www.harmfulalgae.info

The use of molecular approaches in marine
ecosystem health assessment and research in
Malaysia
Po Teen Lim, Chui Pin Leaw, Kieng Soon Hii
Bachok Marine Research Station, Institute of Ocean andEarth Sciences, University of Malaya, Bachok, Kelantan,
Malayisa

www.harmfulalgae.info

Part I
Assessing the diversity of marine phytoplankton acrossthe South China Sea and Malacca Strait using
metabarcoding approach
Kieng Soon Hii, Z. F. Lim, S.N. Tan, Z. Luo, H. Gu, C.P. Leaw, P.T. Lim

Importance of Phytoplankton• They serve as the primary producers in coastal
ecosystem.
• They also act as biogeochemical cycle mediator
for the earth.

Sources: Gulf of Maine research Institute 2010

Sources: Finkel et. al., 2010

• Proliferation of selected phytoplankton taxa in the waters may cause tremendous
impacts on human health and social-economy.
• Toxin producer taxa release biotoxin and • High-biomass producer taxa cause
contaminated seafood or fish kill.
anoxia/hypoxia in waters and lead tomortality of marine life.

Shellfish contamination in Geting River,Kelantan.

Mass mortality of cultured fish inJohor strait due to algal
karlotoxins.

Massive fish mortality in Teluk
Bahang caused by a hypoxiainducing algal bloom

Objective
Investigate phytoplankton community
assemblages across the South China Sea andMalacca Strait by genetic metabarcoding.

Methodology
72 plankton samples were collectedduring research cruise in July-August
2019

DNA isolation and 18S rDNA
V9 region amplified

Illumina Miseq platform anddata analysis

RV Discovery, UMT

Phytoplankton community studies• Metabarcoding is:– A fast-genetic manner to elucidate thecomprehensive taxonomic profiling in theenvironment (Kunin et al., 2008).
– Reliable due to the taxonomic assignment are
based on the sequences similarity of thereferences genomes in huge database such as
Ref seq (Tatusova et al., 2014), PR2 (Cole et al., 2014)
and SILVA (Quast et al., 2013).

• Highly accurate genetic distinction approach,metabarcoding are useful to monitor thephytoplankton/HABs species.

www.harmfulalgae.info

Phytoplankton diversity• A total of 386
phytoplankton species
taxa was revealed.
• High species diversity
was observed at offshore
sampling station.
– This could be due tolower nutrient at
offshore.
– High nutrient tends toshift the high-species
phytoplankton
diversity tomonospecific.

www.harmfulalgae.info

OTUs Abundance of phytoplankton
PredominatedThalassiosiraceae

South China Sea

Coscinodiscaceae

Chaetocerotaceae

Rhizosoleniaceae

Chaetocerotaceae

Chaetocerotaceae

Malacca Strait
Rhizosoleniaceae• High OTU abundancesof phytoplankton
were found at shore
sampling stations ofMalacca Strait.
• This was likely
influenced by highnutrient loading in
the region.

www.harmfulalgae.info

Harmful algal bloom species and its threats• A total of 28 harmful
algal bloom (HAB)
species wasannotated from OTU,
with 7 new records in
Malaysian waters.
• High OTU abundanceof HAB species weremainly found in
Malacca strait.

www.harmfulalgae.info

Conclusion• This study provides valuable baseline inventory of marine
phytoplankton diversity to evaluate long term ofphytoplankton communities along the coast of Malaysia• Metabarcoding approach is useful for HAB species detection
and monitoring.

Acknowledgements•••

Thanks to RV Discovery scientific cruise fromUMT.
HICoE-2014c to PT Lim.
Thanks to Prof Gu, H. and Dr Luo, Z., TIO for
the Illumina Miseq analysis.

www.harmfulalgae.info

Revealing the Harmful Algal Community ShiftAlong the Johor Strait Using Metabarcoding
ApproachHii, K.S., Mohd-Din, M., Tan, S.N., Lim, Z.F., Lee, L.K., Luo, Z., Gu,
H., Leaw, C.P., Lim, P.T.

Study Sites: Johor Strait (JS)
ST3: Cage Fish Farm; ST5 and ST10 : Cage Fish and Green Mussel Farm

ST13-16 : Green mussel farm
Eastern
Western
Johor Strait is divided
by Johor-Singapore
crossway to western(20km) and Easternside (33km)

ST14-15 : Cargo port

ST1: Port of Tanjung
PelepasJohor-Singapore
BridgeST2, ST7-ST8: Near the
Merabong Island

JohorSingapore
Crosswaywww.harmfulalgae.info••

Chl-a in the Johor Strait were in the rangeof 48.82- 3.15 µg/L.
The highest Chl-a (34-48.32 µg/L) content
were observed in inner JS, where highbiomass/ bloom of several diatom
species in the water were observed.

www.harmfulalgae.info

Species composition based on metabarcoding•

A total of 130 phytoplankton taxa wererevealed from OTUs taxonomic
assignment.
– 64 species of Dinoflagellates– 66 species of diatoms•

16 know and potential HAB speciesi) Alexandrium affine, ii) A.
minutum/peruvianum/ostenfeldii/insuetum, iii) A.
pseudogoniaulax/hiranoi, iv) A. tamiyavanichii, v)
A. leei, vi) Chattonella marina, vii) C. subsalsa, viii)
Dinophysis miles/ caudata/norvegica/acuminate,ix) Gymnodinium catenatum, x) Heterosigma
akashiwo, xi) Karenia selliformis, xii) Pseudonitzschia americana, xiii) P. brasiliana, xiv)P.
cuspidata/lineola/turgidula/australis, xv) P.
multiseries/Pungens, xvi) P. australis

www.harmfulalgae.info

Taxonomic assignment
Highest Blast hit
OTU21914 Dinophysis miles
Otu4410 D. caudata
D. norvegica
D.acuminata
Otu15209 Pseudo-nitzschia pungens
Otu3139 P. multiseries
Otu22780 Alexandrium minutum
Otu4439 A. peruvianum
Otu4441 A. ostenfeldii
A. insuetum
Pseudo-nitzschia cuspidata
Otu3088 P. lineola
P.turgidula
Otu7064 Alexandrium pseudogoniaulax
Otu135 A. hiranoi
Otu16641 Pseudo-nitzschia australis
Otu21833 Karenia mikimotoi
Otu23722 Heterosigma akashiwo
Otu24287 Gymnodinium catenatum
Otu4409 Alexandrium affine
Otu4480 Alexandrium leei
Otu4492 Alexandrium tamiyavanichii
Otu4421 Chattonella subsalsa
Otu6704 Chattonella minima
Otu299 Pseudo-nitzschia brasiliana
Otu3526 Pseudo-nitzschia americana

Identity (%)
99.23

Species assignment
Dinophysis miles/caudata/norvegica/acuminata

•
98.47

Pseudo-nitzschia multiseries/pungens

98.46

Alexandrium
minutum/peruvianum/ostenfeldii/insuetum

Pseudo-nitzschia cuspidata/lineola/turgidula
97.67
98
99.24
97.69
99.2
99.23
100
97.3
100
100
100
98.48
100

Alexandrium pseudogoniaulax/hiranoi
Pseudo-nitzschia australis
Karenia mikimotoi
Heterosigma akashiwo
Gymnodinium catenatum
Alexandrium affine
Alexandrium leei
Alexandrium tamiyavanichii
Chattonella subsalsa
Chattonella minima
Pseudo-nitzschia brasiliana
Pseudo-nitzschia americana•

Only OTUs that had> 97% similarity to
NCBI Nt databasewere included in theanalysis.
However, some of
the HABs species stillcannot be
distinguished to
species level byusing V9 of 18S
rDNA region.

www.harmfulalgae.info

HAB species distribution in Johor Strait•

Overall HABs species relative abundance throughout May2018-Jan2019

Eastern•

Western••••

The highest HAB species diversity
and relative abundance werediscovered in outer western andEastern JS.
A. leei, A. pseudogoniaulax/hiranoi,
A. tamiyavanichii, P. americana, P.
australis and P.
cuspidata/lineola/turgidula were
found in outer Western JS.
A. affine and C. minima weredetected in outer of Eastern JS.
Dinophysis sp. was found in outerparts of Western and Eastern JS.
C. subsalsa, G. catenatum and P.
brasiliana were discovered in innerEastern JS.
H. akashiwo and P.
multiseries/punges were detected
throughout the stations in JS.

www.harmfulalgae.info

HAB community assemblages and composition shift• HABs species diversity increased in wet season during the Northeast
monsoon.

www.harmfulalgae.info

Physico-chemical variability•••

Salinity and pH in JS were in the rangeof 23-32 and 7.2-8.2, respectively.
The lowest salinity were observed in
the inner JS, likely due to freshwaterplumes and discharge of freshwaterfrom reservoirs.
The highest salinity and pH weredetected in Jan 2019 during flood tide,
intrusion of the seawater from South
China Sea and Strait Of Malacca.
Water current movement during Jan 2019

Eastern

Western

Singaporewww.harmfulalgae.info• Temperature in JS were observed in the range of 28-33°C.
• The highest temperatures (31-33 °C) were observed in the InterMonsoon (Oct 2018) and Southwest Monsoon.

www.harmfulalgae.info• The highest concentration of micronutrients, nitrate, ammonia, phosphate and silicate were observed
during the Northeast Monsoon (Nov-Jan), postulate the nutrient sources was discharged from rivers andrunoff from land in urban areas during rain.

www.harmfulalgae.info

Canonical Correlation Analysis (CCA)•••

Relative abundance of A. pseudogoniaulax/hiranoi, A. tamiyavanichii, A. leei, P. americana, P.
multiseries/pungens and Dinophysis sp. were associated with increasing DIN: DIP and salinity.
H. akashiwo, A. affine and A. minutum/peruvianum/ostenfeldii/insuetum were related to increasing
phosphate and temperature.
G. catenatum and C. subsalsa were highly correlated with temperature.

Part III
Exploring the diversity ofzooxanthellae associated withcorals of Perhentian Islands
Marine Park by deep
sequencing approach
Po Teen LIM, Lee Kiat LEE, Zhen Fei LIM, Kieng Soon HII, Haifeng GU, ZhaoheLuo, Albert A. CHAN, Chui Pin LEAWThe coasts of Malaysia harbored coral reef ecosystem spanning for up
to 4000km2 in term of area (Wikinson, 2008), with estimated 500
hermatypic coral species, constituting 60% of the world described
hermatypic coral species (Veron et al., 2009).
Climate change and other local stressors (eutrophication, siltation,
habitat destruction and overfishing) has caused mass bleaching ofcoral species. eg., in year 2010.

Reef corals are hosts to a group of exceptionallydiverse dinoflagellate symbionts in the family
Symbiodiniaceae.
These symbionts are critical components of coral
reef ecosystem. Loss of these symbionts in theircoral hosts during stress-related “bleaching”
events can lead to mass mortality of the coral
hosts and collapse of the associated reef
ecosystem.

Zooxanthellae factSheet







Single-cell dinoflagellates
Mutualistic endosymbionts
Autotroph (Photosynthetic)
Containing chloroplasts (chlorophyll a,c, peridinin and diadinoxanthin) andpyrenoid
Coccoid in hosts; free living flagellated
cell

Coccoid

6 to

13 µm

Mastigote© Leaw

Motile flagellated cell

The symbiont is housed in a
specialized compartment (perivacuolar membrane) of host cell,
called symbiosome.
They give corals their vibrant colors.
Zooxanthellae will be expelled when
the coral hosts are exposed toenvironmental stressors, and thiscaused the coral to bleach.

High species and functional
diversity of zooxanthellae
Zooxanthellae is a highly diverse
group, with divergent clades (of which
comprise of multiple subclade types)that are now partitioned into a total ofseven genera.
Different clades have different
resistance to various environmental
stresses.
Clade A – high/variable light
Clade D – thermal tolerance
Clade E- Free living-non symbiotic
LaJeunesse et al. (2018)

Goal:
Exploring the zooxanthellae diversity of
Malaysia

Assessing the symbiont compositionin different corals is essential tounderstand the resilience andsusceptibility of the reefs

To investigate the diversity ofzooxanthellae in Malaysiancommon coral species,
To assess the coral resilience bycomparing the zooxanthellae
communities in the host species.

Perhentian Islands Marine Park

Coral fragments with the size of <5 cm
were collected and placed in smallbags or tubes underwater by SCUBA
diving.
The coral colonies were photographedwith a digital underwater camera prior
to collection for coral species
identification.

Molecular characterization
Samples were processed prior to genomic DNA extraction bymagnetic bead technique.
Gene amplification of the nuclear ribosomal gene (ITS2)
was performed. The amplicons were purified andquantified prior to deep sequencing

NGS deep sequencing
NGS deep sequencing was performed on ILUMINA MiSeq
platform.

Gene amplifications

DNA extraction

NGS deep sequencing

Zooxanthellae diversity• In total, 24 coral samples were analyzed.
• 1,232,181 reads were obtained with median length of 292
bp.
• 900,055 reads were retained with an average 37,503
sequences per sample after sequences filtering.
• Two dominant genetic clades, viz. Clades C and D wereidentified from these coral samples.
• A total of 134 subclades C and 14 subclades D wereconfirmed.

Zooxanthellae diversity
in Galaxea species

D3
Sym11

Sym6

D lineage

C3a

Sym27

D1a

Sym5

High %The high abundances andvarying distributions of D
lineage in the zooxanthellae
communities of Galaxea
species might indicated recent
bleaching of this coral species,
since Clade D is often
associated with recently
bleached and/or recoveringcorals (Baker et al., 2004), but
can be outcompeted following
recovery (Thornhill et al.,2006).

D3
D1a = D. trenchii

Zooxanthellae diversity in Tubinaria species
Specimens from shallower depth exhibit at least 10% higher proportion of D
lineage compared to the deeper counterpart.
Clade C
Clade G
Clade D
Others

Shallower depth
Sym16

deeper depth
Sym13

0

20

40

60

80

100

Relative abundance (%)

C3u

C39

It is interesting to note that the
deeper Tubinaria hosted only C39
lineage.
This lineage has been shown to beprevalent and restricted to the Red
Sea and the Sea of Oman as
potential type that display
increased thermotolerance(Zigeler et al., 2017).

A reconstruction of how thermal stress influenced
symbiont populations among shallow and deepcolonies of Montastraea annularis during and after
the 2005 Caribbean bleaching event.

Thermal stress-tolerance
ecotypes
Opportunistic symbionts may impart
rapid adaptation to environmental
change by their hosts

Opportunistic species may proliferate during
periods of physiological stress and displace thenormal resident symbiont and remainabundant in the host’s tissues for months toyears before being replaced by the original
symbiont.

= tolerate stressful environments +accelerate recovery

LaJeunesse et al. (2009)

Zooxanthellae communities in the coral species of Perhentian Islands did
not show significant trend in depth distribution.
The abundances of thermally tolerant D lineage in various coral species
was not associated with depth distribution, presumably shallower depth
experienced wider ranges of thermal fluctuations and exposed to highest
temperatures.
4.9 m

17.7 m

100.0
80.0
60.0
40.0
20.0

C

D

G

Sym12

Sym13

Sym19

Sym25

Sym20

Sym24

Sym5

Sym2

Sym7

Sym1

Sym29

Sym14

Sym28

Sym10

Sym16

Sym18

Sym27

Sym6

Sym4

Sym8

Sym26

Sym3

Sym11

0.0

The zooxanthellae communities differed significantly among various coral species.

This difference appears to be driven by higher relative abundances of lineages C3u, C115, C18,
and C30_type 1.
For example, the most abundant C lineage in Leptoseris sp. Sym8 was C30_type 1, with up to 97%
of relative abundance, while in Echinopora sp. Sym10, a relatively higher proportion of C3u wasdetected. This supported previous findings of host-specific associations of the endosymbionts(Finney et al., 2010).

Corals with complex structure(branching) hosted mainly
zooxanthellae community in the C
lineages while D lineages areassociated with some robust corals(massive)branchingmassive

100
80
60
40
20

Sym5

Sym25

Sym27

Sym29

Sym6

Sym11

Sym28

G

Sym2

Sym7

D

Sym10

Sym20

C

Sym24

Sym12

Sym16

Sym4

Sym13

Sym8

Sym26

Sym3

Sym1

Sym14

Sym18

Sym19

0

Synthesis
Numerous studies have shown that
fast-growing branching coral species(e.g. Acropora, Millepora,
Pocillopora) suffer higher bleachingmortality than slow-growing massive
corals species (e.g. Favites, Porites,
Turbinaria) (Marshall and Baird,
2000; McClanahan et al., 2004).

The resistance mechanisms ofdifferent coral species are notcompletely understood, but thisstudy showed that many coral
species with robust structure(massive) hosted mainly
zooxanthellae community in the
stress-tolerant D lineages.

Likelihood of thermal tolerance

Sym 14

Sym 26

Sym 4

Sym 3

Acropora

Sym 18

Increasing Likelihood of thermal tolerance

Galaxea

Sym1
1

Sym6

Sym2
7

Sym5

Based on the results of
the relative abundancesof the thermally tolerant D
lineage in our coral
species, we proposedthat zooxanthellae
diversity to be considered
as one of the suite ofcoral resilience indicators

Conclusion
Our understanding on the diversity ofzooxanthellae in term of species- andfunctionality in Malaysian coral reefs has
been severely limited by the lack ofcomprehensive description on theirdiversity.
This study, by utilizing next-generation
sequencing of ITS2 genetic marker,
provides insights into the interspecificdiversity of zooxanthellae and its
community in various coral species of the
Perhentian Island Marine Park.
The results obtained provide acomprehensive inventory of zooxanthellae
diversity as a baseline assessment to study
coral resilience in our coral reef ecosystem.

Part IV
Distribution, abundance andpopulation genetic diversityof the giant clams, Tridacnasquamosa and T. maxima(Cardiidae) in the Perhentian
Islands Marine Park, Malaysia
Chui Pin Leaw,
Li Keat Lee, Zhen Fei Lim, Kieng Soon
Hii, Haifeng Gu, Mei Lin Neo, Po Teen
LimABUNDANCES OF T. SQUAMOSA and T. MAXIMA
in three different size classes

1.62 ind. 100 m-2

juveniles

4.08 ind. 100 m-2

Lee et al., Marine Biodiversity, submitted

mt COI marker

H = 0.842 (0.67–0.93)
π = 6.2% (8.9–17.9%)

H = 0.624 (0.29–0.8)
π = 3.5% (1.4–21.2%)

Population structuring of T. squamosa in the
Malay Archipelago
Perhentian
population shared adegree of genetic
homogeneity withother populations
from Philippines,
Singapore andIndonesia.

Perhentian populations

Population structuring of T. maxima in
the Malay Archipelago
Perhentian populations
showed some degrees
of genetic
heterogeneity.

Conclusion
1. An update data inventory on the
distribution and abundance oftwo giant clams in Perhentian
Marine Park.
2. Insights into the population
genetic connectivity of the two
species of Tridacna of Malaysia.

acknowledgements
This work was funded by Dept. of Marine Park
Malaysia [GA003-2017] and MOE FRGS [FP0582019A]

Concluding
remarks• Molecular methods are high precisionand powerful tools in assessing
ecosystem health status of marine
ecosystem (for short-term and longterm) changes.
• These methods are useful, not only toassess ecosystem health and changes,
but also an enabler wide geographicalregion observation and comparison.
• Standardization of methods(qualitatively and quantitatively) andbetter curated database will be criticalto ensure the success of molecularapproaches in biological observationand monitoring.