The genus Tephrosia is a pantropical taxa with about 400
species (Geesink, 1981) distributed chiefly in Asia, Africa, Australia
and America. Tephrosia belongs to tribe Millettieae (formerly Tephrosieae),
of the family Fabaceae (Leguminosae) and has earlier been classified by
many taxonomists either into sections or subgenera, based mainly on the
morphological traits. De Candolle (1825) classified Tephrosia into
four sections namely Mundulea, Brissonia, Craccoides and Reineria.
Of these only Mundulea and Reineria were represented in
India. Later Bentham (1865) and Baker (1876) have classified the genus
into three subgenera as Macronyx, which includes T. tenuis,Brissonia
that includes T. candida and Reineria, which includes rest
of the species of Tephrosia.
Wood (1949) categorized the new world species of Tephrosia into
two groups, one with a glabrous style and the other with a pubescent style.
Gillett (1971) adopted this classification in African species of Tephrosia.
Subsequently Brummitt (1981) divided Tephrosia into two subgenera,
subg. Tephrosia with a glabrous style (according to which T.
hamiltonii, T. pumila, T. purpurea, T. spinosa, T. strigosa, T. villosa
etc were included) and subg. Barbistyla with a trichiferous
style (which includes T. candida, T. maxima, T. pulcherrima, T. tinctoria
etc.). The above classification was criticized since some of the taxa
(T. pumila,T. maxima etc.) showed both glabrous and trichiferous
The general systematic framework of the genus Tephrosia has been
focused primarily from morphological data, which is adequate and does
probably reflect grouping of a phyletic nature. But there are species,
whose relationships are difficult to judge, even after comprehensive morphological
study. Phytochemical studies especially flavonoids might prove critical
for such taximetric problems, provided the biosynthetic pathways of such
phytoconstituents is known. Therefore, the data in themselves might be
particularly impressive in taxonomic point of view by further understanding
of the genus through genetic characterization using molecular markers.
As such biochemical data and morphological data pooled together with molecular
data would be helpful in sorting out the interrelationships between the
various species of Tephrosia distributed in a particular geographical
area. Despite, years of extensive research on the genus Tephrosia,
very little is known about its phylogeny. Raina et al. (1985) studied
the species variation cytogenetically (based on G+C Content) in eight
species of Tephrosia. Untill to-date, no work using molecular data
(DNA fingerprinting technique) has been carried out in evaluating the
infrageneric relationships among the various species of the genus Tephrosia,
the only exception being the usage of the molecular data, while evaluating
the tribe Millettieae (Acharya et al., 2004).
For a better understanding of the systematic relationships within the
genus Tephrosia, RAPD markers were used to study the variation
among twelve taxa distributed in the state Andhra Pradesh, India. Random
amplified polymorphic DNA dubbed RAPD, has become a powerful tool for
finger printing a variety of organisms; of bacteria, plants, animals and
humans (Williams et al., 1990). RAPD`s arise by PCR with short
oligonucleotides of arbitrary sequences that prime DNA synthesis from
genomic sites, which they fortuitously match or almost match. The high-density
genetic maps constructed through RAPD`s have closely spaced DNA markers,
which are very useful for genome analysis (Williams et al., 1993).
RAPD analysis in particular has found widespread application in systematics
since a large number of markers can be quickly generated and scored (Rieseberg
and Ellstrand, 1993). They have been successfully applied to assess genomic
variability in plant species like Fragaria spp. (Thongthieng and
Prasartporn, 2003), Orobanche spp. (Roman et al., 2003),
Leucaena spp. (Hawkins and Harris, 1998), Oxytropis spp.
(Janet Jorgensen et al., 2003), Afgekia spp. (Prathepha
and Baimai, 2003) etc.
The RAPD fingerprinting technique using decanucleotide primers has presently
been used to reevaluate the systematic status of the genus Tephrosia
Pers, which represents one of the best examples of the complexities, where
the species definitions and relationships within the genus were unclear.
Further to analyze the affinities and phylogenetic relationships of the
various species of the genus Tephrosia and also to trace the progenitor-
Monophyletic/polyphyletic origin of the species.
MATERIALS AND METHODS
Twelve species of the genus Tephrosia have been collected from
various regions of Andhra Pradesh State,India and two species, Crotalaria
verrucosa (Fabaceae), Sorghum bicolor (Gramineae) were chosen
as out groups (Table 1). All these specimens (Fig.
1) were collected and identified with the help of recent floras (Gamble
and Fischer, 1918; Pullaiah and Chennaiah, 1997) and herbarium specimens
available in the Department of Botany, Andhra University. Further all
the above plant species were authenticated by Prof. T. Pulliah, Taxonomist,
Department of Botany, Sri Krishnadevaraya University (SKU), Anantapur,
India. All the voucher specimens were deposited in the Herbarium, Depatment
of Botany, Andhra University, Visakhapatnam, India in April 2005.
DNA Extraction, Amplification and Electrophoresis
DNA was extracted from fresh/dried leaves of the various species of Tephrosia
as described in Reineke et al. (1998) with little modifications.
DNA amplification was done using decamer (10 nucleotide length) with a G+C content
of above 50% as that they form strong hybrids with target DNA and to withstand
72°C of polymerase reaction. Twenty OPC (Operon Biotechnologies GmbH, Germany)
primers were used.
||The various Tephrosia species collected from different regions
of Andhra Pradesh, India
||Plant species collected from the different areas of Andhra
Pradesh authenticated and voucher specimens were deposited in the Herbarium,
Department of Botany, Andhra University, Visakhapatnam
||Primers code, sequence, G+C content, type and number of bands
amplified in the RAPD analysis of the genus Tephrosia
The nucleotide sequence of these primers is shown in the Table
2. Of these twenty primers, six primers were chosen that gave a reasonable
number of polymorphic bands, which were scorable and consistent among the duplicates
of reactions. Control reaction without genomic DNA was run for all these primer
reactions. Duplicate reactions were run with DNA extracted from independent
DNA extractions for each isolate.
The RAPD analysis was performed as per the standard methods of Williams
et al. (1990). Each amplification reaction mixture of 25μL
consisted of 10 μL of DNA (20 ng), 2.5 μL of 10X assay buffer
(100 mM Tris-HCl, pH 8.3, 0.5 M KCl and 0.01% gelatin, 1.5 mM MgCl2),
0.5 μL of DNTP mix, 1 μL of primer (20 pm) and 1 μL of
1 U Taq DNA polymerase (Bangalore Genei Pvt.Ltd., Bangalore, India) and
10 μL of sterile double distilled water. Amplification reactions
were performed using a thermocycler-Eppendorf master cycler gradient (Eppendorf
Netheler-Hinz, GMBH, Hamburg, Germany), programmed as: initial denaturation
for 5 min at 95°C, followed by 45 cycles of denaturation for 1 min
at 95°C, annealing for 1 min at 37°C and extention for 2 min at
72°C and a final extension for 7 min at 72°C using the fastest
available temperature transitions. A control reaction without DNA was
setup for every set of reactions.
Amplified DNA fragments along with a size marker Lambda DNA/Hind III
digest, (Bangalore Genei Pvt.Ltd., Bangalore, India) for band size comparison,
were resolved electrophoretically on 1.5% Agarose gel stained with ethidium
bromide (10 mg mL-1) at 50 V/cm for 5 h using 1X TAE buffer
and photographed under UV illumination with a Kodak DC 120 digital camera
mounted on a dark hood. The photographs were transferred to a PC using
Kodak digital transfer software. The gels were scored conservatively.
The reproducibility of all initially scored bands were rechecked by comparing
banding patterns of individual plants that were rerun to check the cross
comparability among gels.
The banding patterns obtained from the RAPD fingerprinting were analyzed
to estimate the genetic relationship among the twelve species of the genus
Tephrosia. A 0/1 data matrix was constructed for each primer by
scoring each species for the presence (1) or absence (0) of each band.
To minimize errors RAPD fingerprints were evaluated four times with time
intervals by different individuals. The less intense high molecular weight
minor bands (which may be a result of primer annealing to template regions
with fortuitous matching) were marked separately.
To account for the dominant nature of RAPD markers, allele frequencies
were estimated using two different methods (Krauss, 2000), a Bayesian
method with uniform prior distribution of allele frequencies (Zhivotovsky,
1999) and the square root method of the null homozygote frequency (Nei
and Li, 1979). Calculations were performed using the AFLP-SURV 1.0 software
(Vekemans et al., 2002). To examine relationships among species
of Tephrosia, a cluster analysis was conducted based on the similarity
matrix between each pair of individuals in the dataset using the Unweighted
Pair Group Method with Arithmetic average UPGMA (Nei and Li, 1979) with
the help of PHYLIP software ver. 3.65 (Felsenstein, 1989). The tree thus
generated was explored using the programme Tree Explorer of MEGA version
3.0 (Kumar et al., 2004).
A dendrogram (Fig. 3) was constructed from the
similarity values obtained in pair wise comparisons among the species
using data, pooled from all the six primers. Cluster analysis revealed
rooted groups with a similarity of 25% among the various species of the
genus Tephrosia. Out of the twenty primers tested, six primers
gave reproducible, clear polymorphic bands (Fig. 2).
A total of 172 Polymorphic bands were identified with an average of 27
bands per primer. The size of the amplified fragments ranged from 560
kb to 200 bp (Table 2). Less intense bands which were
considered as minor bands, the bands which were shared by not more than
four isolates were considered as rare and those bands which were represented
alone were considered as unique. The dendrogram constructed excluding
minor bands did not differ much from the cluster diagram that was constructed,
including both the major and minor bands (Fig. 3). The
dendrogram was constructed considering all bands which resulted into five
groups with an overall similarity of just 24% among all the sixteen OTU`s
(Operational Taxonomic Units). Group II was the largest with six OTU`s,
which was further grouped into IIA and IIB with a similarity of 35%. The
outgroups Sorghum bicolor and Crotalaria verrucosa were
clustered with Tephrosia strigosa with a similarity of 5%.
Group 1: It includes two OTU`s T. purpurea and T. spinosa
with a similarity of 20%.
Group 2: It is the largest of all with a similarity of 35% including
six OTU`s which were further grouped into two sub-groups designated as
Group 2A and Group 2B. Group 2A includes T. pumila, T. villosa and
T. hirta and Group IIB includes T. pulcherima, T. tinctoria
and T. maxima.
Group 3: It includes two OTU`s, T. procumbens and T.
calophyla with a 25% similarity.
Group 4: This group includes only one species T. hamiltonii,
which showed much diversity with the rest of the species.
||Ethidium bromide stained agarose gels showing RAPD-PCR Banding
patterns of the genomic DNA of various species of Tephrosia with
primers (a) OPC-02 and (b) OPC-08 (Operon Technologies Inc. Almeda, CA)
De Candolle (1825) classified Tephrosia into four sections,
Mundulea Brissonia, Craccoides and Reineria. Of these only
two sections Mundulea and Reineria were represented in India.
In the present work, of the 12 taxa studied, seven were assignable to
the De Candolle`s system (1825), while others were new inclusions. T.
spinosa, T. tinctoria, T. maxima, T. pumila, T. villosa, T. hirta and
T. purpurea that belongs to the Reineria section of De Candolle`s
classification, were also justified with the present RAPD fingerprinting
Subsequently, Brummitt (1981) categorized Tephrosia into two subgenera,
subgenus Tephrosia with a glabrous style and subgenus Barbistyla with
a trichiferous style. According to this classification, T. hamiltonii, T.
pumila, T. purpurea, T. spinosa, T. strigosa and T. villosa of the
present study were included in the sub-genus Tephrosia, while T. tinctoria,
T. maxima and T. pulcherrima belong to the subgenus Barbistyla.
|| Dendrogram showing the genotypic relationship among the species
of the genus Tephrosia
The present study has much deviated from the above classification, since only
T. pumila and T. villosa were grouped together, but were clustered
along with T. tinctoria, T. maxima, T. pulcherrima as a major Group 2
and more over the species T. maxima and T. pulcherrima showed
the presence of both glabrous and trichiferous style. Therefore, the results
obtained through RAPD fingerprinting are quiet contrasting with Brummitt`s classification.
Recently Subba Rao and Shanmukha Rao (1993, 1995) classified the genus
Tephrosia on the basis of differential distribution of the non-protein
amino acids and numerical taxonomy (based on epidermis, leaf architecture,
seed morphology and chemotaxonomy) covering 18 taxa distributed in the
South Indian regions. Of the 18 species, 9 species were included in our
present study and the rest of the two species T. procumbens and
T. hirta are of new inclusion. According to the numerical taxonomy,
the genus Tephrosia was clustered into 11 groups that showed no
clear demarcation and indication of interrelationship among the various
species. The RAPD analysis supported the above classification in the separation
of T. purpurea from T. hamiltonii and inclusion of T.
maxima and T. pumila in the same group.
Relation of the RAPD Fingerprints to the Morphological Traits
Tephrosia strigosa which was earlier treated separately (in
macronyx) by Bentham (1865) and Baker (1876) on the basis of its
simple leaf, in contrast to the typical compound leaves of Tephrosia,
was separated along with the out groups Sorghum (Gramineae) and
Crotalaria (Fabaceae) in the dendrogram (Fig. 3)
justifying its diversification from the rest of the taxa. Thus the RAPD
fingerprinting relationships co-related with the morphological classification.
Group 1: It includes T. spinosa and T. purpurea,
the present clustering was in agreement with morphological traits, since
both the species are mostly morphologically similar except for the presence
of stipulous spines, flower colour and pod characters of T. spinosa.
Group 2A: This cluster includes T. pumila, T. villosa and T.
hirta. The species T. villosa and T. hirta which were
treated earlier as synonyms in different floras (Gamble and Fischer, 1918;
Pullaiah and Chenniah, 1997) has showed only 75% similarity.
However, further evidence is required, since RAPD fingerprinting alone
cannot justify the treatment of both the species as distinct., T. villosa
and T. pumila clustered in RAPD dendrogram was in agreement
with morphological traits since both of them share many morphological
characters (leaf-let size, flower and pod characters).
Group 2B: It includes T. pulcherrima, T. tinctoria and
T. maxima. RAPD dendrogram is slightly in contrast with morphological
traits, since T. tinctoria and T. maxima, which were clustered
together showed differences in leaflet size and flower colour and the
T. tinctoria showed more morphological similarity with T. pulcherrima
when compared to T. maxima.
Group 3: It includes T. procumbens and T. calophylla,
both of which show quiet contrasting morphological characters. T.
procumbens is a creeper with compound leaves and white flowers. T.
calophylla is endemic to South India and was included in the sub-genus
Reineria by Baker (1876). This taxon is found to possess a number
of unique characters namely woody herb, simple leaves, tuberous root,
brick red flowers etc. Therefore the RAPD fingerprinting is not in agreement
with morphological traits in the above grouping.
It includes only a single species T. hamiltonii. The RAPD dendrogram
in this case is in agreement with numerical and chemotaxonomic evidence (Subba
Rao and Shanmukha Rao, 1990, 1995) where T. hamiltonii is shown to be
much diverged from T. purpurea, though they morphologically reassembled
each other. This was further strengthened by the isolation of a new flavonoid
flemichapparin-B from T. hamiltonii, that which was not reported in T.
purpurea (Rajani and Sarma, 1988). Therefore the present evidence from the
RAPD cluster analysis also supports the isolation of T. hamiltonii not
only from T. purpurea but also from the rest of Tephrosia studied;
as such T. hamiltonii represents the lone constituent of a separate Group
The inter-relationship between the various species of the genus
Tephrosia has been a matter of debate. Some of the species, which
are traditionally considered to be closely related, showed much diversification
in chemotaxonomic and numerical taxonomic evaluation (T. pulcherrima
and T. tinctoria, T. villosa and T. pumila etc.). Our
present study has justified to a great extent co-relating with classification
based on the morphological traits, however a distinction between some
members of the genus Tephrosia (T. calophylla and T.
procumbens, T. maxima and T. tinctoria etc.) is still
a difficult task. As such, further analyses are needed to determine the
correct infrageneric taxonomic treatment of the genus Tephrosia,
since it represents one of the largest and most complex groups in the
core tribe Millettieae of the family Fabaceae.
This study represents the first approach in using nuclear DNA finger
print markers as a tool to study molecular systematics in the genus Tephrosia.
Pers. The analysis of additional species and the use of different nuclear
molecular markers, such as ITS (Internal Transcribed Spacers) or plastid/
mitochondrial DNA (RbCL, trn K, mat K etc.,) sequences will improve the
accuracy of resolution of genetic relationships and contribute to a more
accurate classification of the genus Tephrosia.
One of the authors (L P) is thankful to University Grants Commission
(UGC), New Delhi, for a fellowship (SRF/ No. F.17-116/98(SA-I).