(zingiberaceae) from - International Journal of Plant, Animal and

Volume-6, Issue-4 Oct-Dec-2016 Coden: IJPAJX-CAS-USA, [email protected]
Received: 10thAug-2016
Revised: 10th Sept -2016
Accepted: 11th Sept-2016
DOI: 10.21276/Ijpaes http://dx.doi.org/10.21276/ijpaes
Research article
MORPHOLOGICAL CHARACTERS AND RANDOM AMPLIFIED POLYMORPHIC DNA
BASED GENETIC DIVERSITY ANALYSIS OF CURCUMA SPECIES (ZINGIBERACEAE)
FROM INDIA
Rajesh Kumar T1. Santhosh kumar R2. and Yusuf A1.
Interuniveristy Center for Plant Biotechnology, Department of Botany, University of Calicut, Kerala,
India-673 635
ABSTRACT: Genetic diversity of 19 Curcuma species was assessed using morphological characters and random
amplified polymorphic DNA. A dichotomous key and a dendrogram for the species were developed using the
selected morphological characters. The dendrogram derived from the morphological characters consists of III
groups. C.bhati showed distinction from all the other species with a separate node.C.montana and
C.psuedomontana shared the same node in group IIB indicating that both are synonyms and the need for
reassessing their separate species status. Twenty RAPD primers produced a total of 2226 scorable bands in the 19
species of Curcuma, out of which 1025 were polymorphic. The percentage polymorphism ranged from 56.7% to
36.5% within the species. Based on UPGMA clustering, the 19 species were grouped into two main groups. The
group IV contained one node for Curcuma bhatii with a similarity coefficient of 0.23 with the all the other species
clarifying the questions raised against the circumscription of C.bhatii from Paracautleya bhatii. The third group
contained 7 species in which C. montana and C. pseudomontana were grouped together with a similarity
coefficient of 0.33 thusare synonyms. Group II contained 6 species with a similarity coefficient of 0.29 to 0.33.
Group I consists of 5 species, among them,C.amadashowed distinct node justifying its special features. The results
obtained show that by using morphological characters and RAPD the genetic diversity of the Curcuma species can
be assessed.
Key words Curcuma, Species diversity, Taxonomy, RAPD, UPGMA dendrogram
*Corresponding author: Yusuf A. Interuniveristy Center for Plant Biotechnology, Department of Botany,
University of Calicut, Kerala, India-673 635
Copyright: ©2016 Yusuf A. This is an open-access article distributed under the terms of the Creative Commons
Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited
INTRODUCTION
The family Zingiberaceae, generally known as ‘Spice family’, forms an important group with considerable
economic potential, with genera such as Aframomum, K.Schum., Alpinia Roxb., Amomum Roxb., Curcuma L.,
Elettaria Maton., Kaempferia L. and Zingiber Boehm. Many members of this family have been used in the
ayurvedic system of medicine from time immemorial and are well-known spices. The genus Curcuma L., with
around 120 species distributed in tropical and subtropical Asia consists of a rather homogenous group of
rhizomatous perennials [1].
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Zingiberaceae is a taxonomically neglected group mainly because of thespecial floristic characters and
inaccessible nature of the wet evergreen forests in which they grow. The short flowering period coincides with the
monsoon season, makes their collection much difficult. Moreover, huge vegetative and massive underground parts
are major constraints in herbarium preparation. Consequently, most herbarium specimens are fragmentary and the
conclusions in most of the floras, based on dried specimens, are truncated accounts. The Delicate nature of flowers,
loss of colour and formation of a gummy mass soon after collection makes the floral-morphology based
phylogenetic evaluation much difficult for the genus Curcuma. The major problem with taxonomic descriptions is
the lack of, type specimens, illustrations of old species,protologues and complete details in literature. The flowers
are evanescent and even flower specimens in the herbaria cannot be of much help to a critical taxonomist. The
determination of correct species identityis necessary for proper utilization and conservation of Zingiberaceous
crops. Proper characterization of the taxa by using molecular and phytochemical methods is needed to solve the
problem of species delimitation. The vulnerability of many of these species and imminent danger of their
extinction makes it more urgent.
The main identifying character of the genus is its inflorescence, a spike with prominent spiral bracts, which
is laterally fused or adnate to the peduncle and form pouches, each subtending a cincinnus of flowers and a cluster
of, often coloured, sterile, terminal bracts called ‘coma’. Baker [2] described 29 species in the Flora of British
India and subdivided the genus into three sections - Exantha, Mesantha and Hitcheniopsis. Hitcheniopsis differed
from the rest of the genus in its spurless anthers. Schumann [3] rejected the sectional classification based on spike
position and suggested two subgenera [Eucurcuma and Hitcheniopsis (Baker) K. Schum.] based on the presence or
absence of spur on the anthers. Fischer [4] reported eight species. But this was proved as a gross underestimate by
the subsequent addition of twelve more species, bringing the total number of South West Indian species to 20 of
which 12 taxa are endemic to the Indian subcontinent [5]. The species are naturally found in mixed deciduous
tropical forests and tropical broad-leaved evergreen forests. The members of the genus in these regions are
important resources and have great potential in terms of commercial values as a source of spices, medicines and
horticultural products [6, 7,8,9,10].
Taxonomic characterization is mainly dependent on the comparative morphological characters influenced by
environmental factors. Phenotype plasticity is being misunderstood as a taxonomic character in identifying species
of this genus.Confusion prevails in the classification of Zingiberaceous members,due to the difficultyof
conventional taxonomic toolsto discriminate the species. Studies based on the morphological, molecular,
anatomical and biochemical characterization of some Curcuma species have been attempted earlier [11, 12, 13,
14]. Relying much on the morphological characters alone in species delimitation has its own disadvantages since
they are not always completely representing the genetic structure. Conventional taxonomic methods in conjunction
with molecular and biochemical tools will provide accurate and powerful methods for analyzing the genetic
relationship among the species in the family Zingiberaceae. However, concerted efforts are lacking in the
molecular characterization in Curcuma species. Molecular markers assume greater significance, as these methods
detect polymorphisms by assaying subsets of the total amount of DNA sequence variation in a genome [15].
The objective of the present study was to examine the genetic diversity of selected Indian Curcuma species
by using morphological and molecular markers, which has relevance in the present context of the ongoing
taxonomic revision of the genus. The development of highly reliable molecular marker system for assessing the
genetic diversity within the genus could help in crop improvement program through molecular breeding.
MATERIALS AND METHODS
Morphological characters
Curcuma species selected for the study were C. aeruginosa, C. inodora, C. raktakanta, C. longa, C. amada, C.
neilgherrensis, C. oligantha, C. coriacea, C. aurantiaca, C.zanthorrhiza, C. decipiens, C. vamana.
C.karnatakensis, C.mutabilis,C.harita, C.aromatica, C.montana, C.psuedomontana and C. bhatii. Whole plants
and their underground parts such as rhizome, roots and root tubers were collected from different parts of South
India. The rhizomes collected from different regions were planted and maintained in the Calicut University
Botanical Garden (CUBG). Eight morphological characters were taken into consideration inassessing the
morphological similarities/dissimilarities. The characters selected were, floral (spike position, the color of calyx
and color of corolla) Rhizome ( color of rhizome, aroma and taste of rhizome) and aerial ( color of leaf sheath and
leaf midrib). Specimens were identified with the help of Floras, Revisions and Monographs and also referred to
experts for accurate identification.
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The nomenclatural corrections were made according to ICBN [16] and for the abbreviation of periodicals; the BPH
[17] was followed. The abbreviation of authors is according to Brumitt and Powell [18]. The types and authentic
materials available in Central National Herbarium (CAL), Kolkata and Madras Herbarium (MH) at Coimbatore
were studied. Voucher specimens used for this investigation are deposited in the University of Calicut Herbarium
(CALI). The morphological characters were marked as presence and absence and loaded inthe binary matrix of
NTSYS-pc program and computed to derive the genetic similarity index and the dendrogram was constructed by
using UPGMA [19].
RAPD analysis
Plant material
The experimental material comprised of 19 Curcuma species, collected from different geographical locations and
maintained in the field gene bank of Botanical Garden, University of Calicut (CUBG), Kerala, India.
DNA extraction
Genomic DNA was isolated from the young leaves by using the modified CTAB method [20]. The protocol used
for the extraction of DNA worked for all the species, however, interspecific variations were observed in the quality
and quantity of DNA. DNAwas extracted from uppermost first and second leaves of all the Curcuma species. The
extraction buffer contained 100mM Tris - HCl (pH 8.0), 20mM EDTA (pH 8.0), 1.4 M NaCl, 2% (w/v) Cetyl
trimethyl ammonium bromide (CTAB) and 0.2% (v/v) β-mercaptoethanol.
Polymerase Chain Reaction
Polymerase chain reactions were carried out in 25.0 µl reaction volume containing 10X PCR buffer, 0.5U Taq
DNA polymerase, 200µM dNTPs, 2.0mM MgCl2 and 10pmoles of random decamer primer [21]. Amplification
cycles comprised of an initial temperature of 920C for 5 min for complete denaturation.The second step consisted
of 35 cycles of denaturation at 920C for 1 min., annealing at 380C for 1min., extension at 720 C for 1 min. and a
final extension at 720C 10 min. The reactions were carried out in a 96 well thermal cycler with gradient block
(Eppendorf mastercycler pro S, Germany).
Electrophoresis of PCR products
The PCR amplified products were run on an agarose gel (1%w/v) kept in 1X TAE buffer for about 1.5 hrs. at 120
Volts. The gels were stained with ethidium bromide (0.5 µg/ml) and documented using chemiluminescence
imaging system (Cell-bio Science, U.S.A.). The bands were scored by using a 1Kb DNA ladder ( New England
Biolabs)as a marker.
Data scoring and analysis
The electrophoretic patterns were visually analysed and scored as 1/0 for the presence or absence of bands for each
primer. To avoid taxonomic ambiguities, the intensity of the bands was not taken into consideration, only the
presence of intense and reproducible bands was considered. The data were entered into excel spreadsheet and the
matrix obtained was loaded into the NTSYS-PC program [19] and Jaccard’s similarity index (JSI) between the
species were calculated.A UPGMA dendrogram was constructed based on the derived JSI. The frequency of the
RAPD bands detected with the 20 polymorphic RAPD primers was calculatedand estimates of genetic diversity
(H) were obtained using Shannon’sinformation measure modified for RAPD by Chakraborthy and Rao [21] as
H= ∑ ki=l PilogePi where Piis the frequency of the ith RAPD bands.
RESULTS AND DISCUSSION
Morphological evaluation
Based on the morphological characters a dichotomous taxonomic key was developed for all the 19 Curcuma
species studied.
1.
Anther lobes ecalcarate ...................................................................................... C. aurantiaca
1.
Anther lobes spurred ............................................................................................................... 2
2.
Leafy shoot 15-60 cm high...................................................................................................... 3
2.
Leafy shoot 65-125 cm high .................................................................................................. 11
3.
Inflorescence with or without inconspicuous coma ................................................................ 4
3.
Inflorescence with well-developed coma ................................................................................ 8
4.
Rhizome stoloniferous; flowers shorter than the bracts ..................................... 18. C. vamana
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4.
Rhizome ovoid; flowers longer than the bracts ....................................................................... 5
5.
Leaves 0.7-1.5 cm broad; inflorescence only central ............................................... 5 C. bhatii
5.
Leaves 7-14 cm broad; inflorescence both lateral and central ................................................ 6
6.
Leafy shoot upto 35 cm tall; flowers 5-5.7 cm long ........................................ 15 C. oligantha
6.
Leafy shoot upto 60 cm tall; flowers 4.5-6 cm long ................................................................ 7
7.
Labellum white with a median bright yellow band; anther 4.5 mm long......10. C. karnatakensis
7.
Labellum yellow or white with yellow center; anther 3.5-4 mm long ............ 13. C. mutabilis
8.
Root tubers cylindrical, 10-18 cm long; leaves coriaceous, densely pubescent on both sides6. C. coriacea
8.
Root tuber small, spherical, ovoid or oblong; leaves not coriaceous, glabrous or sparsely pubescent 9
9.
Flowers equal to the bracts; lip purple .................................................................................. 10
9.
Flowers longer than bracts; lip yellow ..................................................... 14. C. neilgherrensis
10.
Leaves broadly ovate, sub cordate at base; fertile bracts recurved; lip purple towards base7. C. decipiens
10.
Leaves elliptic, base oblique; fertile bracts not recurved; lip deep purple with a bright yellow band 9. C. inodora
11.
Leaves with purple patch along the midrib ........................................................................... 12
11.
Leaves without a purple patch along midrib ......................................................................... 13
12.
Rhizome blue within; leaves with a purple patch on the distal half on the upper side only1. C. aeruginosa
12.
Rhizome yellow to deep yellow within; leaves with purple patch on both sides along the whole length of the midrib
.................................................................................................................... 19. C. zanthorrhiza
13.
Lateral staminodes with a patch of glandular hairs at centre ................................................. 14
13.
Lateral staminoides without glandular hairs .......................................................................... 15
14.
Pseudostem reddish purple; leaves spreading, oblong-lanceolate, glabrous below 17. C. raktakanta
14.
Pseudostem green with a few light pink dots; leaves erect, semiplicate, ovate-elliptic, densely pubescent on the lower
surface .................................................................................................................... 8 C. haritha
15.
Rhizome with the smell of green mango ................................................................ 2. C. amada
15.
Rhizome without the smell of green mango .......................................................................... 16
16.
Rhizome with sessile tubers .................................................................................................. 17
16.
Rhizome without sessile tubers ............................................................. 16. C. pseudomontana
17.
Rhizome deep orange-yellow within; lip light yellow with a median dark yellow band4. C. longa
17.
Rhizome yellow or light orange yellow within; lip deep yellow ........................................... 18
18.
Rhizome light orange-yellow within; corolla white; calyx 8 mm long ...............12C. montana
18.
Rhizome greyish-yellow within; corolla pinkish white; calyx 2 cm long…………..3. C. aromatica
Taxonomic classifications based onmorphological characters in Curcuma species rely on the position of
the spike, presence or absence of root tubers and the colour of coma. Roxburgh [22] pointed out that the positional
difference of spikes is affected by the flowering season; the early spikes develop laterally and later spikes develop
terminaly. Santapau [23,24] added that in C. pseudomontana a large lateral spike emerges from the sides of the
leaves in the beginning of the rainy season (June-July). Gradually by the beginning of August, this spike decays
and a central spike appear surrounded by leaves, resulting in a central and lateral spikes in the same plant.
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C. aurantiaca shows diverse floral plasticity, which is the distinguishing feature of this species. The plant is
medium sized with small rhizomes and tubers. The inflorescence is central in position and the main feature is the
exceptionally coloured inflorescence. The bract colour varies from green, white, brown, purple to rose, justifying
its specific epithet (Fig.1).
The distinguishing feature of C.inodora is its beautiful and attractive spike. The inflorescence has variations in the
shape of comma bracts,the labellum has colour variations from dark purple, white, yellow or golden (Fig.2). The
plant produces two inflorescences in a year; one lateral and one central. The plant undergoes a dormant phase
during the summer; new sprouts with fresh leaves emerge by the end of April hence called as hidden purple
ginger. The species shares morphological features with C. decipiensbut differs in the number of flowers equaling
the bracts, 3-4 flowered cincinni, purple corolla and staminodes and labellum with a dark yellow band in the
center.
Identification of the species to some extent is possible by the qualitative observation of leaf morphology, rhizome
colour, size,etc. Highest leaf length and breadth in the descending order was observed from C. zanthorrhiza, C.
aromatica, C. aeruginosa, C. longa, C.amada, C.coriacea, C.haritha, C.raktakanta and C.pseudomontana. In C.
zanthorrhiza,the midrib has a purple colour throughout the midrib of the leaf whereas in C. aeruginosa the purple
colour is present only towards the upper half of the leaves. C.raktakanta closely resembles C.aeruginosa however,
differs in the yellowish to gray colour of the rhizomes instead of blue colour in the center, purple coloured
pseudostem and peduncle and absence of a purple patch on the leaves. C. amada is closely related to C.longa, but
the characteristic smell and pale yellow colour of the rhizome, pale yellow flowers and light violet coma in C.
amada displays some degree of differentiation. The characteristic camphor smell of the rhizome and the densely
pubescent lower surface of the leaf are differentiating characters of C.aromatica. C.coriacea is endemic to Kerala,
with coriaceous dense pubescent leaves, lateral inflorescence and bright yellow corolla and C. decipiens can be
distinguished from other species of Curcuma by the deep purple corolla and coma and two flowered cincinni. From
its close ally, C.inodora, the differences arein flowers being longer than the bracts, 2-3 flowered cincinni, labellum
purple with deep yellow bands.
C. vamana has the smallest flowers among the different Curcuma species reported so far from India. It has a sub
equal leaf base, much shorter, condensed spike and bracts, few flowered cincinni, absence of anther crest and
presence of spurs on fertile anther. C. karnatakensis closely resembles C.oligantha but differs from it in the large
size of the plants and large white flowers with a median yellow band [25]. C. oligantha was described as a new
species under the name C. cannanorensis [26]. This plant is closely akin to C. albiflora but differs from it in the
smaller size of leaves with acuminate or apiculate apex; the petiole shorter than the lamina, shorter spike and larger
size of the flowers. It is also related to C. neilgherrensis but differs mainly in the absence of a coma and smaller
bracteoles. Bhat [27] studied this species in detail and confirmed it as C. oligantha. Velayudhan et al., [28]
retained it as C. cannanorensis but the type specimen and protologue of C.oligantha were not taken into account
and rejected.
C. mutabilis is the most interesting species due to the variations in flowercolour (Fig.3). Corolla color varies from
whitish pink, pink-red, reddish orange, dark pink to dark violet; labellum and lateral staminodes are pure white,
white with yellow or reddish streaks in the throat of labellum or base of the lateral staminodes, different shades
ranging from creamy, light yellow to deep yellow. Since there is no correlation between the color of the corolla
and staminodes, the combination of these two characters makes each individual a slightly different look.
Velayudhan et al.[29] described this species as C. nilamburensis based on a collection from Nilambur, Kerala,
India. Unfortunately, the species was not validly published according to the St. Louis code [30] as the description
lacked a Latin diagnosis and a type was not designated. In addition, the publication by Velayudhan et al., [29] is of
limited circulation. Hence, the species was validly published under C. mutabilis [31].
C. haritha is closely related to C. aromaticaSalisb., but differs in the yellowish-gray, non-aromatic rhizome,
leathery, semiplicate, erect leaves, white corolla, and light yellow lip with a median dark yellow band. It also
resembled C. raktakanta but differs in having green pseudostem with light pink spots, white corolla lobes and
swollen placenta.
C.bhatii showed variations from all other species which was originally described as another genus Paracautleya in
1977 by R. M. Smith. Skornickova and Sabu [31] observed the characters were not sufficient to provide a separate
genus status, hence merged Paracautleya bhatii R. M. Smith with Curcuma bhatii on the basis of type specimen
collected by K.G. Bhat from Udupi, Karnataka, South India. The genus Paracautleya was established by Smith
[32] based on the same specimen.
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The affinity of Paracautleya congruent with Curcuma, but pointed out that Curcuma flowers are borne in cincinni
within pouches formed by adnate bracts and confirmed the close affinity of Paracautleya with Cautleya and
Roscoea mainly based on the singly borne ebracteolate flowers and leafy stems. However, the assigned generic
status of this taxon is mainly based on its stemless habit, elongated naked peduncle and ovules attached at the base
of the ovary.
The dendrogram constructed by computing the selected morphological characters of the 19 species using UPGMA
clustering produced a phylogenetic tree with three prominent groups (Fig.4). The groups diverged at a JSI of 0.37
and C. bhati showed distinctiveness from the rest of the species. The group I comprised of a distinct node for C.
aeruginosa with 0.45 JSI from the other species. C.inodora and C.raktakanta shared a common node with a JSI of
0.53 with all the other species. C.longa formed a separate node with a JSI of 0.474 justifying its amphidiploid
nature.Group IIA comprised of C. amada with 0.53 JSI from the rest of the species indicating its special
biochemical features. C.aurantiaca and C.neilgherensis shared a common node with a JSI of 0.84 with all the
other species. C.oligantha showed a JSI of 0.68 and C.coriacea with 0.65 JSI with the other species. C.aromatica
and C.mutabilis showed the same JSI as they shared the same node. Group IIB comprised of C.montana, C.
pseudomontana, C.haritha, C.karnatakensis and C. zanthorrhiza. C.montana, C. pseudomontana showed
maximum similarity among all the species studied hence can be considered as synonyms. Group IIC comprised of
C.decipiens and C.vamana which showed a JSI of 0.47. Group III comprised of C.bhatiwith 0.37 JSI, which raises
the question in the circumscription of P. bhati.
Fig.1. Inflorescence bract colour variations in C.aurantiaca.
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Fig.2. Phenotypic plasticity in the flower colors of C.inodora
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Fig-4: Dendogram of 19 Curcuma Species from India resulting from a UPGMA cluster analysis obtained
from various morphological characters
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Fig-6: Dendrogram of 19 Curcuma Species from India resulting from a UPGMA cluster analysis obtained
from 20 RAPD primers
Molecular characterization
Polymorphic bands were produced by 20 random decamer primers in PCR for all the 19 species of Curcuma
(Fig.5). The remaining 22primers produced monomorphic bands and were not considered for phylogenetic
analysis. The sequence of the RAPD primers used for the molecular characterization, total number of bands
produced by each primer, number of polymorphic bands, percentage polymorphism and genetic diversity are
presented (Table 2).
The scoring was done by running the PCR products on an agarose gel, where the bands were clearly
visible and reproducible in three replicated amplification reactions. Amplified fragments were manually scored for
presence (1) and absence (0) and the binary matrices were subjected to statistical analyses using NTSYS-pc
[Numerical Taxonomic Multivariate Analysis System version 2.1 [33]. The 20 random decamer RAPD primers
produced a total of 2226 scorable bands in all the species of Curcuma studied, out of which 1025 were
polymorphic. The percentage polymorphism ranged from a maximum of 56.7% to a minimum of 36.5%. Highest
genetic diversity was demonstrated by the OPA 14 primer (Table 2).
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Table-2: Total number of bands generated, number of polymorphic bands and percentage polymorphism
exhibited by the RAPD primers in 19Curcuma Sp.
S.No.
1.
2.
3.
4.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Primer
OPA 05
OPA 07
OPA 08
OPA 10
OPA 11
OPA 12
OPA 14
OPA 15
OPA 17
OPA 18
OPA 19
OPB09
OPB 12
OPB 16
OPC 07
OPC 08
OPC 09
OPC 10
OPC 11
OPC 12
Sequence(5’
3’)
AGGGGTCTTG
GAAACGGGTG
GTGACGTAGG
GTGATCGCAG
CAATCGCCGT
TCGGCGATAG
TCTGTGCTGG
TTCCGAACCC
GACCGCTTGT
AGGTGACCGT
CAAACGTCGG
TGGGGGACTC
CCTTGACGCA
TTTGCCCGGA
GTCCCGACGA
TGGACCGGTG
CTCACCGTCC
TGTCTGGGTG
AAAGCTGCGG
TGTCATCCCC
Total
Total
no. of
bands
97
116
116
109
97
111
134
132
133
116
80
120
137
89
117
80
76
133
122
111
2226
No. of
polymorphic
bands
46
43
48
58
47
60
76
57
59
60
41
59
50
34
44
32
40
61
54
56
1025
Polymor
phism
(%)
47.42
37.06
41.37
53.21
48.45
54.05
56.71
43.18
44.36
51.72
51.25
49.16
36.50
38.20
37.61
40
52.63
45.86
44.26
50.45
46.04
Genetic Diversity
0.0448
0.0419
0.0468
0.0565
0.0458
0.0585
0.0741
0.0556
0.0575
0.0585
0.0400
0.0575
0.0487
0.0331
0.0429
0.0312
0.0390
0.0595
0.0526
0.0546
To compute pairwise genetic similarities, Jaccard’s similarity coefficients (J ij) were calculated by using
the formula, Jij= a/(n-d) where a is the number of RAPD bands present in both i and j accessions, d is the number
of bands absent in both I and j accessions, and n is the total number of RAPD bands. The similarity matrices
were computed and the corresponding dendrogram of genetic relatedness was constructed by applying Unweighted
Pair Group Method with Arithmetic Mean (UPGMA) clustering algorithm (Fig.-6 and Table-3).
Table-3.Jaccard’ssimilarity coefficient among 19 species of Curcuma for 20 RAPD polymorphic primers
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The species were grouped by subjecting the similarity values to UPGMA clustering. Amongst the Curcuma species
the lowest genetic similarity was observed between C. aurantiaca and C. oligantha (0.184) and the highest in C.
montana and C. pseudomontana (0.4). Based on the dendrogram developed, all the 19 species were grouped into
four groups. Group IV had only one node representing Curcuma bhatii exhibiting a JSI of 0.23 with all the other
species, indicating the conversion of Paracautleyabhatii to C. bhatii is not correct and deserves a separate genus
status.Group I was formed with five species of which C. karnatakensis and C. mutabilis shared 0.37 JSI between
them. C. neilgherrensis and C.oligantha showed a distinct node with a JSI of 0.36. C. amada shared a 0.28
Jaccard’ssimilarity with the C. neilgherrensis/ C. oligantha cluster and formed a distinct node justifying its special
morphological characters and biochemical features. Group II formed of C. haritha and C. aromatica with ashared
JSI of 0.32 among them. C. aeruginosa and C. zanthorrhizashowed 0.33Jaccard’s similarity coefficient. C.
coriacea and C. longa were grouped together with a similarity coefficient of 0.24. The group III contained 7
species in which C.montana and C. pseudomontana were grouped together with maximum similarity (JSI 0.4)
between them and can be considered as synonyms. C. decipiens and C.inodora showed 0.29 JSI among them and
C. aurantiaca grouped along with C. decipiens/C.inodora cluster with 0.368 similarity. C. vamana showed 0.29
JSI with all the other species in the cluster, and form a separate node. The highest JSI was observed for C.
karnatakensis and C. mutabilis indicating that these two species have the maximum similarity revealed by the
RAPD banding pattern. The two species have diverse geographical existence, C. karnatakensis is reported from
Uttar kannad districts of Karnataka and C. mutabilis was collected from Nilambur of Kerala state. However, the
genetic similarity between these two species revealed by RAPD suggests that the genetic closeness exhibited by
the two species may not have an environmental impact as suggested by [15]. C. amada shared Jaccard’s similarity
coefficient of 0.28 with the C. neilgherrensis/C. oligantha cluster suggesting that C. amada has a divergent
character from the other members of this cluster. Morphological and biochemical features of C. amada bestow it
with a separate species status and the specific aromatic ‘mango’ flavor and secondary metabolites are characters
unique to the species justifying its specific position in cluster I A. Thus the five species which are geographically
close and taxonomically grouped at similar level showed similarities with the members of the same group. C.
amada showed distinctiveness from the other members of the group as described before [11] Cluster IB formed
with 6 species and cluster IC with 7 species. These two sub clusters showed 0.27 Jaccard’s similarity among
themselves. C. aromatica and C. haritha have a common ecological niche in the mid land and base of Western
Ghats of India. C. aromatica is a seed setting species with somatic chromosome number 2n = 42 [34] even though
C. haritha has the same somatic chromosome number this species but never sets seeds [35].The geographical niche
of the two species may also affect the RAPD profile of the species. The group formed together by C. aeruginosa
and C. zanthorrizha has 0.347 JSI, have the implication on the genetic constitution of the species. Both the species
have 2n = 63, 64 chromosomes and this ploidy level may be the basis for the existence of the two species in the
same group.
The most cultivated species C. longa is grouped along with C. coriacea and both of them showed 0.33 Jaccard’s
similarity with each other. The ecological niche of these two species is same and this may be contributing to the
identical banding pattern and similarity of the two species.
Group III comprised of 7 species which shared Jaccard’s similarity index of 0.27. C. montana and C.
pseudomontana shared equal distance among themselves. They were treated as two different species in earlier
taxonomic investigations [36]. However, they showed the maximum JSI (0.4) among the species taken for the
study and can be considered as synonyms.
C. decipiens and C. inodora showed 0.38 Jaccard’s similarity between them and form a common node, however,
the morphological character based classification illustrated the highest level of dissimilarity. C. aurantiaca
showed a JSI of 0.37 between the species is indicative of its relatedness with the other species geographically
located in the same ecological niche. Of all the species C. bhatii forms a clear node, without exhibiting many
similarities with the other species. In earlier classification based on morphological characters, this species was
treated as a separate genus, Paracautleya [32]. However, studies by Skornickova and Sabu [31] shown that
depending on the bracts which forms pouches as in Curcuma circumscribed it as Curcuma bhatii. But RAPD data
showed that C. bhatii exists as a separate entity, showing very less similarity with all the other Curcuma species,
indicating its genetic identity as a separate genus.
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Yusuf et al
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CONCLUSION
This study was aimed at establishing genetic diversity of 19 Curcuma species which shared the morphological
similarity.Though, there wasa dialectical situation of species of un-identical morphology falling in the same group
or vice versa. The study proved that information on the morphological characters alone in species delimitation has
its own limitations for the genus Curcuma. Molecular biology techniques like RAPD markers in conjunction with
conventional taxonomic tools, thus assume significance and can be used for diversity studies of this important
genus and can be used for future breeding programs.
ACKNOWLEDGMENTS
We sincerely thank Dr. Nirmal Babu, Principal Scientist, Indian Institute of Spices Research, Calicut, for
providing the facilities for the data analysis and Director, Interuniversity Centre for Plant Biotechnology,
University of Calicut for the lab facilities provided and extending his support during this work.
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