Plant Sys Evo

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O R I G I N A L A R T I C L E

Morphological and molecular characterization of Thamnocalamus spathiflorus subsp. spathiflorus at population level

Samik Bhattacharya Æ Jayadri Sekhar Ghosh ÆMalay Das Æ Amita Pal

Received: 29 January 2008 / Accepted: 12 August 2008

Ó Springer-Verlag 2009

Abstract Comprehensive morphological characterization

and an incidence of gregarious flowering in Thamnocal-

amus spathiflorus (Trin.) Munro subsp. spathiflorus are

described. Twenty-eight key vegetative characters as well

as reproductive morphology were studied. They are in

gross agreement with prior taxonomic descriptions, yet

more elaborate. Statistical analyses of the quantitative

vegetative characters revealed significant high variability

existing between populations, but not within populations.

However, DNA fingerprinting analyses by applying

highly polymorphic random primers could not detect any

polymorphism either between populations or within

populations. Insignificant within-population variability

indicates possibility of clonal propagation from the

donor(s) possessing similar genetic background and thus

reducing genetic variability.

Keywords Morphology Á Gregarious flowering Á

DNA fingerprinting Á Genetic diversity

Introduction

Thamnocalamus spathiflorus (Trin.) Munro subsp. spath-

iflorus belongs to the subtribe Thamnocalaminae

(=Arundinariinae) under subfamily Bambusoideae. The

group includes several cold-tolerant members (http://www.

bambooresearch.com/Specie/species-thamno.html) and is

usually found in fragile ecosystems like canal banks, steep

terrain and eroding hills. Hence, they play important role in

stabilizing top soil by its widespread rhizomatous rooting

system.

On the basis of the flowering cycle, bamboos have been

categorized in three major groups: annual flowering, spo-

radic or irregular flowering and gregarious flowering

(Brandis 1899). In gregarious flowering, the onset of the

reproductive phase occurs simultaneously in all the mem-

bers of a cohort or species across a wide area and

subsequently dies together. Records of bamboo flowering

are limited in the literature (Siefriz 1950; Filgueiras and

Pereira 1988; Ramanayake and Yakandawala 1998; Bhat-

tacharya et al. 2006). In India, the past incidents of mast

flowering in T. spathiflorus were recorded during 1818–1821

from North-Western Himalayas (Fig. 1, location A), during

1881–1882 from Jaunsar and Tehri Garhwal (Broun 1886;

Fig. 1, location B), in 1942 from Chakrata (Fig. 1, location

C) and in 2002 from Uttaranchal (Naithani et al. 2003;

Fig. 1, location D). The estimated flowering period in

T. spathiflorus is 60 years (Naithani et al. 2003). Here, we

report the incidence of a gregarious flowering event in

T. spathiflorus subsp. spathiflorus growing over an area of

approximately 3.5 km2 in Sikkim, India. This incited us to

re-evaluate the reproductive characteristics of the subspecies

and compare with previous descriptions. Nevertheless, like

most bamboo species, the identification of T. spathiflorus

subsp. spathiflorus is usually dependant on vegetative

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00606-008-0092-1 ) contains supplementarymaterial, which is available to authorized users.

S. Bhattacharya Á J. S. Ghosh Á A. Pal (&)

Plant Molecular and Cellular Genetics Section, Bose Institute,

P 1/12 CIT Scheme VIIM, Kolkata 700054, India

e-mail: [email protected]

M. Das

Department of Plant Pathology, Physiology and Weed Science,

Virginia Polytechnic Institute and State University, Blacksburg,

VA 24061, USA

123

Plant Syst Evol

DOI 10.1007/s00606-008-0092-1

http://www.bambooresearch.com/Specie/species-thamno.html


http://dx.doi.org/10.1007/s00606-008-0092-1

http://dx.doi.org/10.1007/s00606-008-0092-1





characters, which often lead to taxonomic conflicts (Sta-

pleton 1994). Similarly, in a number of instances, we haveidentified significant variability in vegetative features, which

pose difficulty in distinguishing closely allied woody bam-

boo species (Bhattacharya et al. 2006). Hence, in this study,

we attempted to assess the extent of population genetic

diversity in T. spathiflorus by simultaneously utilizing a

wide array of morphological characters and multilocus DNA

fingerprinting profiles. In contrast to widespread interests on

bamboo taxonomy and systematics, efforts on population

level genetic diversity studies are in its infancy, till date (Das

et al. 2008). Our previous study on B. tulda (Bhattacharya

et al. 2006) indicated a low population genetic diversity

existing in this species. Similar trend was reported in

Phyllostachys pubescens from Taiwan (Lai and Hsiao 1997)

and Guadua angustifolia from Colombia (Marulanda et al.

2002). It is likely that only few clones of individual species

acted as the genetic donor within a particular geographic

area resulting in low level among population genetic diver-

sity. On the contrary, relatively higher clonal variation was

found in Sasa senanensis from Japan (Suyama et al. 2000)

and G. amplexifolia from Colombia (Marulanda et al. 2002).

Most possibly, the differential reproductive systems have

influenced the intensity of population genetic diversity, since

it is expected that the allogamous species are usually more

diverse than the autogamous ones.

Materials and methods

Data scoring and collection of materials

Wild stands of Thamnocalamus spathiflorus (Trin.) Munro

subsp. spathiflorus spanning across nine geographical

locations in East Sikkim, India, were surveyed (Fig. 1,

Table 1). Longitude, latitude and altitude of the collection

sites were recorded with a MagellanÒ Sportrack Ò GPSsystem, while temperature and rainfall data were collected

from the local meteorological department.

Vegetative characters

Type of the rhizome was determined by digging the soil or

from the already exposed soil at hill slopes. Among the

selected 28 vegetative characters (qualitative plus quanti-

tative; Das et al. 2007), 14 culm features were recorded at

site, while 14 culm sheath characters were scored after

detailed investigation. Each quantitative morphological

character represents the mean of three randomly chosen

culms from five individual clumps per each population.

Flowering incidences, inflorescence and floral

characters

Flowering was recorded at five distant locations in East

Sikkim, India, during 2006 (Fig. 1). Inflorescences were

collected and key morphological characters of fresh spik-

elets were recorded. Five individual fertile florets were

randomly selected to characterize each population.

Genomic DNA isolation

Genomic DNA was isolated from young leaves of five

randomly selected culms as representatives of each popu-

lation. Surface-sterilized leaf tissues weighing 0.1 g were

sliced into small pieces and homogenized in liquid nitrogen

using mortar and pestle. DNA was extracted using 25 ml

warm CTAB extraction buffer following Doyle and Doyle

(1987). After removal of RNA by RNAase treatment, DNA

concentrations were determined by using an UV

Fig. 1 Map showing past flowering records in T. spathiflorus at

various locations of India and Nepal (reported during A: 1818–1821;

B: 1881–1882; C : 1942; D: 2002; E : 2006). An enlarged view of the

flowering site (E ) depicting nine locations surveyed in the present

study; 1, Namchipong; 2, Khempur; 3, Kyongkhosla Falls; 4, 15 Mile;

5, 14 Mile; 6 , 10 Mile; 7 , 9 Mile; 8, 5 Mile; 9, Gangtok BSI.

Gregarious flowering was observed only at locations 3, 4, 5, 6 and 7

S. Bhattacharya et al.

123



spectrophotometer (Beckman-Coulter, DU-520) and purity

was checked at A260 and A280. An OD of 1 at A260 is

equivalent to 50 lg DNA, and pure DNA, free of protein,

gave a value between 2.0 and 1.9 of A260: A280.

Development of fingerprinting profiles

by random primers

Forty random decamer Operon primers (Operon Technol-

ogies, Germany) were initially screened to select 22

primers that generated distinct and highly reproducible

amplified fragments (primer sequences are given in

Table 4). The amplifications were carried out in a Perkin

Elmer Cetus 2400 thermal cycler with 50 ll reaction

mixture following the protocol of Das et al. (2005). The

ability of these primers to generate reproducible and

polymorphic banding patterns across a wide range of

bamboo species had already been demonstrated in our

laboratory (Das et al. 2007, 2008). Nevertheless, the ability

of these primers to differentiate closely related congeners

was further examined by carrying out amplification reac-

tions using genomic DNA of Arundinaria maling Gamble

[Synonym, Yushania maling (Gamble) Stapleton]. Among

all the temperate, Sino-Himalayan, woody bamboo genera

growing up to 3,000 m, A. maling is phylogenetically

closest to Thamnocalamus (Guo et al. 2002).

The thermal cycler programme was as follows: 4 min at

95°C followed by 35 cycles (45 s at 94°C, 45 s at 37°C and

1 min at 72°C) and finally 10 min at 72°C for elongation.

The amplified PCR products were resolved by

electrophoresis on 1.5% agarose gel in TAE buffer and

recorded with a gel doc 1000 camera using the software

programme Molecular Analyst version 1.5 (Bio Rad,

USA). The exact molecular weights of the PCR-amplified

fragments were calibrated from the standard 100 bp

molecular weight marker (Genei, USA) by the same

software.

Statistical analysis

One-way analysis of variance (ANOVA) was performed to

estimate morphological variability between and within nine

populations of T. spathiflorus subsp. spathiflorus. The

quantitative characters considered for ANOVA analysis

were height and diameter of culm, internode length, ratio of

cavity versus culm diameter, ratio of culm sheath length

versus breadth at base and ratio of culm sheath length to

blade length. Coefficients of variation (CV%) between and

within population for the said parameters were also

computed.

Results

Vegetative characters

The perennial, unicaespitose bamboo has short pachy-

morph rhizome. Culms were 5.8–6.9 m high, usually

straight, terete, glaucous, distally pruinose, yellowish green

in color (Fig. 2a); 22.0–28.0 mm in diameter; internodes

Table 1 Principal geographical features of the collection sites of T. spathiflorus subsp. spathiflorus

Collection

numbera

Place of

collectionb

Latitude Longitude Altitude

(m)

Maximum

temperature

(°C)

Minimum

temperature

(°C)

Average

annual rainfall

(mm)

Soil type

AM/NMC/06/68 Namchipong 27°15.1880N 88°37.1790E 1,321 11.5 2.5 3,245.0 Silty clay

loam

AM/KMP/06/74 Khempur 27°22.8390N 88°41.2580E 2,945 9.0 2.0 3,554.0 Silty clay

loam

JG/KKF/06/045 Kyongkhosla

Fallsc

27°22.3010N 88°42.6990E 3,164 9.0 1.5 3,674.0 Loamy sand

JG/15 M/06/056 15 Milec

27°22.0940N 88°43.0760E 3,200 8.5 1.5 3,688.0 Loamy sand

AM/14 M/06/79 14 Milec

27°22.1590N 88°42.0130E 3,289 8.5 1.5 3,696.0 Loamy sand

JG/10 M/06/062 10 Milec

27°22.210 N 88°41.1700E 3,442 8.0 1.0 3,285.0 Loamy sand

JG/09 M/06/074 9 Milec 27°22.2290N 88°41.0350E 3,122 9.5 1.5 3,195.0 Loamy sand

AM/05 M/06/85 5 Mile 27°22.2270N 88°40.6130E 2,513 10.5 2.5 3,575.0 Silty clay

loam

JG/GBG/06/088 Gangtok BSI 27°20.3290N 88°36.6390E 1,769 11.5 3.5 3,455.0 Silty clay

loam

aCollection number of representative individual of each population

b Locations under different districts of West Bengal, Indiac Gregarious flowering observed

Characterization of Thamnocalamus spathiflorus subsp. spathiflorus

123



straight, 220–280 mm long; ratio of cavity diameter to

culm diameter 0.51–0.59; nodes rarely swollen with sheath

scars and whitish rings. Branching is typically found in the

upper nodes. Culm sheaths were slightly asymmetrical in

shape, deciduous, coriacious and almost glabrous, often

with scanty hairs on abaxial surface (Fig. 2b), vary in size

from base to top of the main culm; ratio of total length to

breadth at the base 2.22–2.77; blade straight, ovate; ratio of

total length to blade length 2.83–3.19; auricles absent;

ligule fimbriate.

Flowering incidence and floral morphology

During August 2006, gregarious flowering of T. spathiflo-

rus subsp. spathiflorus was noticed at five locations in East

Sikkim, India (Fig. 2c, d) at altitudes of 3,122–3,442 m

and stretched over an area of 3.5 km2 (Table 1). Inflores-

cence is a drooping panicle and branching pattern similar to

that of a vegetative culm (Fig. 3a). Rachis was smooth,

jointed, 100–120 mm with two to three spikelets (Fig. 3b)

subtended by shining, chaffy, straw-colored sheathing

Fig. 2 a Vegetative culm and

culm sheath of T. spathiflorus

subsp. spathiflorus;

b photograph of slightly

asymmetric blade of the culm

sheath with scanty hairs on

abaxial surface; c wild stands of

T. spathiflorus subsp.

spathiflorus with gregarious

flowering on stiff mountain

slope; d close-up view of

a flowering branch


123



bracts, 50–60 mm long; empty glumes 13–17 mm, six- to

nine-nerved, ciliate at the tip and edges (Fig. 3c); spikelets

pedicillate, 50–60 mm long, contains four to six fertile

florets with distal floret imperfect (Fig. 3b). Lemma

(Fig. 3d) was 20–25 mm long, acuminate with ciliate tip,

3–4 mm broad at base, glabrous, 11- to 13-nerved, straw-

colored, overlapping with the palea (Fig. 3e); palea sub-

tending a bisexual floret 10–15 mm long, 2–4 mm broad at

the base, four-nerved, penicillate, two-keeled, bearing cil-

iae on the edges (Fig. 3e); lodicules 3, 2.5–3.0 mm long,

ovate acute, hyaline, fimbriate, three-nerved (Fig. 3f);

stamens 3; anthers 7.0–9.0 mm long, basifixed, purple,

emarginated with linear dehiscence (Fig. 3g); ovary

oblong, glabrous, 2–2.5 mm long; style short undivided,

hairy; stigmas 3, 3–4 mm long, yellow, plumose (Fig. 3h).

Seed was not found in any of the populations.

Population variability based on vegetative features

Analyses of variance was significant between population

variations with respect to height and diameter of culm,

internode length, ratios of culm cavity to culm diameter,

Fig. 3 Reproductive

morphology of T. spathiflorus

subsp. spathiflorus:

a inflorescence with fasciculate

arrangement of spikelets;

b a single spikelet; c–h

individual floral parts: c empty

glume; d lemma; e palea;

f lodicule; g stamens;

h pistil


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culm sheath length to breadth at base and culm sheath

length to length of blade (Table 2). The most variable

character was internode length having highest coefficient

of variation between populations (21.03%) with signifi-

cance tested at P\ 0.001, while within-population

variation was reasonably low (CV 1.2–6.3%; Table 3).

Few other quantitative characters also revealed significant

(at P B 0.001 level) variations (Table 2) such as, height of

culm (CV, 14.13%; F = 14.74501), culm diameter (CV,

18.05%; F = 14.69198), ratio of culm cavity to culmdiameter (CV, 11.94%; F = 17.77686), culm sheath length

to breadth at base (CV, 8.47%; F = 77.50478) and ratio of

culm sheath length to length of blade (CV, 3.99%;

F = 144.5635). However, within-population variability

was significantly low (Table 3).

Population genetic diversity based on DNA

fingerprinting analyses

Informative and reproducible DNA fingerprinting patterns

were generated by 22 out of 40 random oligomers

employed. Reproducibility of each fingerprinting pattern

was confirmed by repetition (Fig. 4). Yet again, the ability

of these primers to differentiate closely related congeners

was confirmed by simultaneously carrying out amplifica-

tion reactions with A. maling genomic DNA (Table 4,Figure S1). The approximate size range of the amplified

products in T. spathiflorus subsp. spathiflorus was 134 bp

to 1.38 kb, while in A. maling it was between 201 bp and

1.23 kb (Table 4). The highest number (7) of bands was

obtained with OPOJ-01 and OPA-05, while the lowest (3)

Table 2 Analysis of variance estimated within and between nine populations of T. spathiflorus subsp. spathiflorus for height of culm ( H , in m),

diameter of culm ( D, in mm), internode length ( I , in mm), ratio of culm cavity to culm diameter (CD), ratio of culm sheath length to breadth at

base (LB) and ratio of culm sheath length to length of blade (LL)

Source of variation df Sum of squares

H D I CD LB LL

Within population 4 0.061 0.105 3.111 0.0004 0.002 0.002

Between population 8 2.872 0.774 77.111 0.019 0.893 0.894Error 32 0.779 0.211 28.889 0.004 0.046 0.046

Total 44 3.712 1.090 109.111 0.024 0.942 0.942

Source of variation df Mean squares (MS)

H D I CD LB LL

Within population 4 0.015 0.026 0.778 0.0001 0.0005 0.0003

Between population 8 0.359 0.097 9.639 0.002 0.112 0.065

Error 32 0.024 0.007 0.903 0.0001 0.001 0.0004

Total 44 0.084 0.025 2.480 0.0005 0.021 0.012

Computed F 14.74501a

14.69198a

10.67692a

17.77686a

77.50478a

144.5635a

Coefficient of variation (between population) 14.13% 18.05% 21.03% 11.94% 8.47% 3.99%

aP B 0.001 level

Table 3 Coefficient of variation (CV, in %) estimated within nine populations of T. spathiflorus subsp. spathiflorus for height of culm ( H , in m),

diameter of culm ( D, in mm), internode length ( I , in mm), ratio of culm cavity to culm diameter (CD), ratio of culm sheath length to breadth at

base (LB) and ratio of culm sheath length to length of blade (LL)

Populations H a

Da

I a

CDa

LBa

LLa

Namchipong 1.198486 4.318846 2.828427125 1.543653185 0.879961083 0.518167777Khempur 2.817037 2.828427 6.271632338 1.818181818 3.039867379 1.066142487

Kyongkhosla Falls 1.944039 3.075956 3.63588289 0.76840824 0.958196814 0.404110847

15 Mile 1.792729 3.515378 3.19335888 1.543653185 1.941510436 0.54092787

14 Mile 2.043637 3.074377 2.226514461 2.413022997 1.348073007 0.903843182

10 Mile 1.798384 3.193359 1.98450202 1.437560183 1.275542344 0.563203717

9 Mile 3.983609 4.865076 1.644167631 3.064688742 0.887820616 0.914057667

5 Mile 3.245432 3.521367 4.488879626 3.45359356 0.75920487 0.463762764

Gangtok BSI 1.413277 2.127738 1.150326657 0.649142659 0.318113295 0.198943755

a P B 0.001 level


123



was scored with OPB-02, OPB-19 and PW-02 primers. No

polymorphism could be detected among the nine popula-

tions studied (Fig. 5).

Discussion

Exclusively depending on the culm and culm sheath vari-

ations, three subspecies (spathiflorus, nepalensis and

occidentalis) and two varieties (bhutanensis and crassino-

dous) had previously been described within T. spathiflorus

(Stapleton 1994). The subspecies spathiflorus differs from

others by having short pachymorph rhizome, short culm

node diameter (\3 mm) and slightly asymmetric culm

sheath covered with scanty abaxial hairs (Stapleton 1994;

Fig. 2a, b). Although the nature of rhizome could not be

critically assessed in our study, superficial observation of

the clumping pattern confirms its short pachymorph nature.

The vegetative and inflorescence/floral morphology of

T. spathiflorus subsp. spathiflorus described here are ingross agreements with previous reports (Naithani et al.

2003; Clayton et al. 2006). However, the current study

provides further details of individual floral parts of the

subspecies spathiflorus, which had not been characterized

before.

We also illustrate the spatial and temporal patterns of

gregarious flowering events in the subspecies reported till

date from different climatic zones of India and Nepal

(Fig. 1). Geographically isolated clones adopting variable

flowering cycles have already been recorded in Dendro-

calamus giganteus (Ramanayake and Yakandawala 1998).

The flowering cycle reported was 25 years in South India,40–45 years in North, East, Central India, while 45 years

in Bangladesh and 65 years in West Indies. Another pop-

ulation level study enumerated that clones originating from

the seeds of same flowering event may not necessarily have

the same flowering interval (Isagi et al. 2004). Janzen

(1976) was of opinion that, to resolve the issue, more

Fig. 4 A representative profile of DNA fingerprinting of T. spath-

iflorus subsp. spathiflorus. Lane 1 OPOJ-01; Lane 2 OPOJ-03; Lane 3OPOJ-04; Lane 4 OPOJ-11; Lane 5 OPOJ-18; Lane 6 OPB-01; Lane 7

OPB-02; Lane 8 OPB-03; Lane 9 OPB-04; Lane 10 OPB-05; Lane 11

OPB-06; Lane 12 OPB-07; Lane 13 OPB-08; Lane 14 OPB-18; Lane

15 OPB-19; Lane 16 OPB-20; Lane 17 OPA-02; Lane 18 OPA-04;

Lane 19 OPA-05; Lane 20 OPA-10; Lane 21 PW-01; Lane 22 PW-02;

M1 100 bp DNA ladder marker, M2 1.5 kb ? 100 bp DNA ladder

marker

Table 4 Total number and size

range of amplified products

generated from the genomic

DNA of T. spathiflorus subsp.

spathiflorus, using 22 randomdecamer primers

a Numbers and size range of

amplified products generated

from the genomic DNA of

A. maling are given within

parentheses

Primers Nucleotide

sequence (50–30)

Total number of

amplified fragmentsa

Size ranges of

amplified fragments (bp)a

OPOJ-01 CTGCTGGGAC 7 (5) 451–1,079 (468–1,052)

OPOJ-03 GTAGACCCGT 4 (2) 416–888 (870–1,113)

OPOJ-04 CCTTGACGCA 5 (6) 211–691 (614–1,234)

OPOJ-11 TCCGCTCTGG 4 (3) 134–846 (760–1,064)

OPOJ-18 AGGGAACGAG 4 (3) 260–790 (651–906)

OPB-01 GTTTCGCTCC 6 (1) 387–1,135 (1,015)

OPB-02 TGATCCCTGG 3 (5) 148–790 (566–1,113)

OPB-03 CATCCCCCTG 4 (5) 380–818 (578–1,113)

OPB-04 GGACTGGAGT 5 (4) 310–910 (566–882)

OPB-05 TGCGCCCTTC 6 (4) 529–1,185 (505–1,003)

OPB-06 TGCTCTGCCC 6 (5) 225–790 (274–942)

OPB-07 GGTGACGCAG 5 (4) 366–980 (614–918)

OPB-08 GTCCACACGG 4 (3) 564–825 (493–845)OPB-18 CCACAGCAGT 5 (4) 167–1,022 (687–930)

OPB-19 ACCCCCGAAG 3 (5) 451–888 (675–1,052)

OPB-20 GGACCCTTAC 5 (5) 345–1,164 (444–1,040)

OPA-02 TGCCGAGCTG 6 (5) 176–1,382 (554–1,040)

OPA-04 AATCGGGCTG 6 (5) 416–1,086 (626–1,003)

OPA-05 AGGGGTCTTG 7 (5) 366–966 (420–1,210)

OPA-10 GTGATCGCAG 6 (6) 402–839 (505–1,064)

PW-01 TGGTCACTGA 4 (5) 451–980 (201–748)

PW-02 TGGTCACTGA 3 (5) 543–959 (505–1,076)


123



attention should be paid to ‘habitat variation’ and ‘genetic

versus phenotypic variation.’ The current incidence of

gregarious flowering in T. spathiflorus subsp. spathiflorus

at five sites out of nine sites surveyed in East Sikkim

perhaps suggests the presence of two or more cohorts with

variable intermast period. This could be verified only when

the remaining four populations flower. The flowering cyclein a particular bamboo species/genetic background is pre-

determined. Hence, the prediction of mast flowering in a

particular region is possible only when we know the clonal

history of the donor plants and genetic structure of the

populations.

Statistical analysis of the quantitative morphological

features revealed significant variation between populations

(Table 3). However, these variations did not affect the

identity of the subspecies, since no significant variation was

observed with the subspecies determining culm sheath fea-

tures. Insignificant within-population variability (Table 4)

may suggest clonal propagation from the donor(s) possess-ing similar genetic background and thus reducing genetic

variability. It is also apparent from the fingerprinting studies

(representative Fig. 5a, b). Further studies involving a wide

range of bamboo species from diverse eco-geographical

locations are required to gain a better understanding of the

population genetic diversity existing in bamboos.

Acknowledgments We acknowledge Council of Scientific and

Industrial Research, New Delhi, India, for the financial support [Grant

No. 38 (1062)/03/EMR-II], CSIR-NET fellowships to the author (SB)

and Dr. P. Singh of Botanical Survey of India, Sibpur, Howrah, for

the identification of bamboo material. The help rendered by Dr.

Aparajita Mitra during collection of material is sincerely

acknowledged.

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Fig. 5 Monomorphic fingerprinting profiles of one representative for

each of the nine populations of T. spathiflorus subsp. spathiflorus, in

the order as shown in Table 1, generated using random decamer

primers a OPOJ- 04; b OPA- 04; M, 100 bp DNA ladder in a and

1500 bp DNA ladder marker in b


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