The Systematic of Wild Banana Species (Musa L.) in Sulawesi: Morphology and Molecular Studies
THE SYSTEMATIC OF WILD BANANA SPECIES (MUSA L.)
IN SULAWESI: MORPHOLOGY AND MOLECULAR
STUDIES
LULUT DWI SULISTYANINGSIH
THE GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2013
LETTER OF STATEMENT
I express that thesis entitled:
THE SYSTEMATIC OF WILD BANANA SPECIES (MUSA L.) IN
SULAWESI: MORPHOLOGY AND MOLECULAR STUDIES
Is true represent result of my own research and have never been published. All
information and data that used have been expressed clearly and can be checked its
truth.
Bogor, April 2013
Lulut Dwi Sulistyaningsih
NRP. G353100031
SUMMARY
LULUT DWI SULISTYANINGSIH. The systematic of wild banana species
(Musa L.) in Sulawesi: morphology and molecular studies. Supervised by RITA
MEGIA and ELIZABETH A. WIDJAJA
Information of wild banana species in Sulawesi was lack since there were no
systematic study have been done before. Therefore, the systematic study of wild
banana species (Musa L.) in Sulawesi was carried out. The study consist of (a)
species delimitation of wild banana species (Musa L.) in Sulawesi to provide
information of diversity and distribution of wild banana species in Sulawesi, and
to provide species description and an identification key; (b) phylogenetic studies
based on morphological characters and on internal transcribed spacer (ITS)
regions of nrDNA sequences to determine the phylogenetic relationship between
the species.
A total of 110 specimen consisted of old and new collections deposited in
BO and digital type specimen from MEL were examined morphologically.
Phylogenetic tree used fifty three morphological characters was performed by
Maximum Parsimony (MP) method using PAUP*4.0b10. While phylogenetic tree
reconstruction based on ITS regions sequences used MP and Bayesian (MrBayes
ver. 3.0) methods. Ensete and Musella were selected as the outgroup, whereas the
ingroup represented by Musa species obtained from species delimitation study.
Total DNA was extracted from silica-gel dried leaves by the modified CTAB
method. ITS-5 and ITS-4 primers were used to amplify the ITS regions. Data of
sequences were edited by using ChromasPro programme and were aligned by
Muscle software.
Species delimitation based on morphological characters showed 6 Musa and
1 incompletely known specimen housed in Sulawesi. There were 2 species (Musa
acuminata var. tomentosa and M. celebica) considered as endemic to Sulawesi.
The existence of M. balbisiana and M. itinerans are noted as new records. In
Sulawesi, M. balbisiana represented by a specimen collected from Manado (North
Sulawesi), while M. itinerans represented by specimens collected from Mt.
Nokilalaki (Central Sulawesi), and Lore Lindu National Park (Central Sulawesi).
Observation on the morphology of each species in this study enables the selection
of important characters for species delimitation and identification. Position of
suckers, petiole canal leaf, bract imbrications, bract behavior before falling, bract
colour, fruit shape, and seed surface are important characters for delimiting and
identifying taxa of wild banana species (Musa) in Sulawesi.
Phylogenetic tree used morphological characters showed M. balbisiana and
M. textilis placed in clade I in which they shared some similar characters for
instance bract imbrication and behavior before falling. While Musa acuminata ssp.
banksii, M. acuminata var. tomentosa, M. celebica, and M. itinerans were placed
in clade II. The phylogenetic study based on morphological characters is
incongruent with the phylogenenetic study based on ITS regions of nrDNA
sequences.
Phylogenetic tree used ITS regions of nrDNA sequences using MP and
Bayesian methods showed M. textilis was placed in clade I and separated from M.
balbisiana, M. itinerans, M. celebica, M. acuminata ssp. banksii, and M.
acuminata var. tomentosa. Nevertheless, M. textilis and M. balbisiana have closed
relationship. By using MP analysis, M. itinerans together with M. acuminata ssp.
banksii, M. acuminata var. tomentosa and M. celebica were placed in the same
clade, whereas Bayesian analysis showed M. itinerans was separated from M.
acuminata ssp. banksii, M. acuminata var. tomentosa as well as M. celebica, and
was placed in different clade.
This study revealed that phylogenetic study of wild banana species (Musa)
in Sulawesi based on morphological characters and ITS regions of nrDNA
sequences support the monophyly of genus Musa and also support the taxonomic
status of M. acuminata ssp. banksii as intraspecific taxa of M. acuminata.
Moreover, analysis of ITS regions also showed that sequences of ITS regions of
nrDNA could be used for phylogenetic tree reconstruction at the species level.
Key words: Internal transcribed spacer, phylogenetic, morphology, Musa,
Sulawesi
RINGKASAN
LULUT DWI SULISTYANINGSIH. Sistematika pisang-pisang liar (Musa L.) di
Sulawesi: kajian morfologi dan molekuler. Dibimbing oleh RITA MEGIA dan
ELIZABETH A. WIDJAJA
Ketersediaan informasi mengenai pisang-pisang liar di Sulawesi masih
sangat sedikit mengingat belum ada penelitian sistematika yang pernah dilakukan
sebelumnya. Oleh karena itu, penelitian sistematika pisang-pisang liar (Musa L.)
di Sulawesi ini dilakukan. Penelitian meliputi (a) kajian delimitasi jenis pisangpisang liar (Musa L.) di Sulawesi untuk memberikan informasi keanekaragaman
dan distribusi pisang-pisang liar di Sulawesi, serta menyajikan deskripsi jenis dan
kunci identifikasi; (b) kajian filogenetik berdasarkan karakter morfologi dan
urutan basa nrDNA daerah internal transcribed spacer (ITS) untuk melihat
hubungan kekerabatan antar jenis.
Sebanyak 110 spesimen yang terdiri atas koleksi lama dan baru yang
tersimpan di BO serta gambar digital spesimen tipe dari MEL diperiksa karakter
morfologinya. Konstruksi pohon filogenetik menggunakan 53 karakter morfologi
dianalisis dengan metode Maximum Parsimony (MP) menggunakan program
PAUP*4.0b10. Sementara itu, konstruksi pohon filogenetik berdasarkan urutan
basa daerah ITS menggunakan metode MP dan Bayesian (MrBayes ver. 3.0).
Ensete dan Musella dipilih sebagai outgroup sedangkan ingroup direpresentasikan
oleh jenis-jenis Musa hasil kajian delimitasi jenis. DNA total diekstraksi dari daun
yang tersimpan dalam gel silika menggunakan metode CTAB yang dimodifikasi.
Primer ITS-5 dan ITS-4 digunakan untuk mengamplifikasi daerah ITS. Data
urutan basa diedit dengan program ChromasPro dan disejajarkan dengan software
Muscle.
Kajian delimitasi jenis berdasarkan karakter morfologi menunjukkan 6
Musa dan 1 jenis yang belum diketahui ditemukan di Sulawesi. Terdapat 2 jenis
endemik Sulawesi, yaitu Musa acuminata var. tomentosa dan M. celebica.
Ditemukannya M. balbisiana dan M. itinerans merupakan catatan baru. Di
Sulawesi, keberadaan M. balbisiana direpresentasikan oleh spesimen yang
dikoleksi dari Manado (Sulawesi Utara), sedangkan keberadaan M. itinerans
direpresentasikan oleh spesimen yang dikoleksi dari gunung Nokilalaki (Sulawesi
Tengah) dan Taman Nasional Lore Lindu (Sulawesi Tengah). Dari hasil observasi
karakter morfologi pada masing-masing jenis diperoleh karakter-karakter penting
untuk memberikan batasan jenis dan identifikasi. Posisi tumbuh anakan, lekukan
pada tangkai daun, imbrikata pada braktea, tipe braktea sebelum gugur, warna
braktea, bentuk buah, dan permukaan biji merupakan karakter penting untuk
memberi batasan dan identifikasi jenis pisang-pisang liar (Musa) di Sulawesi.
Pohon filogenetik berdasarkan karakter morfologi menunjukkan M.
balbisiana dan M. textilis berada pada klade I dan mempunyai karakter yang sama
seperti imbrikata pada braktea dan tipe braktea sebelum gugur. Sementara itu, M.
acuminata ssp. banksii, M. acuminata var. tomentosa, M. celebica, dan M.
itinerans berada pada klade II. Hasil kajian filogenetik berdasarkan karakter
morfologi berbeda dengan kajian filogenetik berdasarkan sekuen daerah ITS dari
nrDNA.
Konstruksi pohon filogenetik berdasarkan urutan basa nrDNA daerah ITS
menggunakan metode MP dan Bayesian menunjukkan M. textilis berada pada
klade I dan terpisah dari M. balbisiana, M. itinerans, M. celebica, M. acuminata
ssp. banksii, dan M. acuminata var. tomentosa. Meskipun demikian, M. textilis
berkerabat dekat secara filogenetik dengan M. balbisiana. Dengan menggunakan
metode MP, M. itinerans bersama dengan M. acuminata ssp. banksii, M.
acuminata var. tomentosa dan M. celebica berada pada klade yang sama,
sedangkan metode Bayesian menunjukkan M. itinerans terpisah dari M.
acuminata ssp. banksii, M. acuminata var. tomentosa dan M. celebica serta berada
pada klade yang berbeda.
Penelitian ini menunjukkan bahwa kajian filogenetik pisang-pisang liar
(Musa) di Sulawesi berdasarkan karakter morfologi dan urutan basa nrDNA
daerah ITS mendukung sifat monofiletik dari marga Musa dan juga mendukung
status taksonomi M. acuminata ssp. banksii sebagai infraspesifik taksa dari M.
acuminata. Lebih jauh lagi, analisis daerah ITS juga menunjukkan bahwa urutan
basa nrDNA daerah ITS dapat digunakan untuk rekonstruksi pohon filogenetik
pada tingkat jenis.
Kata Kunci: Filogenetik, internal transcribed spacer, morfologi, Musa, Sulawesi.
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THE SYSTEMATIC OF WILD BANANA SPECIES (MUSA L.)
IN SULAWESI: MORPHOLOGY AND MOLECULAR
STUDIES
LULUT DWI SULISTYANINGSIH
Thesis submitted
As partial fulfillment requirement for the Master
Degree
In Plant Taxonomy
THE GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2013
External examiner: Dr. Nunik Sri Ariyanti, M.Si.
Title
Name
NRP
: The Systematic of Wild Banana Species (Musa L.) in Sulawesi:
Morphology and Molecular Studies
: Lulut Dwi Sulistyaningsih
: G353100031
Certified by
Supervisor Committee
Dr. Rita Megia, D.E.A.
Chairman
Prof. (R.) Dr. Elizabeth A. Widjaja, M.Sc
Member
Approved by
Head of Plant Biology
Study Program
Dean of Graduate School
Dr. Ir. Miftahudin, M.Si
Dr. Ir. Dahrul Syah, MSc. Agr.
Date of Examination:
Date of Graduation:
ACKNOWLEDGEMENT
It would not have possible to write this thesis without the help and support
of many people around me, to only some of whom it is possible to give particular
mention here.
First and foremost, I would like to express my gratitude to my supervisors,
Dr. Rita Megia, D.E.A. and Prof. (R.) Dr. Elizabeth A. Widjaja, M.Sc. for their
valuable advices, guidance, and encouragement throughout my study. Many
thanks also goes to Dr. Nunik Sri Ariyanti, M.Si., Dr. Rugayah, M.Sc., Dr.
Himmah Rustiami, M.Sc. for their suggestions, advices, and criticisms.
I gratefully acknowledge the Karyasiswa Ristek scholarship from Ministry
of Research and Technology (RISTEK); DIPA from Division of Botany, Research
Centre for Biology – LIPI with entitled Revision of Selected Taxa in Sulawesi for
supporting field research; Molecular Systematic Laboratory, Division of Botany,
Research Centre for Biology – LIPI for supporting sequencing fee. I would like to
thank to Dr. Witjaksono, M.Sc., Dr. Yuyu Purba, M.Sc., Fajarudin Ahmad, S.Si.
for their kindly help in DNA materials.
I would like to thank to all taxonomists and technicians of Herbarium
Bogoriense who gave their support and assist when conducted this study. I also
express my gratitude to my colleagues and friends in Research Centre for Biology
– LIPI and IPB.
Last but not the least, my special thanks go to my family, my parents, my
husband Dhani Yudhana and my daughter Nafeeza Armaghan El-Firdausy for
their spirit, patience, and support in my study.
Bogor, April 2013
Lulut Dwi Sulistyaningsih
TABLE OF CONTENTS
LIST OF TABLES
vi
LIST OF FIGURES
vi
LIST OF APPENDICES
vii
1 INTRODUCTION
1
2 LITERATURE REVIEW
2
Taxonomy of Musa L.
General Morphology of Musa L.
Distribution of Musa L.
Internal Transcribed Spacer (ITS)
3 MATERIALS AND METHODS
Species Delimitation of Wild Banana Species (Musa L.) in Sulawesi
Materials
Methods
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi
Based on Morphology Characters
Materials
Methods
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi
Based on Internal Transcribed Spacer Region of nrDNA Sequences
Materials
Methods
DNA Extraction and Amplification
DNA Purification and Sequencing
Phylogenetic Analysis
4 RESULT AND DISCUSSION
Species Delimitation of Wild Banana Species (Musa L.) in Sulawesi
General Morphology of Musa in Sulawesi
Pseudostems
Leaves
Inflorescences
Male Buds
Flowers
Fruits
Seeds
Distribution
Key to the Species and Infraspecific Taxa of Musa from Sulawesi
Key to the Species
Key for Infraspecific Taxa of Musa acuminata Colla
Taxonomic Treatment
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi
2
3
5
7
9
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9
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12
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14
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15
15
16
16
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16
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17
18
Based on Morphology Characters
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi
Based on Internal Transcribed Spacer Region of nrDNA Sequences
DNA Extraction and Amplification
Nucleotide Sequences of ITS Region of nrDNA and Variation
Within Species
Phylogenetic Analysis
General Discussion
35
37
38
42
46
5 CONCLUSION
47
REFERENCES
48
APPENDICES
52
CURRICULUM VITAE
55
LIST OF TABLES
1
2
3
4
5
Morphological characters state used in phylogenetic analysis
Source of ITS sequence utilized in the study
Sequence length variation of ITS region within outgroup and ingroup
Percentages of G+C content of ITS1 and ITS2
Nucleotide sequences variation of ITS region within species showed
unique nucleotides of species from section Eumusa and Australimusa
10
12
39
40
42
LIST OF FIGURES
1
2
3
4
5
6
7
8
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10
11
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29
Variation on leaf base
Variation on leaf canal margin
Variation on bract
Distribution map of Musa sections
Distribution map of Musa sections in Indonesia
Organization of ITS region of nrDNA
Taxonomic level of utility of nuclear DNA regions used in phylogenetic
reconstruction based on angiosperms
Leaf bases shapes
Bract imbrications on male bud
Shape of seeds
Distribution map of wild banana species in Sulawesi
Musa acuminata ssp. banksii (F. Muell.) N.W. Simmonds
Distribution map of M. acuminata ssp. banksii in Sulawesi
Distribution map of M. acuminata var. tomentosa in Sulawesi
Musa balbisiana Colla
Distribution map of M. balbisiana in Sulawesi
Musa celebica Warb. ex K. Schum.
Distribution map of M. celebica in Sulawesi
Musa itinerans Cheesman
Distribution map of M. itinerans
Musa textilis Née
Distribution map of M. textilis in Sulawesi
Distribution map of Musa sp. 1 in Sulawesi
Strict consensus tree resulted from morphological characters
Visualization of PCR product
Nucleotide sequences variation of ITS regions within species that
showed some unique sequences of Musa balbisiana was compared with
other taxa
Nucleotide sequences variation of ITS regions within species that
showed some unique sequences of Musa itinerans was compared with
other taxa
Strict consensus tree of the ITS sequence region resulting from MP
analysis
Strict consensus tree of the ITS sequence region resulting from
Bayesian analysis
4
4
5
6
6
7
8
15
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LIST OF APPENDICES
1 Matrix of morphological characters for phylogenetic analysis
2 The ITS region of nrDNA sequence alignment from position 272 to 373
showed part of the conserved region
3 The ITS region of nrDNA sequence alignment from position 485 to 575
showed part of the variative region
52
53
54
1
1 INTRODUCTION
Bananas belong to Musaceae, a small family which consists of three genera,
Ensete Bruce ex Horan, Musa L., and Musella (Franchet) H.W. Li. In general,
bananas (Musa L.) are grouped into wild seeded bananas that consist of
approximately 70 species (Häkkinen 2008) and edible seedless bananas consisting
of approximately 500 cultivars (Valmayor et al. 2002). Cultivated bananas have
considerable economic value since they have a high level of consumption,
whereas wild banana species have potential value as a genetic resource. Cultivated
bananas have been largely evolved from two wild banana species, Musa
acuminata Colla and M. balbisiana Colla. M. balbisiana is known to be resistant
to drought, while the intraspecific of M. acuminata, namely M. acuminata var.
malaccensis is known to be resistant to fusarium wilt (Nasution 1991).
Elucidating the phylogeny and taxonomy of Musa and its family is important as
phylogenetic relationships can provide valuable information for the collection and
utilization of genetic resources for further banana improvement.
Indonesia has a large number of bananas. This country is the center of
bananas origin (Simmonds 1966) as well as of its diversity (Daniells et al. 2001).
At least 325 cultivars have been recorded in Indonesia (Valmayor et al. 2002),
whereas only 12 wild banana species has been documented (Nasution and
Yamada 2001). Presumably, there are wild banana species that have not been
recorded and well documented.
In Indonesia, wild banana species grow widespread in Sumatra, Java, Lesser
Sunda Islands, Kalimantan, Sulawesi, Moluccas, and Papua. Biogeographycally,
Sulawesi has a unique characteristic because it is located in the Wallace line
which is the transition region between Asia and Australia. Sulawesi is also known
to have a large number of endemic flora and fauna (Mittermier et al. 1999).
Musaceae that have been reported as an endemic flora in Sulawesi are M. celebica
Warb. ex K. Schum. and M. acuminata Colla var. tomentosa (K.Sch.) Nasution
(Nasution 1991; Nasution and Yamada 2001).
As a taxa that can undergo self crossing, hybridization, and mutation,
bananas has a complex genome structure. To analyze bananas genetic diversity,
molecular characterization was required to support morphological characters.
During the past decades, molecular markers such as RFLPs (Gawel and Jaret
1991), AFLP (Wong et al. 2002), HAT-RAPD (Ruangsuttapha et al. 2007), trnLF (Liu et al. 2010; Li et al. 2010), and ITS (internal transcribed spacer) (Liu et al.
2010; Li et al. 2010; Hřibova et al. 2011) have been used for phylogenetic
analysis of Musaceae. Nowadays, ITS is used more often by researchers to
conduct a molecular phylogenetic analysis of plant in order to understand the
diversity and to answer phylogenetic problems. This is because ITS region is easy
to be isolated, amplified, and analyzed, due to its small size (300-800 bp) and high
copy number in the genome (Baldwin et al. 1995).
Taxonomy studies that reveal the morphological diversity of wild bananas
in Central, North, and South Sulawesi has been done by Nasution (1991).
However, in general the systematic studies of wild banana species in Sulawesi are
still rare. It can be seen from the number of identified Musaceae specimen in
Herbarium Bogoriense (BO). It is approximately only 26% of Musaceae
2
specimens from Sulawesi that stored in BO have been identified. Therefore,
systematic studies to reveal the Musaceae diversity in Sulawesi need to be done.
The aims of the study were 1) to provide information of the diversity and
the distribution of wild banana species in Sulawesi, 2) to provide species
description and an identification key and 3) to determine the phylogenetic
relationship between the species using morphological characters and ITS regions
of nrDNA sequences.
2 LITERATURE REVIEW
Taxonomy of Musa L.
APG III (Angiospermae Phylogeny Group) places genus Musa L., together
with Ensete Bruce ex Horan and Musella (Franchet) H.W. Li into family
Musaceae, the member of Zingeberales (APG 2009). Genus Musa is the largest
genus in the family that is firstly established by Linnaeus (1753). Some botanists
believed that the name of genus Musa is thought to be derived from the Arabic
name for the plant (mouz) which in turn may have been applied in honour of
Antonius Musa who is physician to Octavius Agustus, the first emperor of Rome
(Hyam and Pankhurst 1995). Whereas the name “banana” is derived from the
Arabic banan that means finger (Boning 2006) and was thought to be used in
Guinea (West Africa) concomitant with the introduction of the fruit by the
Portuguese and then the name spread to the New World (Cheesman 1948a).
The taxonomic complexity at the family level continues down to the genus
level and there are inconsistencies in the number of sections and number of
species proposed for inclusion in the genus Musa. The first classification made by
Sagot (1887) that divided the genus into three groups: (1) giant bananas (type: M.
ensete J.F. Gmel.), (2) bananas with fleshy fruit and often edible (type: M.
sapientum L.), (3) ornamental bananas with erect inflorescences and brightly
coloured bracts (type: M. coccinea Andrews).
In 1893, Baker divided genus Musa into three subgenera: (1) Physocaulis,
characterized by stem bottle-shaped; flowers many to a bract; petal usually
tricuspidate; fruit not edible. (2) Eumusa, characterized by stem cylindrical;
flower many to a bract; petal ovate-acuminate; bracts green, brown or dull violet;
fruit edible. (3) Rhodoclamys, characterized by stem cylindrical; flower few to a
bract, petal linier; bracts bright-coloured; fruit usually not edible.
Cheesman (1947) elevated the first subgenus to the generic level as the
genus Ensete and then made new classification. The classification is based on the
haploid number of the chromosome that followed by the similarity and the
differences on morphological characters. He treated the genus into four sections as
follows:F
A. Chromosome number x=11
1. Inflorescence pendent or semi-pendent from the first, the fruits reflexing
in development toward the base of the rachis. Flowers many to a bract, in
two series. Bracts commonly dull coloured, green, brownish or dull
3
purple. Pseudostems commonly exceeding three meters high
…...………………………………………………………Section Eumusa
2. Inflorescence erect, or at least at the base, so that the fruits do not reflex
in development but point toward the apex of the rachis. Flowers few to a
bract, usually in a single serie. Bract brightly coloured, often red.
Pseudostems less than three meters high…...........……………...
……………............................................................Section Rhodochlamys
B. Chromosome number x=10
3. Seeds subglobose, or more or less dorsiventrally compressed, smooth,
striate, tuberculate, or irregularly angulate with a marked or obsolete
umbo opposite to the hilum corresponding to small periperm chamber
within…………………………………………........Section Australimusa
4. Seeds cylindrical, barrel-shaped, or top shaped, marked externally by
transverse line or grove, above which are warted, tuberculate, or
variously patterned, below usually smooth; internally a well-developed
perisperm, chamber above the same line, this chamber empty in the ripe
seed…………………………………………………….Section Callimusa
Although this classification was widely accepted by most botanists, its
validity has been questioned, in any case for some sections. Some newly
described species which is already count the chromosome number also appeared
problematic. Argent (1976) proposed to establish one more section in Musa,
Ingentimusa (x=7) to include the single species M. ingens N.W. Simmonds. Based
on morphological and cytological characters, Simmond and Weatherup (1990)
found a very low level of consistency among the characters and suggested that
section Eumusa is heterogeneous and then divided it into two informal subgroups
“Eumusa-1” and “Eumusa-2. Recently, molecular data have been used to solve
such problems in taxonomy of Musa. By using RFLPs data, Gawel and Jaret
(1991) and Gawel et al. (1992) found that a molecular phylogeny was inconsistent
with traditional classification and they proposed that section Rhodochlamys
should be merged with section Eumusa. Wong et al. (2002), by using AFLP data
suggested that section Rhodochlamys should be placed in section Eumusa, and
section Australimusa should be merged in section Callimusa.
General Morphology of Musa L.
Musa is a large perennial herb and they grow in clump with rhizome and
false areal stem, cylindrical pseudostem that consisted of sheath leaves wrapped
together with each other, has a short underground stem (corm). The root system is
adventitious spreading out laterally.
Leaves grow from a cigar leaf into a large, blade oblong, arranged parallel
and pinnate. There are some variations on base shape of leaves: both side
rounded; one side rounded, one pointed; both sides pointed (Figure 1). Leaf canal
margin open with margins spreading, wide with erect margin, straight with erect
margins, margin curved inward or margins overlapping (Figure 2).
4
a
b
c
Figure 1 Variation on leaf base. Both sides rounded (a); one side rounded, one
pointed (b); both sides pointed (IPGRI 1996)
Figure 2 Variation on leaf canal margin. Open with margins spreading (a); wide
with erect margins (b); straight with erect margin (c); margins curved
inward (d) (Nasution and Yamada 2001)
Inflorescence springs from the rhizome and emerges at the top of the stem,
either erect or pendulous; the immature inflorescence is encased inside bracts that
give the appearance of large bud. Bracts are plane or sulcate, revolute or not
revolute before falling, imbricate or not imbricate (Figure 3). Flowers produce
nectar, on each bract there is one or two rows of flower. Basal flower is female or
hemaprodhite, consist of ovary that protected by compound tepal (calyx) and free
tepal (corolla), style and staminode; compound tepal essentially tubular but split
to the base on the adaxial side, 5 toothed at the apex (3-lobed at the apex with 2
accessory teeth between the main lobes; free tepal inserted within the compound
tepal and opposite to it (i.e. in the adaxial position). Male flower have 5 fertile
stamen that falling down with the bract. They are reduced to staminodes in female
flower. Female and male flowers are morphologically indistinguishable until the
inflorescence is about 12 cm long. At this point, the ovary in the male flower fails
to develop any further (Simmonds 1959).
5
a
b
Figure 3 Variation on bract. Not imbricate (a); imbricate (b) (Nasution and
Yamada 2001)
Fruits are berry, some dehiscent and some are not, with numerous seeds
(except in the parthenocarpic form). Each fruit is known as a “finger”. Each
cluster of fruits at node is known as a “hand” and the entire collection of hands is
known as a “bunch”. The number of hands varies each others. The outer
protective layer of each fruit known as the “skin” or “peel” is fusion of the
hypanthium (floral receptacle) and outer layer (exocarp) of the pericarp (fruit wall
derived from the ovary wall). This peel is easily removed from the fleshly pulp
that originates mainly from the endocarp (innermost layer of the pericarp)
(Simmonds 1953). During the development of the fruit from the ovary, the tepals,
style, and staminodes abscise leaving a characteristic calloused scar at the tip of
the fruit. Fruits develop only after pollination. Fruit size depends on the number of
seeds and parenchymatous pulp develops around each seed. The growth volume
curve is sigmoidial (Simmonds 1953). Wild banana species have little flash and is
filled with black or brown seed. The seeds have linier embryos, large amounts of
endosperm and a thick hard testa (Ellis et al. 1985).
Distribution of Musa L.
The section Australimusa is well-known in Brunei Darussalam, Indonesia,
Malaysia, Papua New Guinea, and Philippines, while section Callimusa can be
found in Brunei Darussalam, China, Cambodia, Indonesia, Malaysia, Papua New
Guinea, and Vietnam. The biggest section, Eumusa is widespread in Australia,
Bhutan, Cambodia, China, Eastern and SouthEast India, Indonesia, Japan, Laos,
Malaysia, Papua New Guinea, Philippine, Samoa, Sri Lanka, Thailand, and
Vietnam, wheread section Rhodochlamys grows well in Bangladesh, China,
Malaysia, and Thailand (Figure 4).
6
Rhodochlamys
Pasific Ocean
Eumusa
Callimusa
Australimusa
Indian Ocean
Figure 4 Distribution map of Musa sections.
Eumusa;
Australimusa;
Callimusa;
Rhodoclamys (Pollefeys et al. 2004)
Indonesia is situated in the centre of origin and diversity of Musaceae and
has a large number of both wild banana species and cultivated bananas. The
bananas widespread in Sumatra, Java, Lesser Sunda Islands, Borneo, Sulawesi,
Moluccas, and Papua. Pollefeys et al. (2004) made distribution map of Musa
sections in Indonesia using MGIS (Musa Germplasm Information System) and
DIVA-GIS. Almost all of the accessions that they used come from Nasution’s
(1991) study and the rest come from Musalogue. Because of insufficient
geographical data for some of Nasution’s accessions, no entry is available in
MGIS for Kalimantan (Borneo) although the expedition recorded wild species in
all major islands (Figure 5).
Legend
Australimusa
Callimusa
Eumusa
Figure 5 Distribution map of Musa sections in Indonesia (Pollefeys et al. 2004)
7
Internal Transcribed Spacer (ITS)
The major ribosomal RNA (rRNA) genes of plants are localized in clusters
on highly repeated sequences. Each repeat consist of sequences from 18S, 5.8S
and 25S ribosomal subunits and each copy contains a transcribed region that is
separated by the long non-transcribed intergenic spacer (IGS). These genes show
little sequence divergence between closely related species. Within each repeat,
these conserved regions are separated by internal transcribed spacer (ITS) which
occur in the following order: 5’–18S–ITS-1–5.8S–ITS-2-26S(or 25S)–3’ that
show higher rates of divergence (Figure 6).
ITS Region
Figure 6 Organization of ITS region of nrDNA (Soltis and Soltis 1998)
The internal transcribed spacers including ITS-1 and ITS-2 regions are part
of the nuclear rDNA (nrDNA) transcript but are not incorporated into ribosomes.
In particular, the 5.8S rRNA is separated from 18S, the SSU (small ribosomal
subunit) rRNA, by the first of two ITSs (ITS-1), and from 25-28S, the LSU (large
ribosomal subunit) rRNA, by the second ITS (ITS-2). Sequencing of the ITS
region, however has an exciting potential as a source of nuclear DNA characters
for phylogenetic reconstruction in plants. This promise was heightened recently
by encouraging result from ITS sequence-based phylogenies of protoctistans (Lee
and Taylor 1991), apes and humans (Gonzales et al. 1990). White et al. (1990)
have taken advantage of polymerase chain reaction (PCR) technology to promote
sequencing of nrDNA in fungi. Baldwin (1992) have described the usefulness of
these primers for PCR amplification and sequencing of the ITS region in
angiosperms and also described the utility of ITS DNA sequences as a source of
phylogenetic data in the subtribe Madiinae of Asteraceae.
ITS regions take a role in the maturation of nuclear rRNAs, bring ITS of
18S–26S nrDNA have a particularly valuable marker for phylogenetic analysis at
8
intraspecific level and intergeneric level among angiosperms and other eukaryotes
(Baldwin et al. 1995). In general, the ITS region present some advantages of
being a multicopy locus (100-200 copies), having a small size (300-800 bp),
varying from one taxon to another but highly conserved in size in a given taxon,
and making it a preferred diagnostic target for a universal test (Baldwin et al.
1995).
ITS sequences are proven to be valuable for phylogenetic reconstruction in
angiosperms, algae, and ferns. Recent work indicates that ITS-1 and ITS-2
sequences are inherently G+C rich in which portions of these regions are quite
conserved among angiosperms. Thus, the ITS regions not only possess high
information content at lower taxonomic level, but also exhibit conserved sequence
patterns and high alignability across angiosperms (Figure7).
Figure 7 Taxonomic level of utility of nuclear DNA regions used in phylogenetic
reconstruction based on angiosperms. The shaded box showing the
taxonomic zone that is not presently well covered by nuclear gene
sequences; ? refers to genes that have been rarely used; ----- designates
the approximate upper or lower limits of applicability (Soltis and Soltis
1998)
9
3 MATERIALS AND METHODS
Species Delimitation of Wild Banana Species (Musa L.) in Sulawesi
Materials
A total of 110 sheets (46 number of collections) of herbarium specimens
consist of the dry and spirit collections were used in this study. It composed of all
specimens of Musaceae in Sulawesi which are stored in Herbarium Bogoriense
(BO) and the new specimens collected from Central, North, South, and SouthEast
Sulawesi. The digital data that consist of type specimens from Royal Botanic
Garden (MEL) and JSTOR Plant Science were also evaluated.
Methods
Field research to collect new specimens were carried out by using
exploration method. Sampling method used was purposive random sampling, in
which sampling localities were randomly selected by considering factors that
influence the existence of Musa. Data or information recorded from the field
include location; altitude, longitude and latitude; vernacular name; plant general
habit; pseudostem (height, diameter, colour, appearance, predominant underlying
colour, pigmentation of the underlying pseudostem, sap colour, wax on leaf
sheaths); suckers (number of suckers, position); petiole (blotches at the petiole
base, colour of blotches, length); leaves (length, width, colour, and appearance of
leaf upper surface, colour, and appearance of leaf lower surface, wax, colour of
midrib dorsal and ventral surface); inflorescence/male bud (peduncle length,
bunch position, rachis position, colour of the bract, wax on the bract); flower
(compound tepal basic colour, lobe colour of compound tepal, free tepal colour,
filament colour, anther colour, stigma colour); fruit (number of fruits, length,
immature or mature colour). Morphological comparative study was done in
Herbarium Bogoriense (BO).
All the herbarium specimens were studied based on their morphological
similarity following de Vogel (1987) and Rifai (2008) using comparative
morphology data as main source.
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi Based on
Morphological Characters
Materials
The result of species delimitation study will be used as the ingroup, while
Ensete sp. 1 from Bogor Botanic Garden and Musella lasiocarpa from China were
selected as an outgroup. Fifty three morphological characters selected during
morphological observation of field and herbarium specimens were used in this
study. The characters and characters state followed the descriptor for bananas
(IPGRI 1996) with some modifications (Table 1).
10
Table 1 Morphological characters state used in the phylogenetic analysis
No
Characters
Character s State
1
Habit
0. Single
1. Clumping
2
Sucker
0. Absent
3
Pseudostem height
0.≤ 100 cm
1. Present
1. > 100 cm
4
Pseudostem aspect
0. Slender
1. Robust
5
Pseudostem colour
1. Green
6
Pseudostem
appearance
Predominant
underlying colour
of the pseudostem
Pigmentation of
the underlying
pseudostem
Sap colour
0. Medium
green
0. Dull
(waxy)
0. Green
0. Absent
1. Present
0. Watery
1. Milky
0. Absent
1. Present
1. Straight
with erect
margins
1. Clasping
7
8
9
10
11
Blotches at the
petiole base
Petiole canal leaf
III
2. Red-purple
1. Shiny (not waxy)
1. Greenyellow
2. Pink purple
12
Leaf sheaths
0. Wide with
erect
margins
0. Lax
13
Leaf habit
0. Erect
1. Intermediate
14
Colour of leaf
upper surface
Appearance of leaf
upper surface
0. Medium
green
0. Dull
1. Green
Colour of leaf
lower surface
Appearance of leaf
lower surface
0. Light green
1. Medium
green
1. Shiny
Shape of leaf blade
base
Colour of midrib
dorsal surface
Colour of midrib
ventral surface
Inflorescence habit
0. Both sides
rounded
0. Greenyellow
0. Medium
green
0. Erect
1. Both sides
pointed
1. Light green
0. ≤ 100
1. 101-200
2. ≥201
0. ≤ 30
1. 31-60
2. ≥ 61
24
Inflorescence
length [cm]
Peduncle length
[cm]
Peduncle hairiness
0. Absent
1. Present
25
Bunch appearance
0. Loose
1. Compact
26
Bract appearance
1. Inserted independently on the axis
27
Bract habit
0. Integral
with each
other
0. Persistent
28
Bract behavior
0. Not
revolute
1. Revolute
15
16
17
18
19
20
21
22
23
0. Dull
3.Blue
2. Margins curved inward
2. Dark green
1. Shiny
2. Green
1. Light green
1. Pendulous
1. Deciduous
11
Table 1 Morphological characters state used in the phylogenetic analysis (continue)
No
Characters
Character s State
29
Bract imbrication
1. Imbricate
30
Bract apex shape
31
32
Colour on the
bract apex
Wax on the bract
0. Not
imbricate
0. Slightly
Pointed
0. Not tinted
with yellow
0. Absent
33
Male bud shape
0. Like a top
1. Lanceolate
34
0. Persistent
1. Deciduous
0. Linier
1. Tubular
0. 3 –toothed
1. 5-toothed
0. Yellow
1. Orange
0. Translucent
white
0. Oval
1. White
0. Little or no
visible sign
1. Developed
0. Obtuse
1. Triangular
0. Straight
1. Curved
0. White
1. Cream
0. Straight
1. Arched
45
Male flower
behavior
Compound tepal
of male flower
shape
Lobe of compound
tepal of male
flower
Lobe colour of
compound tepal of
male flower
Free tepal colour
of male flower
Free tepal shape of
male flower
Free tepal apex
development of
male flower
Free tepal apex
shape of male
flower
Style shape of
male flower
Stigma colour of
male flower
Ovary shape of
male flower
Fruit shape
0. Straight
1. Curved
46
Fruit apex
0. Pointed
1. Blunt-tipped
47
Floral relicts
0. Absent
1. Present
48
0. Light green
1. Green
0. Yellow
1. Bright yellow
0. Persistent
1. Deciduous
51
Immature fruit
peel colour
Mature fruit peel
colour
Fruit fall from
hands
Seed diameter
0. >1 cm
1. ≤ 1 cm
52
Seed shape
0. Globular
1. Angular
53
Seed surface
0. Smooth
1. Wrinkled
35
36
37
38
39
40
41
42
43
44
49
50
1. Intermediate
1. Tinted with yellow
1. Present
2. Ovoid
1. Fan-shaped
Methods
Morphological characters were analyzed based on Maximum Parsimony
using PAUP*4.0b10 (Swofford 1998) with Heuristic Search settings vegetative
12
and floral characters were selected for the analysis of which 45 were scored as
binary and 8 as multistate. Some characters were scored as missing data when the
data were unavailable. All characters are treated as unorder data and have an equal
weight. The missing data are symbolized with “?”. Starting tree (S) was obtained
via stepwise addition. Number of trees held at each step during stepwise addition
= 1. Branch swapping algorithm was run by using tree-bisection-reconnection
(TBR) and was evaluated using 100 bootstrap replicates and a complete Hsearch.
Clade support values were obtained by using bootstrap. Bootstrap support (BS)
was categorized as strong (>85%), moderate (70%-85%), weak (50%-69%), or
poor (85%), moderate (70%-85%), weak (50%-69%), or poor (
IN SULAWESI: MORPHOLOGY AND MOLECULAR
STUDIES
LULUT DWI SULISTYANINGSIH
THE GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2013
LETTER OF STATEMENT
I express that thesis entitled:
THE SYSTEMATIC OF WILD BANANA SPECIES (MUSA L.) IN
SULAWESI: MORPHOLOGY AND MOLECULAR STUDIES
Is true represent result of my own research and have never been published. All
information and data that used have been expressed clearly and can be checked its
truth.
Bogor, April 2013
Lulut Dwi Sulistyaningsih
NRP. G353100031
SUMMARY
LULUT DWI SULISTYANINGSIH. The systematic of wild banana species
(Musa L.) in Sulawesi: morphology and molecular studies. Supervised by RITA
MEGIA and ELIZABETH A. WIDJAJA
Information of wild banana species in Sulawesi was lack since there were no
systematic study have been done before. Therefore, the systematic study of wild
banana species (Musa L.) in Sulawesi was carried out. The study consist of (a)
species delimitation of wild banana species (Musa L.) in Sulawesi to provide
information of diversity and distribution of wild banana species in Sulawesi, and
to provide species description and an identification key; (b) phylogenetic studies
based on morphological characters and on internal transcribed spacer (ITS)
regions of nrDNA sequences to determine the phylogenetic relationship between
the species.
A total of 110 specimen consisted of old and new collections deposited in
BO and digital type specimen from MEL were examined morphologically.
Phylogenetic tree used fifty three morphological characters was performed by
Maximum Parsimony (MP) method using PAUP*4.0b10. While phylogenetic tree
reconstruction based on ITS regions sequences used MP and Bayesian (MrBayes
ver. 3.0) methods. Ensete and Musella were selected as the outgroup, whereas the
ingroup represented by Musa species obtained from species delimitation study.
Total DNA was extracted from silica-gel dried leaves by the modified CTAB
method. ITS-5 and ITS-4 primers were used to amplify the ITS regions. Data of
sequences were edited by using ChromasPro programme and were aligned by
Muscle software.
Species delimitation based on morphological characters showed 6 Musa and
1 incompletely known specimen housed in Sulawesi. There were 2 species (Musa
acuminata var. tomentosa and M. celebica) considered as endemic to Sulawesi.
The existence of M. balbisiana and M. itinerans are noted as new records. In
Sulawesi, M. balbisiana represented by a specimen collected from Manado (North
Sulawesi), while M. itinerans represented by specimens collected from Mt.
Nokilalaki (Central Sulawesi), and Lore Lindu National Park (Central Sulawesi).
Observation on the morphology of each species in this study enables the selection
of important characters for species delimitation and identification. Position of
suckers, petiole canal leaf, bract imbrications, bract behavior before falling, bract
colour, fruit shape, and seed surface are important characters for delimiting and
identifying taxa of wild banana species (Musa) in Sulawesi.
Phylogenetic tree used morphological characters showed M. balbisiana and
M. textilis placed in clade I in which they shared some similar characters for
instance bract imbrication and behavior before falling. While Musa acuminata ssp.
banksii, M. acuminata var. tomentosa, M. celebica, and M. itinerans were placed
in clade II. The phylogenetic study based on morphological characters is
incongruent with the phylogenenetic study based on ITS regions of nrDNA
sequences.
Phylogenetic tree used ITS regions of nrDNA sequences using MP and
Bayesian methods showed M. textilis was placed in clade I and separated from M.
balbisiana, M. itinerans, M. celebica, M. acuminata ssp. banksii, and M.
acuminata var. tomentosa. Nevertheless, M. textilis and M. balbisiana have closed
relationship. By using MP analysis, M. itinerans together with M. acuminata ssp.
banksii, M. acuminata var. tomentosa and M. celebica were placed in the same
clade, whereas Bayesian analysis showed M. itinerans was separated from M.
acuminata ssp. banksii, M. acuminata var. tomentosa as well as M. celebica, and
was placed in different clade.
This study revealed that phylogenetic study of wild banana species (Musa)
in Sulawesi based on morphological characters and ITS regions of nrDNA
sequences support the monophyly of genus Musa and also support the taxonomic
status of M. acuminata ssp. banksii as intraspecific taxa of M. acuminata.
Moreover, analysis of ITS regions also showed that sequences of ITS regions of
nrDNA could be used for phylogenetic tree reconstruction at the species level.
Key words: Internal transcribed spacer, phylogenetic, morphology, Musa,
Sulawesi
RINGKASAN
LULUT DWI SULISTYANINGSIH. Sistematika pisang-pisang liar (Musa L.) di
Sulawesi: kajian morfologi dan molekuler. Dibimbing oleh RITA MEGIA dan
ELIZABETH A. WIDJAJA
Ketersediaan informasi mengenai pisang-pisang liar di Sulawesi masih
sangat sedikit mengingat belum ada penelitian sistematika yang pernah dilakukan
sebelumnya. Oleh karena itu, penelitian sistematika pisang-pisang liar (Musa L.)
di Sulawesi ini dilakukan. Penelitian meliputi (a) kajian delimitasi jenis pisangpisang liar (Musa L.) di Sulawesi untuk memberikan informasi keanekaragaman
dan distribusi pisang-pisang liar di Sulawesi, serta menyajikan deskripsi jenis dan
kunci identifikasi; (b) kajian filogenetik berdasarkan karakter morfologi dan
urutan basa nrDNA daerah internal transcribed spacer (ITS) untuk melihat
hubungan kekerabatan antar jenis.
Sebanyak 110 spesimen yang terdiri atas koleksi lama dan baru yang
tersimpan di BO serta gambar digital spesimen tipe dari MEL diperiksa karakter
morfologinya. Konstruksi pohon filogenetik menggunakan 53 karakter morfologi
dianalisis dengan metode Maximum Parsimony (MP) menggunakan program
PAUP*4.0b10. Sementara itu, konstruksi pohon filogenetik berdasarkan urutan
basa daerah ITS menggunakan metode MP dan Bayesian (MrBayes ver. 3.0).
Ensete dan Musella dipilih sebagai outgroup sedangkan ingroup direpresentasikan
oleh jenis-jenis Musa hasil kajian delimitasi jenis. DNA total diekstraksi dari daun
yang tersimpan dalam gel silika menggunakan metode CTAB yang dimodifikasi.
Primer ITS-5 dan ITS-4 digunakan untuk mengamplifikasi daerah ITS. Data
urutan basa diedit dengan program ChromasPro dan disejajarkan dengan software
Muscle.
Kajian delimitasi jenis berdasarkan karakter morfologi menunjukkan 6
Musa dan 1 jenis yang belum diketahui ditemukan di Sulawesi. Terdapat 2 jenis
endemik Sulawesi, yaitu Musa acuminata var. tomentosa dan M. celebica.
Ditemukannya M. balbisiana dan M. itinerans merupakan catatan baru. Di
Sulawesi, keberadaan M. balbisiana direpresentasikan oleh spesimen yang
dikoleksi dari Manado (Sulawesi Utara), sedangkan keberadaan M. itinerans
direpresentasikan oleh spesimen yang dikoleksi dari gunung Nokilalaki (Sulawesi
Tengah) dan Taman Nasional Lore Lindu (Sulawesi Tengah). Dari hasil observasi
karakter morfologi pada masing-masing jenis diperoleh karakter-karakter penting
untuk memberikan batasan jenis dan identifikasi. Posisi tumbuh anakan, lekukan
pada tangkai daun, imbrikata pada braktea, tipe braktea sebelum gugur, warna
braktea, bentuk buah, dan permukaan biji merupakan karakter penting untuk
memberi batasan dan identifikasi jenis pisang-pisang liar (Musa) di Sulawesi.
Pohon filogenetik berdasarkan karakter morfologi menunjukkan M.
balbisiana dan M. textilis berada pada klade I dan mempunyai karakter yang sama
seperti imbrikata pada braktea dan tipe braktea sebelum gugur. Sementara itu, M.
acuminata ssp. banksii, M. acuminata var. tomentosa, M. celebica, dan M.
itinerans berada pada klade II. Hasil kajian filogenetik berdasarkan karakter
morfologi berbeda dengan kajian filogenetik berdasarkan sekuen daerah ITS dari
nrDNA.
Konstruksi pohon filogenetik berdasarkan urutan basa nrDNA daerah ITS
menggunakan metode MP dan Bayesian menunjukkan M. textilis berada pada
klade I dan terpisah dari M. balbisiana, M. itinerans, M. celebica, M. acuminata
ssp. banksii, dan M. acuminata var. tomentosa. Meskipun demikian, M. textilis
berkerabat dekat secara filogenetik dengan M. balbisiana. Dengan menggunakan
metode MP, M. itinerans bersama dengan M. acuminata ssp. banksii, M.
acuminata var. tomentosa dan M. celebica berada pada klade yang sama,
sedangkan metode Bayesian menunjukkan M. itinerans terpisah dari M.
acuminata ssp. banksii, M. acuminata var. tomentosa dan M. celebica serta berada
pada klade yang berbeda.
Penelitian ini menunjukkan bahwa kajian filogenetik pisang-pisang liar
(Musa) di Sulawesi berdasarkan karakter morfologi dan urutan basa nrDNA
daerah ITS mendukung sifat monofiletik dari marga Musa dan juga mendukung
status taksonomi M. acuminata ssp. banksii sebagai infraspesifik taksa dari M.
acuminata. Lebih jauh lagi, analisis daerah ITS juga menunjukkan bahwa urutan
basa nrDNA daerah ITS dapat digunakan untuk rekonstruksi pohon filogenetik
pada tingkat jenis.
Kata Kunci: Filogenetik, internal transcribed spacer, morfologi, Musa, Sulawesi.
Copyright©2013, Bogor Agricultural University
Copyright are protected by law
1. It is prohibited to cite all parts of this thesis without referring to and
mentioning the source
a. Citation only permitted for the sake of education, research, scientific
writing, critical writing or reviewing scientific problems.
b. Citation does not inflict the name and honor of Bogor Agricultural
University.
2. It is prohibited to republish and reproduce all parts of this thesis without
the written permission from Bogor Agricultural University.
THE SYSTEMATIC OF WILD BANANA SPECIES (MUSA L.)
IN SULAWESI: MORPHOLOGY AND MOLECULAR
STUDIES
LULUT DWI SULISTYANINGSIH
Thesis submitted
As partial fulfillment requirement for the Master
Degree
In Plant Taxonomy
THE GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2013
External examiner: Dr. Nunik Sri Ariyanti, M.Si.
Title
Name
NRP
: The Systematic of Wild Banana Species (Musa L.) in Sulawesi:
Morphology and Molecular Studies
: Lulut Dwi Sulistyaningsih
: G353100031
Certified by
Supervisor Committee
Dr. Rita Megia, D.E.A.
Chairman
Prof. (R.) Dr. Elizabeth A. Widjaja, M.Sc
Member
Approved by
Head of Plant Biology
Study Program
Dean of Graduate School
Dr. Ir. Miftahudin, M.Si
Dr. Ir. Dahrul Syah, MSc. Agr.
Date of Examination:
Date of Graduation:
ACKNOWLEDGEMENT
It would not have possible to write this thesis without the help and support
of many people around me, to only some of whom it is possible to give particular
mention here.
First and foremost, I would like to express my gratitude to my supervisors,
Dr. Rita Megia, D.E.A. and Prof. (R.) Dr. Elizabeth A. Widjaja, M.Sc. for their
valuable advices, guidance, and encouragement throughout my study. Many
thanks also goes to Dr. Nunik Sri Ariyanti, M.Si., Dr. Rugayah, M.Sc., Dr.
Himmah Rustiami, M.Sc. for their suggestions, advices, and criticisms.
I gratefully acknowledge the Karyasiswa Ristek scholarship from Ministry
of Research and Technology (RISTEK); DIPA from Division of Botany, Research
Centre for Biology – LIPI with entitled Revision of Selected Taxa in Sulawesi for
supporting field research; Molecular Systematic Laboratory, Division of Botany,
Research Centre for Biology – LIPI for supporting sequencing fee. I would like to
thank to Dr. Witjaksono, M.Sc., Dr. Yuyu Purba, M.Sc., Fajarudin Ahmad, S.Si.
for their kindly help in DNA materials.
I would like to thank to all taxonomists and technicians of Herbarium
Bogoriense who gave their support and assist when conducted this study. I also
express my gratitude to my colleagues and friends in Research Centre for Biology
– LIPI and IPB.
Last but not the least, my special thanks go to my family, my parents, my
husband Dhani Yudhana and my daughter Nafeeza Armaghan El-Firdausy for
their spirit, patience, and support in my study.
Bogor, April 2013
Lulut Dwi Sulistyaningsih
TABLE OF CONTENTS
LIST OF TABLES
vi
LIST OF FIGURES
vi
LIST OF APPENDICES
vii
1 INTRODUCTION
1
2 LITERATURE REVIEW
2
Taxonomy of Musa L.
General Morphology of Musa L.
Distribution of Musa L.
Internal Transcribed Spacer (ITS)
3 MATERIALS AND METHODS
Species Delimitation of Wild Banana Species (Musa L.) in Sulawesi
Materials
Methods
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi
Based on Morphology Characters
Materials
Methods
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi
Based on Internal Transcribed Spacer Region of nrDNA Sequences
Materials
Methods
DNA Extraction and Amplification
DNA Purification and Sequencing
Phylogenetic Analysis
4 RESULT AND DISCUSSION
Species Delimitation of Wild Banana Species (Musa L.) in Sulawesi
General Morphology of Musa in Sulawesi
Pseudostems
Leaves
Inflorescences
Male Buds
Flowers
Fruits
Seeds
Distribution
Key to the Species and Infraspecific Taxa of Musa from Sulawesi
Key to the Species
Key for Infraspecific Taxa of Musa acuminata Colla
Taxonomic Treatment
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi
2
3
5
7
9
9
9
9
11
12
13
13
14
14
14
15
15
15
16
16
16
16
17
17
18
Based on Morphology Characters
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi
Based on Internal Transcribed Spacer Region of nrDNA Sequences
DNA Extraction and Amplification
Nucleotide Sequences of ITS Region of nrDNA and Variation
Within Species
Phylogenetic Analysis
General Discussion
35
37
38
42
46
5 CONCLUSION
47
REFERENCES
48
APPENDICES
52
CURRICULUM VITAE
55
LIST OF TABLES
1
2
3
4
5
Morphological characters state used in phylogenetic analysis
Source of ITS sequence utilized in the study
Sequence length variation of ITS region within outgroup and ingroup
Percentages of G+C content of ITS1 and ITS2
Nucleotide sequences variation of ITS region within species showed
unique nucleotides of species from section Eumusa and Australimusa
10
12
39
40
42
LIST OF FIGURES
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Variation on leaf base
Variation on leaf canal margin
Variation on bract
Distribution map of Musa sections
Distribution map of Musa sections in Indonesia
Organization of ITS region of nrDNA
Taxonomic level of utility of nuclear DNA regions used in phylogenetic
reconstruction based on angiosperms
Leaf bases shapes
Bract imbrications on male bud
Shape of seeds
Distribution map of wild banana species in Sulawesi
Musa acuminata ssp. banksii (F. Muell.) N.W. Simmonds
Distribution map of M. acuminata ssp. banksii in Sulawesi
Distribution map of M. acuminata var. tomentosa in Sulawesi
Musa balbisiana Colla
Distribution map of M. balbisiana in Sulawesi
Musa celebica Warb. ex K. Schum.
Distribution map of M. celebica in Sulawesi
Musa itinerans Cheesman
Distribution map of M. itinerans
Musa textilis Née
Distribution map of M. textilis in Sulawesi
Distribution map of Musa sp. 1 in Sulawesi
Strict consensus tree resulted from morphological characters
Visualization of PCR product
Nucleotide sequences variation of ITS regions within species that
showed some unique sequences of Musa balbisiana was compared with
other taxa
Nucleotide sequences variation of ITS regions within species that
showed some unique sequences of Musa itinerans was compared with
other taxa
Strict consensus tree of the ITS sequence region resulting from MP
analysis
Strict consensus tree of the ITS sequence region resulting from
Bayesian analysis
4
4
5
6
6
7
8
15
15
16
17
19
20
22
24
25
27
28
30
31
32
33
34
36
38
41
41
44
45
LIST OF APPENDICES
1 Matrix of morphological characters for phylogenetic analysis
2 The ITS region of nrDNA sequence alignment from position 272 to 373
showed part of the conserved region
3 The ITS region of nrDNA sequence alignment from position 485 to 575
showed part of the variative region
52
53
54
1
1 INTRODUCTION
Bananas belong to Musaceae, a small family which consists of three genera,
Ensete Bruce ex Horan, Musa L., and Musella (Franchet) H.W. Li. In general,
bananas (Musa L.) are grouped into wild seeded bananas that consist of
approximately 70 species (Häkkinen 2008) and edible seedless bananas consisting
of approximately 500 cultivars (Valmayor et al. 2002). Cultivated bananas have
considerable economic value since they have a high level of consumption,
whereas wild banana species have potential value as a genetic resource. Cultivated
bananas have been largely evolved from two wild banana species, Musa
acuminata Colla and M. balbisiana Colla. M. balbisiana is known to be resistant
to drought, while the intraspecific of M. acuminata, namely M. acuminata var.
malaccensis is known to be resistant to fusarium wilt (Nasution 1991).
Elucidating the phylogeny and taxonomy of Musa and its family is important as
phylogenetic relationships can provide valuable information for the collection and
utilization of genetic resources for further banana improvement.
Indonesia has a large number of bananas. This country is the center of
bananas origin (Simmonds 1966) as well as of its diversity (Daniells et al. 2001).
At least 325 cultivars have been recorded in Indonesia (Valmayor et al. 2002),
whereas only 12 wild banana species has been documented (Nasution and
Yamada 2001). Presumably, there are wild banana species that have not been
recorded and well documented.
In Indonesia, wild banana species grow widespread in Sumatra, Java, Lesser
Sunda Islands, Kalimantan, Sulawesi, Moluccas, and Papua. Biogeographycally,
Sulawesi has a unique characteristic because it is located in the Wallace line
which is the transition region between Asia and Australia. Sulawesi is also known
to have a large number of endemic flora and fauna (Mittermier et al. 1999).
Musaceae that have been reported as an endemic flora in Sulawesi are M. celebica
Warb. ex K. Schum. and M. acuminata Colla var. tomentosa (K.Sch.) Nasution
(Nasution 1991; Nasution and Yamada 2001).
As a taxa that can undergo self crossing, hybridization, and mutation,
bananas has a complex genome structure. To analyze bananas genetic diversity,
molecular characterization was required to support morphological characters.
During the past decades, molecular markers such as RFLPs (Gawel and Jaret
1991), AFLP (Wong et al. 2002), HAT-RAPD (Ruangsuttapha et al. 2007), trnLF (Liu et al. 2010; Li et al. 2010), and ITS (internal transcribed spacer) (Liu et al.
2010; Li et al. 2010; Hřibova et al. 2011) have been used for phylogenetic
analysis of Musaceae. Nowadays, ITS is used more often by researchers to
conduct a molecular phylogenetic analysis of plant in order to understand the
diversity and to answer phylogenetic problems. This is because ITS region is easy
to be isolated, amplified, and analyzed, due to its small size (300-800 bp) and high
copy number in the genome (Baldwin et al. 1995).
Taxonomy studies that reveal the morphological diversity of wild bananas
in Central, North, and South Sulawesi has been done by Nasution (1991).
However, in general the systematic studies of wild banana species in Sulawesi are
still rare. It can be seen from the number of identified Musaceae specimen in
Herbarium Bogoriense (BO). It is approximately only 26% of Musaceae
2
specimens from Sulawesi that stored in BO have been identified. Therefore,
systematic studies to reveal the Musaceae diversity in Sulawesi need to be done.
The aims of the study were 1) to provide information of the diversity and
the distribution of wild banana species in Sulawesi, 2) to provide species
description and an identification key and 3) to determine the phylogenetic
relationship between the species using morphological characters and ITS regions
of nrDNA sequences.
2 LITERATURE REVIEW
Taxonomy of Musa L.
APG III (Angiospermae Phylogeny Group) places genus Musa L., together
with Ensete Bruce ex Horan and Musella (Franchet) H.W. Li into family
Musaceae, the member of Zingeberales (APG 2009). Genus Musa is the largest
genus in the family that is firstly established by Linnaeus (1753). Some botanists
believed that the name of genus Musa is thought to be derived from the Arabic
name for the plant (mouz) which in turn may have been applied in honour of
Antonius Musa who is physician to Octavius Agustus, the first emperor of Rome
(Hyam and Pankhurst 1995). Whereas the name “banana” is derived from the
Arabic banan that means finger (Boning 2006) and was thought to be used in
Guinea (West Africa) concomitant with the introduction of the fruit by the
Portuguese and then the name spread to the New World (Cheesman 1948a).
The taxonomic complexity at the family level continues down to the genus
level and there are inconsistencies in the number of sections and number of
species proposed for inclusion in the genus Musa. The first classification made by
Sagot (1887) that divided the genus into three groups: (1) giant bananas (type: M.
ensete J.F. Gmel.), (2) bananas with fleshy fruit and often edible (type: M.
sapientum L.), (3) ornamental bananas with erect inflorescences and brightly
coloured bracts (type: M. coccinea Andrews).
In 1893, Baker divided genus Musa into three subgenera: (1) Physocaulis,
characterized by stem bottle-shaped; flowers many to a bract; petal usually
tricuspidate; fruit not edible. (2) Eumusa, characterized by stem cylindrical;
flower many to a bract; petal ovate-acuminate; bracts green, brown or dull violet;
fruit edible. (3) Rhodoclamys, characterized by stem cylindrical; flower few to a
bract, petal linier; bracts bright-coloured; fruit usually not edible.
Cheesman (1947) elevated the first subgenus to the generic level as the
genus Ensete and then made new classification. The classification is based on the
haploid number of the chromosome that followed by the similarity and the
differences on morphological characters. He treated the genus into four sections as
follows:F
A. Chromosome number x=11
1. Inflorescence pendent or semi-pendent from the first, the fruits reflexing
in development toward the base of the rachis. Flowers many to a bract, in
two series. Bracts commonly dull coloured, green, brownish or dull
3
purple. Pseudostems commonly exceeding three meters high
…...………………………………………………………Section Eumusa
2. Inflorescence erect, or at least at the base, so that the fruits do not reflex
in development but point toward the apex of the rachis. Flowers few to a
bract, usually in a single serie. Bract brightly coloured, often red.
Pseudostems less than three meters high…...........……………...
……………............................................................Section Rhodochlamys
B. Chromosome number x=10
3. Seeds subglobose, or more or less dorsiventrally compressed, smooth,
striate, tuberculate, or irregularly angulate with a marked or obsolete
umbo opposite to the hilum corresponding to small periperm chamber
within…………………………………………........Section Australimusa
4. Seeds cylindrical, barrel-shaped, or top shaped, marked externally by
transverse line or grove, above which are warted, tuberculate, or
variously patterned, below usually smooth; internally a well-developed
perisperm, chamber above the same line, this chamber empty in the ripe
seed…………………………………………………….Section Callimusa
Although this classification was widely accepted by most botanists, its
validity has been questioned, in any case for some sections. Some newly
described species which is already count the chromosome number also appeared
problematic. Argent (1976) proposed to establish one more section in Musa,
Ingentimusa (x=7) to include the single species M. ingens N.W. Simmonds. Based
on morphological and cytological characters, Simmond and Weatherup (1990)
found a very low level of consistency among the characters and suggested that
section Eumusa is heterogeneous and then divided it into two informal subgroups
“Eumusa-1” and “Eumusa-2. Recently, molecular data have been used to solve
such problems in taxonomy of Musa. By using RFLPs data, Gawel and Jaret
(1991) and Gawel et al. (1992) found that a molecular phylogeny was inconsistent
with traditional classification and they proposed that section Rhodochlamys
should be merged with section Eumusa. Wong et al. (2002), by using AFLP data
suggested that section Rhodochlamys should be placed in section Eumusa, and
section Australimusa should be merged in section Callimusa.
General Morphology of Musa L.
Musa is a large perennial herb and they grow in clump with rhizome and
false areal stem, cylindrical pseudostem that consisted of sheath leaves wrapped
together with each other, has a short underground stem (corm). The root system is
adventitious spreading out laterally.
Leaves grow from a cigar leaf into a large, blade oblong, arranged parallel
and pinnate. There are some variations on base shape of leaves: both side
rounded; one side rounded, one pointed; both sides pointed (Figure 1). Leaf canal
margin open with margins spreading, wide with erect margin, straight with erect
margins, margin curved inward or margins overlapping (Figure 2).
4
a
b
c
Figure 1 Variation on leaf base. Both sides rounded (a); one side rounded, one
pointed (b); both sides pointed (IPGRI 1996)
Figure 2 Variation on leaf canal margin. Open with margins spreading (a); wide
with erect margins (b); straight with erect margin (c); margins curved
inward (d) (Nasution and Yamada 2001)
Inflorescence springs from the rhizome and emerges at the top of the stem,
either erect or pendulous; the immature inflorescence is encased inside bracts that
give the appearance of large bud. Bracts are plane or sulcate, revolute or not
revolute before falling, imbricate or not imbricate (Figure 3). Flowers produce
nectar, on each bract there is one or two rows of flower. Basal flower is female or
hemaprodhite, consist of ovary that protected by compound tepal (calyx) and free
tepal (corolla), style and staminode; compound tepal essentially tubular but split
to the base on the adaxial side, 5 toothed at the apex (3-lobed at the apex with 2
accessory teeth between the main lobes; free tepal inserted within the compound
tepal and opposite to it (i.e. in the adaxial position). Male flower have 5 fertile
stamen that falling down with the bract. They are reduced to staminodes in female
flower. Female and male flowers are morphologically indistinguishable until the
inflorescence is about 12 cm long. At this point, the ovary in the male flower fails
to develop any further (Simmonds 1959).
5
a
b
Figure 3 Variation on bract. Not imbricate (a); imbricate (b) (Nasution and
Yamada 2001)
Fruits are berry, some dehiscent and some are not, with numerous seeds
(except in the parthenocarpic form). Each fruit is known as a “finger”. Each
cluster of fruits at node is known as a “hand” and the entire collection of hands is
known as a “bunch”. The number of hands varies each others. The outer
protective layer of each fruit known as the “skin” or “peel” is fusion of the
hypanthium (floral receptacle) and outer layer (exocarp) of the pericarp (fruit wall
derived from the ovary wall). This peel is easily removed from the fleshly pulp
that originates mainly from the endocarp (innermost layer of the pericarp)
(Simmonds 1953). During the development of the fruit from the ovary, the tepals,
style, and staminodes abscise leaving a characteristic calloused scar at the tip of
the fruit. Fruits develop only after pollination. Fruit size depends on the number of
seeds and parenchymatous pulp develops around each seed. The growth volume
curve is sigmoidial (Simmonds 1953). Wild banana species have little flash and is
filled with black or brown seed. The seeds have linier embryos, large amounts of
endosperm and a thick hard testa (Ellis et al. 1985).
Distribution of Musa L.
The section Australimusa is well-known in Brunei Darussalam, Indonesia,
Malaysia, Papua New Guinea, and Philippines, while section Callimusa can be
found in Brunei Darussalam, China, Cambodia, Indonesia, Malaysia, Papua New
Guinea, and Vietnam. The biggest section, Eumusa is widespread in Australia,
Bhutan, Cambodia, China, Eastern and SouthEast India, Indonesia, Japan, Laos,
Malaysia, Papua New Guinea, Philippine, Samoa, Sri Lanka, Thailand, and
Vietnam, wheread section Rhodochlamys grows well in Bangladesh, China,
Malaysia, and Thailand (Figure 4).
6
Rhodochlamys
Pasific Ocean
Eumusa
Callimusa
Australimusa
Indian Ocean
Figure 4 Distribution map of Musa sections.
Eumusa;
Australimusa;
Callimusa;
Rhodoclamys (Pollefeys et al. 2004)
Indonesia is situated in the centre of origin and diversity of Musaceae and
has a large number of both wild banana species and cultivated bananas. The
bananas widespread in Sumatra, Java, Lesser Sunda Islands, Borneo, Sulawesi,
Moluccas, and Papua. Pollefeys et al. (2004) made distribution map of Musa
sections in Indonesia using MGIS (Musa Germplasm Information System) and
DIVA-GIS. Almost all of the accessions that they used come from Nasution’s
(1991) study and the rest come from Musalogue. Because of insufficient
geographical data for some of Nasution’s accessions, no entry is available in
MGIS for Kalimantan (Borneo) although the expedition recorded wild species in
all major islands (Figure 5).
Legend
Australimusa
Callimusa
Eumusa
Figure 5 Distribution map of Musa sections in Indonesia (Pollefeys et al. 2004)
7
Internal Transcribed Spacer (ITS)
The major ribosomal RNA (rRNA) genes of plants are localized in clusters
on highly repeated sequences. Each repeat consist of sequences from 18S, 5.8S
and 25S ribosomal subunits and each copy contains a transcribed region that is
separated by the long non-transcribed intergenic spacer (IGS). These genes show
little sequence divergence between closely related species. Within each repeat,
these conserved regions are separated by internal transcribed spacer (ITS) which
occur in the following order: 5’–18S–ITS-1–5.8S–ITS-2-26S(or 25S)–3’ that
show higher rates of divergence (Figure 6).
ITS Region
Figure 6 Organization of ITS region of nrDNA (Soltis and Soltis 1998)
The internal transcribed spacers including ITS-1 and ITS-2 regions are part
of the nuclear rDNA (nrDNA) transcript but are not incorporated into ribosomes.
In particular, the 5.8S rRNA is separated from 18S, the SSU (small ribosomal
subunit) rRNA, by the first of two ITSs (ITS-1), and from 25-28S, the LSU (large
ribosomal subunit) rRNA, by the second ITS (ITS-2). Sequencing of the ITS
region, however has an exciting potential as a source of nuclear DNA characters
for phylogenetic reconstruction in plants. This promise was heightened recently
by encouraging result from ITS sequence-based phylogenies of protoctistans (Lee
and Taylor 1991), apes and humans (Gonzales et al. 1990). White et al. (1990)
have taken advantage of polymerase chain reaction (PCR) technology to promote
sequencing of nrDNA in fungi. Baldwin (1992) have described the usefulness of
these primers for PCR amplification and sequencing of the ITS region in
angiosperms and also described the utility of ITS DNA sequences as a source of
phylogenetic data in the subtribe Madiinae of Asteraceae.
ITS regions take a role in the maturation of nuclear rRNAs, bring ITS of
18S–26S nrDNA have a particularly valuable marker for phylogenetic analysis at
8
intraspecific level and intergeneric level among angiosperms and other eukaryotes
(Baldwin et al. 1995). In general, the ITS region present some advantages of
being a multicopy locus (100-200 copies), having a small size (300-800 bp),
varying from one taxon to another but highly conserved in size in a given taxon,
and making it a preferred diagnostic target for a universal test (Baldwin et al.
1995).
ITS sequences are proven to be valuable for phylogenetic reconstruction in
angiosperms, algae, and ferns. Recent work indicates that ITS-1 and ITS-2
sequences are inherently G+C rich in which portions of these regions are quite
conserved among angiosperms. Thus, the ITS regions not only possess high
information content at lower taxonomic level, but also exhibit conserved sequence
patterns and high alignability across angiosperms (Figure7).
Figure 7 Taxonomic level of utility of nuclear DNA regions used in phylogenetic
reconstruction based on angiosperms. The shaded box showing the
taxonomic zone that is not presently well covered by nuclear gene
sequences; ? refers to genes that have been rarely used; ----- designates
the approximate upper or lower limits of applicability (Soltis and Soltis
1998)
9
3 MATERIALS AND METHODS
Species Delimitation of Wild Banana Species (Musa L.) in Sulawesi
Materials
A total of 110 sheets (46 number of collections) of herbarium specimens
consist of the dry and spirit collections were used in this study. It composed of all
specimens of Musaceae in Sulawesi which are stored in Herbarium Bogoriense
(BO) and the new specimens collected from Central, North, South, and SouthEast
Sulawesi. The digital data that consist of type specimens from Royal Botanic
Garden (MEL) and JSTOR Plant Science were also evaluated.
Methods
Field research to collect new specimens were carried out by using
exploration method. Sampling method used was purposive random sampling, in
which sampling localities were randomly selected by considering factors that
influence the existence of Musa. Data or information recorded from the field
include location; altitude, longitude and latitude; vernacular name; plant general
habit; pseudostem (height, diameter, colour, appearance, predominant underlying
colour, pigmentation of the underlying pseudostem, sap colour, wax on leaf
sheaths); suckers (number of suckers, position); petiole (blotches at the petiole
base, colour of blotches, length); leaves (length, width, colour, and appearance of
leaf upper surface, colour, and appearance of leaf lower surface, wax, colour of
midrib dorsal and ventral surface); inflorescence/male bud (peduncle length,
bunch position, rachis position, colour of the bract, wax on the bract); flower
(compound tepal basic colour, lobe colour of compound tepal, free tepal colour,
filament colour, anther colour, stigma colour); fruit (number of fruits, length,
immature or mature colour). Morphological comparative study was done in
Herbarium Bogoriense (BO).
All the herbarium specimens were studied based on their morphological
similarity following de Vogel (1987) and Rifai (2008) using comparative
morphology data as main source.
Phylogenetic Study of Wild Banana Species (Musa L.) in Sulawesi Based on
Morphological Characters
Materials
The result of species delimitation study will be used as the ingroup, while
Ensete sp. 1 from Bogor Botanic Garden and Musella lasiocarpa from China were
selected as an outgroup. Fifty three morphological characters selected during
morphological observation of field and herbarium specimens were used in this
study. The characters and characters state followed the descriptor for bananas
(IPGRI 1996) with some modifications (Table 1).
10
Table 1 Morphological characters state used in the phylogenetic analysis
No
Characters
Character s State
1
Habit
0. Single
1. Clumping
2
Sucker
0. Absent
3
Pseudostem height
0.≤ 100 cm
1. Present
1. > 100 cm
4
Pseudostem aspect
0. Slender
1. Robust
5
Pseudostem colour
1. Green
6
Pseudostem
appearance
Predominant
underlying colour
of the pseudostem
Pigmentation of
the underlying
pseudostem
Sap colour
0. Medium
green
0. Dull
(waxy)
0. Green
0. Absent
1. Present
0. Watery
1. Milky
0. Absent
1. Present
1. Straight
with erect
margins
1. Clasping
7
8
9
10
11
Blotches at the
petiole base
Petiole canal leaf
III
2. Red-purple
1. Shiny (not waxy)
1. Greenyellow
2. Pink purple
12
Leaf sheaths
0. Wide with
erect
margins
0. Lax
13
Leaf habit
0. Erect
1. Intermediate
14
Colour of leaf
upper surface
Appearance of leaf
upper surface
0. Medium
green
0. Dull
1. Green
Colour of leaf
lower surface
Appearance of leaf
lower surface
0. Light green
1. Medium
green
1. Shiny
Shape of leaf blade
base
Colour of midrib
dorsal surface
Colour of midrib
ventral surface
Inflorescence habit
0. Both sides
rounded
0. Greenyellow
0. Medium
green
0. Erect
1. Both sides
pointed
1. Light green
0. ≤ 100
1. 101-200
2. ≥201
0. ≤ 30
1. 31-60
2. ≥ 61
24
Inflorescence
length [cm]
Peduncle length
[cm]
Peduncle hairiness
0. Absent
1. Present
25
Bunch appearance
0. Loose
1. Compact
26
Bract appearance
1. Inserted independently on the axis
27
Bract habit
0. Integral
with each
other
0. Persistent
28
Bract behavior
0. Not
revolute
1. Revolute
15
16
17
18
19
20
21
22
23
0. Dull
3.Blue
2. Margins curved inward
2. Dark green
1. Shiny
2. Green
1. Light green
1. Pendulous
1. Deciduous
11
Table 1 Morphological characters state used in the phylogenetic analysis (continue)
No
Characters
Character s State
29
Bract imbrication
1. Imbricate
30
Bract apex shape
31
32
Colour on the
bract apex
Wax on the bract
0. Not
imbricate
0. Slightly
Pointed
0. Not tinted
with yellow
0. Absent
33
Male bud shape
0. Like a top
1. Lanceolate
34
0. Persistent
1. Deciduous
0. Linier
1. Tubular
0. 3 –toothed
1. 5-toothed
0. Yellow
1. Orange
0. Translucent
white
0. Oval
1. White
0. Little or no
visible sign
1. Developed
0. Obtuse
1. Triangular
0. Straight
1. Curved
0. White
1. Cream
0. Straight
1. Arched
45
Male flower
behavior
Compound tepal
of male flower
shape
Lobe of compound
tepal of male
flower
Lobe colour of
compound tepal of
male flower
Free tepal colour
of male flower
Free tepal shape of
male flower
Free tepal apex
development of
male flower
Free tepal apex
shape of male
flower
Style shape of
male flower
Stigma colour of
male flower
Ovary shape of
male flower
Fruit shape
0. Straight
1. Curved
46
Fruit apex
0. Pointed
1. Blunt-tipped
47
Floral relicts
0. Absent
1. Present
48
0. Light green
1. Green
0. Yellow
1. Bright yellow
0. Persistent
1. Deciduous
51
Immature fruit
peel colour
Mature fruit peel
colour
Fruit fall from
hands
Seed diameter
0. >1 cm
1. ≤ 1 cm
52
Seed shape
0. Globular
1. Angular
53
Seed surface
0. Smooth
1. Wrinkled
35
36
37
38
39
40
41
42
43
44
49
50
1. Intermediate
1. Tinted with yellow
1. Present
2. Ovoid
1. Fan-shaped
Methods
Morphological characters were analyzed based on Maximum Parsimony
using PAUP*4.0b10 (Swofford 1998) with Heuristic Search settings vegetative
12
and floral characters were selected for the analysis of which 45 were scored as
binary and 8 as multistate. Some characters were scored as missing data when the
data were unavailable. All characters are treated as unorder data and have an equal
weight. The missing data are symbolized with “?”. Starting tree (S) was obtained
via stepwise addition. Number of trees held at each step during stepwise addition
= 1. Branch swapping algorithm was run by using tree-bisection-reconnection
(TBR) and was evaluated using 100 bootstrap replicates and a complete Hsearch.
Clade support values were obtained by using bootstrap. Bootstrap support (BS)
was categorized as strong (>85%), moderate (70%-85%), weak (50%-69%), or
poor (85%), moderate (70%-85%), weak (50%-69%), or poor (