Genetic diversity of populations of the malesian moss, acanthorrhynchium papillatum, as measured by microsatellite markers and ITS2 sequences

GENETIC DIVERSITY OF POPULATIONS OF THE
MALESIAN MOSS,
ACANTHORRHYNCHIUM PAPILLATUM,
AS MEASURED BY MICROSATELLITE MARKERS
AND ITS2 SEQUENCES
ALFREDO AMIEL P. LEONARD
´
IA
(M.Sc., Leiden University)
A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF BIOLOGICAL SCIENCES
THE NATIONAL UNIVERSITY OF SINGAPORE
2007
i
ACKNOWLEDGEMENTS
A PhD project –particularly one that involves both field and lab work– is never a
solitary endeavor. Moreover, contributions to its completion are direct and indi-
rect, physical and metaphysical, immediate, long-drawn, early, recent, last-minute.
Though I labored to set down the names of all those I remember who helped, much
time has passed since the project’s inception that I may have inadvertently left
out a person or two despite my best efforts. To them I sincerely apologize and to
all I offer my deep est gratitude.
My sincere and deepest thanks to my supervisors, Associate Professor Benito
C. Tan and Associate Professor Prakash P. Kumar. Thank you for letting me
experiment on microsatellites to near-disastrous results. Thank you for your pa-
tience and continuous supp ort. And thank you to the Tan Chin Kee Foundation,
for funding my flight to Indonesia to start off my studies on mosses. Prof. Ben,
it took me some time to figure this out, but I have not forgotten and I am truly
grateful.
To Professor Lam Toong Jin, my sincerest thanks for teaching me to be (more)

critical and for the suggestion to focus on one species.
To the National University of Singapore and the Department of Biological
Sciences, for funding my research through my research scholarship and various
research grants. This study would literally not be possible without your support.
And to Ee Ling, my loving wife, for never tiring nor failing to teach me how
to use the pH meter. O flog my feeble brain!
And to those who welcomed me at the Cryptogam laboratory, I celebrate our
great times and our friendship. To Boon Chuan, may you b e the best muscologist
of our generation. To Chang Ying, thank you for the (heated) discussions on
ii
cladistics and for your friendship. To Farida, for teaching me how to be more
respectful. To Meng-Shyan, for the early and fruitful discussions. To Su-Fern,
for the seed game. To Mr. Chua, for Linux
TM
and more. To Serena and Hwee
Boon, for the sushi. To Yao Hui, for painting with light the beauty of mosses.
To Claudia and Kai Yang, for the jigsaw and company in Maxwell hill. To Woon
Ping, for help with Pogonatum.
And to the geneticists at the Plant Morphogenesis laboratory, thank you for
allowing some bench space to a moss guy. To Wang Yu, for the pBluescript. Ping
Yu for genome walking. To Li Ang, for the discussions on ISSRs. To Mandar,
for our (unfulfilled) vow and for the biscuits. To Dileep, for drinking water in the
drawer and for (re)teaching me humility. Lian-Lian and Jaclyn and Xin Ying, how
you all changed the lab and made it better. To Mrs. Ang, for your patience and
help.
To Professor Haji Mohamed, my deepest gratitude for facilitating the collec-
tions in Malaysia as part of an existing research collaboration between your lab
and Prof. Ben’s. To the frighteningly impressive Yong Kien Thai, I look forward
to seeing the wonderful places you and Boon Chuan will be bringing Southeast
Asian bryology.

To Dr. Helena Korpelainen and her lab, for kindly hosting my short visit to
her lab in the University of Helsinki.
To the National Parks Board, my sincere thanks for allowing us to do our
collections in Singapore.
And to the 7th Floor mafia. Carol for commiserating. Serena for the crosslinker.
Daryl for badminton. Teng Seah for badminton. Wee Kee for badminton.
To Goh Wee Kee. For being my MCB mother. Twice.
To Lars and Cecilia, it’s been so long since we trudged around Maxwell Hill,
thanks for the memories and the s upport in Stockholm.
iii
And to my field mates, how we had great times together! Tzi Ming for taking
me along on your trips and for sparking an interest in herps. To Su Lee for the
same (minus the herps). Cheng Puay, for your boundless enthusiasm. Eunice for
being such a sport. Bicky for demonstrating that Eunice is a sport. Jeremy for
the same –and for being so darn calm all the time. Reuben for translating and
for the maps. Norman for showing us what it’s like to be a field pro. Matthew for
Tioman and the RAM. Tommy, for Tioman and the crab. Heok Hui for being so
impressive. Swee Hee for being so knowledgeable. Darren for being such a natural.
Zeehan for being so effective. And Ngan Kee for being the mother to us all.
To Joelle, for planting the seed of microsatellites. See what you got me into?
To my Filipino barkada, for restoring the faith. Ming, for being. JC for the
music. Arvin for the “Oh, S. . . !”
To Chye Fong and Bee Ling, for allowing me do fragment analysis. To Joan,
Reena, Sally, Mrs. Chan, and Laurence, no graduate student could survive without
you. Thank you for everything! To Ann Nee, thank you for DreamWeaver and all
the great posters. To Mr. Soong, thank you for always processing our orders so
promptly.
To Vaane and Kavita, Ee Ling has finally shown me the ice cream in Sunset
Way. Here’s to great times past and in the future.
To the makers of ZipLoc bags. Though your products be humble, they proved

to be most indispensable.
To Donald Knuth, Leslie Lamport, Peter Borg and Richard Koch: A botanist
wrote his thesis with L
A
T
E
X on a notebook with a glowing apple using a text editor
iconificated (sic) by a wild strawberry. How strange is that?
To Ee Ling’s family, for welcoming me and for badminton.
To my family. No words can capture or express. But you know you’re all
beyond that.
iv
And again to Ee Ling. You taught me more than just how to use a pH meter.
Your love and patience sustained me through the most trying times spent working
on this project. But since you can read my mind then you already know that,
hunh?
And finally, although this thesis project, in its mostly-quiet, sometimes-angry
all-consuming relentlessness, has taken 16% of my life thus far, may I never forget
what and Who really matter.
v
TABLE OF CONTENTS
Acknowledgements i
Table of Contents v
Summary xii
List of Tables xiv
List of Figures xvii
List of Abbreviations and Symbols xix
Chapter 1: General Introduction and Review of Literature 1
1.1 General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Background of the study . . . . . . . . . . . . . . . . . . . . 1

1.1.2 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1.3 Scope and limitations . . . . . . . . . . . . . . . . . . . . . . 4
1.2 Review of Literature . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.1 Diversity of moss populations . . . . . . . . . . . . . . . . . 5
1.2.2 Selection of molecular markers for this study . . . . . . . . . 25
1.2.3 Description of Acanthorrhynchium papillatum . . . . . . . . 27
Chapter 2: Development of Microsatellite Markers for Acanthor-
rhynchium papillatum 32
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.2.1 Field collection and processing of moss samples . . . . . . . 35
vi
2.2.2 Genomic DNA extraction . . . . . . . . . . . . . . . . . . . 38
2.2.3 Genomic DNA digestion . . . . . . . . . . . . . . . . . . . . 39
2.2.4 Trimming and dephosphorylation of fragment ends . . . . . 40
2.2.5 Ligation of SNX linkers to MBN/CIP-treated fragments . . 41
2.2.6 Enriching for microsatellite-bearing fragments . . . . . . . . 43
2.2.7 PCR amplification of fragments enriched for microsatellites . 45
2.2.8 Trimming SNX linker ends of the amplified fragments . . . . 46
2.2.9 Insertion of trimmed fragments into cloning vector . . . . . . 46
2.2.10 Transformation of competent cells and culture of transfor-
mants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.2.11 Differences in protocol between APMS and APBMS libraries 47
2.2.12 Screening colonies for inserts . . . . . . . . . . . . . . . . . . 47
2.2.13 Culture and miniprep of colonies with inserts . . . . . . . . 49
2.2.14 Sequencing inserts . . . . . . . . . . . . . . . . . . . . . . . 49
2.2.15 Contig assembly and identification of microsatellite-bearing
inserts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.2.16 Primer design and testing . . . . . . . . . . . . . . . . . . . 50
2.2.17 Characterizing the microsatellite markers . . . . . . . . . . . 53

2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.3.1 Construction of microsatellite-enriched libraries . . . . . . . 54
2.3.2 Differences between the APMS and APBMS libraries . . . . 58
2.3.3 Types of microsatellite loci isolated . . . . . . . . . . . . . . 60
2.3.4 Construction of multiplex PCR sets . . . . . . . . . . . . . . 60
2.3.5 Evaluation of markers through preliminary genotyping . . . 63
2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
2.4.1 Library construction . . . . . . . . . . . . . . . . . . . . . . 69
2.4.2 Marker characterizations . . . . . . . . . . . . . . . . . . . . 73
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2.4.3 Long microsatellites . . . . . . . . . . . . . . . . . . . . . . . 77
2.4.4 Comparison with other microsatellite-marker studies . . . . 78
2.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Chapter 3: Genetic Diversity Among Clumps of Acanthorrhyn-
chium papillatum as Measured by Microsatellite Markers 82
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
3.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.2.1 Sampling areas . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.2.2 Collection dates . . . . . . . . . . . . . . . . . . . . . . . . . 91
3.2.3 Field selection and collection . . . . . . . . . . . . . . . . . . 91
3.2.4 Post-collection processing . . . . . . . . . . . . . . . . . . . 94
3.2.5 Lab sampling . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3.2.6 Genomic DNA extraction . . . . . . . . . . . . . . . . . . . 94
3.2.7 Fragment analysis with microsatellite markers . . . . . . . . 95
3.2.8 Size-calling and allele-scoring . . . . . . . . . . . . . . . . . 96
3.2.9 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 98
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
3.3.1 Collected samples . . . . . . . . . . . . . . . . . . . . . . . . 103
3.3.2 Revised marker statistics . . . . . . . . . . . . . . . . . . . . 104
3.3.3 Total microsatellite diversity . . . . . . . . . . . . . . . . . . 107

3.3.4 Microsatellite diversity within sampling areas . . . . . . . . 111
3.3.5 Multi-locus genotypic diversity within sampling areas . . . . 123
3.3.6 Genetic distances . . . . . . . . . . . . . . . . . . . . . . . . 130
3.3.7 Population differentiation . . . . . . . . . . . . . . . . . . . 137
3.3.8 Population genetic structure . . . . . . . . . . . . . . . . . . 139
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
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3.4.1 Allelic diversity explains much of the diversity in Acanthor-
rhynchium papillatum . . . . . . . . . . . . . . . . . . . . . 142
3.4.2 Microsatellite loci of Acanthorrhynchium papillatum experi-
ence high rates of mutation . . . . . . . . . . . . . . . . . . 144
3.4.3 Differences in diversity among sampling areas and popula-
tion genetic structure . . . . . . . . . . . . . . . . . . . . . . 146
3.4.4 Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
3.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Chapter 4: Genetic Diversity Among Clumps of Acanthorrhyn-
chium papillatum as Measured by Variation in ITS 2 Sequences150
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
4.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.2.1 Design of primers for ITS2 amplification and sequencing . . 151
4.2.2 PCR amplification . . . . . . . . . . . . . . . . . . . . . . . 154
4.2.3 Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
4.2.4 Basecalling, contig assembly and alignment . . . . . . . . . . 155
4.2.5 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 155
4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
4.3.1 Sequencing difficulties . . . . . . . . . . . . . . . . . . . . . 156
4.3.2 Characteristics of ITS2 in Acanthorrhynchium papillatum . . 157
4.3.3 Nucleotide diversity . . . . . . . . . . . . . . . . . . . . . . . 161
4.3.4 Haplotype diversity . . . . . . . . . . . . . . . . . . . . . . . 161
4.3.5 Genetic distance . . . . . . . . . . . . . . . . . . . . . . . . 168

4.3.6 Population differentiation and genetic structure . . . . . . . 168
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
4.4.1 High rates of mutation are seen in the ITS 2 region of Acanthor-
rhynchium papillatum . . . . . . . . . . . . . . . . . . . . . 173
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4.4.2 ITS2 tree topology . . . . . . . . . . . . . . . . . . . . . . . 175
4.4.3 Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
4.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Chapter 5: Preliminary Study of Genetic Diversity Within Clumps
of Acanthorrhynchium papillatum as Measured by Microsatellite
Markers 177
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
5.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 179
5.2.1 Collection dates . . . . . . . . . . . . . . . . . . . . . . . . . 179
5.2.2 Sampling within clumps to determine variation . . . . . . . 181
5.2.3 Sample naming . . . . . . . . . . . . . . . . . . . . . . . . . 182
5.2.4 Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
5.3.1 Null alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
5.3.2 Multi-locus genotypes . . . . . . . . . . . . . . . . . . . . . 184
5.3.3 Pairwise genetic distances . . . . . . . . . . . . . . . . . . . 186
5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
5.4.1 Null alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
5.4.2 Extent of vegetative reproduction . . . . . . . . . . . . . . . 194
5.4.3 Presence of multiple genets in a clump . . . . . . . . . . . . 196
5.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Chapter 6: General Conclusions and Future Perspectives 198
References 203
Appendix A: “Dirty” sequences 220
Appendix B: Replicated sequences 229

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Appendix C: Sequences without microsatellites 232
Appendix D: Sequences without flanks 245
Appendix E: Sequences with failing primers 253
Appendix F: Acanthorrhynchium papillatum Microsatellite Mark-
ers 257
Appendix G: Characterizing candidate microsatellite markers 263
Appendix H: Computation of linkage disequilibrium 265
Appendix I: Report on microsatellite markers for Acanthorrhyn-
chium papillatum 267
Appendix J: Interclump data 270
Appendix K: Aligned ITS2 sequences 274
Appendix L: Within-clump data - Main 303
Appendix M: Within-clump data - Supplementary 306
Appendix N: Pairwise genetic distances within a clump, main data
- Method 1 308
Appendix O: Pairwise genetic distances within a clump, supple-
mentary data - Method 1 309
Appendix P: Pairwise genetic distances within a clump, main data
- Method 2 310
Appendix Q: Pairwise genetic distances within a clump, supple-
xi
mentary data - Method 2 311
xii
SUMMARY
Mosses are important yet neglected inhabitants of tropical rain forests. In this
study I investigated the genetic diversity of the Malesian moss species, Acanthor-
rhynchium papillatum (Harv.) Fleisch. using microsatellite markers and DNA
sequences from the second internal transcribed spacer region, ITS2, of ribosomal
DNA. Moss samples for analysis were collected from five sampling areas: three

areas in Peninsular Malaysia, two in Singapore. These sampling areas differ from
each other in habitat quality. Genetic diversity was assessed at several scales:
within clumps of a moss, among moss clumps in one sampling area and among
sampling areas.
Eight microsatellite markers were newly developed for Acanthorrhynchium
papillatum for this project. Rigorous tests on these markers revealed that they
were suitable for use in population genetic studies of the moss. These were the
first markers reported for a tropical moss species.
The microsatellite markers revealed high levels of allelic and haplotypic di-
versity among clumps of Acanthorrhynchium papillatum in each of the sampling
areas. Although a reduction in both allelic and haplotypic levels of diversity were
detected in sampling areas considered to be more ecologically disturbed, diversity
at both allelic and haplotypic levels in these areas was still generally high. High
levels of diversity are hypothesized to stem from high mutation rates in the mi-
crosatellite markers used as evidenced by several observations in the data. Allelic
diversity of A. papillatum was also found to be high in areas that were quali-
tatively thought to be more disturbed. Genotypic diversity was lower in these
areas suggesting that vegetative reproduction is more important in these areas for
maintaining the population numbers of this moss.
xiii
Genetic variation among clumps of Acanthorrhynchium papillatum was also
seen in another marker, ITS 2. Considerable levels of diversity among popula-
tions of A. papillatum were seen with this marker. Similar to the results using
microsatellite markers, ITS 2 haplotype diversity was lower in areas deemed to be
more ecologically disturbed. Compared to the diversity levels in the microsatellite
markers, however, diversity levels in ITS 2 were lower, emphasizing the utility and
importance of microsatellite markers in population genetic studies.
The microsatellite markers were also used to examine genetic diversity within
clumps of Acanthorrhynchium papillatum. Most clumps studied had very low
levels of diversity indicating that vegetative reproduction was more important

within clumps than sexual reproduction. However, multilocus genotypes of sam-
ples within some of the clumps studied were not all alike, providing evidence again
of high microsatellite mutation rates or of occasional sexuality within this species.
The results obtained provide baseline information on the genetic diversity of
Acanthorrhynchium papillatum in Singapore and Malaysia and are hoped to form
one of the first of a series of studies on bryophyte genetic diversity in Southeast
Asia.
xiv
LIST OF TABLES
Table Title Page
1.1 Summary characteristics of molecular techniques . . . . . . . . . . . 26
2.1 Acanthorrhynchium papillatum samples used for library construction 37
2.2 Oligos used in the construction of microsatellite libraries . . . . . . 42
2.3 Protocol differences between APMS and APBMS libraries . . . . . 48
2.4 Attrition of microsatellite-enriched inserts . . . . . . . . . . . . . . 62
2.5 Multiplex PCR primer combinations . . . . . . . . . . . . . . . . . 65
2.6 Characteristics of Acanthorrhynchium papillatum microsatellites . . 68
2.7 Comparison with other microsatellite marker projects . . . . . . . . 80
3.1 Distances between sampling areas . . . . . . . . . . . . . . . . . . . 92
3.2 Collection dates of Acanthorrhynchium papillatum used to study
inter-clump variation . . . . . . . . . . . . . . . . . . . . . . . . . . 93
3.3 Custom run module parameters for fragment analysis runs . . . . . 97
3.4 Revised microsatellite marker characteristics . . . . . . . . . . . . . 105
3.5 Numbers of rare alleles . . . . . . . . . . . . . . . . . . . . . . . . . 110
3.6 Allelic frequencies for each sampling area . . . . . . . . . . . . . . . 112
3.7 Size ranges of alleles . . . . . . . . . . . . . . . . . . . . . . . . . . 113
3.8 Allelic richness without rarefaction . . . . . . . . . . . . . . . . . . 114
3.9 Allelic richness with rarefaction . . . . . . . . . . . . . . . . . . . . 116
xv
3.10 Private alleles without rarefaction . . . . . . . . . . . . . . . . . . . 118

3.11 Private alleles with rarefaction . . . . . . . . . . . . . . . . . . . . . 119
3.12 Gene diversities of each sampling site . . . . . . . . . . . . . . . . . 121
3.13 Details on samples with null alleles . . . . . . . . . . . . . . . . . . 122
3.14 Matching multi-locus genotypes . . . . . . . . . . . . . . . . . . . . 124
3.15 Numbers of unique multi-locus genotypes . . . . . . . . . . . . . . . 126
3.16 Theoretical maximum number of genotypes . . . . . . . . . . . . . . 128
3.17 Power of discrimination and match probabilities . . . . . . . . . . . 129
3.18 Results of AMOVA for microsatellite data . . . . . . . . . . . . . . 140
3.19 Pairwise F
ST
s between populations using microsatellite markers . . 141
4.1 GenBank accessions used for ITS2 primer design . . . . . . . . . . 152
4.2 Primers for ITS2 amplification . . . . . . . . . . . . . . . . . . . . 153
4.3 Numbers of samples with unambiguous ITS2 sequences . . . . . . . 158
4.4 Summary statistics of ITS2 variation . . . . . . . . . . . . . . . . . 160
4.5 Nucleotide diversity in ITS 2 sequences . . . . . . . . . . . . . . . . 162
4.6 Haplotype diversity of ITS2 . . . . . . . . . . . . . . . . . . . . . . 163
4.7 ITS2 haplotype distribution . . . . . . . . . . . . . . . . . . . . . . 166
4.8 ITS2 haplotype gene diversity . . . . . . . . . . . . . . . . . . . . . 167
4.9 Results of AMOVA from ITS2 sequence data . . . . . . . . . . . . 170
4.10 Pairwise F
ST
s between populations using ITS 2 sequences . . . . . . 172
5.1 Collection dates of Acanthorrhynchium papillatum for within-clump
variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
xvi
5.2 Multi-locus genotypes within clumps . . . . . . . . . . . . . . . . . 185
5.3 Summary of pairwise distances within clumps – Method 1 . . . . . 187
5.4 Summary of pairwise distances within clumps – Method 2 . . . . . 190
G.1 Microsatellite marker alleles of samples used to characterize candi-

date markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
J.1 Microsatellite marker alleles of samples for among-clump diversity
studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
L.1 Within-clump microsatellite data (main) . . . . . . . . . . . . . . . 303
M.1 Within-clump microsatellite data (supplementary) . . . . . . . . . . 306
N.1 Pairwise genetic distances within clumps, main data - Method 1 . . 308
O.1 Pairwise genetic distances within a clump, supplementary data -
Method 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
P.1 Pairwise genetic distances within clumps, main data - Method 2 . . 310
Q.1 Pairwise genetic distances within clumps, supplementary data -
Method 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
xvii
LIST OF FIGURES
Figure Title Page
1.1 Location of ITS in ribosomal DNA . . . . . . . . . . . . . . . . . . 21
1.2 Habit and gross morphology of Acanthorrhynchium papillatum . . . 30
1.3 Leaf anatomy of Acanthorrhynchium papillatum . . . . . . . . . . . 31
2.1 Collection localities of Acanthorrhynchium papillatum samples used
for library construction . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2 Genomic DNA for library construction . . . . . . . . . . . . . . . . 56
2.3 Linker-ligated, enriched DNA fragments . . . . . . . . . . . . . . . 57
2.4 Screening for inserts . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.5 Polymorphism of PCR products . . . . . . . . . . . . . . . . . . . . 61
2.6 Multiplex PCR amplifying microsatellite loci . . . . . . . . . . . . . 64
2.7 Raw data from PCR multiplex set A . . . . . . . . . . . . . . . . . 66
2.8 Raw data from PCR multiplex set B . . . . . . . . . . . . . . . . . 67
3.1 Location of sampling areas of Acanthorrhynchium papillatum in
Malaysia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.2 Habitat pictures of sampling areas in Malaysia . . . . . . . . . . . . 86
3.3 Location of sampling areas of Acanthorrhynchium papillatum in Sin-

gapore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.4 Habitat pictures of sampling areas in Singapore . . . . . . . . . . . 90
3.5 Allelic frequencies for all samples . . . . . . . . . . . . . . . . . . . 108
xviii
3.6 Neighbor-joining tree of SBRF samples . . . . . . . . . . . . . . . . 131
3.7 Neighbor-joining tree of GBFR samples . . . . . . . . . . . . . . . . 132
3.8 Neighbor-joining tree of KTP samples . . . . . . . . . . . . . . . . . 133
3.9 Neighbor-joining tree of BTNR samples . . . . . . . . . . . . . . . . 134
3.10 Neighbor-joining tree of MACR samples . . . . . . . . . . . . . . . 135
3.11 Neighbor joining trees of pairwise distances among sampling areas . 138
4.1 Neighbor-joining tree of ITS2 haplotypes . . . . . . . . . . . . . . . 169
xix
LIST OF ABBREVIATIONS AND SYMBOLS
Chemicals and reagents
X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactoside
rATP adenosine triphosphate
BSA bovine serum albumin
CTAB cetyltrimethyl (hexadecyltrimethyl) ammonium bromide
DMSO dimethyl sulfoxide
Na
2
EDTA disodium ethylene diamine tetraacetic acid
DTT dithiothreitol
IPTG isopropyl-β-D-thiogalactopyranoside
LB Luria-Bertani
SDS sodium dodecyl sulfate
SSC standard saline citrate
TAE Tris-acetate, EDTA
TBE Tris-borate, EDTA
TE Tris-EDTA

xx
Molecular techniques
RAPD random amplified polymorphic DNA
ISSR inter-simple sequence repeats
Sampling areas
BTNR Bukit Timah Nature Reserve
GBFR Gunung Belumut Forest Reserve
KTP Kota Tinggi Waterfalls Resort
MACR MacRitchie Reservoir
SBRF Sungei Bantang Recreational Forest
Statistics
A alleles per locus
AMOVA analysis of molecular variance
d.f. degrees of freedom
F
CT
fixation index – between groups of populations
F
SC
fixation index – among populations within groups
F
ST
fixation index – among individuals among populations and
among groups
P
M
match probability
h Nei’s gene diversity
xxi
D Nei’s genetic distance

D
A
Nei’s net genetic distance
ˆ
h Nei’s unbiased gene diversity
P D power of discrimination
P probability
s.d. sample standard deviation
Units and measurements
g acceleration due to gravity
bp base pairs
cm centimeter
◦
C degrees celsius
g gram
ha hectare
h hour
kV kilovolt
km kilometer
m meter
µA microampere
µg microgram
xxii
µL microliter
µm micrometer
µM micromolar
mg milligram
mL milliliter
mm millimeter
mM millimolar

min minute
M molar
ng nanogram
nM nanomolar
OD optical density
rpm revolutions per minute
s second
cm
2
square centimeter
U unit
v/v volume per volume
W watt
w/v weight per volume
xxiii
Others
DNA deoxyribonucleic acid
dNTP deoxyribonucleic acid triphosphate
GPS global positioning system
T
M
melting temperature
PCR polymerase chain reaction
RNA ribonucleic acid
rDNA ribosomal DNA
ts. transition
tv. transversion
1
Chapter 1
GENERAL INTRODUCTION AND REVIEW OF

LITERATURE
1.1 General Introduction
1.1.1 Background of the study
Mosses and other bryophytes are integral components of f orests throughout the
world. Although small and often unnoticed, they provide many significant ecolog-
ical functions.
Bryophytes are among the first plants to colonize newly exposed surfaces
(Hallingb¨ack & Hodgetts, 2000). They stabilize the soil crust and help in the
accumulation of humus, paving the way for the growth of other plants. They
regulate moisture in the forests, helping control erosion and flash floods. Their
contribution to water regulation is particularly significant in highland environ-
ments where they can form a major proportion of the above-ground biomass.
Hallingb¨ack & Hodgetts (2000) state that “they are critical to the survival
of a tremendous diversity of organisms”. These include arthropods and other
invertebrates that depend on bryophytes for habitat or for food. Bryophytes also
provide seedbeds for several tree species (Glime & Saxena, 1991). Some bryophytes
even provide substrates for the growth of Cyanobacteria and thus indirectly help
in fixing atmospheric nitrogen (Glime & Saxena, 1991; Saxena & Harinder, 2004).
Despite the ubiquity and importance of mosses, little is known of the popu-
lation biology of many moss species in the tropics. Most studies on bryophyte
population biology were done on temperate species. Except for an early study