Internal Transcribed Spacer (ITS) Fails Barcoding of the Genus Neotinea Rchb.f. (Orchidaceae)

Kaan Hürkan,Kemal Melih Taşkin

doi:10.15832/ankutbd.615848

Abstract

Internal Transcribed Spacer (ITS) is one of the most used barcoding regions for the molecular phylogenetics and barcoding of orchids. Our aim in this study is to test the reliability of ITS on barcoding of closely related Neotinea spp., including Neotinea tridentata, Neotinea ustulata subsp. ustulata and Neotinea ustulata subsp. aestivalis, by comparing it to the accD-psaI intergenic spacer of the plastid DNA. Both ITS and accD-psaI regions were amplified by specific primer sets and sequenced. Phylogenetic trees were regenerated by using Maximum Parsimony approach. The results showed that ITS separated some N. tridentata samples of Turkish, Greek, Hungarian and Croatian samples from the others on the phylogenetic trees due to the incomplete lineage sorting. In contrast to ITS, the accD-psaI marker could successfully separate N. tridentata and N. ustulata samples according to a priori species classification. Our findings refer to a hybridisation story between some N. tridentata and N. ustulata. We propose not to use ITS sequences directly as a barcode and to reconstruct the phylogeny of the Neotinea group. Instead, the inclusion of other nuclear regions such as LFY, ADH, etc., or utilisation of whole genome sequencing could give better barcoding results.

Highlights

The orchid family (Orchidaceae) is the second largest flowering plant family represented by 899 genera and 27801 species
Evolving DNA regions are needed for species-level barcoding et al 2011; Zimmer & Wen 2012)
There are new technological ways of securing correct reconstruction of molecular phylogenetic trees and barcoding the closely related species, e.g. utilization of Generation Sequencing (NGS) techniques like RAD-seq

Summary

Introduction

The orchid family (Orchidaceae) is the second largest flowering plant family represented by 899 genera and 27801 species (ThePlant List 2013). It is necessary to develop a reliable molecular identification method. Since the 1990s, using molecular markers on the phylogenetic studies of orchids have been increasing incrementally (Chase et al 2000; Bateman et al.2003; Sramko et al 2014). Plant taxonomists have used short sequences of both ribosomal deoxyribonucleic acid (rDNA) and chloroplast deoxyribonucleic acid (cpDNA) as molecular markers to identify species and to reconstruct phylogeny Evolving DNA regions are needed for species-level barcoding et al 2011; Zimmer & Wen 2012). The Internal Transcribed Spacer of the nuclear ribosomal 18S-5.8S-26S cistron (ITS) is one of the most extensively used molecular marker in orchid identification and molecular phylogenetic since the 1990s

Methods

Results

Discussion

Conclusion