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. 2024 Apr 5;19(4):e0301346. doi: 10.1371/journal.pone.0301346

Complete plastid genome of Iris orchioides and comparative analysis with 19 Iris plastomes

Tae-Young Choi 1, Soo-Rang Lee 1,*
Editor: Branislav T Šiler2
PMCID: PMC10997070  PMID: 38578735

Abstract

Iris is a cosmopolitan genus comprising approximately 280 species distributed throughout the Northern Hemisphere. Although Iris is the most diverse group in the Iridaceae, the number of taxa is debatable owing to various taxonomic issues. Plastid genomes have been widely used for phylogenetic research in plants; however, only limited number of plastid DNA markers are available for phylogenetic study of the Iris. To understand the genomic features of plastids within the genus, including its structural and genetic variation, we newly sequenced and analyzed the complete plastid genome of I. orchioides and compared it with those of 19 other Iris taxa. Potential plastid markers for phylogenetic research were identified by computing the sequence divergence and phylogenetic informativeness. We then tested the utility of the markers with the phylogenies inferred from the markers and whole-plastome data. The average size of the plastid genome was 152,926 bp, and the overall genomic content and organization were nearly identical among the 20 Iris taxa, except for minor variations in the inverted repeats. We identified 10 highly informative regions (matK, ndhF, rpoC2, ycf1, ycf2, rps15-ycf, rpoB-trnC, petA-psbJ, ndhG-ndhI and psbK-trnQ) and inferred a phylogeny from each region individually, as well as from their concatenated data. Remarkably, the phylogeny reconstructed from the concatenated data comprising three selected regions (rpoC2, ycf1 and ycf2) exhibited the highest congruence with the phylogeny derived from the entire plastome dataset. The result suggests that this subset of data could serve as a viable alternative to the complete plastome data, especially for molecular diagnoses among closely related Iris taxa, and at a lower cost.

Introduction

The genus Iris L. (~280 species; Iridaceae), a collection of perennial herbs, is widespread across the globe. It is the most diverse taxonomic group within the family, with a presence throughout the Northern Hemisphere [1–3]. Iris displays significant cytological variability, typically with x = 10 or 12 chromosomes, though some taxa exhibit varying numbers due to substantial polyploidy and aneuploidy [4, 5]. The genus also shows high morphological diversity, which can be partly explained by its diverse habitat types, from mesic to xeric [6]. Geophytic adaptations, such as rhizomes (found in subg. Iris and Limniris), bulbs (found in subg. Xiphium, Scorpiris = Juno, and Hermodactylodes), and occasionally swollen storage roots (found in subg. Nepalensis; [8]), are evident, particularly in xeric habitats, accompanied by slender, linear leaves and recessed stomata [1, 2, 7]. Notably, Iris also exhibits unique floral characteristics, such as petaloid-style branches, discrete perianth whorls, and a floral tube with basal nectary tissue [3, 7, 8]. Renowned for their exquisite beauty, Iris flowers are highly sought after as ornamental plants and have been utilized for culinary, medicinal, and horticultural purposes since ancient times, tracing back to ancient Greece [9].

Iris is one of the most taxonomically challenging groups. The genus comprises six subgenera and 12 sections, delineated based on characteristics such as sepal beards, crests, seed arils, and underground storage organs [7, 10–13]. However, recent molecular studies have documented discrepancies between the morphological and molecular data in terms of phylogenetic relationships [2, 6, 14, 15]. Certain taxa, grouped together due to morphological similarities, often fail to form cohesive genetic clusters in molecular phylogenies [2, 6, 14, 15]. In addition, the key morphological characteristics used for taxonomic classification lack homology or synapomorphy [2, 6, 14, 15], further complicating the taxonomy. These taxonomic difficulties are likely further complicated by frequent hybridization among congeners [16–18]. In fact, inter- and intra-specific hybridization has been strongly associated with the taxonomic issues in Iris [18–20]. Considering these taxonomic challenges, comprehensive and updated investigations into the phylogeny of Iris are required. However, genomic information that can be applied to solve these taxonomic difficulties is limited. For instance, the most recent molecular phylogenetic study on Iris largely depended on just a few plastid markers, including matK, ndhF, trnL-trnF, trnQ-rps16, and trnS-trnfM, leading to many unresolved taxonomic relationships [21].

Owing to the conserved nature of the plastid genome among angiosperms, it offers useful molecular tools for phylogenetic analyses, particularly at higher taxonomic levels [22, 23]. Starting with rbcL [24–26] and followed by atpB [27, 28], dozens of plastid regions, such as ndhF, matK, trnL-trnF, and trnS-trnG, have been widely applied in phylogenetic studies [23, 29]. More recently, employing complete plastid genome sequences in phylogenetic studies has become feasible. In fact, plastome phylogenies have shown promising potential in resolving complex phylogenetic relationships among problematic taxa [23]. One compelling example is the phylogenetic position of Amborella in angiosperms, as demonstrated by Drew et al. [30]. In accordance with recent genomic advances, several studies have been conducted on the plastid genomes of Iris. However, the Iris plastomes exhibited relatively conservative sizes and structures [31, 32]. Given the significance of plastid markers with maternally inherited characteristics, identifying plastid markers with varying levels of polymorphism is of great importance to understanding the complexity of the phylogenetic relationships among Iris taxa.

With the advent of advanced sequencing techniques, over 90 Iris plastomes have recently been characterized (http://www.ncbi.nlm.nih.gov/genomes) [31, 33–36]. Genome-scale markers provide good insight into phylogenetic relationships, as observed in many plastome phylogenies [37]. In this study, we aimed to 1) determine the complete structure of the plastome of Iris orchioides (endemic to Central Asia [38]), 2) characterize the architecture and molecular evolution of Iris plastomes via comparative analysis with known Iris plastomes, and 3) identify appropriate plastid markers for distinguishing the phylogenetic relationships among Iris taxa.

Methods

Ethics approval and consent to participate

The plant material in this study was obtained from the wild with a legal permission for sample collection. This study protocol complies with relevant institutional, national, and international guidelines and legislation. This study protocol also complies with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.

Sampling, DNA isolation, and sequencing

In this study, we sequenced and assembled the complete plastid genome of I. orchioides. Young I. orchioides leaves were collected from Yangiabad, Uzbekistan (N 41°08′32.2″, E 70°07′30.4″). The voucher specimen was prepared and deposited at the Herbarium of the Korea National Arboretum (KH) under accession number KHB1544459. We identified the species according to the key morphological characters described by Sennikov et al. [38]. The collected leaf samples were quickly dried with silica gel in a zip-lock plastic bag and stored at room temperature until further use. Genomic DNA was isolated using the DNeasy Plant Mini Kit, following the manufacturer’s protocol (Qiagen, Hilden, Germany). We checked the quantity of the isolated DNA using a NanoDrop ND1000 system (Thermo Fisher Scientific, MA, USA; quality cutoff, OD 260/280 ratio between 1.7–1.9). The isolated DNA was visualized using 1% agarose gel electrophoresis. We constructed the Illumina paired-end libraries of I. orchioides with insert sizes of 300 bp and sent to LABGENOMICS (http://www.labgenomics.co.kr, Sungnamsi, Korea) for sequencing. The prepared libraries were sequenced on MiSeq platform (Illumina Inc., San Diego, CA, USA). We filtered poor quality reads (Phred score, Q < 20) with trim function implemented in CLC Assembly Cell package v. 4.2.1 (CLC Inc., Denmark).

Genome assembly and annotation

The complete plastid genome of I. orchioides was assembled by employing the low-coverage whole-genome sequence (dnaLCW) method [39] with the CLC de novo assembler (CLC Assembly Cell package) and SOAPdenovo (SOAP package v. 1.12). We used default setting for all parameter values for the pipeline run. The Gapcloser function (SOAP package) was applied for gap filling. We also performed a reference-based genome assembly using the complete genome sequence of I. domestica (GenBank accession number: PP475539). For the reference-based assembly, the contigs obtained from the primary de novo assemblies were aligned to the reference plastid genome. The aligned contigs were then assembled in Geneious v. 2019.0.4 (http://www.geneious.com).

We annotated the assembled plastid genome using the Dual Organellar GenoMe Annotator (DOGMA [40]) with a few modifications for the start and stop codons. Plastid-bacterial genetic codes were used to determine the protein-coding genes. To confirm the tRNA boundaries, we scanned the tRNAs using tRNAscan-SE with the default settings [41]. The circular plastome map was visualized using OGDRAW (http://ogdraw.mpimp-golm.mpg.de/). The assembled and annotated plastid genome sequences of I. orchioides were deposited in GenBank (MT254070.1).

Genome structure and comparative analysis

We compared the genome structure, size, gene content, and number of repeats across the 20 Iris taxa including I. orchioides. To simplify the comparison process among the numerous available samples (approximately 90 accessible via https://www.ncbi.nlm.nih.gov/genome/organelle/), we opted to focus on one or a few taxa at the section level. All plastid genomes, excluding that of I. orchioides, were downloaded from GenBank (S1 Table). The GC content was determined using Geneious software. All plastome sequences of the 20 Iris taxa were aligned on MAFFT (http://mafft.cbrc.jp/alignment/server/) with the default settings. The aligned sequences were then visualized using the Shuffle-LAGAN mode in mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml). We used the complete and annotated plastome of I. gatesii as a reference to plot the mVISTA results. We further visualized IR boundaries using IRscope to compare among 20 Iris species (Amiryousefi et al. 2018). Sequence divergence (π) among the 20 Iris taxa was computed in DnaSP v. 6.0 [42], using a 600-bp window size and a 200-bp step size. We used CodonW (http://codonw.sourceforge.net/) to analyzed the distribution of the codon usage with RSCU ratio for all protein-coding genes.

Repeat elements were identified using the two following approaches: 1) using web-based SSR finder MISA-web (https://webblast.ipk-gatersleben.de/misa/) with varying thresholds (10 for mono-, 5 for di-, 4 for tri-, and 3 for tetra-, penta-, and hexa-nucleotide repeats); 2) REPuter was used to determine the size and type of repeats [43]. For REPuter analysis, the parameters were set as follows: minimal repeat size, 30 bp; hamming distance, 3 kb; sequence identity, ≥ 90%.

Identifying plastid markers and testing the utility in phylogenetic inferences

We further assessed the phylogenetic informativeness (PI) of each protein coding gene and 5 Intergenic Spacer (IGS) regions using PhyDesign [44]. Plastomes typically contain over 100 Intergenic Spacer (IGS) regions, and the positions of each IGS can sometimes overlap with other genes. To simplify the analysis and avoid complexity, we opted to include only the 5 most variable Intergenic Spacer regions (IGSs) based on the π values estimated from DnaSP for the PhyDesign analysis. The HyPhy algorithm and per-site profile approach were used to calculate substitution rates per site with the default settings [45]. We inferred ML trees for 20 Iris taxa to identify high PI regions based on the concatenated sequence data of 79 protein coding genes and the 5 hypervariable IGSs. Subsequently, we transformed the ML tree into an ultrametric tree using the chronos function implemented in the ape R package [46]. Following the ML tree inference and ultrametric tree construction, we proceeded to estimate phylogenetic informativeness and selected the 10 most informative regions (5 genes and 5 IGSs). To assess the effectiveness of the selected regions, we conducted Maximum Likelihood (ML) tree inference for each of the 10 regions individually. Additionally, we created concatenated datasets from these 10 regions and inferred ML phylogenies to evaluate the performance of the combined dataset. In total, we generated 1,023 ML trees from the concatenated data, employing various combinations.

We reconstructed a phylogeny from complete plastome sequences of 73 Iris taxa with 84 accessions obtained from GenBank, along with two species of Moraea (Iridaceae) as outgroups (genome sizes and GenBank accession numbers are listed in S1 Table). The 86 plastome sequences were aligned using MAFFT with default settings and were then manually edited for ambiguous locations using the Geneious alignment viewer. Gaps in the sequences were treated as missing data. We inferred the phylogeny using and ML methods. To determine the best-fitting substitution model, the Akaike information criterion, implemented in jModelTest v. 2.1.10 [47], was used. The ML phylogeny was constructed using RAxML v. 8.2.4 based on the GTR GAMMA model with 1,000 rapid bootstrap replicates for the node support.

We used the plastome tree to test utility of the selected plastid regions in phylogenetic analyses. The trees inferred from the high PI regions were compared with the complete plastome phylogeny as a reference. If a tree inferred from one of the selected regions exhibited congruence with the plastome tree, we regarded the selected region as a viable alternative to the entire plastome dataset for phylogenetic reconstruction. To evaluate this congruence, we utilized TreeDist, an R script (https://github.com/ms609/TreeDist) that measures the topological differences between pairs of trees using generalized Robinson-Foulds distances, which compare bipartitions between trees.

Results

Plastid genome assembly and genome annotation of Iris orchioides

The genomic library of I. orchioides produced 10 million high-quality 300-bp paired-end reads. The average number of reads after initial trimming was approximately 9 million, and the average per-base coverage was 241 (Table 1). The final plastid genome of I. orchioides assembled in the current study showed a typical quadripartite structure divided into four regions, including a pair of inverted repeats (IRs; 25,508 bp), a large single-copy region (LSC; 82,271 bp), and a short single-copy region (SSC; 18,335 bp; Fig 1 and Table 1). The plastid genome of I. orchioides contained 113 genes comprising 79 protein-, 30 tRNA-, and 4 rRNA-coding genes (Table 2). Of the 113 genes, 20 genes (all four rRNA-, eight of the tRNA-, and six of the protein-coding genes, as well as two conserved ORFs) were duplicated, resulting in a total of 133 genes (Table 2). We identified 15 genes carrying a single intron and three genes (rps12, ycf3, and clpP) with two introns in I. orchioides (Table 2); one gene (psbZ) contained three introns (Table 2). No pseudogenes were observed in the I. orchioides plastid genome (Table 2).

Table 1. Summary of the plastid genome characteristics and sample sources of the 20 Iris taxa used in this study.

Collection- and assembly-related information is only presented for the newly sequenced Iris orchioides plastome.

Subgeneric classification Species NCBI accession No. Total length (bp) LSC length (bp) SSC length (bp) IRa length (bp) IRb length (bp) Total GC content (%) Total number of genes
subg. Iris sect. Iris Iris germanica MZ571477 158,816 82,596 18,522 28,849 28,849 38.1% 113
subg. Iris sect. Psammiris Iris bloudowii PP069562 153,322 82,364 18,524 26,217 26,217 37.9% 113
subg. Iris sect. Oncocyclus Iris afghanica OR098702 153,234 82,517 18,373 26,172 26,172 37.9% 113
subg. Iris sect. Oncocyclus Iris gatesii KM014691 153,441 82,659 18,376 26,203 26,203 37.9% 113
subg. Iris sect. Iris (= Pardanthopsis) Iris dichotoma MK593157 153,573 83,071 18,140 26,181 26,181 37.9% 113
subg. Iris sect. Iris (= Belmacanda) Iris domestica MT001880 153,724 83,127 18,169 26,214 26,214 37.9% 113
subg. Scorpiris Iris orchioides PP475539 151,622 82,271 18,335 25,508 25,508 38.0% 113
subg. Scorpiris Iris hippolyti OK138594 151,171 81,938 18,307 25,463 25,463 38.0% 113
subg. Scorpiris Iris pseudocapnoides OM990831 151,393 82,228 18,239 25,463 25,463 38.0% 113
subg. Xiphium Iris rutherfordii OP715666 153,239 82,500 18,383 26,178 26,178 38.2% 113
subg. Hermodactyloides Iris tuberosa OP715674 153,239 82,500 18,371 26,184 26,184 37.9% 113
subg. Limniris sect. Limniris ser. ensatae Iris lactea MT740331 152,409 82,257 18,102 26,025 26,025 38.0% 113
subg. Limniris sect. Limniris ser. Tenuifoliae Iris loczyi MT254070 150,940 80,907 17,853 26,090 26,090 38.3% 113
subg. Limniris sect. Limniris ser. Longipetalae Iris missouriensis MH251636 153,084 82,405 18,255 26,212 26,212 37.9% 113
subg. Limniris sect. Limniris ser. Laevigatae Iris pseudacorus MK593164 152,562 82,786 17,880 25,948 25,948 37.9% 113
subg. Limniris sect. Limniris ser. Ruthenicae Iris ruthenica MK593167 152,287 82,311 18,136 25,920 25,920 38.2% 113
subg. Limniris sect. Limniris ser. Sibiricae Iris sanguinea KT626943 152,408 82,340 18,016 26,026 26,026 38.0% 113
subg. Limniris sect. Limniris ser. Chinenses Iris speculatrix OK274247 152,368 82,003 17,941 26,212 26,212 38.0% 113
subg. Limniris sect. Lophiris Iris japonica OK448493 152,443 83,237 18,490 25,358 25,358 37.9% 113
subg. Limniris sect. Lophiris Iris tectorum MW201731 153,253 82,833 18,562 25,929 25,929 37.9% 113
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Fig 1. Gene map of the complete plastid genome of Iris orchioides.

Fig 1

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The conserved plastid genes are presented in the colored boxes. Genes located inward from the black-lined circle are transcribed clockwise, whereas the ones outside the circle are transcribed counter-clockwise. The gray bars shown in the inner circle indicate the GC content.

Table 2. List of genes found in the Iris orchioides plastome.

×2 indicates the genes that were duplicated in the IR regions. Asterisks refer to genes with one (*), two (**), or three introns (***).

Genes groups Genes names
Transcription & translation Large subunit ribosomal proteins rpl2(×2)*, rpl14, rpl16*, rpl20, rpl22, rpl23(×2), rpl32, rpl33, rpl36
Small subunit ribosomal proteins rps2, rps3, rps4, rps7(×2), rps8, rps11, rps12(×2)**, rps14, rps15, rps16*, rps18, rps19(×2)
RNA polymerase rpoA, rpoB, rpoC1*, rpoC2
ribosomal RNAs rrn16(×2), rrn23(×2), rrn4.5(×2), rrn5(×2)
Transfer RNAs trnA-UGC(×2)*, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC*, trnH-GUG(×2), trnI-CAU(×2), trnI-GAU(×2)*, trnK-UUU*, trnL-CAA(×2), trnL-UAA*, trnL-UAG, trnM-CAU, trnN-GUU(×2), trnP-UGG, trnQ-UUG, trnR-ACG(×2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC(×2), trnV-UAC*, trnW-CCA, trnY-GUA, trnfM-CAU
Photosynthesis Photosystem_I psaA, psaB, psaC, psaI, psaJ, ycf3**, ycf4
Photosystem_II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ***
NADH oxidoreductase ndhA*, ndhB(×2)*, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Cytochrome b6/f petA, petB*, petD*, petG, petL, petN
ATP synthase atpA, atpB, atpE, atpF*, atpH, atpI
RUBISCO rbcL
ATP-dependent protease subunit P clpP**
Other genes Plastid envelope membrane protein cemA
Maturase matK
c-Type ccsA
Translation initiation factor infA
Subunit acetyl-CoA-carboxylate accD
Conserved ORFs ycf1(×2), ycf2(×2)
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Comparative analysis of plastid genome structure and polymorphism

The complete I. orchioides plastid genome length was 151,622 bp. The GC content was 38%, similar to that in other Iris species (37.9–38.3%, average = 38.0%; Table 1). The IR region was slightly shorter in three taxa of subg. Scorpiris [I. orchioides (25,508 bp), I. hippolyti (25,463 bp), I. pseudocapnoides (25,463 bp)] than in the other 17 Iris species (> 25,929 bp; Table 1). In contrast, I. germanica had significantly large IRs (28,849 bp). The size of each quadripartite section varied among the 20 Iris taxa, which was mainly attributed to pronounced variation in the LSC region [80,907 (I. loczyi)–83,237 bp (I. japonica)]. Overall, we found high sequence divergence among the 20 Iris taxa in the non-coding regions, whereas nearly no sequence variation was observed in the untranslated regions (Fig 2). There was little variation in the sequences of the coding genes across the 20 Iris taxa; however, some genes, such as ycf1, ycf2, and rpl16, harbored relatively higher levels of sequence divergence (Fig 2). The highest sequence polymorphism was found in I. loczyi, particularly for ycf2 (Fig 2).

Fig 2. mVISTA plot of the 20 Iris taxa.

Fig 2

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The bar indicates the percent sequence identity of the 20 Iris species. I. gatesii was used as a reference sequence to estimate the percent sequence identities.

The sequence variability among the 20 taxa was further explored using nucleotide polymorphism (π). The mean sequence diversity was 0.018 (ranging from 0.0005–0.06; Fig 3). The SSC region showed the highest mean sequence diversity (mean π = 0.031), whereas that estimated for the two IR regions was the lowest (mean π = 0.007; Fig 3). We identified 6 hypervariable regions (π ≈ or > 0.04) including 5 IGS regions: ycf1 and rps15-ycf1 IGS (π = 0.049–0.056), rpoB-trnC IGS (π = 0.044), petA-psbJ IGS (π = 0.042), ndhG-ndhI IGS (π = 0.04), and psbK-trnQ IGS (π = 0.04; Fig 3).

Fig 3. Nucleotide diversity (π) plot.

Fig 3

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The nucleotide diversity among 20 Iris taxa was estimated. The dashed lines are the borders of the LSC, SSC, and IR regions.

The gene content organization and gene size showed a few distinctive variations, although most of the genes were structurally conserved across the 20 Iris taxa (Fig 4). Expansions and contractions were observed in the IR regions of the 20 Iris taxa (IR size = 25,463–28,849 bp; Table 1). All 20 Iris taxa had trnH-GUG in the IR region, as observed in many monocot species (Fig 4). The border between the LSC and IRb regions was between rpl22 and rps19, except in I. japonica. The border in I. japonica was placed within the rps19 gene. The distance between rps19 and the border region varied from 21–45 bp (Fig 4). In I. dicotoma, the border between the IRb and SSC regions was located within the gene ycf1, showing a distinct 180-bp contraction (Fig 4). Although we observed IR region contractions and expansions, these changes did not affect the positions of the genes near the borders (Fig 4).

Fig 4. Comparison of the LSC, SSC, and IR region boundaries among the plastid genomes of 20 Iris taxa.

Fig 4

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Codon usage pattern

On average among the 20 Iris taxa, the coding sequence (CDS) length was ~79,680 bp. We found 79 genes encoded by 26,560 codons that were classified into 64 types. Of the 20 amino acids (AAs), leucine (2661–2752 encoding codons, 10.3%; S2 Table) was the most abundant, followed by isoleucine (1983–2302 encoding codons, 8.6%; S2 Table). The least abundant AA in the 20 Iris taxa was cysteine, encoded by 265–318 codons (1.2%; S2 Table). ATG was the initiator codon for most protein-coding genes, with three exceptions (rps19-GTG, rpl2-ACG, and ndhD-ACG). Relative synonymous codon usage (RSCU) did not differ significantly among the 20 Iris taxa (S2 Table). Two AAs, methionine (AUG) and tryptophan (UGG), were at usage equilibrium (RSCU = 1; S2 Table). The highest RSCU value was observed for codon AGA (~ 1.9), encoding arginine, whereas the lowest RSCU value was for codon AGC (~ 0.31), encoding serine. Iris dichotoma, I. ruthenica, and I. speculatrix had 32 codons with a RSCU > 1. In the remaining Iris species, 31 codons were more frequently used than expected at equilibrium (RSCU > 1). The codons mostly ended with A (~ 31%) or U (~ 37%).

Repeats

In total, 176 simple sequence repeats (SSRs) were identified in I. orchioides, with a minimum repeat number of 10 (Table 3). SSR numbers varied slightly across the 20 Iris taxa, ranging from 167 (I. rutherfordii) to 203 (I. lactea; Table 3). We identified seven repeat motif types (mono- to hexa-nucleotide SSRs and compound SSRs; Table 3). Among all 20 Iris taxa, the most common repeat motif was the hexa-nucleotide SSR, whereas the least frequently observed repeat motif was the penta-nucleotide SSR (Table 3). Nearly all the mono-nucleotide repeats were composed of A or T, except for the one with C located in ycf1 and/or in psbK-psbI IGS (S4 Table). Over 80% of the di-nucleotide SSRs comprised “AT,” and the repeat numbers varied from 10–20 (S4 Table). The number of tri- and tetra-nucleotide SSRs showed little variation among the 20 Iris taxa (Table 3). The repeat numbers of the penta- and hexa-nucleotide SSRs were mostly 10 and 12, respectively; however, 15 penta- and 18 hexa-nucleotide SSRs were observed in a few taxa (S4 Table).

Table 3. Summary of simple sequence repeats (SSRs) across varying unit sizes in 20 Iris taxa.

c denotes compound SSRs comprising more than two adjacent SSRs.

Number of SSRs
Species Unit size Total
1 2 3 4 5 6
Iris germanica 31 10 2 4 1 128 176
Iris afghanica 27 12 3 4 2 131 179
Iris bloudowii 32 12 3 4 2 132 185
Iris gatesii 31 12 3 4 3 135 188
Iris dichotoma 32 9 2 4 1 129 177
Iris domestica 45 10 3 4 1 128 191
Iris orchioides 34 7 3 5 1 126 176
Iris hippolyti 22 10 4 4 1 144 185
Iris pseudocapnoides 25 14 3 5 4 131 182
Iris rutherfordii 20 9 1 7 0 130 167
Iris tuberosa 33 11 2 6 1 141 194
Iris japonica 30 8 4 5 1 128 176
Iris lactea 34 20 2 7 1 139 203
Iris loczyi 28 10 4 5 1 122 170
Iris missouriensis 18 13 3 7 1 139 181
Iris pseudacorus 33 9 3 5 2 126 178
Iris ruthenica 36 14 2 7 1 130 190
Iris sanguinea 37 10 3 6 1 130 187
Iris speculatrix 38 11 4 3 3 139 198
Iris tectorum 30 9 2 6 1 123 171
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Identifying plastid markers and testing the utility in phylogenetic inferences

We inferred the plastome phylogeny and compared the results with the phylogeny inferred from the marker regions selected in our study. In the plastome phylogeny, Iris formed a monophyletic clade distinct from the outgroup taxa (Fig 5). Overall, the phylogeny of Iris was consistent with that proposed by Wilson (2011), with a few exceptions. Henceforth, we interpreted the results in accordance with Wilson’s taxonomic system. Taxa within Iris were divided into two well supported clades (Fig 5). The first clade was composed of taxa from subgenera Limniris, Xiphium, and Hermodactyloides, while the second clade harbored the taxa classified into the subgenera Iris, Scorpiris and Limniris (Fig 5; bootstrap support (BS) > 98). According to the PhyDesign results, we identified five genes with the highest phylogenetic informativeness (PI): matK, ndhF, rpoC2, ycf1, and ycf2 (Fig 6). Additionally, in combination with these five high PI genes, the five hypervariable Intergenic Spacer (IGS) regions (rps15-ycf, rpoB-trnC, petA-psbJ, ndhG-ndhI and psbK-trnQ; Fig 3) also demonstrated high informativeness for phylogenetic analysis.

Fig 5. Phylogenetic relationships among 84 accessions of Iris inferred using maximum likelihood (ML) methods based on whole-plastid genomes.

Fig 5

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The values presented on each node indicate the bootstrap support.

Fig 6. Phylogenetic informativeness profiles of 79 coding sequences of Iris estimated in PhyDesign.

Fig 6

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The five most informative regions are marked in color.

Based on the TreeDist score, identified the most effective marker for molecular diagnosis. The TreeDist result indicated that a concatenated dataset comprising a combination of three regions (rpoC2, ycf1 and ycf2) (TreeDist = 0.88; S5 Table, S2 Fig) displayed the highest congruence score with the complete plastome phylogeny. In addition, overall tree topology of the ML phylogeny inferred from the selected data was fairly congruent with that of the plastome tree (see the dashed lines for the concordances in Fig 7). Upon closer examination, we observed minimal incongruences within major clades (refer to Fig 7). In the ML tree constructed using the selected marker, the monophyly of the genus Iris was strongly supported. Additionally, the two main clades identified in the complete plastome tree formed monophyletic groups with robust node supports in the ML tree of the selected data (BS > 98). Consistent with the plastome phylogeny (Fig 7), two subgenera (subg. Iris and Juno) were found to be monophyletic with strong node supports (BS = 100), while others did not exhibit nested relationships. Minor discordances were primarily observed within sect. Oncocyclous (indicated by the blue colored clade in Fig 7). However, notably, the bootstrap support for each node in the tree generated from the selected marker was slightly lower compared to the plastome tree (Fig 7).

Fig 7. Phylogenetic relationships among 84 Iris accessions, inferred using maximum likelihood (ML) methods based on the whole plastome (left) and the best diagnostic marker, the concatenated data with the three high PI genes (rpoC2, ycf1, ycf2) (right).

Fig 7

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Dashed lines connect the same samples in both data set. Colors in the trees indicate a subgenus to which the clade belong. Blue- subg. Iris, Red-subg. Limniris, Purple- subg. Scorpiris, Green- subg. Hermodactyloides, Yellow- subg. Xiphium.

Discussion

In this study, we provided information for the whole plastid genome of I. orchioides, an endemic to Central Asia, and compared those of 20 Iris plastomes. We found that three Iris taxa (I. orchioides, I. hippolyti, and I. pseudocapnoides) showed larger structural and size variations than the other Iris taxa. We isolated molecular markers, such as SSRs and regions with high polymorphism, that could potentially be used to study Iris population genetics (Fig 3). More importantly, we pinpointed 5 genes (matK, ndhF, rpoC2, ycf1, and ycf2) with high PI values and 5 hypervariable IGS regions (rps15-ycf, rpoB-trnC, petA-psbJ, ndhG-ndhI IGS, and psbK-trnQ) in Iris, offering valuable tools for phylogenetic analysis (see Fig 6).

Like most angiosperm plastomes, the 20 Iris plastomes showed relatively conserved genomic structures, characterized by typical quadripartite structures [48]. The plastome size ranged from 120–170 kbp, falling within the expected size range for angiosperms [48]. Plastid genome structure primarily depends on the organization of IRs, as their size and arrangement often vary [49, 50]. Our comparative analysis revealed size and structural variations in the Iris plastomes, particularly in the IR size (Figs 2 and 4). According to the mVISTA result, five species (I. orchioides, I. japonica, I. hippolyti, I. tectorum, and I. pseudocapnoides) show a 500-bp deletion in the intergenic region around the ycf2 gene (Fig 4). Notably, these five species formed a monophyletic clade in the inferred plastome phylogeny (Fig 5). The ycf2 gene also showed a high PI values likely stemming from the notable size variation observed in this gene (Fig 6). Accordingly, this sequence deletion might contribute strongly to the close phylogenetic relationships among the five species, highlighting the efficacy of the gene as a phylogenetic marker in Iris. With a few exceptions, such as tobacco (171 kbp) and geraniums (217 kbp), length variations in complete plastid genomes are not common in angiosperms [23, 51]. However, similar to our observations, previous studies have reported large sequence variations in ycf2 genes and their neighboring regions [52, 53]. Unfortunately, the mechanism driving this prominent structural variation in ycf2 is still under investigation. Regardless of the causal mechanism, the variation is worth investigating in all Iris taxa, as such large deletions could help to avoid the homoplasy-related challenges in Iris phylogeny [54].

Although the divergence of plastome sequences varies among taxa, coding regions are generally more conserved than non-coding regions, e.g., introns and intergenic spacers [55]. Likewise, in the study, the sequence divergence was lower in the coding regions (mean π of coding sequences = 0.019) than in the non-coding regions (0.021). Overall, the sequence divergences were larger in the 20 Iris plastomes than those observed in other taxa (average π = 0.009 in three Papaver species, average π = 0.003 in three Cardiocrinum species, and average π = 0.0007 in six Hosta species) [56–58]. High sequence divergence was expected, considering the taxonomic characteristics of Iris. The genus Iris is one of the most heterogeneous genera and is endowed with several infrageneric groups showing high morphological and molecular variations, e.g., subg. Scorpiris or Belmacanda. A recent study [15] divided the genus into 23 different genera based on the high molecular variation found in 10 plastid genome regions. Similaly, we isolated six hypervariable sites located in the LSC and SSC regions (rpoB-trnC, petA-psbJ, and ycf1; Fig 3) that can be employed to elucidate shallow-level phylogenies among closely related taxa or in population genetic studies. Sequence alignment (S1 Fig) revealed that I. loczyi had the largest number of polymorphic sites in ycf1, the gene with the highest sequence divergence (π = 0.05; S3 Table). The ycf1 gene encodes a protein crucial for plant viability. Ironically, despite its essential function, the gene exhibits high polymorphism and proves to be useful in both shallow and deep phylogenies [59, 60]. Our data analysis found that ycf1 in I. loczyi exhibited two large deletions (~100 bp; S1 Fig). Collectively, this gene could be an excellent marker for identification and phylogenetic inferences in closely related Iris taxa. Variations such as the ycf1 indel should be further investigated using more Iris taxa to identify variations that could shed light on species and population diversification in the genus.

Codon assignment bias over individual codons is a predominant phenomenon in all living taxa [61]. The two primary factors characterizing codon usage patterns are genome composition and selection toward increased translation efficiency [62, 63]. We observed significant bias toward AU (69% of all codons) in the 20 Iris plastid genomes, which is consistent with previous reports on plastid genomes (Campbell and Gori, 1990). In general, the plastid genomes were extremely AT-rich and relatively GC-limited (30–40% of total sequences). It is likely that the primary cause of codon usage bias observed in the 20 Iris plastid genomes was the AU-rich compositional bias.

We isolated 167–203 SSRs from the 20 Iris taxa, which is rather higher than the numbers previously reported in angiosperm species (105 SSRs in Betula, [64] 130 in Paris, [65] 50 in Chenopodium, [66] 250 in Aconitum, [67] and 48 in Fagopyrum [68]). Given the plethora, neutrality, and high variability, SSRs are the most frequently employed molecular markers in population genetics [69]. Regardless of utility, identifying applicable SSRs is a costly and time-consuming process [70, 71]. Over 1,000 nuclear SSR markers in Iris have been developed [72], although the number of SSRs that can be applied may differ across the targeted taxa, owing to varying polymorphism and amplification levels. However, maternally inherited plastid markers applicable for population-level genetic studies are scarce. The plastid SSR markers we identified in this study offer a useful molecular tool to investigate genetic patterns of maternal inheritance at the population level. In future studies, the SSR markers newly documented in our study should further be tested for rate of polymorphism at population level with many genotype samples to determine the applicability.

Iris is one of the most notorious groups for taxonomic complications owing to the large morphological variation and frequent gene flow among congeners [7, 12, 13, 16, 18, 19]. Numerous classification schemes have been proposed for approximately 280 Iris taxa [7, 11–13, 16, 18, 19, 21], with many relying on floristic characteristics such as sepal beards, crests, seed arils, and subterranean organs, alongside a limited number of plastid markers [7, 21]. However, despite these efforts, the taxonomy of Iris remains elusive, particularly when considering commercial taxa [38]. To address these taxonomic challenges, comprehensive sampling of diverse molecular markers is essential.

When inferring a phylogeny of targeted taxa, selecting appropriate molecular markers is of great concern, as the selected markers can strongly affect overall topology and divergence time estimates [73]. Recently, phylogenetic informativeness (PI), which quantitatively predicts phylogenetic signal based on estimated mutation rates, has gained prominence and is commonly employed in phylogenetic studies across various taxonomic groups [44, 74–76]. We estimated PI and identified the 10 most highly informative regions. Among them, rpoC2 and ycf1 stood out, offering improved overall tree topology with higher node support compared to other high PI regions (Fig 7). In fact, rpoC2 and ycf1 have recently been recognized for their phylogenetic utility among angiosperms [77]. Accordingly, the rpoC2 and ycf1 genes hold promise as useful tools for inferring Iris phylogeny.

In our analysis, we systematically tested various combinations involving 5 high PI genes and 5 hypervariable IGSs to identify the optimal combination for the phylogeny of the 84 Iris taxa. Remarkably, our selection process resulted in selecting the concatenated dataset of three regions (rpoC2, ycf1 and ycf2; Fig 7, S5 Table). The phylogeny inferred using the selected dataset remained largely consistent with the whole plastome phylogeny, and the node support for most clades was robust. Section Oncocyclus exhibited notable incongruence between the phylogeny of the selected data and that of the complete plastome. Taxa within this section are known for their high morphological variability, and certain species have been found to be non-monophyletic in previous studies [2]. Additionally, subgenus Xiphium was found to be non-monophyletic in both the plastome tree and the chosen combination tree (Fig 7). This finding is consistent with recent studies indicating that subgenus Xiphium remained unresolved in plastome data [78]. The results suggest that concatenating the three high PI regions as phylogenetic markers can serve as a cost-effective alternative to sequencing the entire plastome. Applying whole-plastome data for phylogenetic analysis may not always be the most efficient approach, as it can be costly. Moreover, relying solely on whole-plastome phylogeny may not consistently provide the best resolution due to certain limitations inherent to this approach. The over-representation of regions with high variation in whole-plastome data can potentially introduce errors when inferring phylogeny [79, 80]. By employing the high PI regions as markers without the need to sequence the entire plastid genome, we can potentially address phylogenetic complexities and enhance molecular diagnoses between closely related Iris taxa at a reduced cost.

Supporting information

S1 Fig. Sequence alignment of the ycf1 region across 20 Iriss species.

(JPG)

pone.0301346.s001.jpg (255.9KB, jpg)
S2 Fig. Phylogenetic relationships among 84 accessions of Iris inferred using maximum likelihood (ML) methods based on each 10 high PI regions.

(PDF)

pone.0301346.s002.pdf (450.8KB, pdf)
S1 Table. The summary of the plastid genome sequences downloaded from GenBank for phylogenetic analysis.

(XLSX)

pone.0301346.s003.xlsx (12.8KB, xlsx)
S2 Table. Codon usage pattern of 20 Iris species.

(XLSX)

pone.0301346.s004.xlsx (71.2KB, xlsx)
S3 Table. Sequence divergence (π) among the 20 Iris species.

(XLSX)

pone.0301346.s005.xlsx (40KB, xlsx)
S4 Table. Simple sequence repeats (SSRs) across varying unit sizes in 20 Iris species.

(XLSX)

pone.0301346.s006.xlsx (70.6KB, xlsx)
S5 Table. TreeDist distances among concatenated 10 high PI regions.

(XLSX)

pone.0301346.s007.xlsx (33.3KB, xlsx)

Acknowledgments

We thank Dr. K. Sh. Tojibaev for their help in collecting samples and in formal identification.

Data Availability

Raw genomic data used in the analyses are deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) with accession number, PP475539.

Funding Statement

This research was funded by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00212808).

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Decision Letter 0

Branislav T Šiler

23 Jan 2024

PONE-D-23-41497Complete chloroplast genome of Irisorchioides and comparative analysis with 13 Iris plastomesPLOS ONE

Dear Dr. Lee,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Both reviewers questioned the reason for selecting 13 additional plastid genomes among the 40 reported within the genus Iris. Please provide theoretical background for this decision. Additionally, parameters for the selection of 5 genes for further use in phylogenetic analyses in the genus Iris are not clearly presented. Some disagreements have also been noted by both reviewers between the M&M and the Results sections. The authors should put additional effort to clarify these inconsistences and to properly discuss them in the Discussion section.

Please use the term "plastid" instead of "chloroplast" and "CP" throughout the text (including the main title), because various plastid forms (chloroplasts, chromoplasts, leucoplasts, amyloplasts, and elaioplasts) contain the same genome as chloroplasts and from the section "Sampling, DNA isolation, and sequencing", I see the authors did not isolated chloroplasts from the other plastids prior to DNA extraction.

Abstract: I could not understand the statement "genomic information for the Iris chloroplast is limited". Can you please rephrase it to clarify its meaning. In the next sentence, the phrase "of the Iris chloroplast" implies that all Iris species contain the same chloroplast. Would "of plastids within the genus Iris" be better? In the same sentence, please shorten the genus name in "Iris orchioides" as "I. orchioides".

Further in Abstract: what "five selected sites" is related to? Where are these sites? Can you be more precise? In the next sentence, "five identified genes" does not tell much. First, how were they identified? Maybe selected? And, of course, please list the selected genes and criteriums for their selection here.

Introduction: Please italicize "Juno", as it presents a synonym for the genus.

==============================

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We look forward to receiving your revised manuscript.

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Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: N/A

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The researchers successfully assembled the chloroplast genome of Iris orchioides and conducted comparative genomic analysis with the chloroplast genomes of 13 previously published Iris species. They identified highly polymorphic gene regions within the Iris chloroplast genome, utilizing these as markers to identify the phylogeny of Iris. The data analysis appears to be robust, lending substantial support to the majority of the conclusions drawn in this study. However, the study still presents several issues.

The key issues identified in the study are:

1. The paper states that 40 chloroplast genome sequences of Iris species are available. However, the reason for selecting 13 of these for comparative analysis with the I. orchioides chloroplast genome sequence is not clear. The text should explicitly justify this selection.

2. The interpretation of the constructed phylogenetic tree is missing, as is a discussion on how these results contribute to differentiating Iris species. Furthermore, there is no comparison of these findings with prior phylogenetic analyses based on morphology and nuclear genes.

3. The absence of population-level chloroplast genome data precludes inferring the suitability of the identified markers for population data.

4. The methodology for narrowing down the ten regions with high Pi values to five candidate sites remains unclear. Detailed explanation in the text is required.

5. The conclusion drawn from Figure 7, suggesting that the ten genes approximated the entire chloroplast genome in inferring the phylogeny of Iris, requires further clarification and evidence.

The minor issues identified in the study are:

1. Numerous textual errors are present notably in the method section (specific line numbers not provided) and line 182.

2. Figure 1 lacks an indication of the transcription direction.

3. The font of Table 1 should be modified.

4. The identification of outgroups in Figures 5 and 6 is unclear, and the significance of the various colors used is not delineated.

5. In Figure 2, areas of high polymorphism should be emphasized to enhance visibility for readers.

6. The manuscript’s English language quality requires substantial improvement.

Reviewer #2: In this study, the authors newly sequenced the cp genome of Iris orchioides and comparatively analyzed the cp genomes of other Iris species. This study had a scientific significance. However, some questions need to be revised as follows:

1. In the introduction section, S, and some groups fail to form monophyletic groups in most molecular phylogenies?? The phylogenetic relationships were not solved using some molecular markers? Please clarify it in detail. The classification of genus Iris should be detailed, and the information of subgenus was mentioned in the main text.

2. The cp genomes of 40 Iris species were released. Why only 13 species were used to compare in this study?

3. In the method section, the results of phylogenetic analysis were not consistent with the description of the method.

4. In the table 1, the classification of each species had better to be added.

5. In the results section, the description of the figure 6 and 7 was not consistent with the results. In addition, figure 5 showed the phylogenetic tree based on the cp genome data. However, another tree had also included this tree. Figure 5 should be deleted.

6. The author selected ten regions with high-pi values, their phylogenetic tree should be also shown in the text.

7. What method was used to select the five regions? Whether was the five markers suit for these analyzed species? How about other species?

8. Where was the Table S5?

9. The title of axis should be added in figure 7.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2024 Apr 5;19(4):e0301346. doi: 10.1371/journal.pone.0301346.r002

Author response to Decision Letter 0

13 Mar 2024

We appreciate the insightful comments from anonymous reviewers and the handling editor. The manuscript has been improved based on the comments. To navigate the updates in the revised manuscript, please use the review function in MS word. We made the changes available using the track and change function in MS word.

Attachment

Submitted filename: Response to reviewers_PLOS_RV_1 (1).docx

pone.0301346.s008.docx (23.4KB, docx)

Decision Letter 1

Branislav T Šiler

15 Mar 2024

Complete plastid genome of Iris orchioides and comparative analysis with 19 Iris plastomes

PONE-D-23-41497R1

Dear Dr. Lee,

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,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Branislav T Šiler

24 Mar 2024

PONE-D-23-41497R1

PLOS ONE

Dear Dr. Lee,

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on behalf of

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Academic Editor

PLOS ONE

Associated Data

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

    Supplementary Materials

    S1 Fig. Sequence alignment of the ycf1 region across 20 Iriss species.

    (JPG)

    pone.0301346.s001.jpg (255.9KB, jpg)
    S2 Fig. Phylogenetic relationships among 84 accessions of Iris inferred using maximum likelihood (ML) methods based on each 10 high PI regions.

    (PDF)

    pone.0301346.s002.pdf (450.8KB, pdf)
    S1 Table. The summary of the plastid genome sequences downloaded from GenBank for phylogenetic analysis.

    (XLSX)

    pone.0301346.s003.xlsx (12.8KB, xlsx)
    S2 Table. Codon usage pattern of 20 Iris species.

    (XLSX)

    pone.0301346.s004.xlsx (71.2KB, xlsx)
    S3 Table. Sequence divergence (π) among the 20 Iris species.

    (XLSX)

    pone.0301346.s005.xlsx (40KB, xlsx)
    S4 Table. Simple sequence repeats (SSRs) across varying unit sizes in 20 Iris species.

    (XLSX)

    pone.0301346.s006.xlsx (70.6KB, xlsx)
    S5 Table. TreeDist distances among concatenated 10 high PI regions.

    (XLSX)

    pone.0301346.s007.xlsx (33.3KB, xlsx)
    Attachment

    Submitted filename: Response to reviewers_PLOS_RV_1 (1).docx

    pone.0301346.s008.docx (23.4KB, docx)

    Data Availability Statement

    Raw genomic data used in the analyses are deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) with accession number, PP475539.

    Articles from PLOS ONE are provided here courtesy of PLOS

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