Current advances in plant molecular biology techniques and approaches provide a convenient and rapid evaluation of the differences in informational content, derived from DNA sequence data, of related individuals [1,2]. Polymerase chain reaction (PCR)-based techniques have been used comprehensively as plant molecular markers for significant phylogenetic presumption among relatively closely related species [3]. The impact of genetic data from chloroplast DNA (cpDNA) and nuclear ribosomal DNA (nrDNA) can be clearly seen in the fields of plant phylogenetics, systematics, population genetics and molecular biology [4,5].

Polygonum minus Huds., commonly known as kesum in Malaysia, is currently classified in the Polygonum section Persicaria. In 1967, Ridley [6] reported and described P. minus and another eight species of Polygonum from Malay Peninsula. No additional information on P. minus was available until Anjen et al. [7] and Freeman and Reveal [8] reported Polygonum kawagoeanum Makino and Persicaria minor (Huds.) Opiz, respectively, as being synonyms of P. minus. In 2010, we reported the chemical composition of essential oils in P. minus using two dimensional gas chromatography time of flight mass spectrometry (GC×GC-TOF MS) and found a significantly high level of aldehydes, especially decanal and dodecanal as the dominant aldehydes. Thus, P. minus appears to be more similar to Polygonum odoratum Lour., the Vietnamese coriander and Persicaria hydropiper L., known as the laksa plant in Singapore [9–11]. More recently, for the first time, we reported peltate glandular trichomes and conical brushlike clustered trichomes on both the adaxial and abaxial surfaces of P. minus and revealed potentially important distinctive features of the foliar micromorphology of the genus Polygonum [12]. Previously, Lerstern and Curtis [13] noted only capitate trichomes on the epidermis of P. minus, and they suggested trichome structure as a significant taxonomic feature in the genus Polygonum.

The taxonomic classification of the Polygonum genus has been debated since the work of Steward [14] and has presented a great taxonomic challenge [15]. Using cpDNA and nuclear ITS sequences, molecular phylogenetic studies within section Persicaria revealed that it is monophyletic and is most closely related to sections Tovara and Echinocaulon within the genus Polygonum [16]. To our knowledge, there has been no report on the genetic differentiation of P. minus individuals from Peninsular Malaysia. Therefore, in this study, we aim to highlight the potential impact of molecular genetic marker on selected P. minus accessions from Peninsular Malaysia based on the trnL-trnF and nuclear ribosomal ITS sequences in order to clarify the taxonomic status of P. kawagoeanum and P. minor as synonyms to P. minus.

PCR amplification of the trnL-trnF yielded a PCR product with a single-band of 380 bp in size, indicating the suitability of the universal PCR primers for P. minus. The trnL-trnF sequences among the four P. minus accessions were identical. This result implies that the trnL-trnF region from the plastid genome was not suitable to differentiate individuals of P. minus. The BLAST analysis against the GenBank database showed that the trnL-trnF sequences of P. minus had 100% similarity with sequences of Persicaria pubescens (EU197040.1), Persicaria hydropiper (EF653805.1) and Persicaria longiseta (EU109597.1). Therefore, the plastid DNA variation among members of the Persicaria section was low. However, plastid DNA variation has been successfully utilized in population genetics and biosystematic studies of other plants [17–19]. Okaura and Harada [20] reported the intraspecific variation in three non-coding regions of the plastid DNA in 21 Japanese beech (Fagus crenata Blume) populations and revealed the highly structured geographical distribution of cpDNA haplotypes.

The amplification of the ITS region produced a specific DNA band with a size of 650 bp. No sequence heterogeneity was detected among DNA clones from the same individual. The ITS sequences of the P. minus accessions were deposited into the GenBank database (JN709855-JN709858). The ITS sequence divergence among the four P. minus accessions was 1%, and it was more variable compared to the trnL-trnF sequences of P. minus. Generally, the plant nuclear genome evolves faster than the chloroplast genome [21]. The phylogenetic relationships among the P. minus accessions and selected Persicaria species based on the ITS sequences are shown in the neighbor-joining (NJ) tree [Figure 1(A)]. In the maximum parsimony (MP) analysis, 22.92% sites within the ITS region were found to be parsimony-informative characters. The MP analysis produced 18 most parsimonious trees with a tree length of 359 steps. One of the most parsimonious trees is shown in Figure 1B. The phylogenetic characteristics of ITS sequences from the MP analysis are summarized in Table 1. The topology in the Bayesian inference was similar to the NJ and MP trees (Figure 1).

The genetic distances between the P. minus (CH, GH, UY and FH), P. kawagoeana, Persicaria minor P1 and P. minor P2 were determined based on kimura 2-parameter model in PAUP. Genetic distance values are shown in Table 2 as percentages.

The tree topologies from the NJ, MP and Bayesian analyses were generally congruent with each other. The Polygonum taxa formed monophyletic lineages according to section Persicaria (P. punctata, P. pubescens, P. hydropiper, P. lapathifolia, P. longiseta, P. kawagoeana, P. macrantha and P. minor), section Cephalopilon (P. capitata, P. runcinata and P. nepalensis) and section Echinocoulon (P. arifolia, P. maackiana, P. meisneriana and P. sagittata). All four of the P. minus accessions [Cameron Highlands (CH), Genting Highland (GH), Fraser’s Hill (FH) and Ulu Yam (UY)] from Peninsular Malaysia formed a clade that was strongly supported by bootstrap value and posterior probabilities (Figure 1). The MP tree topology showed that the Peninsular Malaysian P. minus clade formed a close relationship to a clade containing P. pubescens, P. hydropiper and P. punctata (Figure 1).

Although Persicaria minor and Polygonum kawagoeanum are treated as synonyms of Polygonum minus [7,8], these three taxa did not cluster together in any of the constructed trees (Figure 1). The three species were found to have great genetic distances (Table 2). Polygonum minus had the highest genetic distance from P. kawagoena and P. minor. Therefore, P. minus of Peninsular Malaysia is exclusive and unique, and it might be distinct from the synonymous P. minor and P. kawagoeanum.

In P. minus, the ITS region from the nuclear genome was more variable than the trnL-trnF region from the plastid genome. The ITS sequences were informative enough to infer the phylogenetic relationships of P. minus and the related Polygonum taxa. The topology of the NJ, MP and Bayesian consensus trees showed that P. minus from Peninsular Malaysia did not form a cluster with P. kawagoeana and P. minor, which were previously reported to be synonyms to P. minus. These findings suggest that further investigation is needed to clarify the taxonomic status of these three taxa.