MOLECULAR PHYLOGENETIC RELATIONSHIP AMONG ENDEMIC BALSMS OF WESTERN GHATS USING CHLOROPLAST atpB-rbcL SPACER SEQUENCES

Shajitha P.P1, Dhanesh N. R1, Aneesha Devassy1, 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, contact number: 9447511352 *Corresponding author: linumathew@mgu.ac.in, 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.

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). 25l PCR reaction was set by mixing 1 l template DNA (25ng), 0.5 l each of forward and reverse primer (10M), 2.5 l of Taq buffer, 0.6 l MgCl2 (25M), 0.5 l of dNTP mix (10M) and 0.5 l of Taq DNA polymerase (3U/l) 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 55oC and 30sec.extention at 72oC 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 7001032bp. 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.