Comparative Chloroplast Genomics and Phylogenetic Analysis of Persicaria amphibia (Polygonaceae)

Abstract

Persicaria amphibia (L.) Delarbre, also known as water knotweed, belongs to the Polygonaceae family and has two types: terrestrial and aquatic. We report the chloroplast genome of P. amphibia obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. We analyzed the complete chloroplast (cp) genome of P. amphibia and found it to be 159,455 bp in length, with a large single-copy region (LSC, 84,281 bp), a small single-copy region (SSC, 13,258 bp), and a pair of inverted repeats (IR, 30,956 bp). It contains 79 protein-coding, 29 tRNA and 4 rRNA genes. Comparative analysis of nine Persicaria cp genomes showed a similar genome structure and gene content. However, ycf3 intron II was lost in three Persicaria species (P. hydropiper, P. japonica, and P. pubescens) and the SC/IR regions of four species (P. amphibia, P. hydropiper, P. japonica, and P. pubescens) included the rps19 gene. Phylogenetic analysis of the nine Persicaria species revealed that P. amphibia is sister to P. hydropiper, P. japonica, and P. pubescens. Moreover, we found sequence divergence regions; the largest were rps16-trnQ, trnQ-psbK, trnW-trnP, ndhF-rpl32, and rpl32-trnL regions. This study could be useful for phylogenetic tree analysis of Persicaria and for the identification of Persicaria species.

1. Introduction

Persicaria Mill. is a genus in the tribe Persicarieae (family Polygonaceae) that contains approximately 130 species distributed worldwide [1,2]. Based on the macromorphological classification, Persicaria is included in the genus Polygonum [3]. However, ribulose-bisphosphate carboxylase large subunit (rbcL) analysis of Polygonaceae showed that the Polygonoideae subfamily is paraphyletic, and is subdivided into two tribes: Polygoneae and Persicarieae [4].

Persicaria amphibia, a species belonging to the genus Persicaria, is native to the Northern Hemisphere. It has been described in both aquatic and terrestrial forms, and exhibits complex patterns in leaf morphological variation [5]. P. amphibia can grow in aquatic environments as well as terrestrial habitats and has different leaf types. The terrestrial form is erect and has hairy leaves with wavy edges. The aquatic form is hairless with oblong flat leaves. Previous phylogenetic analyses of Persicaria used markers of the internal transcribed spacer (ITS) region, and chloroplast DNA (cpDNA) rbcL and trnL-F [6], which showed that P. amphibia formed a clade and was sister to sect. Echinocaulon in two cp markers, whereas it was sister to sect. Tovara in the ITS marker.

Chloroplasts provide energy to almost all green plants. The chloroplast genome is typically quadripartite in structure, containing a large single copy region (LSC) and a small single copy region (SSC), separated by a pair of inverted repeats (IR) [7] in which organization, gene contents and structures are highly conserved [8]. However, some plants have unique structures owing to gene loss [9,10,11], rearrangement [12,13,14], and inversions [15,16]. Recently, cp genome sequences have been used for evolutionary [17,18], taxonomic [19], DNA marker [20,21], and phylogenetic analyses [22,23,24]. NCBI has uploaded and studied the chloroplast genomes of Persicaria species such as P. chinensis (NC_050358) [25], P. pubescens (MK234901), P. hydropiper (MK234902), P. japonica (NC_056952), P. filiformis (NC_058319), P. aviculare (NC_058892), P. perfoliata (NC_060649), P. runcinata (NC_061176), and P. maackiana (NC_061657).

In this study, we sequenced the complete cp genome of P. amphibia and compared it with previously published cp genomes of Persicaria species. The aims of this study were to: (1) evaluate the phylogenetic position of P. amphibia, (2) evaluate the sequence divergence, and (3) suggest useful markers for future phylogenetic studies in Persicaria species.

2. Materials and Methods

2.1. Sampling, DNA Extraction and Sequencing

Fresh leaves of P. amphibia were were collected from Gyodongdo, Ganghwa, South Korea, and specimens were deposited in the Herbarium of the Nakdonggang National Institute of Biological Resources (NNIBR). The total DNA content was extracted using the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). Genomic DNA was sequenced using the Illumina Truseq Nano DNA kit (Illumina, San Diego, CA, USA), as per the manufacturer’s protocol. Approximately 4.0 Gb of raw data were generated. A total of 31,825,998 reads of the 150 bp paired-end sequence were generated.

2.2. Assembly and Annotation

The chloroplast genome was assembled using GetOrganelle [26]. The GeSeq [27] was used to annotate the P. amphibia genome. tRNA gene sequences were obtained using tRNAscan-SE [28]. Finally, all published plastid genomes of Persicaria were compared using Geneious Prime [29]. OrganellarGenomeDRAW (OGDRAW) was used to draw a circular map of the chloroplast genome of P. amphibia [30]. The chloroplast genome of P. amphibia was deposited in GenBank (

Table 1. Comparison of features of nine Persicaria cp genomes.

	Persicaria amphibia	Persicaria pubescens	Persicaria hydropiper	Persicaria chienesis	Persicaria japonica	Persicaria filiformis	Persicaria perfoliata	Persicaria runcinata	Persicaria maackiana
Length
Total	159,455 bp	159,502 bp	159,054 bp	159,981 bp	159,747 bp	159,741 bp	160,585 bp	159,220 bp	160,595 bp
LSC	84,281 bp	84,555 bp	83,835 bp	84,347 bp	85,013 bp	84,432 bp	85,439 bp	84,461 bp	85,376 bp
SSC	13,258 bp	13,385 bp	13,357 bp	12,890 bp	13,178 bp	13,073 bp	12,879 bp	12,807 bp	13,055 bp
IR	30,956 bp	30,781 bp	30,931 bp	30,872 bp	30,778 bp	31,118 bp	31,135 bp	30,884 bp	31,082 bp
Genes
Total	113	113	113	113	113	113	113	113	113
Protein-coding genes	79	79	79	79	79	79	79	79	79
tRNA	29	29	29	29	29	29	29	29	29
rRNA	4	4	4	4	4	4	4	4	4
GC contents	38.2%	38.2%	38.2%	38.0%	38.1%	37.8%	37.5%	37.9%	37.9%
Accession number	This study (ON938209)	MK234901	MK234902	NC_050358	NC_056952	NC_058319	NC_060649	NC_061176	NC_061657

).

2.3. Repeat and Divergence Hotspot Analysis

REPuter was used to visualize forward, palindrome, reverse, and complement sequences with a minimum repeat size of 30 base pairs (bp) and a sequence identity greater than 90% [31]. Simple sequence repeats (SSRs) were detected using MISA [32]. SSRs with minimum numbers of repetitions of 10, 5, 4, 3, 3, 3 for mono-, di-, tri-, tetra-, penta- and hexa-nucleotides were detected. The alignment of nine Persicaria complete chloroplast genome sequences was visually compared using the Shuffle-LAGAN model of mVISATA [33]. The nucleotide diversity (Pi) was determined using DnaSP [34]. The step size was set to 200 bp and the window length was set to 600 bp.

2.4. Phylogenetic Analysis

Seventy-seven protein-coding genes of nine Persicaria species (Table 1) and one outgroup (NC_58892, Polygonum aviculare) were compiled into a single file of 66,731 bp and aligned using MAFFT [35]. Maximum likelihood (ML) analyses were conducted using RAxML v.8 [36] with the GTR+GAMMA I model with 1000 bootstrap replications.

3. Results

3.1. Characteristic of the P. amphibia cp Genome

The complete cp genome of P. amphibia (NCBI accession number: ON938209) comprises 159,455 bp with a quadripartite structure and two IRs (30,956 bp) separated by the LSC (84,281 bp) and SSC (13,258 bp) regions (Figure 1, Table 1). The average GC content was 38.2%. The cp genome of P. amphibia encoded a total of 113 genes, including 79 protein-coding genes, 29 tRNA genes, and 4 rRNA genes (Table 1). The IR region contained 18 duplicated genes, including seven protein-coding genes (ycf1, rps7, ndhB, ycf2, rpl23, rpl2, and rps19), seven tRNA genes (trnN-GUU, trnR-ACG, trnA-UGC, trnI-GAU, trnV-GAC, trnL-CAA, and trnI-CAU), and four rRNA genes (rrn5, rrn4.5, rrn23, and rrn16).

Fifteen genes (rps16, atpF, rpoC1, petB, petD, rpl16, rpl2, ndhB, ndhA, trnA-UGC, trnI-GAU, trnV-UAC, trnL-UAA, trnG-UCC, and trnK-UUU) had one intron, whereas three genes (ycf3, clpP, and rps12) contained two introns.

3.2. Comparison of Other Persicaria Species

Nine Persicaria cp genomes showed a typical quadripartite structure, consisting of a pair of IRs (30,778–31,135 bp), separated by the LSC (84,281–85,439 bp) and SSC (12,879 13,385 bp) regions (Table 1). The cp genome of P. aviculare exhibits the longest genome, and P. hydropiper is smaller than other Persicaria species (Table 1). Nine Persicaria species had the same number of genes (79 protein-coding genes, 30 tRNA, and 4 rRNA genes). However, the second intron of ycf3 was lost in three species (P. pubescens, P. japonica and P. hydropiper Figure 2).

The IR/LSC and IR/SSC junctions of nine Persicaria cp genomes were compared. We found two different junction types: IR/LSC and IR/SSC. Type 1 in four species (P. amphibia, P. filiformis, P. perfoliate, and P. maackiana) of IR included the intact rps19 gene. Type 2 in the other Persicaria species (except for P. amphibia, P. filiformis, P. perfoliate, and P. maackiana) of IR included the partial rps19 gene. The intact ycf1 gene was duplicated in IR regions (IRa and IRb). The junctions of IRb and SSC were located in the ndhF gene, and the junction of SSC/IRA was located between the rps15 and ycf1 genes (Figure 3). However, the total length of IR regions in all of the species was similar (Table 1).

3.3. Repeat Sequence Analysis

Simple sequence repeats (SSRs) were detected using MISA [32]. P. amphibia had 38 repeats. P. chinensis had the highest number of SSRs (59), whereas P. amphibia and P. pubescens (38) had a smaller number of SSRs than other Persicaria species. Most SSRs were A/T mononucleotide repeats (Figure 4A).

Repeats (forward, palindrome, reverse, and complement) were identified using REPuter [31]. A total of 25 repeats were present in P. amphibia (13 palindrome and 12 forward repeats). The SSRs of other Persicaria species were in the range of 19–28. Most of the repeats ranged in size from 30 to 48 bp. P. japonica had two long repeats, 154 and 156 (Figure 4B).

3.4. Sequences Divergence Hotspots in Persicaria

The cp genomes of nine Persicaria species were compared using mVISTA [33]. The results show that protein-coding genes were more conserved than the non-coding regions, and SC regions had lower variation than IR regions (Figure 5).

The number of polymorphic sites and nucleotide diversity were determined using DnaSP [34]. In the complete cp genome, there were 8470 polymorphic sites, and nucleotide diversity (Pi) was found to be 0.02746. The Pi value was calculated with 200 bp steps. These values ranged from 0 to 0.08875 (Figure 6). The high divergence regions were rps16-trnQ, rpl32-trnL, trnW-trnP, trnQ-psbK, and ndhF-rpl32, three of which (rps16-trnQ, trnW-trnP, and trnQ-psbK) were located in the LSC region, and two (rpl32-trnL and ndhF-rpl32) were located in the SSC region. The rps16-trnQ region exhibited the highest nucleotide diversity (0.0875, Figure 6).

3.5. Phylogenetic Analysis

The ML phylogenetic tree of the nine Persicaria species was constructed based on 77 protein-coding genes (66,732 bp, Figure 7). Persicaria is monophyletic (BS = 100), and is divided into two clades: (1) P. runcinata and P. chinensis; and (2) P. filiformis, P. amphibia, P. hydropiper, P. japonica, and P. pubescens. P. amphibia was sister to P. hydropiper, P. japonica, and P. pubescens, with a high bootstrap value (BS = 100).

4. Discussion

Chloroplast genomes are highly conserved in structures and genes. However, previous studies have reported that gene loss in the chloroplast genome occurs in several lineages, including Geraniaceae [37,38], Orobanchaceae [39,40,41,42], Fabaceae [43,44], and Pinaceae [45]. Studies have also shown that introns of clpP [44,46], rps12 [46], and rpl16 [44] are missing in angiosperms. The gene of ycf3 normally contains three exons and two introns located in the LSC region of the chloroplast genome. The ycf3 gene encodes photosystem I, a complex in the thylakoid membrane of plants [47,48,49]. A previous study reported the loss of ycf3 intron II in Grammica and Cuscuta nitida [50]. Our results reveal that among the nine Persicaria species, three species (P. pubescens, P. japonica, and P. hydropiper) lost ycf3 intron II, and P. amphibia is sister to these three species. Longevialle et al. [47] suggested that splicing of ycf3 intron II has an impact on gene ORGANELL TRANSCRIPT PROCESSING 51 (OPT51) that specifically promotes the splicing of the only group II intron.

IR/SC junctions are variable sites in the cp genome, such as IR expansion and contraction [51,52,53,54]. Comparison of SC/IR junctions in the nine species showed two types: First, an intact rps19 gene in the IR region of four species (P. amphibia, P. hydropiper, P. japonica, and P. pubescens); and second, a partial rps19 gene in the IR region of the remaining five species. Previous studies have suggested that partial gene duplication or the contraction and expansion of IR regions causes variable types of rps19 gene [55,56,57]. However, the IR regions in the nine Persicaria are extremely similar in length (Table 1). This suggests that rps19 in Persicaria is more likely to be partially duplicated.

Previous analysis of cpDNA regions (rbcL, trnL-F, and matK) of Persicaria [6] revealed that P. amphibia is a sister species to the sect. Eupersicaria, such as P. punctate, and P. hydropiper. However, P. amphibia did not show the same results using the nuclear ribosomal DNA (nrDNA) ITS data, indicating that P. amphibia is sister to sect. Tovara (P. filiformis and P. virginiana). Our results show that P. amphibia is a sister species to P. hydropiper, P. japonica, and P. pubescens.

Persicaria plants are highly variable in their morphological characters. For example, P. amphibia has variable morphological characteristics, and some authors have reported two varieties, such as P. amphibia var. emersa and P. amphibia var. stipulacea. Additionally, the genus Persicaria was reported to exhibit self-fertilization and hybridization [1,58]. Partridge [59] suggested that P. amphibia has economic qualities as feed for wild animals, herbal medicine and as an ornamental garden plant. Recently, molecular phylogenetic analysis was used to identify plants [60,61]. Previously, cpDNA (matK, rbcL) and nrDNA ITS regions have been used for species identification and phylogenetic analyses [52,53,54,55,56,57,58,59,60,61,62,63,64,65]. In Persicaria, previous studies have used cpDNA (matK, rbcL), nrDNA ITS, and LEAFY regions. However, the relationships between the genus Persicaria are not well supported [1,4,5,6]. Our results reveal that the highest sequence divergence regions were rps16-trnQ, trnQ-psbK, trnW-trnP, ndhF-rpl32, and rpl32-trnL in Persicaria species. These variable DNA regions could be used as molecular markers and will be helpful in the phylogenetic analysis of Persicaria species.

5. Conclusions

In this study, we analyzed the complete cp genome sequence of P. amphibia. It comprises an LSC (84,281 bp), an SSC (13,258 bp), and two IR regions (30,956 bp), and contains 79 protein-coding genes, 29 tRNA genes, and four rRNA genes. Comparative cp genome analysis of the nine Persicaria species showed that all have a similar gene structure and content. However, ycf3 intron II is lost in P. hydropiper, P. japonica, and P. pubescens. Additionally, we found that the SC/IR junctions in four species (P. amphibia, P. hydropiper, P. japonica, and P. pubescens) had an intact rps19 gene. Phylogenetic relationships based on 77 protein-coding genes showed that P. amphibia is sister to P. hydropiper, P. japonica, and P. pubescens. We found five sequence divergence regions (rps16-trnQ, trnQ-psbK, trnW-trnP, ndhF-rpl32, and rpl32-trnL) that will be useful markers for the further phylogenetic analysis of the genus Persicaria.