micaela hellström
TRANSCRIPT
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 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