MOLECULAR PHYLOGENETIC RELATIONSHIP AMONG ENDEMIC

BALSMS OF WESTERN GHATS USING CHLOROPLAST atpB-rbcL

SPACER SEQUENCES

Shajitha PP1, Danesh NR1, Jomy Augustine2, Linu Mathew1,*

1 School of Biosciences, M.G. University, Kottayam, Kerala, India, 686560

2 Department of Botany, St. Thomas college, Pala, Kerala, India

*corresponding author, Email ID linumathew@mgu.ac.in, Contact number: 9447505690

INTRODUCTION

Balsaminaceae are annual or perennial herbs with flowers that exhibit a remarkable

diversity. The family consists of more than 1,000 species (Grey-Wilson 1980a; Clifton 2000), but

only two genera are recognized. Impatiens is a very large genus of over 1,000 species, while

Hydrocera triflora is the only member of the genus Hydrocera. Impatiens usually has three free

sepals and always five petals, four of which are united into two lateral pairs (Steven et al.2006).

The genus Impatiens contains over 210 species in India, more than half of which occur in the

Western Ghats in South India. At least 103 species of Impatiens are endemic to the Western

Ghats including the new species (Bhaskar., 2012) . Some Impatiens species are of horticultural

importance, e.g., the popularly grown flowers, the “Balsam”, I. balsamina, the “Busy Lizzie”, I.

walleriana, and the “New Guinea Hybrid Impatiens”, I. hawkeri. Impatiens is a well-known

example of a taxonomically difficult group (Hooker et al., 1859; Grey Wilson, 1980b). Here we

conducted molecular phylogenetic study with chloroplast atpB-rbcL sequence data and propose a

preliminary reconstruction of the intrageneric relationships in Impatiens.

Key Words:Impatiens, chloroplast atpB-rbcL spacer, N-J Analysis, PCR

MATERIALS AND METHODS

Taxon Sampling and out group choice

We sampled genus Impatiens of family Balsaminacea. Twenty two species of Impatiens

were collected from Munnar,Vagamon hills and Wayanad and identified by Dr.Jomy Augustine,

Taxonomist, St.Thomas college Pala, Kerala, India. We took sequences of Hydrocera triflora

(DQ 147896.1), Marcgravia maguirei (DQ 147896.1), Marcgravia umbeiiata(DQ 147897.1),

Impatiens campanulata (DQ 147822.1) as out group from GENBANK.

Molecular Protocols

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted using plant DNA isolation kit (Sigma Aldrich, St.

Louis, USA). Quality and quantity of DNA was estimated by Agarose Gel Electrophoresis

(0.8%). Specific primers for the atpB-rbcL spacer in Balsaminaceae were synthesized. IMP-atpB

(5-ACATCTAGTACCGGACCAATGA-3) and IMP-rbcL (5-AACACCAGCTTTGAATCCAA-

3) (Steven et al.2006). 25µl PCR reaction was set by mixing 1 µl template DNA (25ng), 0.5 µl

each of forward and reverse primer (10µM), 2.5 µl of Taq buffer, 0.6 µl MgCl2 (25µM), 0.5 µl of

dNTP mix (10µM) and 0.5 µl of Taq DNA polymerase (3U/µl) and volume made with milliQ

water. PCR conditions were as follows 3min. initial denaturation at 95oC and 35 cycles of 30

sec.denaturation at 95oC, 30 sec. primer annealing at 55oC and 30sec.extention at 72oC with a

final extension of 72oC for 10 minutes. (Agilent sure cycler 8800, Agilent Technologies). The

PCR product was visualized by 1.5% AGE with 100 bp ladder.The PCR product directly

sequenced (Scigenome Labs Pvt Ltd., Cochin). Sequencing of amplicon with forward and

reversed primers was carried out in ABI 3730xl cycle sequencer.

Sequence alignment

The obtained atpB-rbcL sequences were initially aligned with Clustal W applying the

default parameters and edited with Bio Edit 7.0.0.(Hall,1984)

Neighbor-joining (NJ) analysis

A distance analysis applying NJ optimality criteria (Saitou & Nei, 1987) was conducted

on reduced data matrices by using MEGA Version 5.05 (Tamura K. et al., 2011). Bootstrap

values were obtained from 1000 replicate NJ analysis.

RESULS AND DISCUSSION

The length of the unaligned atpB- rbcL fragments of the ingroup taxa range from 700-

1032bp. The full atpB-rbcL data set including out group and ingroup taxa consisted 848

characters. Out of 226 were variable 128 were parsimony informative 95 were single tons.

Percentage of T, C and A were 36.1, 13.3, 35.7 respectively and AG dinucleotide were 15

percentage.

Phylogeny inferred from atpB- rbcL region of South Indian Impatiens

Sister genus Hydrocera formed a separate clade with 100% boot strap support showing

the monophyly of Impatiens. It was found that Impatiens goughi is closely related to Impatiens

viscosa with boot strap of 96%. This clade showed relation to I. viscida. I. gughi, I. viscosa and I.

viscida formed a clade with boot strap of 98%.Similarly I. uncinata and I. cordata formed a

clade with boot strap support of 94%. I. wightianae and I. muculata formed a clade with boot

strap of 98%. I. dasysperma and I. flaccida formed a single clade with boot strap of 87%. These

two species shows very similar morphological characters. The molecular analysis also shows the

close relation of these two species. I. scabriusculata and I. mysorense formed a clade with boot

strap of 88% and I. leucida and I. gardneria formed a clade with boot strap of 86%.I. herbicola

shows a close similarity to I .ligulata with boot strap of 100%. It is observed that the traditional

taxonomy based on morphology was reproduced also in the atpB-rbcL based phylogeny. The

relatedness of I. dasysperma and I. flaccida requires further investigation using additional

sequence information namely ITS, matK, trnL-trnF spacers to unequivocally proove the

taxonomic status of these two species of Impatiens.

Baskar V.Taxonomic monograph on Impatiens L. (Balsaminaceae) of Western Ghats,South India

The key Genus for Endemism(2012),Centre for Plant Taxonomic Studies, Bangalore.

Clifton,R. 2000. Geraniales species checklist series: vol. 6. Geraniaceae Bentham and Hooker,

tribe VII Balsamineae. Dover: The Geraniaceae Group.

Grey Wilson,C. 1980a. Impatiens of Africa. Rotterdam: Balkema. 1980 b. Hydrocera triflora, its

floral morphology and relationship with Impatiens. Studies in Balsaminaceae. Kew Bulletin 34:

221–227.

Grey Wilson,C. 1980b. Impatiens of Africa. A. A. Balkema, Rotterdam 1980b. Hydrocera

triflora, its floral morphology and relationship with Impatiens. Studies in Balsaminaceae. Kew

Bulletin 34: 221–227.

Hooker, J. D. and Thompson,T.1859. Praecursores ad floram Indicam. Balsaminaceae. Bot. J.

Linn. Soc. 4: 106–157.

REFERENCES

Steven Janssens, Koen Geuten, Yong-Ming Yuan, Yi Song, Philippe Kupfer, Erik Smets.

Phylogenetics if Impatiens and Hydrocera(Balsaminaceae) using Chloroplast atpB-rbcL Spacer

Sequences. Systematic Botany(2006). 31(1):171-180.