Structural and phylogenetic insights from complete chloroplast genomes of seven Vicia species

Mohammad Mehdi Golchini; Aboozar Soorni

doi:10.1371/journal.pone.0340630

. 2026 Mar 4;21(3):e0340630. doi: 10.1371/journal.pone.0340630

Structural and phylogenetic insights from complete chloroplast genomes of seven Vicia species

Mohammad Mehdi Golchini ¹, Aboozar Soorni ^1,^*

Editor: Md Mahmudul Hasan²

PMCID: PMC12959699 PMID: 41779816

Abstract

The legume genus Vicia L. (Fabaceae) is of significant ecological and agronomic importance, comprising species widely utilized as forage crops, green manure, and sources of valuable phytochemicals. Despite this, a comprehensive genomic understanding of many species, particularly those endemic to underrepresented regions like Iran, remains limited. To address this, we employed a high-throughput sequencing and comparative genomics approach to elucidate the chloroplast (cp) genome architecture and evolutionary relationships of seven previously uncharacterized Iranian Vicia species including V. hirsuta, V. hybrida, V. lathyroides, V. lutea, V. narbonensis, V. peregrina, and V. villosa. Total genomic DNA was sequenced on an Illumina HiSeq 2000 platform, and the cp genomes were assembled de novo using GetOrganelle, followed by comprehensive annotation with a suite of bioinformatic tools. The analysis revealed considerable size variation, ranging from 118,660–130,223 bp, and a key structural divergence involving the loss of one inverted repeat (IR) region in six species, consolidating their placement within the IR-lacking clade (IRLC), while V. villosa retained the ancestral quadripartite structure. Lineage-specific gene losses were documented, including accD in V. lathyroides and ycf2 in V. narbonensis. Microsatellite analysis identified a predominance of A/T-rich mononucleotide simple sequence repeats (SSRs), with V. hybrida exhibiting the highest SSR density. Nucleotide diversity (Pi) analysis across coding regions identified clpP (Pi = 0.19772) and ycf1 (Pi = 0.16964) as hypervariable loci, while the ribosomal protein genes rps7 and rpl20 were validated as highly effective phylogenetic barcodes. Maximum likelihood phylogenetic reconstruction, based on a concatenated alignment of 86 shared protein-coding genes, resolved the species into well-supported clades, providing a robust evolutionary framework. This study delivers essential genomic resources that deepen the understanding of cp genome evolution in the IRLC and provides powerful molecular tools for future research in Vicia systematics, conservation genetics, and precision breeding.

Introduction

The genus Vicia L., a member of the Fabaceae (Leguminosae) family, represents the third-largest group of flowering plants globally. Comprising approximately 150–210 species, it is widely distributed across Europe, Asia, and North America, with the highest species concentration found in the Mediterranean region [1,2]. Indeed, the Mediterranean is recognized as the primary center of diversification for Vicia, with Turkey and northwest Asia exhibiting the greatest species diversity [3,4]. Vicia species are predominantly cultivated as winter forage legumes due to their high nutritional value, serving as green manure, pasture, silage, and hay. Their adaptability to diverse climatic and soil conditions makes them particularly suitable for intercropping with cereals, where they contribute to disease suppression and soil improvement [5]. Additionally, these plants exhibit shade tolerance and possess nitrogen-fixing capabilities, enriching the soil with bioavailable nitrogen that benefits neighboring crops [6]. Beyond their agricultural significance, many Fabaceae species hold considerable economic value as sources of food, herbal medicine, industrial materials, and animal feed [7]. Notably, Vicia seed protein concentrate has recently emerged as a viable raw material for producing vacuum thermoformed bioplastics, demonstrating acceptable mechanical resistance and stability [8]. Furthermore, Vicia seeds contain a diverse array of bioactive compounds, including phenolic acids, flavonoids, organic acids, hydroxybenzoic aldehydes, amino acids, lignans, and terpenes. These phytochemicals underscore their potential applications in pharmaceuticals and functional food additives [9–14]. Scientific investigations have confirmed that Vicia seeds exhibit significant antioxidant properties, nitric oxide scavenging activity, metal chelation capacity, and enzyme inhibitory effects, further supporting their therapeutic potential [6].

Given the ecological and economic significance of Vicia species, a deeper understanding of their genetic diversity and evolutionary relationships is essential for optimizing their agricultural and biotechnological applications. Chloroplasts, as central organelles in plant metabolism, play a pivotal role in this context. They are indispensable for plant survival, converting solar energy into chemical energy through photosynthesis, while their genomes encode critical genes for photosynthesis and other metabolic processes [15–18]. Advances in cp genomics have profoundly enriched plant biology, offering insights into species diversity, evolutionary dynamics, and functional adaptation, knowledge that is instrumental in crop improvement and biotechnology. Structurally, the cp genome of land plants is characterized by a conserved quadripartite organization, comprising a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeat (IR) regions. Although typically circular and ranging between 107 kb and 218 kb in size, these genomes exhibit notable structural variability, including IR loss or gene family deletions, despite their overall stability. They commonly harbor 120–130 genes, predominantly associated with photosynthesis, transcription, and translation, reflecting their functional specialization. The utility of cp genome sequences extends across multiple disciplines. In phylogenetics, they serve as robust markers for resolving evolutionary relationships among species, while domestication studies leverage them to trace the origins and genetic shifts in cultivated plants. Chloroplast-derived DNA barcodes are particularly valuable for cultivar identification and the conservation of genetic resources, aiding in the protection of agrobiodiversity. Beyond traditional breeding, cp genomes are increasingly exploited in biotechnology, where their engineered variants enable the development of stress-resistant crops and the production of recombinant proteins, including vaccines and biopharmaceuticals, in edible plant systems [19–21].

Comparative genomic analyses of Vicia species demonstrate a consistent departure from the typical quadripartite chloroplast structure, primarily due to the loss of IRs. This structural simplification results in a tripartite genome organization, exemplified by V. bungei (130,796 bp), V. sepium (124,095 bp), and V. faba (122,569 bp) [22,23]. IR loss represents a key evolutionary adaptation in Vicia, contributing to both genome size reduction and structural rearrangements [22,24]. Notably, the cp genome of V. sepium illustrates this pattern, containing 110 genes and one pseudogene alongside specific gene and intron losses (ycf4, clpP intron, and rpl16 intron deletions) and insertions (rpl20 and ORF292) [24]. This trend extends to other Vicia species: V. ramuliflora (124,682 bp) maintains 109 genes [25], V. kulingana (125,696 bp) possesses 102 genes [26], and V. cracca (126,272 bp) retains 108 genes [27], all lacking IR regions. Indeed, despite the absence of IRs, core gene content remains remarkably conserved across these species, with protein-coding genes (75–77), tRNAs (28–30), and rRNAs (4) showing limited variability. In contrast, simple sequence repeat (SSR) abundance displays striking interspecific divergence; for instance, V. bungei contains 432 SSRs, over sixfold more than V. sepium (66 SSRs) [22,24], highlighting species-specific microsatellite accumulation patterns. These genomic modifications, particularly IR loss and SSR variability, likely influence evolutionary trajectories by altering gene dosage effects and mutation rates [28]. Collectively, these findings underscore the dynamic nature of cp genome evolution in Vicia, where structural simplification coexists with functional conservation and species-specific repeat diversification.

Despite significant progress in cp genome research, numerous economically and medicinally valuable Vicia species, particularly those indigenous to Iran, remain genomically uncharacterized. Notably, key species including V. hirsuta, V. hybrida, V. lathyroides, V. lutea, V. narbonensis, V. peregrina, and V. villosa currently lack complete cp genome sequences. This knowledge gap substantially hinders the comprehensive understanding of their genomic architecture, evolutionary relationships, and potential biotechnological utility. To address this critical research need, we present the first complete cp genome sequences for seven Iranian Vicia species, employing advanced sequencing and bioinformatic approaches. Our comprehensive investigation included: (1) high-quality genome assembly and annotation, (2) detailed structural characterization, and (3) comparative genomic analyses to identify both conserved and hypervariable regions with potential as molecular markers. Furthermore, we conducted systematic examinations of repetitive elements and codon usage patterns to elucidate evolutionary dynamics and functional adaptations. Phylogenetic reconstructions were performed to clarify taxonomic relationships within the genus and resolve evolutionary histories. This work establishes an essential genomic foundation for future studies on Vicia biodiversity, conservation genetics, and applied research. The newly generated cp genome data will facilitate: (1) precise species identification, (2) investigations of population genetics, and (3) exploration of adaptive mechanisms in this ecologically and economically significant legume genus. Moreover, these resources enable comparative genomic studies across Fabaceae and support efforts to harness Vicia genetic potential for agricultural improvement and medicinal applications.

Materials and methods

Plant material and DNA extraction

We collected leaf samples from seven Vicia species during their peak flowering period. The sampling encompassed distinct geographical locations across northern and central Iran. Specifically, four species, V. hirsuta, V. hybrida, V. lathyroides, and V. lutea, were sampled in the Golestan National Park near Gorgan. The remaining three species included V. narbonensis from Jajrood, Tehran Province, V. peregrina from Tapeh Abbas Abad, Hamedan Province, and V. villosa from Varamin, Tehran Province. No special collection permits were required as these species are neither endangered nor protected in the sampling locations, and all collections were conducted in accordance with local regulations. Total genomic DNA was isolated from 100 mg of fresh leaf tissue using the DNeasy Plant Mini Kit (QIAGEN, Germany). The DNA purity and concentration were evaluated using 1% agarose gel electrophoresis and a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA). Only high-quality DNA samples were selected for library preparation, which was carried out according to the manufacturer’s protocol. The sequencing was conducted on an Illumina HiSeq 2000 platform (Illumina Inc., USA), generating paired-end reads of 150 bp in length.

Cp genome assembly and annotation

The raw paired-end reads (150 bp in length) from each accession were initially evaluated for quality using FastQC v0.11.9 (available at: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter sequences and low-quality reads were removed using Trimmomatic v0.39 [29]. The processed high-quality reads were then assembled into complete cp genomes using GetOrganelle v1.7.7.1 [30]. To confirm genome integrity, the assembly graphs were examined and verified using Bandage [31]. For annotation, three tools were employed: CPGAVAS2 [32], GeSeq [33], and PGA [34]. Finally, circular visualizations of the cp genomes were generated with OGDRAW [35].

Genome feature characterization

In this study, Simple Sequence Repeats (SSRs) were detected using the MISA Perl Script [36] with threshold parameters set to a minimum of eight repeats for mononucleotide SSRs, four repeats for di- and trinucleotide SSRs, and three repeats for tetra-, penta-, and hexanucleotide SSRs. Long repetitive sequences were further analyzed using REPuter [37](available at http://bibiserv.techfak.uni-bielefeld.de/repeater/), with a minimum repeat size of 30 bp and a Hamming distance of three to ensure stringent identification of homologous regions. To assess codon usage bias, Relative Synonymous Codon Usage (RSCU) values were calculated using MEGA6 [38]. The RSCU patterns were visualized using an interactive RSCU plot generated with the RSCU-Plot Shiny app (available at: https://pcg-lab.shinyapps.io/RSCU-Plot/). Additionally, nucleotide diversity (Pi) for each gene was computed using CPStools [39], and the resulting data were visualized using R to facilitate comparative analysis of genetic variation.

To evaluate the evolutionary pressures acting on the chloroplast genomes, we conducted a selection pressure analysis across the shared protein-coding genes of the 16 Vicia species. This was performed using EasyCodeML [40], implementing a suite of site models to detect signatures of positive selection. We compared four nested model pairs (M0 vs. M3, M1a vs. M2a, M7 vs. M8, and M8a vs. M8) and employed likelihood ratio tests (LRTs) with a significance threshold of *p* < 0.05 to identify genes under diversifying selection. For each gene, the nonsynonymous-to-synonymous substitution rate ratio (ω = dN/dS) was calculated. Genes and specific codon sites were inferred to be under positive selection based on a combination of significantly elevated ω values and statistical support from the LRTs.

Comparative cp genomic analysis

A robust comparative genomic analysis was conducted to elucidate cp genome structure across 16 Vicia species. The study encompassed seven newly sequenced Vicia species, supplemented with data from nine additional species retrieved from public databases: V. bungei (MT362055) [18], V. costata (NC_057995), V. cracca (MW266076) [27], V. faba (MT120813), V. kulingana (PQ576733) [26], V. ramuliflora (MN758738) [25], V. sativa (NC_027155), V. sepium (NC_039595) [24], and V. tibetica (OR491712). Cp genome organization was compared and visualized using the BLAST Ring Image Generator (BRIG) software [41]. Furthermore, the Mauve multiple genome alignment algorithm [42] was employed to identify structural variations, including rearrangements, and assess collinearity, among the Vicia cp genomes.

Phylogenetic analysis

To elucidate the evolutionary relationships within the genus Vicia, we conducted a comprehensive phylogenetic analysis using coding sequences obtained from our sequenced species, supplemented with nine additional cp genomes retrieved from the NCBI database, collectively representing 16 Vicia species. Cicer arietinum was selected as the outgroup to root the phylogenetic tree. Initial sequence alignment was performed using MUSCLE v3.8.1551 [43] under default parameters to generate high-quality multiple sequence alignments. To enhance alignment accuracy, poorly aligned regions and gaps were removed using trimAl v1.4 [44] with stringent filtering criteria (“-gt 0.95 -st 0.001”). The refined alignments were subsequently concatenated into a single sequence matrix using SequenceMatrix [45], providing a unified dataset for phylogenetic reconstruction. Maximum likelihood analysis was executed in IQ-TREE [46] under the GTR + Gamma nucleotide substitution model, selected for its robustness in handling sequence evolution. Node support was evaluated through 1000 bootstrap replicates to assess the reliability of the inferred topology. The resulting phylogenetic tree was visualized and annotated using the Interactive Tree of Life (iTOL) platform [47], facilitating clear interpretation of evolutionary relationships among the studied taxa.

Assessment of hypervariable marker efficacy

To assess the phylogenetic applicability of the proposed barcode regions rps7 and rpl20, these loci were retrieved from a representative dataset spanning 16 Vicia species, with C. arietinum designated as the outgroup. Sequence alignments were generated, followed by stringent quality trimming and phylogenetic analysis using established bioinformatics workflows, as outlined in the “phylogenetic analysis” section.

Results

Cp genome assembly and annotation

High-quality sequencing reads were assembled de novo to generate complete, circularized cp genomes for all seven investigated Vicia species. The assembled genomes exhibited size variation ranging from 118,660 bp (V. peregrina) to 130,223 bp (V. hybrida), with intermediate sizes observed for V. hirsuta (124,717 bp), V. lathyroides (123,358 bp), V. lutea (123,404 bp), V. narbonensis (124,953 bp), and V. villosa (125,979 bp) (Fig 1). Structural annotation demonstrated distinct organizational patterns among the species. Vicia villosa maintained the ancestral angiosperm chloroplast structure, featuring a quadripartite organization comprising duplicate inverted repeat regions (IRa/IRb; 1,746 bp each) separating the large single-copy (LSC; 103,653 bp) and small single-copy (SSC; 18,831 bp) regions. In contrast, the remaining species (V. hirsuta, V. hybrida, V. lathyroides, V. lutea, V. narbonensis, and V. peregrina) exhibited the derived inverted-repeat-lacking clade (IRLC) architecture, characterized by complete loss of one IR copy. This structural reduction aligns with established evolutionary patterns observed in IRLC legumes, supporting the phylogenetic placement of these Vicia species within this clade.

Fig 1 — Key features, including gene locations and structural elements, are annotated to provide a comprehensive overview of the genome architecture.

The cp genomes of seven Vicia species were found to contain a conserved set of photosynthetic genes (Table S1), including five photosystem I subunits (psaA, psaB, psaC, psaI, psaJ), fourteen photosystem II subunits (psbA-K), six ATP synthase components (atpA-F), and eleven NADH dehydrogenase genes (ndhA-K). The cytochrome b/f complex was consistently represented by six genes (petA-D, petG, petL, petN), while the large subunit of Rubisco (rbcL) was present in all species. The self-replication machinery showed high conservation, with eight large ribosomal subunit proteins (rpl2, rpl14, rpl16, rpl20, rpl23, rpl32, rpl33, rpl36) and 11 small subunit proteins (rps2–4, rps7–8, rps11–12, rps14–15, rps18–19) identified across species. The DNA-dependent RNA polymerase subunits (rpoA, rpoB, rpoC1, rpoC2) were uniformly well-annotated. Transfer RNA complement ranged from 29–35 genes, with V. villosa showing the highest count (35) due to duplications in trnM-CAU (5 copies) and trnN-GUU (3 copies). The standard set of ribosomal RNAs (rrn5S, rrn4.5S, rrn16S, rrn23S) was complete in all species. accD was annotated in all species except V. lathyroides, where it appears to be a lost gene. The hypothetical ORF ycf2 was identified as a lost gene in V. narbonensis, while being properly annotated in other species. Other conserved functional genes included ccsA, cemA, clpP (with intron status varying by species), and matK, all of which were reliably annotated across the genus.

Genomic feature comparison

Microsatellite characterization across seven Vicia species (Fig 2) demonstrated conserved genomic architecture with marked mononucleotide predominance (74–87% of total SSRs), particularly A/T-rich repeats, reflecting the genus’ high AT-content. While V. hybrida contained the highest SSR density (77 total, including 16 compound microsatellites), all species shared similar repeat-type hierarchies (mono- > di- > tri-nucleotides) and dinucleotide bias toward AT/TA motifs (71–100% of dinucleotides). Notably, V. hybrida and V. peregrina exhibited elevated complex SSR counts (16 each), suggesting greater genomic plasticity, whereas V. lutea showed the simplest profile (45 mononucleotides, only 3 dinucleotides). The single hexanucleotide occurrence in V. villosa (CTCTTC) represents a rare departure from the predominant short-repeat architecture, potentially indicating species-specific transposable element activity. These patterns collectively highlight both deep conservation in microsatellite organization and subtle lineage-specific modifications in Vicia genome evolution.

Our analysis of repeat distributions across seven Vicia species revealed significant interspecific variation in repeat class composition and length distributions (Fig 3). The forward (F) class repeats dominated in most species, particularly in V. hybrida (n = 31 loci), V. lutea and V. villosa (n = 27), while V. peregrina showed the least number of F-class repeats (n = 19). V. hybrida exhibited the greatest length variation, with both extremely long (826 bp) and numerous short P-class repeats. Reverse (R) repeats were rare overall but showed species-specific patterns, with only two loci detected in V. hirsuta and complete absence in other species.

Fig 3 — Points show individual loci; labels indicate counts (n=) per class-species group.

Analysis of RSCU in seven Vicia species revealed strong biases toward A/T-ending codons, consistent with the high AT content typical of plant genomes (Fig 4). Notably, leucine (UUA, RSCU ≈ 2.00) and isoleucine (AUU, RSCU ≈ 1.50–1.57) were universally preferred, while phenylalanine (UUU, RSCU ≈ 1.39–1.43) and tyrosine (UAU, RSCU ≈ 1.56–1.63) also showed high usage. Stop codon preferences varied slightly, with UAA dominant in most species (RSCU ≈ 1.12–1.35) but UGA elevated in V. lathyroides and V. lutea (RSCU ≈ 1.14–1.15). Arginine codons exhibited the strongest divergence, with AGA highly favored (RSCU ≈ 1.61–1.83) and CGG underrepresented (RSCU ≈ 0.52–0.64). Minor species-specific differences were observed, such as higher UCG (serine) usage in V. villosa (RSCU = 0.58) compared to V. peregrina (RSCU = 0.55), and slightly elevated GCG (alanine) in V. hybrida (RSCU = 0.51) versus V. narbonensis (RSCU = 0.47).

Fig 4 — Codons are color-coded, and their corresponding amino acids are labeled below the plot.

Nucleotide diversity analysis

Analysis of cp genomes across seven Vicia species (Fig 5) revealed striking variation in nucleotide diversity (Pi), with the protease gene clpP (Pi = 0.19772) and hypothetical reading frame ycf1 (Pi = 0.16964) showing the highest divergence. Ribosomal protein genes exhibited extreme variability (rps7 [Pi = 0.09247], rpl20 [Pi = 0.08962]), while core photosynthetic genes were highly conserved (psbA [Pi = 0.00646], rbcL [Pi = 0.01331]). Transfer RNAs displayed a broad range (trnF-GAA [Pi = 0.02712] vs trnR-ACG [Pi = 0.00386]), as did NADH dehydrogenase subunits (ndhB [Pi = 0.04259] vs ndhE [Pi = 0.00996]). RNA polymerase subunits (Pi = 0.02653–0.03861) and cytochrome b/f complex genes (Pi = 0.00501–0.03671) showed intermediate variation.

Positive selection analysis of cp genes in Vicia species

Analysis of the chloroplast protein-coding genes across 16 Vicia species revealed significant signatures of positive selection in several genes, indicating a history of adaptive evolution (Table 1). The most pronounced signals were identified in genes encoding ribosomal proteins and components of photosynthetic and respiratory complexes, underscoring key functional categories that have undergone diversifying selection. Notably, multiple ribosomal protein genes exhibited strong evidence of positive selection, characterized by elevated nonsynonymous-to-synonymous substitution rate ratios (ω) and numerous codon sites with high posterior probabilities. These include rps2, rps3, rps4, rps7, and rps18, each harboring over ten positively selected sites. For instance, rps18 contained several sites under near-certain positive selection (e.g., 33Q, 37L, 87R, 121L), suggesting adaptive fine-tuning of the small ribosomal subunit. Similarly, the cemA gene, involved in carbon dioxide uptake and cytochrome c biogenesis, displayed one of the most extensive patterns of positive selection, with 20 identified sites, including four with extremely high confidence (181Q, 185V, 215V, 221T). Beyond ribosomal and metabolic genes, significant positive selection was also detected in subunits of the NADH dehydrogenase complex. The ndhF gene contained four positively selected sites, with 652M and 742L showing particularly strong support. This pattern points toward potential adaptive evolution in the chloroplast’s photosynthetic electron transport chain.

Table 1. The results of positive selective pressure analysis in M7 vs. M8 model.

Gene	LnL	ω	LRT p-value	Positively Selected Sites
cemA	−2370.63	3.89	< 0.001	2 A 0.896, 45 S 0.906, 55 F 0.989, 75 H 0.945, 98 C 0.943, 136 L 0.976, 150 L 0.905, 156 I 0.921, 162 K 0.944, 181 Q 0.993, 185 V 0.995, 190 L 0.981, 196 T 0.975, 197 L 0.961, 201 S 0.989, 208 R 0.997, 215 V 1.000, 221 T 1.000**
rps18	−1061.66	5.45	< 0.001	6 S 0.986, 28 L 0.970, 33 Q 1.000, 35 L 0.984*, 37 L 0.999, 77 S 1.000, 78 L 0.982, 80 A 0.976, 85 E 0.996, 87 R 1.000, 98 N 0.996
rps2	−1965.27	4.41	< 0.001	15 K 1.000, 20 F 0.999, 23 Y 0.975, 36 L 0.985, 37 G 0.962, 63 Y 0.996, 79 K 0.982, 83 S 0.969, 116 R 0.992, 117 Q 0.958, 124 E 1.000, 127 T 0.976, 168 V 0.987, 195 S 0.990, 215 R 0.980*
rps3	−1648.06	4.88	< 0.001	3 L 0.992, 5 N 0.999, 6 L 0.996, 9 N 0.999, 10 P 0.988, 11 E 0.996, 12 I 1.000, 17 H 0.997, 30 I 0.998, 31 N 0.952, 33 A 0.990, 36 T 0.979, 53 G 0.998, 60 P 0.979*, 134 I 0.999
rps4	−1631.34	6.86	< 0.001	26 R 0.985, 31 G 1.000, 34 Q 0.995, 59 Q 0.976, 68 A 0.967, 146 S 0.996, 147 A 0.990, 150 K 0.999, 159 P 0.998, 160 S 1.000*
rps7	−1796.03	6.98	< 0.001	11 S 0.996, 48 Y 0.984, 56 Y 0.969, 63 R 1.000, 64 E 1.000, 68 T 1.000, 70 A 0.995, 89 S 1.000, 110 Q 0.997, 121 L 1.000, 146 A 0.992, 147 F 0.982*, 150 I 0.999
rps11	−1467.02	5.55	< 0.001	106 Y 1.000, 107 R 0.998
rps19	−822.52	5.94	0.0045	84 T 0.996**
ndhF	−5020.78	3.88	< 0.001	46 L 0.964, 652 M 0.992, 742 L 0.998, 744 L 0.990*
ndhH	−2167.36	4.97	< 0.001	195 R 0.995**
petB	−1219.30	3.56	< 0.001	1 S 0.997**

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*: p < 0.05,

**: p < 0.01

Comparative analyses of cp genomes of Vicia species

A comprehensive comparison of sequence identity among 16 Vicia species identified distinct patterns of conservation and divergence, with protein-coding regions exhibiting significantly higher sequence identity (BLASTP) than noncoding regions (BLASTN), consistent with strong purifying selection on functional domains (Fig 6). Notably, several genes and intergenic regions displayed pronounced variability, including rps11, rpoA, rps18, rpl32, rpl33, rpl23, ycf1, ycf2, rpoC1, clpP, and accD, which contained segments with less than 70% sequence identity, suggesting potential hotspots for species-specific adaptation. Among noncoding regions, the spacers rps15-ycf1, ycf1-trnN-GUU, rrn16-rps12, ycf2-trnI-CAU, and psbB-petL were particularly divergent, likely due to reduced evolutionary constraints. Additionally, intronic regions such as the rpl16 intron exhibited high variability, further highlighting the dynamic nature of noncoding sequences.

Fig 6 — The reference genome is represented by the outer circle, while subsequent concentric circles illustrate pairwise sequence identity between *V. costata* and the 15 remaining species. **(A)** Nucleotide-level BLAST comparison. **(B)** Protein-level BLAST comparison.

Despite the overall conservation of cp genomes, the comparative analysis of 16 Vicia species using Mauve (Fig S1) revealed multiple structural rearrangements, including inversions, translocations, and localized collinearity breaks. These variations are particularly pronounced in the Papilionoideae subfamily, to which Vicia belongs, and provide critical insights into the evolutionary history of this group. The most pronounced rearrangements were observed between distantly related species (V. narbonensis vs V. villosa), whereas closely related pairs (V. sativa vs V. faba) maintained near-identical synteny.

Phylogenetic analysis of Vicia species

The seven Vicia species sequenced in this study (V. hirsuta, V. hybrida, V. lathyroides, V. lutea, V. narbonensis, V. peregrina, and V. villosa) alongside additional Vicia species retrieved from public databases, and Cicer arietinum used as an outgroup, were analyzed based on 86 common genes to depict the evolutionary relationships (Fig 7). The results revealed that V. peregrina, V. lutea, V. hybrida, and V. narbonensis formed a strongly supported monophyletic clade (bootstrap support = 100), indicating that these species shared a recent common ancestor. This clade clustered closely with V. faba, a widely cultivated species, suggesting potential genetic similarities and shared evolutionary traits. Similarly, V. hirsuta and V. villosa formed another well-supported monophyletic group (bootstrap = 100), indicating a distinct lineage within Vicia. These two species clustered with V. cracca and V. bungei, suggesting that they belong to a broader evolutionary group with climbing or vining growth habits. In contrast, V. lathyroides was positioned separately from these clades but remained within the main Vicia lineage. Its placement, along with V. sepium and V. sativa, suggested a more divergent evolutionary history, possibly due to differences in ecological adaptation or genome evolution. Despite its separate clustering, the tree topology confirmed that V. lathyroides still belonged to the Vicia genus. The overall phylogenetic structure supported Vicia as a monophyletic genus, with high bootstrap values reinforcing the reliability of these relationships. However, the placement of certain species suggested possible paraphyly within specific subgroups, particularly in relation to species not included in this study. For example, V. costata, V. tibetica, and V. ramuliflora formed a distinct lineage, which diverged earlier from the other Vicia species, indicating a separate evolutionary trajectory. The consistently high bootstrap values (mostly 100) confirmed the robustness of these evolutionary inferences.

Phylogenetic utility of rps7 and rpl20 in Vicia species

The phylogenetic trees reconstructed from the rps7 and rpl20 loci demonstrated strong congruence with the whole cp genome phylogeny (Fig 8), supporting their effectiveness as DNA barcodes for Vicia species. Both markers resolved the relationships among the seven studied species in a manner consistent with the reference tree. Notably, V. narbonensis and V. peregrina formed a well-supported clade across all analyses, while V. villosa and V. cracca consistently appeared as sister taxa and showed close relationship with V. hirsuta. The placement of V. lathyroides close to V. sativa and V. sepium further aligned with the whole-genome topology, reinforcing taxonomic relationships. Although minor variations in branch lengths were observed, the overall structure of the rps7 and rpl20 trees matched the whole-genome phylogeny, particularly in distinguishing major lineages. These findings confirm that both loci are reliable for species discrimination and phylogenetic reconstruction in Vicia, offering practical alternatives to full cp genome sequencing for future barcoding studies.

Discussion

The structural divergence observed in Vicia cp genomes, notably the loss of one IR in six of the seven species, strongly supports their classification within the IR-lacking clade and highlights a key genomic synapomorphy for this group. This aligned with earlier studies on V. sepium (124,095 bp) and V. bungei (130,796 bp), which also exhibited IR loss [18,22]. The results indicated that the size differences are primarily driven by expansions or contractions in protein-coding regions, particularly in accD, rps12, and ycf1, as previously reported in Fabeae [48]. The loss of IR regions in Vicia species likely contributes to accelerated cp genome evolution through several mechanisms, including increased mutation rates, facilitated genome rearrangements, and altered selection pressures on intron structure [49]. This phenomenon is well-documented across legumes, with numerous studies reporting gene and intron losses similar to those observed in our study. For instance, chickpea (Cicer arietinum) shows loss of introns from rps12 and clpP genes [50], Trifolium species have lost the accD gene [51,52], and Vicia species exhibit losses of rpl22, rps16, and one clpP intron [18]. These patterns of gene loss and structural simplification appear to be particularly prevalent in IRLC legumes, suggesting shared evolutionary trajectories following IR loss.

The lineage-specific losses of accD and ycf2 further illustrate the genomic plasticity following IR loss, a phenomenon where non-essential genes are often pseudogenized or functionally relocated to the nucleus, as seen across the IRLC [53,54]. The variation in tRNA complement, including notable duplications in V. villosa, underscores the increased structural plasticity in IR-lacking genomes, a pattern also reflected in the unique ORF and pseudogene profiles of species like V. sepium [22]. These findings supported the hypothesis that IR loss led to increased structural plasticity, as seen in other IRLC taxa like Lathyrus and Pisum [50]. The loss of one clpP intron in the studied species (except V. villosa) further corroborated the IRLC’s distinct evolutionary trajectory, as this reduction was widespread in the clade [55]. Similar intron losses in rpl16 (observed in V. faba) and clpP (in Glycyrrhiza glabra) suggested that these modifications might have arisen from relaxed selection pressures following functional gene transfers to the nucleus [56]. Additionally, the low GC content (34.7–35.1%) across Vicia cp genomes, including V. sepium and V. ramuliflora, may have reflected higher mutational rates in IR-lacking species [57].

The nucleotide diversity analysis revealed striking patterns of molecular evolution across the cp genomes of Vicia species. The protease gene clpP (π = 0.19772) and hypothetical reading frame ycf1 (π = 0.16964) emerged as the most divergent loci, consistent with previous reports of their hypervariability in legumes [58]. These findings support their continued use as primary DNA barcodes, while the intermediate variability observed in ribosomal protein genes rps7 (π = 0.09247) and rpl20 (π = 0.08962) identifies them as valuable supplementary markers. The phylogenetic trees reconstructed from these ribosomal protein genes showed strong congruence with the whole cp genome phylogeny, demonstrating their utility for species discrimination. The clpP gene, while useful for distinguishing some Actinidiaceae species [59], lacks universal resolution. Similarly, the ycf1 gene, which showed the second-highest π value in our analysis, has proven valuable for species identification in diverse plant groups [60] including Pinus [61] and Orchidaceae [62]. While the full-length ycf1 (approximately 7000 bp) is too long for conventional barcoding applications [58], our results support the use of rps7 and rpl20 that have shown high discrimination power across land plants.

Our findings offer tangible genomic resources that can directly inform and enhance conservation strategies for Vicia biodiversity. The high-resolution phylogenetic framework and the validated DNA barcodes (rps7 and rpl20) provide a reliable method for the precise identification of species and evolutionarily significant units (ESUs), which is a critical first step in prioritizing conservation targets [63]. The hypervariable regions identified, such as clpP and ycf1, along with the characterized SSR markers, are powerful tools for conducting population genetic studies. These markers can be used to assess genetic diversity, population structure, and gene flow across natural populations, identifying those that are genetically depauperate or isolated. Consequently, the complete chloroplast genomes presented here serve as a foundational reference for genotyping germplasm bank accessions, guiding the selection of material for seed banking (ex situ conservation) and enabling the management of wild populations (in situ conservation) to maximize the preservation of genetic diversity. By applying these genomic tools, conservation efforts can move beyond morphology-based identification and adopt a molecular-driven strategy to safeguard the adaptive potential and long-term survival of Vicia species.

Comprehensive phylogenetic analyses of Vicia species, integrating complete cp genome data and the well-supported barcoding loci rps7 and rpl20, elucidated complex evolutionary relationships with significant taxonomic implications. Our whole-genome and multi-locus phylogenetic reconstructions consistently identified V. sepium and V. sativa as sister species, corroborating previous findings by Li et al. (2020) [24]. The sister relationship between V. sepium and V. sativa aligns with their shared morphological characteristics, including similar floral morphology and the presence of adaxially hairy styles, as previously documented by Schaefer et al. (2012) [64]. Notably, V. faba formed a stable clade with these species across all analyses, suggesting shared evolutionary pathways. Furthermore, the early-diverging lineage comprising V. ramuliflora, V. tibetica, and V. costata was robustly supported, reinforcing their status as phylogenetically distinct from core Vicia taxa. The basal position of V. ramuliflora, V. tibetica, and V. costata mirrors earlier proposals that these species represent an ancestral lineage with unique ecological adaptations [64]. The strong concordance between our chloroplast-based phylogenies and prior plastome studies [24,64] underscores the continued value of cp genome data for resolving phylogenetic relationships at shallow to moderate evolutionary depths within Vicia. However, the persistent challenges in determining species boundaries and resolving deeper nodes highlight the need for complementary nuclear genomic data to provide a more comprehensive understanding of the genus’s evolutionary dynamics.

Conclusion

This study successfully delineated the complete chloroplast genomes of seven previously uncharacterized Iranian Vicia species, achieving its primary aim of expanding genomic resources for this economically significant genus. Our analyses uncovered substantial structural divergence, primarily driven by the predominant loss of one inverted repeat region in six species—a derived feature aligning them with IRLC, while V. villosa retained the ancestral quadripartite structure. Through comparative genomics, we identified clpP and ycf1 as hypervariable regions and established rps7 and rpl20 as highly effective barcoding markers, providing robust tools for species discrimination. Phylogenetic reconstruction based on 86 shared genes yielded a well-resolved topology, clarifying evolutionary relationships and affirming monophyletic groupings with high statistical support. The lineage-specific gene losses, including accD in V. lathyroides and ycf2 in V. narbonensis, further underscore the dynamic nature of chloroplast evolution in Vicia. To translate these findings into applied outcomes, we recommend: (1) adopting rps7 and rpl20 as standard markers for phylogenetic and barcoding studies; (2) integrating the characterized SSRs and variable loci into population genetic and diversity assessments; and (3) prioritizing functional investigation of divergent genes such as clpP, ycf1, accD, and ycf2 to elucidate their roles in adaptive and agronomic traits. This study establishes a critical genomic foundation for future research in Vicia systematics, conservation, and molecular breeding. However, to move from sequence correlation to functional understanding, future studies should employ comparative transcriptomics to assess RNA editing patterns and proteomic analyses to verify the expression and functional status of the proteins encoded by these variable genes.

Supporting information

S1 Table. Genes predicted in the chloroplast genome of seven Vicia species#: Intron number, (n): Gene copy number.

(PDF)

pone.0340630.s001.pdf^{(23.7KB, pdf)}

S1 Fig. Gene map and MAUVE alignment of 16 Vicia chloroplast genomes.

(PDF)

pone.0340630.s002.pdf^{(117.6KB, pdf)}

Data Availability

The assembled and annotated genomes are accessible in NCBI database under the research accessions PV364429, and PV480546-PV480551.

Funding Statement

The author(s) received no specific funding for this work.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: Weaknesses of the Article

1. Limited Scope of Species: The study focuses on only seven species of the Vicia genus, which may limit the generalizability of the findings. A broader sampling could provide a more comprehensive understanding of the genetic diversity within the genus.

2. Lack of Functional Analysis: While the article provides structural and phylogenetic insights, it lacks a detailed functional analysis of the genes identified. Understanding how these genes contribute to the phenotypic traits of Vicia species would enhance the study's relevance.

3. Absence of Comparative Data: Although the study discusses structural variations and gene losses, it could benefit from a more extensive comparison with chloroplast genomes of other legumes or related genera, which would contextualize the findings within a larger evolutionary framework.

4. Potential Methodological Limitations: The reliance on specific bioinformatics tools and methods for genome assembly and analysis may introduce biases. A discussion of the limitations of these tools and any potential impact on the results would strengthen the manuscript.

5. Nucleotide Diversity Analysis: While nucleotide diversity was assessed, the implications of this diversity on species adaptation and evolution are not fully explored. A deeper analysis could provide insights into how these variations affect the ecological success of the species.

6. Limited Discussion on Conservation Implications: Although the study mentions biodiversity conservation, it could expand on specific strategies or applications for conservation based on the genomic data presented.

7. Ethical Considerations: While the study indicates compliance with ethical guidelines, more detail on the ethical approval process for plant sampling could enhance transparency.

Conclusion

Addressing these weaknesses could improve the manuscript's robustness and provide a more comprehensive understanding of the implications of the research findings.

Reviewer #2: The manuscript was beautifully written with a unique study gap. Please refer to the uploaded manuscript with my few comments. Recommended for acceptance with minor revision. Correct the figure numberings.

**********

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Reviewer #1: Yes: Girma Abebe Adelo

Reviewer #2: No

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PLoS One. 2026 Mar 4;21(3):e0340630. doi: 10.1371/journal.pone.0340630.r002

Author response to Decision Letter 1

20 Oct 2025

Dear Editor and Reviewers,

I hope this message finds you well. We truly appreciate the time and effort you invested in thoroughly assessing our work. Your insightful comments and constructive criticisms have immensely contributed to improving the quality and clarity of our research. I want to assure you that we have carefully reviewed each of the comments provided by the reviewers and have made revisions to address concerns and suggestions. Below, we outline how we have incorporated their feedback into the revised manuscript:

Reviewer Comments:

Reviewer #1:

Comment 1: Limited Scope of Species: The study focuses on only seven species of the Vicia genus, which may limit the generalizability of the findings. A broader sampling could provide a more comprehensive understanding of the genetic diversity within the genus.

Response: We sincerely thank the reviewer for this insightful comment regarding the scope of our study. We agree that a broader sampling would enhance the generalizability of the findings. In response, we extended the scope of our phylogenetic analysis by incorporating chloroplast genome data from nine additional Vicia species obtained from public databases (NCBI), thereby strengthening the phylogenetic inferences presented in the manuscript. We fully acknowledge that sequencing additional, novel species would be ideal. However, the process of field collection (which is season-dependent), precise taxonomic identification, DNA extraction, sequencing, and the subsequent integrated re-analysis of the entire dataset constitutes a major, multi-year research project beyond the scope of the current study. Therefore, while we have maximized the analytical scope using all available genomic data, the practical constraints prevent the inclusion of newly sequenced specimens at this stage. We believe the current study, with its seven newly sequenced genomes and nine supplementary genomes, provides a significant and robust contribution to the genomic resources and phylogenetic understanding of the Vicia genus.

Comment 2: Lack of Functional Analysis: While the article provides structural and phylogenetic insights, it lacks a detailed functional analysis of the genes identified. Understanding how these genes contribute to the phenotypic traits of Vicia species would enhance the study's relevance.

Response: We thank the reviewer for this insightful comment regarding the functional implications of our genomic findings. We agree that understanding the phenotypic contributions of the identified genes, such as accD and ycf2, which showed lineage-specific losses, is a fascinating and important next step. However, the primary aim of this study was to establish a foundational genomic resource and elucidate the evolutionary relationships within Vicia through chloroplast genome sequencing and comparative analysis. Functional characterization of chloroplast genes typically requires extensive transgenic experiments, which fall outside the scope of this descriptive genomic work. Instead, our study successfully identified key candidate genes and highly variable regions that exhibit signatures of evolutionary selection. We have now explicitly stated in the manuscript that these specific genes represent high-priority targets for future functional studies to link sequence variation to phenotypic traits such as environmental adaptation and agronomic performance.

Comment 3: Absence of Comparative Data: Although the study discusses structural variations and gene losses, it could benefit from a more extensive comparison with chloroplast genomes of other legumes or related genera, which would contextualize the findings within a larger evolutionary framework.

Response: We thank the reviewer for this valuable suggestion. To strengthen the phylogenetic scope and evolutionary context of our study, we expanded our analyses by incorporating nine additional Vicia species obtained from public databases. Furthermore, we included Cicer arietinum, a member of a related legume genus, as an outgroup to root the phylogenetic tree and provide a broader evolutionary framework. The expanded analysis, now encompassing 16 Vicia species and one outgroup, has been integrated into the comparative genomics and phylogenetic sections.

Comment 4: Potential Methodological Limitations: The reliance on specific bioinformatics tools and methods for genome assembly and analysis may introduce biases. A discussion of the limitations of these tools and any potential impact on the results would strengthen the manuscript.

Response: We thank the reviewer for raising this important point regarding methodological limitations. In response, we have revised the Conclusion to explicitly acknowledge that our genomic and in silico analyses represent a foundational step. We have added a sentence stating that "future studies are needed to move from sequence correlation to functional understanding, specifically through comparative transcriptomics to assess RNA editing patterns and proteomic analyses to verify the expression and functional status of the identified variable genes, particularly in species with gene losses like accD and ycf2.

Comment 5: Nucleotide Diversity Analysis: While nucleotide diversity was assessed, the implications of this diversity on species adaptation and evolution are not fully explored. A deeper analysis could provide insights into how these variations affect the ecological success of the species.

Response: We thank the reviewer for this valuable suggestion. To address the evolutionary implications of nucleotide diversity, we have now performed a comprehensive selection pressure analysis on the protein-coding genes. The methods and results of this analysis have been incorporated into the respective sections of the manuscript. Specifically, we used EasyCodeML to assess selective pressure across the chloroplast genomes, employing site models to identify genes and specific amino acid sites under positive selection. This addition provides critical insights into how sequence variations may have contributed to adaptive evolution and ecological success in the studied Vicia species.

Comment 6: Limited Discussion on Conservation Implications: Although the study mentions biodiversity conservation, it could expand on specific strategies or applications for conservation based on the genomic data presented.

Response: We thank the reviewer for this valuable suggestion to elaborate on the conservation implications of our findings. In response, we have expanded the Discussion section to include more specific strategies for biodiversity conservation. The revisions now explicitly discuss how the identified hypervariable regions (e.g., clpP, ycf1) and the newly developed barcodes (rps7, rpl20) can be directly applied to accurately identify species and delineate evolutionarily significant units (ESUs) within Vicia. Furthermore, we highlight how the complete chloroplast genomes serve as essential references for genotyping germplasm collections, enabling the assessment of genetic diversity and the identification of unique haplotypes to prioritize populations for in situ and ex situ conservation efforts.

Comment 7: Ethical Considerations: While the study indicates compliance with ethical guidelines, more detail on the ethical approval process for plant sampling could enhance transparency.

Response: We thank the reviewer for this suggestion. We have revised the 'Plant material and DNA extraction' section to provide more detail, confirming that the collection of these common, non-protected plant species from public areas complied with all relevant local guidelines and did not require specific ethical permits.

Reviewer #2:

The manuscript was beautifully written with a unique study gap. Please refer to the uploaded manuscript with my few comments. Recommended for acceptance with minor revision. Correct the figure numberings.

Comment 1: Abstract is written as one paragraph. Kindly rewrite it following the authors guide.

Response: Thank you for the suggestion. We have revised the abstract to follow the journal’s author guidelines and presented it as a single, well-structured paragraph.

Comment 2: Please note that the section you provided explains the justification of the study rather than its objective. Kindly restate it clearly as a specific research objective, outlining what the study aims to achieve rather than why it was conducted

Response: Thank you for your valuable comment. We have rewritten the abstract to clearly state the specific research objective, emphasizing what the study aimed to achieve rather than the justification for conducting it.

Comment 3: Kindly use keywords not included in the title and arrange alphabetically.

Response: We thank the reviewer for the comment. The keywords have been revised accordingly.

Comment 4: This figure should be labeled as Figure 4, not Figure 5. Kindly correct the numbering both in the figure caption and throughout the corresponding text where it is referenced.

Response: We appreciate the reviewer's careful attention to detail. The numbering has been corrected as suggested

Comment 5: Please revise the discussion section to avoid presenting detailed results. Summarize findings briefly and focus on interpreting and explaining the results rather than restating them

Response: We thank the reviewer for this important feedback. The Discussion section has been thoroughly revised to reduce the restatement of detailed results and to strengthen the interpretation and synthesis of our findings in the context of existing literature.

Comment 6: Kindly revise the conclusion to align more closely with the main aim of the study and include clear, concise recommendations based on the findings.

Response: We thank the reviewer for this valuable suggestion. The Conclusion has been revised to align more closely with the main aims of the study and to include clear, concise recommendations based on our findings.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0340630.s005.docx^{(16.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0340630.r003

Decision Letter 1

Md Mahmudul Hasan

20 Nov 2025

Dear Dr. Soorni,

Please submit your revised manuscript by Jan 04 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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We look forward to receiving your revised manuscript.

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Md. Mahmudul Hasan, PhD

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The manuscript titled "Structural and phylogenetic insights from complete chloroplast genomes of seven Vicia species" offers significant contributions to the understanding of chloroplast genomes within the Vicia genus. However, several weaknesses and areas for improvement merit attention:

Weaknesses:

Lack of Specificity in Objectives: The abstract does not clearly define the specific research questions or aims, which could leave readers uncertain about the focus of the study. A more precise articulation would help frame the research within the broader context of legume phylogenetics.

Limited Contextual Background: The abstract lacks sufficient background on the importance of Vicia species. Providing context about their ecological and agricultural significance would underscore the relevance of the findings.

Insufficient Detail on Findings: While the abstract summarizes key results, it does not effectively convey their significance in advancing the understanding of chloroplast genome structures or evolutionary dynamics. Highlighting these contributions would enhance reader engagement and interest.

Omission of Practical Applications: The potential implications of identified genomic resources for conservation and agricultural practices are not addressed in the abstract. Including these applications can demonstrate the practical relevance of the research.

Absence of Methodological Overview: The absence of a brief mention of the methods used for genome sequencing and analysis limits the transparency and rigor of the findings. Providing this information would enhance the credibility of the study.

Additional Comments:

Ethics Considerations: It would be beneficial for the authors to include detailed information regarding ethical approvals for plant collection and research protocols. This transparency is crucial for maintaining ethical standards in research.

Concerns about Dual Publication: The authors should ensure that the research is not simultaneously submitted to other journals to prevent issues of dual publication. Transparent communication regarding submission status is vital.

Publication Ethics: A clear description of data accessibility, including how the chloroplast genomes can be accessed by the scientific community, should be included. Ensuring that research findings are made available and usable by others is an important aspect of publication ethics

Reviewer #2: (No Response)

**********

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Reviewer #1: Yes: Girma Abebe Adelo

Reviewer #2: No

**********

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Attachment

Submitted filename: COMMENT 12.docx

pone.0340630.s006.docx^{(13.5KB, docx)}

PLoS One. 2026 Mar 4;21(3):e0340630. doi: 10.1371/journal.pone.0340630.r004

Author response to Decision Letter 2

22 Nov 2025

Dear Editor and Reviewers,

Reviewer Comments:

Reviewer #1:

Comment 1: Lack of Specificity in Objectives: The abstract does not clearly define the specific research questions or aims, which could leave readers uncertain about the focus of the study. A more precise articulation would help frame the research within the broader context of legume phylogenetics.

Response: We sincerely thank the reviewer for this comment. We refined the abstract to clearly state the research aim

Comment 2: Limited Contextual Background: The abstract lacks sufficient background on the importance of Vicia species. Providing context about their ecological and agricultural significance would underscore the relevance of the findings.

Response: We thank the reviewer for this insightful comment. The opening sentence has been strengthened to immediately establish the genus's significance: "The legume genus Vicia L. (Fabaceae) is of significant ecological and agronomic importance, comprising species widely utilized as forage crops, green manure, and sources of valuable phytochemicals."

Comment 3: Insufficient Detail on Findings: While the abstract summarizes key results, it does not effectively convey their significance in advancing the understanding of chloroplast genome structures or evolutionary dynamics. Highlighting these contributions would enhance reader engagement and interest.

Response: We thank the reviewer for this valuable suggestion. We now present key findings with greater contextual significance in abstract.

Comment 4: Omission of Practical Applications: The potential implications of identified genomic resources for conservation and agricultural practices are not addressed in the abstract. Including these applications can demonstrate the practical relevance of the research.

Response: We thank the reviewer for raising this important point. We completely revised abstract to address this point.

Comment 5: Absence of Methodological Overview: The absence of a brief mention of the methods used for genome sequencing and analysis limits the transparency and rigor of the findings. Providing this information would enhance the credibility of the study.

Response: We thank the reviewer for this valuable suggestion. We significantly expanded the methodological description to enhance transparency and rigor.

Comment 6: Ethics Considerations: It would be beneficial for the authors to include detailed information regarding ethical approvals for plant collection and research protocols. This transparency is crucial for maintaining ethical standards in research.

Response: We thank the reviewer for this comment. As noted in the Material and methods section under "Plant material and DNA extraction," the following statement was already included to address ethical considerations:

"No special collection permits were required as these species are neither endangered nor protected in the sampling locations, and all collections were conducted in accordance with local regulations."

This statement confirms that the plant collection adhered to all relevant guidelines and that no specific permits were necessary, ensuring full ethical compliance.

Comment 7: Concerns about Dual Publication: The authors should ensure that the research is not simultaneously submitted to other journals to prevent issues of dual publication. Transparent communication regarding submission status is vital.

Response: We thank the reviewer for raising this important standard of academic integrity. We confirm that this manuscript is original and has not been published elsewhere. Furthermore, it is not currently under consideration for publication in any other journal. We are fully committed to avoiding any form of dual submission or publication.

Comment 8: Publication Ethics: A clear description of data accessibility, including how the chloroplast genomes can be accessed by the scientific community, should be included. Ensuring that research findings are made available and usable by others is an important aspect of publication ethics

Response: We thank the reviewer for underscoring the importance of data accessibility. As detailed in the "Availability of Data and Materials" section, the complete chloroplast genome sequences generated in this study have been deposited in the NCBI database and are publicly accessible under the research accessions PV364429, and PV480546-PV480551. This ensures that all data supporting the findings of this study are fully available to the scientific community.

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Submitted filename: Response to Reviewers V2.docx

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PLoS One. doi: 10.1371/journal.pone.0340630.r005

Decision Letter 2

Md Mahmudul Hasan

23 Dec 2025

Structural and phylogenetic insights from complete chloroplast genomes of seven Vicia species

PONE-D-25-42003R2

Dear Dr. Soorni,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Md. Mahmudul Hasan

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0340630.r006

Acceptance letter

Md Mahmudul Hasan

PONE-D-25-42003R2

PLOS One

Dear Dr. Soorni,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

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PLOS One

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Genes predicted in the chloroplast genome of seven Vicia species#: Intron number, (n): Gene copy number.

(PDF)

pone.0340630.s001.pdf^{(23.7KB, pdf)}

S1 Fig. Gene map and MAUVE alignment of 16 Vicia chloroplast genomes.

(PDF)

pone.0340630.s002.pdf^{(117.6KB, pdf)}

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Submitted filename: commentt GGG.docx

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Submitted filename: PONE-D-25-42003.pdf

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Submitted filename: COMMENT 12.docx

pone.0340630.s006.docx^{(13.5KB, docx)}

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Submitted filename: Response to Reviewers V2.docx

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Data Availability Statement

The assembled and annotated genomes are accessible in NCBI database under the research accessions PV364429, and PV480546-PV480551.

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PERMALINK

Structural and phylogenetic insights from complete chloroplast genomes of seven Vicia species

Mohammad Mehdi Golchini

Aboozar Soorni

Roles

Abstract

Introduction

Materials and methods

Plant material and DNA extraction

Cp genome assembly and annotation

Genome feature characterization

Comparative cp genomic analysis

Phylogenetic analysis

Assessment of hypervariable marker efficacy

Results

Cp genome assembly and annotation

Fig 1. Circular representations of Vicia cp genomes, generated using OGDRAW.

Genomic feature comparison

Fig 2. The types and distribution of SSRs along the chloroplast genomes of seven Vicia species.

Fig 3. Repeat length distributions by species and repeat class (Forward (F)=blue, Palindromic (P)=green) in Vicia taxa.

Fig 4. The bar plot of the Relative Synonymous Codon Usage (RSCU) values for each amino acid, grouped by species.

Nucleotide diversity analysis

Fig 5. Nucleotide diversity (Pi) across coding regions in the cp genomes of seven Vicia species.

Positive selection analysis of cp genes in Vicia species

Table 1. The results of positive selective pressure analysis in M7 vs. M8 model.

Comparative analyses of cp genomes of Vicia species

Fig 6. Comparative sequence identity analysis among 16 Vicia species.

Phylogenetic analysis of Vicia species

Phylogenetic utility of rps7 and rpl20 in Vicia species

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Md Mahmudul Hasan

Roles

Author response to Decision Letter 1

Decision Letter 1

Md Mahmudul Hasan

Roles

Author response to Decision Letter 2

Decision Letter 2

Md Mahmudul Hasan

Roles

Acceptance letter

Md Mahmudul Hasan

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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