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i
MORPHOLOGY, TAXONOMY AND GENETICS OF TROPICAL
DIATOM PSEUDO-NITZSCHIA SPECIES
(BACILLARIOPHYCEAE) IN MALAYSIAN WATERS
Lim Hong Chang
A thesis submitted
In fulfilment of the requirement for the degree of
Doctor of Philosophy
(Aquatic Science)
Faculty of Resource Science and Technology
UNIVERSITI MALAYSIA SARAWAK
2014
ii
DECLARATION
I hereby declare that the work in this thesis is my own except for quotations and
summaries which have been duly acknowledged.
2014 LIM HONG CHANG
11011542
iii
ACKNOWLEDGEMENTS
I am most grateful to my supervisors, Associate Prof. Dr. Po-Teen Lim and Dr.
Chui-Pin Leaw, who provided me with many research opportunities, careful guidance,
advices, patience and tireless effort to help me succeed through many professional and
personal difficulties. They were my earliest inspirations. They had taught me the
importance of collaborative science and never stop the momentum in research. They
have always tried to be sure that their students have no any forms of difficulties during
the studies, thank you for providing us such research environment that is conducive.
They had made me possible to be developed as a scientist and writer. Without the
presence of Dr. Lim and Dr. Leaw I am not sure whether this thesis could have been
completed. I only hope I can make them proud to say that they are my supervisors. I
am hopeful that I will be able to continue the collaborative research with Dr. Lim and
Dr. Leaw in the future research endeavours for many years to come.
Thanks are also due to Prof. Yasuwo Fukuyo for his comments during the early
stage in developing ideas for this work. I would also like to thank Dr. Nina Lundholm
and Dr. Stephen Bates for their comments and suggestions in improving part of the
analyses and writing skills. I am grateful to Dr. Peter Boyce for his help in improving
the English of this work. My undergraduate supervisor, Dr. Ruhana Hassan and high
school teachers also deserved great thanks. Thanks also to Science Officer, Mdm.
Woei Ting for her technical contribution for the use of electron microscopy facilities
and all of the staff of Institute of Biodiversity and Environmental Conservation (IBEC)
especially Ms. Rahah Mohd Yakup and Mr. Mohd Hasri Al-Hafiz Haba for their helps
in many ways. Thank you, to all the members in the IBEC Molecular Laboratory since
the year 2007, Sing-Tung Teng, Toh-Hii Tan, Kieng-Soon Hii and many more. Thanks
also to the funding provided by Malaysian Government in terms of Scholarship
(MyBrain15 MyPhD) as well as research grants to make this work possible.
Finally, I am eternally grateful to my parents, Chong-Teong Lim and Yik-
Cheng Song as well as my siblings Sin-Hwa, Hong-Hui and Sin-Min. My family has
always encouraged me to pursue my goals throughout my entire life. Lastly, I would
like to acknowledge the enduring support from my „wife-to-be‟ Chai-Ying Tan who
has always been by my side to share my failures and success.
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MORPHOLOGY, TAXONOMY AND GENETICS OF TROPICAL DIATOM
Pseudo-nitzschia SPECIES (BACILLARIOPHYCEAE) IN MALAYSIAN
WATERS
Lim Hong Chang
Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak
ABSTRACT
Pseudo-nitzschia is a marine pennate diatom known to be responsible for Amnesic
shellfish poisoning (ASP) due to domoic acid (DA) production, a type of neurotoxin.
Field studies were conducted from Malaysian Borneo and Straits of Malacca,
Peninsular Malaysia to determine the species occurrence, and distribution of brackish
to marine diatom Pseudo-nitzschia. A total of four species were identified based on a
combination of distinct morphological characteristics and supporting molecular
evidence: P. brasiliana, P. dolorosa, P. micropora, and P. pungens. One morphotypes
from Sarawak; however showed significant morphological distinction from the known
species while similar to that of P. caciantha. Most notably this morphotypes possessed
a characteristic pore arrangement in the poroids with the fine pores arranged in
circular close to the margin of the poroid hymen. The large subunit (LSU) rDNA of
this morphotype and closely related P. caciantha Lundholm, Moestrup & Hasle,
showed 2.7% genetic divergence. Phylogenetic analyses strongly supported the
monophyly of the morphotype. Based upon these supporting data it is here described
as a new species, Pseudo-nitzschia circumpora sp. nov. Molecular signatures for all
species were established based on structural comparisons of the second internal
transcribed spacer (ITS2) rRNA transcripts. In addition, another three new species
were proposed from cultures obtained from the Straits of Malacca based on
morphological data combined with molecular evidences; viz. P. batesiana sp. nov., P.
lundholmiae sp. nov., and P. fukuyoi sp. nov. The three new species closely resemble
species in the P. pseudodelicatissima complex sensu lato. Morphologically, P.
batesiana differs from other species in the complex (i.e. P. fukuyoi) by having a
smaller part of cell overlapping in the chain, whereas P. lundholmiae differs by having
fewer poroid sectors and P. fukuyoi by having a distinct type of poroid sectors.
Nucleotide sequences of the LSU rDNA of the three new species reveal significant
nucleotide sequence divergence from each other and from the other species in the P.
pseudodelicatissima complex s.l. The three species show 2-3 compensatory base
changes (CBCs) in their ITS2 transcripts when compared to closely related species.
The three species are phylogenetically closely related to species in the P.
pseudodelicatissima complex, with P. batesiana appearing as a sister taxon to P.
circumpora, P. caciantha and P. subpacifica; while P. lundholmiae and P. fukuyoi are
more closely related to P. pseudodelicatissima and P. cuspidata. The effectiveness of
using ribosomal gene of LSU rDNA (domain 1-3), the whole region of ITS (ITS1-
5.8S-ITS2) and ITS2 together with structural alignment to discriminate Pseudo-
nitzschia to species level were shown in this study. To examine the genetic diversity of
Pseudo-nitzschia in Malaysia, two commonly found Pseudo-nitzschia i.e. P.
brasiliana and P. pungens were selected. Hence, the genetic marker of internal
transcribed spacer (ITS) of nuclear-encoded ribosomal DNA of this two species was
used to clarify diversity and population structure. In P. brasiliana, high genetic
homogeneity was detected (0.07-0.54%) but no clear trend on geographical localities,
v
phylogenetically. The low geographical patterns lead to the conclusion that there‟s no
speciation although P. brasiliana from Malaysian waters was geographically isolated.
Whereas in P. pungens, phylogenetic tree from Profile Neighbour Joining (PNJ)
analysis revealed that there are three ITS entities (Clade I to Clade III), in which
strains in clade I found to be distributed widely in Northern and Southern Hemisphere,
clade II confined only to NE Pacific and clade III restricted to tropical and subtropical
waters. The result also showed that there is no gene flow between P. pungens from
warm tropical water and cold temperate water and the populations were genetically
structured based on AMOVA (ФST=0.7495) and STRUCTURE analysis. The
evolutionary diversification patterns of P. pungens were first revealed based on these
analyses. Additionally, we present a phylogeographical hypothesis in which the
evolutionary of P. pungens is triggered by ecological differentiation due to the closure
of Central American Seaway and the global distribution is driven by thermohaline
circulation. These hypotheses are consistent with the formation of Isthmus of Panama.
In conclusion, high diversity Pseudo-nitzschia was documented in this study with the
description of four new species. Species diversification and phylogeography of P.
pungens served as a model demonstrated in this study in understanding speciation in
marine diatom in particularly the potentially toxic Pseudo-nitzschia species.
Key words: Pseudo-nitzschia; morphology; molecular signature; ITS2 transcript;
population structure; phylogeography
vi
MORFOLOGI, TAKSONOMI DAN GENETIK SPESIES DIATOM TROPIKA
Pseudo-nitzschia (BACILLARIOPHYCEAE) DI PERAIRAN MALAYSIA
Lim Hong Chang
Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak
ABSTRAK
Pseudo-nitzschia adalah diatom pinnate yang diketahui telah menyebabkan keracunan
kerang-kerangan amnesia (ASP) dengan penghasilan sejenis toksin saraf, asid domoik.
Kajian lapangan telah dijalankan untuk menentukan kehadiran dan taburan diatom
Pseudo-nitzschia di perairan air payau dan marin di Borneo dan Selat Melaka,
Malaysia. Sejumlah 4 species iaitu, P. brasiliana, P. dolorosa, P. micropora, and P.
pungens telah dicam dengan yakin berdasarkan kombinasi pencirian morfologi dan
sokongan data molekular. Satu morfotaip Pseudo-nitzschia dari Sarawak
menunjukkan ciri menyerupai P. caciantha tetapi dengan perbezaan ciri morfologi
yang bermakna, khususnya penyusunan liang di poroid dalam bentuk lingkaran
berdekatan dengan himen poroid. Subnit besar rDNA (LSU) morfotaip ini juga
menunjukkan ia berkait-rapat dengan P. caciantha dengan pencapahan genetik 2.7%.
Analisis filogenetik menunjukkan sokongan kuat kedudukan monofilik morfotaip ini.
Morfotaip ini didirikan sebagai spesies baru P. circumpora sp. nov berdasarkan data-
data ini. Tanda molekular untuk semua spesies juga didirikan berdasarkan
pembandingan struktur transkrip ITS2. Tiga spesies Pseudo-nitzschia yang baru juga
dicadang berdasarkan pencirian morfologi dan bukit molekular ke atas kultur klonal
yang diperolehi dari perairan Selat Melaka, termasuk P. batesiana sp. nov., P.
lundholmiae sp. nov., and P. fukuyoi sp. nov. Ketiga-tiga spesies ini menyerupai spesies
dalam kompleks P. pseudodelicatissima sensu lato. Pseudo-nitzschia batesiana berbeza
dengan spesies lain dalam kompleks ini dengan pertindihan sel yang kecil, manakala P.
lundholmiae berbeza dengan bilangan sektor poroid yang kurang dan P. fukuyoi dengan
sektor poroid yang unik. Jujukan nukleotida LSU rDNA juga menunjukkan pencapahan
jujukan yang bermakna berbanding dengan spesies lain dalam kompleks P.
pseudodelicatissima s.l. Tiga spesies tersebut juga menunjukkan 2-3 perubahan bes
gantian (CBC) berbanding dengan spesies yang berhubungan rapat. Pseudo-nitzschia
batesiana adalah takson persaudaraan kepada P. circumpora, P. caciantha and P.
subpacifica; P. lundholmiae dan P. fukuyoi adalah lebih berkaitan dengan P.
pseudodelicatissima dan P. cuspidata. Keberkesanan gen ribosomal LSU rDNA (domain
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1-3), kawasan ITS dan ITS2 dengan penjajaran struktur telah berjaya mengasingkan
spesies-spesies Pseudo-nitzschia seperti yang ditunjukkan dalam kajian ini. Untuk
menentukan kepelbagaian genetic Pseudo-nitzschia di Malaysia, dua spesies yang kerap
dijumpai, Pseudo-nitzschia i.e. P. brasiliana and P. pungens telah digunakan dalam kajian
ini. Penanda genetik ITS rDNA dalam pengkodan nukleus telah digunakan untuk
menperjelaskan kepelbagaian dan struktur populasi dua spesies tersebut. Keseragaman
genetik yang tinggi (0.07-0.54%) telah dikesan dalam P. brasiliana dan tiada arah aliran
berpandukan kedudukan geografi. Ini membawa kepada kesimpulan tiada penspesiesan
berlaku walaupun berlaku pemencilan geografi. Pokok filogenetik analisa PNJ P. pungens
menunjukkan tiga entiti ITS (Klad 1 –Klad 3), di mana strain dalam Klad I tertabur luas
di hemisfera utara dan selatan, Klad II hanya didapati di Timur Laut Lautan Pasifik dan
Klad III terhad di peraiaran tropika dan subtropika. Keputusan juga menunjukkan tiada
aliran gen antara P. pungens antara perairan tropika dan temperat, struktur populasi
terasing secara genetik berdasarkan analisa AMOVA (ФST=0.7495) dan STRUCTURE.
Corak pempelbagaian evolusi P. pungens juga didedahkan buat kali pertama berdasarkan
analisa tersebut. Dengan hipotesis filogeografi, adalah dipercayai bahawa evolusi P.
pungens dicetuskan oleh pembezaan ekologi akibat penutupan laluan lautan Amerika
tengah dan penyebaran spesies ini secara global adalah didorong oleh penedaran
thermohaline. Hipotesis-hipotesis ini adalah konsisten dengan pembentukan Segenting
Panama. Secara kesimpulan, kepelbagaian Pseudo-nitzschia yang tinggi telah didokumen
dalam kajian ini dengan pemerihalan empat spesies baru. Pempelbagaian spesies dan
filogeografi P. pungens sebagai model telah berjaya digunakan memperbaiki pemahaman
penspesiesan diatom marin khususnya spesies Pseudo-nitzschia yang berpotensi toksik.
Kata kunci: Pseudo-nitzschia; morfologi; tanda molekular; transkrip ITS2; struktur
populasi; filogeografi
viii
TABLE OF CONTENTS Page
DECLARATION ii
ACKNOWLEDGEMENT iii
ABSTRACT iv
ABSTRAK vi
CONTENTS viii
LIST OF TABLES xi
LIST OF FIGURES xiii
LIST OF ABBREVIATIONS xviii
CHAPTER I GENERAL INTRODUCTION 1
CHAPTER II OCCURRENCE, MORPHOLOGY AND
PHYLOGENETICS OF Pseudo-nitzschia
(BACILLARIOPHYCEAE) SPECIES IN MALAYSIAN
WATERS
7
2.1 Morphology and molecular characterization of Pseudo-
nitzschia from Malaysian Borneo, including the new species
Pseudo-nitzschia circumpora sp. nov.
7
2.1.1 Introduction 7
2.1.2 Materials and Methods 9
2.1.2.1 Samples and cultures 9
2.1.2.2 Species identification 11
2.1.2.3 DNA extraction, amplification and sequencing
of rDNA
14
2.1.2.4 Sequence alignment 15
2.1.2.5 Molecular signatures 16
2.1.2.6 Phylogenetic reconstruction 16
2.1.2.7 Toxin analysis 21
2.1.3 Results 22
2.1.3.1 Pseudo-nitzschia brasiliana Lundholm, Hasle
& Fryxell 2002
22
2.1.3.2 Pseudo-nitzschia cf. cuspidata Hasle (Hasle)
emend. Lundholm, Moestrup & Hasle 2003
25
2.1.3.3 Pseudo-nitzschia dolorosa Lundholm &
Moestrup 2006
27
2.1.3.4 Pseudo-nitzschia micropora Priisholm,
Moestrup & Lundholm 2002
29
2.1.3.5 Pseudo-nitzschia pungens (Grunow & Cleve)
Hasle 1993 var. pungens
31
2.1.3.6 Pseudo-nitzschia circumpora H.C. Lim, C.P.
Leaw et P.T. Lim sp. nov.
33
2.1.3.7 Secondary structure of ITS2 38
2.1.3.8 Phylogenetic inferences 40
2.1.3.9 Toxin analysis 41
2.1.4 Discussion 44
2.1.4.1 Comparison of Pseudo-nitzschia circumpora
to its sister species
44
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2.1.4.2 Pseudo-nitzschia in the Southeast Asia 45
2.1.4.3 Key to species of Pseudo-nitzschia
(Bacillariophyceae) in Southeast Asia
49
2.1.4.4 ITS2 transcript as barcode for Pseudo-
nitzschia
52
2.2 Three novel species in the Pseudo-nitzschia
pseudodelicatissima complex: P. batesiana sp. nov., P.
lundholmiae sp. nov. and P. fukuyoi sp. nov.
(Bacillariophyceae), from the Straits of Malacca, Malaysia
53
2.2.1 Introduction 53
2.2.2 Materials and Methods 55
2.2.2.1 Samples and culture collection 55
2.2.2.2 Morphological observation 57
2.2.2.3 DNA extraction, amplification, and
sequencing of rDNA
57
2.2.2.4 ITS and LSU rDNA phylogenetic analyses 58
2.2.2.5 ITS2 transcript phylogenetic analyses 59
2.2.3 Results 66
2.2.3.1 Pseudo-nitzschia batesiana H.C. Lim, S.T.
Teng, C.P. Leaw et P.T. Lim sp. nov.
66
2.2.3.2 Pseudo-nitzschia lundholmiae H.C. Lim, S.T.
Teng, C.P. Leaw et P.T. Lim sp. nov.
70
2.2.3.3 Pseudo-nitzschia fukuyoi H.C. Lim, S.T.
Teng, C.P. Leaw et P.T. Lim sp. nov.
73
2.2.3.4 Molecular datasets 80
2.2.3.5 Phylogenetic inferences 82
2.2.3.6 ITS2 secondary structures 83
2.2.4 Discussion 93
2.2.4.1 Description of three novel species in the
P. pseudodelicatissima complex
93
2.2.4.2 Nucleotide sequence alignment strategies
100
2.2.4.3 ITS2 transcript as a better marker to resolve
Pseudo-nitzschia phylogeny
101
CHAPTER III POPULATION GENETIC STRUCTURE OF TWO
Pseudo-nitzschia SPECIES
103
3.1 Genetic diversity of Pseudo-nitzschia brasiliana
(Bacillariophyceae) from Malaysia
103
3.1.1 Introduction 103
3.1.2 Materials and Methods 105
3.1.2.1 Sampling, culture and morphological
Observation
105
3.1.2.2 Total genomic DNA extraction 108
3.1.2.3 Gene amplification and sequencing 108
3.1.2.4 Secondary structure prediction and sequence-
structure alignment
109
3.1.2.5 Phylogenetic analysis based on ITS structural 110
x
information
3.1.2.6 Haplotype determination 110
3.1.3 Results 113
3.1.3.1 Morphology 113
3.1.3.2 Secondary structures of the ITS region 115
3.1.3.3 Genetic diversity of P. brasiliana 117
3.1.4 Discussion 122
3.2 Evolution diversification of a diatom, Pseudo-nitzschia
pungens (Bacillariophyceae)
125
3.2.1 Introduction 125
3.2.2 Materials and Methods 128
3.2.2.1 Sampling and cultures collections 128
3.2.2.2 DNA extraction, PCR and taxon sampling 128
3.2.2.3 ITS folding 136
3.2.2.4 Genetic diversity and population
differentiation
136
3.2.2.5 Population structure inference 137
3.2.3 Results 138
3.2.3.1 ITS folding and structural information 138
3.2.3.2 Diversity within the nuclear encoded ITS
Region of P. pungens
141
3.2.3.3 Haplotype diversity and geographic
distribution of ITS rDNA variation
144
3.2.3.4 Population structure 155
3.2.4 Discussion 158
3.2.4.1 Global patterns of genetic variation 159
3.2.4.2 Phylogeographic hypothesis and
diversification of P. pungens
160
CHAPTER IV CONCLUSION 164
REFERENCES 166
xi
LIST OF TABLES
Table Page Table 2.1.1 Species and culture strains of Pseudo-nitzschia established in
this study
11
Table 2.1.2 List of LSU rDNA sequences of Pseudo-nitzschia used in
phylogenetic studies. (* indicate strains obtained in this
study)
18
Table 2.1.3 List of ITS2 rRNA sequences of Pseudo-nitzschia used in
phylogenetic studies. (* indicate strains obtained in this
study)
20
Table 2.1.4 Morphometric data of Pseudo-nitzschia circumpora sp. nov.
compared to its sister taxa, and other Pseudo-nitzschia
species. Number showed in [ ] brackets indicated the number
of cells measured.
36
Table 2.1.5 Pseudo-nitzschia species found in Southeast Asia region 48
Table 2.2.1 Strain designation, localities and date when samples were
collected for cultures of Pseudo-nitzschia established for the
present study. (* indicate sequence obtained from type
strains; bold indicate strains obtained in present study).
56
Table 2.2.2 List of ITS-5.8S-ITS2 and LSU rDNA sequences of Pseudo-
nitzschia used in phylogenetic studies. (* indicate sequence
obtained from type strains; bold indicate strains obtained in
present study)
61
Table 2.2.3 Morphometric data of Pseudo-nitzschia batesiana, P.
lundholmiae and P. fukuyoi compared to its closest species.
Brackets indicate number of cells measured in this study.
78
Table 2.2.4 Corrected p-distances of P. batesiana, P. lundholmiae and P.
fukuyoi for the LSU rDNA dataset compared to their closely
related species.
81
Table 2.2.5 Numerical and statistical values of the secondary structures
(ITS2) of selected Pseudo-nitzschia species; * indicate the
type strains, bold indicated the strains obtained in present
study
88
Table 2.2.6 Comparisons of the detailed secondary structures of P.
batesiana, P. lundholmiae and P. fukuyoi to its closest
species. Number in bracket indicates the number of changes
of the nucleotides.
90
Table 3.1.1 List of P. brasiliana strains used in the present study 111
xii
Table 3.1.2 Detailed morphometric characteristics of P. brasiliana as
observed in Malaysian strains compared with data from
literature, n indicates number of cells examined
114
Table 3.1.3 Nucleotides composition in the ITS region (bp) in P.
brasiliana from different locations obtained in this study.
Variations were in comparison to haplotype h1. Sites with
identical nucleotide to the reference haplotype are indicated
with “.”. Numbering of nucleotides start from ITS1 until
ITS2 region of strain PnSm19 (HQ111405)
119
Table 3.1.4 Genetic distances in the ITS region of P. brasiliana.
Diagonal elements represent the average number of pairwise
differences within population (PiX) [100%], and the lower
diagonal represents the percentage of average number of
pairwise differences between populations (PiXY) [100%]
121
Table 3.2.1 List of P. pungens strains used in the present study. Boldface
indicates strains collected in this study. Strain marked with *
indicate P. pungens var. aveirensis while # indicate P.
pungens var. cingulata
130
Table 3.2.2 Sequence divergence (uncorrected p-values) within and
among the three clades, shown as minimum-maximum
(average)
143
Table 3.2.3 Nucleotide composition in the ITS1-5.8S-ITS2 region (in
base pairs) in P. pungens from different geographical regions
included in this study. Variations were in comparison to
haplotype h1. Sites with identical nucleotide to the reference
haplotype are indicated with “.”. Numbering of nucleotides
starts from ITS1 until ITS2 of h1
145
Table 3.2.4 Genetic divergence in ITS1-5.8S-ITS2 within P. pungens 154
xiii
LIST OF FIGURES
Figure Page Figure 2.1.1 Map of Malaysian Borneo showing the sampling sites. (A)
Santubong, Samariang Batu and Muara Tebas in Kuching, (B)
Kudat of Sabah, (C) Pulau Mamutik, Jetty and Teluk Likas,
(D) Tasik Sitompok in Kuala Penyu
10
Figure 2.1.2 Pseudo-nitzschia brasiliana: (A) SEM. Acid-washed valve
showing fibulae, striae, and symmetrical valve. Scale bar = 10
µm. (B) LM, phase contrast. Girdle view of live cells in chain,
overlap of cell ends c. 1/11 of total cell length. Scale bar = 10
µm. (C) TEM. Valve shows slight tapering towards the tip.
Scale bar = 1 µm. (D) TEM. Cingular bands. Scale bar = 1
µm. (E) TEM. Close-up of valve showing the simple hymenate
velum of the poroids (inset)
24
Figure 2.1.3 Pseudo-nitzschia cf. cuspidata: (A) LM, phase contrast. Girdle
view of live cells in chain, overlap of cell ends 1/6 of total cell
length. Scale bar = 10 µm. (B) SEM. Acid-washed valve
showing fibulae, central interspace, and symmetrical valve.
Scale bar = 10 µm. (C) TEM. Central part of valve showing
presence of central interspace. Scale bar = 1 µm. (D) TEM.
Cingular band, valvocopula. Scale bar = 1 µm. (E) Detail of
poroids, with hexagonal pattern on the hymens. Close-up
showing a poroid possessing a centrally-located sector (inset).
Scale bar = 0.5 µm. (F) TEM. Poroid perforation, 2–4 sectors.
Scale bar = 0.5 µm
26
Figure 2.1.4 Pseudo-nitzschia dolorosa: (A) SEM. Acid cleaned frustules
showing the valve of the cell. Note that the valve margins are
asymmetrical, one side straight, the other convex. Scale bar =
10 µm. (B) Central part of the cell showing the presence of a
large central interspace, and eccentric raphe divided by a
central nodule. Scale bar = 1 µm. (C) TEM. Parts of the valve,
showing one row of square poroids. Scale bar = 1 µm
28
Figure 2.1.5 Pseudo-nitzschia micropora: (A) SEM. Cell frustules showing
the valve. Note that valve margins are symmetrical. Scale bar
= 10 µm. (B & C) TEM. Valve showing two rows of poroids.
Scale bar = 1 µm. (D) TEM. Cingular band showing
valvocopula, second and third band. Scale bar = 1 µm. (E)
TEM. Valvocopula, biseriate band striae with two poroids
height. Scale bar = 1 µm
30
Figure 2.1.6 Pseudo-nitzschia pungens var. pungens: (A – C) SEM. Acid-
cleaned frustule showing coarsely structured valve. Note that
valve margins are symmetrical. Scale bar = 10 µm. (A) Wild
sample from Santubong, Sarawak. (B) Cultured sample
collected from Santubong, Sarawak, strain PnSb54. (C)
32
xiv
Culture sample collected from Kudat, Sabah, strain PnKd01.
(D) LM, phase contrast. Girdle view of live cells in chain,
overlap of cell ends c. ¼ of total cell length. Scale bar = 10
µm. (E) SEM. Ends of one valve from a clonal culture,
showing the overall shape of pointed cell ends. Scale bar = 1
µm. (F) TEM. Central part of cell showing absence of a central
interspace. Striae with two rows of poroids. Scale bar = 1 µm.
(G) TEM. Valve with round hymenate velum of the poroids
(inset). (H & I) TEM. Details of cingular bands. Scale bar = 1
µm
Figure 2.1.7 Pseudo-nitzschia circumpora: (A) SEM. Acid-cleaned valve
showing fibulae, central interspace, and asymmetrical valve.
Scale bar = 10 µm. (B) LM, phase contrast. Girdle view of live
cells in chain, overlap of cell ends c. 1/8 of total cell length.
Scale bar = 10 µm. (C) TEM. Apical end of cell. (D) TEM.
Central part of valve showing presence of central interspace.
Scale bar = 1 µm. (E & F) TEM. Detail of fibulae and striae,
note the poroids irregularly spaced. (G) TEM. Detail of
poroids, with hexagonal pattern on the hymens. Close-up
showing each sector with further perforation. Scale bar = 1
µm. (H) TEM. Detail of valvocopula, VC and second cingular
band, II. Scale bar = 1 µm
35
Figure 2.1.8 Molecular signatures of Pseudo-nitzschia species found in
Malaysian Borneo. (A) P. brasiliana, (B) P. dolorosa, (C) P.
micropora, (D) P. pungens and (E) P. circumpora. Rectangle
showing the CBC while arrow indicate the hemi-CBC
39
Figure 2.1.9 Phylogenetic tree via maximum parsimony (MP) analysis of
Pseudo-nitzschia spp. based on the D1-D3 LSU rDNA from
694 aligned positions. Tree length= 498 steps; CI= 0.6265; and
RI= 0.7597. The vertical bars denote the two main clades (I
and II). Values on the nodes correspond to bootstrap values of
MP (1000 replicates), likelihood (GTR+I+G model; 100
replicates of bootstrap) and Bayesian (PP), only values > 0.50
are shown. Taxa in bold designate sequences obtained in this
study
42
Figure 2.1.10 Profile Neighbour-Joining (PNJ) tree inferred by ITS2
sequence-structure information using ProfDistS with ITS2
specific general time reversible (GTR) substitution model. The
nodal supports are bootstrap support values from 1000 pseudo-
replicates. Cylindrotheca closterium (AF289049) was used as
outgroup
43
Figure 2.2.1 Pseudo-nitzschia batesiana sp. nov. (A) Cell valve view, scale
bar = 10 µm. (B) LM micrograph: Girdle view. Note cell
overlap at 1/10. Scale bar = 10 µm. (C) Striae, poroid structure
of valve and mantle. Note central interspace. Scale bar = 1 µm.
69
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(D) Valve ends. Scale bar = 1 µm. (E) Detail of poroids. Note
poroid hymen divided into two to three sectors. (F) Part of
valve. Note irregularly spaced fibulae. Scale bar = 1 µm. (G)
Details of cingulum. Note valvocopula and second band. Scale
bar = 1 µm. (H) Detail of third band. (I) The close-up of
valvocopula and second band. Scale bar = 2 µm
Figure 2.2.2 Pseudo-nitzschia lundholmiae sp. nov. (A) Whole valve. Scale
bar = 10 µm. (B) LM micrographs: Girdle view. Note cell
overlap at 1/6. Scale bar = 10 µm. (C) Striae, poroid structure
of valve and mantle. Note central interspace. Scale bar = 0.5
µm. (D) Valve ends. Scale bar = 1 µm. (E and F) Detail of
poroids. Note poroids hymen with one to three sectors. Scale
bar = 0.2 µm. (G) Details of valvocopula. Scale bar = 0.5 µm.
(H) Valve striae structure. Scale bar = 10 µm
72
Figure 2.2.3 Pseudo-nitzschia fukuyoi sp. nov. (A) Whole valve. Scale bar
= 10 µm. (B) LM micrograph: Girdle view. Note cell overlap
at 1/6. Scale bar = 10 µm. (C and D) Striae, poroid structure of
valve and mantle. Note missing poroids and sectors. Scale bar
= 1 µm. (E and F) Valve ends with slightly difference shapes.
Scale bar = 10 µm. (G and H) Detail of poroids. Note poroids
hymens with two to four sectors. Scale bar = 0.2 µm. (I)
Details of valvocopula. Scale bar = 0.2 µm. (J) The three
cingular bands delimited by horizontal lines. From left: VC =
valvocopula, II = second cingular band, III = third cingular
band. Scale bar = 0.2 µm
76
Figure 2.2.4 Secondary structures of ITS2 and the molecular signatures of
(A) P. batesiana, (B) P. lundholmiae and (C) P. fukuyoi.
Rectangles indicate compensatory base changes (CBCs) and
arrows point to hemi compensatory base changes (HCBCs)
77
Figure 2.2.5 (A) Phylogenetic tree from maximum likelihood (ML)
analysis based on Pseudo-nitzschia whole ITS1-5.8S-ITS2
region of the ribosomal DNA (403 characters included). The
tree is rooted (outgroup not shown). (B) Phylogenetic tree
from ML analysis based on the Pseudo-nitzschia D1-D3 LSU
rDNA (697 characters included). The tree is rooted (outgroup
not shown). Asterisks indicate types strains used in species
references. Both trees search used 100 random-addition
replications and TBR branch-swapping
84
Figure 2.2.6 Profile neighbor joining tree based on ITS2 with orthologous
sequence-structure alignment information (612 positions
included) using ProfDistS. The tree was rooted with
Fragilariopsis kerguelensis (EF660061) as the outgroup. The
nodal supports are bootstrap values from 1,000 pseudo-
replications. Posterior probabilities of more than 0.95 are
marked with thick lines
85
xvi
Figure 2.2.7 Morphometric data on cell width (A), fibulae (B), striae (C),
poroid (D), perforation sectors (E), and density of band striae
(F) of species classified under the P. pseudodelicatissima
complex: P. batesiana (bat), P. caciantha (cac), P. circumpora
(cir), P. lundholmiae (lund), P. fukuyoi (fuk), P. cuspidata
(cus), P. pseudodelicatissima (pse), P. calliantha (cal), P.
fryxelliana (fry), P. hasleana (has), and P. mannii (man). The
data on P. batesiana, P. lundholmiae and P. fukuyoi are from
the present study; data on P. caciantha, P. calliantha, P.
cuspidata and P. pseudodelicatissima are from Lundholm et
al., (2003); Trainer et al., (2009); Moschandreou and
Nikolaidis, (2010a); Lundholm et al., (2012); data on P.
fryxelliana and P. hasleana are from Lundholm et al., (2012);
and P. mannii from Amato and Montresor, (2008). Boxes
indicate the standard error, line in box indicates the median, +
in box showed the mean while the whiskers showed the min
and max. Dark grey boxed represent the three new species
described in present study
98
Figure 2.2.8 Morphological characters mapped onto the ITS ML tree of
Pseudo-nitzschia. Several clades were collapsed to triangles
with the number on the right (in bracket) indicates the number
of taxa. Drawings represent the number of dividing sectors
99
Figure 3.1.1 Sampling locations in Malaysia where P. brasiliana strains
were collected: (A) Santubong and Samariang Batu in
Kuching, (B) Port Dickson in Negeri Sembilan, (C) Pulau
Mamutik and Jetty in Kota Kinabalu
107
Figure 3.1.2 Pseudo-nitzschia brasiliana: (A) SEM. Valve view of frustule.
(B) LM. 1/10 cells overlap Scale bar = 10 µm. (C-E) TEM. (C)
Broad round end apices (D) Two rows of poroid in each striae
(E) simple poroid, hexagonal arrangement (inset). Scale bar =
1 µm
113
Figure 3.1.3 Secondary structure of ITS1-5.8S-ITS2 rDNA of P. brasiliana
strain PnSm07. Major helices were labeled as I – V in the
ITS1 transcript and I – IV, IIa in the ITS2 transcript. Numbers
in brackets next to boxes and circles indicate the base changes
occurred among sequences of P. brasiliana in Malaysian
waters
116
Figure 3.1.4 (A) Profile neighbor joining (PNJ) tree of P. brasiliana
derived from ITS nucleotide sequences with structural
information. The tree was rooted by P. pungens (HQ111412)
and P. multistriata (EF636677) with outgroup not shown.
Values on the nodes correspond to posterior probability (PP),
only values > 0.50 are shown. (B) Minimum spanning tree of
the 20 haplotypes found among 36 strains of P. brasiliana.
120
xvii
Each circle represents a haplotype, and scaled with number of
strain as frequency
Figure 3.2.1 The ITS1 consensus secondary structure of all P. pungens
analyzed. Major helices were labeled in ITS1 (helix I-V).
Rectangles indicate hemi compensatory base changes
(HCBCs). Circles represent single nucleotide polymorphisms
(SNPs)
139
Figure 3.2.2 The ITS2 consensus secondary structure of all P. pungens
analyzed. Major helices were labeled in ITS2 (helix I-IV and
pseudo-helix IIa). Rectangles indicate hemi-CBC and shaded
rectangle indicates CBC. Circles represent single nucleotide
polymorphisms (SNPs)
140
Figure 3.2.3 Profile neighbor joining tree of P. pungens derived from ITS1-
5.8S-ITS2 nucleotide sequence with structural information.
The tree was rooted with P. multiseries (DQ062664), outgroup
not shown
142
Figure 3.2.4 Median-joining network based on 722 bp of P. pungens ITS1-
5.8S-ITS2 rDNA. Each circle represents a haplotype.
Haplotypes are designated by numbers. Each line on the line
connecting haplotypes represents a mutational step. Red dots
represent median vectors. The haplogroups were assigned to
G1, G2 and G3 where G1 corresponding to sub-clade Ia in Fig.
3, G2 to clade II and G3 to clade III. Dotted circle corresponds
to sub-clade Ib
153
Figure 3.2.5 The best K selected based on Pritchard et al. (2000) 156
Figure 3.2.6 Geographical distribution of P. pungens and the population
structure. (A) Colored line represents global thermohaline
circulations (Conveyor Belt), blue line indicates cold, salty
dense water and red line indicates warm shallow water.
Colored circle indicated the strains verified based on ITS
sequences while black dot indicated morphology based-record
of P. pungens. Modified from Thessen, 2007 (B) Population
structure based on ITS gene estimated by Bayesian cluster
analysis implemented in STRUCTURE. Assignment of 164
individuals to K = 7 genetically distinct group. Each individual
is represented by a vertical bar colored according to the
assigned cluster
157
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LIST OF ABBREVIATIONS
AMOVA analysis of molecular variance
ARISA automated ribosomal intergenic spacer analysis
ASP Amnesic shellfish poisoning
BI Bayesian
bp base pair
CBC compensatory base change
CIS central interspace
CNS central nervous system
CTAB cetyl-trimetyl ammonium bromide
D1-D3 domain 1 to domain 3
DA domoic acid
dNTP deoxyribonucleotide triphosphate
EDTA ethelyenediaminetetraacetate
EM electron microscopy
FMOC 9-fluorenylmethylchlorofromate
GTR general time reversal
HAB harmful algae bloom
HCBC hemi-compensatory base change
HCl hydrochloric acid
HPLC high performance liquid chromatography
ITS internal transcribed spacer
ITS2 second internal transcribed spacer
KMnO4 potassium manganate
L:D light:dark
LM light microscope
LSU large subunit
MCMC Markov chain Monte Carlo
MgCl2 magnesium chloride
ML maximum likelihood
MP maximum parsimony
Mya million years ago
xix
LIST OF ABBREVIATIONS
NaCl sodium chloride
NE north east
NW north west
OTU operational taxonomic unit
PAUP phylogenetic analysis using parsimony
PCR polymerase chain reaction
PNJ profile neighbour joining
PP posterior probabilities
PSP paralytic shellfish poisoning
rbcL RUBISCO large subunit
rDNA ribosomal deoxyribonucleic acid
rRNA ribosomal ribonucleic acid
RUBISCO ribulose-1,5-bisphosphate carboxylase oxygenase
S south
SEM scanning electron microscope
SNP single nucleotide polymorphism
sp. nov. species novel
TBR tree-bisection reconnection
TEM transmission electron microscope
var. variety
VC valvocopula
W west
1
CHAPTER I
GENERAL INTRODUCTION
Pseudo-nitzschia species had become one of the study interests after the first incident
of human intoxication reported at Prince Edward Island since 1987, where 100 people
were hospitalized, and four persons died after ingestion of contaminated shellfish
mollusks, blue mussels (Mytilus edulis) (Bates et al., 1989). Autopsies revealed brain
lesions, some of the survivors suffered permanent loss of short-term memory, and
Amnesic shellfish poisoning (ASP) was named based on the syndrome (Bird and
Wright 1989). The causative organism and the source of neurotoxin domoic acid (DA)
were found to be the marine diatom, Pseudo-nitzschia multiseries (formerly Nitzschia
pungens f. multiseries) which bloomed in the mussels cultivation area (Bates et al.,
1991). Domoic acid is a tricarboxylic acid, that disrupts normal neurochemical
transmission in the brain by binding to certain glutamate receptors in mammalian
central nervous system (CNS) (Bird and Wright 1989).
More than thirty species of Pseudo-nitzschia had been described thus far. Half
of the species were known to produce DA and Pseudo-nitzschia seriata, Pseudo-
nitzschia australis and Pseudo-nitzschia multiseries were the most potent neurotoxin
producers. Subsequently, benthic diatom Amphora coffeaeformis (Shimizu et al., 1989)
and Nitzschia navis-varingica (Kotaki et al., 2000) were also reported as DA producer.
In Malaysia, the most prominent HAB-related intoxication is Paralytic
Shellfish Poisoning (PSP) which has caused human intoxication including several
fatalities (Lim et al., 2004). Intensive monitoring on PSP producing dinoflagellates
2
and shellfish management has been conducted by relevant agencies. However, other
form of shellfish poisoning including Amnesic Shellfish Poisoning (ASP) has little
attention paid to it.
Pseudo-nitzschia species are of particular interests because very little is
known on their occurrence and distribution in Malaysian coastal waters. The Pseudo-
nitzschia had been well documented in Vietnam with thirteen species found but not in
other countries in Southeast Asia region (Larsen and Nguyen, 2004). In order to gain
better understanding on occurrence, taxonomy, seasonal distribution and physiology of
Pseudo-nitzschia species in Malaysian waters, various studies have been conducted
since the year 2007. So far, four species of Pseudo-nitzschia viz. P. brasiliana, P.
dolorosa, P. micropora and P. pungens had been confirmed in Malaysian waters (Lim
2011; Suriyanti 2011). Although P. brasiliana and P. pungens was reported elsewhere
as potential toxin producer of domoic acid (Whyte et al., 1995; Sahraoui et al., 2011;
Moschandreou et al., 2012), but none of the strains found in Malaysia showed
detectable level of domoic acid (Lim et al. 2010). The ability to produce DA under
different physiological condition might contribute to the discrepancy (Pan et al., 1998;
Ramsey et al., 1998). The blooms of this DA producing diatom cause bioaccumulation
of the toxin in filter feeder shellfish molluscs. Contamination of DA is shellfish
mollusks had been reported in Vietnam (Dao et al., 2006; 2009; 2012; Takata et al.,
2009). Shellfish poisoning incident might happen if human, marine mammals or
marine bird ingested the contaminated shellfish.
Well documentation of occurrence and distribution of toxic Pseudo-nitzschia
documented may provide useful and critical information for mitigation management,
mitigation of HABs occurrence and minimize threat to public health. Till to date, our
understanding on the species occurrence and distribution of Pseudo-nitzschia species
3
were severely limited by the lack of a quantitative and comprehensive description on
their population dynamics. Apart from that the taxonomy of the genus is also far from
resolve and this partly hampered the effort in development of species-specific
molecular oligonucleotide probes. This is mainly due to close morphological
similarity/crypticity among species especially within P. delicatissima complex and P.
pseudodelicatissima complex (Lundholm et al., 2003; Amato and Montresor, 2008;
Quijano-Scheggia et al., 2009b; Lundholm et al., 2012; Lim et al., 2012a). Noteworthy,
with the presence of high levels of genetic diversity (Parsons et al., 1999), toxic and
nontoxic strains present within the same morphospecies (Orsini et al., 2002; Evans et
al., 2004).
The taxonomy of P. pseudodelicatissima complex is always challenging in
terms of morphology and genetic delineation especially P. pseudodelicatissima and P.
cuspidata (Lundholm et al., 2012). The entities of these two species and also the other
species within this complex were analysed using sequences available from temperate
countries though GenBank. However, the information on tropical strains might
provide value clue to solve the puzzle. Under appropriate geographic and
environmental conditions especially separation in the Pleistocene due to climate
changes may lead speciation to a population structure and hence plays an important
role in species diversification of marine phytoplankton.
Setting up of the clonal laboratory cultures were carried out in parallel in this
study and the cultures were used for taxonomy, toxicity and population genetics study.
Information on the species present and their capability to produce toxins would be
very important in the selection of aquaculture sites and monitoring activities for the
country. Hence, the goals of this study was aimed to understand the morphology,
phylogenetic relationship among the species and genetic diversity of Pseudo-nitzschia
4
originated from Malaysian coastal waters in comparison with strains from other
geographical origin. The approach provides fundamental information on the genetic
variability among different populations of Pseudo-nitzschia at different spatial and
temporal scales.
Objectives of this study were listed as below:
i. To examine and characterize the morphology of Pseudo-nitzschia species in
Malaysian coastal waters,
ii. To determine the morphological variations and taxonomy of Pseudo-nitzschia
within P. pseudodelicatissima complex sensu lato,
iii. To analyze the phylogenetic relationships of Pseudo-nitzschia using several
selected genetic markers,
iv. To characterize the inter- and intraspecific variations in Pseudo-nitzschia using
ITS1-5.8S-ITS2 rDNA, and
v. To investigate the genetic structure and gene flow of Pseudo-nitzschia natural
populations.
This dissertation is divided into four chapters. Chapter I provided the brief
introduction with emphasis on the gap lack of Pseudo-nitzschia in tropical waters.
Several research problems identified and research questions to be answered in the end
of this study. In Chapter II, the taxonomy unambiguity within P. pseudodelicatissima
complex was re-examined in attempt to resolve the morphological and also molecular
data using the strains of Pseudo-nitzschia collected along the coastal waters of
Malaysia throughout the study. A morphotype collected from Santubong was
described as a new species P. circumpora, based on morphological and molecular
5
evident. More samples were collected from the Strait of Malacca to clarify the species
richness of Pseudo-nitzschia in Malaysia. Three more new morphotypes were found
closely related to P. pseudodelicatissima and P. cuspidata and the taxonomic status
within this complex were further clarified using morphologically and genetically tools.
In Chapter III, the population genetic structure of P. brasiliana and P. pungens
were examined as this two species were found widely distributed not only in
Malaysian coastal water but also distributed globally. The genetic diversity and
geographic distribution pattern of Malaysian P. brasiliana was evaluated using ITS1-
5.8S-ITS2 rDNA with inference of secondary structure information. A similar
approach was taken in attempt to evaluate the genetic variation of P. pungens in global
population with more inputs from tropical waters to answer the gaps in the dispersal of
this cosmopolitan species. A summary of dissertation was drawn to conclude the
findings of this study in the last chapter.