REPORT

Photobiology of Symbiodinium revisited: bio-physicaland bio-optical signatures

S. J. Hennige Æ D. J. Suggett Æ M. E. Warner ÆK. E. McDougall Æ D. J. Smith

Received: 9 May 2008 / Accepted: 20 October 2008 / Published online: 16 November 2008

� Springer-Verlag 2008

Abstract Light is often the most abundant resource

within the nutrient-poor waters surrounding coral reefs.

Consequently, zooxanthellae (Symbiodinium spp.) must

continually photoacclimate to optimise productivity and

ensure coral success. In situ coral photobiology is becom-

ing dominated by routine assessments using state-of-the-art

non-invasive bio-optical or chlorophyll a fluorescence (bio-

physical) techniques. Multiple genetic types of Symbiodi-

nium are now known to exist; however, little focus has

been given as to how these types differ in terms of char-

acteristics that are observable using these techniques.

Therefore, this investigation aimed to revisit and expand

upon a pivotal study by Iglesias-Prieto and Trench (1994)

by comparing the photoacclimation characteristics of dif-

ferent Symbiodinium types based on their bio-physical

(chlorophyll a fluorescence, reaction centre counts) and

bio-optical (optical absorption, pigment concentrations)

‘signatures’. Signatures described here are unique to Sym-

biodinium type and describe phenotypic responses to set

conditions, and hence are not suitable to describe taxo-

nomic structure of in hospite Symbiodinium communities.

In this study, eight Symbiodinium types from clades and

sub-clades (A–B, F) were grown under two PFDs (Photon

Flux Density) and examined. The photoacclimation

response by Symbiodinium was highly variable between

algal types for all bio-physical and for many bio-optical

measurements; however, a general preference to modifying

reaction centre content over effective antennae-absorption

was observed. Certain bio-optically derived patterns,

such as light absorption, were independent of algal type

and, when considered per photosystem, were matched

by reaction centre stoichiometry. Only by better under-

standing genotypic and phenotypic variability between

Symbiodinium types can future studies account for the

relative taxonomic and physiological contribution by

Symbiodinium to coral acclimation.

Keywords Symbiodinium � Fluorescence �Photosystem II � Pigments � Light absorption

Introduction

Symbiosis between the microalga Symbiodinium spp. and

cnidarians enables an increase in productivity through a

mutual exchange of growth-limiting nutrients (Falkowski

et al. 1990). Photosynthesis by Symbiodinium produces

organic matter that supplements host metabolism whilst

inorganic nutrients excreted by the host in turn sustains

photosynthesis (Muscatine 1990; Dubinsky and Berman-

Frank 2001). Within nutrient-poor waters surrounding

coral reefs, light is the most abundant resource available.

Therefore, optimisation of photosynthesis by Symbiodini-

um is crucial to the stability of this symbiosis and

the ultimate success of corals. Given the importance of

corals, both ecologically and economically (Moberg and

Communicated by Environment Editor Prof. Rob van Woesik

S. J. Hennige � D. J. Suggett (&) � D. J. Smith

Coral Reef Research Unit, Department of Biological Sciences,

University of Essex, Colchester, Essex CO4 3SQ, UK

e-mail: dsuggett@essex.ac.uk

M. E. Warner

College of Marine Studies, University of Delaware,

700 Pilottown Rd. Lewes, Delaware 19958, USA

K. E. McDougall

Environmental Research Institute, North Highland College,

UHI Millennium Institute, Castle Street, Thurso,

Caithness KW14 7JD, UK

123

Coral Reefs (2009) 28:179–195

DOI 10.1007/s00338-008-0444-x

Folke 1999) much effort has been invested in attempting to

understand the photobiology of Symbiodinium both in ho-

spite in corals and in vitro (Iglesias-Prieto and Trench

1994, 1997; Gorbunov et al. 2001; Lesser 2004; Lesser and

Farrell 2004; Robison and Warner 2006). Consequently,

although this study is relevant for numerous cnidarian-

Symbiodinium studies, particular attention is drawn

towards the implications for coral–Symbiodinium research.

Conventional techniques for assessment of photobiology

confine many studies to the laboratory since measurements

require concentrations of material far in excess of that found

in nature. Symbiodinium spp. reside within the gastrodermis

of host corals and collection of the microalgae requires

destruction of the coral. To avoid such practices, researchers

have turned to unobtrusive optical-based approaches that

exploit (1) the absorption properties of whole corals (as

surface reflectance; Mazel 1997; Hochberg et al. 2006) or (2)

the fluorescence emission by photosystem II of Symbiodi-

nium (via active chlorophyll a fluorescence induction; Ralph

et al. 1999; Gorbunov et al. 2001). The latter is strictly a bio-

optical measurement but is often labelled as bio-physical

since it is based on the bio-physical processes that separate

light harvesting (absorption) from fluorescence (emission).

Florescence represents the efficiency of light transfer from

all light harvesting pigments to the core chlorophyll a mol-

ecule and the subsequent probability of excitons being

utilised for charge separation in the reaction centres or re-

emission as either heat or fluorescence through the antennae

bed (Falkowski and Raven 1997; Suggett et al. 2004).

Both bio-optical and bio-physical approaches are highly

attractive to coral research since measurements can be

made rapidly and conveniently in situ with the potential to

scale measurements up to remotely sensed platforms

(Kolber et al. 2005; Hochberg et al. 2006). Not surpris-

ingly, their application has grown, and continues to grow

rapidly, since the introduction of commercially available

diving-based spectrometers and induction fluorometers

10 years ago. However, despite this surge of interest, rel-

atively little research has been invested in ascertaining

whether photobiological (bio-optical and bio-physical)

signals are the same for all coral–Symbiodinium associa-

tions that exist in nature. Similarly, the basis for these

photobiological signals has received little attention. Con-

sequently, interpretation of these results when collected

across various environmental gradients remains highly

questionable, since the coral–Symbiodinium association

may change and the different Symbiodinium communities

(which are often unaccounted for) may have different

photobiological characteristics (Robison and Warner

2006). This paper specifically addresses the extent and

nature of the variability observed in these bio-optical and

bio-physical signals by focussing on photoacclimation,

which is relatively well studied in Symbiodinium.

Photoacclimation characteristics of Symbiodinium

Photoacclimation describes the phenotypic response of

algae to changes in the light environment (Falkowski and

LaRoche 1991) and occurs from alterations to a number of

components of the photosynthetic apparatus (Falkowski

and LaRoche 1991; MacIntyre et al. 2002). Light har-

vesting photosystems I (PSI) and II (PSII) each consist of

pigment–protein complexes coupled to reaction centre

(RC) complexes. Efficient transfer of light from the pig-

ment-proteins to the RCs is ensured through modification

of both the species and organisation of pigment (Falkowski

and Raven 1997; Suggett et al. 2007). However, excitation

pressure upon the photosystems can be further optimised

by altering the number of RC complexes in each photo-

system (Falkowski et al. 1981; Mauzerall and Greenbaum

1989). In both cases, the effective absorption of PSI (rPSI)

and PSII (rPSII) (the target ‘size’ for light interception

per unit RC, see Table 1) will change (Mauzerall and

Greenbaum 1989; Falkowski and Raven 1997; Suggett et al.

2007). Maximum photosynthesis, and hence productivity, is

dependent upon the efficiency of these mechanisms to

successfully capture and utilise light energy. Therefore,

photoacclimation can also induce alterations of cell size,

Rubisco content and total cellular carbon (Sukenik et al.

1987; Geider et al. 1998; MacIntyre et al. 2002).

Any one genotype of algae has an optimised environ-

mental range that results from evolutionary selective

pressure. However, algal communities in nature are rarely

composed of a single genotype (Medlin and Kaczmarska

2004) but instead represent a complex mix of species, each

of which is characterised by a specific phenotypic response

to environmental change (Suggett et al. 2007). Natural

assemblages of Symbiodinium are no exception. Nuclear

and chloroplast molecular markers have shown and con-

tinue to show that the genus Symbiodinium is characterised

by considerable genetic diversity, with the genus currently

split into eight major lineages or clades designated A

through H (Coffroth and Santos 2005). Evidence to date

suggests that reef forming corals contain a highly con-

served assemblage of symbionts which may contain more

than one Symbiodinium genetic type in some cases (Ulstrup

and van Oppen 2003; LaJeunesse et al. 2004a; Goulet

2006). Some flexibility to environmental change can be

ensured through ‘shuffling’ of the dominant zooxanthellae

type within the assemblage (Berkelmans and van Oppen

2006), which may yield discrete patterns of zonation for

Symbiodinium assemblages with depth (Iglesias-Prieto

et al. 2004; Warner et al. 2006) and across broad geo-

graphic scales within a single reef complex (Garren et al.

2006). However, the extent of Symbiodinium zonation

between corals and depths is also disputed, with some

studies finding little or no variability across a reef system

180 Coral Reefs (2009) 28:179–195

123

(Goulet 2006) with two of the most prevalent types

(Caribbean to Indo-Pacific), often types A and B (LaJeu-

nesse 2002; Baker 2003; Suggett et al. 2008).

Previous laboratory investigations have confirmed

photoadaptation amongst different Symbiodinium types

when grown under identical conditions (Chang et al. 1983;

Iglesias-Prieto and Trench 1994, 1997; Savage et al. 2002;

Rodriguez-Roman and Iglesias-Prieto 2005; Robison and

Warner 2006; Suggett et al. 2008). Most of these investi-

gations also demonstrated that a range of photoacclimatory

responses are observed at the ‘cladal’ level. Likewise, such

phenotypic plasticity to environmental change also appears

to extend to the intracladal level (Tchernov et al. 2004;

Robison and Warner 2006; Suggett et al. 2008). Of major

consequence is that optical-based measurements made in

situ across gradients of Symbiodinium genetic types may be

erroneously identified as acclimatory (phenotypically)

rather than natural adaptively (genetically) driven vari-

ability. Therefore, it is crucial that the taxonomic

variability of optical ‘signatures’ determined by the geno-

type of Symbiodinium present is characterised to accurately

interpret the response of coral photobiology to environ-

mental conditions, such as light availability, as either

genetic community changes (taxonomic) or phenotypic

responses.

The earlier work by Iglesias-Prieto and Trench (1994,

1997), which described photobiology for three Symbiodi-

nium types, was revisited with key additional techniques,

such as chlorophyll a fluorescence, which are highly rele-

vant to current in hospite Symbiodinium photoacclimation

studies. Bio-optical (optical absorption, pigment concen-

trations) and bio-physical (chlorophyll a fluorescence,

reaction centre counts) characteristics or ‘signatures’ for

eight Symbiodinium types from clades and sub-clades

Table 1 Table of terms, their definitions and their units

Terms Definition Units

Optical

E Excitation PFD of FIRe fluorometer lmol photons m-2 s-1

PFD Photon Flux Density lmol photons m-2 s-1

F730 Fluorescence excitation at 730 nm IU

F* Chlorophyll a-specific fluorescence excitation at 730 nm IU mg chl a-1

a(k) Optical absorption coefficient (k) m-1

a* (k) Chlorophyll a-specific optical absorption coefficient (k) m2 mg chl a-1

a*PSI (II) (k) Chlorophyll a-specific optical absorption coefficient (k) associated with PSI (II) m2 mg chl a-1

a* PSI (PSII) Effective a*PSII (I) (k) weighted to the spectra of the growth lamps m2 mg chl a-1

aTpig(k) Chlorophyll a-specific absorption spectra for all cellular pigments m2 mg chl a-1

aPSC Chlorophyll a-specific absorption spectra for all photosynthetic pigments weighted

to the excitation spectra of the FIRe fluorometer LEDs

m2 mg chl a-1

PSC Photosynthetic Carotenoids

PPC Photoprotectant Carotenoids

ai (k) In vivo weight-specific absorption coefficient m2 mg-1

ci Concentration of pigment i mg m-3

PI Packaging Index Dimensionless

Biophysical

PSI (II) Photosystem I (or II)

RCI (II) Reaction Centres associated with Photosystem I (or II)

Fo Minimum fluorescence Relative fluorescence

FmST (MT) Maximum fluorescence; Single Turnover or Multiple Turnover Relative fluorescence

Fv Variable fluorescence (Fm - Fo) Relative fluorescence

Fv/Fm Maximum photochemical quantum efficiency Dimensionless

Fo0 Minimum fluorescence under actinic light Relative fluorescence

F0 Fluorescence under actinic light Relative fluorescence

FmST0 (MT) Maximum fluorescence under actinic light; Single Turnover or Multiple Turnover Relative fluorescence

NPQ Non-photochemical quenching (Fm - Fm0)/Fm

0 Dimensionless

rPSII (I) Functional absorption cross section of PSII (I) A2 quanta-1

rPSII* Independent measure of absorption cross section of PSII (see Eq. 8) A2 quanta-1

rPSII0 Functional absorption cross section of PSII under ambient light A2 quanta-1

Coral Reefs (2009) 28:179–195 181

123

(A–B, F) grown under two PFDs (Photon Flux Density)

were examined to determine between-type variability.

These observations are discussed within the context of

(1) previous studies of Symbiodinium photoacclimation and

(2) how such adaptive variability may confound the inter-

pretation of photobiology when measured across gradients

of Symbiodinium types in nature. Ultimately, understanding

of this variability is crucial for future in hospite Symbiodi-

nium photoacclimation studies, which utilise methods

described here, since taxonomic shifts in the Symbiodinium

community may otherwise be misinterpreted as photoacc-

limation. Signatures described here are not definitive

identifiers of in hospite Symbiodinium types, but rather

demonstrate important functional similarities and dispari-

ties between algal types, crucial for future photoacclimation

studies.

Materials and methods

Growth, experimental conditions and sampling regime

Eight Symbiodinium strains designated by the internal tran-

scribed spacer 2 (ITS2) typing nomenclature (LaJeunesse

2001, 2002) as A1, A1.1, A2, A3, B1 (see Robison and

Warner 2006), B1*, B1** and F2 were grown as described

previously (Suggett et al. 2007, 2008). Briefly, cultures were

maintained in climate-controlled cabinets at 26�C on a 14:10

light: dark cycle at two photon flux densities (100 and

650 lmol photons m-2 s-1) as semi-continuous cultures in

2 l flasks of artificial seawater media ASP-8A (Provasoli

et al. 1957). Cultures were bubbled gently with air passed via

a carbon column to maintain constant pH (Suggett et al.

2008). Superscripts * and ** indicate types of B1 isolated

from a different hosts (Table 2) and were included to test

whether the same Symbiodinium type isolated from different

hosts would yield similar photobiology. Cells were moni-

tored by daily cells counts (Haemocytometer and Beckman

Coulter counter and size analyser) and minimum fluores-

cence, and harvested during exponential growth from

sequential generations. Samples were either used uncon-

centrated or concentrated gently by gravity filtration (see

Suggett et al. 2007).

Photosynthetic unit sizes

These are conventionally defined as the ratio of chlorophyll

a to either RCI or RCII (Suggett et al. 2007), and consid-

ered to be indicative of the concentration of functional

reaction centres (RCs), i.e. those that are capable of charge

separation and hence the release of an electron through the

electron transport chain (ETR) from absorbed photons.

Chlorophyll a (referred to as chl a in subsequent equations

and ratios) per RCII was determined from the product of

the Emerson & Arnold number, mol chl a (mol O2)-1, and

the minimum number of electron transfer steps required

to evolve 1 mol O2, an assumed constant of 0.25 mol O2

(mol RCII)-1, using an oxygen electrode and custom-made

LED array as described in Suggett et al. (2003, 2006).

Chlorophyll a per PSI was determined from redox changes

in P700 chlorophyll a as measured from a PAM 101

coupled with a Dual-Wavelength Emitter-Detector Unit

ED-P700DW (Suggett et al. 2007).

Optical light absorption and spectrofluorescence

A U-3000 spectrophotometer with / - 60 integrating

sphere (Hitachi) was used to determine sample optical

density as described in Suggett et al. (2007, 2008). For

spectrofluorometric measurements, DCMU was added to

the concentrated algal sample to yield a final concentration

of 30 mM to prevent oxidation of QA (quinine acceptor)

(see Suggett et al. 2004). Emission was measured at

730 nm with excitation at 1 nm intervals between 400 and

715 nm to yield a PSII-specific fluorescence excitation

spectrum (termed F730, Suggett et al. 2004, 2007). Sample

Table 2 Growth (l day-1) and chl a cell-1 (g cell-1 (Exp-13)) (±SE) for Symbiodinium types under LL (100 lmols photons m-2 s-1) and HL

(650 lmols photons m-2 s-1) including isolation origin of host species

Algal type Host Host origin Growth (l day-1) chl a cell-1 (g cell-1 (Exp-13))

LL HL LL HL

A1 Cassiopeia xamachana Florida 0.28 (0.03) 0.52 (0.05) 6.25 (0.70) 3.00 (0.82)

A1.1 Condylactis gigantea Jamaica 0.20 (0.04) 0.40 (0.05) 4.02 (0.02) 1.74 (0.36)

A2 Montastrea spp. Florida 0.22 (0.02) 0.46 (0.06) 6.56 (0.49) 1.13 (N/A)

A3 Tridacna maxima Palau 0.25 (0.03) 0.46 (0.04) 4.09 (0.62) 4.02 (0.55)

B1 Aiptasia pallida Bermuda 0.23 (0.03) 0.45 (0.03) 3.05 (0.09) 1.63 (0.88)

B1* Aiptasia puchella Hawaii 0.19 (0.03) 0.39 (0.04) 3.32 (0.65) 2.07 (0.29)

B1** Acropora spp. Aquarium (UK) 0.21 (0.02) 0.39 (0.03) 1.42 (0.19) 0.95 (0.10)

F2 Meandrina meandrites Jamaica 0.32 (0.04) 0.76 (0.06) 7.99 (0.61) 4.63 (1.15)

182 Coral Reefs (2009) 28:179–195

123

absorption (a, m-1) and emission at 730 nm (F730) were

normalised to chlorophyll a to yield a* (m2 mg chl a-1)

and F*(IU mg chl a-1). Chlorophyll a concentrations for

each sample concentrate were determined using a 4�C

methanol extraction measured spectrophotometrically,

since methanol is an effective extractor of chlorophyll

(Porra et al. 1989).

PSII fluorescence measurements

A Satlantic Inc. (Halifax, Canada) Fluorescence Induction

and Relaxation (FIRe) fluorometer was used to generate

single turnover (ST) and multiple turnover (MT) fluores-

cence transients (full fluorescence profiles over a pre-

determined time period (Fig. 1)) to characterise photosys-

tem II (PSII) photochemistry on unconcentrated algae as

described by Suggett et al. (2008). Briefly, the FIRe was

programmed to deliver acquisitions each comprising a 4-

step sequence analogous to the modified fast repetition rate

fluorescence technique of Kolber et al. (1998) (Fig. 1). The

4 steps included (1) a 100 ls ST excitation pulse, (2)

relaxation following the ST event from a weak modulated

light over 500 ms, (3) MT excitation from a 600 ms pulse

and (4) relaxation following the MT event over 1 s. Twenty

iterations of the 4-step sequence were averaged into a

single fluorescence transient to increase the signal-to-noise

ratio for each sample. Similarly, instrument gain was

modified so as to ensure that the MT fluorescence transient

was always ca. 70% of the saturating signal. FIRe sample

excitation was from high luminosity blue and green light

emitting diodes (450 nm and 500 nm peak emission,

respectively, with 30 nm bandwidth) with a combined

emission of ca. 0.03 mol photons m-2 s-1 (Suggett et al.

2008). Fluorescence emission was detected at 680 nm

(20 nm bandwidth) using a sensitive avalanche photodiode.

All FIRe measurements were performed in a climate-con-

trolled room that matched the temperature for growth

throughout.

Values of the minimum, maximum ST and maximum

MT fluorescence yields and the functional absorption cross

section of PSII (which describes the rise from minimum to

maximum fluorescence yields) were determined by fitting

the bio-physical (KPF) model of Kolber et al. (1998) to

each fluorescence transient using FIREPRO software pro-

vided by Satlantic Inc. Under dark-adapted conditions,

these parameters are termed Fo, FmST and Fm

MT (dimen-

sionless) and rPSII, respectively. Here, rPSII describes the

rise from Fo to FmST and is output in relative values. FIRe-

fluorescence transients were also collected under actinic

light exposure during light–response curves. These light–

response curves were performed on each sample, where

actinic PFDs between 5 and 500 lmols photons m-2 s-1

were delivered for periods of 4 mins using a calibrated

actinic light source (ALS, Satlantic Inc.) as detailed by

Suggett et al. (2008). Under actinic light, the analogous

fluorescence parameters are termed Fo0, Fm

ST0, FmMT0 and

rPSII0, respectively. To adjust values of effective absorption

to absolute values with units of A2 quanta-1, values here

were compared with those obtained from a calibrated FIRe

fluorometer (see Suggett et al. 2008). The maximum PSII

photochemical efficiency was determined as (Fm – Fo)/

FmST = Fv/Fm

ST or (Fm – Fo)/FmMT = Fv/Fm

MT following the

terminology of Kromkamp and Forster (2003). Non-photo-

chemical quenching, NPQ, was calculated as (FmST – Fm

ST0)/Fm

ST0, and informed of the quantum yield of non-photo-

chemical quenching (Gorbunov et al. 2001). Non-

linearities of the instrument response were determined for

each gain setting from an extract of chlorophyll a prior to

experimentation. Blanks were run on filtrates of each

sample. Both the instrument response and blank were

subtracted from the sample fluorescence transient at the

time of fitting the KPF model.

Pigments and packaging

Aliquots of dark-adapted samples from each culture

were collected and filtered through Whatman GF/F filters

and stored at -80�C (Suggett et al. 2003) for 4 months.

Filtrates were extracted in methanol with ultrasonifica-

tion (McDougall et al. 2006) and then centrifuged

to clarify. Subsequent HPLC analysis was performed

using an integrated Thermoseparations High-Performance

Liquid Chromatography (HPLC) system as described by

McDougall et al. (2006). Pigments were identified and

Time

Rel

ativ

e flu

ores

cenc

e yi

eld

(F/F

o)

1.0

1.5

2.0

2.5

Fo

FmST

FmMT

σ PS

II

(a) (b) (d)(c)

Fig. 1 Example of fluorescence induction trace using a FIRe

fluorometer. Fluorescence parameters of minimum fluorescence, Fo,

maximum fluorescence (single turnover) FmST, maximum fluorescence

multiple turnover FmMT, and effective cross-section absorption of PSII

(rPSII) are illustrated. Letters a–d refer to fluorescence induction

stages; (a) describes the rise in fluorescence following a 100 ls single

turnover (ST) event, (b) is the 500 ms relaxation stage following the

ST event, (c) is the fluorescence rise from a multiple turnover (MT)

flash over 600 ms and (d) is the relaxation stage following the MT

event over 1 s

Coral Reefs (2009) 28:179–195 183

123

quantified (mg m-3) via co-elution with pigment standards

for chlorophyll a, chlorophyll c2, peridinin, diadinoxanthin

(Dd), diatoxanthin (Dt) and b-carotene.

An absorption spectrum was reconstructed from the

HPLC pigment data according to Moore et al. (2005)

(Eq. 1), since reconstructed spectra provide absorption

where pigments are not packaged within the thylakoid

membranes; in contrast, optical absorption measured using

a spectrophotometer accounts for pigment packaging.

aTpig kð Þ ¼X

ai kð Þci ð1Þ

aTpig(k) accounts for the spectral absorption for all

pigments, ai (k) is the in vivo weight-specific absorption

coefficient and c is the concentration of each pigment

(i).Values for aTpig(k) were calculated using data for ai (k)

digitised from Levy et al. (2003), where chlorophyll a,

chlorophyll c2, peridinin, diadinoxanthin and diatoxanthin

were extracted from Symbiodinium spp. from Favia favus

and Plerogyra sinuosa. ai (k) for b-carotene was obtained

from Bidigare et al. (1990). Importantly, aTpig(k) does not

account for how pigments are packaged within the

thylakoid membranes; therefore, a pigment ‘Packaging

Index’ (PI) could be estimated from the ratio of absorption

determined optically (a*) and reconstructed from pigment

concentrations in vivo (aTpig)

PI ¼400700

Pa� kð Þ

400700

PaTpig kð Þ ð2Þ

Calculating PSII and PSI specific absorption

The wavelength-specific absorption cross section for PSII,

rPSII(k), was calculated according to Suggett et al. (2007),

whereby F* spectra were normalised according to the

corresponding value of rPSII measured by the FIRe at peak

excitation in the blue at 478 nm (Eq. 3)

rPSII kð Þ ¼ rPSIIð478Þ=F*(478)

� �� F* kð Þ ð3Þ

a*PSII (k) (m2 mg chl a-1) was then calculated using

wavelength-specific absorption as

a�PSII kð Þ ¼ rPSIIðkÞm2

mol RCII�mol RCII

mg chl a� 0:00675

� �� 1�Fv=FST

m

ð4Þ

where 1/Fv/FmST is used to account for the potential

absorption by non-photosynthetic pigments (Suggett

et al. 2007) and 0.00675 accounts for the conversion

of mol RCII mg chl a-1 to mol RCII mol chl a-1 and

rPSII from A2 quanta-1 to m2 mol RCII-1. a*PSI (k)

(m2 mg chl a-1) was calculated from the difference

between chlorophyll a-specific light absorption a* (k)

and a*PSII (k) (m2 mg chl a-1).

a� kð Þ ¼ a�PSII kð Þ þ a�PSI kð Þ ð5Þ

Chlorophyll a-specific absorption coefficients from 400 to

700 nm for both photosystems were then calculated by

weighting a*PSII (PSI) (k) to the spectrum of the lights used

for growth PFD as a*PSII (PSI) (m2 mg chl a-1) according to

Suggett et al. (2007), Eq. 6.

�a�PSIIðPSIÞ ¼P

a�PSIIðPSIÞ kð Þ � PFD kð ÞP

PFD kð Þ ð6Þ

The absorption coefficients for all photosynthetic

pigments, aPSC, were calculated by weighting the sum of

weight-specific absorption coefficients, (ai) to the FIRe LED

spectra and pigment concentrations (ci) for photosynthetic

pigments (Eq. 7). The product of aPSC, chl a: RCII and PI

was subsequently used to calculate an independent measure

of rPSII (rPSII*, Eq. 7) in m2 mol RCII-1, where 892500

accounts for the conversion of mol chl a: mol RCII to mg chl

a: mol RCII, 1/6023 for the conversion of m2 mol RCII-1 to

A quanta-1 and 1/2 for the assumption that absorption for

photosynthetic pigments is equal between photosystems.

rPSII� ¼P

ai kð Þ � ci kð Þ � EFIRe kð ÞPEFIRe kð Þ

� �� chl a

RCII� PI�

892500 � 1

6023� 12

ð7Þ

Statistical analysis

Heteroscadistic data and data deviating from a normal

distribution were transformed (log n ? 1, Zar 1996) prior

to statistical analyses. Analyses of variance (ANOVA)

were used to examine differences between multiple sample

means. One-way ANOVA was used followed by a Tukey

Post Hoc comparison. Pearson product-moment correla-

tions were used to analyse associations between variables

and linear regressions to predict pigment concentrations

from optical absorption.

Results

Growth

Algal type F2 exhibited the highest values for both the

growth rate (l day-1) and chl a cell-1 under LL growth

(Tables 2, 3). B1 algal types generally exhibited the lowest

l day-1 and chl a cell-1 under LL growth. Growth

increased and chl a cell-1 decreased between LL and HL

(Table 2) for all algal types by approximately a factor of 2.

A3 was an obvious exception, which exhibited a doubling

of growth with no corresponding decrease of chl a cell-1

with increased PFD.

184 Coral Reefs (2009) 28:179–195

123

Photosynthetic unit size and concentration

Values of photosynthetic unit ‘size’, chl a: RCII and chl a:

RCI were also highly variable with algal type (Table 4),

with statistical groupings not correlating with overall clade

type (Table 3). However, under LL, highest and lowest

values of both chl a: RCI and chl a: RCII were observed for

types F2 and A1, respectively. Under HL, most algal types

exhibited a decrease in chl a: RCII (mean across all types

ca. 20%); however, algal type A1.1. exhibited an increase

whilst type A2 exhibited no change in chl a: RCII. Changes

to chl a: RCI with increased growth PFD were also highly

variable between types, either increasing (A1.1), decreas-

ing (A2, B1, B1*, F2) or exhibiting little or no change (A1,

A3, B1**). Similarly, cellular quotas of both RCI and

RCIIs were highly variable between algal types, and under

HL, most types decreased cellular RC content. RCII cell-1

decreased by ca. 50% and RCI cell-1 by ca. 60%. F2 was

the only type to increase cellular RC content from LL to

HL. Consequently, RC stoichiometry of PSII to PSI (RCII:

RCI) did not vary in a consistent pattern for algal type or

growth PFD, even though mean values of chl a: RCI and

chl a: RCII across all algal types decreased by a similar

extent from LL to HL (ca. 20%) and the mean RC stoi-

chiometry was unchanged.

Pigment concentrations

Peridinin (per) followed by chloropyll c (chl c) were the

most abundant accessory pigments for all algal types

(compared to chlorophyll a) (Table 5). Since PI and cell

size influence absorption (Laurion et al. 2004; Moore et al.

2005), algal pigments were expressed relative to chloro-

phyll a to enable cross comparison between types.

Relatively little variability was observed (x% from the

mean) between algal types for any of the pigments under

LL; per: chl a (8.9 %), chl c: chl a (5.1 %), Dt ? Dd: chl a

(13.2 %), and b-carotene: chl a (7.8 %). Similarly, little

variability was observed under HL; per: chl a (15.1 %), chl

c: chl a (7.5 %), Dt ? Dd: chl a (12.5 %), and b-carotene:

chl a (11.4 %). However, significant variability was

observed in the de-epoxidation state (Dt/(Dd ? Dt)) of

algal types. Overall, no single pattern of variability was

evident amongst algal types for any pigments (Tables 3, 5).

The ratio of ‘photosynthetically active’ pigments (peridinin,

chlorophyll c) to chlorophyll a decreased whilst the ratio of

‘photoprotective’ pigments (diadinoxanthin, diatoxanthin,

b-carotene) increased between LL and HL growth condi-

tions. On average, across all clades, per: chl a and chl c: chl

a decreased by ca. 30% and 15% whilst Dt ? Dd: chl a and

b-carotene: chl a increased by ca. 35% from LL to HL. The

greatest increases of the de-epoxidation state with increased

growth PFD were observed for all types A and F2.Ta

ble

3S

tati

stic

alT

uk

eyg

rou

pin

gs

foll

ow

ing

AN

OV

As

of

all

Sym

bio

din

ium

typ

esfo

rm

easu

red

var

iab

les

un

der

LL

and

HL

Lo

wli

gh

tH

igh

lig

ht

Gro

wth

(ld

ay-

1)

F7,1

6=

4.6

2,

P\

0.0

05

(B1

*,

A1

.1,

B1

**

)(A

2,

B1

)(A

3)

(A1

,F

2)

F7,1

6=

4.6

2,

P\

0.0

05

(B1

*,

B1

**

)(A

1.1

)(B

1)

(A2

,A

3)

(A1

)(F

2)

chl

ace

ll-

1(g

cell

-1(e

xp

-13))

F7,1

6=

9.2

9,

P\

0.0

01

(B1

**

)(B

1,

B1

*,

A1

.1)

(A3

,A

1,

A2

)(F

2)

F7,1

4=

5.7

8,

P\

0.0

05

(B1

**

,A

2)

(B1

,A

1.1

,B

1*

,A

1,

A3

)(F

2)

chl

a:

RC

IIF

7,1

6=

3.9

3,

P\

0.0

5(A

1,

A1

.1)

(A2

,B

1*

*,

A3

,B

1*

,B

1)

(F2

)F

7,1

4=

4.1

2,

P\

0.0

5(F

2,

A1

,B

1)

(B1

**

,A

3,

A2

,B

1*

)(A

1.1

)

chl

a:

RC

IF

7,1

6=

38

.2,

P\

0.0

01

(A1

,A

1.1

,B

1,

A2

)(B

1*

*)

(A3

,B

1*

)(F

2)

F7,1

4=

6.1

6,

P\

0.0

05

(A2

,B

1,

A1

,A

1.1

)(B

1*

,B

1*

*)

(F2

,A

3)

RC

II:

RC

IF

7,1

6=

18

.7,

P\

0.0

01

(B1

)(B

1*

*,

A1

,A

1.1

,A

2)

(B1

*,

A3

)(F

2)

F7,1

4=

5.3

1,

P\

0.0

1(A

1.1

,B

1*

,A

2,

B1

)(A

1,

A3

,B

1*

*)

(F2

)

per

:ch

la

F7,1

6=

49

.2,

P\

0.0

01

(A1

.1)

(A1

,B

1*

*,

B1

*)

(B1

)(A

2,

A3

,F

2)

F7,1

4=

14

4,

P\

0.0

01

(A1

.1)

(B1

*,

B1

**

)(A

1,

B1

)(F

2)

(A2

,A

3)

chl

c:ch

la

F7,1

6=

40

.0,

P\

0.0

01

(A1

,A

1.1

,B

1)

(B1

**

,A

3,

B1

*,

A2

,F

2)

F7,1

4=

14

.5,

P\

0.0

01

(B1

*,

A1

.1,

B1

**

,A

1)

(B1

)(A

2,

F2

,A

3)

Dd

?D

t:ch

la

F7,1

6=

38

.67

,P

\0

.00

1(A

3,

F2

,A

2,

A1

,A

1.1

)(B

1)

(B1

*)

(B1

**

)F

7,1

4=

51

.3,

P\

0.0

01

(A1

,F

2,

A2

)(A

3)

(B1

*,

B1

,A

1.1

)(B

1*

*)

b-c

ar:

chl

aF

7,1

6=

7.3

4,

P\

0.0

01

(F2

)(A

2,

A3

,B

1,

B1

**

,B

1*

)(A

1)

(A1

.1)

F7,1

4=

19

.0,

P\

0.0

01

(A3

)(A

1,

A2

,F

2)

(A1

.1)

(B1

*,

B1

**

,B

1)

Dt/

(Dd

?D

t)F

7,1

6=

24

.7,

P\

0.0

01

(A1

,F

2)

(A2

,A

3,

B1

,A

1,1

)(B

1*

,B

1*

*)

F7,1

4=

37

.4,

P\

0.0

01

(B1

*,

F2

,B

1)

(B1

**

)(A

1,

A1

.1,

A3

)(A

2)

Fv/F

mST

F7,1

6=

59

.0,

P\

0.0

01

(B1

,B

1*

)(B

1*

*,

A1

.1,

A3

,F

2)

(A2

,A

1)

F7,1

6=

35

.8,

P\

0.0

01

(B1

)(B

1*

*,

B1

*,

A1

.1)

(A3

)(A

2,

F2

)(A

1)

rP

SII

F7,1

6=

15

.62

,P

\0

.00

1(F

2)

(A2

,A

1,

A1

.1)

(B1

,A

3)

(B1

*,

B1

**

)F

7,1

6=

3.8

1,

P\

0.0

5(A

1.1

,B

1*

)(B

1)

(F2

,A

1,

A2

)(A

3)

(B1

**

)

All

gro

up

sre

pre

sen

tst

atis

tica

lly

sim

ilar

typ

es,

incl

ud

ing

ov

erla

pp

ing

gro

up

s.G

rou

ps

and

ind

ivid

ual

typ

esar

eo

rder

edle

ftto

rig

ht

fro

mlo

wes

tto

hig

hes

td

ata

val

ues

Coral Reefs (2009) 28:179–195 185

123

Light absorption

Unfortunately, type F2 frequently formed aggregates, in

particular during the gravity filtration for concentrating

material thereby making all bio-optical measurements

problematic. Consequently, F2 data were omitted from the

bio-optical data sets.

Chlorophyll a-specific absorption, as determined from

both optical measurements and pigment reconstructions

(Eq. 1), was greatest in the blue–green region (450–525 nm)

Table 4 PSU size (chl a: RCII (I) (mol: mol)), cellular RC content (exp-19) and reaction centre stoichiometry (RCII: RCI, dimensionless)

(±SE) for all Symbiodinium types at LL and HL

Algal type chl a: RCII (mol: mol) chl a: RCI (mol: mol) RCII cell-1 RCI cell-1 RCII: RCI

Low light A1 392 (41.0) 775 (56.5) 17.9 9.13 1.96

A1.1 405 (9.73) 821 (61.6) 11.1 5.49 2.02

A2 453 (32.9) 1020 (41.8) 16.1 7.09 2.27

A3 513 (21.3) 1440 (60.0) 8.92 3.16 2.82

B1 590 (14.4) 983 (149) 5.79 3.47 1.67

B1* 557 (28.3) 1430 (60.0) 6.67 2.49 2.68

B1** 495 (144.8) 1010 (455) 31.9 16.4 1.94

F2 635 (48.7) 3536 (35.8) 14.1 2.51 5.62

Mean 505 1380 14.1 6.22 2.62

High light A1 323 (19.0) 854 (53.3) 10.4 3.94 2.64

A1.1 639 (35.2) 916 (4.40) 3.04 2.11 1.44

A2 454 (180) 650 (132) 6.95 3.78 1.84

A3 439 (50.4) 1390 (19.1) 10.2 3.51 2.91

B1 327 (51.8) 676 (182) 1.51 0.76 2.00

B1* 505 (20.8) 921 (63.4) 2.54 1.39 1.83

B1** 313 (63.0) 1124 (61.2) 2.97 0.79 3.78

F2 273 (20.3) 1274 (52.7) 18.9 4.01 4.71

Mean 409 976 7.06 2.53 2.64

Table 5 Pigment ratios (±SE) normalised to chlorophyll a for peridinin, chlorophyll c, diadinoxanthin ? diatoxanthin (Dd ? Dt), b-carotene

and Dt/(Dd ? Dt) for all Symbiodinium types at LL and HL

Algal type per: chl a chl c: chl a Dd ? Dt: chl a b-car: chl a Dt/(Dd ? Dt)

Low light A1 0.535 (0.010) 0.242 (0.003) 0.176 (0.011) 0.026 (0.001) 0.005 (0.000)

A1.1 0.418 (0.005) 0.250 (0.001) 0.188 (0.004) 0.028 (0.000) 0.019 (0.007)

A2 0.608 (0.003) 0.284 (0.001) 0.171 (0.009) 0.022 (0.001) 0.013 (0.002)

A3 0.621 (0.002) 0.279 (0.001) 0.165 (0.001) 0.022 (0.001) 0.013 (0.002)

B1 0.555 (0.006) 0.257 (0.002) 0.198 (0.003) 0.023 (0.000) 0.017 (0.002)

B1* 0.548 (0.013) 0.283 (0.002) 0.214 (0.006) 0.025 (0.000) 0.056 (0.004)

B1** 0.535 (0.021) 0.275 (0.004) 0.277 (0.004) 0.023 (0.001) 0.060 (0.006)

F2 0.658 (0.014) 0.289 (0.004) 0.166 (0.002) 0.021 (0.002) 0.005 (0.001)

Mean 0.560 0.270 0.194 0.024 0.024

High light A1 0.365 (0.011) 0.215 (0.002) 0.212 (0.003) 0.030 (0.002) 0.105 (0.010)

A1.1 0.265 (0.002) 0.207 (0.001) 0.287 (0.003) 0.034 (0.001) 0.120 (0.001)

A2 0.461 (n/a) 0.242 (n/a) 0.228 (n/a) 0.030 (n/a) 0.204 (n/a)

A3 0.470 (0.008) 0.255 (0.002) 0.260 (0.002) 0.026 (0.001) 0.136 (0.003)

B1 0.369 (0.007) 0.222 (0.011) 0.286 (0.003) 0.039 (0.001) 0.065 (0.008)

B1* 0.312 (0.001) 0.202 (0.002) 0.275 (0.004) 0.036 (0.001) 0.055 (0.001)

B1** 0.318 (0.003) 0.210 (0.003) 0.330 (0.007) 0.037 (0.001) 0.079 (0.001)

F2 0.406 (0.003) 0.245 (0.004) 0.213 (0.009) 0.030 (0.001) 0.058 (0.001)

Mean 0.365 0.215 0.212 0.030 0.105

186 Coral Reefs (2009) 28:179–195

123

(e.g. Fig. 2). Similarly, estimates of packaging using the PI

(Eq. 2) were highest (=PI lowest) at these wavelengths.

Packaging was lowest and highest for algal types B1 and A2,

respectively, and both the absorption coefficients and values

of PI typically increased from LL to HL by ca. 10% for most

algal types (Table 6). Notable exceptions to this general

trend were B1 types which exhibited minimal packaging at

either growth PFD.

Chlorophyll a-specific absorption coefficients at wave-

lengths of peak absorption (peridinin, k = 474 nm;

chlorophyll c, k = 446 nm; chlorophyll a k = 430 and

662 nm; diadinoxanthin, k = 448 nm; diatoxanthin, k =

454 nm) were compared between those measured optically

(a) and those reconstructed from pigment concentrations

(aTpig). Both coefficients were significantly correlated for

all pigment types (Table 7) suggesting that absorption

variability (Table 6) between types and growth PFD was

determined by variability of pigment content (Table 5).

Photosystem-specific absorption

Chlorophyll a-specific absorption (a*) was partitioned into

a* specific to PSI and PSII (Eqs. 4, 5). Typically, a*PSII

comprised ca. 50% and 75% of the total absorption (a*) at

the red chlorophyll a peak, ca. 680 nm, and in the blue

green region, 400–500 nm, respectively (Fig. 3b). The ratio

of a*PSII(k) to a*PSI(k) (weighted to the spectral quality of

light used for growth, a*PSII: a*PSI) was highest for type A3

and lowest for type A2 under LL growth (Table 6). All

values remained above 1 suggesting preferential absorption

by PSII throughout. A decrease of a*PSII: a*PSI was

observed for A1, A2 and B1* types between LL and HL by

Wavelength (nm)

450 500 550 600 650 700

Wavelength (nm)

400 450 500 550 600 650 700

a* (

m2 m

g ch

l a-1

)

0.00

0.01

0.02

0.03

0.04

0.05

0.06B1**B1*B1A1.1A3A1A2

(a) (b)Fig. 2 Optical absorption

normalised to chlorophyll a, a*

(m2 mg chl a -1), from 400 to

715 nm for all Symbiodiniumtypes except F2 under (a) LL

and (b) HL

Table 6 Packaging index (PI),

growth-spectra weighted

photosystem-specific absorption

coefficient stoichiometry

(a*PSII: a*PSI), absorption

coefficients from 400 to 700 nm

(a*(400–700)) and absorption per

cell a*(400–700) cell-1 (m2 cell-1

(exp-12)) (±SE) for all

Symbiodinium types at LL and

HL

Algal type Packaging index a*PSII: a*PSI a*(400–700)

m2 mg chl a-1a*(400–700) cell-1

(m2 cell-1 (exp-12))

Low light A1 0.75 1.967 0.0084 (0.0002) 5.30 (0.85)

A1.1 0.9 1.819 0.0092 (0.0005) 5.80 (2.52)

A2 0.63 1.476 0.0081 (0.0002) 7.08 (1.96)

A3 0.75 2.933 0.0092 (0.0001) 3.79 (0.61)

B1 0.98 1.497 0.0116 (0.0001) 3.46 (0.21)

B1* 1 2.043 0.0116 (0.0001) 3.82 (6.58)

B1** 1 2.153 0.0133 (0.0006) 1.83 (0.07)

F2 N/A N/A 0.0071 (0.0002) 3.62 (2.60)

Mean 0.859 1.984 0.0098 4.34

High light A1 1 1.157 0.0097 (0.0001) 2.94 (0.84)

A1.1 1 0.87 0.0113 (0.0001) 1.98 (0.44)

A2 0.7 0.86 0.0107 (0.0024) 1.91 (n/a)

A3 0.85 2.066 0.0092 (0.0003) 3.65 (0.30)

B1 1 2.110 0.0162 (0.0001) 4.33 (0.12)

B1* 1 1.016 0.0144 (0.0014) 1.65 (0.64)

B1** 1 2.749 0.0167 (0.0001) 1.38 (0.22)

F2 N/A N/A 0.0088 (0.0002) 4.03 (0.95)

Mean 0.936 1.546 0.0121 2.73

Coral Reefs (2009) 28:179–195 187

123

up to 50%. B1 and B1** were the only types that increased

a*PSII: a*PSI between light conditions. For types A1.1 and

A2, values of a*PSII: a*PSI decreased below a value of 1

suggesting preferential absorption to PSI at HL. These

changes of absorption between PSII and PSI were closely

matched by the simultaneous changes of RCII: RCI

(Fig. 4a), so algal types and growth conditions which

favoured more RCII than RCI content exhibited a greater

capacity for absorption by PSII.

The product of chlorophyll a-specific absorption (m2 mg

chl a-1) and the inverse of the PSU size (RC mol chl a-1)

was subsequently used to calculate the effective absorption

per RC (m2 mol RC-1) for PSI and PSII (Fig. 4b). Here,

changes in LL cultures were closely proportionate to 1:1

line, suggesting a balance of maintaining light harvesting

capacity per unit reaction centre between photosystems

throughout. However, at HL this balance decreased, with a

bias towards increased absorption per RCI. As demon-

strated in Fig. 4a, algal types with more RCII than RCI

content had a greater capacity for PSII absorption. To

further demonstrate this at the cellular level, PSII and PSI

absorption per cell was calculated from the product of

absorption per RC (m2 mol RCII-1) and RC per cell (mol

RCII cell-1, Fig. 4c). Clearly, absorption by PSII was

consistently higher than absorption by PSI at LL but not at

HL across these Symbiodinium types. At HL, most algal

types had higher absorption per cell by RCI thereby indi-

cating greater excitation of PSI under HL. The only two

exceptions were types A2 and A3 which had preferential

absorption cell-1 by PSII under HL (Fig. 4c).

Dark-adapted physiology

Measurements of Fv/FmST and rPSII were used to charac-

terise physiological state and to calculate the proportion of

absorption directed to PSII and PSI (Eq. 4). Under steady

Table 7 Regression equations for a (optical (m-1)) for pigments

characterised by Levy et al. (2003)a. P (m-1) denotes the predictor

(concentration of pigment (mg m-3) � pigment peak absorption

coefficient (m2 mg-1) � 50, to account for dilution factors between

variables)

Pigment Regression equation Test results

Chl ared 3.70 ? (0.44 * P) (96.8) P \ 0.001, F1,11 = 328

Chl ablue 24.9 ? (0.91 * P) (92.5) P \ 0.001, F1,11 = 137

Chl c 26.8 ? (2.54 * P) (86.8) P \ 0.001, F1,11 = 72.5

Diadinoxanthin 43.8 ? (1.41 * P) (72.0) P \ 0.001, F1,11 = 28.3

Diatoxanthin 72.9 ? (12.9 * P) (37.4) P = 0.026, F1,11 = 6.57

Peridinin 33.7 ? (0.64 * P) (78.8) P \ 0.001, F1,11 = 40.9

b-carotene 13.2 ? (1.32 * P) (89.9) a P \ 0.001, F1,11 = 98.1

chl ared denotes chlorophyll peak absorption in the red (662 nm)

region and chl ablue in the blue region (ca. 430 nm). r2 values are

superscripted in parentheses. ab-carotene peak absorption coefficients

were taken from Bigidare et al. (1990)

Wavelength (nm)

a * (

m2 m

g ch

l a-1

)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

a* opticala*PSII optical

a* HPLC

Wavelength (nm)

Perc

enta

ge

0

20

40

60

80

100

Pack

agin

g In

dex

0.0

0.2

0.4

0.6

0.8

1.0

a*PSII % of a*

Packaging Index

(a) (b)

400 450 500 550 600 650 700 400 450 500 550 600 650 700

Fig. 3 a Optical and HPLC

reconstructed a* and aTpig*

(m2 mg chl a -1) from 400 to

715 nm. a*PSII of a*optical

shown as a dashed line. b a*PSII

expressed as a percentage total

of a*optical and packaging

index (PI) calculated from

pigment concentrations

*PS

I · R

CI

(m2 m

ol R

CI-1

) *

106

RCII: RCI0 1 2 3 4 5

0

1

2

3

4

5

LLHL

(PSII) m2 cell-1*10-120 2 4 6 8

(PSI

) m

2 cel

l-1*1

0-12

0

2

4

6

8

0 2 4 6 80

2

4

6

8

(a) (b) (c)

*PS

II:

*PS

I

* PSII · RCII (m2 mol RCII-1) * 106

Fig. 4 a Mean ratio of a*PSII: a*PSI against RCII: RCI. Average S.E.

LL 9%, HL 18%. b The product of chlorophyll a-specific absorption

(m2 mg chl a-1) and the inverse of the PSU size (RC mol chl a-1) for

PSI and PSII to give effective absorption per RC (m2 mol

RC-1(Exp 6)) with a 1:1 line included. c Results from (b) normalised

to RC cell-1 to give RC absorption per cell (m2 cell-1 (Exp -12))

188 Coral Reefs (2009) 28:179–195

123

state growth, values for both Fv/FmST and rPSII were highly

variable between algal type and growth PFD (Table 8). Fv/

FmST remained highest and lowest for types A1 and B1 at

both growth PFDs. Mean values for Fv/FmST and rPSII were

ca. 25% and 5% lower under HL than LL, respectively, but

magnitude of change was type-dependent and the statistical

groupings of both parameters were not identical under both

growth irradiances (Table 3). When Fv/FmST and growth

rates were compared, two general clusters (LL and HL)

existed from trends of increased Fv/FmST and decreased

growth under LL conditions, consistent with previous algal

studies (Robison and Warner 2006). However, at each

growth PFD, changes in Fv/FmST were independent of the

corresponding changes of growth rate between algal types

(Fig. 5a). This supports that variability of Fv/FmST was dri-

ven by adaptive differences and not by differences in

nutrient availability between cultures, since trends

observed between algal types at either growth PFD should

be matched when compared across both growth PFDs.

Similarly, Fv/FmST, which is indicative of RCII activity, did

not negatively correlate with changes in rPSII (Pearson

correlation = 0.001, P = 0.998), a pattern that would be

expected in response to nutrient limitation (Kolber et al.

1988) (Fig. 5b). Measurements of Fv/Fm derived from the

single turnover induction measurements were compared to

Fv/Fm derived from the multiple turnover induction (also

measured by the FIRe (Fig. 1)). Values of Fv/FmMT were

very strongly correlated with those of Fv/FmST (Pearson

correlation = 0.962, P \ 0.001), but were consistently

higher by ca. 15% (Fig. 5c).

Values of rPSII are determined by the concentration,

type and intracellular arrangement of pigments, as well as

RCII concentration (Suggett et al. 2004). An independent

measure of rPSII was calculated using Eq. 7 as rPSII*.

When compared with rPSII, all algal types and growth

conditions yielded values of rPSII* that accounted for ca.

53% of the variability expressed in rPSII (Fig. 5d). In

general, HL and LL data were clustered but some overlap

was observed.

Physiology under the growth irradiance

NPQ was determined from fluorescence yields measured

during the actinic light–response curve at the PFD corre-

sponding to that used for growth. All PFDs from the light-

response curve were spectrally corrected to growth cabinet

spectra (Suggett et al. 2008). At LL, all algal types had

little NPQ, whereas at HL, NPQ was variable between

types. Changes in NPQ did not correlate with changes in

rPSII consistently between low and high light (Fig. 6a).

Values of rPSII were also compared with the de-epoxida-

tion state of xanthophyll pigments (Fig. 6b), where an

increase in Dt/(Dd ? Dt) (and hence increased Dt relative

to Dd) equated to an increase in non-photochemical

quenching, as more diadinoxanthin had been de-epoxidated

to diatoxanthin. Similar clustering of LL and HL algal

types was observed when rPSII was compared to NPQ and

to Dt/(Dd ? Dt).

In part, the lack of co-variability between rPSII and the

de-epoxidation state of xanthophyll pigments may reflect

that dark-adapted measures of rPSII may not account for

additional rapid mechanisms that act to quench PFD.

Therefore, values of rPSII at PFDs which corresponded to

the algal growth irradiance (rPSII0) were normalised

against dark-adapted values, as rPSII0/rPSII. This provides

a specific measure of antennae-bed non-photochemical

quenching (Gorbunov et al. 2001, Suggett et al. 2007). In

this case, two clusters were formed dependent on growth

irradiance; LL algal types had low antennae-bed

quenching (high rPSII0/rPSII) and low NPQ/de-epoxidation

state of xanthophyll pigments, whereas HL types had high

antennae-bed quenching (low rPSII0/rPSII) and increased

NPQ or Dd de-epoxidation. HL algal types exhibited

substantial antennae-bed quenching variability (Fig. 6b,

c), but this variability significantly correlated with the de-

epoxidation of xanthophyll pigments for both growth

irradiances (Pearson’s correlation = -0.594, P \ 0.05)

suggesting that increased antennae-bed quenching likely

occurred under high growth PFD, and was directly

determined by the potential of xanthophyll cycling via de-

epoxidation.

Discussion

Symbiodinium photoacclimation strategy

Substantial variability was observed between algal type

and growth PFD for almost every bio-optical and

Table 8 Maximum Single Turnover PSII photochemical efficiency

(Fv/FmST) (±SE) and effective cross-section absorption of PSII (r PSII)

for all Symbiodinium types at LL and HL

Algal type Low light High light

Fv/FmST r PSII Fv/Fm

ST r PSII

A1 0.54 (0.03) 410 (10.0) 0.42 (0.02) 405 (7.88)

A1.1 0.46 (0.02) 411 (13.3) 0.32 (0.03) 378 (5.69)

A2 0.53 (0.02) 406 (10.2) 0.38 (0.02) 400 (6.9)

A3 0.46 (0.03) 463 (0.67) 0.35 (0.02) 434 (21.4)

B1 0.33 (0.02) 450 (8.51) 0.26 (0.02) 372 (18.0)

B1* 0.39 (0.02) 490 (13.3) 0.32 (0.01) 378 (2.03)

B1** 0.44 (0.03) 511 (30.8) 0.31 (0.02) 423 (7.75)

F2 0.48 (0.02) 258 (33.2) 0.37 (0.02) 397 (6.67)

Mean 0.45 425 0.34 403

Coral Reefs (2009) 28:179–195 189

123

bio-physical measurement suggesting adaptive differences

for regulating light harvesting machinery. Several photo-

acclimation strategies inherent to light harvesting are

known to exist for microalgae (Falkowski and Owens

1980; Falkowski and Raven 1997; Suggett et al. 2007):

cells can preferentially favour a change either of compo-

nents comprising the light harvesting antennae (PSU size)

or of the reaction centre pool size (PSU content) available

for processing harvested light (Falkowski and Owens 1980;

Six et al. 2008). The ‘choice’ of strategy varies for different

microalgal species (summarised in Suggett et al. 2007) but

is thought to reflect nutrient and light availability (Moore

et al. 2006; Six et al. 2008). Ultimately, the two predom-

inant strategies lead to significant changes in the antennae

size or RCII content, respectively, and so have recently

been termed r-versus n-type acclimation (Iglesias-Prieto

and Trench 1994; Suggett et al. 2007; Six et al. 2008).

Symbiodinium spp. were previously shown to modify their

RCs in response to a change in light environment (Iglesias-

Prieto and Trench 1994), but the magnitude of RC change

and the primary photosystem modified varied with Sym-

biodinium type. This same variability of ‘strategy’ applied

here both within algal types (types A) and for the same

algal type at the intracladal level (e.g. B1) (Table 3).

Predominance of the ‘‘n-type’’ strategy amongst

Symbiodinium would suggest the role of a generalist algal

strategy for algae which are subject to varying light

intensities and are not nutrient limited (Six et al. 2008).

Modifying RC concentrations over antennae size is bene-

ficial under conditions liable to cause photoinhibition, such

as high light (as experienced on reef flats by corals), by

enabling a ‘compensatory effect’ within cells to increase

electron turnover rates in non-photoinhibited RCs to

maintain maximum photosynthetic rate (Behrenfeld et al.

1998; Kana et al. 2002). However, some types investigated

here (B1 types and the F2 isolate) also exhibited a r-type

acclimation strategy (compared to other types, ca 15–30%

change in rPSII between growth PFDs), in addition to an

n-type strategy. Of course, there might be an advantage to

Symbiodinium types that are able to utilise both strategies,

that is to maximise photoacclimatory ability under variable

light conditions (n-type) and to potentially lower energetic

costs (r-type) under more stable light regimes (Six et al.

2008). Such differences in strategy may provide some

adaptive reasoning for niche separation amongst Symbi-

odinium types, which has been demonstrated to occur

(Futuyma and Moreno 1988; Iglesias-Prieto and Trench

1997; LaJeunesse 2002; LaJeunesse et al. 2004a; Sampayo

Growth (µ day−1)

0.1 0.2 0.3 0.4 0.5 0.6

0.2 0.3 0.4 0.5 0.6

Fv/F

mS

T

0.2

0.3

0.4

0.5

0.6LLHL

Fv /FmMT

Fv/F

mS

T

0.2

0.3

0.4

0.5

0.6

σPSII

σ PS

II*

200

400

600

800

σPSII

350 400 450 500 550

350 400 450 500 550F

v/F

mS

T

0.2

0.3

0.4

0.5

0.6

(b)(a)

(d)(c)

Fig. 5 (a) A comparison

between Fv/FmST and growth

under LL and HL, (b) rPSII

against Fv/FmST (c) a comparison

of Fv/Fm measured using ST and

MT techniques from the FIRe

fluorometer and (d) rPSII

measured fluorometrically

against an independent

assessment of the effective

cross-section absorption, rPSII*

190 Coral Reefs (2009) 28:179–195

123

et al. 2007). However, in order to assess advantages and

disadvantages of both acclimation strategies fully, other

factors such as Symbiodinium cell size and cost-benefit

analysis specific to the host-Symbiodinium symbiosis need

to be considered.

Of key importance from the results was the observation

for preferential absorption of cellular RCII over RCI at LL,

but preferential absorption of RCI at HL. Previous data

have shown that genetic variants of Emiliania huxleyii

balance absorption between photosystems despite differ-

ences in acclimation ‘strategy’ (Suggett et al. 2007).

However, most Symbiodinium types here clearly differ

between growth PFD; the observed preferential absorption

of PSI cell-1 under HL conditions (Fig. 4c) may be a form

of photoprotection, by increasing cyclic electron transport

around PSI (Warner and Berry-Lowe 2006), as cyclic

electron transport around PSI may be important in devel-

oping non-photochemical quenching (NPQ) (Munekaga

et al. 2004). This is complemented by recent evidence that

in some Symbiodinium there is a link between light har-

vesting complex dissociation from PSII and increased

cyclic electron transport around PSI under bleaching

conditions (Reynolds et al. 2008).

Antennae bed variability

The data clearly demonstrated that reaction centre stoichi-

ometry co-varied with light absorption stoichiometry. RCII

content was one of the major factors that regulated the

antennae size in many algae (Iglesias-Prieto and Trench

1994) and consequently also likely accounted for much of

the variability of rPSII (Fig. 5d). However, only ca. 50% of

rPSII variability was accounted for by the independent

measure of rPSII, rPSII* (Fig. 5d). The use of Eq. 7 to derive

rPSII* is limited by the assumption that aPSC is equal between

photosystems. This is not the case for many Symbiodinium

types and consequently rPSII can be overestimated because

of the assumption that aPSC is split equally between photo-

systems (Eq. 7). However, this may not be the case for all

Symbiodinium types (Reynolds et al. 2008). Pigment type

and packaging also act to drive variability of the antennae

size. Whilst these pigment-driven modifications to the

antennae bed size may not be as important for photoaccli-

mation in Symbiodinium as RC content, changes to the

pigment profile and packaging were still observed across

algal types. When rPSII was compared to both NPQ and the

de-epoxidation of xanthophyll pigments, two general

0.00 0.05 0.10 0.15 0.20 0.25400

500

600

700

800

900

1000

1100

0 1 2 3 4

σ PSII

(gro

wth

)

400

500

600

700

800

900

1000

1100

NPQ (Fm -Fm' / Fm')

0 1 2 3 4

σ PSII '(

grow

th)

/ σPS

II

0.4

0.6

0.8

1.0

Low LightHigh Light

Dt / (Dd + Dt)

0.00 0.05 0.10 0.15 0.20 0.25

0.4

0.6

0.8

1.0

(a) (b)

(c) (d)

Fig. 6 NPQ calculated at

growth irradiance and the de-

epoxidation state of xanthophyll

pigments compared to rPSII,

weighted to low and high light

growth spectra (panels a and b,

respectively), and antennae-bed

quenching (rPSII0/rPSII)

measured at growth irradiance

(c and d)

Coral Reefs (2009) 28:179–195 191

123

clusters according to algal growth PFD were observed

(Fig. 6a, b), where low light had lower non-photochemical

quenching (according to both NPQ and Dt/(Dd ? Dt) than at

HL. However, the variability expressed in the de-epoxida-

tion of xanthophyll pigments between algal types was not

matched by rPSII variability (Fig. 6b), which again illus-

trates how antennae size is driven by multiple factors so that

rPSII values may not necessarily correlate with any one

driving factor, such as the proportion of photoprotective

pigments or even PSU size. When rPSII was used to calculate

antennae-bed quenching, rPSII0/rPSII (Gorbunov et al. 2001),

the de-epoxidation state of xanthophyll pigments (and NPQ)

was generally matched by rPSII0/rPSII, meaning that anten-

nae-bed quenching, and its modification under different

growth irradiances, needs to be considered when using rPSII

to infer changes in PSU efficiencies. The change in photo-

synthetic and non-photosynthetic pigments per chlorophyll a

between growth PFDs was also consistent with previous

microalgae observations (Harris et al. 2005; Suggett et al.

2007), where the decrease in photosynthetic pigments (PSC)

relative to photoprotectant pigments (PPC) acts to reduce

excitation energy cascading to the RCs, ultimately pre-

venting photodamage at high light.

All types also followed the general trend of decreased

packaging (increased PI) under high light, consistent with

other Symbiodinium and microalgae studies where a

decline in chlorophyll a and photosynthetic accessory

pigments (from increased irradiance) can ultimately reduce

packaging (Harris et al. 2005; Robison and Warner 2006;

Suggett et al. 2007). Low packaging is also associated with

smaller microalgal cells (Laurion et al. 2004) but Symbi-

odinium cell size remained relatively constant (ca. 4.5 lm

diameter). Given that Symbiodinium absorption increased

between LL and HL, cell size showed little variability and

that packaging was relatively low for most types (at both

light levels) data here suggests that the contribution of

spectrally averaged packaging between types to light

absorption was low, but dependent on algal type.

One of the key results was in demonstrating that the

variability of pigment concentration could largely account

for the variability of optical absorption and was indepen-

dent of algal type or environmental growth conditions

(Tables 5, 6, 7). Data here are consistent with previous

studies, which examined absorption or reflectance in rela-

tion to pigment content across a wide variety of corals

(Hochberg et al. 2006; Apprill et al. 2007). Differences

observed between pigments with regard to their relation-

ship with absorption may be explained by different

packaging between pigments (Harris et al. 2005; Robison

and Warner 2006), hence data here confirm that zooxan-

thellae absorption can be confidently ‘reconstructed’ from

pigment concentrations alone without the need for knowl-

edge of Symbiodinium taxonomic composition.

PSII fluorescence

As with previous investigations that have grown Symbi-

odinium isolates under nutrient replete conditions,

measurements of Fv/Fm in this study varied widely between

types (Tchernov et al. 2004; Robison and Warner 2006).

Differences in media type may account for discrepancies

between data for algal types here and for Tchernov et al.

(2004) (Rodriguez-Roman and Iglesias-Prieto 2005).

Importantly, taxonomic regulation of Fv/Fm reduces the

confidence with which this parameter can be used as a

gauge of environmental response without having full

knowledge of the in hospite community structure of Sym-

biodinium. Since Fv/Fm of in hospite Symbiodinium changes

over light gradients driven by depth and turbidity (Hennige

et al. 2008), it is crucial to account for Symbiodinium

community structure when assessing photoacclimation over

environmental gradients using genetic techniques.

In cultured Symbiodinium studies, variability in Fv/Fm

consistent with observations here were reported in A1,

A1.1, B1 and F2 by Robison and Warner (2006). These

authors measured Fv/Fm using an MT protocol (Fv/FmMT),

and thus yielded values that were typically ca. 15–25%

higher than measures of Fv/FmST in this study, a trend con-

sistent in the comparison of FIRe-based measures of Fv/FmST

and Fv/FmMT. This relationship between Fv/Fm measured

using ST or MT protocols provides a clear demonstration

that Fv/Fm can be readily inter-converted between proto-

cols. However, similar comparisons are still warranted for

comparing Fv/Fm generated from different commercially

available instruments, such as the Diving PAM and FRR.

Environmental versus taxonomic variability

Where variability exists between algal types, the question

is whether genetic variability is larger than that observed

between environmental conditions. This is particularly

important when studying photobiology of corals under

different environmental conditions, such as light environ-

ment, as the algal community may not be constant

(LaJeunesse and Trench 2000; LaJeunesse et al. 2004b; van

Oppen et al. 2005; Warner et al. 2006).

Bio-optical descriptions of microalgal photobiology are

well documented, in particular, for pigmentation and

absorption (Dubinsky et al. 1986; Berner et al. 1989;

MacIntyre et al. 2002). Various studies have previously

determined pigment ratios and optical absorption spectra

for Symbiodinium both in hospite (Enriquez et al. 2005;

Stambler and Dubinsky 2005) and grown in culture

(Dustan 1979; Falkowski et al. 1990; Iglesias-Prieto et al.

1991). However, these reports are hard to compare since

growth conditions such as light and nutrition, which are

known to modify optical characteristics (Dubinsky et al.

192 Coral Reefs (2009) 28:179–195

123

1990; Apprill et al. 2007), vary substantially both between

in hospite studies and between in hospite and cultured

studies.

As a group, changes in bio-optical properties (for exam-

ple pigment concentrations (Table 3)) were usually greater

in response to environmental change (LL and HL) than

variability expressed between Symbiodinium types. All

Symbiodinium types have a taxonomic contribution to their

bio-optical signature but modification between different

light environments acts to ‘dial out’ this variability. Clearly,

some parameters (such as a*) can be used with confidence to

assess acclimation of Symbiodinium types in in situ studies,

regardless of whether the genetic signal of the assemblage

changes. In clear contrast to the bio-optical signatures,

variability in bio-physical signatures between Symbiodini-

um types was often equal to or greater than change between

growth conditions. This of course creates difficulty in using

bio-physical signatures, for example Fv/Fm and rPSII, to

identify acclimation in Symbiodinium assemblages between

growth PFDs without identifying the genetic type.

It is notable that B1 Symbiodinium is considered a

‘‘generalist’’ symbiont on many Caribbean reefs, residing

in several different host taxa across a wide light/depth

range while there tends to be a predominance in clade A

Symbiodinium distribution towards shallow water locations

(LaJeunesse 2002; Warner et al. 2006). With regard to

host-Symbiodinium specificity, Symbiodinium genetic var-

iability may thus reflect how different coral species may

ultimately provide a unique environment for their associ-

ated Symbiodinium through modification of pigmentation

(Apprill et al. 2007), light scatter by the skeleton (Enriquez

et al. 2005) and through resources available to the algae

(Falkowski et al. 1993; Rodriguez-Roman and Iglesias-

Prieto 2005). At present, the understanding of coral–Sym-

biodinium specificity and the benefits to a host of being

adapted to harbour certain algal types that will inevitably

exhibit different photobiology is poor (Lesser and Farrell

2004; Suggett et al. 2008). A systematic set of experiments

including cultured and in hospite Symbiodinium types

would be needed to address this.

In summary, substantial taxonomic variability was

demonstrated to underlie the photobiological characteris-

tics of Symbiodinium. In spite of this variability, general

features are maintained across Symbiodinium as a species,

in particular, greater change of RC content over size for

acclimating to different light conditions. However, the

underlying reasons for this ‘choice’ of strategy are not

clear. Although signatures reported here cannot be used to

assess in hospite Symbiodinium community composition,

the demonstrated adaptive variability between types high-

lights the potential of using chlorophyll a fluorescence, in

conjunction with genetic identification (ITS2), to assay the

phenotypic and taxonomic responses of in hospite

Symbiodinium populations to environmental changes.

Coupled with host-algae specificity and the role of the host

to modify the light environment, it is not surprising that

certain coral species are more suited to certain environ-

ments than others. Future eco-physiological studies will

have to consider all these aspects to draw meaningful

ecological conclusions, but by using a combined bio-

physical and bio-optical approach, coupled with knowledge

of Symbiodinium taxonomic composition, future studies

will have powerful tools to assess bio-physical and bio-

optical responses, to ultimately determine the contribution

of Symbiodinium types to coral photoacclimation.

Acknowledgements The authors wish to thank Phil Davey and

Maxim Gorbunov for technical support of the growth facilities and

FIRe, respectively. This manuscript was greatly improved by com-

ments from two anonymous reviewers and Robert van Woesik. This

work was funded through a National Environmental Research Council

postdoctoral fellowship to DJS, a NERC studentship to SJH, High-

lands and Islands Enterprise (The Scottish Funding Council) and

European Regional Development Funding to KEM, and a National

Science Foundation grant (IOB 544765) to MEW.

References

Apprill AM, Bidigare RR, Gates RD (2007) Visibly healthy corals

exhibit variable pigment concentrations and symbiont pheno-

types. Coral Reefs 26:387–397

Baker AC (2003) Flexibility and specificity in coral-algal symbiosis:

diversity, ecology and biogeography of Symbiodinium. Annual

Review of Ecology Evolution and Systematics 34:661–689

Behrenfeld MJ, Prasil O, Kolber ZS, Babin M, Falkowski PG (1998)

Compensatory changes in Photosystem II electron turnover rates

protect photosynthesis from photoinhibition. Photosynth Res

58:259–268

Berkelmans R, van Oppen MJH (2006) The role of zooxanthellae in

the thermal tolerance of corals: a ‘nugget of hope’ for coral reefs

in an era of climate change. Proc R Soc Lond B Biol Sci

273:2305–2312

Berner T, Dubinsky Z, Wyman K, Falkowski PG (1989) Photoadap-

tation and the package effect in Dunaliella-Tertiolecta(Chlorophyceae). J Phycol 25:70–78

Bidigare RR, Ondrusek ME, Morrow JH, Kiefer DA (1990) In vivo

absorption properties of algal pigments. In: Spinrad RW (ed)

Ocean Optics X (Proceedings Volume 1302). SPIE, Bellingham,

pp 290–302

Chang SS, Prezelin BB, Trench RK (1983) Mechanisms of photo-

adaptation in three strains of the symbiotic dinoflagellate

Symbiodinium microadriaticum. Mar Biol 76:219–229

Coffroth MA, Santos SR (2005) Genetic diversity of symbiotic

dinoflagellates in the genus Symbiodinium. Protist 156:19–34

Dubinsky Z, Berman-Frank I (2001) Uncoupling primary production

from population growth in photosynthesizing organisms in

aquatic ecosystems. Aquat Sci 63:4–17

Dubinsky Z, Falkowski PG, Wyman K (1986) Light harvesting

and utilization by phytoplankton. Plant Cell Physiol 27:1335–

1349

Dubinsky Z, Stambler N, Benzion M, McCloskey LR, Muscatine L,

Falkowski PG (1990) The effect of external nutrient resources on

the optical-properties and photosynthetic efficiency of Stylopho-ra-Pistillata. Proc R Soc Lond, B 239:231–246

Coral Reefs (2009) 28:179–195 193

123

Dustan P (1979) Distribution of zooxanthellae and photosynthetic

chloroplast pigments of the reef-building coral Montastrea-Annularis Ellis and Solander in relation to depth on a West-

Indian coral-reef. Bull Mar Sci 29:79–95

Enriquez S, Mendez ER, Iglesias-Prieto R (2005) Multiple scattering

on coral skeletons enhances light absorption by symbiotic algae.

Limnol Oceanogr 50:1025–1032

Falkowski PG, LaRoche J (1991) Acclimation to spectral irradiance

in algae. J Phycol 27:8–14

Falkowski PG, Owens TG (1980) Light-shade adaptation; two

strategies in marine-phytoplankton. Plant Physiol 66:592–595

Falkowski PG, Raven JA (1997) Aquatic photosynthesis. Blackwell

Science, Oxford

Falkowski PG, Owens TG, Ley AC, Mauzerall DC (1981) Effects

of growth irradiance levels on the ratio of reaction centers

in 2 species of marine-phytoplankton. Plant Physiol 68:969–

973

Falkowski PG, Jokiel PL, Kinzie RA III (1990) Irradiance and corals.

In: Dubinsky Z (ed) Coral reefs ecosystems of the world.

Elsevier Science Publication, Amsterdam, pp 89–107

Falkowski PG, Dubinsky Z, Muscatine L, McCloskey L (1993)

Population-control in symbiotic corals. Bioscience 43:606–611

Futuyma DJ, Moreno G (1988) The evolution of ecological special-

ization. Annu Rev Ecol Syst 19:207–233

Garren M, Walsh SM, Caccone A, Knowlton N (2006) Patterns of

association between Symbiodinium and members of the Mon-tastraea annularis species complex on spatial scales ranging

from within colonies to between geographic regions. Coral Reefs

25:503–512

Geider RJ, MacIntyre HL, Kana TM (1998) A dynamic regulatory

model of phytoplanktonic acclimation to light, nutrients, and

temperature. Limnol Oceanogr 43:679–694

Gorbunov MY, Kolber ZS, Lesser MP, Falkowski PG (2001)

Photosynthesis and photoprotection in symbiotic corals. Limnol

Oceanogr 46:75–85

Goulet TL (2006) Most corals may not change their symbionts. Mar

Ecol Prog Ser 321:1–7

Harris GN, Scanlan DJ, Geider RJ (2005) Acclimation of Emilianiahuxleyi (Prymnesiophyceae) to photon flux density. J Phycol

41:851–862

Hennige SJ, Smith DJ, Perkins R, Consalvey M, Paterson DM,

Suggett DJ (2008) Photoacclimation, growth and distribution of

massive coral species in clear and turbid waters. Mar Ecol Prog

Ser 369:77–88

Hochberg EJ, Apprill AM, Atkinson MJ, Bidigare RR (2006) Bio-

optical modeling of photosynthetic pigments in corals. Coral

Reefs 25:99–109

Iglesias-Prieto R, Trench RK (1994) Acclimation and adaptation to

irradiance in symbiotic dinoflagellates. 1. Responses of the

photosynthetic unit to changes in photon flux-density. Mar Ecol

Prog Ser 113:163–175

Iglesias-Prieto R, Trench RK (1997) Acclimation and adaptation to

irradiance in symbiotic dinoflagellates. II. Response of chloro-

phyll-protein complexes to different photon-flux densities. Mar

Biol 130:23–33

Iglesias-Prieto R, Govind NS, Trench RK (1991) Apoprotein

composition and spectroscopic characterization of the water-

soluble peridinin chlorophyll alpha-proteins from 3 symbiotic

dinoflagellates. Proc R Soc Lond, B 246:275–283

Iglesias-Prieto R, Beltran VH, LaJeunesse TC, Reyes-Bonilla H,

Thome PE (2004) Different algal symbionts explain the vertical

distribution of dominant reef corals in the eastern Pacific. Proc R

Soc Lond, B 271:1757–1763

Kana R, Lazar D, Prasil O, Naus J (2002) Experimental and

theoretical studies on the excess capacity of photosystem II.

Photosynth Res 72:271–284

Kolber Z, Zehr J, Falkowski P (1988) Effects of growth irradiance and

nitrogen limitation on photosynthetic energy-conversion in

photosystem-II. Plant Physiol 88:923–929

Kolber ZS, Prasil O, Falkowski PG (1998) Measurements of variable

chlorophyll fluorescence using fast repetition rate techniques:

defining methodology and experimental protocols. Biochim

Biophys Acta Bioenerg 1367:88–106

Kolber Z, Klimov D, Ananyev G, Rascher U, Berry J, Osmond B

(2005) Measuring photosynthetic parameters at a distance: laser

induced fluorescence transient (LIFT) method for remote

measurements of photosynthesis in terrestrial vegetation. Photo-

synth Res 84:121–129

Kromkamp JC, Forster RM (2003) The use of variable fluorescence

measurements in aquatic ecosystems: differences between

multiple and single turnover measuring protocols and suggested

terminology. Eur J Phycol 38:103–112

LaJeunesse TC (2001) Investigating the biodiversity, ecology, and

phylogeny of endosymbiotic dinoflagellates in the genus Sym-biodinium using the ITS region: In search of a ‘‘species’’ level

marker. J Phycol 37:866–880

LaJeunesse TC (2002) Diversity and community structure of symbi-

otic dinoflagellates from Caribbean coral reefs. Mar Biol

141:387–400

LaJeunesse TC, Trench RK (2000) Biogeography of two species of

Symbiodinium (Freudenthal) inhabiting the intertidal sea anem-

one Anthopleura elegantissima (Brandt). Biol Bull 199:126–134

LaJeunesse TC, Thornhill DJ, Cox EF, Stanton FG, Fitt WK, Schmidt

GW (2004a) High diversity and host specificity observed among

symbiotic dinoflagellates in reef coral communities from Hawaii.

Coral Reefs 23:596–603

LaJeunesse TC, Bhagooli R, Hidaka M, DeVantier L, Done T,

Schmidt GW, Fitt WK, Hoegh-Guldberg O (2004b) Closely

related Symbiodinium spp. differ in relative dominance in coral

reef host communities across environmental, latitudinal and

biogeographic gradients. Mar Ecol Prog Ser 284:147–161

Laurion I, Blouin F, Roy S (2004) Packaging of mycosporine-like

amino acids in dinoflagellates. Mar Ecol Prog Ser 279:297–303

Lesser MP (2004) Experimental biology of coral reef ecosystems. J

Exp Mar Biol Ecol 300:217–252

Lesser MP, Farrell JH (2004) Exposure to solar radiation increases

damage to both host tissues and algal symbionts of corals during

thermal stress. Coral Reefs 23:367–377

Levy O, Dubinsky Z, Achituv Y (2003) Photobehavior of stony

corals: responses to light spectra and intensity. J Exp Biol

206:4041–4049

MacIntyre HL, Kana TM, Anning T, Geider RJ (2002) Photoaccli-

mation of photosynthesis irradiance response curves and

photosynthetic pigments in microalgae and cyanobacteria. J

Phycol 38:17–38

Mauzerall D, Greenbaum NL (1989) The absolute size of a

photosynthetic unit. Biochim Biophys Acta 974:119–140

Mazel CH (1997) Diver-operated instrument for in situ measurement

of spectral fluorescence and reflectance of benthic marine

organisms and substrates. Opt Eng 36:2612–2617

McDougall KE, Gibb SW, Boyd KG, Brown BE (2006) ‘Chlorophyll-

like’ compounds as novel biomarkers of stress in corals. Mar

Ecol Prog Ser 325:137–144

Medlin LK, Kaczmarska I (2004) Evolution of the diatoms: V.

Morphological and cytological support for the major clades and

a taxonomic revision. Phycologia 43:245–270

Moberg F, Folke C (1999) Ecological goods and services of coral reef

ecosystems. Ecol Econ 29:215–233

Moore CM, Lucas MI, Sanders R, Davidson R (2005) Basin-scale

variability of phytoplankton bio-optical characteristics in rela-

tion to bloom state and community structure in the Northeast

Atlantic. Deep-Sea Res Part I 52:401–419

194 Coral Reefs (2009) 28:179–195

123

Moore CM, Suggett DJ, Hickman AE, Kim YN, Tweddle JF, Sharples

J, Geider RJ, Holligan PM (2006) Phytoplankton photoacclima-

tion and photoadaptation in response to environmental gradients

in a shelf sea. Limnol Oceanogr 51:936–949

Munekaga Y, Hashimoto M, Miyaka C, Tomizawa KI, Endo T,

Tasaka M, Shikanai T (2004) Cyclic electron flow around

photosystem I is essential for photosynthesis. Nature 429:

579–582

Muscatine L (ed) (1990) The role of symbiotic algae in carbon and

energy flux in reef corals. Elsevier Science Publishing Company,

Amsterdam

Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of

accurate extinction coefficients and simultaneous-equations for

assaying chlorophyll-a and chlorophyll-b extracted with 4

different solvents - verification of the concentration of chloro-

phyll standards by atomic-absorption spectroscopy. Biochim

Biophys Acta 975:384–394

Provasoli L, McLaughlin JJA, Droop MR (1957) The development of

artificial media for marine algae. Arch Mikrobiol 25:392–428

Ralph PJ, Gademann R, Larkum AWD, Schreiber U (1999) In situ

underwater measurements of photosynthetic activity of coral

zooxanthellae and other reef-dwelling dinoflagellate endos-

ymbionts. Mar Ecol Prog Ser 180:139–147

Reynolds JM, Bruns BU, Fitt W, Schmidt GW (2008) Enhanced

photoprotection pathways in symbiotic dinoflagellates of shal-

low-water corals and other cnidarians. Proc Natl Acad Sci USA

105:13674–13678

Robison JD, Warner ME (2006) Differential impacts of photoaccli-

mation and thermal stress on the photobiology of four different

phylotypes of Symbiodinium (Pyrrhophyta). J Phycol 42:568–

579

Rodriguez-Roman A, Iglesias-Prieto R (2005) Regulation of photo-

chemical activity in cultured symbiotic dinoflagellates under

nitrate limitation and deprivation. Mar Biol 146:1063–1073

Sampayo EM, Franceschinis L, Hoegh-Guldberg O, Dove S (2007)

Niche partitioning of closely related symbiotic dinoflagellates.

Mol Ecol 16:3721–3733

Savage AM, Trapido-Rosenthal H, Douglas AE (2002) On the

functional significance of molecular variation in Symbiodinium,

the symbiotic algae of Cnidaria: photosynthetic response to

irradiance. Mar Ecol Prog Ser 244:27–37

Six C, Finkel ZV, Rodriguez F, Marie D, Partensky F, Campbell DA

(2008) Contrasting photoacclimation costs in ecotypes of the

marine eukaryotic picoplankter Ostreococcus. Limnol Oceanogr

53:255–265

Stambler N, Dubinsky Z (2005) Corals as light collectors: an

integrating sphere approach. Coral Reefs 24:1–9

Suggett DJ, Oxborough K, Baker NR, MacIntyre HL, Kana TM,

Geider RJ (2003) Fast repetition rate and pulse amplitude mod-

ulation chlorophyll a fluorescence measurements for assessment

of photosynthetic electron transport in marine phytoplankton.

Eur J Phycol 38:371–384

Suggett DJ, MacIntyre HL, Geider RJ (2004) Evaluation of biophys-

ical and optical determinations of light absorption by

photosystem II in phytoplankton. Limnol Oceanogr 2:316–332

Suggett DJ, Moore CM, Maranon E, Omachi C, Varela RA, Aiken J,

Holligan PM (2006) Photosynthetic electron turnover in the

tropical and subtropical Atlantic Ocean. Deep-Sea Res Part II

53:1573–1592

Suggett DJ, Le Floc’H E, Harris GN, Leonardos N, Geider RJ (2007)

Different strategies of photoacclimation by two strains of

Emiliania huxleyi (Haptophyta). J Phycol 43:1209–1222

Suggett DJ, Warner ME, Smith DJ, Davey P, Hennige SJ, Baker NR

(2008) Photosynthesis and production of hydrogen peroxide by

Symbiodinium (Pyrrhopyta) phylotypes with different thermal

tolerances. J Phycol 44:948–956

Sukenik A, Bennett J, Falkowski P (1987) Light-saturated photosyn-

thesis - limitation by electron-transport or carbon fixation.

Biochim Biophys Acta 891:205–215

Tchernov D, Gorbunov MY, de Vargas C, Yadav SN, Milligan AJ,

Haggblom M, Falkowski PG (2004) Membrane lipids of

symbiotic algae are diagnostic of sensitivity to thermal bleaching

in corals. Proc Natl Acad Sci USA 101:13531–13535

Ulstrup KE, van Oppen MJH (2003) Geographic and habitat

partitioning of genetically distinct zooxanthellae (Symbiodinium)

in Acropora corals of the Great Barrier Reef. Mol Ecol 12:3477–

3484

van Oppen MJH, Mieog JC, Sanchez CA, Fabricius KE (2005)

Diversity of algal endosymbionts (zooxanthellae) in octocorals:

the roles of geography and host relationships. Mol Ecol

14:2403–2417

Warner ME, Berry-Lowe S (2006) Differential xanthophyll cycling

and photochemical activity in symbiotic dinoflagellates in

multiple locations of three species of Caribbean coral. J Exp

Mar Biol Ecol 339:86–95

Warner ME, LaJeunesse TC, Robison JD, Thur RM (2006) The

ecological distribution and comparative photobiology of symbi-

otic dinoflagellates from reef corals in Belize: Potential

implications for coral bleaching. Limnol Oceanogr 51:1887–

1897

Zar JH (1996) Biostatistical analysis, 3rd edn. Prentice-Hall Inc, N.J

Coral Reefs (2009) 28:179–195 195

123