A TAXONOMIC STUDY ON RAFFLESIA SPECIES FROM TANJUNG DATU NATIONAL PARK, SARAWAK

Yong Choi Yen

(39423)

QK Bachelor of Science with Honours 495 (Plant Resource Science and Management)RI5 2015 Y55 2015

l'usat Khidmar Maldumat Aka« . . UNIVERSITl MALAYSIA SARA~r; : ,

A Taxonomic Study on Rafflesia species from Tanjung Datu National Park, Sarawak

Y ong Choi Yen

39423

A report submitted in partial fulfilment of the requirement for the degree of

Bachelor of Science with Honour

(Plant Resource Science and Management)

Supervisor: Dr. Aida Shafreena Ahmad Puad

Plant Resource Science and Management

Department of Plant Science and Environmental Ecology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

.' 2015

I

APPROVAL SHEET

Name of candidate Y ong Choi Yen (39423)

Title A Taxonomic Study on Rafflesia specIes from Tanjung Datu

National Park, Sarawak

~

(Dr. Aida Shafreena Ahmad Puad)

Supervisor

-------------------.------------------------­

(Dr. Rebicca Edward)

Coordinator Plant Resource Science and Management

Department of Plant Science and Environmental Ecology

Faculty of Resource Science and Technology

DECLARATION

This Final Year Project 2014 is based on my original work except for quotation and

citation which have been acknowledged. I also declared that it has not been submitted

previously or currently to any other degree at Universiti Malaysia Sarawak or any other

institution.

(Yong Choi Yen)

Plant Resource Science and Management

Department of Plant Science and Environmental Ecology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak (UNIMAS)

III

Acknowledgement

First and most of all, I am grateful to the God for giving me strength and determination to

complete my [mal year project. Second, I also would like to express my sense of gratitude

to my supervisor, Dr. Aida Shafreena binti Ahmad Puad for valuable guidance and advices

extended to me. Without her guidance and persistent help, this thesis would not have been

possible.

In addition, I would like to express my sincere thanks to Wan Nuur Fatiha binti Wan

Zakaria and Siti Noor Aishah binti Mohd Noor, postgraduates who always helping me in

this study especially in collecting and interpreting raw data as well as valuable guidance on .

doing }ab work. I take this opportunity to record my thanks to all the faculty members of

Department of Plant Science and Environmental Ecology for their help and encouragement.

I sincerely thanks to my lab mates, Muhammad Raimi bin Mohd Fauzi, Ibrahim Izzat bin

Zabidin and Muhammad Aiman bin Mohammad Rahimi for their support and help. I also

would like to express my appreciation to all my course mates for their constant

encouragement and support . ..

Finally, I would like to express the deepest gratitude to my lovely family for their

continuous moral support and encouragement. Not to forget to all my friends for their

constant encouragement, SUppOlt and understanding.

IV

Table of Contents

Title page I

Approval II

Declaration III

Acknowledgement IV

Table of Contents V

List of Abbreviations VII

List of Appendices VIII

List of Figures IX

List of Tables X

Abstract

Chapter 1 Introduction & Objective 2

Chapter 2 Literature Review 4

2.1 Rafflesiaceae 4

2.2 Morphology of RajJlesia R. Brown 4

2.3 Distribution and Ecology of RajJlesia 7

2.4 RajJlesia species in Sarawak. 8

2.4.1 RajJlesia kethii Meijer, Blumea 30 (1984): 211 8

2.4.2 RajJlesiapricei Meijer, Blumea 30 (1984): 214 9

2.4.3 RajJlesia tuan-mudae Beccari, Atti. Soc. Ita!' Sc. Nat. J J 10

(1868): 197-198

2.5 Biology of RajJlesia 11

2.6 The Host Plant-Tetrastigma (Miq.) Planch. 14

2.7 Phylogeny of RajJlesia 14

Chapter 3 Materials and Methods 16

3.1 Taxonomic Description 16

3.2 DNA Extraction and Amplification 17

3.3 Amplification of Targeted Sequence Using Polymerase Chain 18

Reaction (PCR)

3.4 Pairwise Sequence Alignment 20

Chapter 4 Results 21

4.1 Unknown RajJlesia species in Sarawak . 21

4.2 Key to the RajJlesia in Sarawak 21

V

4.3 Description of Unknown RajJlesia species from Tanjung Datu 22

4.4 Morphological Comparison between Rafflesia hasseltii and 25

Unknown Rafflesia species from Tanjung Datu

4.5 Molecular Anaiysis of Nuclear Region (ITS) and Plastid Gene 27

(16S rDNA)

4.5.1 DNA isolation of total plant genome and amplification of 27

nuclear sequence (ITS region) and plastid sequence (16S

rDNA)

4.5.2 Pairwise sequence alignment 28

Chapter 5 Discussion 31

Chapter 6 Conclusion and Recommendations 33

References 34

Appendices 36

•.

VI

bp

CIA

CTAB

DNA

dNTP

H20

ITS

MgCh

PCR

PL

rDNA

Taq polymerase

.'

List of Abbreviations

Base pair

Chloroform-isoamylalcohol

Cetyltrimethyl ammonium bromide

Deoxyribonucleic acid

Dideoxyl nucleotide

Water

Internal Transcribed Spacer

Magnesium chloride

Polymerase Chain Reaction

Perigonelobe

Nuclear ribosomal DNA cistron

Themus aquatiqus polymerase

VII

List of Appendices

Page

Appendix 1 Measurements were taken in the laboratory. A and B. 36 Perigone lobe diameter. C. Diaphragm diameter. D. Length of upper type ramenta. E. Length of middle type ramenta. F. Length of lower type of ramenta. G. Bract diameter. H. Length of process

Appendix 2 Table showing the data for speCllTIens examined In the 37 laboratory

Appendix 3 Morphological comparison among Rafflesia keithii, Rafflesia 38 pricei, Rafflesia tuan-mudae, Rafflesia hasseltii and unknown Ra.fJlesia species from Tanjung Datu

Appendix 4 Morphological comparison between Rafflesia hasseltii and 46 unknown Rafflesia species from Tanjung Datu

•.

VIII

Figure I

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

I,::

Figure 10

Figure 11

I

List of Figures

.' Page

The floral morphology of RafJlesia female flower (Adapted 4 from Nais, 2001)

Diagrammatic sketch showing three concentric zones of 5 RafJlesia: (a) outer zone, (b) middle zone, (c) innermost zone (Adapted from Nais, 2004)

The floral morphology ofRafJlesia male flower (Adapted from 6 Nais, 2001)

RafJlesia keithii (Adapted from Nais, 2001) 9

RafJlesia pricei (Adapted from Nais, 2001) 10

RafJlesia tuan-mudae (Adapted from Nor-Izah, unpublished) 11

The life cycle of RafJlesia (Adapted from Nais, 2001) 13

ITS region that used for phylogenetic analysis (Adapted from 18 Soltis & Soltis, 1998)

RafJlesia species is showing A. Young and mature bud. B. 24 Open flower with 5 peri gone lobes. C. Diaphragm (shown with black arrow) and window (shown with white arrow) . D. Disk (shown with arrow). E. (a) Lower type (b) Middle type (c) Upper type (shown with arrow). F. Middle type ramenta. G. Annulus with presence of anther shown with arrow

Sequence alignment for ITS region between R. hasseltti and 29 RafJlesia sp. from Tanjung Datu National Park

Sequence alignment for Raffl6S between R. hasseltti and 30 RafJlesia sp. from Tanjung Datu National Park

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IX

List of Tables

Page

Table 1 Distribution of Rafflesia species in Southeast Asia 7

Table 2 Differences in floral characteristics between the unknown Rafflesia hasseltii and Rafflesia species from Tanjung Datu

26

Table 3 GenBank accession number for unknown Rafflesia species from Tanjung Datu National Park

27

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x

A Taxonomic Study on Rafflesia species from Tanjung Datu National Park, Sarawak

Y ong Choi Yen Plant Resource Science and Management Programme

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Rafflesia (Rafflesiaceae) is the largest flower in the world and it is an iconic species in Sarawak. Three Rafflesia species can be found in Sarawak, namely R. keithii, R. pricei, and R. tuan-mudae. An unknown Rafflesia species was discovered from Tanjung Datu National Park, Sarawak. This species morphologically resembled to R. hasseltii from Sumatra, Indonesia. In order to identify the unknown Rafflesia species, a morphological and molecular analysis were conducted. In this study, a detailed description of the unknown Rafflesia species and a taxonomic key to the Rafflesia species were made. Molecular analyses were carried out by sequencing two DNA regions (ITS and l6S rDNA) of the unknown Rafflesia species and compare with sequences of R. hasseltii. There are some morphological differences between the unknown Rafflesia species and R. hasseltii. In addition, molecular analyses showed that genetically these two species are quite distinct. Therefore, the unknown Rafflesia species may probably be a novel species. However, further molecular analysis is needed to support these [mdings.

Key words: Rafflesia, Raffiesiaceae, ITS, l6S rDNA, Tanjung Datu

ABSTRAK

Rafflesia (Rafflesiaceae) merupakan bunga yang terbesar di dunia dan ia adalah spesies .flora yang ikonik di Sarawak. riga spesies Rafflesia boleh didapati di Sarawak, iaitu R. keithii. R. pricei. dan R. tuan-mudae. Spesies Rafflesia yang tidak dikenali telah ditemui dari Taman Negara Tanjung Datu. Sarawak. Spesies ini menyerupai kepada R. hasseltii dari Sumatera dalam segi m01fologi. Untuk men genal pasti spesies Rafflesia yang tidak diketahui. analisis morfologi dan molekul telah dijalankan. Dalam kajian ini, penerangan terperinci meng~nai spesies Rafflesia yang tidak diketahui dan kunci taksonomi kepada spesies Rafflesia telah dibuat. Analisis molekul telah dijalankan dengan membuat urutan pada dua bahagian DNA (ITS dan 16S rDNA) spesies Rafflesia yang tidak diketahui dan bandingkan dengan urutan R. hasseltii. Terdapat beberapa perbezaan antara spesies Rafflesia yang tidak diketahui dan R. hasseltii dalam sefi morfologi. Di samping itu, analisis molekul menunjukkan bahawa genetik kedua-dua spesies adalah agak berbeza. Oleh itu, spesies Rafflesia yang tidak diketahui berkemungkinan adalah spesies baru. Walau bagaimanapun. analisis molekul yang lebih lanjut diperlukan untuk menyokong penemuan ini.

Kata kunci: Rafflesia. Rafflesiaceae, ITS, 16S rDNA, Tanjung Datu

I

Chapter 1

Introduction

Tropical rainforest is well known as the most complex terrestrial ecosystem on Earth and

rich in biodiversity (Nais, 2004). Therefore, tropical rainforest becomes a typical habitat

for many flora and fauna. Rafflesia is one of the unique and amazing flora species that is

endemic to tropical rainforest in Southeast Asia (Nais, 2004). Rafflesia is the largest flower

in the world (Nais, 2001). Rafflesia is rare and it is threatened with extinction mainly by

habitat destruction (Nais, 2004). Thus, it becomes an icon of plant ecology conservation

and protection especially tropical rainforests (Nais, 2004).

Rafflesia plant consists of flower without stem, leaves and roots (Nais, 2004). However,

Rafflesia has haustoria to penetrate the stem or root tissues of its host plant (Nais, 2004).

Rafflesia is an endophytic holoparasitic plant that grows completely embedded within its

host, Tetrastigma (Vitaceae), which is also widely distributed in tropical rainforests

(Barkman et ai., 2008). Rafflesia obtains nutrients and water from its host plant since this

parasitic plant is without photosynthetic pigments to carry out photosynthesis (Nais, 2001).

The pollination of Rafflesia depends on pollinators such as carrion flies through the

emission of rotten, smell from its flower (Beaman et ai., 1988). Rafflesia also relies upon

dispersal agents such as squirrel and tree-shrew to disperse its seeds for establishing the

next generation (Nais, 2001).

2

I

I

The rarity and uniqueness of Rajjlesia arises the interests of botanists and ecologists to

conduct research on Rajjlesia for scientific and education purposes (Nais, 2001). There are

many Rajjlesia species that have been described and studied since 1821. Out of the 18

species of Rajjlesia that have been described and studied before 2002, eight species were

discovered in Borneo and five in Sumatra, two from Peninsular Malaysia, three from Java

and two from the Philippines, and one species can be found in Thailand (Nais, 2001).

However, since 2002, eleven more new species were described and studied, making the

total number ofrecognised Rajjlesia species becomes 32 (Barcelona et al., 2009; Balete et

al. , 2010; Haji-Adam et al., 2013; Wong et al. , 2009).

Problem Statement and Objective

In Sarawak, there are three Rajjlesia species recorded, namely R. tuan-mudae, R. keith ii,

and R. pricei (Nais, 2004). Recently, an ullknown Rajjlesia species was discovered at

Tanjung Datu National Park, Sarawak. The floral morphology of unknown Rajjlesia

species is almost similar to R. hasseltii. Rajjlesia hasse/iii is found in Sumatra and is

believed to also be found in Samunsam, Sarawak. However, there is lack of well

documentation of this species found in Sarawak been published. Therefore, detailed

morphological characteristics and molecular analysis are necessary to detennine this

.' unknown Rajjlesia species. Therefore, the objective of this study is to identify the

unknown Rajjlesia species from Tanjung Datu National Park based on morphological and

molecular data.

3

Chapter 2

Literature Review

2.1 Rafflesiaceae

Rafflesiaceae is a holoparasitic plant family that contains eight genera which are

Apodanthes, Bdallophyton, Cythinus, Mitrastemol1 , Pilostyles, Rizanthes, Sapria, and

Rafflesia (Nais, 2001; Nais, 2004). All representatives of Rafflesiaceae are holoparasitic

plants, which mean they are without chlorophyll (Barkman et aI., 2004; Bendisksby et aI.,

2010). They totally depend on their host plant for water and nutrients (Barkman et aI.,

2004; Bendisksby et aI., 2010). Members of Rafflesiaceae also exist as endophytes (Nais,

2001). They grow completely embedded within the stem or root tissues of their host plant,

Tetrasiigma (Vitaceae) (Barkman et aI., 2004; Bendisksby et aI., 2010).

2.2 Morphology of Rafflesia R. Brown.

Rafflesia is exclusively characterised with its floral morphology as shown in Figure 1 (Nais,

2001; Nais, 2004). Rafflesia flower basically has three concentric zones: outer zone,

middle zone and innermost zone (Figure 2) (Nais, 2001; Nais, 2004).

Diaphragl'T1

Stigmatic are a ~(---~

Cupula ~---....".-

Perigonelobe

~-~ Ramenta

--::-lO""'--.;:-+--~)o Central col umn

Figure 1. The floral morphology of Rafflesia female flower (Adapted from Nais, 2001)

4

Figure 2. Diagrammatic sketch showing three concentric zones of Rafflesia: (8) outer zone, (b) middle zone, (c) innermost zone (Adapted from Nais, 2004)

The outer zone is made up of perigone lobes resembling petal, usually in five and

sometimes six, or even seven. The colour and pattern of perigone lobes indicates the

differences among species of Rafflesia (Nais, 2001; Nais, 2004).

The middle zone is called diaphragm that is ornamented by various pattern of white warts,

which differ among species. The lower face of diaphragm is covered by ramenta. Ramenta

differs among species based on their position at perigone tube of Rafflesia flower. There

are three types of ramenta: upper type, middle type and lower type. The upper type is

positioned near or on the lower surface of the diaphragm, whereas the middle type is on the

vertical centre of the perigone tube. The lower type is located on or near the base of the

perigone tube (Nais, 2001; Nais, 2004).

The innermost zone is known as diaphragm aperture. The lower face feature of aperture,

around the opening is called window. Window also ornamented by white warts, whereby

5

the arrangement pattern is important to characterise different Rafflesia species (Nais, 2001;

Nais, 2004).

The centre of the flower has a central column supporting a circular disc. The top of the

circular disc has projecting processes (Mat-SaUeh, 1991). According to Mat-Salleh (1991),

the central colurrm bears the sexual reproductive organs of Rafflesia, holding either ovaries

(Figure 1) or anthers (Figure 3).

Anther ~-----+'I"""""--'r--:iII .....~"'--dil'-->'i'---~ Annulus interior

Bristles

Figure 3. The floral morphology of Rafflesia male flower (Adapted from Nais, 2001 )

6

2.3 Distribution and Ecology of Rafflesia

Rajjlesia is restricted to tropical rainforests of Southeast Asia. The 32 species of Rajjlesia

are distributed from Thailand, throughout Peninsular Malaysia, Sumatra, Java, Borneo to

the Philippines (Bendiksby et aI., 2010). The distribution of Rajjlesia species in Southeast

Asia is presented in the following table (Table l).

Table 1. Distribution ofRajJIesia species in Southeast Asia (Adapted from Nais, 2001)

Localities Species

Borneo

Java

Peninsular Malaysia

Philippines

Sumatra

Thailand

R. arnoldii R. Brown, R. borneensis Koorders, R. ciliata Koorders, R. keithii Meijer, R. pricei Meijer, R. tengku­adlinii Mat Salleh & Latiff, R. tuan-mlldae Beccari, R. witkampii Koorders

R. patma Blume, R. rochussenii Teijsmann & Binnendjik, R. zollingeriana Koorders

R. cantleyi Solms, R. kerrii Meijer, R. azlanii Latiff & M. Wong, R. su-meiae Wong, N ais, & Gan, R. sharifah­hapsahiae Adam, Mohamed, Aizat-Juhari & Wan

R. balatei Barcelona & Cajano, R. banahaw Barcelona, Pelser & Cajano, R. leonardi Barcelona & PeIser, R. lobata R. Galang & Madulid, R. manillana Tescehemacher, R. mira Fernando & Ong, R. philippensis Blanco, R. schadenbergiana Geoppert, R. speciosa Bercelona & Fernando, R. verrucosa Balete, PeIser, Nickrent & Barcelona

R. arnoldii R. Brown, R. gadutensis Meijer, R. hasseltii Suringar, R. micropylora Meijer, R. patma Blume, R. rochussenii TeijsmaIpl & Binnendjik

R. ken'ii Meijer

7

RafJlesia is often found in both primary and secondary forests (Nais, 2001). Rafflesia

distribution is reliant on that of the typical habitat of Tetrastigma which is also found in

primary and secondary forests (Nais, 2001). Ralflesia populations are usually found near to

the stream banks and high elevation areas (Nais, 2001). Nais (2001) stated that the

altitudinal distribution of Rafflesia differs according to species, from as low as 5 m above

sea level (R. patma in Java), to as high as 1400 m or above (R. pricei in Sabah).

2.4 RaJjlesia species in Sarawak

In Sarawak, there are three species of Rafflesia present, namely R. keithii, R. pricei and R.

luan-mudae (Nais, 2004). Rafflesia keithii was found at Gunung Hose, whereas R. pricei

was discovered at Bario Highlands in Sarawak (Nais, 2001). Ralflesia tuan-mudae which is

an endemic species of Sarawak was first discovered at Mount Pueh in 1868 by Odoardo

. Beccari (Nais, 2001). At present, this species can be found in Gunung Gading National

Park, Lundu (Sarawak) (Nais, 2004). Rajjlesia keithii and R. pricei were also found in

Sabah.

2.4.1 Rafflesia keithii Meijer, Blumea 30 (1984): 211

Type: Madani s.n. (SAN); Sabah, Sungai Melaut, along the Ranau-Tambunan road.

Rafflesia keithii is one of the larger-flowered species. Its open flower can reach up to 80-94

cm in diameter. Ralflesia species commonly having five perigone lobes but R. keithii has

been discovered with six perigone lobes at Kampung Kokob, Ranau (Sabah). Perigone

warts of R. keitlzii are interspaced to each other. White warts on diaphragm are contrasting,

isolated and in 5 concentric rings. The number of anthers is always 40. Rafflesia keithii is

8

-------

widely distributed in Borneo (Sabah; probably also Kalimantan and Sarawak). It grows in

primary and secondary hill dipterocarp forest, normally near streams or rivers (Nais, 200 1).

Figure 4. RafJlesia keithii (Adapted from Nais, 200 1)

. 2.4.2 Rafflesia pricei Meijer, Blumea 30 (1984): 214

Type: Price S.n. (K), Sabah, Mount Kinabalu, Mamut Copper Mine, 1300 m a.s.l., 4 May

1967.

Rafflesia pricei is the smallest among RajJlesia species in Sarawak. The diameter of open

flower is about 25-30 cm, with aperture about 5-6 cm wide. The perigone warts of R. price;

are white, matted-like and not interspaced to each other. The diaphragm has 4-5 irregular

rings of white warts smaller than those of the peri gone lobes. The number of anthers is 20.

This species usually found in Sabah and probably also in north-eastern Kalimantan and

Sarawak (Borneo). The typical habitats of R. pricei are in primary and secondary hill

dipterocarp forest, normally near streams or rivers (Nais, 200 I).

9

--------

Figure 5. Ra.fflesia pricei (Adapted from Nais, 200 I)

2.4.3 Rafflesia tuan-mudae Beccari, Atti. Soc. Ital. Sc. Nat. 11 (1868): 197-198

Type: Beeeari S.n. (FI-B), Gunung Poe (Pueh), Sarawak.

RajJlesia tuan-mudae blooms up to 56 cm in diameter, with aperture about 15-18 cm wide.

However, the diameter can reach up to 72 cm as recorded by Nor-Izah (unpublished).

RajJlesia tuan-mudae has distinct brick-red colouration of its perigone lobes, and the apex

tapering into an obtuse tip, giving it an oblong shape, makes it easy to identify. The white

warts on perigone lobes are less numerous and interspaced to each other. The warts on

diaphragm arranged in 2 concentric rings. The number of anthers is 25. This species is

endemic to Sarawak but it also can be found in Kalimantan (eagar Alam Gunung Rara

Pasi near Singkawang). RajJlesia tuan-mudae grows in lowland and hill dipterocarp forest

(Nais, 2001).

10

Figure 6. RajJlesia tuan-mudae (Adapted from Nor-Izah, unpublished)

2.5 Biology ofRafj1esia

The biological system of Rafflesia is unique and incredible (Nais, 2004). Rajjlesia is not

only endophytic and holoparasitic, but also depends upon animals for pollination and

dispersal to establish next generation (Nais,.2004).

Blooming Rafflesia flower emits rotten smell to attract the pollinators (Nais, 2004). The

main pollination agent of Rafflesia is carrion flies from the genera of Lucilia and

Chrysoma (Mat-Salleh, 1991). Carrion flies are lured by the smell and crawl into the anther

grooves in male flowers to find food sources (Mat-Salleh, 1991). They are precisely guided

by ridges armed with bristles (Beaman et aI., 198~). In the process of crawling upward

searching for food, the back of the flies come into contact with the pollen, and the sticky

mass attached to its back (Nais, 2001). However, there is no such food provided by

Rafflesia for the pollinators but deceives them by an apparent offering of food and possibly

brood place (Beaman et aI., 1988). Hence, pollen-loaded flies leave the male flower, and

searching for another flower. If they visit the female flower, the flies enter the sulcus of

female flower where the stigmatic area is positioned (Beaman et al., 1988). On entering the

11

,.

sulcus, the sticky mass pollen attached at flies' back comes into contact with the stigmatic

area, and rubbed offthe pollen, affecting pollination (Nais, 2001).

The dispersal of Rafflesia is dependent upon that of the small animals such as squirrel and

tree-shrew (Mat-Salleh, 1991). Species of squirrel and tree-shrew that has been reported to

disperse seeds of Rafflesia is Callsociurus notatus and Tupaia tana (Nais, 2004).

According to Nais (2004), the remaining fruit and seeds that attached at the mouth and feet

of squirrel and tree-shrew will be anchored on stem or root of Tetrastigma when they are

climbing the plant. The seed then wiU be germinated, to start over the life cycle of

RajJlesia (Nais, 2004).

RajJlesia takes 46 months to complete its life cycle (Nais, 2004). The life cycle of Rafflesia

follows a sequence of stages: seed germination, development of the Rafflesia plant inside

the host plant, emergence of flower buds, flower buds development, flowering, pollination,

fruit and seed set, seeds dispersal, and seed germination for the establishment of next

generation (Nais, 2001) (Figure 7).

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~

I'

I'

I

Seed ge

Stage VIII

nnmation & inoculation 1'----7of host

(46 months)

Stage VII

Seed dispersal

(1-2 days)

Stage VI

Mature fruits

(6-8 months)

Stage V

Flo wer fully open for

4~8 days to open

Figure 7. The life eye Ie of RajJlesia (Adapted from Nais, 200 I)

13

Stage I

Host organ swollen

(6 days)

Stage II

Emergence from host

(160 days)

\

Stage III

Bract abscission

(8 days)

Stage IV

Flower beginning

(24-48 hours)