Full Paper Shajitha

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MOLECULAR PHYLOGENETIC RELATIONSHIP AMONG ENDEMIC

BALSMS OF WESTERN GHATS USING CHLOROPLAST atpB-rbcL

SPACER SEQUENCES

Shajitha P.P1, Dhanesh N. R

1, Aneesha Devassy

1, Jomy Augustine

2, Linu Mathew

1*

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

2 Department of Botany, St. Thomas College, Pala, Kerala, India, contact number: 9447511352

*Corresponding author: [email protected], Contact number: 9447505690

Topic area: Plant Biodiversity

1. Introduction

Balsaminaceae are annual or perennial herbs and consist of more than 1,000 species

with only two genera Impatiens and Hydrocera. Impatiens consists of more than 1000 species.

Out of which 210 species are found in India and at least 103 species of Impatiens are endemic

to the Western Ghats (Bhaskar., 2012) .Taxonomic studies on Balsaminaceae have encountered

several difficulties, leaving numerous relationships within Impatiens unresolved.

This paper reports molecular phylogenetic study with chloroplast atpB-rbcL sequence

data and propose a preliminary reconstruction of the intrageneric relationships in 22 Impatiens

species.

2. Materials and methods

i. Taxon Sampling and out group choice

Twenty two species of Impatiens were collected from Munnar,Vagamon hills and Wayanad and

identified by Dr. Jomy Augustine. Hydrocera triflora (DQ 147896.1) , Marcgravia maguirei

(DQ 147896.1) and Marcgravia umbellata (DQ 147897.1) were selected as out group from

GenBank.

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ii. DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted (GenEluteTM

, Sigma Aldrich, St. Louis, USA).

Specific primers for the atpB-rbcL spacer in Balsaminaceae were synthesized (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 3’. initial denaturation at 95oC and 35 cycles of 30 sec.

denaturation at 95oC, 30 sec. primer annealing at 55

oC and 30sec.extention at 72

oC with a final

extension of 72oC for 10’. (Agilent sure cycler 8800). The PCR product was visualized by 1.5%

AGE with 100 bp ladder. The PCR product were directly sequenced (Scigenome Labs Pvt Ltd.,

Cochin) in ABI 3730xl cycle sequencer.

iii. Neighbor-joining (NJ) analysis

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

default parameters and edited with Bio Edit 7.0.0 A distance analysis applying NJ optimality

criteria was conducted on reduced data matrices by using MEGA Version 5.05 with 1000

Bootstrap values.

3. Results 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 singletons.

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 (Fig1)

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 prove the

taxonomic status of these two species of Impatiens.

Fig. 1. Phylogenetic tree of Impatiens with N-J Analysis



Fig.2.

Impatiens scapiflora Impatiens muculata Impatiens cordata

Impatiens viscosa Impatiens goughi Impatiens flaccida

REFERENCES

Baskar, V. 2012 Taxonomic monograph on Impatiens L. (Balsaminaceae) of Western Ghats,

South India the key Genus for Endemism. Bangalore: Centre for Plant Taxonomic Studies.

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

2006. Phylogenetics of Impatiens and Hydrocera (Balsaminaceae) using Chloroplast atpB-rbcL

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

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