3)

Vis

range

(m)

North 182 1 .Cat

Ba IS I

20°45'N,

107°04'E 0.8 2.8 25.5 17-32 2.4

0.84-

5.94 0.5-4

2. Cat

Ba IS

II

20°47'N,

107°06'E 0.1 2.2 25.5 17-32 2.4

0.84-

5.94 0.5-4

3. Cat

Ba OS

I

20°37'N,

107°09'E 14.2 6.7 25.5 17-32 1.6

0.45-

3.00

1.5-

10

4. Cat

Ba

OSII

20°37'N,

107°08'E 13 2.8 25.7 17-32 1.6

0.45-

3.00

1.5-

10

North

Central 334 5. Hue

16°14'N,

108°12'E 0.3 6 27.2 18-32 2.4

0.65-

7.28

1.5-

10

6.Hoi

An

15°56'N,

108°29'E 12 12 27.1 20-32 1.2

0.21-

6.23

1.0-

30

Central 397

7. Nha

Trang

IS I

12°12'N,

109°13'E 0.04 4.2 27.8 24-30 0.5

0.05-

1.96 0.5-4

8. Nha

Trang

IS II

12°10'N,

109°12'E 0.01 4 27.8 24-30 0.5

0.05-

1.96

1.5-

15

9. Nha

Trang

OSI

12°05'N,

109°18'E 8.3 8.6 27.6 24-31 0.5

0.16-

2.47

4.0-

30

10.Nha

Trang

OS II

12°04'N,

109°18'E 8.3 10 27.6 24-31 0.5

0.16-

2.47

4.0-

30

South 404 11.Phu

Quoc

09°55'N,

103°59'E 11 12 29.4 27-32 1.5

0.42-

3.30

2.0-

15

Methods Paper I: Colony Measurements To test the hypotheses that the basal circumference is a more precise meas-

ure 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

25

displacement in formalin). The best predictor of colony volume was ex-

amined using linear regression.

Figure 3. Illustration of the measurements of the colony size made for (a) Sarcophyton ele-

gans, (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 in-

cluded 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 maturi-

ty. 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 seawa-

ter (v/v) for three days and then transferred to 70% ethanol for further analy-

sis. 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

26

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, ma-

ture 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 obser-

vations 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 colo-

ny 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.

27

Methods Paper IV: Genetic distribution of G. fascicularis and its asso-ciated 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 fur-

ther 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.

28

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 be-

tween 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 (la-

goon or exposed), replicate (transect 1, 2 or 3 within each site), signs of co-

lony 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 refer-

ence 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

29

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‘)

H‘ = - Σ si = 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 as-

sessed 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 vari-

able 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 differ-

ent ITS2 types (with n > 30) was also tested separately by using a general-

ized linear model (GLM). As the ITS2 type data were binary (pres-

ence/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

30

linear model was the Akaike‘s Information Criterion (AIC—Chambers and

Hastie, 1997).

31

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 maxi-

mum 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 reproduc-

tive 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.

32

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 reproduc-

tion 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 ooge-

nesis. Spermatogenesis takes 10-12 months (Fig 6b, 7b).

The majority of gametes are released during the annual austral mass spawn-

ing 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 reproduc-

tion, 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 co-

lonies 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.

33

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.

34

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 oo-

cyte, 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 sperma-

tids. 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 indi-

cate 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 con-

trol 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-

35

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 colo-

nies. (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 the-

sis showed that soft corals exhibit an even larger spectrum of reproductive

traits than described before in the order (Paper II).

36

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

37

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 in-

shore 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.

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).

LA ORIGINAL

LD cytb+ND2

LE cytb+ND2

L01

L02

LC cytb+ND2

LB cytb+ND2

SB cytb+ND2(7)

SC cytb+ND2(8)

SA ORIGIN cytb+ND2(6)

S01

S02

S03

S04

30

23

25

58

32

23

72

87

0.001

38

Figure 11. Observed geographic distribution patterns of Galaxea fascicularis mtDNA inter-

genic 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 haplo-

types 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 sig-

nificant 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 distribu-

tion (Fig 12). The diversity indices of the symbionts showed a strong latitu-

dinal 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).

39

Figure 12. Genetic diversity of G. fascicularis based on mtDNA (non-coding intergenic re-

gion 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 resi-

lient 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.

40

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.

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.

0,00

1,00

2,00

3,00

4,00

5,00

6,00

North

IS1

North

IS2

North

OS1

North

OS2

Hue Hoi

An

Nha

Trang

IS 1

Nha

Trang

IS2

Nha

Trang

OS2

Nha

Trang

OS1

Phu

Quoc

Div

eris

ty i

nd

ex D

Sites

41

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 differenc-

es 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 ap-

proximately 50% of G. fascicularis colonies in the southern South China Sea

harboured clade D due to inshore-offshore patterns possibly created by anth-

ropogenic 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, differ-

ent 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 environ-

mental differences and not to genetic characteristics in the coral G. fascicu-

laris.

42

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 ex-

hibit 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 envi-

ronmental 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 combina-

tion 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.

43

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 dis-

tribution 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 communi-

ties, schools, entrepreneurs (local scientists, industry, fishermen, dive opera-

tors, 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?

44

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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.

53

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  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 de-

sign.

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