Phylogenetic and evolutionary relationships in selected Pinus species using rbcL and matK chloroplast genes

The genetic diversity of Pinus plants has been assessed in various phylogenetic studies that reveal the polymorphism directly at DNA levels. The rbcL and matK are the most commonly used markers for phylogenetic studies of Pinus sp. that exhibit a diverse geophysical adaptiveness and geographical variations across different regions as a result of genotypic modifi cations. This study evaluated usefulness of rbcL and matK genes for molecular identifi cation and phylogenetic study among various species of Pinus. Maximum Likelihood (ML) and Neighbor Joining (NJ) analysis of the data obtained from rbcL gene belonging to ten Pinus species revealed four clusters. First Cluster included P. Wallichiana (PW), P.Elliottii (PEl) and P.Greggii (PG). Second cluster included P. Thunbergii (PT), P. Echinata (PE) and P.merkusii (PM). Third cluster included P.taeda (Ptd) and P. khasya (PK) while fourth cluster included P. Roxburghii(PR) and P. patula (PP). Similarly ML and NJ analysis of the data obtained from matK gene belonging to ten Pinus species revealed four cluster and one outgroup. First Cluster included P. Thunbergii (PT), P.Greggii (PG) and P.Elliottii (PEl) Second cluster included P. roxburghii(PR) and P.merkusii (PM)P. Echinata (PE) and third cluster included P. Wallichiana (PW) and P.taeda (Ptd) while fourth cluster included P. Patula (PP) and P. Khasya (PK). P.echinata remained out clustered in this analysis. Received: 20 May, 2021 Accepted: 21 June, 2021 Published: 22 June, 2021 *Corresponding author: Gauri Saxena, Department of Botany, University of Lucknow, Lucknow -226001, India, Tel: 09415182051; E-mail:


Introduction
Pinus (Pinaceae) is the largest genus of order Coniferales widely distributed throughout temperate zones in the Northern Hemisphere, and restricted to high elevation in the tropics and subtropics [1,2] Pines differ from other members of family Pinaceae and are easily identifi able by their dimorphic long and short shoots called fascicles. These fascicles bear long, narrow needle-like leaves mostly present in groups of two to fi ve. Due to its great economical and ecological importance, phylogeny and systematics of this genus has received great attention based on morphology, anatomy, ethnobotanical values, karyotypic analysis, secondary products, isozymes, pharmacognosy and, most recently, molecular studies [2].

Research Article
Phylogenetic and evolutionary relationships in selected Pinus species using rbcL and matK chloroplast genes Citation: Singh  Based on morpho-anatomical and molecular data, Pinus has been divided into two monophyletic subgenera: Haploxylon (subgenus Strobus, with single fi brovascular bundle in the needle, also known as "Soft pines") and Diploxylon (subgenus Pinus, with double fi brovascular bundles in the needle, also known as "Hard pines") [3][4][5][6]. These two subgenera namely Haploxylon and Diploxylon have further been divided into many sections and subsections [1,4].
In this study, we analyzed sequences from rbcL and matK, for ten species of the Pinus namely P.merkusii, P.khasya,

Collection of plant material
The leaves (needles) of 10 pine species were collected in September, 2016, from a wild population in the region of Ranikhet (located at 357 km NSE of New Delhi, latitude 29°39'52.2" (N); longitude 79°28'40.9" (E); altitude 1,727 m).
The site received median rainfall, had very low winter temperature and low soil fertility. Voucher specimen of all the species selected for study was deposited in the herbarium of the National Botanical Research Institute, Lucknow (Table   1). Prior to extract preparation, study material was dried at room temperature and ground to fi ne powder in a blender.
The collected plant material was brought to the laboratory and stored at -80 0 C.

DNA extraction, amplifi cation and sequencing
DNA was extracted from 100 mg of dried needles using a modifi ed CTAB method, treated with RNase, and purifi ed by phenol. Two cpDNA regions (rbcL and matK) were sequenced using the primers ( Table 2)  Alignments were performed using Clustal X [8]. The phylogenetic analyses were completed using single data sets.
The phylogenetic and evolutionary analyses were performed with MEGA X using neighborjoining (NJ) and maximum likelihood (ML) algorithms. Gel electrophoresis representing DNA bands of rbcL and matK are represented in Figures 1,2 respectively.

Phylogenetic relationships among selected species of Pinus
The results of the tree construction were midpoint rooting based on partial sequences of rbcL gene ( Figure 3   while fourth cluster included P. Patula (PP) and P. Khasya (PK).
P.echinata remained out clustered in this analysis (Figure 4).

Discussion
Analyzing the amounts of nucleotide and patterns of nucleotide variation between and within species is important to understand the mechanisms of evolution by which processes of genetic polymorphisms within species become transformed into genetic divergence between species and genetic diversity is maintained. Such diversities are infl uenced by evolutionary processes, such as selection, recombination, mutation and population structures.
In one of the earliest studies by Govindaraju, et al. 1992  Lately, [12] and [13] used matK, rbcL, and other genes to understand the phylogeny of the Pinus sp. According to [12] P. taeda, P. echinata, P. greggii, P. patula, and P. elliottii were all     [7,9,11,19]. However, these studies have been limited in terms of taxonomic sampling and/or geographic scope, particularly in subgenus Pinus and pine species growing in Europe and American continents.
The study by [11] using chloroplast DNA (cpDNA) analyzed 18 Diploxylon pines species of North America. Other studies such as analysis of ITS sequences by [5] involved a broad sampling

Conclusion
Further studies involving as many as species possible using multiple marker, including both subgenera and all their sections as well as subsection are need to be studied to develop a complete database that can be used for a full proof classifi cation and identifi cation of Pinus up to species level.

Authors contribution
LS was a major contributor in writing the manuscript, GS and PCV proposed the research idea, conceptualized the research design and prepared the fi nal manuscript including the original experimental work, and data recording. PJ, RS, SP and AS was involved with material collection and identifi cation.