International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 2(5) pp.

119-128, May, 2011 Available online http://www.interesjournals.org/IRJPS Copyright 2011 International Research Journals

Full length Research Paper

Phylogenetic relationships of Crotalaria species based on seed protein polymorphism revealed by SDS-PAGE
L. Joelri Michael Raj1*, S.John Britto1, S.Prabhu1, and S.R.Senthilkumar2.
1

The Rapinat Herbarium and Center for Molecular Systematics, St. Josephs College, Tiruchirappalli 620 002, South India. 2 Department of Plant Biology and Plant Biotechnology, St. Josephs College, Tiruchirappalli 620 002, South India.
Accepted 13 May 2011

Seed protein profile of 12 accessions belonging to 9 species of genus Crotalaria were investigated through SDS-PAGE. Intra and inter specific relationship was estimated using Nei and leis genetic distances. A dendrogram based on UPGMA revealed the inter relationship of species within Crotalaria. Most species had distinctly different protein patterns but close association was found between C.pallida and C.laburnifolia. The Morphological variation observed between the two specimens of C. grahamiana collected from two different geographical regions showed good variations in their protein profiles also, that are enough to give it the rank of sub species of C. grahamiana. PCA had grouped C.verrucosa and C.beddomeana together where both are specifically characterized by the presence of well developed stipules indicating it as a phylogenetically important character. The present study provides useful markers for the identification of the taxa, their relationship and the delimitation of their taxonomic status both at interspecific and intraspecific levels.The phylogenetic tree obtained by DAMBE predicts sequence of evolution within the genus. Keywords: Crotalaria; Seed proteins; SDS-PAGE; UPGMA; PCA; Phylogenetic tree. INTRODUCTION Crotalaria is a species complex genus and is considered to be the third largest genus of Papilionoideae, with around 600 species distributed throughout the tropics and subtropics (Polhill 1982). They are used as a source of fibers, silage and green manure which are its agronomic traits (Cook and White, 1996; Ramos et al., 2001; Sakala et al., 2006). Crotalaria species can be used as forage for horses and cattle owing to the large amounts of water soluble gums and proteins in their seeds(Purseglove, 1981; Pandey and Srinivatsva, 1990). Their ability to fix nitrogen enables these plants to be used for enhancing soil fertility (Samba et al., 2002). Moreover these leguminous plants develop a high resistance to main species of root knot nematodes which cause severe
*Corresponding author Email: joelriraj@gmail.com

damage to crops (Antonio and Neumaier, 1986). Crotalaria species are used as food plants for larvae of Lepidoptera species (Eisner et al., 2000). The toxic alkaloids produced by some members of this genus are known to be incorporated by these larvae and used to secure their defense from predators (Bernays et al., 2003). Alkaloid monocrotaline, a pyrrolizidine alkaloid is the main toxic principle of genus Crotalaria which is used to induce experimental pulmonary hypertension in laboratory animals (Werchan et al., 1989). Thus there arises a great need to unravel the phylogenetic relationship of such an agronomic ethnomedicinal and horticulturally important genus. Moreover morphological markers used in the past are insufficient for their correct and proper identification. Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) technology is widely used to study plant taxonomy,

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affinities and genetic diversity. SDS-PAGE of total seed proteins has found wide application in resolving systematic relationships and for inter and intra specific studies (Karihaloo et al., 2002). When the plants are highly variable and contain large number of hybrids, identification based on morphological characters is quite difficult. SDS-PAGE have extensively been used to study genome structure in various crops of agronomic importance like wheat, maize, soybean, sunflower, etc (Weber et al., 2005, Zhu et al., 2005). Seed protein profile obtained by electrophoresis has been successfully used to resolve the taxonomic problems of Solanum and Capsicum species (Edmonds and Glidewell ,1977; Zubaida et al., 2006). SDS-PAGE is most widely used due to its validity and simplicity for describing genetic structures of group of plants (Sher et al., 2010) rather relying on DNA based markers which are expensive and time consuming (Wang et al., 2006) . However the potentials of DNA markers to resolve inter and intraspecific variations is well known and hence could be used to study variations that could not be revealed by protein markers. Moreover proteins are expressed form of genome which can be biomarkers for identification of botanic drugs (Muhammad et al, 2010). The mature seed provides a stable and convenient system for biochemical analysis to establish relationship in parents and hybrids (Gangwar and Bajpa, 2006). The genetic similarity information could make decisions regarding the choice for selecting superior genotypes for improvement or to be used as parents for the development of future cultivars through hybridization. Legumes have played a primary role in the search for vegetable sources of proteins owing to the high protein content of the seed (Valizadeh, 2001). The comparative study of protein variation in these species is not well demonstrated. Hence, it is desirable to increase our knowledge of the genetic resemblance among the most important genus Crotalaria by employing variations in seed storage proteins, which are their main common characteristics. The present study was initiated to find genetic similarity on the basis of seed protein profile and its relationship with agronomic traits in genus Crotalaria. It was also used to study the Intra specific variations to identify the elite variety. For example wild species of C.grahamiana RHT56077 has specific features such as drought tolerance, high seed protein, high pod set and viable large seeds whereas C.grahamiana RHT56085 lacks these features. Since genetic differences are reflected in shifts of seed protein patterns, the present study was under taken on biochemical characterization of seed storage proteins in order to see variation for seed protein in the segregating populations due to environmental pressures. Boatwright et al (2008) has did a systematic study of generic

relationships in the tribe Crotalarieae based on ITS and rbcL sequences but the inter and intra specific relations among one of the genus Crotalaria in the tribe has not specifically dealt with. Thus the present investigation aims to determine the taxonomic relationship by using SDS-PAGE of seed proteins of 12 taxa at interspecific and intraspecific levels. MATERIALS AND METHODS Collection of samples Two accessions of C.verrucosa, C.retusa and C.grahamiana from widely separated geographical regions were chosen for study to measure the range of divergence that had occurred in the protein banding patterns within the species.RHT (Rapinat Herbarium, Tiruchirappalli) 56077, C.grahamiana and RHT56060, C.beddomeana were collected from the Western Ghats of,SouthIndia at high altitudes. Similarly C. pallida RHT56081 and RHT56083 were collected from foothills of kollihills and Puliyancholai hills respectively. The other accessions were from plains of Central Tamil Nadu south India (Table 1). The voucher specimens were identified at the Rapinat Herbarium and Center for Molecular Systematics, St. Josephs College Tiruchirappalli, Tamil Nadu, India. The morphological identification and comparison was done based on The Flora of the Tamilnadu Carnatic (Matthew, 1981) and The Flora of the Palni hills (Matthew, 1999). Mature seeds only were collected and stored at room temperature for protein analysis. Sample extraction Seed proteins were extracted by modified procedure of Miller et al (1972). Total seed proteins were extracted from 2.0 g of seed flour using 10ml of extraction buffer that contained 125mM Tris-HCl pH 7.2, 0.2% SDS, 0.5M NaCl, and 1% Mercaptoethanol. Seed flour was thoroughly mixed with buffer by vortexing. The extracted protein was separated by centrifuging the sample at the rate of 15000rpm for 10 minutes. The supernatant was the protein source. Protein estimation and sample preparation Total soluble protein in extracted samples was estimated using Perkin Elmer spectrum lambda35 UV spectrophotometer at 280nm. The protein solution was

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Table 1. List of species within the Genus Crotalaria taken for study
S.No 1 Accession No. RHT56077 Botanical Name (Leaf Morphology) C.grahamiana ( 5- 7 foliate) Latitude 10.27083 N Longitude 77.56944 E Location and altitude Kodaikanal Ghat road, 1835m.

2 3

RHT56060 RHT56075

C. beddomeana ( Unifoliate) C. retusa ( Unifoliate)

10.2372 N 10.85444 N

77.4714 E 78.66417 E

Kodaikanal S.H college, 1327m. Kambarasampettai, 68m.

4 5

RHT56078 RHT56079

C. verrucosa( Unifoliate) C. juncea( Unifoliate)

10.835 N 10.85545 N

78.73222 E 78.66518 E

Keelmulaikudi, 71m. Kambarasampettai, 72m.

RHT56080

C.paniculata ( Unifoliate)

10.85570 N

78.66558 E

Kambarasampettai, 73m.

7 8

RHT56081 RHT56082

C. pallida ( Trifoliate) C. laburnifolia ( Trifoliate)

10.75354 N 10.85345 N

78.43291 E 78.66398 E

Kolli hills, 754m. Kambarasampettai, 73m.

9 10 11 12

RHT56083 RHT56084 RHT56076 RHT56085

C. pallida ( Trifoliate) C. retusa ( Unifoliate) C. verrucosa ( Unifoliate) C.grahamiana ( 5- 7 foliate)

10.73543 N 10.83543 N 10.85450 N 10.85491 N

78.77219 E 78.73291 E 78.66554 E 78.66468 E

Puliancholai hills, 350m. Cauvery river banks 65m. Kambarasampettai, 73m. Kambarasampettai, 73m.

diluted two-fold with 125mM Tris buffer pH 7.2 so that the concentration is well within the accurate range of the instrument. Accuracy is greatest, approximately at 0.3 absorbance. (Aitken and learmonth, 2002) This gives an accurate estimate of the protein content by removing the contribution to absorbance by nucleotides at 280 nm, by measuring the A-260 which is largely owing to the latter (Aitken and learmonth, 2002). Protein (mg/mL) = 1.55 A280 -0.76 A260 All the samples were further diluted according to the yield and brought to 1mg/ml concentration. The samples were then mixed with equal volumes of sample buffer (Jha and Ohri, 2002) and equal amount 50l (25g) of protein was loaded in each well. Protein profiling using SDS PAGE Electrophoresis was carried out in a discontinuous SDSPAGE system of Laemmli (1970) using 12% acrylamide gel. A Protein Molecular Weight Marker Medium range (Genei, Bangalore) was also run along with protein

samples as standard (M.wt 97.4 - 14.3 KDa). SDS PAGE was performed at constant voltage (100 V). Gels were stained using Coomassie brilliant blue dye R-250 (Hames and Rickwood, 1990) and documented by UVidoc Gel Documentation System. The banding patterns were analysed in GEL DOC software (UVItec, UK). The experiment was repeated thrice to check the consistency of the data. Band scoring and analysis Only the unambiguous bands were coded for presence / absence (1/0) and analysed in POPGENE ver.3.5 software. The UPGMA dendrogram was generated based on the Nei and leis genetic distances (Nei, 1978). The tree was viewed in TREE VIEW software and later imported in MEGA ver. 4.0 for branch swapping and modification. The data was also analysed using MVSP 3.1 (Multi Variate Statstical Package) (Kovah, 2005) for calculating Jaccards similarity coefficient, Squared Euclidean distances and PCA (Principal Component

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Analysis) was done based on the Euclidean distances. The distance matrix was also subjected to analysis in DAMBE 5.1.2. (Data Analysis for Molecular Biology and Evolution) to trace the phylogenetic relationship (Xia, 2001). RESULTS AND DISCUSSION Protein markers for Crotalaria The SDS PAGE analysis had generated 32 protein markers for these species (Figure 1, Table 2).These banding patterns clearly segregated the simple leafed species from compound leafed species with one exception where C.grahamiana, with compound leaves (RHT56077, RHT56085) shared specific bands at 124kDa, 73kda, 63kDa, 60kDa and 42kDa with the simple leafed ones (Table 2). Some specific markers at 120kDa, 92kDa and 46kDa were identified for the trifoliate species C.pallida (RHT56081, RHT56083) and C.laburnifolia (RHT56082). Another important marker was observed at 30kDa common to all the species of Crotalaria studied. Except for C.retusa (RHT56075, RHT56084) the intensity of this band was observed to be prominent for all the species studied (Figure 1). These are suspected as lectin proteins that has a molecular weight of 30kDa. The results obtained for the lectins of C. juncea (Ersson et al., 1973, Ersson 1977), C. striata (Khang et al., 1990), and C. pallida (Rego et al., 2002) shows that most Leguminosae lectins consist of four subunits of 30 kDa each, which are held together by noncovalent interactions to yield a tetramer of 110 kDa (Sharon and Lis, 1990). The lectin shows a single band of 30 kDa in SDS-PAGE (Luzia et al., 2004). Moreover previous studies indicated that there are many unique features identified for each lectin proteins especially lectins obtained for C.paulina seeds. These are observed to have a peculiar feature capable of agglutinating erythrocytes in the absence of divalent ions ( Luzia et al., 2004) unlike the lectins obtained from C. striata (Khang et al., 1990) and C. pallida (Rego et al., 2002). Our work gives a scope to analyse the variations in the activity of lectins from each species that can be specifically identified by the protein markers identified in this study (Table 2) Genetic Similarity The Jaccards similarity coefficient ranged between 0.063 -1.00 (Table 3). The minimum similarity value of 0.063 was observed between C.retusa (RHT56075) and

C.laburnifolia (RHT56082) and also between C.retusa (RHT56084) and C.pallida (RHT56081, RHT56082). The maximum similarity value was 1.00 showing no allelic variations between the two accessions of C.pallida collected from two different geographical regions (Table 1). Both the species did not show much variation in their morphology too. This shows the genetic stability of seed storage proteins that could be used to derive a meaningful phylogeny. But on the contrast similarity value of the two accessions of C.grahamiana was 0.75. But this accession had many morphologically variant features which was well depicted by the protein banding patterns showing allelic variations at 143kDa, 99kDa and 60kDa (Table 2). RHT56085 also has some morphologically dominant characters like large seeds and fleshy leaves with silky pubescent hairs. This may be due to the difference in geographical regions that these two accessions grow. Moreover the intensity of the shared bands of RHT56077 with RHT56085 is much higher indicating richness of the seed proteins in RHT56085 which is considered to be an elite variety. Another fact about this species is that its occurrence is reported only in the higher altitudes of palni hills (e.g. C.grahamiana RHT56077. But RHT56085 was found in the plains of Kambarasampettai which is the first report on its occurrence at the hay fields (Table 1). These differences are enough to give it the rank of sub species of C.grahamiana. Moreover specific markers for identification of C.grahamiana was also identified at 90kDa and 40 KDa which was common to both these accessions indicating that it was the variant of the elite one in higher altitudes which could have been introduced by native farmers a decade back to improve the fertility of their lands. So it is emphasized that more work should be done to identify the agronomic potentials of the elite variety of C.grahamiana (RHT56077). Baijukya et al. (2006) reports that the percentage fertiliser equivalency (%FE) of legumes ranged between 25 and 59% with higher values recorded with C. grahamiana. Cluster analysis The cluster analysis of the taxa based on Nei and leis genetic distances was represented by an UPGMA dendrogram in Figure 2. The protein patterns divided the twelve species of Crotalaria into two broad clusters. All the species in the cluster II were trifoliate ones. The genetic relatedness of unifoliate ones like C.juncea with C.retusa was much closer than that with trifoliate ones like C.pallida and C.laburnifolia is precisely depicted by the data generated by protein markers.Our studies are in congruence with the study on four species of Crotalaria based on EST SSR markers (Wang et al ., 2006). Thus

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Table 2. Molecular weight values for various bands electrophoresed in seed proteins of Crotalaria species.

143 kDa

139 kDa

136 kDa

134 kDa

124 kDa

120 kDa

114 kDa

99 kDa

92 kDa

90 kDa

86 kDa

81 kDa

76 kDa

73 kDa

68 kDa

66 kDa

63 kDa

60 kDa

56 kDa

53 kDa

50 kDa

48 kDa

46 kDa

42 kDa

38 kDa

37 kDa

35 kDa

34 kDa

33 kDa

31 kDa 0 0 1 1 1 1 0 0 0 1 1 0

Acc.No. RHT56077 RHT56060 RHT56075 RHT56078 RHT56079 RHT56080 RHT56081 RHT56082 RHT56083 RHT56084 RHT56076 RHT56085

1 0 0 0 0 0 0 0 0 0 0 0

0 0 0 1 0 0 0 0 0 0 1 0

0 1 0 0 0 0 0 0 0 0 0 0

0 1 0 0 0 0 0 0 0 0 0 0

1 0 0 1 0 0 0 0 0 0 1 1

0 0 0 0 1 0 1 1 1 0 0 0

0 1 0 1 0 0 0 0 0 0 1 0

0 1 0 1 1 0 1 0 1 0 1 1

0 0 0 0 0 0 1 1 1 0 0 0

1 0 0 0 0 0 0 0 0 0 0 1

0 0 0 0 1 0 0 0 0 0 0 0

0 0 0 0 0 0 0 1 0 0 0 0

1 0 1 1 1 0 1 0 1 1 1 1

1 1 1 1 1 1 0 0 0 1 1 1

1 0 0 0 1 0 1 1 1 0 1 1

0 0 0 0 1 1 1 1 1 0 0 0

1 1 1 1 1 0 0 0 0 1 1 1

1 1 0 1 0 0 0 0 0 0 1 0

0 1 0 1 1 0 1 1 1 0 1 0

0 0 0 0 0 1 1 1 1 0 0 0

1 1 0 1 1 0 1 1 1 0 1 1

0 1 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 1 1 1 1 0 0 0

1 0 1 0 1 0 0 0 0 1 0 1

1 0 0 0 0 0 0 0 0 0 0 1

0 1 0 1 0 0 0 0 0 0 1 0

0 0 0 0 0 0 0 1 0 0 0 0

0 1 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 1 0 1 0 0 0

0 0 1 0 1 1 1 1 1 0 0 0

1 1 1 1 1 1 1 1 1 1 1 1

protein markers are simple and effective in revealing the inter specific relationships.The interesting fact revealed by protein markers is that it had clustered C.grahamiana; a 5-7 foliate species along with the simple leafed species whereas the classical taxonomic key clusters C.grahamiana with the compound leafed species which is not supported by the protein data. Since seed storage proteins are the products of gene expression with genetic stability and not been affected much by environment, the protein banding patterns poses a much validated phylogenetic relationship than relying on few morphological characters (Ghafoor et al., 2002; Panigrahi et al., 2007). Within the Cluster I, C.grahamiana is clustered within Cluster IA along

with C.juncea, C.retusa and C.paniculata. So C.grahamiana is expected to have evolved from any one of these these simple leafed species and also is expected to share many agronomic traits with C. juncea which is one of much exploited species agronomically as a potential green manure worldwide, particularly in the Asian countries. C. grahamiana is already exploited as a green manure in many African countries (Tumuhairwe et al., 2007; Niang et al., 2002). Phylogenetic tree obtained by DAMBE analysis clearly predicts the sequence of evolution in morphology that could have occurred within this genus. The relationships revealed by protein markers could be well explained by the presence or absence of stipules which is declared to be an

evolutionarily important character in genus Crotalaria where it had clustered the prominent stipulated species C.verrucosa and C.beddomeana together. The morphological features are added to the tree for better understanding of phylogenetic relationships. (Figure 3). PCA analysis The Principal component analysis (PCA) of the protein data represented as Figure 4, had also grouped C verrucosa and C.beddomeana in the same axis. This result is in congruence with the classical taxonomic key, where both these

30 kDa

40 kDa

Mol.wt.

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Figure 1. Seed protein profiles of 12 Crotalaria species showing inter and intra specific relationships. M= Marker (medium range). Numbers 1-12 as in Table - 1.The 30kDa marker which was observed common to all the 12 species is indicated below the white line

0.000 8.495 24.408 8.495 11.362 4.567 3.824 6.845 9.775 15.929 1.587 1.587 0.000

RHT56081 C.pallida RHT56083 C.pallida RHT56082 C.laburnifolia RHT56080 C.paniculata RHT56075 C.retusa RHT56084 C.retusa
Cluster I A Cluster II

RHT56079 C.juncea
4.922

RHT56077 C.grahamiana RHT56085 C.grahamiana RHT56078 C.verrucosa RHT56076 C.verrucosa RHT56060 C.beddomeana
Cluster I B

Cluster I

14.832 6.304 13.861

4.922 1.587 1.587

11.151

15.448

Figure 2. Dendrogram for Protein markers based on Nei's (1978) Genetic distance: Method = UPGMA . Modified from NEIGHBOR procedure of PHYLIP Version 3.5 Tree Caption. The tree with sum of branch lengths = 164.4436 is shown. The branch length in which the associated taxa are clustered together in the bootstrap test (1000) replicates are shown next to the branches. The tree is drawn to scale with branch lengths in same units as those of evolutionary distances used to infer the phylogenetic tree.

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Table 3. Similarity matrix based on Jaccards coefficient.

RHT560 77 (C.graha miana) 1 0.2 0.308 0.333 0.333 0.063 0.15 0.1 0.15 0.333 0.389 0.75 1 0.118 0.529 0.238 0.056 0.136 0.091 0.136 0.125 0.5 0.211 1 0.267 0.462 0.333 0.125 0.063 0.125 0.833 0.25 0.333 1 0.368 0.118 0.19 0.091 0.19 0.286 0.929 0.353 1 0.267 0.471 0.333 0.471 0.385 0.421 0.438 1 0.286 0.308 0.286 0.222 0.111 0.067 1 0.643 1 0.063 0.238 0.222 1 0.643 0.05 0.136 0.105 1 0.063 0.238 0.222 1 0.267 0.364 1 0.412 1 RHT560 60 (C.bedd omeana) RHT560 75 (C.retus a) RHT560 78 (C.verru cosa) RHT560 79 (C.junce a) RHT560 80 (C.panic ulata) RHT560 81 (C.pallid a) RHT560 82 (C.labur nifolia) RHT560 83 (C.pallid a) RHT560 84 (C.retus a) RHT560 76 (C.verru cosa) RHT560 85 (C.graha miana)

S.No. RHT56077 (C.grahamiana) RHT56060 (C.beddomeana) RHT56075 (C.retusa) RHT56078 (C.verrucosa) RHT56079 (C.juncea) RHT56080 (C.paniculata) RHT56081 (C.pallida) RHT56082 (C.laburnifolia) RHT56083 (C.pallida)( RHT56084 (C.retusa) RHT56076 (C.verrucosa) RHT56085 C.grahamiana)

species also share some specific morphological features such as presence of angular branchlets, prominent and foliaceous, semi-lunate stipules. (Mathew, 1999). These two species shares many protein markers at 114, 99, 73, 63, 60, 56, 50 and 38kDa. The 38kDa marker was very specific to these two species and were absent in other species of Crotalaria studied. Two markers at 136 and 134 kDa were specific in identifying

C.beddomeana exclusively from other species. Similarity values of C.beddomeana with other species were also very low indicating it to be a unique species. The PCA has its advantage in delimiting some in precise manner. For example, it had grouped C.juncea and C.paniculata together which gives some scope to test the agronomic potentials of C.paniculata a hitherto under utilized species. PCA has also pictured the

close association of C.pallida with C.laburnifolia, where the latter is expected to share similar chemical profiles to the former which is much reported for its specific pyrrolizidine alkaloids like retrorsine and a senecionine associated with hepatotoxicity (Artz and mount, 1999).

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RHT56079 C.juncea

Rooted ( stipules 0)

RHT56082 C.laburnifolia RHT56081 C.pallida RHT56083 C.pallida RHT56080 C. paniculata RHT56075 C.retusa
Stipules linear subulate Stipules linear subulate Trifoliate species ( stipules 0)

RHT56084 C.retusa RHT56077 C.grahamiana

5- 7 foliate species ( linear stipules)

RHT56085 C.grahamiana RHT56078 C.verrucosa RHT56076 C.verrucosa RHT56060 C beddomeana

Presence of foliaceous semi-lunate Stipules

0.05

Figure 3. Phylogenetic reconstruction with DAMBE 5.1.2 using Euclidean distance matrix. Tree caption: Phylogenetic tree from a user-supplied distance matrix, based on the FastME algorithm (Desper and Gascuel 2002, 2004), with branch evaluation by the balanced method and with the initial tree generated by the GME method. The final tree results from evaluating candidate trees generated by extensive NNI (nearest neighbor interchange). The tree length (sum of branch lengths) is 1.65201. The tree is rooted by outgroup RHT56079. Phylogenetic reconstruction is done with DAMBE 5.1.2(Xia 2001; Xia and Xie 2001).

Figure 4. Principle Component analysis (PCA) using Euclidean distances for 12 accessions based on 32 protein

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CONCLUSION SDS-PAGE was found effective for phylogenetic studies in Crotalaria species. It had proved its advantage over morphological identification and had also detected specific proteins of which lectins are tools of medical and biological research which can be further analysed by 2D gel electrophoresis. Although this study added new findings to the literature, it is somewhat limited to the known species and subspecies distributed in the central Tamilnadu. Moreover the overall similarity values seemed to be very low showing wide range of divergence between the species (Table 3). A comprehensive study covering a few more economically important species of Crotalaria from the Western Ghats seems to be necessary to construct a more satisfactory classification and interpretation of evolutionary relationships. The intraspecific variations between the two accessions of C.pallida could be resolved only by DNA based techniques like RAPD and ISSR markers. Sequence analysis of chloroplast genes could obtain a much more resolved phylogeny for genus Crotalaria.
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