Investigation of the relationship between Honeysuckle species (Lonicera spp.) in Iran using DNA barcoding and morphological markers

The genus Lonicera belongs to the Caprifoliaceae and in Persian, it is called Plakhor, Shung, or Honeysuckle. Understanding inter-species relationships based on morphological and biochemical characteristics sometimes have conflicting results. In this study, morphological markers, and DNA of nuclear and chloroplast barcodes (including ITS and matK) along with several other sequences from NCBI gene bank database were used to identify, differentiate and determine the phylogenetic relationships of 12 Lonicera species in Iran. Genomic DNA was extracted using existing kits and PCR reaction was performed to amplify the two gene regions and finally the purified samples were sequenced. Cluster analysis based on morphological characteristics divided the species into two large groups, the first group consisting of L. sempervirens and the second group consisting of 11 other species. The closest genetic relationship was observed between L. floribunda and L. nummulariifolia and the least similarity was related to L. sempervirens and L. caprifolium. Principle component analysis (PCAs) of morphological data showed that the first three principal components explaining 71.1% of total variation. The first principal component is controlled by fluffy stem cover, leaflet shape, leaf margin, leaflet tip shape, flower color, fluffy leaf cover, leaf base and fruit color, petiole length and internode distance explaining 38.8% of variation. Based on molecular studies, the length of the matK region was about 1110-1135 nucleotides. Parsimony analysis of this area showed 944 protected sites, 109 variable sites and 19 Parsimony sites. In the phylogenetic tree created by the matK region, all species were isolated except two species, L. korolkovii and L. maackii, and were placed in separate branches. The genetic diversity and distance between species were observed in the matK region with a genetic distance of 0.00 to 0.057. This study could not provide a satisfactory answer from ITS primers. Therefore, the matK region was identified as the best region in terms of the ability to show genetic diversity between species due to its maximum diversity and easy reproduction, which can be used in future research.